Rat Models of Central Nervous System Injury

C H A P T E R

29 Rat Models of Central Nervous System Injury Blythe H. Philips1, Kevin D. Browne2, D. Kacy Cullen3, Samer M. Jaber4,5 1

University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA, United States; 2Neurosurgery, University of Pennsylvania, Philadelphia PA, United States; 3Neurosurgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; 4Department of Animal Medicine, University of Massachusetts Medical School, Worcester, MA, United States; 5Department of Pathology, University of Massachusetts Medical School, Worcester, MA, United States

I. OVERALL INTRODUCTION The central nervous system (CNS) plays a pivotal role in virtually all mammalian functions, including cognitive behavior, motility, perception, and homeostasis/ regulation. Trauma to the CNS can therefore have devastating consequences due to the disruption of complex neural circuitry underlying these critical functions, which is compounded by the limited regenerative capacity of the brain and spinal cord. In particular, traumatic brain injury (TBI) and spinal cord injury (SCI) are unique from other neurological afflictions in that they are induced by a discrete physical event, and therefore occur in patients across the spectrum of age, sex, and baseline health. Indeed, given the complexity of the CNS, the variability of traumatic injury conditions (no two traumas are the same) and patient heterogeneity, it is clearly a daunting challenge to understand and treat the resulting sequelae. For at least 100 years, animal models have proven to be an invaluable resource in improving our understanding of trauma-induced pathophysiology and functional consequences, as well as evaluating the efficacy of potential neuroprotective or restorative treatments. In particular, rodent models have seen widespread utility in providing a basic and economical platform to study TBI and SCI. There are multiple established rat models of both TBI and SCI, each intended to replicate various features of clinical CNS trauma based on injury mechanisms, pathophysiological features, and underlying assumptions relating rat anatomy, physiology, and genetic underpinning to those of humans. In this chapter, we present descriptions, methods, experimental

The Laboratory Rat, Third Edition https://doi.org/10.1016/B978-0-12-814338-4.00029-5

considerations, and limitations for the laboratory implementation of the most common rat models of TBI and SCI.

II. SPINAL CORD INJURY A. Introduction to Spinal Cord Injury The major goals of translational SCI research are to gain insight into pathological processes and recovery mechanisms after trauma to develop therapies that improve functional outcomes in human patients (Fouad et al., 2013; Reier et al., 2012; Cheriyan et al., 2014). The incidence of new SCIs is 12,000 cases per year in the United States with a prevalence of approximately 230,000e260,000 living SCI patients in the United States and up to 40 million worldwide (Sekhon and Fehlings, 2001; Hook, 2013). In addition to the impact on patient physical and emotional health, lifetime monetary healthcare costs for cervical injury can exceed US$2 million during the patient’s lifetime and the impact on society can be compounded due to the loss of economic productivity from inability to work (Sekhon and Fehlings, 2001). Motor vehicle accidents account for approximately 40% of the SCI cases in human patients, followed by sports injuries, acts of violence, and falls (Hook, 2013; Sekhon and Fehlings, 2001). SCI-associated pathology can be divided into primary and secondary injuries. Primary injury is due to the mechanical disruption of the cells and tissue of the spinal cord due to physical forces, whereas secondary injuries are subsequent evolving events that lead to further

1023

Copyright © 2020 Elsevier Inc. All rights reserved.

1024

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

damage to the CNS, such as inflammation, excitotoxicity, edema, vascular abnormalities, and neurodegeneration (LaPlaca et al., 2007; Kwon et al., 2004). Primary and secondary injuries both contribute to outcomes from SCI, including permanent motor and sensory impairment (Grill, 2005). Functional deficits in sensory and motor ability are the primary clinical signs after SCI. The American Spinal Injury Association (ASIA) categorizes the degree of functional and sensory impairment into five categories from A (completedno sensory or motor function) to E (normal function), with categories BeD representing varying degrees of partial functional deficits (Lukovic et al., 2015; Ditunno et al., 1994). Sequelae of SCI include chronic pain, motor dysfunction (including increased muscle tone/spasticity, or decreased absent muscle tone), sensory dysfunction (including hypo- and hypersensitivity), muscle atrophy, decubitus, urinary tract infections, and sexual dysfunction (Kjell et al., 2013; Kjell and Olson, 2016; Adams and Hicks, 2005). Chronic pain is often refractory to treatment and can lead to work loss independent of functional deficits, as well as severe depression and suicide (Mills et al., 2001; Nakae et al., 2011; Cao et al., 2014). Up to 80% of SCI patients suffer from some degree of chronic pain postinjury (Nakae et al., 2011; Siddall et al., 2003; Finnerup et al., 2001). Recovery after SCI is complicated by the spinal cord’s limited ability to regenerate (Young, 2014). No current therapies are sufficiently effective at restoring preinjury levels of function and there is an urgent need to discover novel therapeutic strategies (Lukovic et al., 2015; Baptiste and Fehlings, 2007). The limited treatments currently available mainly focus on patient stabilization and mitigation of secondary injury progression (Kwon et al., 2004). This can include nonpharmaceutical approaches, such as decompression surgery (Fehlings et al., 2012) or physical rehabilitation (Baptiste and Fehlings, 2007), pharmaceutical approaches such as inflammation-modulating compounds, as well as nextgeneration experimental treatments such as cell-based therapies (Baptiste and Fehlings, 2007; Kwon et al., 2004).

B. Rats as Models of Human SCI: Anatomy and Response to Injury Rats have become the primary model from which knowledge of the pathophysiology of SCI and response to experimental treatments is derived (Vogelaar, 2016; Kjell and Olson, 2016; Cheriyan et al., 2014; Onifer et al., 2007b). Rats were used in >70% of published animal model SCI studies identified by one literature review (Sharif-Alhoseini et al., 2017). Rats are popular models for SCI research because they are widely

available, inexpensive, easily handled and cared for, trainable, present a low reported incidence of surgical site infection, and a plethora of well-validated methodologies have been established in this species (Lukovic et al., 2015; Cheriyan et al., 2014). Rat models replicate features of the biochemical, morphological, and functional outcomes seen in people following SCI (Onifer et al., 2007b; Metz et al., 2000a). While there are many advantages to using rats to study SCI, it is important to identify the areas where model limits exist and improvements can be made. Many of these limitations will be discussed in this chapter; for more in-depth discussion, the reader is referred to any of the excellent reviews that have been written on this topic (Onifer et al., 2007b; Cheriyan et al., 2014; Fouad et al., 2013; Reier et al., 2012; Kwon et al., 2002). A detailed anatomical comparison of the rat and human spinal anatomy is beyond the scope of this chapter and is available elsewhere, but investigators should be mindful of interspecies differences that may contribute to variations in outcome (Watson et al., 2009). For example, the spinal cord of the rat ends at the L3 vertebra, whereas the human spinal cord ends near L1/L2, which may have implications for researchers planning for surgery or imaging. The number and distribution of spinal segments is also different between rats and humans. Rats have a total of 34 segments, whereas humans have only 31. The make-up of the spinal regions is also somewhat different; although both rats and humans have eight cervical segments, rats have one more thoracic (13) and lumbar (6) segment, two more coccygeal segments (3), and one less sacral (4) segment than humans (Watson et al., 2009). Another obvious difference between the rat and human spinal cord is size; the human spinal cord is four times longer than the length of the entire rat CNS (Akhtar et al., 2008). This size difference has real implications for the use of rats in translational spinal cord research. Because axonal growth rates are generally approximated to be 1 mm per day, rats could regenerate axons to bridge functional gaps between the brain and distal sites in months, whereas in humans it would take years (da Costa et al., 2008; Dobkin and Havton, 2004). Similarly, rat spinal cords have much smaller white matter tracts (roughly 800,000 axons vs. the human spinal cord, which contains 20 million), so rats may reach the 5% regeneration threshold (the lower limit of where a degree of functional recovery is possible) more easily than human patients (Fehlings and Tator, 1995). Lastly, some have argued that differences in spinal cord circuitry between quadrupeds and bipeds, such as a higher reliance on peripheral neural stimulation during the recovery of locomotion following SCI in quadrupeds, may limit the translatability of conclusions drawn in rodent SCI studies (Saunders et al., 2017).

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

1025

FIGURE 29.1 Histological differences in wound cavitation in mice and rats at 0 (A and B), 2 (C and D), 8 (E and F) and 15 days post injury (G and H). In rats, SCI lesions form into cavitations that extend rostral and caudal to the injury site over time, similarly to what happens in humans. In contrast, in mice the lesion fills in with solid fibrous scar tissue. Hematoxylin and eosin staining. Scale bars = 200 mm; arrowheads in D, F, and H are cell-free areas in rat SCI cavities. Reproduced from Surey, S., Berry, M., Logan, A., Bicknell, R., Ahmed, Z., 2014. Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 275, 62e80.

Although greater availability of genetically modified mice may preserve a role for that species in SCI research, especially when interrogating specific genetic influences on disease and recovery, the rat maintains a number of advantages over the mouse. Rats may be anatomically and behaviorally more well suited than mice for SCI research. They are easier to handle and train for behavioral tasks, and have larger anatomy for surgery and imaging (Ellenbroek and Youn, 2016; Kjell and Olson, 2016). Additionally, new technologies may be closing the gap in

ability to manipulate the rat genome compared to mice, as discussed in Chapter 7 of this text. Pathology after SCI in the rat spinal cord resembles the human condition more compared to mice (Metz et al., 2000a). In rats and humans, SCI lesions form cavitations that extend rostral and caudal from the injury site over time, whereas in mice the lesion fills in with solid fibrous scar tissue (Fig. 29.1) (Byrnes et al., 2010; Surey et al., 2014). The difference in tissue response to injury may be attributed to inflammatory cell populations infiltrating the injury site,

RATS AS RESEARCH MODELS

1026

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

secretion of factors promoting wound healing, angiogenesis, and differences in bloodespinal cord barrier permeability. All of these are important players in the pathophysiology of injury, and many have argued that rats are a superior platform for the study of the pathophysiology of human SCI due to their tendency to develop cavitation bordered by a defined glial scar at the injury site (Surey et al., 2014; Guth et al., 1999; Byrnes et al., 2010; Sroga et al., 2003).

C. Rat Spinal Cord Injury Models The ideal rat model for traumatic human SCI would reliably and reproducibly mimic aspects of the human condition, including both primary injury and secondary or downstream effects (Onifer et al., 2007b). It is desirable to have models that approximate millisecond loading rates similar to those observed in human SCIs (Nightingale et al., 1996). Additional desirable characteristics include allowing for gradability of injury severity so that structural and functional elements of injury and recovery can be compared. The use of injury models with titratable severity also allows investigators to refine procedures so that animals are injured “enough” to study treatment interventions without the possibility of spontaneous recovery, but not so severely that evidence of functional recovery is masked or unattainable (Onifer et al., 2007b; Dabney et al., 2004). Several institutions offer training on the creation and evaluation of SCI animal models. In addition to considering how closely the injury itself approximates the human condition and how tightly controlled the injury can be, investigators should consider how closely their model approximates the environment in which SCIs occur within the patient population. For example, the vast majority of rat SCI models require the performance of a laminectomy for investigators to access the spinal cord. The removal of parts of the spinal column can confound these models in several ways. First, removal of muscle and bone during the surgical approach to the spinal cord may create spinal column instability, which could confound injury as well as recovery. Second, laminectomy is an invasive surgical procedure that not only carries the risks of any major surgery (such as hemorrhage, iatrogenic meningeal or cord trauma, infection, or anesthetic death), but also introduces additional pain and stress beyond that of the experimental injury. Any of these could confound the interpretation of outcome measures, in particular behavioral ones. Lastly, the removal of elements of the rigid vertebral column effectively opens what was previously a closed, volume-fixed space, which can lead to vast reductions in the pressures faced by the tissues at the injury site (Marcol et al., 2012; Hook, 2013; Akhtar

et al., 2008). Vertebral decompression not only has the potential to alter pathology at the site of injury, it has also been used therapeutically in a clinical setting and could vastly change clinical outcomes (Hook, 2013; Kjell et al., 2013). Another major difference between experimental and naturally occurring SCI is that virtually all animal models of trauma are performed on anesthetized animals for ethical reasons. While the community agrees that this is necessary for the welfare of the animals, it is important to keep in mind that the anesthetics used and their effects on animal muscle tone and systemic physiology may influence injury parameters in unexpected ways, some of which will be discussed later in this chapter (Blickman and Brossia, 2009). Finally, it is important to note that in the vast majority of human SCIs, damage to the cord is more multifactorial than the tightly controlled injuries that are created in most animal models. Humans commonly suffer comorbidities such as fractures, hemorrhage, infection, shock, and organ failure, and are typically administered multiple drugs that may have contradictory systemic effects (Akhtar et al., 2008; Lukovic et al., 2015). Furthermore, human injuries are generally complex combinations of injuries, as opposed to pure “contusion” or “compression” injuries (Habgood, 2011). As such, the utility of animal models in SCI is not so much to capture the vast complexity of the human condition, but rather to help investigators piece together different elements of spinal cord pathophysiology to better pursue relevant interventions. As with any CNS injury model, injury location is of critical importance. Injuries to each of the four main regions of the spinal cord (cervical, thoracic, lumbar, sacral) are associated with different clinical signs. Higher-level injuries generally result in more severe outcomes, although injuries in any region can have grave implications for quality of life. For example, injuries in the cervical region generally lead to quadriplegia, and are considered more clinically relevant to humans due to their high prevalence in the patient populations, as well as their relationship with maintenance of fine motor hand function (Choo et al., 2009; Onifer et al., 2007b; Hodgetts et al., 2009; Hook, 2013). High cervical injuries are often fatal due to disruption of the respiratory center (Sharif-Alhoseini and Rahimi-Movaghar, 2014; Hook, 2013). In contrast, low thoracic injuries often result in hindlimb paralysis, sensory deficits, and below-injury autonomic dysfunction largely due to white matter disruption. Lumbosacral lesions are typically associated with poor lower limb control, incontinence, and sexual dysfunction, whereas sacral injuries may yield deficits limited to just the tail (Kjell and Olson, 2016; Hook, 2013; Bennett et al., 1999). In addition to the research goals, investigators must carefully consider the expected clinical outcomes and resultant supportive care that animals will require postinjury before choosing a region of

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

spinal cord for their focus. In addition to differences in clinical signs developed following injury to each region, parameters like blood supply, cord diameter, tract directionality, and cell population all vary by spinal cord level and can directly influence the outcome of injury, which limits comparisons of injuries and interventions between regions (Akhtar et al., 2008; Sharif-Alhoseini and Rahimi-Movaghar, 2014; Cheriyan et al., 2014). The most common human SCIs are mixed contusione compression type injuries to the ventral (anterior) cord (Hook, 2013). Despite this, historically, rat SCI models have been overwhelmingly (81%) focused on injury to dorsal tracts within the thoracic region, in part because this region is easily accessible, but also out of concerns regarding heightened morbidity and mortality and welfare concerns related to animals with many types of cervical injury (Cheriyan et al., 2014; Akhtar et al., 2008; Habgood, 2011; Sharif-Alhoseini and RahimiMovaghar, 2014; Zhang et al., 2014a, 2014b; SharifAlhoseini et al., 2017). To address this, as better tools become available and injury techniques are refined, cervical-level rat injury models and injury to the ventral aspect of the spinal cord are becoming more commonplace (Dunham et al., 2010; Pearse et al., 2005; Geissler et al., 2013; Hook, 2013). The vast majority of human SCIs are traumatic in nature, caused by motor vehicle accidents, falls, athletic injuries, or violence (Habgood, 2011). The type of spinal cord trauma can further be classified into three main categories according to mechanism of injury, including contusion, compression, and laceration/transection (Cheriyan et al., 2014). Injuries may also be categorized into complete (generally limited to complete transection injuries, which are rare in humans) and incomplete. Completeness of injury has obvious implications for severity of outcome, because complete injuries always require intensive nursing care, while incomplete injuries may vary from mild to severe (Hodgetts et al., 2009). Given the traumatic and unpredictable nature of these injuries, naturally occurring human SCIs are generally very complex, and a single patient will often experience multiple different categories of insult following a single traumatic event (Habgood, 2011). Efforts to replicate subsets of human injury in rats have led to the development of models in each of these categories (Choo et al., 2009; Dabney et al., 2004; Fiford et al., 2004; Grill, 2005; Akhtar et al., 2008, Puckett et al., 2009). Details of how the injuries are created, as well as some benefits and drawbacks associated with different strategies, are outlined next. 1. Contusion Contusions are the most common category of investigational rat SCI (Sharif-Alhoseini and RahimiMovaghar, 2014; Hook, 2013; Sharif-Alhoseini et al., 2017). Spinal cord contusions are created when a transient

1027

force is applied to the cord, resulting in trauma and subsequent tissue damage. This type of injury has been shown to reliably replicate many aspects of spinal cord pathology seen in humans with similar injuries, including the creation of glial scarring and cavitary lesions that can complicate healing, making contusions more directly clinically relevant than transection injuries (Hodgetts et al., 2009; Choo et al., 2009; Habgood, 2011; Kwon et al., 2002). There are a number of techniques that have been described to create spinal cord contusions in rats. The primary difference between these models is the mechanism by which the injury is created and controlled. Some of the most commonly used rat spinal contusion models are (1) weight drop, (2) force controlled, (3) displacement controlled, and (4) air-gun impactor (Table 29-1) (Habgood, 2011; Cheriyan et al., 2014). a. Weight Drop The Allen weight-drop technique was the first animal model for spinal contusion injury, developed in dogs in 1911 (Allen, 1911). Over the subsequent century, in response to the need for a reproducible highthroughput platform to study treatment interventions for SCI and the rising popularity of rodents in biomedical research, this technique was adapted for use in the rat. Weight-drop paradigms continue to be the most popular strategy for creating experimental contusion injuries in rats today (Sharif-Alhoseini et al., 2017; Marcol et al., 2012; Wrathall et al., 1985; Gale et al., 1985). While weight-drop devices vary somewhat, the principles are the same for each of them: an injury is created when an object of set mass is dropped from a defined height onto the rat’s exposed (laminectomized) spinal cord (Jakeman et al., 2009). Initial weight-drop models had high variability, due to differences in forces encountered by the weight during “freefall” down the guide tube, differences in vertebral stabilization during injury, and differences in whether the weight was dropped directly onto the cord or onto an impounder plate (Onifer et al., 2007b; Koozekanani et al., 1976; Khan and Griebel, 1983). In part, these concerns led to attempts to more tightly control injury protocols, such as the creation of the NYU impactor model: a weight-drop apparatus that allowed for automated recording of parameters such as velocity of weight at impact, cord compression, and height of weight drop. This automated data acquisition allowed investigators to have greater confidence in the consistency of the injury produced, and also allowed for exclusion of animals with anomalous values (Gruner, 1992; Kwon et al., 2002; Onifer et al., 2007b). A National Institutes of Health-funded effort used the NYU impactor device for a large-scale Multicenter Animal Spinal Cord Injury Study (MASCIS) to create a validated, thoroughly standardized model platform for the study of treatment interventions. In addition to

RATS AS RESEARCH MODELS

TABLE 29.1

Comparison of devices used to create experimental spinal cord contusion injuries in rats. Adapted by permission from Macmillan Publishers Ltd: Cheriyan, T., Ryan, D.J., Weinreb, J.H., Cheriyan, J., Paul, J.C., Lafage, V., Kirsch, T., Errico, T.J., 2014. Spinal cord injury models: a review. Spinal Cord 52, 588e595. % of Animal SCI Contusion Studies Using This Technique (1946e2016)a

Injury Device

Factor Driving Injury

Strengths

Drawbacks

Weight drop/NYU/ MASCIS

Height/mass of weight dropped

More recent versions very reproducible

Requires laminectomy; force of impact and degree/duration of compression not controlled; "bounce" injury possible

IH

Force applied directly to cord

Reproducible; Requires laminectomy; degree and duration 21 commercially available; of cord compression not controlled; no bounce injury difficulty stabilizing vertebral column

Scheff and Roberts (2009), Scheff et al. (2003), Popovich et al. (2012), Sandrow et al. (2008), Dunham et al. (2010)

OSU/ESCID

Displacement of cord

Reproducible; no bounce injury

Air-gun impactor

Force of air ejected onto Relatively noninvasive; cord no bounce injury

65 (37 NYU/MASCIS, 28 other weight drop)

Representative References Gruner (1992), Dunham et al. (2010), Soblosky et al. (2001), Gensel et al. (2006), Simard et al. (2007)

Requires laminectomy; potential for priming injury; complexity of device

4

Jakeman et al. (2009) , Noyes (1987), Stokes (1992), Yeo et al. (2004), Pearse et al. (2005)

Not yet well validated

0.2

Marcol et al. (2012)

ESCID, Electromagnetic Spinal Cord Injury Device; IH, Infinite Horizon impactor; MASCIS, Multicenter Animal Spinal Cord Injury Study impactor; NYU, New York University impactor, OSU, Ohio State University impactor. a Based on the data in Sharif-Alhoseini et al. (2017); includes nonrat studies. More than 72% of SCI research using animal models in this timeframe was performed in rats.

II. SPINAL CORD INJURY

standardizing the device inducing injury itself, the MASCIS protocol carefully standardized every aspect of injury, including age and stock of rat and anesthetic used (Basso et al., 1996; Cheriyan et al., 2014; Kwon et al., 2002; Young, 2009). Today, the terms MASCIS impactor and NYU impactor are often used interchangeably to describe similar devices, which are probably the most widely used weight-drop injury devices used to create spinal contusions in rats (Fig. 29.2) (Kwon et al., 2002). Subsequent versions of the MASCIS impactor have incorporated controls, such as electromagnetic push-button weight release, to further reduce variability from experiment to experiment (Cheriyan et al., 2014). While weight-drop devices have predominantly been used to create thoracic contusions, cervical injuries can also be created with some modifications to the technique; for example, creation of a unilateral hemicontusion spares function on the side contralateral to the injury, thus reducing morbidity and mortality. This strategy also allows for a within-subject control, because there will be both injured and noninjured tissue in the same spinal cord segment (Soblosky et al., 2001; Simard et al., 2007; Cheriyan et al., 2014; Gensel et al., 2006; Dunham et al., 2010). The MASCIS impactor has also been used for the production of lumbosacral contusions (Wen et al., 2015). A technique has also been described that uses custom-made Teflon spacers placed into the dorsal epidural space in conjunction with the MASCIS impactor to study the concurrent effects of contusion along with spinal cord narrowing, which may occur following trauma (Dimar et al., 1999). Weight-drop models have been touted for their ability to replicate functional, morphologic, and electrophysiologic outcomes of human chronic SCI, and are relatively

FIGURE 29.2 Representative image of the MASCIS impactor, which is used to create experimental spinal cord contusions in rats. Image courtesy of Dr. Wise Young, W. M. Keck Center for Collaborative Neuroscience, Rutgers, The State University of New Jersey.

1029

easily controlled and reproducible. They are also considered gradable, with severity of injury generally correlated with the height from which the weight is released (Metz et al., 2000a; Kwon et al., 2002). These devices are simple and inexpensive to assemble relative to some other injury apparatuses, which contribute to their continued widespread use in the field (Gruner, 1992; Habgood, 2011). However, there are several drawbacks to using weightdrop models to create spinal cord contusions. First, these models generally require laminectomy, which is not ideal for reasons previously discussed. Second, the injury is primarily focused on the dorsal tracts of the spinal cord, whereas human contusion injuries may involve dorsal, ventral, or both tracts (Hook, 2013; Metz et al., 2000a). While factors like the mass dropped, velocity at time of impact, and height of fall can be tightly regulated, others, such as force of impact, degree of compression, and duration of compression, are typically out of the control of the user and subject to variation based on factors such as cord diameter and viscoelasticity (Gruner, 1992; Habgood, 2011). Furthermore, while more recent weight-drop models have improved in this regard, they still carry the potential for the weight to “bounce” off the surface of the spinal cord, which can lead to an increased severity of injury through rebound (Habgood, 2011; Cheriyan et al., 2014; Onifer et al., 2007b). b. Force Controlled Force-controlled contusion devices create spinal cord contusions through the application of a controlled force to the surface of the rat’s exposed spinal cord (Scheff and Roberts, 2009; Kwon et al., 2002). One such device is the widely used and commercially available Infinite Horizon (IH) impactor. This device is available for purchase from a commercial vendor, relatively lightweight, and was designed to be “user friendly,” which has greatly driven popularity in recent years (Silva et al., 2014; Scheff et al., 2003). In contrast to the weight-drop mechanisms, where the injury is driven by gravity and the height from which the weight is dropped, these injuries are driven by an electric stepper motor that deploys the impounder tip toward the intact dura on the exposed spinal cord. In the case of the IH impactor, once the desired force is applied to the spinal cord, the impactor autoretracts, preventing the type of “after bounce” or rebound injury that commonly occurs with weight-drop paradigms. This same feature helps reduce motion artifact, such as that which may stem from animal respirations (Habgood, 2011; Scheff and Roberts, 2009; Scheff et al., 2003). Like the weightdrop model, this style of device has more recently been adapted for use in the cervical spine (Dunham et al., 2010; Popovich et al., 2012; Sandrow et al., 2008). Similar to weight-drop models, force-controlled devices do not control the degree or duration of cord compression (Habgood, 2011). The relatively slow speed

RATS AS RESEARCH MODELS

1030

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

at which the IH impactor executes its injury (0.1 m/s) has been criticized as not relevant to the more rapid, naturally occurring human injury (Zhang et al., 2014b; Nightingale et al., 1996). In addition, these models require laminectomy. Lastly, some users report difficulty stabilizing the vertebral column when using the IH impactor (Cheriyan et al., 2014), which in some cases has been addressed by engineering-enhanced vertebral stabilization systems (Lee et al., 2012; Streijger et al., 2013). c. Displacement Controlled Both force of impact and spinal cord displacement factor heavily in determining the severity of SCI (Grill, 2005). In contrast to the previously described injury mechanisms, several devices have been designed to provide the user precise control of spinal cord displacement. The most well known of these devices is the Ohio State University impactor, which was later refined and renamed the electromagnetic spinal cord injury device (ESCID) (Stokes, 1992; Jakeman et al., 2009; Noyes, 1987; Cheriyan et al., 2014; Kwon et al., 2002; Yeo et al., 2004). These devices use computer feedback to deploy a vertical shaft to apply a user-defined displacement to the exposed (laminectomized) spinal cord through the intact dura. Like the IH impactor, the ESCID has been engineered to autoretract following impact, eliminating the bounce injury that is a concern for weight-drop models (Kwon et al., 2002). Investigators can control the velocity of the impact, the depth of cord depression, and the duration that the cord remains compressed (Habgood, 2011). In addition, they can measure the force of impact and other parameters of interest (Jakeman et al., 2009). Like the other contusion models, these devices were originally designed for use in the thoracic region, but have also been adapted for use in the rat cervical cord (Pearse et al., 2005; Choo et al., 2009). A similar device, known as the LISA (Louisville Injury System Apparatus), also uses cord displacement at the primary driver of injury, but utilizes a novel vertebral stabilization technique to allow for more rapid impact velocity that its developers argue is more clinically relevant to human injury (Zhang et al., 2008). Like weight-drop and force-controlled contusion devices, the use of displacement-controlled contusion devices requires laminectomy. In addition, since displacement is controlled by the user, it is very important to consistently get an accurate “zero” value for the position of the impactor at baseline without inadvertently applying force to the spinal cord (Cheriyan et al., 2014). The ESCID version of the device incorporates a “Touch” signal to aid users in establishing a zero point, and while there is no evidence of behavioral or histologic deficits in animals due to the Touch stage of injury, some minimal deformation of the cord is expected with this technique and may be seen as a potential source of what has been called “priming

injury.” In contrast, the LISA device uses laser technology to measure the distance from the impactor to the spinal cord prior to creation of injury, eliminating this concern (Zhang et al., 2008). These devices are relatively complex to engineer and are not currently commercially available, which may make implementation challenging for new users (Onifer et al., 2007b; Jakeman et al., 2009). d. Air-gun Impactor In efforts to circumvent some of the limitations of laminectomy-based contusion models, Marcol et al. (2012) developed an “air-gun impactor” designed to contuse the spinal cord via delivery of a precisely controlled stream of air. While this technique still requires a surgical approach, the injury is made through a 2 mm burr hole (dura intact) rather than a complete laminectomy, making it less invasive and potentially more replicative of the circumstances surrounding human SCI than most other commonly used contusion models. This technique appears to create histologic lesions similar to those seen in contused human cord, including cavitations. Despite the reportedly straightforward operation and less invasive surgery, this technique is not yet in widespread use. Locomotor deficits induced with the air-gun impactor do not appear to track as clearly with injury severity as histologic lesions (Cheriyan et al., 2014; Marcol et al., 2012). Further validation will be needed before generalizations can be made regarding its utility in comparison with other contusion methods. 2. Compression In contrast to contusion injuries, which are created by an instantaneous, acute impact, compression injuries are created by the application of forces to the cord over a defined period of time (Cheriyan et al., 2014). As the majority of human SCIs involve some degree of lasting cord compression, be it from fractured vertebrae, swelling, or hemorrhage, animal models of spinal cord compression are often considered more relevant to human injury than contusions (Cheriyan et al., 2014; Choo et al., 2009; Kwon et al., 2002). The duration of compression, in addition to other factors such as the amount of compressive force, are the primary drivers of injury severity. Researchers have used compression injury models to demonstrate that rapid decompression can improve outcome (Kjell and Olson, 2016; Kwon et al., 2002). A number of variations on techniques for inducing spinal cord compression in rats exist; some of the more common techniques are described next. a. Acute Clip Impact/Clip Compression Originally developed in 1978, with this technique the investigator applies an aneurysm clip to the rat’s exposed (laminectomized) spinal cord, compressing it dorsoventrally for a defined period of time (Rivlin and Tator, 1978; Tator and Poon, 2009; Weaver et al., 2001;

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

Nashmi and Fehlings, 2001; Cheriyan et al., 2014). This technique is the most common way to induce compression injury in animal models (Sharif-Alhoseini et al., 2017). There are two components to this injury: the initial, acute impact that the clip imparts as it is applied to the cord, as well as the sustained compression created by the clip as it is maintained in site on the cord. Because the majority of human SCIs are created from a combination of impact force and sustained compression, and many spinal cord compression injuries carry a component of anteroposterior compression, this model is thought to be replicative of the mechanism by which many human injuries occur. Subsequent studies have adapted the acute clip impact/clip compression technique to allow for lateral compression of the cord as well (Onifer et al., 2007b; von Euler et al., 1997). One of the greatest advantages of this technique is its flexibility. While originally described for application to the thoracic region of the spinal cord, the clip compression model has been adapted for use in all regions of the cord (Cheriyan et al., 2014). Additionally, this model provides gradability of injury severity. Depending on the force of clip used and the duration that the clip is applied, injuries ranging from ASIA grades BeD are possible (Tator and Poon, 2009; Ditunno et al., 1994). The clip can be applied to the cord for what might be considered “clinically relevant” periods of time to better replicate human injuries. Clips can be used multiple times following resterilization, making this one of the less expensive techniques for creating spinal cord compression in rats. Investigators should periodically recalibrate clips to confirm that the desired force will be applied (Tator and Poon, 2009). The simplicity of the technique also leads to some drawbacks, however. A laminectomy is required for the surgeon to gain adequate access to the spinal cord. Furthermore, while the user controls the clip force and compression duration, there is not a mechanism to measure the velocity of impact, degree of cord compression, or the actual compressive force applied at the level of the cord (Tator and Poon, 2009; Cheriyan et al., 2014). This leads to some limitations regarding control of injury, which may lead to variability in injury severity and difficulty in identifying/excluding anomalous injuries (Kwon et al., 2002; Zhang et al., 2014b). In 1996 a similar technique known as the calibrated forceps compression model was described in the rat (Gruner et al., 1996). Modified from work done in guinea pigs, the surgical approach is similar, also requiring laminectomy, but the forceps create a larger, more broadly compressive injury than aneurysm clips (Onifer et al., 2007b; Blight, 1991). Because this model lacks the initial impact, it less represents injury seen in human clinical cases compared to the aneurysm clip model (Cheriyan et al., 2014). In addition, the forceps are typically applied in the lateral direction in contrast to most naturally

1031

occurring human SCIs in which dorsoventral compression is more common (McDonough et al., 2015). b. Balloon Compression Balloon compression injuries were first developed in larger animals and were adapted to rats later, as smaller balloon catheters became available. With this technique, a balloon catheter is placed into the rat’s epidural (Vanicky et al., 2001; Khan and Griebel, 1983; Chung et al., 2013) or subdural (Martin et al., 1992) space, and the severity of injury is dictated by the volume of either air or fluid injected into the balloon, as well as the duration of time that the balloon is inflated. The technique has been performed through the aid of a laminectomy (Khan and Griebel, 1983), either at the site of injury or caudal to it, but has also been performed without laminectomy, gaining entry to the epidural space by means of a small burr hole (Vanicky et al., 2001) or percutaneously with fluoroscopic guidance. These latter two techniques have clear benefits for animal welfare, as well as for enhancing comparisons to human injuries since they better maintain the anatomy of the spinal canal relative to techniques requiring laminectomy (Chung et al., 2013). One potential downside to this method is its lack of an acute injury component seen with techniques like acute clip impact or clip compression (Cheriyan et al., 2014; Hook, 2013). In addition, the rat’s relatively small epidural space makes this technique somewhat challenging and may limit its utility (Zhang et al., 2014b). c. Blocking Weight Compression Perhaps the simplest technique for the creation of spinal cord compression injury is weight blocking. First described in rats in the late 1980s, with this technique, weights are placed on a platform connected to a rod with a curved baseplate that rests in contact with the rat’s exposed thoracic spinal cord to create a sustained compressive injury on the region of interest (Holtz et al., 1989; Nystrom and Berglund, 1988; Perdiki et al., 1998). The duration that the weights are in place and the mass that is applied together determine the severity of injury. This type of injury is thought to mimic the forces applied to the spinal cord in injuries such as vertebral wedge fractures, and has been useful in evaluating the time to decompression on functional impairment. A similar technique that uses direct placement of a custom weight with a concave bottom in contact with the dura has been adapted for cervical injury as well (Ohta et al., 1999). While weight block injuries are relatively simple to perform, they require extensive laminectomy. In addition, factors such as the degree of cord compression are not typically quantified. d. Spinal Cord Strapping A novel, more recently described spinal cord compression technique has been referred to as “strapping.” This technique has the significant advantage of being relatively

RATS AS RESEARCH MODELS

1032

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

noninvasive, because it requires no laminectomy. Briefly, the user passes a curved cardiovascular needle with attached suture through the epidural space of the rat. The ends of the suture are attached to a device known as the “SC-STRAPPER,” essentially a pulley system attached to a mass. The mass can be lowered to create gradable spinal cord compression over a period of time controlled by the user. In contrast to the previously discussed techniques that compress the cord in a single direction, this technique creates more circumferential compression, which may better replicate some classes of human injury. This technique can be performed with relatively simple and inexpensive equipment, and the developers of the model report low morbidity and mortality (da Costa et al., 2008; Cheriyan et al., 2014). While this technique in many ways offers significant refinements over more invasive injuries, it provides the user with less control and fewer opportunities to measure parameters such as cord compression. In addition, since this technique was somewhat more recently developed, additional work is required to determine the reliability and reproducibility of the injury under different conditions, and it is not yet in wide use (Cheriyan et al., 2014). 3. Distraction and Dislocation Many human spinal injuries involve subluxation or dislocation of vertebrae, which can lead to spinal cord contusion, compression, stretching, or shearing forces, or any combination thereof (Habgood, 2011; Fiford et al., 2004; Choo et al., 2009). Until the early 2000s, investigators had focused their attention primarily on pure animal models of contusion and compression injuries. Since then, some researchers have developed techniques to create injuries that apply forces to the vertebral column (as opposed to the spinal cord itself) that can lead to perturbations of the spinal cord analogous to what a human might experience during a traumatic injury. In addition to more faithfully modeling the mechanism of human injury, white matter is less frequently spared in the context of distraction/dislocation injury (unlike contusion injury). This gives these techniques a potential role in studying preservation strategies for keeping white matter alive following SCI (Choo et al., 2009). While these techniques provide an intriguing perspective on evaluation of the complexity of SCI, one drawback is that the actual forces applied to the cord are not as tightly controlled or measured as they are with some contusion and compression injuries. Techniques for distraction and dislocation injuries in rats are briefly summarized next (Fig. 29.3). i. Distraction Several techniques have been described for the creation of distraction SCI in rats. One of the best known, the Harrington distractor, was

FIGURE 29.3 Schematic comparison of different rat models of spinal cord distraction injury: (A) Bidirectional distraction using sublaminar hooks and stepper motor (Dabney et al., 2004). (B) Unidirectional distraction using clamps and linear actuators (Choo et al., 2007). (C) Bidirectional distraction using clamps and linear actuators (Seifert et al., 2011). Reproduced from Seifert, J.L., Bell, J.E., Elmer, B.B., Sucato, D.J., Romero, M.I., 2011. Characterization of a novel bidirectional distraction spinal cord injuryanimal model. J. Neurosci. Methods 197, 97e103.

the first technique to attempt to mimic human distraction SCI in rats. Based on a technique developed as a treatment modality for humans with scoliosis, the Harrington distractor was first applied to rats in the 1980s to study the effects of vertebral distractive forces on rats with induced scoliosis (Dabney et al., 1988; Salzman et al., 1988; Donovan, 2007). The technique was adapted in 2004 for the creation of SCI on the premise that a rat model of spinal distraction injury would be informative, given that human SCI often involves both tensile and contusive forces (Dabney et al., 2004). Here, a laminectomy is performed in the thoracic region and hooks are secured in a sublaminar fashion directly cranial and caudal to the site to be injured. The hooks are then attached to a stepping motor device that applies userdefined tensile forces to the spinal column to create longitudinal bidirectional distraction of the segment of interest. This technique is quite invasive, requiring not

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

only laminectomy but removal of facet ligaments flanking the injury to allow for production of gradable injury. Even so, there appears to be significant variability in injury severity between animals (Dabney et al., 2004). Furthermore, some have argued that the velocity of injury may be too slow (1 cm/s) to effectively mimic typical human distraction injuries. Lastly, the technique is new relative to many of the more established SCI models, and therefore more work is needed to better understand how best to create a reproducible injury (Cheriyan et al., 2014). More recently, a device known as the University of Texas Arlington distractor has been developed (Seifert et al., 2011). Briefly, the user surgically approaches the spinal column and applies clips to the vertebral bodies cranially and caudally to the site to be injureddno laminectomy is required. Forces are applied simultaneously to both clips in opposing directions using a linear actuator, creating a bidirectional spinal distraction. Forces administered to the column during the injury itself can be measured to provide some degree of quality control. While this technique has the benefit of being less invasive than the Harrington distractor, both techniques are considered to be slow relative to naturally occurring human SCI, and, as relatively new injury models, have not been fully validated at this time (Nightingale et al., 1996; Cheriyan et al., 2014). ii. Dislocation In contrast to distraction injuries, which apply tensile forces along the longitudinal axis of the spine, displacement injuries apply lateral or dorsoventral forces. These are a common cause for human SCI, in particular in pediatric patients (Clarke and Bilston, 2008). In 2004 Fiford et al. described a technique for the production of a lateral dislocation injury in the rat. This technique was appealing because it can mimic many aspects of human SCI, including ligamentous injury and vertebral perturbations, without the need for laminectomy (Fiford et al., 2004). Briefly, the spinal column is approached surgically (no laminectomy is required), and specialized sliding clamps, which are affixed to rods, are placed onto the lateral aspects of the vertebral bodies flanking the injury. One rod is fixed while the other applies a lateral force to the clamps to create the injury through the use of a linear actuator. The resulting SCI is a product of the interaction of the inside walls of the vertebral canal with the spinal cord during the dislocation event, as is commonly the mechanism for human SCI. Force and degree of lateral displacement can be measured automatically. This paradigm has been used to model human lateral flexion-type injuries, and is highly gradable, with injuries ranging from minimal to complete severance of the cord (Fiford et al., 2004). The device has been used in the thoracic and

1033

thoracolumbar regions (Clarke and Bilston, 2008; Lau et al., 2013), and a variation exists where displacement forces are applied dorsally rather than laterally to approximate certain fractures that occur in people (Choo et al., 2009; Cheriyan et al., 2014). Like the distraction models, displacement models have not yet been standardized or fully validated, which places some limits on their widespread use (Cheriyan et al., 2014). iii. University of British Columbia Multimechanism Device (Contusion, Distraction, Dislocation) The University of British Columbia Multimechanism device has been described as the most advanced rat SCI induction device, but is also one of the more complex to operate (Choo et al., 2009; Marcol et al., 2012). This multifunctional apparatus has the capability to create contusion, distraction, or dislocation injury in rats (Choo et al., 2009; Cheriyan et al., 2014). In contrast to the previously described distraction and dislocation injury devices that generally induce injury at the thoracic level, this device produces a cervical injury, with clamps affixed to the transverse processes of the cervical vertebrae using a complex but well-described mechanism that has been optimized to allow for rapid induction of injury. The speed of injury induction is a strength of this technique, because it reportedly mimics clinical human SCI (Chen et al., 2016; Choo et al., 2009). In contrast to the University of Texas Arlington distractor, the distraction injuries created by this apparatus are unidirectional (Seifert et al., 2011). The device can be engineered to automatically record force of injury as well as cord compression. Like the previously described dislocation and distraction techniques, the use of this apparatus is not yet fully validated (Cheriyan et al., 2014), and laminectomy is required for creation of all three of the injuries, limiting the clinical relevance of the technique (Choo et al., 2009). 4. Transection/Laceration While human spinal cord transection injuries are less common clinically than contusion, compression, distraction, and dislocation injuries, animal models of spinal cord transection remain some of the most common SCI paradigms used today. In fact, transection injuries were second only to contusions in one recent review (Sharif-Alhoseini et al., 2017). Their popularity stems not from helping researchers to understand the pathophysiology of naturally occurring SCI, but rather from their utility in helping investigators develop a better understanding of clinical interventions aimed at the promotion of axonal recovery and function within the spinal cord following insult (Onifer et al., 2007b; Cheriyan et al., 2014; Vogelaar, 2016; Hodgetts et al., 2009; Han et al., 2010; Kwon et al., 2002; Rosenzweig and McDonald, 2004). Transection injuries used in rats can

RATS AS RESEARCH MODELS

1034

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

FIGURE 29.4 (A) Schematic representation of the main ascending sensory tracts (right) and descending motor tracts (left) in a transverse section of the rodent spinal cord. Dashed line indicates the location of the corticospinal tract in humans. (B) Schematic drawings of transverse sections through the spinal cord with motor tracts depicted left and sensory tracts depicted right. Dashed areas represent the extent of tissue damage produced by typical injuries using the different injury paradigms. Adapted from Vogelaar, C.F., Estrada, V., 2016. In: Fuller, H. (Ed.), Experimental SpinalCord Injury Models in Rodents: Anatomical Correlations and Assessment of Motor Recovery. InTech., under CC BY 3.0 license. Available from: https://doi.org/10.5772/62947x.

be divided into two main categories: complete transection and partial transection (Fig. 29.4). i. Complete Transection As the name implies, complete transection paradigms involve complete separation between the rostral and caudal segments of the spinal cord (Heimburger, 2005). This technique is the only SCI model that reliably produces an injury corresponding to ASIA category A in humans, and is frequently used to evaluate true axonal regeneration without the potential for regain of function due to collateral effects of spared axons (Lukovic et al., 2015; Ditunno et al., 1994). This technique is relatively simple to perform and easy to reproduce given that, in contrast to partial transection models, visual inspection alone can confirm an appropriate degree of injury. In general, researchers access the cord via laminectomy and produce the injury using fine scissors or a spinal cord hook (Vogelaar, 2016; Kwon et al., 2002). While the “all or nothing” nature of the complete transection model leads to excellent reproducibility, it also has the drawback that there is no option for gradable injury, and animal mortality is, not unexpectedly, higher with this technique than many less catastrophic injuries (Battistuzzo et al., 2012; Lukovic et al., 2015). Animals undergoing complete transection require significantly greater nursing care than animals undergoing partial transection, for example (Hodgetts et al., 2009). Another complicating factor subsequent to this technique is that some animals appear to have “native locomotor activity,” allowing them to regain some degree of motor function posttransection, even in the absence of substantive spinal cord healing (Hodgetts et al., 2009). For this reason, to confirm that return of motor function is due to clinical improvement and not this phenomenon, it may be necessary to submit animals to a second surgery, retransecting the cord; animals that have truly regained

function that was lost with the initial injury should lose function again, whereas animals that regained function based on compensatory activity will continue as they had before the second transection (Cheriyan et al., 2014; Kwon et al., 2002; Heimburger, 2005). A variation on the complete transection model exists where a segment of spinal cord is resected, rather than simply transected. Resection models allow for the testing of implanted biomaterial scaffolds to promote axonal regrowth, but have been criticized for being far removed from naturally occurring human injuries (Han et al., 2010; Wang et al., 2014a). While complete transection models can be particularly useful to study therapeutic interventions conducive to the recovery of function following severe SCI, it has been recommended that treatments tested in these models be further evaluated in contusion models to increase prediction of translatability (Vogelaar, 2016). ii. Partial Transection While naturally occurring complete spinal cord transections are relatively rare in human patients, partial transections are more clinically relevant and may result from trauma occurring from vertebral fracture. These models require precise disruption of specific target tracts within the spinal cord for transection by way of laminectomy, and allow for study of both axonal repair as well as regain of function due to sprouting from axons that were spared in the initial injury. Tracts targeted for transection are chosen based on accessibility to the surgeon and potential to result in useful outcome measures based on the function expected to be inhibited by interruption of particular tract(s) (Vogelaar, 2016; Kwon et al., 2002; Lee et al., 2012; Silva et al., 2014). While a variety of partial transection models exist, the two most common techniques are dorsal and lateral hemisections. These are commonly performed using

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

fine spring scissors, scalpels, or stereotactic-guided retractable wire knives, although some investigators have recommended avoiding the use of scalpel blades due to damage to the sectioned stump (Wang and Xu, 2009). Out of concerns that these instruments may produce some variation in injury quality, or that they may yield a contusive component to the transection injury, a device known as the Vibraknife has also been developed with the intention of creating a precise, pure laceration injury (Zhang et al., 2004). Dorsal hemisections are most commonly performed in the midthoracic region. At this level, the ascending dorsal columns and descending corticospinal and rubrospinal tracts (the latter depending on the laterality of the injury) are generally transected (Klapka et al., 2005; Vogelaar et al., 2015; Hermanns et al., 2001; Silva et al., 2014). While the functions of these descending (motor) tracts are complex and likely complementary, the corticospinal tract is often considered to be more involved in fine movements of the paws, whereas the rubrospinal tract, which is considered more important in rodents than in humans for control of voluntary movement, is thought to play a more generalized locomotor control role (Vogelaar, 2016; Lee et al., 2012; Kwon et al., 2002; Morris and Whishaw, 2016; Kjell and Olson, 2016). Lateral hemisections are performed in a similar manner to dorsal hemisections, except that all tracts on one side of the cord are transected while the contralateral cord is left intact (Wang and Xu, 2009). In some cases it is possible to use an animal’s uninjured contralateral side as an internal control (Cheriyan et al., 2014; Kwon et al., 2002; Hodgetts et al., 2009; Morris and Whishaw, 2016). In contrast to dorsal hemisections, lateral hemisections are more commonly performed at the cervical level, allowing for simultaneous study of fore and hindlimb recovery, and have also been useful for evaluating the contribution of the contralateral cord in mediating recovery of function (Vogelaar, 2016; Zorner et al., 2014). Some other variations on partial transection models include funiculotomy (dorsolateral or ventrolateral) (Schrimsher and Reier, 1993), pyramidotomy (Vogelaar, 2016; Kathe et al., 2014), aspiration injury (Rosenzweig and McDonald, 2004), and overhemisection (typically unilateral dorsolateral funiculotomy combined with contralateral lateral hemisection) (Firkins et al., 1993; Bernstein and Stelzner, 1983). Partial transections are much more difficult to execute than complete transection models. Care must be taken to ensure that target tracts are completely severed, for example, by use of retrograde axonal tracers to track axons that escaped transection, or the intraoperative use of somatosensory evoked potentials to ensure complete injury (Cheriyan et al., 2014; Kwon et al., 2002; Cloud et al., 2012). It is important to note that timelines for these studies are occasionally quite long, because

1035

evidence exists suggesting that posthemisection rats do not reach a neurologic state analogous to humans with chronic SCI until months after their injury (Leszczy nska et al., 2015). Fortunately, animals that undergo these procedures have lower morbidity and mortality than animals with complete spinal cord transection, and are thus much easier to manage clinically than those with complete transections (Silva et al., 2014; Gill et al., 2014; Liebscher et al., 2005; Cao et al., 2015). 5. Other Techniques The emphasis here has been on traumatic SCIs. It is important to note, however, that the rat has been used extensively in other SCI models outside of the scope of this chapter, including injuries aimed at evaluating secondary, downstream effects of SCI. For instance, other injury paradigms that have been extensively studied in the rat include ischemic injury (Lafci et al., 2013; Vaquero et al., 2007; Fan et al., 2011; Benton et al., 2005), cervical spondylytic myelopathy (Karadimas et al., 2013), vibrational injury (Matloub et al., 2005; Baig et al., 2013), nerve root compression/avulsion (Chu and Wu, 2009; Hubbard and Winkelstein, 2005; Xue et al., 2014; Oprych et al., 2015; Rosenzweig and McDonald, 2004), vertebral facet injury (Shuang et al., 2015; Lee et al., 2004), photochemical injury (Watson et al., 1986; Hao et al., 1991; Bunge et al., 1994), excitotoxic injury (Nakae et al., 2011; Park et al., 2004; Yezierski et al., 1998), electrolytic injury (Masri et al., 2009), demyelination (Jernigan et al., 2009), inflammatory (Popovich et al., 2002), and other chemical injuries (Benton et al., 2005; Dora et al., 1998; Park et al., 2004; Liu et al., 1995). For details on these and other models of rat SCI, we refer the reader to other reviews that have been written on these topics (Cheriyan et al., 2014; Onifer et al., 2007b; Kwon et al., 2002; Hodgetts et al., 2009; Kjell and Olson, 2016; Akhtar et al., 2008; Sharif-Alhoseini et al., 2017; Sharif-Alhoseini and Rahimi-Movaghar, 2014; Hook, 2013; Vogelaar, 2016; Rosenzweig and McDonald, 2004; Silva et al., 2014). D. Outcome Measures for Rat SCI Models A commonly cited reason for failure of translatability from preclinical to clinical trials is inappropriate selection of outcome measures (Fouad et al., 2013). In particular, selection of behavioral assays requires special consideration to ensure the most appropriate test is performed under the right circumstances (Basso, 2004). Standardizing test conditions is crucial for determining whether differences between intervention groups are due to biological changes or chance variability (Basso, 2004; Sedy et al., 2008). Some of the most common and historically relevant techniques used to evaluate outcome following SCI in the rat are summarized next.

RATS AS RESEARCH MODELS

1036

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

1. Motor Function The most visible result of SCI is perturbation of normal locomotion. Therefore motor function tests are commonly employed to evaluate recovery in rodent models of SCI (Sedy et al., 2008). It is important to measure functional outcomes in addition to pure anatomic outcomes, such as histology or imaging, because functional deficits do not always correlate with extent of the lesion (Metz et al., 2000a; Fouad et al., 2013). When the goal of the animal model is to evaluate therapeutic interventions after SCI, tests must be evaluated to ensure they are as objective as possible. It is also helpful to evaluate motor function from quantitative (e.g., speed with which the animal ambulates) and qualitative (e.g., use of each leg) perspectives simultaneously to avoid missing components of the animal’s overall clinical status (Fouad et al., 2013). Furthermore, it is important to be sure that motor function is evaluated in a method through which deficits from the site of the lesion would be evident. High-quality video recordings allow for many of these motor tests to be replayed in slow motion and scored by blinded observers (Soblosky et al., 1997, 2001). While a battery of functional tests is available to investigators interested in identifying recovery of function, for consistency it is important for researchers to focus their studies, targeting behavioral tests specific to their injury model while reporting all significant and nonsignificant results (Geissler et al., 2013). Tarlov and Klinger (1954) developed one of the first scales for the rating of locomotor function in an animal model of SCI (Tarlov and Klinger, 1954). This was a basic observational scale that evaluated joint movement and ability to stand from 0 (no voluntary function) to 4 (normal movement) (Tarlov and Klinger, 1954). This motor scale was adapted by others to monitor locomotive recovery in rats after SCI (Gale et al., 1985). Some have suggested that adding thoracolumbar height to the Tarlov scale may increase sensitivity in detecting changes after mild injury, because this parameter varies with degree of weight bearing and differs between SCI rats and sham-operated controls (van de Meent et al., 1996). Gale et al. also included other scales in their evaluation, creating a Combined Behavior Score (CBS). The CBS added parameters such as toe spread, paw placing and withdrawal, ability to right, swim, walk up an inclined plane (Rivlin and Tator, 1977), and reaction to increasing temperatures on a hot plate (Gale et al., 1985; Panjabi and Wrathall, 1988). The Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale is one of the most common assays for the evaluation of pelvic limb function after thoracic level injury in rats (Fouad et al., 2013; Hook, 2013; Onifer et al., 2007b; Kwon et al., 2002). In this test, rats are placed in an open field for a 4-min observation and evaluated on a scale of 0 (no spontaneous movement) to 21 (normal movement)

based on joint motion, paw placement, weight bearing, and coordination (Basso et al., 1995). This test should be monitored by at least two blinded observers to ensure agreement. This scale has also been adapted to more sensitively detect changes after severe injury, when the high end of the scale (most motor function) is never reached (Estrada et al., 2014; Antri et al., 2002). Although the BBB score was first designed for evaluation of animals after thoracic injury, it has also been used after cervical injury (Geissler et al., 2013; Nguyen et al., 2012). As an alternative, others have developed open-field locomotor rating systems specific to cervical injury. The Forelimb Motor Scale, Forelimb Locomotor Assessment Scale, and University of Provence scale are all based on similar principles used for the BBB scale, such as paw placement, weight bearing, and joint range of motion, but more accurately reflect the types of motion disruption seen in forelimbs after cervical injury (Anderson et al., 2009a; Sandrow et al., 2008; Cao et al., 2008). A BBB subscore has also been developed to help evaluate higher motor function after milder injuries by measuring toe clearance, predominant paw position, instability, and tail position (Lankhorst et al., 1999). Because gait is often affected by spinal injury, investigators frequently evaluate perturbations in rat gait post-SCI. Gait evaluation can be performed using ink and paper techniques (Chan et al., 2005) or automated walkways with sophisticated image acquisition and analysis software (Kunkel-Bagden et al., 1993; Cheng et al., 1997). One of the more common gait analysis systems is CatWalk, a system that uses sophisticated footprint analysis of light deflection caused by paws contacting a glass surface during forward ambulation (Hamers et al., 2001, 2006). Parameters evaluated in this assay include stride length, base of support (distance between right and left limbs), walking speed, duration of swing/stand phases, regularity index, print intensity, and duration of tail or abdominal drags (Hamers et al., 2001). More recently, almost entirely automated systems, such as DigiGait and TreadScan, have further expanded the ability to evaluate gait by including a treadmill with the ability to vary walk speed (Springer et al., 2010; McEwen and Springer, 2006; Ek et al., 2010). Evaluating misplaced steps on various terrains that are more complex than flat surfaces is another strategy for assessing limb coordination and fine motor skill. These tests take advantage of the need for sensory feedback to the supraspinal motor system to accomplish the tasks proficiently. One approach, originally developed to quantify recovery after sensorimotor cortex damage in the rat, is to use a horizontally placed ladder and measure misplaced steps (Soblosky et al., 1997, 2001; Gensel et al., 2006). With this assay, rats walk forward across ladder rungs and misplaced steps are scored. In this technique, factors such as rung spacing and diameter are important design considerations. It is necessary to tailor these parameters to the study to ensure that

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

animals can properly traverse rungs at baseline, but that missteps will be evident following injury. Spacing distance between rungs can be varied to increase complexity and ensure spinal learning during a training phase does not confound outcomes after injury (Ek et al., 2010). Related techniques include evaluating rats walking across beams (Metz et al., 2000b; Jeffery and Blakemore, 1997; Kim et al., 2001; Bradbury et al., 2002; Kunkel-Bagden et al., 1993), grids (Metz et al., 2000b; Hicks and D’Amato, 1975; Schucht et al., 2002; Bresnahan et al., 1987; Behrmann et al., 1992), and ropes (Kim et al., 2001; Anderson et al., 2005; Z’Graggen et al., 1998). Kinematics have also been used to evaluate the components of a rat’s gait after SCI. As opposed to the previously mentioned gait analyses that focus on the rat’s contact points with the ground (primarily the paws), kinematics can evaluate more proximal limb anatomy in motion such as metatarsal, stifle, and hip joints (Alluin et al., 2011; Metz et al., 2000b; Kunkel-Bagden et al., 1993). These techniques involve using video records of rats with anatomical markers, usually over the hip and ankle after thoracic injury, which allow data acquisition of the movements of different points in space and in relation to one another. Software is commercially available to allow for recording and three-dimensional reconstruction for advanced analysis (Shah et al., 2013; Courtine et al., 2008). Some researchers have used swimming tests to gauge function after SCI (Smith et al., 2006a, 2006b; Kim et al., 2001; Xu et al., 2015; Liebscher et al., 2005). The advantage of using swim tests is that unlike some other locomotor scores, weight bearing is not required. Also, it has been proposed that since less cutaneous feedback is required for swimming compared to walking, swimming may be more sensitive for supraspinal motor control in the absence of sensation (Xu et al., 2015). Advanced kinematic analysis can also be performed on swimming rats (Zorner et al., 2010). Despite the advantages, finding an objective measure of performance can be challenging because, although rats normally propel through water with the hindlimbs, forelimb compensation is often seen after injury (Smith et al., 2006a). For this reason, using metrics such as swimming speed or time to finish can be misleading without some other qualitative descriptor. Scores evaluating the angle at which the rat’s body hangs below the surface of the water, trunk stability, tail motion, and use of thoracic compared to pelvic limbs has been shown to correlate with injury severity (Smith et al., 2006a; Xu et al., 2015). As an alternative, evaluation of animals as they wade through shallow water has been used to evaluate function while partial weight bearing (Zorner et al., 2010; Filli et al., 2011). Observation of movement not related to locomotion can lend great insight into outcome following injury.

1037

Grooming is a common spontaneous behavior in rats that may be altered by cervical SCI (Gensel et al., 2006). Evaluating the ability to groom can be either by observation alone or in combination with a sensory stimulus to induce grooming. A standard stimulus (e.g., saline on the back of the neck) is applied, and the rats are then observed for licking, rubbing, and scratching (Gensel et al., 2006). Similarly, a small adhesive sticker can be placed on a limb or head and latency to removal recorded (Kim et al., 2001; Onifer et al., 2005; Bradbury et al., 2002). This has been done to evaluate sensation of the forelimbs, because removal was performed by the rats using their teeth (Schallert et al., 2000), or to evaluate motor function of the forelimbs, when the sticker is placed on the forehead (Schrimsher and Reier, 1992). These techniques are especially useful for evaluating the use of forelimbs after cervical level injuries. Another technique for the evaluation of forepaws allows a rat to explore a vertical Plexiglas cylinder while the investigator observes asymmetries in paw use during rearing and landing behaviors (Onifer et al., 2007b; Geissler et al., 2013; Liu et al., 1999; Soblosky et al., 2001; Schallert et al., 2000; Gensel et al., 2006). Food retrieval tests are useful after cervical SCI for evaluating dexterity in a variety of settings, such as reaching down a staircase (Montoya et al., 1991; Lee et al., 2012; McKenna et al., 2000) or through a fenestrated barrier (Z’Graggen et al., 1998), and with a variety of substrates (Onifer et al., 2007b), including pasta (Ballermann et al., 2001; Allred et al., 2008; Tennant et al., 2010) and cereal (Fig. 29.5) (Irvine et al., 2010, 2014). However, alternative retrieval strategies may be employed by highly motivated rats, leading to possible misinterpretation of data on number of food items retrieved (Fouad et al., 2013). Many of these systems require a prolonged training time commitment by investigators as pretrained animals perform better overall on these tasks (May et al., 2015). Some tests of motor function focus on strength or balance required to maintain a static position despite a changing force. Evaluating the maximum angle that an animal can maintain itself on an inclined plane has been used to assess motor function independent of spinal learning. In this method, a rat is placed on a flat surface and then one end of that surface is raised until the rat cannot maintain its position and falls off the plane (Rivlin and Tator, 1977). Grip strength of the forelimbs can be assessed after cervical SCI by allowing rats to hold a bar as they slowly pull away from said bar (Anderson et al., 2005; Onifer et al., 1997). As distance from the bar increases, so does the amount of force required to maintain the grip. The maximum force applied before that rat releases its grip is recorded and results can be compared between intervention groups.

RATS AS RESEARCH MODELS

1038

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

(A)

(B)

(C)

FIGURE 29.5 An example forepaw adjustment of (AeC) a rat during manipulation of vermicelli during the pasta handling task. (A) The animal’s paws before the adjustment, (B) The forepaws adjusting (asterisk indicates adjusting paws), (C) The replacement of the forepaws onto the pasta piece. Animals with cord lesions may demonstrate perturbations in the total time required to consume a length of pasta, the number of paw adjustments needed to facilitate pasta consumption, or symmetry of paw use. Reproduced with permission from Tennant, K.A., Asay, A.L., Allred, R.P., Ozburn, A.R., Kleim, J.A., Jones, T.A., 2010. The vermicelli and capellini handling tests: simplequantitative measures of dexterous forepaw function in rats andmice. J. Vis. Exp. e2076.

Limb placing tests have been used to test a rat’s sensorimotor and proprioceptive abilities after SCI (Goldberger et al., 1990; Kunkel-Bagden et al., 1993; Geissler et al., 2013). These usually involve manipulating the rat so that either the vibrissae (Schallert et al., 2000; Khaing et al., 2012) or dorsal surface of the paw (Ramon-Cueto et al., 2000) are touched up against a ledge. The normal rat will right the foot and place it in a weight-bearing position onto the flat surface. Rats are scored categorically from 0 to 4 based on degree of successful placement (Geissler et al., 2013), or results may be expressed as a percentage of correct placing events after multiple trials (Schallert et al., 2000). 2. Sensory Testing and Neuropathic Pain Although many of the previously described tests require some component of intact sensory tracts for rats to perform, certain tests are used to more specifically evaluate sensation after SCI, especially as they relate to hyperalgesia (increased pain sensation from a mildly painful stimulus) or allodynia (pain in response to a normally nonpainful stimulus). Careful analysis of the results is required, because SCI models frequently have some components of loss of motor function, loss of sensation, allodynia, or hyperalgesia, all of which may impact the outcomes of these tests. In contrast to locomotor activity that recovers in a graded fashion, return of sensation in rats after SCI seems to be an “all-or-none” phenomenon (Detloff et al., 2010; Kloos et al., 2005). Von Frey filaments are commonly used to assess sensation or mechanical allodynia in spinal-injured rats (Detloff et al., 2010, 2012; Anderson et al., 2009b; Christensen et al., 1996; Hutchinson et al., 2004; Mills

et al., 2001; Chan et al., 2005). This has largely replaced less standardized stimuli, such as pinching with the fingers or towel clamps, as was done in the past (Gale et al., 1985). In one standard form of the assay, rats stand on a mesh surface, and filaments of increasing diameter, and therefore force, are touched to the palmar or plantar surface of the paw until the column buckles. A variation of this test has been developed for use on the dorsum of the paw for spinal-injured rats (Detloff et al., 2010). Application of the filaments to the areas other than the limbs, such as the head, neck, or trunk, may also be used to evaluate those regions for the presence of allodynia (Lindsey et al., 2000). Investigators record avoidance reactions, with the presumption that a brisk withdrawal of the paw indicates an adverse sensation such as pain. In animals with decreased motor function and inability to briskly withdrawal the paw, other reactions may be noted, such as vocalization, movement of the head, neck, or trunk, or display of aggressive behaviors (Anderson et al., 2009b; Christensen et al., 1996). Sensitivity to changing temperature has also been evaluated in SCI rats. Tests evaluating supraspinal reactions, such as licking of the paw, vocalizing, or escape behaviors after application of a hot or cold stimulus to the distal limb, enable the investigator to interrogate the neural sensory connections between the distal limbs and brain. The application of heat has been by hot plate (Christensen et al., 1996; Gale et al., 1985) or infrared beam (Liebscher et al., 2005; Mills et al., 2001), and cold by ethyl chloride spray (Yu et al., 1998; Vaquero et al., 2006), acetone (M’Dahoma et al., 2014; Baastrup et al., 2010), or ice (Lindsey et al., 2000). The tail-flick test similarly uses a focused beam of light onto the tail instead of a paw, and records latency time to moving

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

the tail out of the beam (Gale et al., 1985; Merkler et al., 2001). While most studies have historically evaluated sensory phenomena in post-SCI animals by assessing evoked responses to mechanical or thermal stimulation like those just described, there is growing interest in the evaluation of spontaneous pain in animals following SCI, because such pain is a common and quality-of-lifelimiting sequela to SCI in humans (Mogil and Crager, 2004). Pain is a quintessentially subjective experience, defined by the International Association for the Study of Pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (IASP, 2018), and techniques such as the use of von Frey filaments or thermal testing better evaluate the nociceptive component of the pain cascade than the emotional component (Mogil et al., 2010; Vierck et al., 2008; Murphy et al., 2014; Mogil, 2009). In fact, decerebrate rats that should not be able to process pain in the emotional sense can still respond to nociceptive stimuli to some degree through local reflex arcs (Woolf, 1984). Given the rat’s inability to self-report the experience of pain, spontaneous pain is challenging to measure, and these studies often require some degree of subjectivity. Techniques such as conditioned place preference testing, in which the rat is trained to associate a substance thought to be analgesic with one chamber and a nonpain relieving vehicle in another chamber, then given the choice between chambers to assess strength of the analgesic reward, may help investigators to tease out some of the more subtle, affective components of the painful condition (Davoody et al., 2011). Other techniques that may help researchers evaluate spontaneous, as opposed to evoked, pain following SCI include analgesic self-administration studies (Woller et al., 2014), investigations aimed at correlating chronic pain and depressive-type behaviors, and ethogram-based analyses focusing on behaviors thought to be specific to discomfort (e.g., limb guarding) (Maldonado-Bouchard et al., 2016). 3. Impact of Environmental Enrichment Housing rats in enriched environments may impact the results of both sensory and motor tests that measure animal movement. In particular, housing rats in cages that had elements designed to increase spontaneous locomotion led to improved locomotor function tests with no changes in electrophysiological measures or histology (Lankhorst et al., 2001). Exercise alone also did this to some extent (Van Meeteren et al., 2003). This may be mediated by insulin-like growth factor (IGF), since blocking IGF reduced improvements in recovery that were gained by enriched environments in some locomotor tests (Koopmans et al., 2006). Improvement in

1039

functional tests has even been seen when introduction to the enriched environment is delayed for 3 months after injury (Fischer and Peduzzi, 2007). Although improvements in these measures are modest, and comparison to other therapeutic trials in rats is difficult because of lack of standardization, enrichment has outperformed many other experimental treatments. Of note, however, not all reports show functional improvement in rats in enriched environments, in particular when enrichment was modest and did not include voluntary exercise equipment or varying items in the cage (Erschbamer et al., 2006). 4. Electrophysiology Electrophysiological evaluation can be valuable for testing the integrity of electrical connections between distinct parts of the nervous system. In the case of SCI, the connections of interest are between the peripheral nerves that sense and control muscle fibers and the brain, because these signals are transduced via the spinal cord (Metz et al., 2000b), or within the spinal cord across the focus of injury (Imaizumi et al., 2000; Deng et al., 2013; Pinzon et al., 2001). These techniques generally involve sophisticated equipment for producing controlled electrical or magnetic stimulation, recording the small impulses generated by nerves, and analyzing the subsequent data. They may also be invasive procedures that require anesthesia and a moderate degree of technical skill to accomplish. Anesthesia may alter the results of these conduction studies (Kawaguchi et al., 1996; Keller et al., 1992; Oria et al., 2008; Fishback et al., 1995). Performing multiple analyses over time without anesthesia would add to the understanding of the extent and rate of recovery compared to terminally performed studies under anesthesia (Onifer et al., 2007a; Nashmi et al., 1997). Another limitation of these techniques is that they largely reflect the activity of large diameter fibers, not every axon of interest (Blight, 1992). Despite this limitation, electrophysiological studies are likely to continue to play an important role in evaluating recovery after SCI in rat models (Kwon et al., 2002). The two main categories of electrophysiological recordings used in SCI research are somatosensory evoked potentials (SEPs) and motor-evoked potentials (MEPs). Both correlate to spinal damage and functional outcome, but MEPs may be more sensitive than SEPs in both rats and humans (Metz et al., 2000b). SEPs are recorded from the somatosensory cortex after electric stimulation of distal sites such as the sciatic nerve (Gaviria et al., 2000a, 2000b) or footpad (Onifer et al., 2005). These techniques may be performed in awake rats after surgical implantation of recording electrodes in the brain (Onifer et al., 2005). MEPs are evaluated by stimulating the sensorimotor cortex in the brain and recording electromyograph (EMG) responses in peripheral muscles or

RATS AS RESEARCH MODELS

1040

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

action potentials in the distal spinal cord (Sedy et al., 2008; Fehlings et al., 1987). Alternatively, a subcutaneous cathode can be placed over the skull adjacent to the sensorimotor cortex and anode at the nose (GarciaAlias et al., 2003). A transcranial magnetic MEP approach has been developed that limits invasiveness of the technique. Instead of surgically implanting metallic electrodes in the brain, a coil is placed over the head that generates the impulse to be propagated (Linden et al., 1999; Magnuson et al., 1999). This technique can also be used in the absence of anesthesia (Linden et al., 1999). Using MEP recordings, it is possible to evaluate the differential contribution of certain spinal pathways to functional outcome after injury (Shiau et al., 1992); however, these recordings do not always closely mirror functional recovery (Gruner et al., 1993; Yu et al., 2001; Magnuson et al., 1999). While pyramidal tract ablation is critical to loss of motor function (Park et al., 2007), MEPs are controlled in part by extrapyramidal pathways in rats. As a result, it is possible that animals with SCI and resulting poor motor function may still demonstrate some degree of MEP normalcy, which can confound interpretation (Adamson et al., 1989; Dull et al., 1990). Although most electrophysiological assays used in SCI rats focus on the communication between the brain and peripheral nervous system, the magnetically evoked interenlargement system measures propriospinal axon conduction between separated spinal segments (Beaumont et al., 2006b). In this assessment, EMG recordings of the triceps and masseter muscles are made after sciatic nerve stimulation with a magnetic transducer (Beaumont et al., 2006b). EMG alone can also be used to gauge muscle tone following SCI, and this technique has been used in tandem with degree of response to nerve stimulation in efforts to quantify spasticity of the tail in rats with SCI (Bennett et al., 1999). 5. Autonomic Testing Disruptions of the autonomic nervous system are a major complication of SCI in human patients, with great potential to negatively impact quality of life (Karlsson, 2006; Anderson, 2004). Rat models of SCI also display autonomic dysfunction, and the degree of dysfunction and recovery after treatment can be quantified, with the stipulation that dysfunction depends on both location and severity of the lesion (Inskip et al., 2009). For example, severe thoracic SCI may lead to autonomic dysfunction as evident by distension of the urinary bladder (Kruse et al., 1993), but a bilateral cervical model of moderate SCI showed no urinary bladder dysfunction (Anderson et al., 2009b). Tests assessing urinary bladder function, sexual reflexes, and autonomic dysreflexia have all been developed. As with other functional outcomes after SCI, autonomic testing results are altered as

the injury evolves. For example, after spinal transection the external anal sphincter of rats has no reflex contraction for 24 h due to spinal shock, but later displays hyperreflexive contraction due to loss of inhibitory upper motor neuron control (Holmes et al., 1998). Despite the clinical prioritization of resolution of autonomic dysfunction, SCI research using animal models overwhelmingly evaluates locomotion (Inskip et al., 2009). Inskip et al. reviewed the pathophysiology of autonomic dysfunction in SCI, as well as available methods of assessment in animal models (Inskip et al., 2009). The most common clinical sign of autonomic dysfunction in rats with experimental SCI is urine retention due to detrusoresphincter dyssynergia (Sedy et al., 2008). Although this poses a clinical problem in caring for SCI rats, quantifying this condition may give valuable information regarding functional recovery that is relevant to the human condition. The degree of dysfunction correlates to severity of SCI (Wrathall and Emch, 2006). A rough categorization of urinary bladder function simply as normal or dysfunctional, defined as requiring manual expression by an investigator or caretaker, may elucidate improved recovery in some treatment groups (Liebscher et al., 2005). Almost as simply, calculating ellipsoid volume of the bladder after direct visualization at necropsy (Pikov and Wrathall, 2001), quantifying output of urine in metabolic caging (Chancellor et al., 1994; Mitsui et al., 2003), or measuring volume of urine expelled by manual expression using the Crede´ maneuver (Caggiano et al., 2005) can all be used to evaluate urine retention. More sophisticated techniques, such as cystometrograms, have also been employed to better quantify bladder function (Pikov and Wrathall, 2001, 2002; Yoshiyama and de Groat, 2002; Kruse et al., 1993; Cheng and de Groat, 2004; Cheng et al., 1995; Mitsui et al., 2003; Dolber et al., 2007). These experiments involve intravesicular pressure monitoring with or without external urethral sphincter EMG monitoring during saline infusion into the bladder. Recovery of function, as measured by cystometrograms, occurs in an injury-dependent manner (Pikov and Wrathall, 2001). Choice and dose of anesthetic used during these procedures should be considered to minimize impact on micturition responses (Cheng and de Groat, 2004). However, cystometrograms without EMG recording of the external urethral sphincter can be obtained in conscious rats (Yoshiyama and de Groat, 2002). Sexual dysfunction tests may also give insight into a sequela of SCI. A variety of tests have been developed to measure occurrence and latency to erection and degree of penile engorgement. Ex copula testing has been useful in rat SCI models that have poor motor function such that actual mounting and mating behavior may not be possible (Inskip et al., 2009). During the ex copula

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

test, the rat is placed in dorsal recumbency and the penis is retracted from the preputial sheath. The penis is secured along the ventral abdomen with perforated adhesive tape and observed visually or by telemetry (Nout et al., 2007). Manipulations to induce responses may include pressure at the base of the penis, manipulation of the urethra, or electrical stimulation of the dorsal penile nerve (Inskip et al., 2009). Visually monitored responses include reddening, flaring, or anteroflexion of the glans (Inskip et al., 2009). Noncontact erection testing is performed when a male rat is placed in an enclosure separated from a female by a mesh barrier. This allows the male to sense olfactory and visual cues relative to sexual receptiveness. Contact or mating sexual function tests are similar, although the female is placed within the same enclosure as the male and copulation events are monitored. For the noncontact and contact tests, the female must be in estrus prior to pairing. This can be ensured with hormonal injections. For example, administering estradiol 24 h prior to a progesterone injection at 6 h before pairing (Nout et al., 2007). Telemetric data on corpus spongiosum pressure can be obtained to monitor spontaneous or induced micturition or erectile events in nonanesthetized rats (Nout et al., 2005, 2007). Although sexual impediments are documented in women following SCI (Sramkova et al., 2017; Sipski and Arenas, 2006), animal studies evaluating female sexual dysfunction are much less common. Somatostatin treatment has been evaluated on clitoral and vaginal Doppler blood flow in female rats after SCI (Abdel-Karim and Carrier, 2002). The cardiovascular system is also under supraspinal nervous control and normal functions can be disrupted by SCI. Neurogenic shock may contribute to hypotension and bradycardia in rats immediately after high thoracic or cervical-level injuries (Osborn et al., 1989), but hypertension in response to colorectal distension develops later (Mayorov et al., 2001). This hypertensive response to visceral stimuli is termed autonomic dysreflexia (AD). Rats have been evaluated for AD after SCI in a variety of techniques. These studies usually assess blood pressure, measured invasively via intraarterial catheters, after colonic or urinary bladder distension (Krassioukov and Weaver, 1995; Alan et al., 2010). AD occurs in rats with high, but not low, thoracic injury (Rivas et al., 1995) and can persist for at least 6 weeks after the injury event (Laird et al., 2006). Unlike AD, orthostatic hypotension (OH), another cardiovascular derangement seen in SCI patients, has garnished less attention. OH is a drop in blood pressure seen in SCI patients in the sitting position. Although few researchers have evaluated rats for OH specifically, it may be seen in rats after T3eT4 level injury (Alan et al., 2010). Not surprisingly, the use of anesthetics for evaluation of the cardiovascular system after SCI

1041

can complicate the interpretation of results (Nout et al., 2012). Other autonomic functions have been evaluated after experimental SCI in rats (Inskip et al., 2009; Zimmer et al., 2007). Thermoregulatory apparatus may be impacted, and rats after SCI have lower core body temperature than uninjured controls (Laird et al., 2006). Gastrointestinal motility is frequently altered after SCI. Gastrointestinal tract activity can be monitored in rats via passage of orally administered dyes (Gondim et al., 1999, 2003), EMG (Holmes et al., 1998), manometry (Meshkinpour et al., 1985), acid breath tests (QuallsCreekmore et al., 2010), and strain gauge transducers (Qualls-Creekmore et al., 2010). Respiratory abnormalities and the ability to evaluate them in animal models have been reviewed previously (Zimmer et al., 2007). Methods of assessment include phrenic nerve conduction studies (Baussart et al., 2006; el-Bohy et al., 1998; Zimmer and Goshgarian, 2006; Golder and Mitchell, 2005; Fuller et al., 2006), diaphragmatic EMG (Baussart et al., 2006; Vinit et al., 2006; Nantwi and Goshgarian, 1998), plethysmography (Choi et al., 2005; Fuller et al., 2006; Teng et al., 2003), and pneumotachometry (Golder et al., 2001; Skjodt et al., 2001). In addition to functional tests of the autonomic nervous system, histology of the target innervated organs, such as the diaphragm (Gill et al., 2014), can be performed. 6. Imaging Imaging modalities can uniquely noninvasively examine internal anatomy of a single animal over time. Magnetic resonance imaging (MRI) and positron emission tomography (PET) findings in rats after spinal contusion correlate to lesions seen in postmortem exam and may be valuable for temporal tracking of individual SCI animals exposed to different therapeutic agents (Fraidakis et al., 1998; Nandoe Tewarie et al., 2010; Falconer et al., 1994; Byrnes et al., 2010; Bilgen et al., 2005; Tremoleda et al., 2016; von Leden et al., 2016). In addition to evaluation of the spinal cord parenchyma itself, imaging modalities may help evaluate supporting structures and distal organs that develop dysfunction. Synchrotron microcomputed tomography was used to reveal changes in the vasculature of contused rat spinal cords (Cao et al., 2015). Other imaging modalities can be used to evaluate target organ dysfunction. Urinary bladder volume has been estimated by ultrasound in rats with SCI (Keirstead et al., 2005). In this study, the calculated bladder volume correlated to locomotor function as measured by the BBB scale (Keirstead et al., 2005). 7. Microscopic Tissue Analysis Microscopic examination of the damaged spinal tissue is critical to the evaluation of pathological

RATS AS RESEARCH MODELS

1042

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

processes and recovery after SCI. Hematoxylin and eosin (H&E) staining may be used to determine cell populations and overall microscopic architecture (Byrnes et al., 2010; Akdemir et al., 1992; Ek et al., 2010; Surey et al., 2014; Benveniste et al., 1998). Other standard histological techniques use luxol fast blue for labeling myelin (Basso et al., 1995; Kjell et al., 2013, 2016; Benveniste et al., 1998), eriochrome cyanine for marking of axons (Detloff et al., 2010), silver stains for neurofibers (Khan et al., 1999), and cresyl violet stains for labeling neuronal cell bodies (May et al., 2015; Fischer and Peduzzi, 2007). Immunohistochemistry (IHC) can be used to label specific subpopulations of neurons or inflammatory cells within the spinal cord, based on antibody reactions with localized proteins (Kwon et al., 2002). Common markers of axonal damage include neurofilament (e.g., phosphorylation specific such as SMI-31/32 (Kjell et al., 2013) or general neurofilament-200 (Kjell et al., 2016; Ward et al., 2014; Metz et al., 2000a)) and myelin basic protein (Ward et al., 2014). Neuronal damage can be visualized by using antibodies against neuron-specific enolase (NSE) (Ek et al., 2010). Immunohistochemical markers of glial and inflammatory cell infiltration activation include glial fibrillary acidic protein (GFAP) for astrocytes (Gwak et al., 2012), OX-42 for microglia (Gwak et al., 2012), and ED1 or CD68 for macrophages (Kjell et al., 2013, 2016; Popovich et al., 1997). This list is far from complete, and some studies use many more markers, especially when evaluating supporting structures such as the spinal cord vasculature (Surey et al., 2014). A more comprehensive review of the spinal cord chemoarchitecture can be found elsewhere (Sengul, 2015). Evans blue dye has been used to evaluate the amount of microvascular leakage after SCI, and its content in the spinal cord after intravenous injection is correlated with injury severity (Kaptanoglu et al., 2004). Axonal tracers may be used to assess transport along the axon in either the anterograde (toward axon terminal) or retrograde (toward cell body) direction. After injection and a predetermined latency period to allow for transport to the target area, the animal is euthanized and the tracers are viewed using microscopy alone or in conjunction with IHC. Anterograde tracers are injected near the neuronal cell bodies and after uptake are transported down the axon to its terminal. Biotinylated dextran amine can be used as an anterograde tracer of the corticospinal tracts after injection into the sensorimotor cortex (Anderson et al., 2009b; Chan et al., 2005). Cholera toxin subunit B (CTB) has been used as a retrograde or anterograde tracer to measure large myelinated A fibers of intact spinal projections in SCI rats (Alluin et al., 2011). CTB is injected peripherally, for example, into the sciatic nerve or distal paw (Onifer

et al., 2005), and is then measured in the sensorimotor cortex of the brain with the use of anti-CTB antibodies by IHC. Other retrograde tracers include horseradish peroxidase, FluoroGold, Nuclear Yellow, and Fluororuby, among others (Pikov and Wrathall, 2001; Hill et al., 2001; Boulenguez et al., 2007; Plant et al., 2003; Biernaskie et al., 2007; Kwon et al., 2002). Modified viruses, such as adeno-associated virus (Liu et al., 2014), rabies (Adler et al., 2017), and pseudorabies (Pikov and Wrathall, 2001; Duale et al., 2009; Lee et al., 2014) have also been used as tracers.

E. Special Considerations for Animal Care in SCI Studies 1. Preinjury As with any animal protocol, it is important to begin by carefully considering animal selection. Variations in motor recovery timelines and the development of thermal allodynia have been documented between different stocks of rats (Mills et al., 2001). Differences in behavioral outcomes and even the paths of white matter tracts within the CNS may vary not only by stock or strain, but even within stocks from differing vendors, emphasizing the need for attention to detail in this area (Clarke and Bilston, 2008; Kjell et al., 2013; Clark and Proudfit, 1992; Popovich et al., 1997). For example, Lewis rats differ from Sprague-Dawley in magnitude and duration of macrophage activation and number of T-cells in the lesion epicenter after contusion injury (Popovich et al., 1997). Responses in behavior outcomes may also be seen at baseline and after different types of experimental SCI. After contusion injury, Long-Evans rats may be more sensitive to developing mechanical allodynia, while Sprague-Dawley rats may be more sensitive after transection and different strain/stock-related patterns may be seen with thermal testing (Mills et al., 2001). Recovery of locomotor function after SCI can also progress at different speeds between rat stocks and strains, with Sprague-Dawley rats performing better than Wistar, and Long-Evans recovering slowest in the first 10 days after injury (Mills et al., 2001). Even differences in rat stocks purchased from different vendors may lead to differences in functional outcome after experimental SCI (Kjell et al., 2013). Furthermore, while inbred rat strains are widely available and advantageous due to reduced interanimal variability, some have argued that outbred stocks may be more representative of the diversity seen in the human population, thus improving potential for translation (Mills et al., 2001). In addition to stock, strain, and vendor, researchers may want to consider performing pilot studies whenever new animal types are used to avoid so-called “ceiling and basement” effects in outcome measures (Fouad

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

et al., 2013). Of note, the rat’s spinal cord grows by age of rat, not by body weight, so males and females of the same strain and similar age will have similar spinal cord lengths despite differences in mass (Young, 2009). In addition to understanding the implications of using a certain stock or strain, as always, it is important to use animals of known health status. While some diseases, such as TMEV-GDVII, are known to produce flaccid paralysis similar to that seen following SCI in experimentally infected rats, there is also potential for unexpected outcomes with animals of unknown or poor health status (Rodrigues et al., 2005). One group reports that, after a cohort of animals showed surprisingly good functional and histologic outcomes in an established SCI model, they learned that their animal vendor had experienced a parvovirus outbreak and those animals may have been infected (Kjell et al., 2016). Lastly, environmental stressors such as handling can affect the pathophysiology of injury (Akhtar et al., 2008). In anticipation of the increased handling that will be necessary for postinjury support and monitoring, it is generally recommended to acclimatize animals to handling before injury to reduce stress from postinjury handling. This may include multiple stages of acclimatization spanning several days. For example, rats will benefit from a period of acclimation following arrival in a new facility, with the time to acclimate varying somewhat based on the needs of the study (Conour et al., 2006). Following this, many recommend gradually introducing research animals to the handler and testing apparatus, for example, exposing the animal to frequent gentle handling until the animal no longer demonstrates signs of apprehension in the presence of the handler (Puckett et al., 2009). 2. Injury Procedure Many anesthetics have neuroprotective or neurotoxic properties that may confound neurotrauma studies. Reductions in metabolic rate within the CNS during isoflurane anesthesia, for example, may protect the tissue from some degree of damage (Akhtar et al., 2008; Fouad et al., 2013; Blickman and Brossia, 2009). Historically, some researchers have avoided the use of inhaled anesthetics for animals undergoing CNS injury procedures due to difficulties in delivering such agents given the special constraints of stereotactic equipment and the proximity of the surgical field (e.g., cervical spine) to the drug administration apparatus, but many companies now produce devices that can help overcome these obstacles (Blickman and Brossia, 2009). While barbiturates have historically been used in CNS injury studies, they can have respiratory depressant effects and can impair gastrointestinal motility, which could be problematic for recovering animals. Furthermore, their use may result in prolonged recoveries with some

1043

residual CNS depression, which may complicate postinjury observations (Schantz, 2009). Although the use of anesthesia is required for ethical reasons during induction of experimental SCI in animals, the impact these agents may have on animal responses and outcome measures should be considered during the study design phase, and investigators should strive to maintain consistency in the anesthetic experience between animals (Hodgetts et al., 2009). Furthermore, it is important that researchers maintain noninjured but anesthetized control animals to be certain that outcomes seen are related to injury and not just to the effects of anesthesia (e.g., animals undergoing anesthesia only) or surgery alone (e.g., sham-injured animals) (Hook, 2013). Aside from specific anesthetic drugs, surgical support and monitoring can have profound effects on the outcome in SCI studies. Investigators should pay close attention to anesthetic depth to minimize physiologic effects of deep anesthesia. The use of local anesthetic agents can help investigators to minimize the amount of systemic anesthetics needed to maintain animals at an appropriate plane of anesthesia, and may help mitigate some of the negative physiologic effects of those agents. Maintenance of normothermia is critical, because both hyper- and hypothermia have been used as trial therapies for the treatment of SCI, and active warming to maintain body temperature is possible with medical-grade heating devices or warm fluid bags (Santos-Benito et al., 2006). Hemorrhage from the vertebrae or epidural venous plexus can be controlled with the use of bone wax or gel foam, respectively (Zhang et al., 2014b). If respiratory disruption occurs, for example, following cervical injury, respiratory support may need to be provided, and in some models it may be best to preemptively intubate and ventilate animals throughout their injury event, and it may be beneficial to provide animals with flow-by oxygen during recovery (Ayer et al., 2009). Respiratory rate and mucous membrane color can be useful in approximation of oxygenation status, but precise determination requires the collection of blood gases and alteration of ventilatory parameters as appropriate. As hypotension and hypoxia are both potential outcomes of SCI, it is important to control these parameters as tightly as possible to avoid unintentionally worsening the effects of the injury (Young, 2009; Akhtar et al., 2008). In instances where a strong impact is planned as part of the injury, it may be beneficial to express the urinary bladder prior to injury to reduce the likelihood of bladder trauma (Jakeman et al., 2009), and in anticipation of postinjury locomotor and urinary difficulties it is often recommended to express the urinary bladder and provide warm isotonic fluids prior to the animal recovering (Puckett et al., 2009; Santos-Benito et al., 2006). Immediately following injury, animals may lose some fine control of

RATS AS RESEARCH MODELS

1044

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

blood vessel tone and fluctuations in blood pressure are possible. For this reason, animals should be kept calm and relaxed during the immediate postinjury period to avoid catastrophic drops in blood pressure (SantosBenito et al., 2006). 3. Postinjury The spinal cord plays a number of critical roles in the day-to-day function of an animal. For this reason, animals with experimentally created SCI require careful attention in both preinjury protocol planning as well as specialized support in the days following injury. Animals may experience a variety of maladies due to their expected reduced mobility following SCI, ranging from relatively mild (callouses) to severe (decubitus sores from prolonged recumbency or weight loss and dehydration from inability to prehend food and/or water) (Hodgetts et al., 2009). For this reason, investigators should have a plan in place to carefully monitor weight, body condition, activity level, hydration status, and pressure points to address any of these issues in a timely manner postinjury. It is often helpful to acquire a baseline set of observations on each animal preinjury so that any changes that occur postinjury are more readily apparent. On a similar note, whenever possible the same researcher should be responsible for making the majority of observations on a given animal, thus reducing interobserver variability in subjective measurements (Schantz, 2009). Even in long-term (up to a year) studies, animals benefit from daily clinical exams to identify issues early. Frequent massage of the tissues overlying pressure points may help promote blood flow to these areas and prevent ulceration, and some investigators report that performing a passive range of motion exercises on major muscle groups below the level of injury can help reduce muscle atrophy and joint contracture (Santos-Benito et al., 2006). In most instances, animals will require administration of subcutaneous fluids for at least several days following injury (Puckett et al., 2009; Lukovic et al., 2015; Young, 2009; Lee et al., 2012). In many cases, the monitoring paradigms may differ between the acute (immediate postinjury) and chronic phases, as animals may begin to regain motor or bladder function (Schantz, 2009). As with all animal protocols, investigators should develop detailed humane endpoints allowing for the elimination of unnecessary pain and suffering, and all personnel should be appropriately trained to identify welfare concerns and intervene as necessary (Schantz, 2009). It is important to remember that, in addition to the SCI itself, many of these animals will have undergone major surgeries such as laminectomies. As with all postsurgical animals, rats should be provided postoperative analgesia, monitored carefully for signs of incisional issues or discomfort, and treated with analgesics (Schantz,

2009; Lukovic et al., 2015). Both opioids (such as buprenorphine) and nonsteroidal antiinflammatory drugs are commonly used in animals following SCI; for more invasive surgeries, a combination of these drugs may be necessary to achieve appropriate pain control (Blickman and Brossia, 2009). In addition to the anticipated postsurgical pain, rats may experience dysesthesias following SCI, and self-mutilation, sometimes known as autophagia, is a common clinical sign (Santos-Benito et al., 2006; Geissler et al., 2013). With autophagia, animals may self-bite at dermatomes “below” the level of injury, and wounds can be categorized from stage 1 (mild red skin and hair loss) through 4 (severe exposure of viscera) (Young, 2009). A percentage of rats may also self-traumatize their feet and toes, potentially causing severe injury that has the potential to interfere with locomotor assays in addition to raising welfare concerns for the animal (Fig. 29.6) (Young, 2009). Investigators should plan to monitor areas prone to these lesions, in particular the extremities, and have a mechanism in place for treatment or removal of affected animals from the study. Some investigators have had success providing animals with alternative chewing substrates (e.g., nylon bones), and other techniques that have been attempted to minimize self-trauma include bandaging the affected region of the body and applying aversivetasting substances (e.g., Bitter Apple, Chew Guard,

FIGURE 29.6 Photograph of self-trauma to the digits, as is common due to dysesthesia following some spinal cord injuries. Image courtesy of Dr. Chelsea Wallace, University Laboratory Animal Resources, University of Pennsylvania.

RATS AS RESEARCH MODELS

II. SPINAL CORD INJURY

Mordex Solution, Specicare BitterSpray; a liquid bandage, such as New-Skin, mixed with metronidazole) to the affected extremity, bandaging, or Elizabethan collar placement (Santos-Benito et al., 2006; Brown, 2006; Zhang YP et al., 2001; Puckett et al., 2009). When wounds are present, they should be cleaned as appropriate and kept dry; some authors have suggested routine soaking in Epsom salts (Puckett et al., 2009). Antibiotics and analgesics should be tailored to the individual animal; antibiotics should only be used if there is a high index of suspicion for infection, ideally based on the results of culture and sensitivity testing (Santos-Benito et al., 2006; Puckett et al., 2009). Children’s acetaminophen is highly palatable and reportedly helps alleviate these signs in rats, and antiepileptic drugs such as gabapentin can also alleviate abnormal sensation in rats with SCI; other drugs may be mixed with preferred substances such as chocolate cream to improve the likelihood of voluntary intake (Hulsebosch et al., 2000; Young, 2009; Santos-Benito et al., 2006). Many of these animals will temporarily or permanently lose the ability to urinate and/or defecate independently, which can lead to stagnancy and ascending infection (Geissler et al., 2013). These animals may require regular daily abdominal massage to promote fecal passage, and urinary bladder expression, typically at least two times daily, often up to four times daily, depending on severity of injury (Dabney et al., 2004; Lukovic et al., 2015; Marcol et al., 2012; Young, 2009; Nashmi and Fehlings, 2001; Santos-Benito et al., 2006). The frequency of bladder expressions can be progressively reduced until the animal regains control of bladder function, which may be several months following injury, and some authors suggest continuing with at least one daily bladder expression until the animal’s endpoint to ensure that complete emptying occurs at least daily. In addition to its therapeutic benefits, bladder expressions offer the investigator or clinical staff an opportunity to check the urine visually, by microscopy, or by the aid of a dipstick for signs of early infection (Santos-Benito et al., 2006). Caution must be exercised during bladder expression to prevent rupture of the urinary bladder. These animals also require heightened monitoring for signs of ascending urinary tract infection, such as blood spotting and foul or discolored urine, because this is a major cause of morbidity and mortality in animals with SCI (Young, 2009). Animals with any of these clinical signs should undergo urine culture and should be treated according to the results of sensitivity testing. If urine leaking is present, the perineum should be cleaned regularly to prevent the

1045

development of urine scald (Puckett et al., 2009). If scald develops, treatment with a barrier ointment is sometimes helpful (Puckett et al., 2009). The urinary bladders of female rats are easier to fully express, which makes them somewhat less vulnerable to urinary tract infection and may contribute to a preference for using female rats for some studies (Battistuzzo et al., 2012). A technique for suprapubic urinary catheterization in male rats has been described, and this may help investigators to manage this issue (Robinson et al., 2012; Onifer et al., 2007b). There are a variety of additional nonpharmaceutical supportive measures that can be taken to improve outcome and quality of life for animals that have undergone these types of injuries. For example, animals should be handled gently, and specifically should not be lifted by the tail following SCI due to the potential for exacerbated injury or discomfort. If animals are expected to have poor locomotive ability, they can be provided with clean, soft, deep, absorbent bedding, moistened chow on the cage bottom, and easily accessible water or gel (Santos-Benito et al., 2006). Animals that lose weight after injury, or that are nauseated following administration of pain medication, can be enticed to eat with a highly palatable diet or supplemented with flavored vitamin mixes (e.g., ENSURE) and may require encouragement or hand feeding (Schantz, 2009; Puckett et al., 2009; Santos-Benito et al., 2006; Onifer et al., 2007a), although investigators must bear in mind that some diet changes, including high-fat diets or intermittent fasting, may alter functional outcome following SCI (Streijger et al., 2013; Miyazato et al., 2005). Reductions in environmental noise or light in the housing room along with provision of appropriate environmental enrichment, social interaction, and an appropriate environment for thermoregulation may be stress relieving and have some analgesic effects (Schantz, 2009; National Research Council, 2003). In addition to improving animal welfare, reductions in environmental stress may have the added benefit of reducing variability in some behavioral outcome measures. For example, animals housed in a room with others that had undergone stressful procedures performed differently in a behavioral task than those housed with unstressed animals (Fouad et al., 2013). Some investigators provide opportunities for exercise for rats with SCI, which may help improve outcome and animal wellbeing (Santos-Benito et al., 2006). Lastly, to maximize efficacy, it may be helpful to acclimate animals to these supportive measures (palatable diet, changes in bedding, etc.) prior to injury (Schantz, 2009).

RATS AS RESEARCH MODELS

1046

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

III. TRAUMATIC BRAIN INJURY A. Introduction to Traumatic Brain Injury TBI represents a major health and socioeconomic problem throughout the world. In the United States alone, it is estimated that between 1.7 and 2.8 million people suffer a TBI annually (Taylor et al., 2017; Faul et al., 2010; Langlois et al., 2010; Langlois et al., 2006; Jordan, 2013; Humphreys et al., 2013; Committee on SportsRelated Concussions in Youth, 2014) with those estimates expanded to 1.5e3.8 million when including sports-related TBIs per year (Faul et al., 2010; Jordan, 2013; Centers for Disease Control and Prevention, 2011; Committee on Sports-Related Concussions in Youth, 2014). In addition, TBI has been described as the “signature injury” in soldiers returning from Operations Iraqi Freedom and Enduring Freedom (Hoge et al., 2008). In the clinic, TBI is often classified as mild (also referred to as “concussion”), moderate, or severe, with outcomes ranging from mild cognitive impairment to long-lasting and/or permanent physical and emotional changes and memory loss (Monti et al., 2013; Ryan and Warden, 2003). TBI is set apart from other neurological disorders such as stroke in that it is induced by a discrete physical action, whether it be a blow to the head or rapid acceleration of the brain within the skull (inertial force). This event is referred to as the “primary injury” and is the watershed event leading to downstream changes in neurological function and physiology. The primary injury can result in blood vessel disruption (e.g., ranging from overt bleeds to subtle changes in the bloodebrain barrier), tissue tears caused by shearing, and subcellular damage such as plasma membrane and cytoskeletal disruption. Similar to SCIs, primary TBIs are often complicated by other factors, such as secondary pathophysiological cascades such as excitotoxicity, oxidative stress, inflammation, and/or reactive gliosis (Ruppel et al., 2001; Yi and Hazell, 2006; Clausen et al., 2012; Halstrom et al., 2017; Johnson et al., 2013; Andriessen et al., 2010; Muir, 2006). These sequelae often persist long after the primary injury occurs. In fact, TBI has been linked to neurodegenerative diseases such as Alzheimer’s disease (Johnson et al., 2010; Smith et al., 2013) and chronic traumatic encephalopathy (CTE) (Mez et al., 2017; Huber et al., 2016; Montenigro et al., 2017; Baugh et al., 2012; McKee et al., 2016). Indeed, TBI should be considered as an acute biomechanical trauma that elicits chronic neurodegenerative and inflammatory sequelae in at least some individuals. Given the widespread nature of TBI throughout the United States and the world, it is important to understand the onset and temporal evolution of the changes that occur within the brain. Research into the

neuroanatomical substrates most commonly afflicted as well as the general pathophysiological changes will provide valuable insight into treating the disorder. Thus it is important to have animal models that reproduce the physical and physiological conditions that are associated with TBI. Rodents provide adequate models in that they are easily maintained, inexpensive, highly trainable, and offer the chance to address a series of hypotheses with relative expediency. Furthermore, the creation of genetically modified rat strains provides an avenue to explore more detailed and specific questions, from subcellular organization to afflicted signaling pathways to overall network connectivity changes. Several rat models of TBI have been developed, each presenting specific advantages and disadvantages. All have been extensively reviewed elsewhere, and the reader is referred to those publications for more in-depth analysis of each model (Xiong et al., 2013; Johnson et al., 2015; Estrada-Rojo et al., 2018; Marklund, 2016; Rostami, 2016; Phipps, 2016). As with SCI, our intention in this chapter is not to thoroughly review each model of rat TBI, but rather to provide an introduction and overview to the veterinarian or scientist unfamiliar with models used in the field.

B. Rats as Models for Human TBI Rodent models of TBI provide a powerful platform with which to develop and test hypotheses related to trauma-induced physiological responses and neurodegeneration. Furthermore, they offer an opportunity to test neuroprotective agents prior to moving to models utilizing larger animals or into clinical studies. However, the rat has inherent limitations as a platform for the study of TBI, which are outlined in the following, and therefore is not suitable to mimic all aspects of clinical TBI. Crucial differences between rats and humans (and large animals such as pigs and sheep) must be considered when formulating hypotheses, designing experiments, and interpreting the results in studies using rodent models of TBI. The first key difference is gross anatomy. Perhaps most obviously, in humans and bipeds the brain is arranged at a right angle relative to the spinal cord, whereas in rats and other quadrupeds there is a linear relationship. This difference in orientation could affect the biomechanics of injury as it is applied to the brain. Another major difference is related to the anatomy of the brains themselves: humans and most large mammals have gyrencephalic brains of three-dimensional gyri and sulci and substantial white matter domains, whereas rats have lissencephalic brains that are flat and have significantly less white matter.

RATS AS RESEARCH MODELS

III. TRAUMATIC BRAIN INJURY

Also, human brains exhibit approximately a 60:40 ratio of white matter to gray matter, whereas that ratio is approximately 14:86 in rats (Howells et al., 2010; Bailey et al., 2009; Zhang and Sejnowski, 2000). Given that the predominant neuropathological outcome of closedhead injury is diffuse axonal injury (DAI) (Povlishock and Katz, 2005; Smith and Meaney, 2000; Adams et al., 1982, 1989, 1991; Graham et al., 1988; Povlishock and Becker, 1985; Povlishock et al., 1983), and that cortical neuronal degeneration in closed-head TBI and CTE follows a distinct pattern with respect to macrostructures (e.g., gyri and sulci in the cortex) (McKee et al., 2009), the complex constitution of the human brain may be the key factor in the development of trauma-induced neuropathology (Duhaime, 2006; Smith and Meaney, 2000). Replicating these conditions in a lissencephalic rodent brain is nearly impossible. Second, due to the biomechanical etiology of TBI, it is desirable to apply injury parameters that are scaled from the physical loading experienced by the human brain in TBI, for instance, specific tissue deformation levels applied over rapid timescales, which are well established based on cadaveric studies, computer simulations, and physical models. Brain mass is a key factor in scaling these parameters for closed-head inertial brain injurydthe most common type of TBI, which includes all concussionsdthat are generally caused by rapid head rotation (i.e., described as angular acceleration/ deceleration), with or without concomitant head impact (Holbourn, 1943; Gennarelli et al., 1971, 1972, 1982). Indeed, the large mass of the human brain is a dominant cause of deformation levels and patterns resulting from these closed-head rotational injuries. However, scaling rotational injury parameters from humans to rats is problematic because the angular accelerations in the rat brain would need to approach 8000% of that which would induce injurious tissue strain levels in humans (Meaney et al., 2001). It is a significant challenge to achieve sufficient head rotational acceleration in small rodents to mimic the relevant tissue-level deformations without inducing compression effects or rupture of the vasculature, although some models have been proposed as described later. However, a general workaround for this issue is to employ open-head injury models where the brain tissue itself is impacted. While this approach is able to replicate features of the tissue- and cell-level deformation levels and timescales representative of human TBI, the open cranium often confounds studies of mild TBI that by definition is a closed-head injury. Thus rat models are limited in their ability to be appropriately scaled to achieve mechanical loading parameters that would be analogous to that seen in human TBI (Thibault et al., 1990; Margulies et al., 1990; Meaney et al., 1995; Holbourn, 1943), and therefore have the risk of reporting effects that, while certainly traumatically

1047

induced, are outside the biomechanical loading “dose” that is relevant for TBI in humans. Third, there is significant interest in exploring links between TBI and specific neurodegenerative diseases by utilizing animal models that replicate the conditions and pathology seen in Alzheimer’s disease, Parkinson’s disease, and CTE, among other diseases. Unfortunately, in the absence of genetic modification, rodents do not produce the characteristic tauopathy, synucleinopathy, and/or amyloid beta plaques that are the defining features of these diseases in humans, although notably these changes do emerge in larger animal models, such as swine (Chen et al., 2004). Although genetic manipulation in mice has provided the TBI community with valuable tools to explore the potential link between head trauma and chronic neurodegenerative diseases, the availability of genetically modified rats remains a limiting factor in the field. Moreover, the usage of particular genetic modifications in rodents requires careful interpretation because it is based on a priori assumptions regarding the pathophysiological progression and/or blossomed neuropathological endpoint(s) rather than having such findings naturally emerge as a consequence of the traumatic loading. In summary, rat models of TBI present a useful, highthroughput platform for exploring specific facets of TBI. However, injuries carried out in these animals may fail to fully capture the mechanisms and distribution of pathophysiological responses and neuropathological manifestations. To underscore this point, despite the success of a multitude of neuroprotective agents in rodent models, none have translated to clinical success (Marklund and Hillered, 2011). As such, there should be careful selection of an appropriate rodent TBI model based on the particular scientific question and/or endpoint(s). For example, those research questions that focus on pediatric injury need careful planning in animal age selection and modifications to injury procedures (Prins and Hovda, 2003). Moreover, it may be necessary to further vet the findings in a large animal model with greater biomechanical and physiological fidelity to human TBI to validate pathophysiological mechanisms found using rodents.

C. Rat Traumatic Brain Injury Models 1. Fluid Percussion InjurydLateral and Midline The most widely used model of TBI in rats is fluid percussion injury (FPI). Originally adapted for use in rats in 1987 by Dixon and colleagues (Dixon et al., 1987), the FPI model was designed to systematically examine the neurological, physiological, and histopathological outcomes of a direct mechanical insult to the brain. Over the years, the model has evolved to include

RATS AS RESEARCH MODELS

1048

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

lateral (LFPI) (McIntosh et al., 1989), midline (MFPI) (McIntosh et al., 1987), and parasagittal (Sanders et al., 1999) models of FPI. The FPI device is currently commercially available through AmScien. The FPI model has a simple design: a pendulum strikes a plunger on a fluid-filled cylindrical reservoir that causes a pressure pulse to propagate through sterile saline and across the intact dura via a small craniectomy (Fig. 29.7). The pulse, the severity of which can be controlled by the height/angle of the pendulum, causes a reproducible deformation of the tissue (primary injury) and starts a cascade of physiological changes (secondary injury) in the brain. Small shifts in craniectomy position will afflict different neuroanatomical substrates, and will therefore yield different outcomes. FPI techniques have been successfully adapted for use in juvenile rats, although morbidity may be increased when immature animals are used (Sun et al., 2005; Rowe et al., 2018; Prins et al., 1996; Semple et al., 2016). To perform FPI, the rat must be anesthetized. The most commonly used anesthetics are isoflurane, pentobarbital, or the combination of ketamine/xylazine/acepromazine. Once the animal is anesthetized and prepared for sterile surgery, it is mounted into a stereotactic frame. A midline scalp incision is made and the skin reflected to expose the bregma and lambda landmarks on the skull (Fig. 29.8). For midline FPI, a 5-mm craniectomy is made on the sagittal suture midway between bregma and lambda. For lateral FPI, a 5-mm craniectomy is created 1 mm from the temporal ridge and centered between bregma and lambda. The dura should remain intact. A luer-lock hub is placed in the craniectomy, secured to the skull with cyanoacrylate/dental cement, and filled with sterile saline. At a specified time postsurgery (generally ranging from 1 to 24 h), the

FIGURE 29.7 Representative photograph of a fluid percussion injury device for the creation of TBI in the rat.

(re-)anesthetized rat is placed on the FPI device by connecting the luer hub to the opening on the reservoir. While holding the rat in position, the pendulum is released allowing it to strike the piston on the fluid reservoir. A bolus of saline is transferred from the reservoir to the intact dura creating the pressure wave and associated injurious brain deformation. A pressure transducer attached to the FPI device at or near the point of connection to the rat records the pressure. After the injury is induced, the rat is removed from the device and respirations are measured. Apnea commonly occurs at higher levels of injury, and the duration of respiratory arrest is recorded. The luer and dental cement are removed, and the incision is closed. Here it is preferable to use interrupted suture or staples as rats have a tendency to pick at the suture line, especially if not singly housed, and continuous sutures may unravel if the rats are able to remove the knot or cut the suture anywhere along its length. In a variation of this technique, the rat is anesthetized with isoflurane during injury preparation, but after this the inhalant is withdrawn and the animal monitored closely. When the animal reaches a plane of anesthesia where they are still unconscious but develop a positive toe pinch reflex, the injury is administered. Following injury, the latency for the toe pinch reflex to return has been used as a surrogate for injury severity (Prins et al., 1996). Yet another version of this model exists where the rat is connected to the injury device via polyethylene tubing rather than directly positioned on the Plexiglass cylinder (Kawamata et al., 1992, 1995; Katayama et al., 1990). When using this method, it is important to realize that the pressure at the end of the tubing is never the same as what is being measured by the transducer attached to the end of the Plexiglass cylinder. As noted previously, placement of the craniectomy (and thus the site of injury) is critical and care must be taken when performing the surgery. Use of the LFPI model of injury results in tissue damage predominantly constrained to the ipsilateral side of the brain (although contralateral effects have been studied), whereas MFPI results in bilateral tissue damage and may influence activity of the brainstem (Cernak, 2005). Previous studies examining the placement of the craniectomy have shown that the extent of tissue damage, cell loss, and cognitive deficits are affected by small shifts in position (Vink et al., 2001; Floyd et al., 2002). Furthermore, and perhaps most importantly, it is vital that craniectomy be consistent from animal to animal to ensure reproducibility within the experiment. 2. Controlled Cortical Impact Another widely used impact model is the controlled cortical impact (CCI) model. CCI is similar to FPI in that a mechanical force is applied to the intact dura to create a distinct, highly reproducible injury. However,

RATS AS RESEARCH MODELS

III. TRAUMATIC BRAIN INJURY

(A)

1049

(B)

FIGURE 29.8 Common landmarks for craniectomy in the rat. (A) Schematic demonstrating the location of bregma, lambda, and the interaural line. (B) Photograph of a rat during preparation for craniectomy. Please note: a drape has been excluded to facilitate visualization. Panel A reproduced from Paxinos, G., Watson, C., 2007. Stereotaxic reference system. In: The RatBrain in Stereotaxic Coordinates. Academic Press, Elsevier, Oxford, p. XII.

in this case, a pneumatic piston drives a rigid impactor into the brain (Fig. 29.9) (Dixon et al., 1991). CCI devices are commercially available through Leica Biosystems and Stoelting and can be mounted to stereotaxic frames (Sutton et al., 1993). Traditionally, CCI has been used as a unilateral model of TBI, and the impactor properties may be modified based on diameter, geometry (e.g., rounded or flat bottom), and hardness. Unique to CCI, the impactor can be set to a specific depth and speed, thereby creating a mild injury (slow and/or shallow) or severe injury (rapid and/or deep). As in LFPI, the contralateral hemisphere may be used as a control for changes related to TBI, although investigators should be cautioned that global changes may occur following injury (Xing et al., 2009). Of note, both the FPI and the CCI models induce progressive tissue atrophy and at sufficient severity levels will result in a defined lesion cavity (Smith et al., 1997; Dixon et al., 1999). CCI injuries have been used in both adult and juvenile rats (Prins and Giza, 2006; Huang et al., 2016; Semple et al., 2016).

The surgical preparation of the rat is similar to that in FPI. The animal is anesthetized, prepped for sterile surgery, and mounted into a stereotactic frame. A midline scalp incision is made to expose bregma and lambda. A 3e5-mm craniectomy is created midway between bregma and lambda lateral to the midline suture. Craniectomies can be unilateral or bilateral if desired. Once the craniectomy is complete with the dura intact, the rat is positioned under the piston/impactor such that the tip of the impactor barely touches the dura. Keeping the rat in the stereotactic frame while performing the injury stabilizes the head, minimizing any motion that may confound the injury; however, the rat may be placed on the platform with the head resting on the base. Once the rat is in position and the piston is set at the dura, the piston is raised into its uppermost position and the depth and speed are set. The piston is then fired onto the brain and the incision is closed (McIntosh et al., 1987). Similar to FPI, a version of this technique has been described where isoflurane is discontinued immediately prior to injury and latency to return of toe pinch is measured (Casella et al., 2014). 3. Penetrating

FIGURE 29.9 Representative photograph of a controlled cortical impact device for the creation of traumatic brain injury in the rat.

Penetrating brain injuries (PBIs), such as those caused by bullets, are usually associated with high mortality in humans. However, survival from PBI has been dramatically increased with improved clinical intervention and management; in some cases, recovery is similar to patients that have experienced nonpenetrating injuries (Hotz et al., 2000; Ylioja et al., 2010; Zafonte et al., 2001; Kriet et al., 2005). In humans, PBI is most prevalent among warfighters, although the civilian population is not immune. Self-inflicted gunshots, crime, and terror attacks have all contributed to the number of PBIs reported throughout the world. In ballistic injuries, the projectile carves out a path of destruction that is dependent on the size, shape, and degree of movement (i.e., “wobble”) of the projectile. Early

RATS AS RESEARCH MODELS

1050

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

research on missile injury in cats was helpful in demonstrating that the projectile is also associated with highenergy pressure waves that create a cavity in the tissue as the projectile moves along its track (Carey et al., 1989). With increasing need to improve outcomes for warfighters with penetrating TBI in the wars of the late 20th and early 21st centuries, the need arose to further understand and characterize the anatomical, physiological, and neuropathological changes that result from PBI. To address these questions, a novel technique was developed in rats to model cranial bullet wounds, with the intent to focus on the injury tract created by the bullet path and the large cavity created by the energy of the projectile (Williams et al., 2005). The procedure involves placing an anesthetized rat in a stereotactic frame and creating a craniectomy in the desired location. The injury is produced by inserting a probe (attached to a stereotaxic arm) attached to a balloon into a desired location within the brain and inflating the balloon via a pressure pulse of air. Once the probe is in position, the balloon is rapidly inflated to the appropriate volume using a commercially available device. The probe is withdrawn and the skin sutured closed. Injury severities can be varied by expanding the balloon to different volumes. Angle of insertion and depth of penetration are easily controlled. More recently, another model of PBI was developed that utilizes a probe driven into the brain after being hit by a high-velocity pellet (Plantman et al., 2012). In this model, the anesthetized rat is placed in a stereotactic frame and a burr hole created in the skull at the desired location. The impactor is then positioned directly over the dura. A lead pellet is accelerated by air pressure from a modified air rifle. The projectile then impacts a probe consisting of a metal cylinder with an attached carbon fiber pin. Depth of penetration is controlled by a brass ferrule fitted around the carbon pin. Once the injury is complete, the skin is closed and the animal recovered. 4. Weight Drop In weight-drop models, the head is exposed to a freefalling weight and injury severity can be manipulated by changing the mass of weight or the height from which the weight is dropped (Fig. 29.10). One of the major advantages that the weight-drop TBI model has over the FPI and CCI models is that it is generally employed as a closed-head injury, which as noted previously is more representative of the vast majority of clinical TBIs. This also makes the weight-drop method more amenable to examine repetitive injury to simulate multiple concussions over time, believed to be an important risk factor in the development of CTE. However, the model is versatile because it can be carried out using a closed-skull approach (Shohami et al., 1988; Shapira

FIGURE 29.10 Representative photograph of a weight drop device for the creation of traumatic brain injury in the rat.

et al., 1988; Marmarou et al., 1994) or an open-skull approach with an intact dura (Feeney et al., 1981). For the latter, a craniectomy is created in the skull (similar to as described for FPI or CCI) and the weight is dropped onto the intact dura, creating a contusion in the tissue below the site of impact. Weight-drop TBI models created in adult rats have also been adapted for use in juvenile animals (Mychasiuk et al., 2014; Grundl et al., 1994; Semple et al., 2016). The most common weight-drop methods are closed skull and performed unilaterally (Shapira et al., 1988; Shohami et al., 1988; Feeney et al., 1981) or bilaterally (Marmarou et al., 1994). In the unilateral model, the skull remains unprotected and the weight is dropped directly on the bone while the head is placed on a hard surface. In contrast, the Marmarou method utilizes a protective plate and places the head on a soft surface. The soft surface allows for head motion, thus the Marmarou model combines focal impact with head acceleration. It should be noted that in this model, the composition of the padding is critical because its make-up controls the speed and magnitude of head movement at the time of injury. Although the intent of the model is to mimic the forces that may be associated with falls and motor vehicle accidents, the most common cause of TBI in humans, the extent that head rotational acceleration factors into the effects of the injury is debatable. Feeney et al. designed an apparatus to include a catch mechanism for the weight, preventing it from bouncing on

RATS AS RESEARCH MODELS

III. TRAUMATIC BRAIN INJURY

the plate and creating what is essentially a repetitive injury. A third model of closed-head weight drop, developed by Kilbourne et al., is designed to investigate changes in the brain that are caused by frontal impacts (Kilbourne et al., 2009). This method also incorporates focal and rotational forces but rather than dropping the weight midway between bregma and lambda, the impact is directed at the anterior portion of the skull. Similar to the CCI model of injury, the open-skull weight-drop method requires a craniectomy to expose the intact dura. A weight is dropped from a specified position directly onto the brain. The result is a cortical contusion and focal damage to the white matter tracts deep to the site of impact. The model produces neuronal and microvasculature injury, as well as profuse DAI in the white matter tracts, most notably the corpus callosum and internal capsule (Foda and Marmarou, 1994). Lesion development has also been reported (Shapira et al., 1988). Regardless of the model chosen, surgical preparation for the injury is simple. For closed-skull weight drop, the rat is anesthetized, the surgical site prepared aseptically, and the skin opened via a small midline incision to expose the skull. Once the injury is induced, the skin is closed and the animal recovered. The open-skull weight drop requires a craniectomy, thus the procedure involves the extra step of creating the opening in the skull. Physiological outcomes depend on the force of impact, which determines injury severity, and can include skull fractures (obviously not in the open-skull case), which can complicate the biomechanics of injury and have obvious repercussions for animal welfare. For this reason, animals with skull fractures are commonly excluded. Another complication is respiratory depression, which can be severe enough to result in death if animals are not ventilated (Marmarou et al., 2009). 5. Blast TBI Recent military conflicts have resulted in a large number of blast-exposed warfighters being diagnosed with TBI either along with complex polytrauma or in the absence of other injuries, thought to be due to the effects of blast exposure on the brain (Hoge et al., 2008; Warden, 2006). In an effort to understand the effects of blastinduced TBI, researchers have dedicated extensive resources to modeling explosive conditions in rodents that may represent those encountered by soldiers in the field. The nomenclature to describe the components of blast injuries was developed independently from the description of the pathophysiological phases of conventional TBI, which, unfortunately, has resulted in a confusing overlap in terminology. Briefly, the mechanisms of injury due to blast exposure are described using the terms primary, secondary, tertiary, and quaternary. Primary blast injury refers to injury that is

1051

caused purely by the shockwave, or blast overpressure, generated by the explosion. Effects are dependent on the magnitude, frequency, and timing of the overpressure waves. Early research into shockwave-induced injury focused primarily on fluid- and air-filled organs; however, more attention has been given to the direct effects of blast shockwave propagation on the brain that would cause a blast-specific form of TBI. Secondary blast injury is caused by objects that are propelled into the subject, such as flying debris and shrapnel, which may cause penetrating brain injuries. Tertiary blast injury results when the body is thrown and subsequently impacts a solid object, such as the ground or a wall with brain injury resulting from either direct impact or through inertial forces. Finally, quaternary injuries include burns, chemical exposure, crush injuries, and other maladies that result from the explosion and are often not included in models that address blast-induced TBI. While the contribution of primary blast in isolation on military TBI is being actively debated, it is likely that the combination of primary, secondary, and/or tertiary blast exposure collectively form a unique clinical entity responsible for the majority of the TBI seen in warfighters. Efforts are under way to address this question, and evidence in mice suggests that the contribution of isolated blast wind to head acceleration is a primary mechanism in the development of blast-related TBI and CTE (Goldstein et al., 2012). In a separate study using postmortem tissue from athletes diagnosed with CTE and tissue from rats exposed to blast injury, Lucke-Wold et al. presented findings showing endoplasmic reticulum stress and tau pathology were similar between the two populations (Lucke-Wold et al., 2016). Further work is necessary to completely settle this debate. Various rat models of blast-induced TBI have been developed and generally incorporate the use the explosives in open field or a confined space, or shock tubes that are either gas pressure driven or explosive driven (Cernak et al., 1996; Cheng et al., 2010; Long et al., 2009; Reneer et al., 2011; Kuehn et al., 2011; Sundaramurthy et al., 2012; Risling et al., 2011). However, the majority of studies to date examining pure shockwave-induced TBI have been executed using a gas-driven shock tube. Since this method is most accessible to researchers wishing to conduct this type of research, we will limit our discussion to this model (Fig. 29.11). The shock tube consists of two compartments: one designed to contain compressed gas and the other that remains open to the environment. The chambers are separated by a sacrificial membrane, typically sheet(s) of mylar of a specified thickness. The number of sheets and thickness determine the rupture pressure and therefore the magnitude of the resulting blast pressure wave. Compressed gas is introduced into the gas chamber and upon rupture of the

RATS AS RESEARCH MODELS

1052

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

outfitted with monitoring equipment, such as intracranial pressure or blood flow sensors, no surgery is required to create this injury. The choice of anesthetic should be based on its influence on cerebral blood flow (CBF) and recovery time. 6. Rotation

FIGURE 29.11 A compressed gas shock tube used to create blast TBI in the rat. Reproduced from Mishra, V., Skotak, M., Schuetz, H., Heller, A., Haorah, J., Chandra, N., 2016. Primary blast causes mild, moderate, severe and lethal TBI with increasing blast over pressures: experimental rat injury model. Sci. Rep. 6, 26992.

mylar sheets, a shockwave is created that escapes down the length of tube toward the open end, propagating across an animal near the end of the tube. While the basic design of the shock tube is fairly consistent across laboratories carrying out this type of research, the positioning of the rat may vary based on experimental objectives. The rat may be placed inside the tube, at the opening of the tube, or a distance away from the tube. This positioning has important implications for the type of loading experienced by the animal. For instance, animals placed a distance within the shock tube experience more of a pure shockwave, which propagates through tissues (primary blast) but generally does not induce significant head/body motion (minimal tertiary loading). In contrast, when an animal is placed outside the shock tube, there is an extensive expansion wave and so-called “blast wind” that imparts significant momentum and therefore induces rapid head/body motion (primary þ tertiary loading). Additionally, the rat may be positioned such that the blast wave strikes the rostral, caudal, or lateral aspects of the animal. Furthermore, the rat may be outfitted with a Kevlar vest (or similar protection) to evaluate the effects of blast injury with or without additional bodily protection (i.e., isolating effects on the head/brain without confounding damage to gas-filled organs), or a harness or other restraint device to minimize movement of the animal during the blast wave (Mishra et al., 2016). There is considerable debate within the blast community about body positioning and protection of the rat, and no standard practices yet exist. As such, researchers should be cognizant of the trade-offs and limitations noted previously, and should select the experimental parameters that fit with the objectives of their study. If possible, it is good practice to measure both pressure wave characteristics and head motion in the rat. Unless the animal is

As noted previously, the vast majority of clinical TBIs are closed-head diffuse brain injuries caused by inertial (i.e., rotational) loading to the head (Langlois et al., 2006; Coronado et al., 2012; Ommaya and Gennarelli, 1974; Povlishock, 1992; Adams et al., 1989, 1991). One cause of injurious inertial loading is when the body has significant momentum but subsequently impacts a structure that causes rapid deceleration, such as the dashboard in a motor vehicular collision or the ground in the case of a fall. Alternatively, similar head rotational loading can occur when a person is struck by an object having significant momentum, such as a projectile (e.g., a baseball) or a tackler in American football. Rotational loading of the head can also occur in the absence of impact, such as a restrained occupant in a motor vehicle collision. However, all of these exposure types may lead to the same effect: the generation of rapid deformation patterns within the brain, which bioengineers refer to as dynamic stresses and strains. It is these suprathreshold strain fields (generally, 5%e50% strain over tens of milliseconds) that are believed to be the initial biophysical causation of diffuse brain injury (Holbourn, 1943; Denny-Brown and Russell, 1941). Of note, peak tissue stresses are associated with anatomical transition zones, so the organization of the mammalian brain may create many pockets of mechanical vulnerability, because of the different material properties of white matter tracts, gray matter, cerebrospinal fluid, and ventricles, which has implications for the response of the brain to insult. Given the prevalence of closed-head TBI, especially concussion, several rat models have been developed to examine the contributions of linear and rotational acceleration or pure rotational acceleration alone (Rostami et al., 2013; Kilbourne et al., 2009; Li et al., 2010, 2011; Mychasiuk et al., 2016; Fijalkowski et al., 2007). Models that attempt to evaluate the combination of linear and rotational forces involve the use of an impactor (Marmarou et al., 1994; Kilbourne et al., 2009; Li et al., 2011; Mychasiuk et al., 2016) and are based on the argument that the impact causes acceleration of the brain within the skull in different planes. This latter group includes the Marmarou weight-drop model that was previously mentioned due to the potential for rotational injury following impact. Pure rotational injury in either the coronal or sagittal plane has also been described (Fijalkowski et al., 2007; Li et al., 2010; Rostami et al., 2013). Although these models have been reported to produce

RATS AS RESEARCH MODELS

III. TRAUMATIC BRAIN INJURY

basic histopathological outcomes similar to those seen in human TBI such as neuronal loss, axonal degeneration, and glial activation, they are not expected to recapitulate the tissue-level deformation fields experienced in the human brain caused by rotational loading for the reasoning noted previously. There is little surgical preparation necessary when implementing these models since they are designed to model closed-head TBI. The impact/acceleration approach can be carried out in an anesthetized animal with or without the skin intact and with or without skull protection. Anesthesia has been achieved with pentobarbital, ketamine/xylazine, and isoflurane. While a common outcome of the Marmarou method is skull fracture, the other models seldom produce such a result. Most models will yield some degree of respiratory depression, with the most severe present in the Marmarou model (main cause of mortality).

D. Outcome Measures for Rat TBI Studies 1. Behavioral Tests Common deficits associated with TBI include cognitive impairment (memory loss, learning disability, lack of concentration), motor deficits (problems with motility and balance), and anxiety (Blennow et al., 2012; Ling and Ecklund, 2011; Bazarian et al., 2009). To be clinically relevant, rodent models of TBI should be capable of modeling elements of the anatomical, physiological, and behavioral changes related to TBI in humans. In all the models listed previously, behavioral and cognitive deficits are readily detectable and easily measured. Numerous tests exist to evaluate behavioral outcomes following TBI (Awwad, 2016; Fujimoto et al., 2004). Many of these tests require some degree of acclimation or training for the animal. It is important for investigators to provide a consistent level of training to each animal, because subjects that are “overtrained” may outperform cohortmates regardless of injury status (Wilcott, 1994). In the following, we present an overview of some of the most common tests used in the field. a. Morris Water Maze The Morris Water Maze was developed in 1984 by Richard Morris to assess learning and memory in rodents (Morris, 1984) and was later adopted by the TBI community. Over the years, the paradigm has been slightly modified by individual labs; however, the principle remains the same: rats are placed in a circular pool of water and provided a means of escape onto a hidden platform submerged just below the water line. Visual cues on the walls of the pool (or the walls of the room) are used for the rat to associate a certain region of the pool with the escape platform. The arena is 2e2.3 m in

1053

diameter and the test can be conducted in water that is rendered opaque (using powdered milk or small quantities of white body paint) or in clear water if the tank is constructed of black walls. Alternatively, Styrofoam peanuts can be floated on the surface to mask the location of the platform. Once the water is at the desired temperature (usually 20e22 C), rats are released into the arena from four different locations and given 60 s to locate the platform. Time to reach the platform (latency) is recorded for each trial and averaged. The rat is given four to six trials per day over 3 days and culminates in a probe trial where the platform is removed and the time spent in regions surrounding the absent platform is recorded. Since the development of more advanced video tracking systems and analysis software, researchers have begun collecting data on search strategy. As the animals swim in search of the platform, their activity is recorded and information is gathered on aspects such as swim speed, swim pattern, and average distance traveled over time (Edwards et al., 2015; Sweet et al., 2014; Wagner et al., 2013). The execution of the experiment varies depending on which parameter the researcher wants to measure. In general, the task will measure learning and spatial memory, but can be specifically designed to measure learning and memory or just memory. If learning and memory are desired, the experiment is performed after the TBI is produced and the researcher measures how quickly the rat learns and remembers where the platform is located. If memory is tested, the rat is exposed to the task prior to the TBI, thus acquiring the location of the platform under normal conditions. At a specified time postinjury, the rat is reintroduced into the maze and the trials are conducted, usually without the platform in place. Swimming speed is usually recorded to control for latency changes resulting from motor deficits rather than learning deficits. The Morris Water Maze is a simple task to perform and has been established to be reliable and reproducible. However, researchers should be mindful that age, strain, and sex play an important role in behavioral outcomes (Hamm et al., 1992; Reid et al., 2010; Fujimoto et al., 2004). Furthermore, since trials are conducted over several days, it is important to keep experimental conditions, such as ambient light, water temperature, and environmental cues, as controlled as possible. Rats are natural swimmers and as such take well to the water. On rare occasions, rats may exhibit signs of distress, such as vocalization or lack of swimming (in which case they sink). These situations are easily remedied by removing the rat from the trial. If the situation continues, the subject is usually excluded from the study. Once the rats have completed the trial, it is important to provide thermal support, usually in the form of a heat lamp positioned a safe distance from the animal.

RATS AS RESEARCH MODELS

1054

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Animals should not be returned to the colony until completely dry. b. Elevated Plus Maze People with TBI may suffer from various psychiatric conditions, such as depression, posttraumatic stress disorder, and anxiety (Ahmed et al., 2017; Howlett and Stein, 2016; Zaninotto et al., 2016). The elevated plus maze (EPM) (Walf and Frye, 2007; Carobrez and Bertoglio, 2005) is used by researchers studying the effects of TBI on psychological disturbances and is aimed at measuring the balance of exploratory and security motivations in rats. It capitalizes on the natural behavior tendencies of rats and does not depend on a trigger or noxious stimulus. The primary assessment is the rat’s preference to remain in the dark and its innate curiosity to explore the surrounding environment. The maze consists of four arms of equal length and width that are constructed in the shape of a “þ” and elevated 2e3 ft above the ground. The arms of the maze are either open (“light” areas) or closed (“dark” areas) and are oriented light/dark/light/dark in a clockwise fashion at 90 degrees to each other. An open center square is formed at the intersection of the four arms and represents the starting point for each animal. The closed arms provide the dark areas where rats prefer to remain when anxious. By placing a video camera above the apparatus, the researcher is able to record a number of variables for later analysis, for instance, time spent in each zone, and various behaviors such as rearing, grooming, and head dipping (Awwad, 2016). The EPM is simple to design, build, and implement. It can be constructed of solid, sturdy plastic that is easily cleaned and sanitized after the experiment. Although the trials can be time consuming based on the number of subjects, there is little manipulation on the part of the researcher. It is recommended that the animals be allowed to acclimate to their surroundings when moved from the colony to the experiment room. The time spent acclimating in the home cage is laboratory dependent, but often ranges from 15 to 30 min. c. Rotarod Issues with motor function and balance are common among TBI patients across the spectrum of severities (Kontos et al., 2012; Hoffer et al., 2004; Lau et al., 2011; Gottshall, 2011). The rotarod test has been shown to be highly sensitive to subtle changes that occur even following mild injury (Hamm, 2001; Hamm et al., 1994). The description of the test varies throughout the literature, and presenting the details of each protocol is beyond the scope of this chapter. In general, animals are initially trained to stay on a rotating rod at low speed; those that fall off repeatedly are placed back on the rod until they can stay for a designated time, usually 2e3 min. Trained

rats are then tested on a rotating rod that increases in speed over a set period of time. The rat is tested for balance and motor coordination over three to six trials per day for 4e6 days, depending on the experimental design. The rotarod apparatus and tracking system is commercially available or can be constructed within the lab. The test is well tolerated by the animals and stress on the animal is minimal. As with the EPM, it is recommended that the animals be acclimated to the testing room while in their home cage for 15e30 min prior to performing the test. d. The Whisker Nuisance Task and Analysis of the Whisker Barrel Circuit Rats rely heavily on their whiskers to gather information about their surroundings and navigate their environment. When a whisker senses a cue, an electrical impulse from the whisker is relayed to the barrel cortex of the somatosensory region of the brain. First described in detail in 1970, the “barrels” are anatomically cylindrical in shape with an area of dense cells surrounding the hollow center (Woolsey and Van der Loos, 1970). The barrel field is organized into a topographical map that appears to correspond with the layout of the vibrissae on the facial whisker pads; hence it is believed that these structures are the cortical correlates of the facial vibrissae (Fig. 29.12). Experimental evidence suggests that brain injury induces chronic somatomotor deficits and attenuates metabolic activation by whisker stimulation (Dietrich et al., 1994; Dunn-Meynell and Levin, 1995; Passineau et al., 2000). Furthermore, injury to the circuit is highlighted by changes in glycolysis when the somatomotor cortex is stimulated at a high enough level to produce a visible vibrissa response (Ip et al., 2003). McNamara et al. hypothesized that the whisker circuit could be used to determine somatosensory deficits induced by TBI (McNamara et al., 2010). To interrogate the system, the researchers developed a whisker nuisance task designed to detect TBI-induced sensory sensitivity. Briefly, rats are acclimated to a plastic test cage for 5e10 min. The researcher then manually stimulates the vibrissae of both whisker pads (bilateral stimulation) for three consecutive 5-min periods. The test is recorded for later analysis. Behaviors are divided into eight categories and assigned a score of 0 (absent), 1 (present), or 2 (profound/abnormal) for each of the 5min intervals, yielding a maximum score of 16. The higher scores are interpreted as more abnormal responses such as agitation or freezing. The test has been shown to be able to identify deficits in midline FPI up to 8 weeks postinjury regardless of injury severity, and data indicate that whisker stimulation in brain-injured animals elicits a stress response as measured by corticosterone levels (McNamara et al., 2010). Further work with the whisker barrel circuit has demonstrated its potential as a target for therapy after

RATS AS RESEARCH MODELS

III. TRAUMATIC BRAIN INJURY

1055

FIGURE 29.12 Topography of whisker pad and cortical barrel field. The individual whiskers on the rodent whisker pad are arranged in five rows (Left; Postnatal day 1; P1). This topographic arrangement is replicated in layer IVof somatosensory cortex, where thalamocortical afferents and their target neurons aggregate into clusters termed barrels (Right; CO histochemistry; P10). Image from Wilson, M.A., Johnston, M.V., Goldstein, G.W., Blue, M.E., 2000. Neonatal lead exposure impairs development of rodent barrel field cortex. Proc. Natl. Acad. Sci. 97, 5540e5545. Copyright (2000) National Academy of Sciences, USA.

TBI. Sensory activation of the injured brain is first attenuated and later augmented, suggesting potential restructuring of the circuit following injury, and pathology in the ventral basal complex of the thalamus coincides with the development of late-onset aberrant behavioral responses to whisker stimulation (Hall and Lifshitz, 2010). This is important in the context of neuronal plasticity, which is the ability of the brain to rewire itself and compensate for injury. Studies in various species have shown that remodeling within the brain can occur over several millimeters (Pons et al., 1991; Dancause et al., 2005; Werner and Stevens, 2015; Griesbach and Hovda, 2015; Adkins, 2015). Under normal circumstances, remodeling may improve the quality of existing connections; however, under traumatic conditions, plasticity may be maladaptive, creating aberrant axonal sprouting or synaptogenesis (Chuckowree et al., 2004; Carmichael, 2003). Additionally, midline FPI resulted in significant neuropathology and microglial activation in sensory nuclei that is not present in motor nuclei, as well as progressive neurodegeneration and neuronal nuclear atrophy of the barrel field and ventral basal complex of the thalamus (Miremami et al., 2014; Cao et al., 2012; Lifshitz and Lisembee, 2012). Taken together, the results of these studies confirm that TBI induces persistent and progressive changes at both the cellular and circuit levels, and the whisker circuit offers a discrete anatomical location in which to measure the efficacy of pharmaceutical intervention. e. Electrophysiology While the molecular and morphological changes that occur after TBI are important factors in determining outcome and recovery, there is also a vital need to understand the changes that occur to the connectome, or the complex network of neural connections within an

animal’s brain. The use of electrophysiological recording can help elucidate the effects of TBI on pathways within the brain, as well as provide further insight into pathophysiological mechanisms of circuitry disturbance such as changes in synaptic structure or membrane channel disruptions. Furthermore, electrophysiology can be used to continuously measure chronic changes in brain activity in an awake animal for an extended period of time. The use of electrophysiology in nervous system studies is not new; however, only recently has the field made broad inroads into TBI research. Electrophysiology can be used across the spectrum of rodent TBI models presented; however, it has been applied most frequently to the FPI and CCI models, because those are the most widely used and are both easily adapted for electrophysiological study. In the following, we will introduce the reader to the utility of electrophysiology in rat models of TBI. For a more detailed discussion on how electrophysiology has been used in a rodent TBI setting, as well as for useful information for those seeking to carry out these types of studies, the reader is referred to Reeves and Colley (2012). There are several types of electrophysiological measurements that are used to evaluate brain circuitry following TBI. Two of the most prominent are the electroencephalogram (EEG) and field excitatory postsynaptic potentials (fEPSPs). EEG is used to record the electrical activity in the cortex and can be performed in an anesthetized animal, or with the animal awake, either tethered to recording apparatus or freely moving through the use of wireless technology. To record EEG activity in awake animals, investigators typically perform a surgery in which small screws, functioning as electrodes, are placed into the skull. EEG is capable of detecting seizure-like activity in the brain and may provide

RATS AS RESEARCH MODELS

1056

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

insight into the development of TBI-induced posttraumatic epilepsy (Reeves and Colley, 2012). In contrast, fEPSPs are membrane potentials that will make a postsynaptic neuron more likely to fire an action potential. While EEG can provide an overall picture of the electrical activity of the brain, investigators are often interested in a specific structure within the brain. For example, the hippocampus has been shown to be selectively vulnerable to TBI, displaying a high capacity for neuroplasticity and reorganization after insult. This structure is of high importance since the hippocampus is the seat of memory and learning within the brain, is involved in long-term potentiation (the strengthening of the response of the postsynaptic cell across the synapse with repeated stimulation) that is widely thought to be the basis of long-term memory formation, and has been implicated in posttraumatic epilepsy. For these reasons, there has been great interest in using electrophysiology to probe changes in synaptic connectivity following injury. Within this highly organized structure, EPSPs can provide more detailed information on electrical activity within a specific brain region (subiculum, dentate gyrus, etc.) or layer of cells (stratum oriens, stratum radiatum) within the hippocampus. One can examine network disruptions by measuring field potentials in subregions of the hippocampus, either acutely by dropping an electrode to measure immediate changes or chronically by placing an indwelling electrode to measure changes over time in the awake animal. Aberrations in hippocampal signaling as detected by electrophysiology can indicate changes in synaptic organization or neurotransmitter release, disruptions in long-term potentiation, or changes in the balance between inhibitory and excitatory neurons. A detailed list of references on experiments conducted in the hippocampus can be found in Reeves and Colley (2012). Another region of the brain that has been popular for electrophysiological study following TBI is the corpus callosum, a thick bundle of axons that connects one hemisphere of the brain to the other and serves to integrate motor, sensory, and cognitive information from one area of the cerebral cortex to the same area on the opposite hemisphere. This region of the brain has been shown to be particularly susceptible to diffuse axonal injury. Electrophysiological measurements are commonly made ex vivo in rat brain slices in this region, allowing investigators the opportunity to measure compound action potentials and interrogate the state of axonal conduction. This technique is especially useful for investigators examining novel pharmacologic compounds intended to attenuate axonal dysfunction and/or demyelination. Since multiple slices can be obtained from a single rat brain, this technique provides the investigator with the opportunity to rapidly generate reliable data while minimizing the number of animals needed for the study.

f. Imaging Many of the same imaging modalities used in SCI are also used for TBI. MRI is easily applied to rodent models, and rats are excellent subjects for acquiring highresolution images of cranial neuroanatomy since their heads fit easily into smaller bore, higher magnetic field scanners. Advanced imaging techniques, such as diffusion tensor imaging, susceptibility-weighted imaging, arterial spin labeling, nuclear magnetic resonance spectroscopy, and positron-emission tomography, have proven useful at identifying damage to axonal tracts, identifying microhemorrhages, demonstrating changes in CBF, and measuring variations in metabolite concentrations and alterations in metabolism, respectively, following mild TBI (Tang et al., 2017; Wright et al., 2017; Benson et al., 2012; Shen et al., 2007; Hayward et al., 2010; Bartnik-Olson et al., 2010; Xu et al., 2011; Alessandri et al., 2000). Blood oxygen level-dependent responses and functional MRI (fMRI) have been used to evaluate functional impairments in rat brains following TBI (Niskanen et al., 2013; Mishra et al., 2014). Other tools, such as dynamic susceptibility contrast and single-photon emission computer tomography, may prove useful for identifying neuropathological changes due to TBI (Beaumont et al., 2006a). g. Microdialysis As mentioned previously, secondary cascades related to TBI often include chemical imbalances, such as changes in the levels of neurotransmitters, increases in excitatory amino acids, or alterations in cellular metabolism. One technique in particular, cerebral microdialysis, has gained traction in the field of TBI as a powerful tool for measuring chemical changes within the brain of living animals. Cerebral microdialysis is a technique that enables sampling and quantification of hormones, peptides, neurotransmitters, and other small peptides within the interstitial space. The technique requires insertion of a small probe with a semipermeable membrane at the tip that allows passive diffusion of small analytes. The shaft of the probe is connected to two tubes: one for introduction of solution that closely mimics the composition of the surrounding tissue (usually artificial cerebrospinal fluid) and the other for collection of the analyte(s) of interest. As perfusate is introduced into the shaft of the probe, a diffusion gradient is created and proteins/molecules of interest can be collected at metered time intervals for further analysis (Chefer et al., 2009). Ultimately, the goal of most animal TBI work is either to serve as an avenue to study the mechanisms responsible for the deleterious effects in humans or to test a suitable remedy. Since microdialysis is used routinely in the clinic for human TBI patients, its use in rat models of TBI has enormous potential for translational relevance between rodent and human TBI (Hillered et al.,

RATS AS RESEARCH MODELS

III. TRAUMATIC BRAIN INJURY

2005; Koura et al., 1998). In fact, several studies have laid the foundation for examination of neurotransmitter depletion and recovery (Busto et al., 1997; Shin et al., 2011; Carlson and Dixon, 2018), release and accumulation of harmful excitatory amino acids (Rose et al., 2002; Globus et al., 1995; Palmer et al., 1993), and cellular respiration and metabolism (Geeraerts et al., 2006; Chen et al., 2000; Kilbaugh et al., 2011; Darbin et al., 2006). Furthermore, when coupled with imaging techniques such as PET and spectroscopy, microdialysis data provide a clearer picture of metabolic changes that occur within specific regions of the brain following TBI (Alessandri et al., 2000). h. Microscopic Tissue Analysis and Biomarkers As with SCI, microscopic examination of postmortem tissue is essential to understanding the pathological consequences of TBI. Many of the same stains can be used to identify widespread tissue damage as well as cellular abnormalities that result from TBI. Routine staining and histological dyes are used first followed by IHC techniques to reveal more detailed information about the tissue, and the two approaches should be used complementary to each other to allow for better interpretation of the data. Thousands of rodent TBI studies have used these techniques; rather than delve into the plethora of studies that have examined histological outcomes (Johnson et al., 2015; Xiong et al., 2013; EstradaRojo et al., 2018), we present several useful methods that are commonly employed in TBI studies. H&E staining is widely used to identify tissue atrophy (i.e., lesion area, as shown in Fig. 29.13) as well as

1057

provide a picture of overall tissue architecture. Cresyl violet (CV) stain demonstrates the Nissl substance in neuronal somata and nuclei and is commonly used to identify neuronal structure. It can also be used as a counterstain to Luxol fast blue (LFB), which identifies myelin (KluvereBarrera stain). Silver staining (SS) techniques are also used to visualize degenerating neurons and axons in both acutely and chronically injured rats (Baloyannis, 2015). H&E, CV, SS, and LFB are important tools for researchers studying tissue loss, neuronal degeneration, white matter/demyelination changes, and neuroinflammation. Once the routine histological stains have been implemented, further information may be needed such as type of cell affected or particular mechanism involved in pathogenesis. In this regard, researchers often turn to IHC. By capitalizing on antibodyeantigen specificity, particular proteins can be identified. Neuroinflammation, axonal damage, and neurodegeneration/cell death are all hallmark pathologies induced by TBI. The use of an antibody against GFAP, a protein enriched in astrocytes, can reveal potential inflammation and reactive astrocytosis. If further examination is necessary, Iba1 (ionized calcium binding adaptor molecule 1) can be employed to identify microglia/macrophages. When used together, both stains may provide a clearer picture of the types of cells involved in neuroinflammatory responses. Another important feature of TBI is diffuse axonal injury, the hallmark pathology of closed-head TBI, which is typically observed based on markers for impaired axonal transport and disconnection. Here,

FIGURE 29.13 Representative micrographs of rat brains at time points over one year following lateral fluid percussion injury in the rat. Hematoxylin and eosin staining allows the viewer to evaluate overall brain architecture, and progressive brain atrophy is evident in these sections. Reproduced from Johnson, V.E., Stewart, W., Weber, M.T., Cullen, D.K., Siman, R.,Smith, D.H., 2016. SNTF immunostaining reveals previously undetectedaxonal pathology in traumatic brain injury. Acta Neuropathol. 131, 115e135.

RATS AS RESEARCH MODELS

1058

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

protein buildup at points of transport interruptionsd generally due to loss axonal cytoskeletal continuityd can be identified using IHC usually via antibody to amyloid precursor protein (APP). The resulting accumulations of APP can be readily identified as axonal varicosities and/or diffuse axonal bulbs, and generally lead to axonal disconnection (Gentleman et al., 1993; Sherriff et al., 1994; Blumbergs et al., 1994, 1995; McKenzie et al., 1996; Geddes et al., 1997). Cell death is often classified as following either an apoptotic (active) or necrotic (passive) process, with gradients between these two extremes. Apoptosis is considered to be a programmed, orderly process, while necrosis is considered a nonregulated event resulting from extreme perturbations, often resulting in a release of myriad inflammatory factors. While CV and H&E staining can help identify healthy as well as unhealthy neurons, it does not provide information on the mechanism that may lead to neurodegeneration and loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling is a method for detecting apoptotic DNA fragmentation and can be used to separate cells undergoing apoptosis from those undergoing necrosis based on morphological criteria. Finally, the principal pathology of chronic neurodegenerative pathologies such as Alzheimer’s-like pathology and CTE is aberrant tau expression and accumulation, which is being identified via IHC using antibodies directed against tau protein (McKee et al., 2016). This list is far from exhaustive because there are a multitude of antibodies and techniques available to help researchers identify pathological features of TBI. An exciting avenue of research that shows great clinical promise for the diagnosis and prognosis of TBI patients is the use of serum biomarkers to noninvasively measure pathological features of TBI. Several of the more common serum biomarkers identified are S100B, NSE, GFAP, ubiquitin carboxy-terminal hydrolase L1, and neurofilament protein (Thelin et al., 2017). Several studies have shown the utility of measuring calpaincleaved aII-spectrin N-terminal fragment in identifying mild TBI (Johnson et al., 2016; Siman et al., 2013, 2015).

E. Special Considerations for Animal Care With TBI Studies 1. Preinjury Many of the considerations for the selection and acclimation of rats for TBI research are similar to those outlined in the previous section for SCI (Robinson et al., 2018). Rats should be in good health and with known genetic background and infectious disease status. Staff members performing the injury procedures, behavioral assays, and postinjury care must be adequately trained in advance of the experiments. Acclimation of rats to

the facility, and potentially to the investigators that will be working with them, is important before injury procedures, especially when behavioral tests are to be performed (Kamnaksh et al., 2011). As always, it is important that any shared equipment, such as that used for behavior studies, be sanitized frequently. 2. Injury Procedure Neuroprotective effects of anesthetics discussed for SCI models also pertain to TBI models. The use of anesthetics and analgesics should be considered during the planning phase, and uniform regimens should be used for experimental and control groups (Rowe et al., 2013). No one anesthetic, or anesthetic combination, is ideal for every TBI model. Some anesthetic techniques, such as hypothermia, which is commonly used in neonatal rats, may alter brain morphology and animal behavior and are not recommended for neurotrauma work (Blickman and Brossia, 2009). Ketamine increases CBF and intracranial pressure (ICP), and can alter neuronal responses to injury (Blickman and Brossia, 2009). Ketamine, through its mechanism of action antagonizing the N-methyl-D-aspartate receptor, may also mitigate secondary excitatory injury and change behavioral outcomes (Smith et al., 1993). Isoflurane has the advantage of quick and uncomplicated recovery after experimental TBI (Lifshitz, 2009; Dixon and Kline, 2009; Marmarou et al., 2009). However, it may improve outcome immediately after CCI compared to other anesthetics in rats (Statler et al., 2006). Different inhaled anesthetics may have different effects on ICP and cerebral perfusion pressure. For example, sevoflurane may increase ICP and therefore decrease cerebral perfusion pressure more than isoflurane in a rat model of diffuse TBI (Goren et al., 2001). Propofol may also impact outcomes after brain injury through a variety of mechanisms, including antioxidant effects (Adembri et al., 2007; Ozturk et al., 2005). As with SCI, some investigators have historically used injectable anesthetics during induction of TBI to avoid interference between the nose cone and the stereotactic equipment and sterile field. As previously mentioned, various companies have developed techniques for delivering inhaled agents to animals in stereotactic set-ups, which has facilitated the use of isoflurane for these injuries (Fig. 29.14) (Blickman and Brossia, 2009). When a surgical approach is used, sterility should be maintained to avoid complications from wound infection and the use of perioperative antibiotics is not a substitute for aseptic technique. The injury procedures, by their nature, may induce unwanted noncerebral trauma. Skull fracture and suture diastasis are possible adverse events after induction of the weight-drop injury model (Henninger et al., 2016; Marmarou et al., 2009). These events influence the biomechanics of injury and could lead to other unwanted variables, such as systemic and

RATS AS RESEARCH MODELS

1059

IV. CONCLUSIONS

FIGURE 29.14 Rat anesthetized with isoflurane in stereotactic setup in preparation for craniectomy. Photograph taken prior to complete surgical preparation to allow for better visualization.

localized inflammation (Marmarou et al., 2009). Consideration should be given to exclude animals with these conditions, preferably prior to anesthetic recovery for humane reasons. It is possible to measure CBF intraoperatively in the rat, and the technique has been well described. In instances where investigators monitor this parameter during injury, it may allow a better understanding of the severity of injury and help improve consistency between animals. If monitored, however, CBF must be monitored relative to an individual animal’s baseline CBF, because normal reference ranges are not readily available for this parameter (Cahill and Zhang, 2009). For techniques where a craniotomy is required, interrupted suture patterns are preferred over continuous sutures due to the tendency for rats (in particular, socially housed rats) to unravel sutures postoperatively. 3. Postinjury Whenever there is a surgical approach to induce a disease model in animals, postprocedural care inclusive of monitoring for signs of pain, bleeding, or infection and general wound care is necessary. TBI models are no exception. Similarly to anesthetics, analgesics may change functional or histopathological outcomes after experimental TBI. Nonsteroidal antiinflammatory agents can reduce lesion size and improve function outcomes after TBI (Thau-Zuchman et al., 2012; Cernak et al., 2002; Gopez et al., 2005). However, their chronic use may have opposite effects (Browne et al., 2006). The administration of opioids may also change the natural progression of disease after experimental TBI, because endogenous opiate receptors are upregulated and may play a role in secondary injury (Lyeth et al., 1993; Hayes et al., 1990). Mortality due to respiratory failure may be high following TBI in some cases (Marklund, 2016). Depending

on the model, interventions may be necessary to restore airflow, although not intervening is often scientifically justified when evaluation of apnea is used as an outcome measure. Some investigators do not recommend treatment of apnea (Lifshitz, 2009), while others believe ventilator support is beneficial, especially in models where acute mortality due to respiratory failure may reach 50% (Lifshitz, 2009; Marmarou et al., 2009). Mechanical ventilation to maintain eucapnia or slight hypocapnia may be beneficial in increasing short-term survival. Monitoring of end tidal carbon dioxide, blood gas analysis, CBF, and invasive blood pressure helps guide anesthetic depth, ventilator parameters, and resuscitative measures (such as fluid administration), and better quantifies severity of injury and maintains consistency between animals (Marmarou et al., 2009). Posttraumatic seizures may also occur in rats (Kharatishvili et al., 2006; Nilsson et al., 1994). Treatment of seizures with antiepileptic drugs or other measures will be dependent on the experimental design and research goals.

IV. CONCLUSIONS A. Implementation of Rodent Models of CNS Trauma This chapter presented descriptions, methods, experimental considerations, and limitations for the implementation of the most common models of TBI and SCI using rats. Implementing animal models of CNS injury requires precision, attention to detail, and consistency of execution. Since many of these injuries will require delicate surgery or complex procedures, it is recommended that investigators practice their technique on cadavers under the guidance of persons experienced in the technique prior to attempting work on living animals. Once comfortable with the technique in cadavers, it is generally recommended for the investigator to progress to work on nonrecovered live animals before moving on to survival procedures (Schantz, 2009). As shown in this chapter, there are multiple established rat models of TBI and SCI, each intended to replicate various features of clinical CNS trauma based on injury mechanisms, pathophysiological features, and underlying assumptions relating the anatomy, physiology, and genetic underpinning of rats to those of humans. 1. Caution in Interpreting Results from Rodent Studies and Extrapolating to Human Conditions The models described in this chapter have been and will continue to be an irreplaceable resource in facilitating initial discovery of trauma-induced pathology and putative treatment strategies. However, caution should be employed to avoid overinterpretation of

RATS AS RESEARCH MODELS

1060

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

findings as attempts to extrapolate observations from rodent models to humans have generally been unsuccessful. Indeed, therapeutic interventions identified during preclinical investigations in rodent models have largely fallen short of helping human patients with both SCI and TBI. In TBI alone, a sobering assessment reveals that despite hundreds of treatments reported to show efficacy in rodent models, none have shown efficacy in humans despite over 30 clinical trials strongly supported by preclinical data (Stein, 2015; Kabadi and Faden, 2014). This lack of positive findings in clinical studies reflects the complexity and heterogeneity of human TBI as well as challenges in clinical trial design (Saatman et al., 2008); however, it should also serve as a cautionary tale of the inability of rodent models to replicate the spectrum of pathophysiology and neurodegenerative sequelae found in humans (Smith et al., 2015). Some investigators have attempted to address the complexities of naturally occurring CNS injury in humans by creating models that incorporate concomitant injury or disease (Statler et al., 2001; Wang et al., 2014b; Simon et al., 2016; Thompson et al., 2016). 2. Common Data Elements and Standardization of Practice in the Field The lack of translatability of findings from rodent models has been blamed on a variety of factors, including differences between animals (and in particular rodents) and people, as well as factors relating to experimental consistency between labs. To close this translatability gap, panels of experts in both SCI and TBI have come together to attempt to facilitate comparisons of preclinical studies across institutions and methodologies. Efforts to independently replicate impactful preclinical SCI studies were performed by the Facilities of Research Excellence-Spinal Cord Injury (FORE-SCI). This group was unable to replicate most of the studies attempted (Steward et al., 2012). The FORE-SCI participants had several recommendations for the SCI research community, including standardization for reporting of animal studies of SCI. Minimum information standards for preclinical SCI studies have been proposed. The Minimum Information About Spinal Score Injury Consortium has proposed 250 required data elements across 11 major sections, including the animals, reagents, and statistical analyses involved in the study (Lemmon et al., 2014). The TBI community has also made similar recommendations, calling for the reporting of Common Data Elements (CDEs) in reporting preclinical TBI studies (Smith et al., 2015). These CDEs are subdivided into those Core CDEs that would be applicable to virtually all preclinical TBI studies and Injury-ModelSpecific CDEs that may be useful only for specific injury models. Researchers have used standardized data

reporting systems for clinical trials and holding preclinical studies to similar standards may increase translatability. With the implementation of these standards, there is hope that conclusions drawn from studies on CNS injury in rats will have greater translational success moving forward. 3. Moving Forward: The Role of Rodent Models in CNS Trauma Research Rodent models play an important role in the development of our understanding of the pathophysiology of traumatic CNS injury, and continue to be an important high-throughput platform for the investigation of both insult and interventions. Researchers seeking to use the rat for these types of investigations should carefully design studies to play to the strengths of the rat, while bearing in mind the limitations of the model, including issues relating to size, neuroanatomy, and genetic endowment. With properly tempered interpretation, rodent models can greatly inform the direction of studies to be executed up the phylogenetic chain to eventually impact human health. For instance, researchers have established several large animal models of TBI (Cullen et al., 2016; Margulies et al., 2015; Lafrenaye et al., 2015; Curvello et al., 2017) and SCI (Nardone et al., 2017) that may be an appropriate intermediate between rodents and humans. This multilevel paradigm would benefit the field by allowing hypotheses and treatments to be vetted in more clinically relevant models to better inform costly clinical trial design, treatment windows/ dosages, and patient selectiondthereby increasing the likelihood of successful translation to improve patient recovery and reduce societal burden following devastating SCI and TBI. Overall, rodent modelsdapplied judiciouslydwill continue to play a critical role in this mission as the first line of basic science discovery in the field that can be both mechanism based, economical, and most importantly insightful to sharpen investigations into the human condition.

References Abdel-Karim, A.M., Carrier, S., 2002. Possible improvement of the clitoral and vaginal blood flow using a somatostatin analog in chronic spinalized Sprague-Dawley rats. J. Sex Marital. Ther. 28, 1e9. Adams, J.H., Doyle, D., Ford, I., Gennarelli, T.A., Graham, D.I., McLellan, D.R., 1989. Diffuse axonal injury in head injury: definition, diagnosis and grading. Histopathology 15, 49e59. Adams, J.H., Graham, D.I., Gennarelli, T.A., Maxwell, W.L., 1991. Diffuse axonal injury in non-missile head injury. J. Neurol. Neurosurg. Psychiatry 54, 481e483. Adams, J.H., Graham, D.I., Murray, L.S., Scott, G., 1982. Diffuse axonal injury due to nonmissile head injury in humans: an analysis of 45 cases. Ann. Neurol. 12, 557e563. Adams, M.M., Hicks, A.L., 2005. Spasticity after spinal cord injury. Spinal Cord 43, 577. Adamson, J., Zappulla, R.A., Fraser, A., Ryder, J., Malis, L.I., 1989. Effects of selective spinal cord lesions on the spinal motor evoked

RATS AS RESEARCH MODELS

REFERENCES

potential (MEP) in the rat. Electroencephalogr. Clin. Neurophysiol. 74, 469e480. Adembri, C., Venturi, L., Pellegrini-Giampietro, D.E., 2007. Neuroprotective effects of propofol in acute cerebral injury. CNS Drug Rev. 13, 333e351. Adkins, D.L., 2015. Cortical stimulation-induced structural plasticity and functional recovery after brain damage. In: Kobeissy, F.H. (Ed.), Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects. Boca Raton (FL). Adler, A.F., Lee-Kubli, C., Kumamaru, H., Kadoya, K., Tuszynski, M.H., 2017. Comprehensive monosynaptic rabies virus mapping of host connectivity with neural progenitor grafts after spinal cord injury. Stem Cell Rep. 8, 1525e1533. Ahmed, S., Venigalla, H., Mekala, H.M., Dar, S., Hassan, M., Ayub, S., 2017. Traumatic brain injury and neuropsychiatric complications. Indian J. Psychol. Med. 39, 114e121. Akdemir, H., Pasaoglu, A., Ozturk, F., Selcuklu, A., Koc, K., Kurtsoy, A., 1992. Histopathology of experimental spinal cord trauma. Comparison of treatment with TRH, naloxone, and dexamethasone. Res. Exp. Med. 192, 177e183. Akhtar, A.Z., Pippin, J.J., Sandusky, C.B., 2008. Animal models in spinal cord injury: a review. Rev. Neurosci. 19, 47e60. Alan, N., Ramer, L.M., Inskip, J.A., Golbidi, S., Ramer, M.S., Laher, I., Krassioukov, A.V., 2010. Recurrent autonomic dysreflexia exacerbates vascular dysfunction after spinal cord injury. Spine J. 10, 1108e1117. Alessandri, B., al-Samsam, R., Corwin, F., Fatouros, P., Young, H.F., Bullock, R.M., 2000. Acute and late changes in N-acetyl-aspartate following diffuse axonal injury in rats: an MRI spectroscopy and microdialysis study. Neurol. Res. 22, 705e712. Allen, A.R., 1911. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column: a preliminary report. J. Am. Med. Assoc. LVII, 878e880. Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado, M.A., Kane, J.R., Schallert, T., Jones, T.A., 2008. The vermicelli handling test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229e244. Alluin, O., Karimi-Abdolrezaee, S., Delivet-Mongrain, H., Leblond, H., Fehlings, M.G., Rossignol, S., 2011. Kinematic study of locomotor recovery after spinal cord clip compression injury in rats. J. Neurotrauma 28, 1963e1981. Anderson, K.D., 2004. Targeting recovery: priorities of the spinal cordinjured population. J. Neurotrauma 21, 1371e1383. Anderson, K.D., Gunawan, A., Steward, O., 2005. Quantitative assessment of forelimb motor function after cervical spinal cord injury in rats: relationship to the corticospinal tract. Exp. Neurol. 194, 161e174. Anderson, K.D., Sharp, K.G., Hofstadter, M., Irvine, K.A., Murray, M., Steward, O., 2009a. Forelimb locomotor assessment scale (FLAS): novel assessment of forelimb dysfunction after cervical spinal cord injury. Exp. Neurol. 220, 23e33. Anderson, K.D., Sharp, K.G., Steward, O., 2009b. Bilateral cervical contusion spinal cord injury in rats. Exp. Neurol. 220, 9e22. Andriessen, T.M., Jacobs, B., Vos, P.E., 2010. Clinical characteristics and pathophysiological mechanisms of focal and diffuse traumatic brain injury. J. Cell Mol. Med. 14, 2381e2392. Antri, M., Orsal, D., Barthe, J.Y., 2002. Locomotor recovery in the chronic spinal rat: effects of long-term treatment with a 5-HT2 agonist. Eur. J. Neurosci. 16, 467e476. Awwad, H.O., 2016. Detecting behavioral deficits post traumatic brain injury in rats. In: Kobeissy, F.H., et al. (Eds.), Injury Models of the Central Nervous System: Methods and Protocols. Springer New York, New York, NY, pp. 573e596. Ayer, R., Cahill, J., Sugawara, T., Jadhav, V., Zhang, J.H., 2009. Rodent surgical procedures and tissue collection. In: Chen, J., et al. (Eds.),

1061

Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 19e38. Baastrup, C., Maersk-Moller, C.C., Nyengaard, J.R., Jensen, T.S., Finnerup, N.B., 2010. Spinal-, brainstem- and cerebrally mediated responses at- and below-level of a spinal cord contusion in rats: evaluation of pain-like behavior. Pain 151, 670e679. Baig, H.A., Guarino, B.B., Lipschutz, D., Winkelstein, B.A., 2013. Whole body vibration induces forepaw and hind paw behavioral sensitivity in the rat. J. Orthop. Res. 31, 1739e1744. Bailey, E.L., McCulloch, J., Sudlow, C., Wardlaw, J.M., 2009. Potential animal models of lacunar stroke: a systematic review. Stroke 40, e451e458. Ballermann, M., Metz, G.A., McKenna, J.E., Klassen, F., Whishaw, I.Q., 2001. The pasta matrix reaching task: a simple test for measuring skilled reaching distance, direction, and dexterity in rats. J. Neurosci. Methods 106, 39e45. Baloyannis, S.J., 2015. Staining neurons with Golgi techniques in degenerative diseases of the brain. Neural Regen. Res. 10, 693e695. Baptiste, D.C., Fehlings, M.G., 2007. Update on the treatment of spinal cord injury. Prog. Brain Res. 161, 217e233. Bartnik-Olson, B.L., Oyoyo, U., Hovda, D.A., Sutton, R.L., 2010. Astrocyte oxidative metabolism and metabolite trafficking after fluid percussion brain injury in adult rats. J. Neurotrauma 27, 2191e2202. Basso, D.M., 2004. Behavioral testing after spinal cord injury: congruities, complexities, and controversies. J. Neurotrauma 21, 395e404. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12, 1e21. Basso, D.M., Beattie, M.S., Bresnahan, J.C., Anderson, D.K., Faden, A.I., Gruner, J.A., Holford, T.R., Hsu, C.Y., Noble, L.J., Nockels, R., Perot, P.L., Salzman, S.K., Young, W., 1996. MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J. Neurotrauma 13, 343e359. Battistuzzo, C.R., Callister, R.J., Callister, R., Galea, M.P., 2012. A systematic review of exercise training to promote locomotor recovery in animal models of spinal cord injury. J. Neurotrauma 29, 1600e1613. Baugh, C.M., Stamm, J.M., Riley, D.O., Gavett, B.E., Shenton, M.E., Lin, A., Nowinski, C.J., Cantu, R.C., McKee, A.C., Stern, R.A., 2012. Chronic traumatic encephalopathy: neurodegeneration following repetitive concussive and subconcussive brain trauma. Brain Imaging Behav. 6, 244e254. Baussart, B., Stamegna, J.C., Polentes, J., Tadie, M., Gauthier, P., 2006. A new model of upper cervical spinal contusion inducing a persistent unilateral diaphragmatic deficit in the adult rat. Neurobiol. Dis. 22, 562e574. Bazarian, J.J., Cernak, I., Noble-Haeusslein, L., Potolicchio, S., Temkin, N., 2009. Long-term neurologic outcomes after traumatic brain injury. J. Head Trauma Rehabil. 24, 439e451. Beaumont, A., Fatouros, P., Gennarelli, T., Corwin, F., Marmarou, A., 2006a. Bolus tracer delivery measured by MRI confirms edema without blood-brain barrier permeability in diffuse traumatic brain injury. Acta Neurochir. Suppl. 96, 171e174. Beaumont, E., Onifer, S.M., Reed, W.R., Magnuson, D.S., 2006b. Magnetically evoked inter-enlargement response: an assessment of ascending propriospinal fibers following spinal cord injury. Exp. Neurol. 201, 428e440. Behrmann, D.L., Bresnahan, J.C., Beattie, M.S., Shah, B.R., 1992. Spinal cord injury produced by consistent mechanical displacement of the cord in rats: behavioral and histologic analysis. J. Neurotrauma 9, 197e217. Bennett, D.J., Gorassini, M., Fouad, K., Sanelli, L., Han, Y., Cheng, J., 1999. Spasticity in rats with sacral spinal cord injury. J. Neurotrauma 16, 69e84.

RATS AS RESEARCH MODELS

1062

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Benson, R.R., Gattu, R., Sewick, B., Kou, Z., Zakariah, N., Cavanaugh, J.M., Haacke, E.M., 2012. Detection of hemorrhagic and axonal pathology in mild traumatic brain injury using advanced MRI: implications for neurorehabilitation. NeuroRehabilitation 31, 261e279. Benton, R.L., Woock, J.P., Gozal, E., Hetman, M., Whittemore, S.R., 2005. Intraspinal application of endothelin results in focal ischemic injury of spinal gray matter and restricts the differentiation of engrafted neural stem cells. Neurochem. Res. 30, 809e823. Benveniste, H., Qui, H., Hedlund, L.W., D’Ercole, F., Johnson, G.A., 1998. Spinal cord neural anatomy in rats examined by in vivo magnetic resonance microscopy. Reg. Anesth. Pain Med. 23, 589e599. Bernstein, D.R., Stelzner, D.J., 1983. Plasticity of the corticospinal tract following midthoracic spinal injury in the postnatal rat. J. Comp. Neurol. 221, 382e400. Biernaskie, J., Sparling, J.S., Liu, J., Shannon, C.P., Plemel, J.R., Xie, Y., Miller, F.D., Tetzlaff, W., 2007. Skin-derived precursors generate myelinating Schwann cells that promote remyelination and functional recovery after contusion spinal cord injury. J. Neurosci. 27, 9545e9559. Bilgen, M., Dancause, N., Al-Hafez, B., He, Y.Y., Malone, T.M., 2005. Manganese-enhanced MRI of rat spinal cord injury. Magn. Reson. Imaging 23, 829e832. Blennow, K., Hardy, J., Zetterberg, H., 2012. The neuropathology and neurobiology of traumatic brain injury. Neuron 76, 886e899. Blickman, A., Brossia, L.J., 2009. Anesthesia and analgesia for research animals. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 11e18. Blight, A.R., 1991. Morphometric analysis of a model of spinal cord injury in Guinea pigs, with behavioral evidence of delayed secondary pathology. J. Neurol. Sci. 103, 156e171. Blight, A.R., 1992. Spinal cord injury models: neurophysiology. J. Neurotrauma 9, 147e150. Blumbergs, P.C., Scott, G., Manavis, J., Wainwright, H., Simpson, D.A., McLean, A.J., 1994. Staining of amyloid precursor protein to study axonal damage in mild head injury. Lancet 344, 1055e1056. Blumbergs, P.C., Scott, G., Manavis, J., Wainwright, H., Simpson, D.A., McLean, A.J., 1995. Topography of axonal injury as defined by amyloid precursor protein and the sector scoring method in mild and severe closed head injury. J. Neurotrauma 12, 565e572. Boulenguez, P., Gestreau, C., Vinit, S., Stamegna, J.C., Kastner, A., Gauthier, P., 2007. Specific and artifactual labeling in the rat spinal cord and medulla after injection of monosynaptic retrograde tracers into the diaphragm. Neurosci. Lett. 417, 206e211. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W., McMahon, S.B., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636e640. Bresnahan, J.C., Beattie, M.S., Todd 3rd, F.D., Noyes, D.H., 1987. A behavioral and anatomical analysis of spinal cord injury produced by a feedback-controlled impaction device. Exp. Neurol. 95, 548e570. Brown, C., 2006. Restraint collars. Part I: Elizabethan collars and other types of restraint collars. Lab. Anim. (NY) 35, 23e25. Browne, K.D., Iwata, A., Putt, M.E., Smith, D.H., 2006. Chronic ibuprofen administration worsens cognitive outcome following traumatic brain injury in rats. Exp. Neurol. 201, 301e307. Bunge, M.B., Holets, V.R., Bates, M.L., Clarke, T.S., Watson, B.D., 1994. Characterization of photochemically induced spinal cord injury in the rat by light and electron microscopy. Exp. Neurol. 127, 76e93. Busto, R., Dietrich, W.D., Globus, M.Y., Alonso, O., Ginsberg, M.D., 1997. Extracellular release of serotonin following fluid-percussion brain injury in rats. J. Neurotrauma 14, 35e42.

Byrnes, K.R., Fricke, S.T., Faden, A.I., 2010. Neuropathological differences between rats and mice after spinal cord injury. J. Magn. Reson. Imaging 32, 836e846. Caggiano, A.O., Zimber, M.P., Ganguly, A., Blight, A.R., Gruskin, E.A., 2005. Chondroitinase ABCI improves locomotion and bladder function following contusion injury of the rat spinal cord. J. Neurotrauma 22, 226e239. Cahill, J., Zhang, J.H., 2009. Brain monitoring. In: Chen, J., et al. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 39e45. Cao, T., Thomas, T.C., Ziebell, J.M., Pauly, J.R., Lifshitz, J., 2012. Morphological and genetic activation of microglia after diffuse traumatic brain injury in the rat. Neuroscience 225, 65e75. Cao, Y., Massaro, J.F., Krause, J.S., Chen, Y., Devivo, M.J., 2014. Suicide mortality after spinal cord injury in the United States: injury cohorts analysis. Arch. Phys. Med. Rehabil. 95, 230e235. Cao, Y., Shumsky, J.S., Sabol, M.A., Kushner, R.A., Strittmatter, S., Hamers, F.P., Lee, D.H., Rabacchi, S.A., Murray, M., 2008. Nogo66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat. Neurorehabil. Neural Repair 22, 262e278. Cao, Y., Wu, T., Yuan, Z., Li, D., Ni, S., Hu, J., Lu, H., 2015. Threedimensional imaging of microvasculature in the rat spinal cord following injury. Sci. Rep. 5, 12643. Carey, M.E., Sarna, G.S., Farrell, J.B., Happel, L.T., 1989. Experimental missile wound to the brain. J. Neurosurg. 71, 754e764. Carlson, S.W., Dixon, C.E., 2018. Lithium improves dopamine neurotransmission and increases dopaminergic protein abundance in the striatum after traumatic brain injury. J. Neurotrauma. https:// doi.org/10.1089/neu.2017.5509. Epub ahead of print. Carmichael, S.T., 2003. Plasticity of cortical projections after stroke. The Neuroscientist 9, 64e75. Carobrez, A.P., Bertoglio, L.J., 2005. Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neurosci. Biobehav. Rev. 29, 1193e1205. Casella, E.M., Thomas, T.C., Vanino, D.L., Fellows-Mayle, W., Lifshitz, J., Card, J.P., Adelson, P.D., 2014. Traumatic brain injury alters long-term hippocampal neuron morphology in juvenile, but not immature, rats. Child’s Nerv. Syst. 30, 1333e1342. Centers for Disease Control and Prevention, 2011. Nonfatal traumatic brain injuries related to sports and recreation activities among persons aged
RATS AS RESEARCH MODELS

REFERENCES

Differential histopathological and behavioral outcomes eight weeks after rat spinal cord injury by contusion, dislocation, and distraction mechanisms. J. Neurotrauma 33, 1667e1684. Chen, T., Qian, Y.Z., Di, X., Rice, A., Zhu, J.P., Bullock, R., 2000. Lactate/ glucose dynamics after rat fluid percussion brain injury. J. Neurotrauma 17, 135e142. Chen, X.H., Siman, R., Iwata, A., Meaney, D.F., Trojanowski, J.Q., Smith, D.H., 2004. Long-term accumulation of amyloid-beta, betasecretase, presenilin-1, and caspase-3 in damaged axons following brain trauma. Am. J. Pathol. 165, 357e371. Cheng, C.L., de Groat, W.C., 2004. The role of capsaicin-sensitive afferent fibers in the lower urinary tract dysfunction induced by chronic spinal cord injury in rats. Exp. Neurol. 187, 445e454. Cheng, C.L., Ma, C.P., de Groat, W.C., 1995. Effect of capsaicin on micturition and associated reflexes in chronic spinal rats. Brain Res. 678, 40e48. Cheng, H., Almstrom, S., Gimenez-Llort, L., Chang, R., Ove Ogren, S., Hoffer, B., Olson, L., 1997. Gait analysis of adult paraplegic rats after spinal cord repair. Exp. Neurol. 148, 544e557. Cheng, J., Gu, J., Ma, Y., Yang, T., Kuang, Y., Li, B., Kang, J., 2010. Development of a rat model for studying blast-induced traumatic brain injury. J. Neurol. Sci. 294, 23e28. Cheriyan, T., Ryan, D.J., Weinreb, J.H., Cheriyan, J., Paul, J.C., Lafage, V., Kirsch, T., Errico, T.J., 2014. Spinal cord injury models: a review. Spinal Cord 52, 588e595. Choi, H., Liao, W.L., Newton, K.M., Onario, R.C., King, A.M., Desilets, F.C., Woodard, E.J., Eichler, M.E., Frontera, W.R., Sabharwal, S., Teng, Y.D., 2005. Respiratory abnormalities resulting from midcervical spinal cord injury and their reversal by serotonin 1A agonists in conscious rats. J. Neurosci. 25, 4550e4559. Choo, A.M., Liu, J., Liu, Z., Dvorak, M., Tetzlaff, W., Oxland, T.R., 2009. Modeling spinal cord contusion, dislocation, and distraction: characterization of vertebral clamps, injury severities, and node of Ranvier deformations. J. Neurosci. Methods 181, 6e17. Christensen, M.D., Everhart, A.W., Pickelman, J.T., Hulsebosch, C.E., 1996. Mechanical and thermal allodynia in chronic central pain following spinal cord injury. Pain 68, 97e107. Chu, T.-H., Wu, W., 2009. Spinal root avulsion and repair model. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, p. 487. Chuckowree, J.A., Dickson, T.C., Vickers, J.C., 2004. Intrinsic regenerative ability of mature CNS neurons. The Neuroscientist 10, 280e285. Chung, W.-H., Lee, J.-H., Chung, D.-J., Yang, W.-J., Lee, A.J., Choi, C.B., Chang, H.-S., Kim, D.-H., Chung, H.J., Suh, H.J., Hwang, S.-H., Han, H., Do, S.H., Kim, H.-Y., 2013. Improved rat spinal cord injury model using spinal cord compression by percutaneous method. J. Vet. Sci. 14, 329e335. Clark, F.M., Proudfit, H.K., 1992. Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons. Brain Res. 591, 44e53. Clarke, E.C., Bilston, L.E., 2008. Contrasting biomechanics and neuropathology of spinal cord injury in neonatal and adult rats following vertebral dislocation. J. Neurotrauma 25, 817e832. Clausen, F., Marklund, N., Lewen, A., Enblad, P., Basu, S., Hillered, L., 2012. Interstitial F(2)-isoprostane 8-iso-PGF(2alpha) as a biomarker of oxidative stress after severe human traumatic brain injury. J. Neurotrauma 29, 766e775. Cloud, B.A., Ball, B.G., Chen, B., Knight, A.M., Hakim, J.S., Ortiz, A.M., Windebank, A.J., 2012. Hemisection spinal cord injury in rat: the value of intraoperative somatosensory evoked potential monitoring. J. Neurosci. Methods 211, 179e184. Committee on Sports-Related Concussions in Youth, 2014. In: Graham, R., et al. (Eds.), Sports-Related Concussions in Youth: Improving the Science, Changing the Culture. Washington (DC).

1063

Conour, L.A., Murray, K.A., Brown, M.J., 2006. Preparation of animals for research–issues to consider for rodents and rabbits. ILAR J. 47, 283e293. Coronado, V.G., McGuire, L.C., Sarmiento, K., Bell, J., Lionbarger, M.R., Jones, C.D., Geller, A.I., Khoury, N., Xu, L., 2012. Trends in traumatic brain injury in the U.S. And the public health response: 1995-2009. J. Safety. Res. 43, 299e307. Courtine, G., Song, B., Roy, R.R., Zhong, H., Herrmann, J.E., Ao, Y., Qi, J., Edgerton, V.R., Sofroniew, M.V., 2008. Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury. Nat. Med. 14, 69e74. Cullen, D.K., Harris, J.P., Browne, K.D., Wolf, J.A., Duda, J.E., Meaney, D.F., Margulies, S.S., Smith, D.H., 2016. A porcine model of traumatic brain injury via head rotational acceleration. Methods Mol. Biol. 1462, 289e324. Curvello, V., Hekierski, H., Riley, J., Vavilala, M., Armstead, W.M., 2017. Sex and age differences in phenylephrine mechanisms and outcomes after piglet brain injury. Pediatr. Res. 82, 108e113. da Costa, E.S., Carvalho, A.L., Martinez, A.M., De-Ary-Pires, B., PiresNeto, M.A., de Ary-Pires, R., 2008. Strapping the spinal cord: an innovative experimental model of CNS injury in rats. J. Neurosci. Methods 170, 130e139. Dabney, K.W., Ehrenshteyn, M., Agresta, C.A., Twiss, J.L., Stern, G., Tice, L., Salzman, S.K., 2004. A model of experimental spinal cord trauma based on computer-controlled intervertebral distraction: characterization of graded injury. Spine 29, 2357e2364. Dabney, K.W., Salzman, S.K., Wakabayashi, T., Sarwark, J.F., Gao, G.X., Beckman, A.L., Bunnell, W.P., 1988. Experimental scoliosis in the rat. II. Biomechanical analysis of the forces during Harrington distraction. Spine 13, 472e477. Dancause, N., Barbay, S., Frost, S.B., Plautz, E.J., Chen, D., Zoubina, E.V., Stowe, A.M., Nudo, R.J., 2005. Extensive cortical rewiring after brain injury. J. Neurosci. 25, 10167e10179. Darbin, O., Carre, E., Naritoku, D., Risso, J.J., Lonjon, M., Patrylo, P.R., 2006. Glucose metabolites in the striatum of freely behaving rats following infusion of elevated potassium. Brain Research 1116, 127e131. Davoody, L., Quiton, R.L., Lucas, J.M., Ji, Y., Keller, A., Masri, R., 2011. Conditioned place preference reveals tonic pain in an animal model of central pain. J. Pain 12, 868e874. Deng, L.X., Deng, P., Ruan, Y., Xu, Z.C., Liu, N.K., Wen, X., Smith, G.M., Xu, X.M., 2013. A novel growth-promoting pathway formed by GDNF-overexpressing Schwann cells promotes propriospinal axonal regeneration, synapse formation, and partial recovery of function after spinal cord injury. J. Neurosci. 33, 5655e5667. Denny-Brown, D.E., Russell, W.R., 1941. Experimental concussion: (section of neurology). Proc. R. Soc. Med. 34, 691e692. Detloff, M.R., Clark, L.M., Hutchinson, K.J., Kloos, A.D., Fisher, L.C., Basso, D.M., 2010. Validity of acute and chronic tactile sensory testing after spinal cord injury in rats. Exp. Neurol. 225, 366e376. Detloff, M.R., Fisher, L.C., Deibert, R.J., Basso, D.M., 2012. Acute and chronic tactile sensory testing after spinal cord injury in rats. J. Vis. Exp. e3247. Dietrich, W.D., Alonso, O., Busto, R., Ginsberg, M.D., 1994. Widespread metabolic depression and reduced somatosensory circuit activation following traumatic brain injury in rats. J. Neurotrauma 11, 629e640. Dimar 2nd, J.R., Glassman, S.D., Raque, G.H., Zhang, Y.P., Shields, C.B., 1999. The influence of spinal canal narrowing and timing of decompression on neurologic recovery after spinal cord contusion in a rat model. Spine 24, 1623e1633. Ditunno Jr., J.F., Young, W., Donovan, W.H., Creasey, G., 1994. The international standards booklet for neurological and functional classification of spinal cord injury. American Spinal Injury Association. Paraplegia 32, 70e80.

RATS AS RESEARCH MODELS

1064

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Dixon, C.E., Kline, A.E., 2009. Controlled cortical impact injury model. In: Chen, J., et al. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 385e391. Dixon, C.E., Kochanek, P.M., Yan, H.Q., Schiding, J.K., Griffith, R.G., Baum, E., Marion, D.W., DeKosky, S.T., 1999. One-year study of spatial memory performance, brain morphology, and cholinergic markers after moderate controlled cortical impact in rats. J. Neurotrauma 16, 109e122. Dixon, C.E., Clifton, G.L., Lighthall, J.W., Yaghmai, A.A., Hayes, R.L., 1991. A controlled cortical impact model of traumatic brain injury in the rat. J. Neurosci. Methods 39, 253e262. Dixon, C.E., Lyeth, B.G., Povlishock, J.T., Findling, R.L., Hamm, R.J., Marmarou, A., Young, H.F., Hayes, R.L., 1987. A fluid percussion model of experimental brain injury in the rat. J. Neurosurg. 67, 110e119. Dobkin, B.H., Havton, L.A., 2004. Basic advances and new avenues in therapy of spinal cord injury. Annu. Rev. Med. 55, 255e282. Dolber, P.C., Gu, B., Zhang, X., Fraser, M.O., Thor, K.B., Reiter, J.P., 2007. Activation of the external urethral sphincter central pattern generator by a 5-HT(1A) receptor agonist in rats with chronic spinal cord injury. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R1699eR1706. Donovan, W.H., 2007. Donald Munro Lecture. Spinal cord injury–past, present, and future. J. Spinal Cord Med. 30, 85e100. Dora, C.D., Koch, S., Sanchez, A., Ruenes, G., Liu, S., Yezierski, R.P., 1998. Intraspinal injection of adenosine agonists protect against L-NAME induced neuronal loss in the rat. J. Neurotrauma 15, 473e483. Duale, H., Hou, S., Derbenev, A.V., Smith, B.N., Rabchevsky, A.G., 2009. Spinal cord injury reduces the efficacy of pseudorabies virus labeling of sympathetic preganglionic neurons. J. Neuropathol. Exp. Neurol. 68, 168e178. Duhaime, A.C., 2006. Large animal models of traumatic injury to the immature brain. Dev. Neurosci. 28, 380e387. Dull, S.T., Konrad, P.E., Tacker Jr., W.A., 1990. Amplitude and latency characteristics of spinal cord motor evoked potentials in the rat. Electroencephalogr. Clin. Neurophysiol. 77, 68e76. Dunham, K.A., Siriphorn, A., Chompoopong, S., Floyd, C.L., 2010. Characterization of a graded cervical hemicontusion spinal cord injury model in adult male rats. J. Neurotrauma 27, 2091e2106. Dunn-Meynell, A.A., Levin, B.E., 1995. Lateralized effect of unilateral somatosensory cortex contusion on behavior and cortical reorganization. Brain Res. 675, 143e156. Edwards, C.M., Kumar, K., Koesarie, K., Brough, E., Ritter, A.C., Brayer, S.W., Thiels, E., Skidmore, E.R., Wagner, A.K., 2015. Visual priming enhances the effects of nonspatial cognitive rehabilitation training on spatial learning after experimental traumatic brain injury. Neurorehabil. Neural Repair 29, 897e906. Ek, C.J., Habgood, M.D., Callaway, J.K., Dennis, R., Dziegielewska, K.M., Johansson, P.A., Potter, A., Wheaton, B., Saunders, N.R., 2010. Spatio-temporal progression of grey and white matter damage following contusion injury in rat spinal cord. PLoS One 5, e12021. el-Bohy, A.A., Schrimsher, G.W., Reier, P.J., Goshgarian, H.G., 1998. Quantitative assessment of respiratory function following contusion injury of the cervical spinal cord. Exp. Neurol. 150, 143e152. Ellenbroek, B., Youn, J., 2016. Rodent models in neuroscience research: is it a rat race? Dis. Model. Mech. 9, 1079e1087. Erschbamer, M.K., Pham, T.M., Zwart, M.C., Baumans, V., Olson, L., 2006. Neither environmental enrichment nor voluntary wheel running enhances recovery from incomplete spinal cord injury in rats. Exp. Neurol. 201, 154e164. Estrada-Rojo, F., Martinez-Tapia, R.J., Estrada-Bernal, F., MartinezVargas, M., Perez-Arredondo, A., Flores-Avalos, L., Navarro, L., 2018. Models used in the study of traumatic brain injury. Rev. Neurosci. 29, 139e149. Estrada, V., Brazda, N., Schmitz, C., Heller, S., Blazyca, H., Martini, R., Muller, H.W., 2014. Long-lasting significant functional improvement

in chronic severe spinal cord injury following scar resection and polyethylene glycol implantation. Neurobiol. Dis. 67, 165e179. Falconer, J.C., Narayana, P.A., Bhattacharjee, M.B., Liu, S.J., 1994. Quantitative MRI of spinal cord injury in a rat model. Magn. Reson. Med. 32, 484e491. Fan, L., Wang, K., Shi, Z., Die, J., Wang, C., Dang, X., 2011. Tetramethylpyrazine protects spinal cord and reduces inflammation in a rat model of spinal cord ischemia-reperfusion injury. J. Vasc. Surg. 54, 192e200. Faul, M., Xu, L., Wald, M.M., Coronado, V., Dellinger, A.M., 2010. Traumatic brain injury in the United States: national estimates of prevalence and incidence, 2002e2006. Inj. Prev. 16. A268-A268. Feeney, D.M., Boyeson, M.G., Linn, R.T., Murray, H.M., Dail, W.G., 1981. Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res. 211, 67e77. Fehlings, M.G., Tator, C.H., 1995. The relationships among the severity of spinal cord injury, residual neurological function, axon counts, and counts of retrogradely labeled neurons after experimental spinal cord injury. Exp. Neurol. 132, 220e228. Fehlings, M.G., Tator, C.H., Linden, R.D., Piper, I.R., 1987. Motor evoked potentials recorded from normal and spinal cord-injured rats. Neurosurgery 20, 125e130. Fehlings, M.G., Vaccaro, A., Wilson, J.R., Singh, A.,D.,W.C., Harrop, J.S., Aarabi, B., Shaffrey, C., Dvorak, M., Fisher, C., Arnold, P., Massicotte, E.M., Lewis, S., Rampersaud, R., 2012. Early versus delayed decompression for traumatic cervical spinal cord injury: results of the Surgical Timing in Acute Spinal Cord Injury Study (STASCIS). PLoS One 7, e32037. Fiford, R.J., Bilston, L.E., Waite, P., Lu, J., 2004. A vertebral dislocation model of spinal cord injury in rats. J. Neurotrauma 21, 451e458. Fijalkowski, R.J., Stemper, B.D., Pintar, F.A., Yoganandan, N., Crowe, M.J., Gennarelli, T.A., 2007. New rat model for diffuse brain injury using coronal plane angular acceleration. J. Neurotrauma 24, 1387e1398. Filli, L., Zorner, B., Weinmann, O., Schwab, M.E., 2011. Motor deficits and recovery in rats with unilateral spinal cord hemisection mimic the Brown-Sequard syndrome. Brain 134, 2261e2273. Finnerup, N.B., Johannesen, I.L., Sindrup, S.H., Bach, F.W., Jensen, T.S., 2001. Pain and dysesthesia in patients with spinal cord injury: a postal survey. Spinal Cord 39, 256e262. Firkins, S.S., Bates, C.A., Stelzner, D.J., 1993. Corticospinal tract plasticity and astroglial reactivity after cervical spinal injury in the postnatal rat. Exp. Neurol. 120, 1e15. Fischer, F.R., Peduzzi, J.D., 2007. Functional recovery in rats with chronic spinal cord injuries after exposure to an enriched environment. J. Spinal Cord Med. 30, 147e155. Fishback, A.S., Shields, C.B., Linden, R.D., Zhang, Y.P., Burke, D., 1995. The effects of propofol on rat transcranial magnetic motor evoked potentials. Neurosurgery 37, 969e974. Floyd, C.L., Golden, K.M., Black, R.T., Hamm, R.J., Lyeth, B.G., 2002. Craniectomy position affects morris water maze performance and hippocampal cell loss after parasagittal fluid percussion. J. Neurotrauma 19, 303e316. Foda, M.A., Marmarou, A., 1994. A new model of diffuse brain injury in rats. Part II: morphological characterization. J. Neurosurg. 80, 301e313. Fouad, K., Hurd, C., Magnuson, D.S.K., 2013. Functional testing in animal models of spinal cord injury: not as straight forward as one would think. Front. Integr. Neurosci. 7, 85. Fraidakis, M., Klason, T., Cheng, H., Olson, L., Spenger, C., 1998. Highresolution MRI of intact and transected rat spinal cord. Exp. Neurol. 153, 299e312. Fujimoto, S.T., Longhi, L., Saatman, K.E., Conte, V., Stocchetti, N., McIntosh, T.K., 2004. Motor and cognitive function evaluation following experimental traumatic brain injury. Neurosci. Biobehav. Rev. 28, 365e378.

RATS AS RESEARCH MODELS

REFERENCES

Fuller, D.D., Golder, F.J., Olson Jr., E.B., Mitchell, G.S., 2006. Recovery of phrenic activity and ventilation after cervical spinal hemisection in rats. J. Appl. Physiol. 100, 800e806. Gale, K., Kerasidis, H., Wrathall, J.R., 1985. Spinal cord contusion in the rat: behavioral analysis of functional neurologic impairment. Exp. Neurol. 88, 123e134. Garcia-Alias, G., Verdu, E., Fores, J., Lopez-Vales, R., Navarro, X., 2003. Functional and electrophysiological characterization of photochemical graded spinal cord injury in the rat. J. Neurotrauma 20, 501e510. Gaviria, M., Privat, A., d’Arbigny, P., Kamenka, J., Haton, H., Ohanna, F., 2000a. Neuroprotective effects of a novel NMDA antagonist, Gacyclidine, after experimental contusive spinal cord injury in adult rats. Brain Res. 874, 200e209. Gaviria, M., Privat, A., d’Arbigny, P., Kamenka, J.M., Haton, H., Ohanna, F., 2000b. Neuroprotective effects of gacyclidine after experimental photochemical spinal cord lesion in adult rats: dosewindow and time-window effects. J. Neurotrauma 17, 19e30. Geddes, J.F., Vowles, G.H., Beer, T.W., Ellison, D.W., 1997. The diagnosis of diffuse axonal injury: implications for forensic practice. Neuropathol. Appl. Neurobiol. 23, 339e347. Geeraerts, T., Ract, C., Tardieu, M., Fourcade, O., Mazoit, J.X., Benhamou, D., Duranteau, J., Vigue, B., 2006. Changes in cerebral energy metabolites induced by impact-acceleration brain trauma and hypoxic-hypotensive injury in rats. J. Neurotrauma 23, 1059e1071. Geissler, S.A., Schmidt, C.E., Schallert, T., 2013. Rodent models and behavioral outcomes of cervical spinal cord injury. J. Spine (Suppl. 4). Gennarelli, T.A., Thibault, L.E., Adams, J.H., Graham, D.I., Thompson, C.J., Marcincin, R.P., 1982. Diffuse axonal injury and traumatic coma in the primate. Ann. Neurol. 12, 564e574. Gennarelli, T.A., Thibault, L.E., Ommaya, A.K.(, 1971. Comparison of linear and rotational acceleration in experimental cerebral concussion. In: Proceedings of the 15th Stapp Car Crash Conference. Society of Automotive Engineers, New York, pp. 797e803. Gennarelli, T.A., Thibault, L.E., Ommaya, A.K.(, 1972. Pathophysiologic responses to rotational and translational accelerations of the head. In: Proceedings of the 16th Stapp Car Crash Conference. Society of Automotive Engineers, New York, pp. 296e308. Gensel, J.C., Tovar, C.A., Hamers, F.P., Deibert, R.J., Beattie, M.S., Bresnahan, J.C., 2006. Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats. J. Neurotrauma 23, 36e54. Gentleman, S.M., Nash, M.J., Sweeting, C.J., Graham, D.I., Roberts, G.W., 1993. Beta-amyloid precursor protein (beta APP) as a marker for axonal injury after head injury. Neurosci. Lett. 160, 139e144. Gill, L.C., Ross, H.H., Lee, K.Z., Gonzalez-Rothi, E.J., Dougherty, B.J., Judge, A.R., Fuller, D.D., 2014. Rapid diaphragm atrophy following cervical spinal cord hemisection. Respir. Physiol. Neurobiol. 192, 66e73. Globus, M.Y., Alonso, O., Dietrich, W.D., Busto, R., Ginsberg, M.D., 1995. Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia. J. Neurochem. 65, 1704e1711. Goldberger, M.E., Bregman, B.S., Vierck Jr., C.J., Brown, M., 1990. Criteria for assessing recovery of function after spinal cord injury: behavioral methods. Exp. Neurol. 107, 113e117. Golder, F.J., Mitchell, G.S., 2005. Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. J. Neurosci. 25, 2925e2932. Golder, F.J., Reier, P.J., Davenport, P.W., Bolser, D.C., 2001. Cervical spinal cord injury alters the pattern of breathing in anesthetized rats. J. Appl. Physiol. 91, 2451e2458. Goldstein, L.E., Fisher, A.M., Tagge, C.A., Zhang, X.L., Velisek, L., Sullivan, J.A., Upreti, C., Kracht, J.M., Ericsson, M., Wojnarowicz, M.W., Goletiani, C.J., Maglakelidze, G.M., Casey, N.,

1065

Moncaster, J.A., Minaeva, O., Moir, R.D., Nowinski, C.J., Stern, R.A., Cantu, R.C., Geiling, J., Blusztajn, J.K., Wolozin, B.L., Ikezu, T., Stein, T.D., Budson, A.E., Kowall, N.W., Chargin, D., Sharon, A., Saman, S., Hall, G.F., Moss, W.C., Cleveland, R.O., Tanzi, R.E., Stanton, P.K., McKee, A.C., 2012. Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model. Sci. Transl. Med. 4, 134ra160. Gondim, F.A., Alencar, H.M., Rodrigues, C.L., da Graca, J.R., dos Santos, A.A., Rola, F.H., 1999. Complete cervical or thoracic spinal cord transections delay gastric emptying and gastrointestinal transit of liquid in awake rats. Spinal Cord 37, 793e799. Gondim, F.A., Rodrigues, C.L., Lopes Jr., A.C., Leal, P.R., Camurca, F.L., Freire, C.C., Dos Santos, A.A., Rola, F.H., 2003. Effect of preinjury large bowel emptying on the inhibition of upper gastrointestinal motility after spinal cord injury in rats. Dig. Dis. Sci. 48, 1713e1718. Gopez, J.J., Yue, H., Vasudevan, R., Malik, A.S., Fogelsanger, L.N., Lewis, S., Panikashvili, D., Shohami, E., Jansen, S.A., Narayan, R.K., Strauss, K.I., 2005. Cyclooxygenase-2-specific inhibitor improves functional outcomes, provides neuroprotection, and reduces inflammation in a rat model of traumatic brain injury. Neurosurgery 56, 590e604. Goren, S., Kahveci, N., Alkan, T., Goren, B., Korfali, E., 2001. The effects of sevoflurane and isoflurane on intracranial pressure and cerebral perfusion pressure after diffuse brain injury in rats. J. Neurosurg. Anesthesiol. 13, 113e119. Gottshall, K., 2011. Vestibular rehabilitation after mild traumatic brain injury with vestibular pathology. NeuroRehabilitation 29, 167e171. Graham, D.I., Adams, J.H., Gennarelli, T.A., 1988. Mechanisms of nonpenetrating head injury. Prog. Clin. Biol. Res. 264, 159e168. Griesbach, G.S., Hovda, D.A., 2015. Cellular and molecular neuronal plasticity. Handb. Clin. Neurol. 128, 681e690. Grill, R.J., 2005. User-defined variables that affect outcome in spinal cord contusion/compression models. Exp. Neurol. 196, 1e5. Grundl, P.D., Biagas, K.V., Kochanek, P.M., Schiding, J.K., Barmada, M.A., Nemoto, E.M., 1994. Early cerebrovascular response to head injury in immature and mature rats. J. Neurotrauma 11, 135e148. Gruner, J.A., 1992. A monitored contusion model of spinal cord injury in the rat. J. Neurotrauma 9, 123e128. Gruner, J.A., Wade, C.K., Menna, G., Stokes, B.T., 1993. Myoelectric evoked potentials versus locomotor recovery in chronic spinal cord injured rats. J. Neurotrauma 10, 327e347. Gruner, J.A., Yee, A.K., Blight, A.R., 1996. Histological and functional evaluation of experimental spinal cord injury: evidence of a stepwise response to graded compression. Brain Res. 729, 90e101. Guth, L., Zhang, Z., Steward, O., 1999. The unique histopathological responses of the injured spinal cord. Implications for neuroprotective therapy. Ann. N. Y. Acad. Sci. 890, 366e384. Gwak, Y.S., Kang, J., Unabia, G.C., Hulsebosch, C.E., 2012. Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats. Exp. Neurol. 234, 362e372. Habgood, M., 2011. Experimental models for assessing treatments for spinal cord injury [Video file]. In: The Biomedical & Life Sciences Collection. Henry Stewart Talks. Hall, K.D., Lifshitz, J., 2010. Diffuse traumatic brain injury initially attenuates and later expands activation of the rat somatosensory whisker circuit concomitant with neuroplastic responses. Brain Res. 1323, 161e173. Halstrom, A., MacDonald, E., Neil, C., Arendts, G., Fatovich, D., Fitzgerald, M., 2017. Elevation of oxidative stress indicators in a pilot study of plasma following traumatic brain injury. J. Clin. Neurosci. 35, 104e108. Hamers, F.P., Koopmans, G.C., Joosten, E.A., 2006. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J. Neurotrauma 23, 537e548.

RATS AS RESEARCH MODELS

1066

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Hamers, F.P., Lankhorst, A.J., van Laar, T.J., Veldhuis, W.B., Gispen, W.H., 2001. Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries. J. Neurotrauma 18, 187e201. Hamm, R.J., 2001. Neurobehavioral assessment of outcome following traumatic brain injury in rats: an evaluation of selected measures. J. Neurotrauma 18, 1207e1216. Hamm, R.J., Pike, B.R., O’Dell, D.M., Lyeth, B.G., Jenkins, L.W., 1994. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. J. Neurotrauma 11, 187e196. Hamm, R.J., White-Gbadebo, D.M., Lyeth, B.G., Jenkins, L.W., Hayes, R.L., 1992. The effect of age on motor and cognitive deficits after traumatic brain injury in rats. Neurosurgery 31, 1072e1078. Han, Q., Jin, W., Xiao, Z., Ni, H., Wang, J., Kong, J., Wu, J., Liang, W., Chen, L., Zhao, Y., Chen, B., Dai, J., 2010. The promotion of neural regeneration in an extreme rat spinal cord injury model using a collagen scaffold containing a collagen binding neuroprotective protein and an EGFR neutralizing antibody. Biomaterials 31, 9212e9220. ˚ ., Wiesenfeld-Hallin, Z., Hao, J.X., Xu, X.J., Aldskogius, H., Seiger, A 1991. Allodynia-like effects in rat after ischaemic spinal cord injury photochemically induced by laser irradiation. Pain 45, 175e185. Hayes, R.L., Lyeth, B.G., Jenkins, L.W., Zimmerman, R., McIntosh, T.K., Clifton, G.L., Young, H.F., 1990. Possible protective effect of endogenous opioids in traumatic brain injury. J. Neurosurg. 72, 252e261. Hayward, N.M., Immonen, R., Tuunanen, P.I., Ndode-Ekane, X.E., Grohn, O., Pitkanen, A., 2010. Association of chronic vascular changes with functional outcome after traumatic brain injury in rats. J. Neurotrauma 27, 2203e2219. Heimburger, R.F., 2005. Return of function after spinal cord transection. Spinal Cord 43, 438e440. Henninger, N., Bouley, J., Sikoglu, E.M., An, J., Moore, C.M., King, J.A., Bowser, R., Freeman, M.R., Brown Jr., R.H., 2016. Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1. Brain 139, 1094e1105. Hermanns, S., Reiprich, P., Muller, H.W., 2001. A reliable method to reduce collagen scar formation in the lesioned rat spinal cord. J. Neurosci. Methods 110, 141e146. Hicks, S.P., D’Amato, C.J., 1975. Motor-sensory cortex-corticospinal system and developing locomotion and placing in rats. Am. J. Anat. 143, 1e42. Hill, C.E., Beattie, M.S., Bresnahan, J.C., 2001. Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat. Exp. Neurol. 171, 153e169. Hillered, L., Vespa, P.M., Hovda, D.A., 2005. Translational neurochemical research in acute human brain injury: the current status and potential future for cerebral microdialysis. J. Neurotrauma 22, 3e41. Hodgetts, S.I., Plant, G.W., Harvey, A.R., 2009. Spinal Cord Injury: experimental animal models and relation to human therapy. In: Paxinos, G., Kayalioglu, G. (Eds.), The Spinal Cord. Academic Press, San Diego, pp. 209e237. Hoffer, M.E., Gottshall, K.R., Moore, R., Balough, B.J., Wester, D., 2004. Characterizing and treating dizziness after mild head trauma. Otol. Neurotol. 25, 135e138. Hoge, C.W., McGurk, D., Thomas, J.L., Cox, A.L., Engel, C.C., Castro, C.A., 2008. Mild traumatic brain injury in U.S. Soldiers returning from Iraq. N. Engl. J. Med. 358, 453e463. Holbourn, A.H.S., 1943. Mechanics of head injuries. The Lancet 242, 438e441. Holmes, G.M., Rogers, R.C., Bresnahan, J.C., Beattie, M.S., 1998. External anal sphincter hyperreflexia following spinal transection in the rat. J. Neurotrauma 15, 451e457. Holtz, A., Nystro¨m, B., Gerdin, B., 1989. Spinal cord blood flow measured by 14C-iodoantipyrine autoradiography during and

after graded spinal cord compression in rats. Surg. Neurol. 31, 350e360. Hook, M., 2013. Animal models of spinal cord injury. In: Handbook of Laboratory Animal Science, third ed., vol. III. CRC Press, pp. 1e42. Hotz, G.A., Stewart, K.J., Petrin, D., Villanueva, P.A., Cohn, S.M., Nedd, K.J., Puentes, G., Duncan, R., 2000. Neurobehavioural outcomes of penetrating and tangential gunshot wounds to the head. Brain Inj. 14, 649e657. Howells, D.W., Porritt, M.J., Rewell, S.S., O’Collins, V., Sena, E.S., van der Worp, H.B., Traystman, R.J., Macleod, M.R., 2010. Different strokes for different folks: the rich diversity of animal models of focal cerebral ischemia. J. Cereb. Blood Flow Metab. 30, 1412e1431. Howlett, J.R., Stein, M.B.(, 2016. Post-traumatic stress disorder: relationship to traumatic brain injury and approach to treatment. In: Laskowitz, D., Grant, G. (Eds.), Translational Research in Traumatic Brain Injury. CRC Press/Taylor and Francis Group, Boca Raton (FL). Huang, L., Obenaus, A., Hamer, M., Zhang, J.H., 2016. Neuroprotective effect of hyperbaric oxygen therapy in a juvenile rat model of repetitive mild traumatic brain injury. Med. Gas Res. 6, 187e193. Hubbard, R.D., Winkelstein, B.A., 2005. Transient cervical nerve root compression in the rat induces bilateral forepaw allodynia and spinal glial activation: mechanical factors in painful neck injuries. Spine 30, 1924e1932. Huber, B.R., Alosco, M.L., Stein, T.D., McKee, A.C., 2016. Potential long-term consequences of concussive and subconcussive injury. Phys. Med. Rehabil. Clin. N. Am. 27, 503e511. Hulsebosch, C.E., Xu, G.Y., Perez-Polo, J.R., Westlund, K.N., Taylor, C.P., McAdoo, D.J., 2000. Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin. J. Neurotrauma 17, 1205e1217. Humphreys, I., Wood, R.L., Phillips, C.J., Macey, S., 2013. The costs of traumatic brain injury: a literature review. Clin. Outcomes Res. 5, 281e287. Hutchinson, K.J., Gomez-Pinilla, F., Crowe, M.J., Ying, Z., Basso, D.M., 2004. Three exercise paradigms differentially improve sensory recovery after spinal cord contusion in rats. Brain 127, 1403e1414. IASP. IASP Terminology: Pain terms. Retrieved 7/12/18 2018: http:// www.iasp-pain.org/Education/Content.aspx?ItemNumber¼1698. Imaizumi, T., Lankford, K.L., Kocsis, J.D., 2000. Transplantation of olfactory ensheathing cells or Schwann cells restores rapid and secure conduction across the transected spinal cord. Brain Res. 854, 70e78. Inskip, J.A., Ramer, L.M., Ramer, M.S., Krassioukov, A.V., 2009. Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord 47, 2e35. Ip, E.Y., Zanier, E.R., Moore, A.H., Lee, S.M., Hovda, D.A., 2003. Metabolic, neurochemical, and histologic responses to vibrissa motor cortex stimulation after traumatic brain injury. J. Cereb. Blood Flow Metab. 23, 900e910. Irvine, K.A., Ferguson, A.R., Mitchell, K.D., Beattie, S.B., Beattie, M.S., Bresnahan, J.C., 2010. A novel method for assessing proximal and distal forelimb function in the rat: the Irvine, Beatties and Bresnahan (IBB) forelimb scale. J. Vis. Exp. 46, e2246. Irvine, K.A., Ferguson, A.R., Mitchell, K.D., Beattie, S.B., Lin, A., Stuck, E.D., Huie, J.R., Nielson, J.L., Talbott, J.F., Inoue, T., Beattie, M.S., Bresnahan, J.C., 2014. The Irvine, Beatties, and Bresnahan (IBB) forelimb recovery scale: an assessment of reliability and validity. Front. Neurol. 5, 116. Jakeman, L., McTigue, D.M., Walters, P., Stokes, B.T., 2009. The Ohio state University ESCID spinal cord contusion model. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 433e448. Jeffery, N.D., Blakemore, W.F., 1997. Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination. Brain 120 (Pt 1), 27e37.

RATS AS RESEARCH MODELS

REFERENCES

Jernigan, S.C., Zhang, Y.P., Shields, C.B., Whittemore, S.R., 2009. Rodent spinal cord demyelination models. In: Chen, J., et al. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 471e478. Johnson, V.E., Meaney, D.F., Cullen, D.K., Smith, D.H., 2015. Animal models of traumatic brain injury. Handb. Clin. Neurol. 127, 115e128. Johnson, V.E., Stewart, J.E., Begbie, F.D., Trojanowski, J.Q., Smith, D.H., Stewart, W., 2013. Inflammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136, 28e42. Johnson, V.E., Stewart, W., Smith, D.H., 2010. Traumatic brain injury and amyloid-beta pathology: a link to Alzheimer’s disease? Nat. Rev. Neurosci. 11, 361e370. Johnson, V.E., Stewart, W., Weber, M.T., Cullen, D.K., Siman, R., Smith, D.H., 2016. SNTF immunostaining reveals previously undetected axonal pathology in traumatic brain injury. Acta Neuropathol. 131, 115e135. Jordan, B.D., 2013. The clinical spectrum of sport-related traumatic brain injury. Nat. Rev. Neurol. 9, 222e230. Kabadi, S.V., Faden, A.I., 2014. Neuroprotective strategies for traumatic brain injury: improving clinical translation. Int. J. Mol. Sci. 15, 1216e1236. Kamnaksh, A., Kovesdi, E., Kwon, S.K., Wingo, D., Ahmed, F., Grunberg, N.E., Long, J., Agoston, D.V., 2011. Factors affecting blast traumatic brain injury. J. Neurotrauma 28, 2145e2153. Kaptanoglu, E., Okutan, O., Akbiyik, F., Solaroglu, I., Kilinc, A., Beskonakli, E., 2004. Correlation of injury severity and tissue Evans blue content, lipid peroxidation and clinical evaluation in acute spinal cord injury in rats. J. Clin. Neurosci. 11, 879e885. Karadimas, S.K., Moon, E.S., Yu, W.R., Satkunendrarajah, K., Kallitsis, J.K., Gatzounis, G., Fehlings, M.G., 2013. A novel experimental model of cervical spondylotic myelopathy (CSM) to facilitate translational research. Neurobiol. Dis. 54, 43e58. Karlsson, A.K., 2006. Autonomic dysfunction in spinal cord injury: clinical presentation of symptoms and signs. Prog. Brain Res. 152, 1e8. Katayama, Y., Becker, D.P., Tamura, T., Hovda, D.A., 1990. Massive increases in extracellular potassium and the indiscriminate release of glutamate following concussive brain injury. J. Neurosurg. 73, 889e900. Kathe, C., Hutson, T.H., Chen, Q., Shine, H.D., McMahon, S.B., Moon, L.D.F., 2014. Unilateral pyramidotomy of the corticospinal tract in rats for assessment of neuroplasticity-inducing therapies. J. Vis. Exp. 94, e51843. Kawaguchi, M., Shimizu, K., Furuya, H., Sakamoto, T., Ohnishi, H., Karasawa, J., 1996. Effect of isoflurane on motor-evoked potentials induced by direct electrical stimulation of the exposed motor cortex with single, double, and triple stimuli in rats. Anesthesiology 85, 1176e1183. Kawamata, T., Katayama, Y., Hovda, D.A., Yoshino, A., Becker, D.P., 1992. Administration of excitatory amino acid antagonists via microdialysis attenuates the increase in glucose utilization seen following concussive brain injury. J. Cereb. Blood Flow Metab. 12, 12e24. Kawamata, T., Katayama, Y., Hovda, D.A., Yoshino, A., Becker, D.P., 1995. Lactate accumulation following concussive brain injury: the role of ionic fluxes induced by excitatory amino acids. Brain Research 674, 196e204. Keirstead, H.S., Fedulov, V., Cloutier, F., Steward, O., Duel, B.P., 2005. A noninvasive ultrasonographic method to evaluate bladder function recovery in spinal cord injured rats. Exp. Neurol. 194, 120e127. Keller, B.P., Haghighi, S.S., Oro, J.J., Eggers Jr., G.W., 1992. The effects of propofol anesthesia on transcortical electric evoked potentials in the rat. Neurosurgery 30, 557e560. Khaing, Z.Z., Geissler, S.A., Jiang, S., Milman, B.D., Aguilar, S.V., Schmidt, C.E., Schallert, T., 2012. Assessing forelimb function after

1067

unilateral cervical spinal cord injury: novel forelimb tasks predict lesion severity and recovery. J. Neurotrauma 29, 488e498. Khan, M., Griebel, R., 1983. Acute spinal cord injury in the rat: comparison of three experimental techniques. Can. J. Neurol. Sci. 10, 161e165. Khan, T., Havey, R.M., Sayers, S.T., Patwardhan, A., King, W.W., 1999. Animal models of spinal cord contusion injuries. Lab. Anim. Sci. 49, 161e172. Kharatishvili, I., Nissinen, J.P., McIntosh, T.K., Pitkanen, A., 2006. A model of posttraumatic epilepsy induced by lateral fluidpercussion brain injury in rats. Neuroscience 140, 685e697. Kilbaugh, T.J., Bhandare, S., Lorom, D.H., Saraswati, M., Robertson, C.L., Margulies, S.S., 2011. Cyclosporin A preserves mitochondrial function after traumatic brain injury in the immature rat and piglet. J. Neurotrauma 28, 763e774. Kilbourne, M., Kuehn, R., Tosun, C., Caridi, J., Keledjian, K., Bochicchio, G., Scalea, T., Gerzanich, V., Simard, J.M., 2009. Novel model of frontal impact closed head injury in the rat. J. Neurotrauma 26, 2233e2243. Kim, D., Schallert, T., Liu, Y., Browarak, T., Nayeri, N., Tessler, A., Fischer, Murray, M., 2001. Transplantation of genetically modified fibroblasts expressing BDNF in adult rats with a subtotal hemisection improves specific motor and sensory functions. Neurorehabil. Neural Repair 15, 141e150. Kjell, J., Olson, L., 2016. Rat models of spinal cord injury: from pathology to potential therapies. Dis. Model. Mech. 9, 1125e1137. Kjell, J., Olson, L., Abrams, M.B., 2016. Improved recovery from spinal cord injury in rats with chronic parvovirus serotype-1a infection. Spinal Cord 54, 517e520. Kjell, J., Sandor, K., Josephson, A., Svensson, C.I., Abrams, M.B., 2013. Rat substrains differ in the magnitude of spontaneous locomotor recovery and in the development of mechanical hypersensitivity after experimental spinal cord injury. J. Neurotrauma 30, 1805e1811. Klapka, N., Hermanns, S., Straten, G., Masanneck, C., Duis, S., Hamers, F.P., Muller, D., Zuschratter, W., Muller, H.W., 2005. Suppression of fibrous scarring in spinal cord injury of rat promotes long-distance regeneration of corticospinal tract axons, rescue of primary motoneurons in somatosensory cortex and significant functional recovery. Eur. J. Neurosci. 22, 3047e3058. Kloos, A.D., Fisher, L.C., Detloff, M.R., Hassenzahl, D.L., Basso, D.M., 2005. Stepwise motor and all-or-none sensory recovery is associated with nonlinear sparing after incremental spinal cord injury in rats. Exp. Neurol. 191, 251e265. Kontos, A.P., Elbin, R.J., Schatz, P., Covassin, T., Henry, L., Pardini, J., Collins, M.W., 2012. A revised factor structure for the postconcussion symptom scale: baseline and postconcussion factors. Am. J. Sports Med. 40, 2375e2384. Koopmans, G.C., Brans, M., Gomez-Pinilla, F., Duis, S., Gispen, W.H., Torres-Aleman, I., Joosten, E.A., Hamers, F.P., 2006. Circulating insulin-like growth factor I and functional recovery from spinal cord injury under enriched housing conditions. Eur. J. Neurosci. 23, 1035e1046. Koozekanani, S.H., Vise, W.M., Hashemi, R.M., McGhee, R.B., 1976. Possible mechanisms for observed pathophysiological variability in experimental spinal cord injury by the method of Allen. J. Neurosurg. 44, 429e434. Koura, S.S., Doppenberg, E.M.R., Marmarou, A., Choi, S., Young, H.F., Bullock, R.(, 1999. Relationship between excitatory amino acid release and outcome after severe human head injury. Acta Neurochir. Suppl. 244e246 (Springer, Vienna). Krassioukov, A.V., Weaver, L.C., 1995. Episodic hypertension due to autonomic dysreflexia in acute and chronic spinal cord-injured rats. Am. J. Physiol. 268, H2077eH2083. Kriet, J.D., Stanley Jr., R.B., Grady, M.S., 2005. Self-inflicted submental and transoral gunshot wounds that produce nonfatal brain injuries: management and prognosis. J. Neurosurg. 102, 1029e1032.

RATS AS RESEARCH MODELS

1068

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Kruse, M.N., Belton, A.L., de Groat, W.C., 1993. Changes in bladder and external urethral sphincter function after spinal cord injury in the rat. Am. J. Physiol. 264, R1157eR1163. Kuehn, R., Simard, P.F., Driscoll, I., Keledjian, K., Ivanova, S., Tosun, C., Williams, A., Bochicchio, G., Gerzanich, V., Simard, J.M., 2011. Rodent model of direct cranial blast injury. J. Neurotrauma 28, 2155e2169. Kunkel-Bagden, E., Dai, H.N., Bregman, B.S., 1993. Methods to assess the development and recovery of locomotor function after spinal cord injury in rats. Exp. Neurol. 119, 153e164. Kwon, B.K., Oxland, T.R., Tetzlaff, W., 2002. Animal models used in spinal cord regeneration research. Spine 27, 1504e1510. Kwon, B.K., Tetzlaff, W., Grauer, J.N., Beiner, J., Vaccaro, A.R., 2004. Pathophysiology and pharmacologic treatment of acute spinal cord injury. Spine J. 4, 451e464. Lafci, G., Gedik, H.S., Korkmaz, K., Erdem, H., Cicek, O.F., Nacar, O.A., Yildirim, L., Kaya, E., Ankarali, H., 2013. Efficacy of iloprost and montelukast combination on spinal cord ischemia/reperfusion injury in a rat model. J. Cardiothorac. Surg. 8, 64. Lafrenaye, A.D., Todani, M., Walker, S.A., Povlishock, J.T., 2015. Microglia processes associate with diffusely injured axons following mild traumatic brain injury in the micro pig. J. Neuroinflammation 12, 186. Laird, A.S., Carrive, P., Waite, P.M., 2006. Cardiovascular and temperature changes in spinal cord injured rats at rest and during autonomic dysreflexia. J. Physiol. 577, 539e548. Langlois, J., Rutland-Brown, W., Thomas, K., 2010. Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations, and Deaths. Retrieved 7/27/2018, from Centers for Disease Control and Prevention: https://www.cdc.gov/ traumaticbraininjury/pdf/blue_book.pdf. Langlois, J.A., Rutland-Brown, W., Wald, M.M., 2006. The epidemiology and impact of traumatic brain injury: a brief overview. J. Head Trauma Rehabil. 21, 375e378. Lankhorst, A.J., ter Laak, M.P., van Laar, T.J., van Meeteren, N.L., de Groot, J.C., Schrama, L.H., Hamers, F.P., Gispen, W.H., 2001. Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat. J. Neurotrauma 18, 203e215. Lankhorst, A.J., Verzijl, M.R., Hamers, F.P.T., 1999. Experimental spinal cord contusion injury: comparison of different outcome parameters. Neurosci. Res. Commun. 24, 135e148. LaPlaca, M.C., Simon, C.M., Prado, G.R., Cullen, D.K., 2007. CNS injury biomechanics and experimental models. Prog. Brain Res. 161, 13e26. Lau, B.C., Kontos, A.P., Collins, M.W., Mucha, A., Lovell, M.R., 2011. Which on-field signs/symptoms predict protracted recovery from sport-related concussion among high school football players? Am. J. Sports Med. 39, 2311e2318. Lau, N.-S.S., Gorrie, C.A., Chia, J.Y., Bilston, L.E., Clarke, E.C., 2013. Severity of spinal cord injury in adult and infant rats after vertebral dislocation depends upon displacement but not speed. J. Neurotrauma 30, 1361e1373. Lee, J.H.T., Streijger, F., Tigchelaar, S., Maloon, M., Liu, J., Tetzlaff, W., Kwon, B.K., 2012. A contusive model of unilateral cervical spinal cord injury using the Infinite Horizon impactor. J. Vis. Exp. 65, e3313. Lee, K.E., Thinnes, J.H., Gokhin, D.S., Winkelstein, B.A., 2004. A novel rodent neck pain model of facet-mediated behavioral hypersensitivity: implications for persistent pain and whiplash injury. J. Neurosci. Methods 137, 151e159. Lee, K.Z., Lane, M.A., Dougherty, B.J., Mercier, L.M., Sandhu, M.S., Sanchez, J.C., Reier, P.J., Fuller, D.D., 2014. Intraspinal transplantation and modulation of donor neuron electrophysiological activity. Exp. Neurol. 251, 47e57. Lemmon, V.P., Ferguson, A.R., Popovich, P.G., Xu, X.M., Snow, D.M., Igarashi, M., Beattie, C.E., Bixby, J.L., Consortium, M., 2014. Minimum information about a spinal cord injury experiment: a

proposed reporting standard for spinal cord injury experiments. J. Neurotrauma 31, 1354e1361. Leszczy nska, A.N., Majczy nski, H., Wilczy nski, G.M., Sławi nska, U., Cabaj, A.M., 2015. Thoracic hemisection in rats results in initial recovery followed by a late decrement in locomotor movements, with changes in coordination correlated with serotonergic innervation of the ventral horn. PLoS One 10, e0143602. Li, X.Y., Li, J., Feng, D.F., Gu, L., 2010. Diffuse axonal injury induced by simultaneous moderate linear and angular head accelerations in rats. Neuroscience 169, 357e369. Li, Y., Zhang, L., Kallakuri, S., Zhou, R., Cavanaugh, J.M., 2011. Quantitative relationship between axonal injury and mechanical response in a rodent head impact acceleration model. J. Neurotrauma 28, 1767e1782. Liebscher, T., Schnell, L., Schnell, D., Scholl, J., Schneider, R., Gullo, M., Fouad, K., Mir, A., Rausch, M., Kindler, D., Hamers, F.P., Schwab, M.E., 2005. Nogo-A antibody improves regeneration and locomotion of spinal cord-injured rats. Ann. Neurol. 58, 706e719. Lifshitz, J., 2009. Fluid percussion injury model. In: Chen, J., et al. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 369e384. Lifshitz, J., Lisembee, A.M., 2012. Neurodegeneration in the somatosensory cortex after experimental diffuse brain injury. Brain Struc. Function 217, 49e61. Linden, R.D., Zhang, Y.P., Burke, D.A., Hunt, M.A., Harpring, J.E., Shields, C.B., 1999. Magnetic motor evoked potential monitoring in the rat. J. Neurosurg. 91, 205e210. Lindsey, A.E., LoVerso, R.L., Tovar, C.A., Hill, C.E., Beattie, M.S., Bresnahan, J.C., 2000. An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat. Neurorehabil. Neural Repair 14, 287e300. Ling, G.S., Ecklund, J.M., 2011. Traumatic brain injury in modern war. Curr. Opin. Anaesthesiol. 24, 124e130. Liu, D., Yang, J., Li, L., McAdoo, D.J., 1995. Paraquat–a superoxide generator–kills neurons in the rat spinal cord. Free Rad. Biol. Med. 18, 861e867. Liu, Y., Keefe, K., Tang, X., Lin, S., Smith, G.M., 2014. Use of selfcomplementary adeno-associated virus serotype 2 as a tracer for labeling axons: implications for axon regeneration. PLoS One 9, e87447. Liu, Y., Kim, D., Himes, B.T., Chow, S.Y., Schallert, T., Murray, M., Tessler, A., Fischer, I., 1999. Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J. Neurosci. 19, 4370e4387. Long, J.B., Bentley, T.L., Wessner, K.A., Cerone, C., Sweeney, S., Bauman, R.A., 2009. Blast overpressure in rats: recreating a battlefield injury in the laboratory. J. Neurotrauma 26, 827e840. Lucke-Wold, B.P., Turner, R.C., Logsdon, A.F., Nguyen, L., Bailes, J.E., Lee, J.M., Robson, M.J., Omalu, B.I., Huber, J.D., Rosen, C.L., 2016. Endoplasmic reticulum stress implicated in chronic traumatic encephalopathy. J. Neurosurg. 124, 687e702. Lukovic, D., Moreno-Manzano, V., Lopez-Mocholi, E., RodriguezJime´nez, F.J., Jendelova, P., Sykova, E., Oria, M., Stojkovic, M., Erceg, S., 2015. Complete rat spinal cord transection as a faithful model of spinal cord injury for translational cell transplantation. Sci. Rep. 5, 9640. Lyeth, B.G., Liu, S., Hamm, R.J., 1993. Combined scopolamine and morphine treatment of traumatic brain injury in the rat. Brain Res. 617, 69e75. M’Dahoma, S., Bourgoin, S., Kayser, V., Barthelemy, S., Chevarin, C., Chali, F., Orsal, D., Hamon, M., 2014. Spinal cord transectioninduced allodynia in rats–behavioral, physiopathological and pharmacological characterization. PLoS One 9, e102027. Magnuson, D.S., Trinder, T.C., Zhang, Y.P., Burke, D., Morassutti, D.J., Shields, C.B., 1999. Comparing deficits following excitotoxic and

RATS AS RESEARCH MODELS

REFERENCES

contusion injuries in the thoracic and lumbar spinal cord of the adult rat. Exp. Neurol. 156, 191e204. Maldonado-Bouchard, S., Peters, K., Woller, S.A., Madahian, B., Faghihi, U., Patel, S., Bake, S., Hook, M.A., 2016. Inflammation is increased with anxiety- and depression-like signs in a rat model of spinal cord injury. Brain Behav. Immun. 51, 176e195. Marcol, W., Slusarczyk, W., Gzik, M., Larysz-Brysz, M., Bobrowski, M., Grynkiewicz-Bylina, B., Rosicka, P., Kalita, K., Weglarz, W., Barski, J.J., Kotulska, K., Labuzek, K., Lewin-Kowalik, J., 2012. Air gun impactor–a novel model of graded white matter spinal cord injury in rodents. J. Reconstr. Microsurg. 28, 561e568. Margulies, S.S., Kilbaugh, T., Sullivan, S., Smith, C., Propert, K., Byro, M., Saliga, K., Costine, B.A., Duhaime, A.C., 2015. Establishing a clinically relevant large animal model platform for TBI therapy development: using cyclosporin A as a case study. Brain Pathol. 25, 289e303. Margulies, S.S., Thibault, L.E., Gennarelli, T.A., 1990. Physical model simulations of brain injury in the primate. J. Biomech. 23, 823e836. Marklund, N., 2016. Rodent models of traumatic brain injury: methods and challenges. In: Kobeissy, F.H., et al. (Eds.), Injury Models of the Central Nervous System: Methods and Protocols. Springer New York, New York, NY, pp. 29e46. Marklund, N., Hillered, L., 2011. Animal modelling of traumatic brain injury in preclinical drug development: where do we go from here? Br. J. Pharmacol. 164, 1207e1229. Marmarou, A., Foda, M.A., van den Brink, W., Campbell, J., Kita, H., Demetriadou, K., 1994. A new model of diffuse brain injury in rats. Part I: pathophysiology and biomechanics. J. Neurosurg. 80, 291e300. Marmarou, C.R., Prieto, R., Taya, K., Young, H.F., Marmarou, A., 2009. Marmarou weight drop injury model. In: Chen, J., et al. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 393e407. Martin, D., Schoenen, J., Delre´e, P., Gilson, V., Rogister, B., Leprince, P., Stevenaert, A., Moonen, G., 1992. Experimental acute traumatic injury of the adult rat spinal cord by a subdural inflatable balloon: methodology, behavioral analysis, and histopathology. J. Neurosci. Res. 32, 539e550. Masri, R., Quiton, R.L., Lucas, J.M., Murray, P.D., Thompson, S.M., Keller, A., 2009. Zona incerta: a role in central pain. J. Neurophysiol. 102, 181e191. Matloub, H.S., Yan, J.G., Kolachalam, R.B., Zhang, L.L., Sanger, J.R., Riley, D.A., 2005. Neuropathological changes in vibration injury: an experimental study. Microsurgery 25, 71e75. May, Z., Fouad, K., Shum-Siu, A., Magnuson, D.S., 2015. Challenges of animal models in SCI research: effects of pre-injury task-specific training in adult rats before lesion. Behav. Brain Res. 291, 26e35. Mayorov, D.N., Adams, M.A., Krassioukov, A.V., 2001. Telemetric blood pressure monitoring in conscious rats before and after compression injury of spinal cord. J. Neurotrauma 18, 727e736. McDonough, A., Monterrubio, A., Ariza, J., Martı´nez-Cerden˜o, V., 2015. Calibrated forceps model of spinal cord compression injury. J. Vis. Exp. 98, e52318. McEwen, M.L., Springer, J.E., 2006. Quantification of locomotor recovery following spinal cord contusion in adult rats. J. Neurotrauma 23, 1632e1653. McIntosh, T.K., Noble, L., Andrews, B., Faden, A.I., 1987. Traumatic brain injury in the rat: characterization of a midline fluidpercussion model. Cent. Nerv Syst. Trauma 4, 119e134. McIntosh, T.K., Vink, R., Noble, L., Yamakami, I., Fernyak, S., Soares, H., Faden, A.L., 1989. Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model. Neuroscience 28, 233e244. McKee, A.C., Alosco, M.L., Huber, B.R., 2016. Repetitive head impacts and chronic traumatic encephalopathy. Neurosurg. Clin. N. Am. 27, 529e535.

1069

McKee, A.C., Cantu, R.C., Nowinski, C.J., Hedley-Whyte, E.T., Gavett, B.E., Budson, A.E., Santini, V.E., Lee, H.-S., Kubilus, C.A., Stern, R.A., 2009. Chronic traumatic encephalopathy in athletes: progressive tauopathy following repetitive head injury. J. Neuropathol. Experimental Neurol. 68, 709e735. McKenna, J.E., Prusky, G.T., Whishaw, I.Q., 2000. Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis. J. Comp. Neurol. 419, 286e296. McKenzie, K.J., McLellan, D.R., Gentleman, S.M., Maxwell, W.L., Gennarelli, T.A., Graham, D.I., 1996. Is beta-APP a marker of axonal damage in short-surviving head injury? Acta Neuropathol. 92, 608e613. McNamara, K.C., Lisembee, A.M., Lifshitz, J., 2010. The whisker nuisance task identifies a late-onset, persistent sensory sensitivity in diffuse brain-injured rats. J. Neurotrauma 27, 695e706. Meaney, D.F., Margulies, S.S., Smith, D.H., 2001. Diffuse axonal injury. J. Neurosurg. 95, 1108e1110. Meaney, D.F., Smith, D.H., Shreiber, D.I., Bain, A.C., Miller, R.T., Ross, D.T., Gennarelli, T.A., 1995. Biomechanical analysis of experimental diffuse axonal injury. J. Neurotrauma 12, 689e694. Merkler, D., Metz, G.A., Raineteau, O., Dietz, V., Schwab, M.E., Fouad, K., 2001. Locomotor recovery in spinal cord-injured rats treated with an antibody neutralizing the myelin-associated neurite growth inhibitor Nogo-A. J. Neurosci. 21, 3665e3673. Meshkinpour, H., Harmon, D., Thompson, R., Yu, J., 1985. Effects of thoracic spinal cord transection on colonic motor activity in rats. Paraplegia 23, 272e276. Metz, G.A., CURT, A., van de Meent, H., Klusman, I., Schwab, M.E., Dietz, V., 2000a. Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury. J. Neurotrauma 17, 1e17. Metz, G.A., Merkler, D., Dietz, V., Schwab, M.E., Fouad, K., 2000b. Efficient testing of motor function in spinal cord injured rats. Brain Res. 883, 165e177. Mez, J., Daneshvar, D.H., Kiernan, P.T., Abdolmohammadi, B., Alvarez, V.E., Huber, B.R., Alosco, M.L., Solomon, T.M., Nowinski, C.J., McHale, L., Cormier, K.A., Kubilus, C.A., Martin, B.M., Murphy, L., Baugh, C.M., Montenigro, P.H., Chaisson, C.E., Tripodis, Y., Kowall, N.W., Weuve, J., McClean, M.D., Cantu, R.C., Goldstein, L.E., Katz, D.I., Stern, R.A., Stein, T.D., McKee, A.C., 2017. Clinicopathological evaluation of chronic traumatic encephalopathy in players of American football. J. Am. Med. Assoc. 318, 360e370. Mills, C.D., Hains, B.C., Johnson, K.M., Hulsebosch, C.E., 2001. Strain and model differences in behavioral outcomes after spinal cord injury in rat. J. Neurotrauma 18, 743e756. Miremami, J.D., Talauliker, P.M., Harrison, J.L., Lifshitz, J., 2014. Neuropathology in sensory, but not motor, brainstem nuclei of the rat whisker circuit after diffuse brain injury. Somatos. Mot Res. 31, 127e135. Mishra, A.M., Bai, X., Sanganahalli, B.G., Waxman, S.G., Shatillo, O., Grohn, O., Hyder, F., Pitka¨nen, A., Blumenfeld, H., 2014. Decreased resting functional connectivity after traumatic brain injury in the rat. PLoS One 9, e95280. Mishra, V., Skotak, M., Schuetz, H., Heller, A., Haorah, J., Chandra, N., 2016. Primary blast causes mild, moderate, severe and lethal TBI with increasing blast overpressures: experimental rat injury model. Sci. Rep. 6, 26992. Mitsui, T., Kakizaki, H., Tanaka, H., Shibata, T., Matsuoka, I., Koyanagi, T., 2003. Immortalized neural stem cells transplanted into the injured spinal cord promote recovery of voiding function in the rat. J. Urol. 170, 1421e1425. Miyazato, M., Sugaya, K., Nishijima, S., Ashitomi, K., Morozumi, M., Ogawa, Y., 2005. Dietary glycine inhibits bladder activity in normal rats and rats with spinal cord injury. J. Urol. 173, 314e317.

RATS AS RESEARCH MODELS

1070

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Mogil, J.S., 2009. Animal models of pain: progress and challenges. Nat. Rev. Neurosci. 10, 283e294. Mogil, J.S., Crager, S.E., 2004. What should we be measuring in behavioral studies of chronic pain in animals? Pain 112, 12e15. Mogil, J.S., Graham, A.C., Ritchie, J., Hughes, S.F., Austin, J.S., Schorscher-Petcu, A., Langford, D.J., Bennett, G.J., 2010. Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice. Mol. Pain 6, 34. Montenigro, P.H., Alosco, M.L., Martin, B.M., Daneshvar, D.H., Mez, J., Chaisson, C.E., Nowinski, C.J., Au, R., McKee, A.C., Cantu, R.C., McClean, M.D., Stern, R.A., Tripodis, Y., 2017. Cumulative head impact exposure predicts later-life depression, apathy, executive dysfunction, and cognitive impairment in former high school and college football players. J. Neurotrauma 34, 328e340. Monti, J.M., Voss, M.W., Pence, A., McAuley, E., Kramer, A.F., Cohen, N.J., 2013. History of mild traumatic brain injury is associated with deficits in relational memory, reduced hippocampal volume, and less neural activity later in life. Front. Aging Neurosci. 5, 41. Montoya, C.P., Campbell-Hope, L.J., Pemberton, K.D., Dunnett, S.B., 1991. The "staircase test": a measure of independent forelimb reaching and grasping abilities in rats. J. Neurosci. Methods 36, 219e228. Morris, R., 1984. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47e60. Morris, R., Whishaw, I.Q., 2016. A proposal for a rat model of spinal cord injury featuring the rubrospinal tract and its contributions to locomotion and skilled hand movement. Front. Neurosci. 10, 5. Muir, K.W., 2006. Glutamate-based therapeutic approaches: clinical trials with NMDA antagonists. Curr. Opin. Pharmacol. 6, 53e60. Murphy, N.P., Mills, R.H., Caudle, R.M., Neubert, J.K., 2014. Operant assays for assessing pain in preclinical rodent models: highlights from an orofacial assay. Curr. Topics Behav. Neurosci. 20, 121e145. Mychasiuk, R., Farran, A., Angoa-Perez, M., Briggs, D., Kuhn, D., Esser, M.J., 2014. A novel model of mild traumatic brain injury for juvenile rats. J. Vis. Exp. 94, e51820. Mychasiuk, R., Hehar, H., Candy, S., Ma, I., Esser, M.J., 2016. The direction of the acceleration and rotational forces associated with mild traumatic brain injury in rodents effect behavioural and molecular outcomes. J. Neurosci. Methods 257, 168e178. Nakae, A., Nakai, K., Yano, K., Hosokawa, K., Shibata, M., Mashimo, T., 2011. The animal model of spinal cord injury as an experimental pain model. J. Biomed. Biotechnol. 2011, 11. Nandoe Tewarie, R.D., Yu, J., Seidel, J., Rahiem, S.T., Hurtado, A., Tsui, B.M., Grotenhuis, J.A., Pomper, M.G., Oudega, M., 2010. Positron emission tomography for serial imaging of the contused adult rat spinal cord. Mol. Imaging 9, 108e116. Nantwi, K.D., Goshgarian, H.G., 1998. Effects of chronic systemic theophylline injections on recovery of hemidiaphragmatic function after cervical spinal cord injury in adult rats. Brain Res. 789, 126e129. Nardone, R., Florea, C., Holler, Y., Brigo, F., Versace, V., Lochner, P., Golaszewski, S., Trinka, E., 2017. Rodent, large animal and nonhuman primate models of spinal cord injury. Zoology (Jena) 123, 101e114. Nashmi, R., Fehlings, M.G., 2001. Changes in axonal physiology and morphology after chronic compressive injury of the rat thoracic spinal cord. Neuroscience 104, 235e251. Nashmi, R., Imamura, H., Tator, C.H., Fehlings, M.G., 1997. Serial recording of somatosensory and myoelectric motor evoked potentials: role in assessing functional recovery after graded spinal cord injury in the rat. J. Neurotrauma 14, 151e159. National Research Council, 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. National Academies Press (US), Washington DC. Nguyen, D.H., Cho, N., Satkunendrarajah, K., Austin, J.W., Wang, J., Fehlings, M.G., 2012. Immunoglobulin G (IgG) attenuates

neuroinflammation and improves neurobehavioral recovery after cervical spinal cord injury. J. Neuroinflammation 9, 224. Nightingale, R.W., McElhaney, J.H., Richardson, W.J., Best, T.M., Myers, B.S., 1996. Experimental impact injury to the cervical spine: relating motion of the head and the mechanism of injury. J. Bone Joint Surg. Am. 78, 412e421. Nilsson, P., Ronne-Engstrom, E., Flink, R., Ungerstedt, U., Carlson, H., Hillered, L., 1994. Epileptic seizure activity in the acute phase following cortical impact trauma in rat. Brain Res. 637, 227e232. Niskanen, J.-P., Airaksinen, A.M., Sierra, A., Huttunen, J.K., Nissinen, J., Karjalainen, P.A., Pitka¨nen, A., Gro¨hn, O.H., 2013. Monitoring functional impairment and recovery after traumatic brain injury in rats by fMRI. J. Neurotrauma 30, 546e556. Nout, Y.S., Beattie, M.S., Bresnahan, J.C., 2012. Severity of locomotor and cardiovascular derangements after experimental highthoracic spinal cord injury is anesthesia dependent in rats. J. Neurotrauma 29, 990e999. Nout, Y.S., Bresnahan, J.C., Culp, E., Tovar, C.A., Beattie, M.S., Schmidt, M.H., 2007. Novel technique for monitoring micturition and sexual function in male rats using telemetry. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R1359eR1367. Nout, Y.S., Schmidt, M.H., Tovar, C.A., Culp, E., Beattie, M.S., Bresnahan, J.C., 2005. Telemetric monitoring of corpus spongiosum penis pressure in conscious rats for assessment of micturition and sexual function following spinal cord contusion injury. J. Neurotrauma 22, 429e441. Noyes, D.H., 1987. Electromechanical impactor for producing experimental spinal cord injury in animals. Med. Biol. Eng. Comput. 25, 335e340. Nystrom, B., Berglund, J.E., 1988. Spinal cord restitution following compression injuries in rats. Acta Neurol. Scand. 78, 467e472. Ohta, K., Fujimura, Y., Nakamura, M., Watanabe, M., Yato, Y., 1999. Experimental study on MRI evaluation of the course of cervical spinal cord injury. Spinal Cord 37, 580e584. Ommaya, A.K., Gennarelli, T.A., 1974. Cerebral concussion and traumatic unconsciousness. Correlation of experimental and clinical observations of blunt head injuries. Brain 97, 633e654. Onifer, S.M., Nunn, C.D., Decker, J.A., Payne, B.N., Wagoner, M.R., Puckett, A.H., Massey, J.M., Armstrong, J., Kaddumi, E.G., Fentress, K.G., Wells, M.J., West, R.M., Calloway, C.C., Schnell, J.T., Whitaker, C.M., Burke, D.A., Hubscher, C.H., 2007a. Loss and spontaneous recovery of forelimb evoked potentials in both the adult rat cuneate nucleus and somatosensory cortex following contusive cervical spinal cord injury. Exp. Neurol. 207, 238e247. Onifer, S.M., Rabchevsky, A.G., Scheff, S.W., 2007b. Rat models of traumatic spinal cord injury to assess motor recovery. ILAR Journal 48, 385e395. Onifer, S.M., Rodriguez, J.F., Santiago, D.I., Benitez, J.C., Kim, D.T., Brunschwig, J.P., Pacheco, J.T., Perrone, J.V., Llorente, O., Hesse, D.H., Martinez-Arizala, A., 1997. Cervical spinal cord injury in the adult rat: assessment of forelimb dysfunction. Restor. Neurol. Neurosci. 11, 211e223. Onifer, S.M., Zhang, Y.P., Burke, D.A., Brooks, D.L., Decker, J.A., McClure, N.J., Floyd, A.R., Hall, J., Proffitt, B.L., Shields, C.B., Magnuson, D.S., 2005. Adult rat forelimb dysfunction after dorsal cervical spinal cord injury. Exp. Neurol. 192, 25e38. Oprych, K.M., Kalsi, P.S., Kachramanoglou, C., Choi, D., 2015. Technical improvements to a rat brachial plexus avulsion model via a posterior surgical approach. Plast. Reconstr. Surg. Glob. Open 3, e576. Oria, M., Chatauret, N., Raguer, N., Cordoba, J., 2008. A new method for measuring motor evoked potentials in the awake rat: effects of anesthetics. J. Neurotrauma 25, 266e275. Osborn, J.W., Taylor, R.F., Schramm, L.P., 1989. Determinants of arterial pressure after chronic spinal transection in rats. Am. J. Physiol. 256, R666eR673.

RATS AS RESEARCH MODELS

REFERENCES

Ozturk, E., Demirbilek, S., Kadir But, A., Saricicek, V., Gulec, M., Akyol, O., Ozcan Ersoy, M., 2005. Antioxidant properties of propofol and erythropoietin after closed head injury in rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 922e927. Palmer, A.M., Marion, D.W., Botscheller, M.L., Swedlow, P.E., Styren, S.D., DeKosky, S.T., 1993. Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model. J. Neurochem. 61, 2015e2024. Panjabi, M.M., Wrathall, J.R., 1988. Biomechanical analysis of experimental spinal cord injury and functional loss. Spine 13, 1365e1370. Park, E., Velumian, A.A., Fehlings, M.G., 2004. The role of excitotoxicity in secondary mechanisms of spinal cord injury: a review with an emphasis on the implications for white matter degeneration. J. Neurotrauma 21, 754e774. Park, J.P., Kim, K.J., Phi, J.H., Park, C.K., Kim, J.H., Kang, H.J., Lee, D., Han, K.H., Wang, K.C., Paek, S.H., 2007. Simple measurement of spinal cord evoked potential: a valuable data source in the rat spinal cord injury model. J. Clin. Neurosci. 14, 1099e1105. Passineau, M.J., Zhao, W., Busto, R., Dietrich, W.D., Alonso, O., Loor, J.Y., Bramlett, H.M., Ginsberg, M.D., 2000. Chronic metabolic sequelae of traumatic brain injury: prolonged suppression of somatosensory activation. Am. J. Physiol. Heart Circ. Physiol. 279, H924eH931. Paxinos, G., Watson, C., 2007. Stereotaxic reference system. In: The Rat Brain in Stereotaxic Coordinates. Academic Press, Elsevier, Oxford, p. XII. Pearse, D.D., Lo Jr., T.P., Cho, K.S., Lynch, M.P., Garg, M.S., Marcillo, A.E., Sanchez, A.R., Cruz, Y., Dietrich, W.D., 2005. Histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat. J. Neurotrauma 22, 680e702. Perdiki, M., Farooque, M., Holtz, A., Li, G.L., Olsson, Y., 1998. Expression of endothelial barrier antigen immunoreactivity in blood vessels following compression trauma to rat spinal cord. Temporal evolution and relation to the degree of the impact. Acta Neuropathol. 96, 8e12. Phipps, H.W., 2016. Systematic review of traumatic brain injury animal models. In: Kobeissy, F.H., et al. (Eds.), Injury Models of the Central Nervous System: Methods and Protocols. Springer New York, New York, NY, pp. 61e88. Pikov, V., Wrathall, J.R., 2001. Coordination of the bladder detrusor and the external urethral sphincter in a rat model of spinal cord injury: effect of injury severity. J. Neurosci. 21, 559e569. Pikov, V., Wrathall, J.R., 2002. Altered glutamate receptor function during recovery of bladder detrusor-external urethral sphincter coordination in a rat model of spinal cord injury. J. Pharmacol. Exp. Ther. 300, 421e427. Pinzon, A., Calancie, B., Oudega, M., Noga, B.R., 2001. Conduction of impulses by axons regenerated in a Schwann cell graft in the transected adult rat thoracic spinal cord. J. Neurosci. Res. 64, 533e541. Plant, G.W., Christensen, C.L., Oudega, M., Bunge, M.B., 2003. Delayed transplantation of olfactory ensheathing glia promotes sparing/ regeneration of supraspinal axons in the contused adult rat spinal cord. J. Neurotrauma 20, 1e16. Plantman, S., Ng, K.C., Lu, J., Davidsson, J., Risling, M., 2012. Characterization of a novel rat model of penetrating traumatic brain injury. J. Neurotrauma 29, 1219e1232. Pons, T.P., Garraghty, P.E., Ommaya, A.K., Kaas, J.H., Taub, E., Mishkin, M., 1991. Massive cortical reorganization after sensory deafferentation in adult macaques. Science 252, 1857e1860. Popovich, P.G., Guan, Z., McGaughy, V., Fisher, L., Hickey, W.F., Basso, D.M., 2002. The neuropathological and behavioral consequences of intraspinal microglial/macrophage activation. J. Neuropathol. Exp. Neurol. 61, 623e633. Popovich, P.G., Lemeshow, S., Gensel, J.C., Tovar, C.A., 2012. Independent evaluation of the effects of glibenclamide on reducing

1071

progressive hemorrhagic necrosis after cervical spinal cord injury. Exp. Neurol. 233, 615e622. Popovich, P.G., Wei, P., Stokes, B.T., 1997. Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats. J. Comp. Neurol. 377, 443e464. Povlishock, J.T., 1992. Traumatically induced axonal injury: pathogenesis and pathobiological implications. Brain Pathol. 2, 1e12. Povlishock, J.T., Becker, D.P., 1985. Fate of reactive axonal swelling induced by head injury. Lab. Invest. 52, 540e552. Povlishock, J.T., Becker, D.P., Cheng, C.L., Vaughan, G.W., 1983. Axonal change in minor head injury. J. Neuropathol. Exp. Neurol. 42, 225e242. Povlishock, J.T., Katz, D.I., 2005. Update of neuropathology and neurological recovery after traumatic brain injury. J. Head Trauma Rehabil. 20, 76e94. Prins, M.L., Giza, C.C., 2006. Induction of monocarboxylate transporter 2 expression and ketone transport following traumatic brain injury in juvenile and adult rats. Dev. Neurosci. 28, 447e456. Prins, M.L., Hovda, D.A., 2003. Developing experimental models to address traumatic brain injury in children. J. Neurotrauma 20, 123e137. Prins, M.L., Lee, S.M., Cheng, C.L., Becker, D.P., Hovda, D.A., 1996. Fluid percussion brain injury in the developing and adult rat: a comparative study of mortality, morphology, intracranial pressure and mean arterial blood pressure. Brain Res. Develop. Brain Res. 95, 272e282. Puckett, A., Nunn, C.D., Onifer, S.M., 2009. Animal care and surgery: veterinary care methods for rats and mice in experimental spincal cord injury studies. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 47e64. Qualls-Creekmore, E., Tong, M., Holmes, G.M., 2010. Time-course of recovery of gastric emptying and motility in rats with experimental spinal cord injury. Neuro Gastroenterol. Motil. 22 (62e69), e27e68. Ramon-Cueto, A., Cordero, M.I., Santos-Benito, F.F., Avila, J., 2000. Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia. Neuron 25, 425e435. Reeves, T.M., Colley, B.S., 2012. Electrophysiological approaches in traumatic brain injury. In: Chen, J., et al. (Eds.), Animal Models of Acute Neurological Injuries II: Injury and Mechanistic Assessments, vol. 2. Humana Press, Totowa, NJ, pp. 313e330. Reid, W.M., Rolfe, A., Register, D., Levasseur, J.E., Churn, S.B., Sun, D., 2010. Strain-related differences after experimental traumatic brain injury in rats. J. Neurotrauma 27, 1243e1253. Reier, P.J., Lane, M.A., Hall, E.D., Teng, Y.D., Howland, D.R., 2012. Translational spinal cord injury research: preclinical guidelines and challenges. Handb. Clin. Neurol. 109, 411e433. Reneer, D.V., Hisel, R.D., Hoffman, J.M., Kryscio, R.J., Lusk, B.T., Geddes, J.W., 2011. A multi-mode shock tube for investigation of blast-induced traumatic brain injury. J. Neurotrauma 28, 95e104. Risling, M., Plantman, S., Angeria, M., Rostami, E., Bellander, B.M., Kirkegaard, M., Arborelius, U., Davidsson, J., 2011. Mechanisms of blast induced brain injuries, experimental studies in rats. Neuroimage 54 (Suppl. 1), S89eS97. Rivas, D.A., Chancellor, M.B., Huang, B., Salzman, S.K., 1995. Autonomic dysreflexia in a rat model spinal cord injury and the effect of pharmacologic agents. Neurourol. Urodyn. 14, 141e152. Rivlin, A.S., Tator, C.H., 1977. Objective clinical assessment of motor function after experimental spinal cord injury in the rat. J. Neurosurg. 47, 577e581. Rivlin, A.S., Tator, C.H., 1978. Effect of duration of acute spinal cord compression in a new acute cord injury model in the rat. Surg. Neurol. 10, 38e43. Robinson, M., Jaber, S.M., Piotrowski, S.L., Gomez, T.H., 2018. Preprocedural consideration and post-procedural care for animal

RATS AS RESEARCH MODELS

1072

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

models with experimental traumatic brain injury. In: Srivastava AK, C.C. (Ed.), Pre-Clinical and Clinical Methods in Brain Trauma Research. Springer, New York, pp. 155e172. Robinson, M.A., Herron, A.J., Goodwin, B.S., Grill, R.J., 2012. Suprapubic bladder catheterization of male spinal-cord-injured SpragueDawley rats. J. Am. Assoc. Lab. Anim. Sci. 51, 76e82. Rodrigues, D.M., Martins, S.S., Gilioli, R., Guaraldo, A.M., Gatti, M.S., 2005. Theiler’s murine encephalomyelitis virus in nonbarrier rat colonies. Comp. Med. 55, 459e464. Rose, M.E., Huerbin, M.B., Melick, J., Marion, D.W., Palmer, A.M., Schiding, J.K., Kochanek, P.M., Graham, S.H., 2002. Regulation of interstitial excitatory amino acid concentrations after cortical contusion injury. Brain Research 943, 15e22. Rosenzweig, E.S., McDonald, J.W., 2004. Rodent models for treatment of spinal cord injury: research trends and progress toward useful repair. Curr. Opin. Neurol. 17, 121e131. Rostami, E., 2016. General consideration in using animal laboratory in CNS injury research. In: Kobieissy, F., Dixon, C.E., Hayes, R.L., Mondello, S. (Eds.), Injury Models of the Central Nervous System: Methods and Protocols. Humana, New York, NY, pp. 47e60. Rostami, E., Davidsson, J., Gyorgy, A., Agoston, D.V., Risling, M., Bellander, B.M., 2013. The terminal pathway of the complement system is activated in focal penetrating but not in mild diffuse traumatic brain injury. J. Neurotrauma 30, 1954e1965. Rowe, R.K., Harrison, J.L., Ellis, T.W., Adelson, P.D., Lifshitz, J., 2018. Midline (central) fluid percussion model of traumatic brain injury in pediatric and adolescent rats. J. Neurosurg. Pediatr. 22, 22e30. Rowe, R.K., Harrison, J.L., Thomas, T.C., Pauly, J.R., Adelson, P.D., Lifshitz, J., 2013. Using anesthetics and analgesics in experimental traumatic brain injury. Lab. Anim. (NY) 42, 286e291. Ruppel, R.A., Kochanek, P.M., Adelson, P.D., Rose, M.E., Wisniewski, S.R., Bell, M.J., Clark, R.S., Marion, D.W., Graham, S.H., 2001. Excitatory amino acid concentrations in ventricular cerebrospinal fluid after severe traumatic brain injury in infants and children: the role of child abuse. J. Pediatr. 138, 18e25. Ryan, L.M., Warden, D.L., 2003. Post concussion syndrome. Int. Rev. Psychiatry 15, 310e316. Saatman, K.E., Duhaime, A.C., Bullock, R., Maas, A.I., Valadka, A., Manley, G.T., 2008. Classification of traumatic brain injury for targeted therapies. J. Neurotrauma 25, 719e738. Salzman, S.K., Dabney, K.W., Mendez, A.A., Beauchamp, J.T., Daley, J.C., Freeman, G.M., Fonseca, A., Ingersoll, E.B., Beckman, A.L., Bunnell, W.P., 1988. The somatosensory evoked potential predicts neurologic deficits and serotonergic pathochemistry after spinal distraction injury in experimental scoliosis. J. Neurotrauma 5, 173e186. Sanders, M.J., Dietrich, W.D., Green, E.J., 1999. Cognitive function following traumatic brain injury: effects of injury severity and recovery period in a parasagittal fluid-percussive injury model. J. Neurotrauma 16, 915e925. Sandrow, H.R., Shumsky, J.S., Amin, A., Houle, J.D., 2008. Aspiration of a cervical spinal contusion injury in preparation for delayed peripheral nerve grafting does not impair forelimb behavior or axon regeneration. Exp. Neurol. 210, 489e500. Santos-Benito, F.F., Munoz-Quiles, C., Ramon-Cueto, A., 2006. Longterm care of paraplegic laboratory mammals. J. Neurotrauma 23, 521e536. Saunders, N.R., Dziegielewska, K.M., Whish, S.C., Hinds, L.A., Wheaton, B.J., Huang, Y., Henry, S., Habgood, M.D., 2017. A bipedal mammalian model for spinal cord injury research: the tammar wallaby. F1000Research 6, 921. Schallert, T., Fleming, S.M., Leasure, J.L., Tillerson, J.L., Bland, S.T., 2000. CNS plasticity and assessment of forelimb sensorimotor

outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology 39, 777e787. Schantz, L., 2009. Animal care and surgery: animal protocol. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 3e10. Scheff, S.W., Rabchevsky, A.G., Fugaccia, I., Main, J.A., Lumpp Jr., J.E., 2003. Experimental modeling of spinal cord injury: characterization of a force-defined injury device. J. Neurotrauma 20, 179e193. Scheff, S., Roberts, K.N., 2009. Infinite horizon spinal cord contusion model. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 423e432. Schrimsher, G.W., Reier, P.J., 1992. Forelimb motor performance following cervical spinal cord contusion injury in the rat. Exp. Neurol. 117, 287e298. Schrimsher, G.W., Reier, P.J., 1993. Forelimb motor performance following dorsal column, dorsolateral funiculi, or ventrolateral funiculi lesions of the cervical spinal cord in the rat. Exp. Neurol. 120, 264e276. Schucht, P., Raineteau, O., Schwab, M.E., Fouad, K., 2002. Anatomical correlates of locomotor recovery following dorsal and ventral lesions of the rat spinal cord. Exp. Neurol. 176, 143e153. Sedy, J., Urdzikova, L., Jendelova, P., Sykova, E., 2008. Methods for behavioral testing of spinal cord injured rats. Neurosci. Biobehav. Rev. 32, 550e580. Seifert, J.L., Bell, J.E., Elmer, B.B., Sucato, D.J., Romero, M.I., 2011. Characterization of a novel bidirectional distraction spinal cord injury animal model. J. Neurosci. Methods 197, 97e103. Sekhon, L.H., Fehlings, M.G., 2001. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26, S2eS12. Semple, B.D., Carlson, J., Noble-Haeusslein, L.J., 2016. Pediatric rodent models of traumatic brain injury. Methods Mol. Biol. 1462, 325e343. Sengul, G., 2015. Spinal cord cyto- and chemoarchitecture. In: Paxinos, G. (Ed.), The Rat Nervous System. Academic Press, Elsevier, Oxford, UK, pp. 87e95. Shah, P.K., Garcia-Alias, G., Choe, J., Gad, P., Gerasimenko, Y., Tillakaratne, N., Zhong, H., Roy, R.R., Edgerton, V.R., 2013. Use of quadrupedal step training to re-engage spinal interneuronal networks and improve locomotor function after spinal cord injury. Brain 136, 3362e3377. Shapira, Y., Shohami, E., Sidi, A., Soffer, D., Freeman, S., Cotev, S., 1988. Experimental closed head injury in rats: mechanical, pathophysiologic, and neurologic properties. Crit. Care Med. 16, 258e265. Sharif-Alhoseini, M., Khormali, M., Rezaei, M., Safdarian, M., Hajighadery, A., Khalatbari, M.M., Safdarian, M., Meknatkhah, S., Rezvan, M., Chalangari, M., Derakhshan, P., RahimiMovaghar, V., 2017. Animal models of spinal cord injury: a systematic review. Spinal Cord 55, 714e721. Sharif-Alhoseini, M., Rahimi-Movaghar, V., 2014. Animal models in traumatic spinal cord injury. In: Dionyssiotis, Y. (Ed.), Topics in Paraplegia. InTech, Rijeka, pp. 209e228. Shen, Y., Kou, Z., Kreipke, C.W., Petrov, T., Hu, J., Haacke, E.M., 2007. In vivo measurement of tissue damage, oxygen saturation changes and blood flow changes after experimental traumatic brain injury in rats using susceptibility weighted imaging. Magn. Reson. Imag. 25, 219e227. Sherriff, F.E., Bridges, L.R., Sivaloganathan, S., 1994. Early detection of axonal injury after human head trauma using immunocytochemistry for beta-amyloid precursor protein. Acta Neuropathol. 87, 55e62. Shiau, J.S., Zappulla, R.A., Nieves, J., 1992. The effect of graded spinal cord injury on the extrapyramidal and pyramidal motor evoked potentials of the rat. Neurosurgery 30, 76e84.

RATS AS RESEARCH MODELS

REFERENCES

Shin, S.S., Bray, E.R., Zhang, C.Q., Dixon, C.E., 2011. Traumatic brain injury reduces striatal tyrosine hydroxylase activity and potassium-evoked dopamine release in rats. Brain Research 1369, 208e215. Shohami, E., Shapira, Y., Cotev, S., 1988. Experimental closed head injury in rats: prostaglandin production in a noninjured zone. Neurosurgery 22, 859e863. Shuang, F., Hou, S.-X., Zhu, J.-L., Liu, Y., Zhou, Y., Zhang, C.-L., Tang, J.-G., 2015. Establishment of a rat model of lumbar facet joint osteoarthritis using intraarticular injection of urinary plasminogen activator. Sci. Rep. 5, 9828. Siddall, P.J., McClelland, J.M., Rutkowski, S.B., Cousins, M.J., 2003. A longitudinal study of the prevalence and characteristics of pain in the first 5 years following spinal cord injury. Pain 103, 249e257. Silva, N.A., Sousa, N., Reis, R.L., Salgado, A.J., 2014. From basics to clinical: a comprehensive review on spinal cord injury. Progress Neurobiol. 114, 25e57. Siman, R., Giovannone, N., Hanten, G., Wilde, E.A., McCauley, S.R., Hunter, J.V., Li, X., Levin, H.S., Smith, D.H., 2013. Evidence that the blood biomarker SNTF predicts brain imaging changes and persistent cognitive dysfunction in mild TBI patients. Front. Neurol. 4, 190. Siman, R., Shahim, P., Tegner, Y., Blennow, K., Zetterberg, H., Smith, D.H., 2015. Serum SNTF increases in concussed professional ICE hockey players and relates to the severity of postconcussion symptoms. J. Neurotrauma 32, 1294e1300. Simard, J.M., Tsymbalyuk, O., Ivanov, A., Ivanova, S., Bhatta, S., Geng, Z., Woo, S.K., Gerzanich, V., 2007. Endothelial sulfonylurea receptor 1-regulated NC Ca-ATP channels mediate progressive hemorrhagic necrosis following spinal cord injury. J. Clin. Invest. 117, 2105e2113. Simon, D.W., Vagni, V.M., Kochanek, P.M., Clark, R.S., 2016. Combined neurotrauma models: experimental models combining traumatic brain injury and secondary insults. Methods Mol. Biol. 1462, 393e411. Sipski, M.L., Arenas, A., 2006. Female sexual function after spinal cord injury. Prog. Brain Res. 152, 441e447. Skjodt, N.M., Farran, R.P., Hawes, H.G., Kortbeek, J.B., Easton, P.A., 2001. Simulation of acute spinal cord injury: effects on respiration. Respir. Physiol. 127, 3e11. Smith, D.H., Chen, X.H., Pierce, J.E., Wolf, J.A., Trojanowski, J.Q., Graham, D.I., McIntosh, T.K., 1997. Progressive atrophy and neuron death for one year following brain trauma in the rat. J. Neurotrauma 14, 715e727. Smith, D.H., Hicks, R.R., Johnson, V.E., Bergstrom, D.A., Cummings, D.M., Noble, L.J., Hovda, D., Whalen, M., Ahlers, S.T., LaPlaca, M., Tortella, F.C., Duhaime, A.C., Dixon, C.E., 2015. Pre-clinical traumatic brain injury common data elements: toward a common language across laboratories. J. Neurotrauma 32, 1725e1735. Smith, D.H., Johnson, V.E., Stewart, W., 2013. Chronic neuropathologies of single and repetitive TBI: substrates of dementia? Nat. Rev. Neurol. 9, 211e221. Smith, D.H., Meaney, D.F., 2000. Axonal damage in traumatic brain injury. The Neuroscientist 6, 483e495. Smith, D.H., Okiyama, K., Gennarelli, T.A., McIntosh, T.K., 1993. Magnesium and ketamine attenuate cognitive dysfunction following experimental brain injury. Neurosci. Lett. 157, 211e214. Smith, R.R., Burke, D.A., Baldini, A.D., Shum-Siu, A., Baltzley, R., Bunger, M., Magnuson, D.S., 2006a. The Louisville Swim Scale: a novel assessment of hindlimb function following spinal cord injury in adult rats. J. Neurotrauma 23, 1654e1670. Smith, R.R., Shum-Siu, A., Baltzley, R., Bunger, M., Baldini, A., Burke, D.A., Magnuson, D.S., 2006b. Effects of swimming on functional recovery after incomplete spinal cord injury in rats. J. Neurotrauma 23, 908e919.

1073

Soblosky, J.S., Colgin, L.L., Chorney-Lane, D., Davidson, J.F., Carey, M.E., 1997. Ladder beam and camera video recording system for evaluating forelimb and hindlimb deficits after sensorimotor cortex injury in rats. J. Neurosci. Methods 78, 75e83. Soblosky, J.S., Song, J.H., Dinh, D.H., 2001. Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects. Behav. Brain Res. 119, 1e13. Springer, J.E., Rao, R.R., Lim, H.R., Cho, S.I., Moon, G.J., Lee, H.Y., Park, E.J., Noh, J.S., Gwag, B.J., 2010. The functional and neuroprotective actions of Neu2000, a dual-acting pharmacological agent, in the treatment of acute spinal cord injury. J. Neurotrauma 27, 139e149. Sramkova, T., Skrivanova, K., Dolan, I., Zamecnik, L., Sramkova, K., Kriz, J., Muzik, V., Fajtova, R., 2017. Women’s sex life after spinal cord injury. Sex. Med. 5, e255ee259. Sroga, J.M., Jones, T.B., Kigerl, K.A., McGaughy, V.M., Popovich, P.G., 2003. Rats and mice exhibit distinct inflammatory reactions after spinal cord injury. J. Comp. Neurol. 462, 223e240. Statler, K.D., Alexander, H., Vagni, V., Dixon, C.E., Clark, R.S., Jenkins, L., Kochanek, P.M., 2006. Comparison of seven anesthetic agents on outcome after experimental traumatic brain injury in adult, male rats. J. Neurotrauma 23, 97e108. Statler, K.D., Jenkins, L.W., Dixon, C.E., Clark, R.S., Marion, D.W., Kochanek, P.M., 2001. The simple model versus the super model: translating experimental traumatic brain injury research to the bedside. J. Neurotrauma 18, 1195e1206. Stein, D.G., 2015. Embracing failure: what the Phase III progesterone studies can teach about TBI clinical trials. Brain Inj. 29, 1259e1272. Steward, O., Popovich, P.G., Dietrich, W.D., Kleitman, N., 2012. Replication and reproducibility in spinal cord injury research. Exp. Neurol. 233, 597e605. Stokes, B.T., 1992. Experimental spinal cord injury: a dynamic and verifiable injury device. J. Neurotrauma 9, 129e134. Streijger, F., Plunet, W.T., Lee, J.H., Liu, J., Lam, C.K., Park, S., Hilton, B.J., Fransen, B.L., Matheson, K.A., Assinck, P., Kwon, B.K., Tetzlaff, W., 2013. Ketogenic diet improves forelimb motor function after spinal cord injury in rodents. PLoS One 8, e78765. Sun, D., Colello, R.J., Daugherty, W.P., Kwon, T.H., McGinn, M.J., Harvey, H.B., Bullock, M.R., 2005. Cell proliferation and neuronal differentiation in the dentate gyrus in juvenile and adult rats following traumatic brain injury. J. Neurotrauma 22, 95e105. Sundaramurthy, A., Alai, A., Ganpule, S., Holmberg, A., Plougonven, E., Chandra, N., 2012. Blast-induced biomechanical loading of the rat: an experimental and anatomically accurate computational blast injury model. J. Neurotrauma 29, 2352e2364. Surey, S., Berry, M., Logan, A., Bicknell, R., Ahmed, Z., 2014. Differential cavitation, angiogenesis and wound-healing responses in injured mouse and rat spinal cords. Neuroscience 275, 62e80. Sutton, R.L., Lescaudron, L., Stein, D.G., 1993. Unilateral cortical contusion injury in the rat: vascular disruption and temporal development of cortical necrosis. J. Neurotrauma 10, 135e149. Sweet, J.A., Eakin, K.C., Munyon, C.N., Miller, J.P., 2014. Improved learning and memory with theta-burst stimulation of the fornix in rat model of traumatic brain injury. Hippocampus 24, 1592e1600. Tang, S., Xu, S., Fourney, W.L., Leiste, U.H., Proctor, J.L., Fiskum, G., Gullapalli, R.P., 2017. Central nervous system changes induced by underbody blast-induced hyperacceleration: an in vivo diffusion tensor imaging and magnetic resonance spectroscopy study. J. Neurotrauma 34, 1972e1980. Tarlov, I.M., Klinger, H., 1954. Spinal cord compression studies. II. Time limits for recovery after acute compression in dogs. AMA Arch. Neurol. Psychiatry 71, 271e290. Tator, C., Poon, P., 2009. Acute clip impact-compression model. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of

RATS AS RESEARCH MODELS

1074

29. RAT MODELS OF CENTRAL NERVOUS SYSTEM INJURY

Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 449e460. Taylor, C.A., Bell, J.M., Breiding, M.J., Xu, L., 2017. Traumatic brain injury-related emergency department visits, hospitalizations, and deaths - United States, 2007 and 2013. Morb. Mortal. Wkly. Rep. Surveillance Summ. 66, 1e16. Teng, Y.D., Bingaman, M., Taveira-DaSilva, A.M., Pace, P.P., Gillis, R.A., Wrathall, J.R., 2003. Serotonin 1A receptor agonists reverse respiratory abnormalities in spinal cord-injured rats. J. Neurosci. 23, 4182e4189. Tennant, K.A., Asay, A.L., Allred, R.P., Ozburn, A.R., Kleim, J.A., Jones, T.A., 2010. The vermicelli and capellini handling tests: simple quantitative measures of dexterous forepaw function in rats and mice. J. Vis. Exp. e2076. Thau-Zuchman, O., Shohami, E., Alexandrovich, A.G., Trembovler, V., Leker, R.R., 2012. The anti-inflammatory drug carprofen improves long-term outcome and induces gliogenesis after traumatic brain injury. J. Neurotrauma 29, 375e384. Thelin, E.P., Zeiler, F.A., Ercole, A., Mondello, S., Buki, A., Bellander, B.M., Helmy, A., Menon, D.K., Nelson, D.W., 2017. Serial sampling of serum protein biomarkers for monitoring human traumatic brain injury dynamics: a systematic review. Front. Neurol. 8, 300. Thibault, L.E., Gennarelli, T.A., Margulies, S.S.(, 1990. The strain dependent pathophysiological consequences of inertial loading on central nervous system tissue. In: Proceedings of the International Conference on the Biomechanics of Impact, pp. 191e202. Lyon, France). Thompson, F.J., Hou, J., Bose, P.K., 2016. Closed-head TBI model of multiple morbidity. Methods Mol. Biol. 1462, 521e536. Tremoleda, J.L., Thau-Zuchman, O., Davies, M., Foster, J., Khan, I., Vadivelu, K.C., Yip, P.K., Sosabowski, J., Trigg, W., MichaelTitus, A.T., 2016. In vivo PET imaging of the neuroinflammatory response in rat spinal cord injury using the TSPO tracer [(18)F] GE-180 and effect of docosahexaenoic acid. Eur. J. Nucl. Med. Mol. Imaging 43, 1710e1722. van de Meent, H., Hamers, F.P., Lankhorst, A.J., Buise, M.P., Joosten, E.A., Gispen, W.H., 1996. New assessment techniques for evaluation of posttraumatic spinal cord function in the rat. J. Neurotrauma 13, 741e754. Van Meeteren, N.L., Eggers, R., Lankhorst, A.J., Gispen, W.H., Hamers, F.P., 2003. Locomotor recovery after spinal cord contusion injury in rats is improved by spontaneous exercise. J. Neurotrauma 20, 1029e1037. Vanicky, I., Urdzikova, L., Saganova, K., Cizkova, D., Galik, J., 2001. A simple and reproducible model of spinal cord injury induced by epidural balloon inflation in the rat. J. Neurotrauma 18, 1399e1407. Vaquero, C., Arce, N., Agudo, J., Martinez, R., Garjal, C., Diago, M.V., 2007. Evaluation of ischemic injury of the spinal cord following endoprosthesis implantation in the thoraco-abdominal aorta on a rat model. Rev. Port. Cir. Cardio-Toracica Vasc. 14, 33e37. Vaquero, J., Zurita, M., Oya, S., Santos, M., 2006. Cell therapy using bone marrow stromal cells in chronic paraplegic rats: systemic or local administration? Neurosci. Lett. 398, 129e134. Vierck, C.J., Hansson, P.T., Yezierski, R.P., 2008. Clinical and pre-clinical pain assessment: are we measuring the same thing? Pain 135, 7e10. Vinit, S., Gauthier, P., Stamegna, J.C., Kastner, A., 2006. High cervical lateral spinal cord injury results in long-term ipsilateral hemidiaphragm paralysis. J. Neurotrauma 23, 1137e1146. Vink, R., Mullins, P.G., Temple, M.D., Bao, W., Faden, A.I., 2001. Small shifts in craniotomy position in the lateral fluid percussion injury model are associated with differential lesion development. J. Neurotrauma 18, 839e847. Vogelaar, C.F., Konig, B., Krafft, S., Estrada, V., Brazda, N., Ziegler, B., Faissner, A., Muller, H.W., 2015. Pharmacological suppression of CNS scarring by deferoxamine reduces lesion volume and

increases regeneration in an in vitro model for astroglial-fibrotic scarring and in rat spinal cord injury in vivo. PLoS One 10, e0134371. Vogelaar, C.F., Estrada, V., 2016. In: Fuller, H. (Ed.), Experimental Spinal Cord Injury Models in Rodents: Anatomical Correlations and Assessment of Motor Recovery. InTech. von Euler, M., Seiger, A., Sundstrom, E., 1997. Clip compression injury in the spinal cord: a correlative study of neurological and morphological alterations. Exp. Neurol. 145, 502e510. von Leden, R.E., Selwyn, R.G., Jaiswal, S., Wilson, C.M., Khayrullina, G., Byrnes, K.R., 2016. (18)F-FDG-PET imaging of rat spinal cord demonstrates altered glucose uptake acutely after contusion injury. Neurosci. Lett. 621, 126e132. Wagner, A.K., Brayer, S.W., Hurwitz, M., Niyonkuru, C., Zou, H., Failla, M., Arenth, P., Manole, M.D., Skidmore, E., Thiels, E., 2013. Non-spatial pre-training in the water maze as a clinically relevant model for evaluating learning and memory in experimental TBI. Neurobiol. Learn. Mem. 106, 71e86. Walf, A.A., Frye, C.A., 2007. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat. Protoc. 2, 322e328. Wang, F., Huang, S.-L., He, X.-J., Li, X.-H., 2014a. Determination of the ideal rat model for spinal cord injury by diffusion tensor imaging. Neuroreport 25, 1386e1392. Wang, H.C., Sun, C.F., Chen, H., Chen, M.S., Shen, G., Ma, Y.B., Wang, B.D., 2014b. Where are we in the modelling of traumatic brain injury? Models complicated by secondary brain insults. Brain Inj. 28, 1491e1503. Wang, X., Xu, X.-M., 2009. Spinal cord lateral hemisection and implantation of guidance channels. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, pp. 479e486. Ward, R.E., Huang, W., Kostusiak, M., Pallier, P.N., Michael-Titus, A.T., Priestley, J.V., 2014. A characterization of white matter pathology following spinal cord compression injury in the rat. Neuroscience 260, 227e239. Warden, D., 2006. Military TBI during the Iraq and Afghanistan wars. J. Head Trauma Rehabil. 21, 398e402. Watson, B.D., Prado, R., Dalton Dietrich, W., Ginsberg, M.D., Green, B.A., 1986. Photochemically induced spinal cord injury in the rat. Brain Res. 367, 296e300. Watson, C., Paxinos, G., Kayalioglu, G., Heise, C., 2009. Atlas of the rat spinal cord. In: The Spinal Cord. Academic Press, San Diego, pp. 238e306. Weaver, L.C., Verghese, P., Bruce, J.C., Fehlings, M.G., Krenz, N.R., Marsh, D.R., 2001. Autonomic dysreflexia and primary afferent sprouting after clip-compression injury of the rat spinal cord. J. Neurotrauma 18, 1107e1119. Wen, J., Sun, D., Tan, J., Young, W., 2015. A consistent, quantifiable, and graded rat lumbosacral spinal cord injury model. J. Neurotrauma 32, 875e892. Werner, J.K., Stevens, R.D., 2015. Traumatic brain injury: recent advances in plasticity and regeneration. Curr. Opin. Neurol. 28, 565e573. Wilcott, R.C., 1994. Further investigation of preoperative overtraining, visual cortex lesions and black-white discrimination by the rat. Behav. Brain Res. 62, 103e106. Williams, A.J., Hartings, J.A., Lu, X.C., Rolli, M.L., Dave, J.R., Tortella, F.C., 2005. Characterization of a new rat model of penetrating ballistic brain injury. J. Neurotrauma 22, 313e331. Wilson, M.A., Johnston, M.V., Goldstein, G.W., Blue, M.E., 2000. Neonatal lead exposure impairs development of rodent barrel field cortex. Proc. Natl. Acad. Sci. 97, 5540e5545. Woller, S.A., Malik, J.S., Aceves, M., Hook, M.A., 2014. Morphine selfadministration following spinal cord injury. J. Neurotrauma 31, 1570e1583.

RATS AS RESEARCH MODELS

REFERENCES

Woolf, C.J., 1984. Long term alterations in the excitability of the flexion reflex produced by peripheral tissue injury in the chronic decerebrate rat. Pain 18, 325e343. Woolsey, T.A., Van der Loos, H., 1970. The structural organization of layer IV in the somatosensory region (SI) of mouse cerebral cortex. The description of a cortical field composed of discrete cytoarchitectonic units. Brain Res. 17, 205e242. Wrathall, J.R., Emch, G.S., 2006. Effect of injury severity on lower urinary tract function after experimental spinal cord injury. Prog. Brain Res. 152, 117e134. Wrathall, J.R., Pettegrew, R.K., Harvey, F., 1985. Spinal cord contusion in the rat: production of graded, reproducible, injury groups. Exp. Neurol. 88, 108e122. Wright, D.K., Johnston, L.A., Kershaw, J., Ordidge, R., O’Brien, T.J., Shultz, S.R., 2017. Changes in apparent fiber density and trackweighted imaging metrics in white matter following experimental traumatic brain injury. J. Neurotrauma 34, 2109e2118. Xing, G., Ren, M., Watson, W.A., O’Neil, J.T., Verma, A., 2009. Traumatic brain injury-induced expression and phosphorylation of pyruvate dehydrogenase: a mechanism of dysregulated glucose metabolism. Neurosci. Lett. 454, 38e42. Xiong, Y., Mahmood, A., Chopp, M., 2013. Animal models of traumatic brain injury. Nat. Rev. Neurosci. 14, 128e142. Xu, N., Akesson, E., Holmberg, L., Sundstrom, E., 2015. A sensitive and reliable test instrument to assess swimming in rats with spinal cord injury. Behav. Brain Res. 291, 172e183. Xu, S., Zhuo, J., Racz, J., Shi, D., Roys, S., Fiskum, G., Gullapalli, R., 2011. Early microstructural and metabolic changes following controlled cortical impact injury in rat: a magnetic resonance imaging and spectroscopy study. J. Neurotrauma 28, 2091e2102. Xue, F., Wei, Y., Chen, Y., Wang, Y., Gao, L., 2014. A rat model for chronic spinal nerve root compression. Eur. Spine J. 23, 435e446. Yeo, S.J., Hwang, S.N., Park, S.W., Kim, Y.B., Min, B.K., Kwon, J.T., Suk, J.S., 2004. Development of a rat model of graded contusive spinal cord injury using a pneumatic impact device. J. Korean Med. Sci. 19, 574e580. Yezierski, R.P., Liu, S., Ruenes, G.L., Kajander, K.J., Brewer, K.L., 1998. Excitotoxic spinal cord injury: behavioral and morphological characteristics of a central pain model. Pain 75, 141e155. Yi, J.H., Hazell, A.S., 2006. Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury. Neurochem. Int. 48, 394e403. Ylioja, S., Hanks, R., Baird, A., Millis, S., 2010. Are cognitive outcome and recovery different in civilian penetrating versus nonpenetrating brain injuries? Clin. Neuropsychol. 24, 1097e1112. Yoshiyama, M., de Groat, W.C., 2002. Effect of bilateral hypogastric nerve transection on voiding dysfunction in rats with spinal cord injury. Exp. Neurol. 175, 191e197. Young, W., 2009. MASCIS spinal cord contusion model. In: Chen, J., Xu, Z.C., Xu, X.M., Zhang, J.H. (Eds.), Animal Models of Acute Neurological Injuries. Humana Press, Totowa, NJ, p. 411. Young, W., 2014. Spinal cord regeneration. Cell Transplant. 23, 573e611. Yu, K., Rong, W., Li, J., Jia, L., Yuan, W., Yie, X., Shi, Z., 2001. Neurophysiological evidence of spared upper motor conduction fibers

1075

in clinically complete spinal cord injury: discomplete SCI in rats. J. Neurol. Sci. 189, 23e36. Yu, W., Hao, J.X., Xu, X.J., Saydoff, J., Haegerstrand, A., Hokfelt, T., Wiesenfeld-Hallin, Z., 1998. Long-term alleviation of allodynialike behaviors by intrathecal implantation of bovine chromaffin cells in rats with spinal cord injury. Pain 74, 115e122. Z’Graggen, W.J., Metz, G.A., Kartje, G.L., Thallmair, M., Schwab, M.E., 1998. Functional recovery and enhanced corticofugal plasticity after unilateral pyramidal tract lesion and blockade of myelinassociated neurite growth inhibitors in adult rats. J. Neurosci. 18, 4744e4757. Zafonte, R.D., Wood, D.L., Harrison-Felix, C.L., Valena, N.V., Black, K., 2001. Penetrating head injury: a prospective study of outcomes. Neurol. Res. 23, 219e226. Zaninotto, A.L., Vicentini, J.E., Fregni, F., Rodrigues, P.A., Botelho, C., de Lucia, M.C., Paiva, W.S., 2016. Updates and current perspectives of psychiatric assessments after traumatic brain injury: a systematic review. Front. Psychiatry 7, 95. Zhang, K., Sejnowski, T.J., 2000. A universal scaling law between gray matter and white matter of cerebral cortex. Proc. Natl. Acad. Sci. USA 97, 5621e5626. Zhang, N., Fang, M., Chen, H., Gou, F., Ding, M., 2014a. Evaluation of spinal cord injury animal models. Neural Regen. Res. 9, 2008e2012. Zhang, Y.P., Burke, D.A., Shields, L.B., Chekmenev, S.Y., Dincman, T., Zhang, Y., Zheng, Y., Smith, R.R., Benton, R.L., DeVries, W.H., Hu, X., Magnuson, D.S., Whittemore, S.R., Shields, C.B., 2008. Spinal cord contusion based on precise vertebral stabilization and tissue displacement measured by combined assessment to discriminate small functional differences. J. Neurotrauma 25, 1227e1240. Zhang, Y.P., Iannotti, C., Shields, L.B., Han, Y., Burke, D.A., Xu, X.M., Shields, C.B., 2004. Dural closure, cord approximation, and clot removal: enhancement of tissue sparing in a novel laceration spinal cord injury model. J. Neurosurg. 100, 343e352. Zhang YP, O.S., Burke, D.A., Shields, C.B., 2001. A topical mixture for preventing, abolishing, and treating autophagia and self-mutilation in laboratory rats. Contemp. Top. Lab. Anim. Sci. 40, 35e36. Zhang, Z., Zhang, Y.P., Shields, L.B.E., Shields, C.B., 2014b. Technical comments on rodent spinal cord injuries models. Neural Regen. Res. 9, 453e455. Zimmer, M.B., Goshgarian, H.G., 2006. Spinal activation of serotonin 1A receptors enhances latent respiratory activity after spinal cord injury. J. Spinal Cord Med. 29, 147e155. Zimmer, M.B., Nantwi, K., Goshgarian, H.G., 2007. Effect of spinal cord injury on the respiratory system: basic research and current clinical treatment options. J. Spinal Cord Med. 30, 319e330. Zorner, B., Bachmann, L.C., Filli, L., Kapitza, S., Gullo, M., Bolliger, M., Starkey, M.L., Rothlisberger, M., Gonzenbach, R.R., Schwab, M.E., 2014. Chasing central nervous system plasticity: the brainstem’s contribution to locomotor recovery in rats with spinal cord injury. Brain 137, 1716e1732. Zorner, B., Filli, L., Starkey, M.L., Gonzenbach, R., Kasper, H., Rothlisberger, M., Bolliger, M., Schwab, M.E., 2010. Profiling locomotor recovery: comprehensive quantification of impairments after CNS damage in rodents. Nat. Methods 7, 701e708.

RATS AS RESEARCH MODELS