Potentials and limitations of peripheral nerve injury models in rodents with particular reference to the femoral nerve

Annals of Anatomy 193 (2011) 276–285

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Annals of Anatomy journal homepage: www.elsevier.de/aanat

Invited review

Potentials and limitations of peripheral nerve injury models in rodents with particular reference to the femoral nerve Andrey Irintchev ∗ Neuroscience Laboratory, Department of Otorhinolaryngology, University Jena, Germany

a r t i c l e

i n f o

Article history: Received 9 January 2011 Received in revised form 16 February 2011 Accepted 24 February 2011

Keywords: Facial nerve Motion analysis Outcome measures Peripheral nerve regeneration Recovery of function Sciatic nerve

s u m m a r y Restoration of function after peripheral nerve repair in humans is unsatisfactory. Various causes of poor recovery have been proposed. Still, we do not understand which of these potential factors are indeed detrimental and do not know how to manipulate them experimentally in a clinically feasible way. Future success largely depends on methodological improvement in rodent models. An example of recent progress is the introduction of new functional and anatomical outcome measures in the facial nerve injury paradigm which led to novel insights into facial nerve regeneration and a new therapeutic concept. Less success can be ascribed to the use of the classical spinal nerve model, the sciatic nerve paradigm, not least because of its anatomical and functional complexity making assessment of recovery challenging. A simpler alternative to the sciatic nerve is the femoral nerve model. It offers, alongside with its known usefulness for studies on precision of motor reinnervation, the possibility of reliable functional assessments and a straightforward search of anatomical substrates of dysfunction. The structure-function approach in the femoral nerve paradigm has been useful for testing of novel therapeutic means and analyses of regeneration in mutant mice. The potential of the method has still not been really exploited and its more extensive use may contribute to better understanding of nerve regeneration. © 2011 Elsevier GmbH. All rights reserved.

Contents 1. 2. 3. 4. 5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors limiting recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The facial nerve model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The sciatic nerve model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The femoral nerve model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Specificity of motor reinnervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Single-frame motion analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Generalization of the SFMA approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Functional and anatomical outcome of femoral nerve repair in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Testing of therapeutic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Potential factors limiting recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Correspondence address: Department of Otorhinolaryngology, Friedrich Schiller University Jena, Lessingstrasse 2, D-07740 Jena, Germany. Tel.: +49 3641 935 354; fax: +49 3641 932 5847. E-mail address: [email protected] 0940-9602/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2011.02.019

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1. Introduction Despite increasing knowledge of cellular and molecular mechanisms involved in peripheral nerve regeneration (Chen et al., 2007) and optimized surgical treatments, the clinical outcome of nerve repair in adults remains disappointing (Guntinas-Lichius et al., 2007; Lee and Wolfe, 2000; Lundborg, 2003; Lundborg and Rosén, 2007; Robinson, 2000; Scholz et al., 2009). Precise estimates of the overall frequency of serious permanent disabilities are difficult because recovery depends on multiple clinically relevant factors including severity, type and level of the lesion, timing of repair and reconstruction technique, type of the injured nerve and age of the patient (Lundborg and Rosén, 2007). It is generally considered that only half of the patients with complete nerve transection regain useful function and that full recovery occurs in less than 10% of the cases (Brushart, 1988; Sunderland, 1991). Serious nerve injuries are frequent, affecting 2–3% of the trauma patients, and associated with high treatment and care costs (Jaquet et al., 2001; Kreiger et al., 1981; Noble et al., 1998; Robinson, 2000). All these facts show that restoration of function after nerve lesions remains a major challenge to clinical medicine. Effective therapeutic measures do not exist and the search for novel treatments requires improvement in existing experimental models and better understanding of the cellular and molecular mechanisms limiting functional regeneration (Höke, 2006).

2. Factors limiting recovery In cases of large tissue defects requiring nerve grafting, the primary cause of poor recovery is restricted regrowth of axons into the distal nerve stump and target organs. Therefore, in such cases the strategy to improve regeneration to a level achievable by direct nerve suture is to develop conduits using tissue engineering and novel biomaterials, which support axonal regrowth over large distances and are superior to the gold standard; i.e. the autologous nerve graft (Dahlin et al., 2009; Muir, 2010; Siemionow and Brzezicki, 2009). But why is functional recovery deficient when the severed nerve is immediately repaired by microsurgical end-to-end suture (primary repair) and large numbers of axons reinnervate the distal stump and reach target organs? Experimental studies have identified several potential causes in the periphery including misdirected regrowth of axons into inappropriate targets (Brushart and Mesulam, 1980; Sumner, 1990), diminished trophic support by Schwann cells in the distal stump (Fu and Gordon, 1997), poor myelination and aberrant functional properties of regenerated axons (Hildebrand et al., 1985; Kocsis and Waxman, 1983; Mert et al., 2004), and polyneuronal innervation of muscle fibers (Guntinas-Lichius et al., 2005b; Ijkema-Paassen et al., 2002). The most widely accepted and easily comprehensible cause of functional deficits is axonal misdirection. Injury and suture of a major nerve providing motor and sensory innervation to different muscles, joints and skin areas inevitably result in regrowth of axons to false targets. For example, motor axons reinnervate skin or incorrect, sometimes functionally antagonistic muscles, while afferent axons regrow to muscles or inappropriate receptors (Brushart and Mesulam, 1980; Koerber et al., 1989; Sumner, 1990). It is apparent that false peripheral rewiring cannot be beneficial to functional recovery. The question, however, remains of the quality of the proof of primary detrimental role of axonal misdirection (Sunderland, 1991). The fact that restoration of function after nerve repair is worse than after crush injury of the same nerve (de Ruiter et al., 2008; Sunderland, 1991) provides no real proof of this concept. Compared with crush, transection is also more challenging in terms of reparative demands at the site of injury, axonal guid-

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ance and trophic support, regenerative responses of axotomized neurons and neural plasticity (Moran and Graeber, 2004; Navarro et al., 2007). Thus, in addition to reduced precision of reinnervation, nerve repair results in a number of structural deficits including, for example, a more pronounced deafferentation of motoneurons (Svensson et al., 1991) as well as poorer myelination and smaller diameters of the regenerated axons compared with crush (de Ruiter et al., 2008). It is, therefore, impossible to estimate the contribution of each of several anatomical variables to the functional outcome in these two types of injury. Moreover, the results of experiments in which axonal misdirection after nerve repair has been successfully manipulated question its crucial role in determining the functional outcome (Guntinas-Lichius et al., 2005b; Hamilton et al., 2011). Similar to misdirection, the contribution of the other potential factors in the periphery to failures of functional restoration remain uncertain. The question of factors limiting recovery becomes even more complicated if we consider the immense complexity of central nervous system (CNS) reorganization after peripheral nerve injury (Navarro et al., 2007). Structural, functional and molecular changes occur at all levels in the affected sensorimotor system including motor and sensory neurons, reflex pathways, spinal and brainstem circuitries, as well as thalamic and cortical representations. For example, post-traumatic rearrangements of cortical motor representations in rodents are long-lasting if not permanent (Franchi, 2000; Sanes et al., 1990). Years after upper limb nerve repair in humans, the primary and secondary somatosensory cortices contralateral to the injury are thinned and measures of sensory recovery are negatively correlated with the cortical atrophy (Taylor et al., 2009). Motoneurons are acutely deafferented after nerve injury (“synaptic stripping”, Blinzinger and Kreutzberg, 1968) and the synaptic inputs after peripheral target reinnervation are incompletely restored (Brännström and Kellerth, 1999). Stretch areflexia after successful muscle reinnervation, which is inevitably present and incapacitates sensing and controlling limb position, appears to be a phenomenon related to active suppression of sensory information by central neural circuits (Haftel et al., 2005). All these phenomena are potential contributors to poor functional outcome after peripheral nerve injury. In addition, recovery of motor function appears to be limited by extracellular matrix components concentrated in perineuronal nets (Fawcett, 2009) like the glycoprotein tenascin-R (TNR) and chondroitin sulfate proteoglycans (CSPG). The evidence for this notion comes from experiments showing that mice deficient in TNR recover better from facial nerve repair than wild-type mice (Guntinas-Lichius et al., 2005a) and that digestion of CSPG in the spinal cord with chondroitinase ABC improves the outcome of medial and ulnar nerve injuries in rats (Galtrey et al., 2007). The above considerations suggest that multiple mechanisms contribute to the success or failure of motor recovery after nerve injury. For development of novel treatment strategies, the challenge is to identify primary detrimental factors that could be experimentally manipulated in a manner feasible to clinical settings. Crucial to the success of such efforts is the availability of reproducible, discriminative and meaningful quantitative methods of assessment of neurological dysfunction (Basso, 2004). In addition, the search for substrates of dysfunction requires adequate, functionally relevant anatomical measures. And finally, it has to be considered that successful functional regeneration may depend on different factors in different experimental models. To substantiate these statements, some characteristics and outcome measures of two most commonly used models in rodents, the facial and the sciatic nerve models, as well as some recent findings relevant to understanding of functional regeneration, are briefly considered in the following two sections.

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3. The facial nerve model After its exit from the stylomastoid foramen of the temporal bone, the facial nerve in rodents is a “pure” motor nerve containing axons of alpha-motoneurons supplying innervation to numerous facial muscles which are devoid of proprioceptors (McComas, 1998; Rice et al., 1997; Welt and Abbs, 1990; Whitehead et al., 2005). The sensory input to the facial motoneurons is provided by the trigeminal nerve via direct ipsilateral connections between trigeminal sensory nuclei and the facial nucleus (Erzurumlu and Killackey, 1979; Isokawa-Akesson and Komisaruk, 1987; Nguyen and Kleinfeld, 2005). Thus, lesions of the facial nerve in rats and mice provide a unique opportunity to study motor axon regeneration and motor recovery in the absence of damage to sensory neurons. Extensive use of the facial model over decades has provided an enormous amount of data on cellular and molecular responses of motoneurons and their environment to different insults (Moran and Graeber, 2004). More recently, the introduction of a kinematic method for quantitative estimation of vibrissal movements after facial nerve injury in rodents (Tomov et al., 2002) added novel value to the established injury paradigm. One important finding obtained by the motion analysis approach was that functional recovery after primary facial nerve repair (facial–facial anastomosis, FFA) in rats and mice is very deficient. The amplitude of vibrissal movements on the side of injury reaches a maximum of 30–40% compared with the non-injured contralateral side or non-injured animals (Angelov et al., 2007; Guntinas-Lichius et al., 2005a,b; Kiryakova et al., 2010; Tomov et al., 2002). More importantly, the functional assessment combined with retrograde tracings and morphological analysis of endplates to estimate axonal misdirection and quality of endplate reinnervation, respectively, allowed insight into structural determinants of the functional outcome. Improved recovery after FFA in rats and mice as a result of genetic defects or treatments is not paralleled by improved precision of reinnervation (Angelov et al., 2007; Guntinas-Lichius et al., 2005a; Tomov et al., 2002). Vice versa, reducing axonal misdirection by applying antibodies against growth factors to the site of injury does not improve functional outcome (Guntinas-Lichius et al., 2005b). In contrast to axonal misrouting, the frequency of aberrant endplate innervation by multiple axons is correlated to the level of functional restoration (Angelov et al., 2007; Guntinas-Lichius et al., 2005b). Attempts to reduce endplate poly-innervation by mechanical stimulation (MS, daily rhythmic stroking of the vibrissa pads for 5 min during the first two months after FFA) was not only successful but led to a striking improvement of whisking function (Angelov et al., 2007; Lindsay et al., 2010). These findings demonstrate the potential of a well-designed structural–functional approach to unravel mechanisms restricting functional recovery and leading to the design of a clinically feasible therapeutic approach. On the other hand, the failure of MS to improve the outcome of median nerve injury (Sinis et al., 2008) suggests that mechanisms of recovery and treatment effects differ in different nerve injury models.

4. The sciatic nerve model The sciatic nerve, like most other peripheral nerves, is a “mixed” one; i.e. it contains motor and sensory axons. Therefore, injury to the sciatic nerve, of a major branch such as the tibial nerve or even to a small muscle branch like the one to the soleus muscle, leads to damage of motor and sensory axons. Sciatic lesions greatly challenge the regenerative potential of the peripheral nervous system. Motor axons have to regrow to appropriate muscles, proprioceptive afferents have to find their way to the correct muscles and joints, and other sensory axons of different modalities have to regenerate to diverse targets. Therefore, it is not surprising that motor recov-

ery after sciatic nerve injury is deficient, reaching, as estimated by walking track analysis in rats, a maximum of 40% of normal function (Nichols et al., 2005). The sciatic nerve injury model is doubtlessly an important and valuable model and its use in rats has provided most of the data on regeneration of spinal nerves up to now (Nichols et al., 2005). However, it is a very complex model in terms of anatomical and functional circuitries and has several limitations which must be considered. Sciatic nerve repair leads, as a rule, to development of neuropathic pain in rodents manifested by abnormal response to thermal and tactile stimuli; i.e. thermal hyperalgesia and tactile allodynia (Eaton, 2003; Wall et al., 1979). This is an advantage of the model as neuropathic pain is a common consequence of nerve damage in humans. A disadvantage is the high incidence of complications like autotomy (self-mutilation), skin ulcerations and limb contractures which lead to exclusion of animals during the observation time period and hamper functional assessments (Nichols et al., 2005; Varejão et al., 2004). Another problem is assessment of motor recovery after sciatic nerve injury in rodents. For nearly three decades, the walking track (footprint) analysis of de Medinaceli and its numerous modifications has been the method of choice for functional analyses in rats (de Medinaceli et al., 1982; Nichols et al., 2005; Varejão et al., 2004) and, more recently, in mice (see Fey et al., 2010). The method is based on measurements of footprints of walking animals, and nerve function is estimated by the sciatic functional index or, in case of lesions of the tibial and peroneal branches of the sciatic nerve, by the tibial and peroneal functional indices, respectively. Although still widely used, the assay has several limitations the most serious of which is low precision and, thus, limited discriminative power (Fey et al., 2010; Schiaveto de Souza et al., 2004; Shenaq et al., 1989; Urbanchek et al., 1999; Valero-Cabré and Navarro, 2002; Varejão et al., 2003b; Vleggeert-Lankamp, 2007; Yu et al., 2001). Awareness of the insufficiencies of the track analysis has led researchers to seek novel approaches, and several kinematical parameters like gait-stance duration, ankle angle, toe out angle, limb angle and limb length, have been used for evaluation of sciatic nerve function in rats (Hamilton et al., 2011; Santos et al., 1995; Varejão et al., 2003a,b; Walker et al., 1994; Yu et al., 2001). Kinematic analyses provide quantitative data but these methods require special knowledge and expensive equipment and are, thus, limited to specialized laboratories (Basso, 2004; Varejão et al., 2004). In addition, the small size of laboratory rodents, the short extremities bent under the body and the skin movements around joints limit the accuracy of joint and limb segment identification (Basso, 2004; Filipe et al., 2006; Muir and Webb, 2000; Varejão et al., 2004). A recently introduced quantitative approach, the CatWalk gait analysis (Vogelaar et al., 2004), allows simultaneous measurements of numerous static and dynamic gait parameters (Deumens et al., 2007) and has been proposed as a complementary approach to other behavioral tests for assessment of functional recovery (Bozkurt et al., 2008). Limitations of the CatWalk are the high costs of the equipment and the need for extensive training of the animals including food restriction and rewarding. In addition to functional (gait) analyses, objective electrophysiological methods have been used as functional measures in the sciatic nerve paradigm. For example, measurements of compound muscle action potentials using electromyography (EMG) provide the opportunity to estimate the time course of reinnervation and degree of recovery of individual muscles (de Ruiter et al., 2008; Krarup et al., 2002; Valero-Cabré and Navarro, 2002; Wolthers et al., 2005). Another method, muscle tension recording, allows estimates of muscle contractile properties including muscle force, degree of muscle reinnervation and maturation of endplate transmission (Badke et al., 1989; Chipman et al., 2010; Herbison et al., 1981; Irintchev et al., 1990, 1991; Yeagle et al., 1983). Similar to EMG, tension recordings concentrate on specific muscles and mus-

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cle properties without consideration of limb functions and behavior of the animals. The correlations between functional tests and electrophysiological measures in the sciatic nerve model are poor which can be explained by limitations of the methods and/or estimation of different aspects of recovery (Kanaya et al., 1996; Martins et al., 2006; Munro et al., 1998; Wolthers et al., 2005). Similarly, poor correlations have been found between commonly used morphological estimates of regeneration, for example, number of myelinated axons in the distal nerve stump, and functional and electrophysiological measures (Munro et al., 1998). The only exception are significant correlations between thickness or myelination of the regenerated axons, on the one hand, and functional measures like sciatic functional index or ankle angle, on the other hand (de Ruiter et al., 2008; Kanaya et al., 1996; Martins et al., 2006). In conclusion, the sciatic nerve model challenges researchers with many variables and several methodological uncertainties. It is conceivable that the complexity of the model rather than lack of adequate outcome measures sets limits to this injury paradigm. Up to now, the model has provided evidence against rather than in favor of axonal misdirection as a major limitation to functional restoration after nerve repair (Hamilton et al., 2011; Tomita et al., 2007; Valero-Cabré and Navarro, 2002). An alternative for regeneration studies using a major hindlimb nerve is the femoral nerve model which is discussed in the following sections. 5. The femoral nerve model The femoral nerve shares major similarities with the sciatic nerve: It is a mixed hindlimb nerve supplying motor innervation to several muscles and sensory innervation to muscles and skin. One anatomical feature of this nerve renders the model a specific importance for regeneration research. Close to the inguinal ligament, the femoral nerve divides into two roughly similar in diameter terminal branches, a muscle branch to the quadriceps muscle and a cutaneous branch, the saphenous nerve. Injury immediately proximal to the bifurcation of the two branches affects a single muscle, the quadriceps muscle. After nerve repair, the quadriceps motor axons have an equal opportunity to regrow into the original pathway, the quadriceps branch, or choose the wrong pathway, the purely sensory saphenous nerve. This choice situation allows analyses of cellular and molecular mechanisms involved in specificity of motor reinnervation (Section 5.1). In addition, the model is interesting because injury causes functional disability of a single muscle, the quadriceps, which has a unique function in a major hindlimb joint, knee extension. In contrast, the sciatic nerve controls multiple functions of agonistic and antagonistic muscles some of which require higher precision of motor control compared with knee extension like, for example, ankle dorsiflexion and plantar flexion, toe flexion and toe extension. The simplicity of the motor deficit after femoral nerve injury allows a simple approach for functional (gait) analysis (Section 5.2). Complications like self-mutilations, limb contractures and skin ulcerations after femoral nerve repair have not been reported and, in my experience, do not occur. 5.1. Specificity of motor reinnervation The femoral nerve was “discovered” as a contemporary model for regeneration research when Thomas Brushart reported that after femoral nerve repair in juvenile and adult rats, the quadriceps motor axons regenerate preferentially into the nerve branch to the quadriceps muscle as opposed to the sensory saphenous branch (Brushart, 1988, for a review see Madison et al., 2007). This “preferential motor reinnervation” (PMR) proceeds in a staggered fashion (Al-Majed et al., 2000; Brushart, 1993; Brushart et al., 2002). At

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2 weeks after nerve repair, similar numbers of motor axons reinnervate the correct (motor) and the incorrect (skin) pathway. The initially random regrowth is followed by a gradual increase in the correct, but not incorrect projections. Finally, by the 10th week, about 60–70% of the motoneurons project correctly to the quadriceps muscle, 20–30% project to the skin and only a few percent innervate both targets. One hypothesis of PMR is that differences in cellular and molecular constituents in motor versus sensory pathways favor the correct targeting of regenerating motor axons (Brushart, 1993). Two carbohydrates have been identified as potential molecular determinants of PMR. One of these carbohydrates is the human natural killer (HNK) cell glycan known as HNK-1 epitope and expressed on different glycoproteins (Kleene and Schachner, 2004). The HNK-1 epitope is expressed by myelinating Schwann cells associated with motor but not sensory axons during development and is re-expressed after nerve injury in adult mice (Martini and Schachner, 1986; Martini et al., 1992, 1994). In vitro, substrate-coated HNK-1 carbohydrate enhances neurite outgrowth from motoneurons but not from sensory neurons (Martini et al., 1992). Finally, the findings that application of HNK-1 mimetic peptides to the injured femoral nerve improves PMR and motor recovery in adult mice (Simova et al., 2006) indicate the functional significance of HNK-1 mediated responses in vivo. The second molecule which appears to be involved in PMR is the glycan ␣2,8 polysialic acid (PSA), a carbohydrate predominantly expressed on the neural cell adhesion molecule (NCAM, Rutishauser, 2008). After femoral nerve injury in adult mice, PSA-NCAM is up-regulated in the motor and less so in the sensory nerve branch and PMR is abolished if (1) PSA-NCAM is genetically ablated, (2) PSA is enzymatically removed or (3) the motoneuron pool fails to up-regulate PSA expression after injury (Franz et al., 2005, 2008). PMR has been consistently observed after femoral nerve repair in rats (Brushart, 1988, 1993; Madison et al., 1996). In contrast, PMR does not appear to be a consistent feature of the model in mice. Both pronounced PMR as in the rat (Franz et al., 2005, 2008; Guseva et al., 2009) and lack of PMR (Ahlborn et al., 2007; Mears et al., 2003; Mehanna et al., 2009; Robinson and Madison, 2003; Simova et al., 2006) have been reported after nerve repair in adult wild-type mice. No explanation for the variability of PMR in mice has yet been found. Importantly, however, experiments by Roger Madison and colleagues have led to identification of factors that influence PMR in both mice and rats: presence or absence of end organ contact, length of the terminal branches and Schwann cell numbers in the distal nerve branches (Madison et al., 2009; Robinson and Madison, 2003, 2004, 2005, 2006). If the regenerating motor axons have access to both muscle and skin, they regrow predominantly into the motor branch; i.e. PMR occurs. However, when muscle contact is prevented by a ligature placed at the distal end of the quadriceps branch, motor axons preferentially reinnervate the inappropriate pathway to skin (Robinson and Madison, 2004). Furthermore, the preferential regrowth of motor axons into a particular terminal branch positively correlates with the length of that terminal branch and number of Schwann cells wthin it (Madison et al., 2009). These findings have led to an alternative hypothesis of PMR (Madison et al., 2007, 2009). According to this hypothesis, the motor and sensory branches of the femoral nerve do not differ in their intrinsic ability to support motor axon regeneration. Motoneuron regeneration accuracy is determined by the relative level of trophic support by the terminal pathways with muscle contact providing the most potent support, followed by number or density of Schwann cells in the distal nerve branch. It is apparent that selectivity of motor reinnervation and the underlying mechanisms remain a matter of controversy. Knowledge about molecular and cellular factors that influence the behavior of regenerating axons in a choice situation or different

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environments is of general interest and further studies on the PMR in the femoral model will certainly provide important insights into these issues.

5.2. Single-frame motion analysis

Fig. 1. Single-frame motion analysis parameters. Single video frames from recordings of beam walking showing intact animals (A,B,E,F) and animals with crush injury of the left femoral nerve at 7 days after lesion (“inj” in C,D,G,H). The lines drawn in the images show the heels–tail angle (HTA, A–D) and the foot–base angle (FBA, E–H). For further explanations see the main text. Adapted from Irintchev et al. (2005).

Until recently, an appropriate method for functional analysis after femoral nerve injury in rodents was not available. Therefore, most of the studies employing this model have been limited to use of anatomical measures of regeneration, primarily retrograde labeling of regenerated motoneurons. A few years ago, we introduced a video-based method for analysis of functional deficits after femoral nerve injury in mice designated single-frame motion analysis (SFMA, Irintchev et al., 2005). The reliability and sensitivity of this quantitative approach have been subsequently demonstrated in studies on nerve regeneration in knockout mice (Eberhardt et al., 2006; Guseva et al., 2009; Malin et al., 2009) and experiments testing novel treatment strategies (Ahlborn et al., 2007; Mehanna et al., 2009; Simova et al., 2006). The principle of the method is simple though unconventional. Rather than continuously tracing joints in video sequences to define limb segments and analyze their movements as required for kinematic analyses, measurements of limb segments’ positions are performed using single video frames in which the motor deficit is best manifested. Similar to humans, injury of the femoral nerve in mice impairs knee extension and, thus, leads to inability to bear body weight during single-support phases required for the swing of the contralateral leg during walking. To assess this deficit, the animals are video recorded during beam walking using an ordinary video camera. The beam walking test is a commonly used behavioral test in which the animal walks along a long narrow platform towards its home cage. The nerve lesion induces two characteristic changes detectable in rear-view video frames. During single-support phases of the injured limb, when the contralateral limb is at maximal swing altitude, the sole of the foot is not visible on the intact side (Fig. 1A–C) but visible on the injured side (Fig. 1D). The second aberration in gait is visible at toe-off position at which the paw of intact limbs is externally rotated in the transverse plane (Fig. 1E,F,H). On the injured side, the paw is internally rotated (Fig. 1G). These abnormalities are directly related to the impaired knee extension function. Increased body weight load in the stance phase causes abnormal bending in the knee and consequent plantar flexion in the ankle joint, lifting of the heel and

Fig. 2. Functional recovery after femoral nerve injuries in rats and mice. Shown are mean values ± SEM (standard error of mean) of heel-tail angles (A) and foot–base angles (B,C) prior to injury (day 0) and at various time points after femoral nerve crush (A,B) or transection and surgical repair (A–C) in 2-month-old female C57BL/6 mice (A,B) and 2-month-old male Wistar rats (C). The femoral nerve was repaired with a small (2-mm) gap using a polyethylene cuff in mice and by direct end-to-end suture in rats. Data are from 6–8 animals per group. Asterisks indicate significant differences compared with pre-operative value in the same group (p < 0.05, one-way analysis of variance, ANOVA, for repeated measurements with Tukey’s post hoc test). Data from Irintchev et al. (2005, A,B) and unpublished data (C).

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Fig. 3. Outcome of sciatic nerve crush in NCAM-deficient (NCAM−/− ) mice and wild-type (NCAM+/+ ) litter mates. Shown are mean values ± SEM of sciatic functional indices (A) and overall recovery indices (B) calculated from beam walking parameters (Table 1) prior to injury (day 0) and 5–90 days after injury in NCAM−/− mice (N = 7) and NCAM+/+ mice (N = 8). Indicated are significant differences between the genotypes at a given time-point (asterisks) and values at time periods longer than 30 days (dashed vertical line) significantly different from preoperative values (cross-hatches, p < 0.05, ANOVA for repeated measurements with Tukey’s post hoc test). The recovery index is an individual estimate which can be calculated for each parameter, for example, foot–base angle (Fig. 2B) as [(Y1+n − Y1 )/(Y0 − Y1 )] × 100, where Y0 , Y1 and Y1+n are values prior to operation, 1 week (5 or 7 days, see also Fig. 4) after injury, and a time-point n days after the first week, respectively. This measure estimates gain of function after the first week [(Y1+n − Y1 )] as a fraction of the functional loss [(Y0 − Y1 ) induced by the operation. Overall recovery indices are calculated as means of recovery indices for two or more parameters. A recovery index of 100% indicates complete recovery. Data from Fey et al. (2010).

internal rotation of the paw. Two parameters are used to estimate the gait abnormalities. The first one is the heels–tail angle (HTA, Fig. 1A–D) which is formed by the lines connecting the heels with a clearly discernible sagittal point on the animal’s body, the external urethral orifice in female mice. Lesion of the left femoral nerve leads to changes of the left HTA (Fig. 1D) but not of the right HTA (Fig. 1C). The second parameter is designated foot–base angle (FBA) and is measured at toe-off position at which the sole is parallel to the transverse plane (Fig. 1E–H). The angle is formed by the line symmetrically dividing the sole into two halves and the horizontal line. Functional recovery can be estimated using, in addition to the absolute angle values, normalized estimates of recovery, recovery indices. The recovery index is an individual measure normalizing “gain” of function after the first week to “loss” of function between 0 and 1 week estimated by a given parameter (see legend of Fig. 3). Overall recovery indices can be calculated, on an individual animal basis, as means of recovery indices for individual parameters. The two gait parameters, HTA and FBA, allow precise estimation of functional impairment and recovery after, for example, femoral nerve injuries of different severity. This is illustrated by data comparing the effects of nerve crush and nerve repair in adult C57BL/6 mice (Fig. 2A,B). The inter- and intra-individual variability of the measures is low. The coefficient of variation (standard deviation/mean value), which estimates the biological variability and experimental error, is below 10% (typically 3–7%) meaning that the approach has a high discriminative power. In addition to precision, the method has several other advantages. It is simple with respect to the equipment and does not require special knowledge. The only requirement is to strictly apply the criteria for selecting video frames and the definitions of the parameters. The approach relies on the performance of a task (beam walking) which is not stressful and does not require any special training or rewarding of the mice. Finally, the method is efficient as the time required for recording and analysis is reasonably short.

5.3. Generalization of the SFMA approach One of the characteristics which a valid behavioral test is expected to fulfill is generalization (Basso, 2004). This means, the test should be translatable to other species and other types and

severities of injury. The SFMA fulfills this requirement in several respects. First, it is applicable to mice (Fig. 2A,B) and rats (Fig. 2C and additional unpublished data) with femoral injuries of different severity. Second, the SFMA principles have been successfully translated to a compression spinal cord injury model in the mouse (Apostolova et al., 2006) and the SFMA for spinal cord injury has shown reproducibility and reliability (Chen et al., 2007; Chen et al., 2010; Jakovcevski et al., 2007; Lee et al., 2009; Mehanna et al., 2010). And third, the approach has been translated to the sciatic nerve crush model in mice (Fey et al., 2010). This latter assay deserves a brief mentioning considering the previous discussion on functional tests in the sciatic nerve model (Section 4). The sciatic SFMA uses a battery of measures, including the aforementioned foot–base angle, which estimate plantar flexion and toe spreading during beam walking and inclined ladder climbing (Table 1). This battery of parameters allows versatile functional analysis under different conditions including variable body weight loads and skill requirements. Analysis of C57BL/6 mice showed that different motor abilities recover completely over quite variable time periods after injury (Table 1). The overall recovery estimated using all 6 parameters reached 100% (full recovery) at 3 months after injury (Fig. 3B). In contrast, the classical walking tract analysis showed full functional restoration within 3 weeks (Fig. 3A). The footprint test was also unable to detect a deficit in functional recovery in neural cell adhesion molecule (NCAM)-deficient mice compared with wild-type mice (Fig. 3A) while the SFMA data showed a lateappearing significant deficit in the mutant mice (Fig. 3B). The SFMA approach for sciatic nerve injury appears sensitive and promising but its reproducibility and usefulness await further proof.

5.4. Functional and anatomical outcome of femoral nerve repair in mice For the first time, the SFMA has allowed for an estimation of the functional outcome after femoral nerve injury in mice. Considering the “simple” function of the quadriceps muscle which does not require high precision of motor control as well as lack of innervation by foreign motoneurons, a good recovery was expected. However, the results of several experiments performed using C57BL/6 mice showed that functional restoration is deficient reaching a max-

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Fig. 4. Effects of brief electrical stimulation (A) and HNK-1 mimetic application (B) on the time course and degree of functional recovery after femoral nerve repair in adult female C57BL/6J mice (N = 8 per group). Animals subjected to electrical stimulation (Stim) are compared with sham-stimulated (Sham) mice. The effects of the HNK-1 peptide mimetic (PEP) application are compared with application of a control peptide (CON). Shown are stance recovery indices ± SEM calculated as means from the recovery indices for the heels–tail and the foot–base angles. Asterisks indicate significant differences between the mean values at a given time point (p < 0.05, one-way ANOVA with Tukey’s post hoc test). Data from Ahlborn et al. (2007, A) and Simova et al. (2006, B).

imum of 60–70% of normal function (100%) at 3 months after injury (Fig. 4A,B, Ahlborn et al., 2007; Mehanna et al., 2009; Simova et al., 2006). Morphological analyses revealed several anatomical deficits in regeneration at 3 months after injury. The diameters of myelinated axons and the degree of axonal myelination (estimated by the g-ratio; i.e. axon-to-fiber diameter ratio) in the distal quadriceps nerve branch were, expectedly (see Section 4), reduced compared with intact nerves. Retrograde tracing showed that only one-third of the quadriceps motoneurons reinnervated the muscle branch. Approximately equal numbers of motoneurons projected incorrectly into the saphenous nerve branch and very few motoneurons reinnervated both branches of the femoral nerve; i.e. there was no PMR. The reduction in the number of regenerated motoneurons compared with the number of motoneurons originally innervating the quadriceps muscle was primarily due to motoneuron death. Control experiments revealed that more than 30% of the motoneurons labeled at the time of nerve repair died after the first post-operative week and were phagocytosed by microglial cells (Simova et al., 2006). In contrast to mice, femoral nerve repair in adult rats does not cause motoneuron loss (Xu et al., 2010). Enhanced death of axotomized motoneurons in mice as opposed to rats has also been documented in other models, e.g. after facial and hypoglossal nerve repair (Moran and Graeber, 2004; Kiryu-Seo et al., 2005) and attributed to apoptosis triggered by Fas and proceeding via the mitochondrial death pathway (Kiryu-Seo

et al., 2006; Martin et al., 2005; Ugolini et al., 2003). In the rat, anti-apoptotic signaling apparently dominates over pro-apoptotic mechanisms leading to motoneuron survival. For example, experimental evidence has suggested that the amino acid transporter EAAC1 (neuronal glutamate transporter excitatory amino-acid carrier 1) has an anti-apoptotic function and its down-regulation after nerve injury in adult mice and up-regulation in rats contribute to cell death and neuroprotection, respectively (Kiryu-Seo et al., 2006). In the context of this review, it is important to emphasize the existence of species differences in the response to axotomy, as exemplified here for cell death, which has to be considered in the design and interpretation of regeneration experiments. 5.5. Testing of therapeutic approaches The deficient functional recovery after femoral nerve repair in mice provides a good opportunity to test effects of therapeutic treatments. For example, one experiment tested the effect of brief electrical stimulation (20 Hz, 1 h) of the proximal nerve stump prior to nerve repair (Fig. 4A, Ahlborn et al., 2007). By that time, it was known that this treatment significantly shortens the period of asynchronous, staggered axonal regrowth and accelerates preferential motor reinnervation after femoral nerve lesion in rats (Al-Majed et al., 2000; Brushart et al., 2002) but the functional effects were unknown. In the mouse, the stimulation indeed

Table 1 Overview of the single-frame motion analysis parameters for sciatic nerve injury in mice (Fey et al., 2010). Parameter

Abbreviation

Step cycle phase, ipsilateral

Step cycle phase, contralateral

Body weight load

Type of detected nerve injury

Detects dysfunction in

Full recovery in C57BL/6 mice after

Beam walking Foot–base angle Lateral foot–base angle Toe-spread angle

FBA LFB TSA

Take-off Take-off Early swing

Early stance Early stance Mid-stance

Low Low No

Sciatic, tibial Sciatic, tibial Sciatic, tibial, peroneal

Plantar flexion Plantar flexion Toe spreading

21 days 21 days >50 days

Ladder climbing Ladder step angle Ladder toe spread

LSA LTS

Stance Early stance

End-swing Stance

High Low

Plantar flexion Toe spreading

>50 days >28 days

Ladder spread index

LSI

End-swing

Stance

No

Sciatic, tibial Sciatic, tibial, peroneal Sciatic, tibial, peroneal

Toe spreading

>50 days

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Table 2 Functional and anatomical outcome of femoral nerve injury in mice treated with electrical stimulation or mimetic peptides. Treatment

Functional outcome

1. Electrical stimulation 2. HNK-1 mimetic 3. PSA mimetic

= ↑ ↑

Anatomical outcome MN number

MN size

Axon number

Axon diameter

Myelination

= ↑ =

↑ ↑ =

= = =

↑ = =

↓ ↑ ↑

Summary of results from studies on mice subjected to femoral nerve repair and intra-operative: (1) brief electrical stimulation of the proximal nerve stump (1 h, 20 Hz, Ahlborn et al., 2007), (2) application of a HNK-1 mimetic peptide (Simova et al., 2006) or (3) application of a PSA mimetic peptide (Mehanna et al., 2009). The outcome at 3 months after injury is compared with appropriate control animals: ↑ – improved/increased; ↓ – impaired/decreased; = – no difference. MN number and MN size: numbers and soma areas of motoneurons labeled by application of a retrograde tracer to the quadriceps nerve branch; Axon number and diameter: total numbers and diameters of myelinated axons in the quadriceps nerve branch; Myelination: relative degree of myelination (g-ratio) of axons in the quadriceps nerve branch.

resulted in an accelerated recovery but the final outcome was not significantly improved as compared with sham-stimulated mice (Fig. 4A). It can be speculated that the limited effect of the treatment is related to failure to enhance targeting of motoneurons to the quadriceps muscle and to impairment, as compared with sham stimulation, of remyelination (Table 2). Another experiment analyzed, considering the potential role of the HNK-1 epitope in PMR (Section 5.1), the effects of a HNK-1 mimetic application to the injured nerve (Simova et al., 2006). In contrast to electrical stimulation, the mimetic did not influence the initial rate of recovery but significantly improved the final outcome (Fig. 4B) as well as the correct targeting of motoneurons and axonal myelination compared with control peptide application (Table 2). Finally, a PSA mimetic showed a functional result similar to that of the HNK-1 mimetic (Mehanna et al., 2009) but the only histologically detectable effect was improved myelination (Table 2). These results indicate that acceleration of regeneration in a small animal model does not lead to a beneficial outcome. Furthermore, the data suggest, not unexpectedly, that different anatomical aberrations contribute to restricted functional recovery.

5.6. Potential factors limiting recovery Systematic analyses of the anatomical substrates of recovery or its failure have not yet been done using the femoral nerve model. Nevertheless, the experiments performed until now point to potential limiting factors in the mouse model. One of these limitations seems to be the poor degree of myelination of the regenerated axons as indicated by a parallelism between functional outcome and g-ratio, an estimate of the degree of myelination relative to axonal thickness (Table 2). Other likely causes are motoneuron death, exhaustion of the regenerative potential of the surviving motoneurons and reinnervation of the muscle by a low number of motoneurons (Guseva et al., 2009; Simova et al., 2006). Functional regeneration may also be limited by inhibition of Schwann cell proliferation and, thus, limited trophic support of regenerating motoneurons, by molecules up-regulated in the distal nerve stump like the cell adhesion molecule L1 (Guseva et al., 2009). For further understanding of function-structure relationships, reconsideration and refinement of the traditional anatomical measures of regeneration are required. For example, the total number of axons in a regenerated nerve may be an appropriate measure in cases of poor axonal regeneration after conduit repair of a large nerve gap (Vleggeert-Lankamp, 2007). In cases of good axonal regeneration, however, this is an ambiguous estimate of several variables; i.e. numbers of different types of axons which depend on the survival and sprouting potential of different classes of motor and sensory neurons. It is, therefore, not surprising that this parameter does not correlate with functional outcome (Section 4, Table 2). A more rational approach will be to analyze properties (numbers, spatial distribution, diameters, myelination) of defined classes of axons, for example, cholinergic motor axons (Lago and Navarro, 2006). In

addition, apparently more attention should be paid to plasticity of motoneuron afferents in the spinal cord (Guseva et al., 2009), quality of muscle reinnervation (Guntinas-Lichius et al., 2005b) and, not least, CNS responses to peripheral nerve injury (Section 2). 6. Conclusions and perspectives The femoral nerve paradigm is promising for use in regeneration research. The model has already demonstrated usefulness for testing novel therapeutic means. The relative simplicity of the model and the availability of precise functional measures combined with refined anatomical outcome estimates can significantly aid the search of factors limiting functional recovery after nerve repair. The approach is applicable to mice and rats allowing, on the one hand, studies in two species which differ in responses to nerve injury and, on the other hand, taking advantage of one or other feature of the animals like larger body size of the rat or availability of genetic mouse models. References Ahlborn, P., Schachner, M., Irintchev, A., 2007. One hour electrical stimulation accelerates functional recovery after femoral nerve repair. Exp. Neurol. 208, 137–144. Al-Majed, A., Neumann, C., Brushart, T., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602–2608. Angelov, D., Ceynowa, M., Guntinas-Lichius, O., Streppel, M., Grosheva, M., Kiryakova, S., Skouras, E., Maegele, M., Irintchev, A., Neiss, W., Sinis, N., Alvanou, A., Dunlop, S., 2007. Mechanical stimulation of paralyzed vibrissal muscles following facial nerve injury in adult rat promotes full recovery of whisking. Neurobiol. Dis. 26, 229–242. Apostolova, I., Irintchev, A., Schachner, M., 2006. Tenascin-R restricts posttraumatic remodeling of motoneuron innervation and functional recovery after spinal cord injury in adult mice. J. Neurosci. 26, 7849–7859. Badke, A., Irintchev, A., Wernig, A., 1989. Maturation of transmission in reinnervated mouse soleus muscle. Muscle Nerve 12, 580–586. Basso, D.M., 2004. Behavioral testing after spinal cord injury: congruities, complexities, and controversies. J. Neurotrauma 21, 395–404. Blinzinger, K., Kreutzberg, G., 1968. Displacement of synaptic terminals from regenerating motoneurons by microglial cells. Z. Zellforsch. Mikrosk. Anat. 85, 145–157. Bozkurt, A., Deumens, R., Scheffel, J., O’Dey, D., Weis, J., Joosten, E., Führmann, T., Brook, G., Pallua, N., 2008. CatWalk gait analysis in assessment of functional recovery after sciatic nerve injury. J. Neurosci. Methods 173, 91–98. Brushart, T., 1988. Preferential reinnervation of motor nerves by regenerating motor axons. J. Neurosci. 8, 1026–1031. Brushart, T., 1993. Motor axons preferentially reinnervate motor pathways. J. Neurosci. 13, 2730–2738. Brushart, T., Hoffman, P., Royall, R., Murinson, B., Witzel, C., Gordon, T., 2002. Electrical stimulation promotes motoneuron regeneration without increasing its speed or conditioning the neuron. J. Neurosci. 22, 6631–6638. Brushart, T.M., Mesulam, M.M., 1980. Alteration in connections between muscle and anterior horn motoneurons after peripheral nerve repair. Science 208, 603–605. Brännström, T., Kellerth, J.O., 1999. Recovery of synapses in axotomized adult cat spinal motoneurons after reinnervation into muscle. Exp. Brain Res. 125, 19–27. Chen, J., Joon Lee, H., Jakovcevski, I., Shah, R., Bhagat, N., Loers, G., Liu, H.Y., Meiners, S., Taschenberger, G., Kügler, S., Irintchev, A., Schachner, M., 2010. The extracellular matrix glycoprotein tenascin-C is beneficial for spinal cord regeneration. Mol. Ther. 18, 1769–1777. Chen, J., Wu, J., Apostolova, I., Skup, M., Irintchev, A., Kügler, S., Schachner, M., 2007a. Adeno-associated virus-mediated L1 expression promotes functional recovery after spinal cord injury. Brain 130, 954–969.

284

A. Irintchev / Annals of Anatomy 193 (2011) 276–285

Chen, Z.L., Yu, W.M., Strickland, S., 2007b. Peripheral regeneration. Annu. Rev. Neurosci. 30, 209–233. Chipman, P.H., Franz, C.K., Nelson, A., Schachner, M., Rafuse, V.F., 2010. Neural cell adhesion molecule is required for stability of reinnervated neuromuscular junctions. Eur. J. Neurosci. 31, 238–249. Dahlin, L., Johansson, F., Lindwall, C., Kanje, M., 2009. Chapter 28: future perspective in peripheral nerve reconstruction. Int. Rev. Neurobiol. 87, 507–530. de Medinaceli, L., Freed, W., Wyatt, R., 1982. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp. Neurol. 77, 634–643. de Ruiter, G.C., Malessy, M.J., Alaid, A.O., Spinner, R.J., Engelstad, J.K., Sorenson, E.J., Kaufman, K.R., Dyck, P.J., Windebank, A.J., 2008. Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model. Exp. Neurol. 211, 339–350. Deumens, R., Jaken, R., Marcus, M., Joosten, E., 2007. The CatWalk gait analysis in assessment of both dynamic and static gait changes after adult rat sciatic nerve resection. J. Neurosci. Methods 164, 120–130. Eaton, M., 2003. Common animal models for spasticity and pain. J. Rehabil. Res. Dev. 40, 41–54. Eberhardt, K., Irintchev, A., Al-Majed, A., Simova, O., Brushart, T., Gordon, T., Schachner, M., 2006. BDNF/TrkB signaling regulates HNK-1 carbohydrate expression in regenerating motor nerves and promotes functional recovery after peripheral nerve repair. Exp. Neurol. 198, 500–510. Erzurumlu, R.S., Killackey, H.P., 1979. Efferent connections of the brainstem trigeminal complex with the facial nucleus of the rat. J. Comp. Neurol. 188, 75–86. Fawcett, J., 2009. Molecular control of brain plasticity and repair. Prog. Brain Res. 175, 501–509. Fey, A., Schachner, M., Irintchev, A., 2010. A novel motion analysis approach reveals late recovery in C57BL/6 mice and deficits in NCAM-deficient mice after sciatic nerve crush. J. Neurotrauma 27, 815–828. Filipe, V.M., Pereira, J.E., Costa, L.M., Maurício, A.C., Couto, P.A., Melo-Pinto, P., Varejão, A.S., 2006. Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats. J. Neurosci. Methods 153, 55–61. Franchi, G., 2000. Changes in motor representation related to facial nerve damage and regeneration in adult rats. Exp. Brain Res. 135, 53–65. Franz, C., Rutishauser, U., Rafuse, V., 2008. Intrinsic neuronal properties control selective targeting of regenerating motoneurons. Brain 131, 1492–1505. Franz, C.K., Rutishauser, U., Rafuse, V.F., 2005. Polysialylated neural cell adhesion molecule is necessary for selective targeting of regenerating motor neurons. J. Neurosci. 25, 2081–2091. Fu, S., Gordon, T., 1997. The cellular and molecular basis of peripheral nerve regeneration. Mol. Neurobiol. 14, 67–116. Galtrey, C.M., Asher, R.A., Nothias, F., Fawcett, J.W., 2007. Promoting plasticity in the spinal cord with chondroitinase improves functional recovery after peripheral nerve repair. Brain 130, 926–939. Guntinas-Lichius, O., Angelov, D., Morellini, F., Lenzen, M., Skouras, E., Schachner, M., Irintchev, A., 2005a. Opposite impacts of tenascin-C and tenascin-R deficiency in mice on the functional outcome of facial nerve repair. Eur. J. Neurosci. 22, 2171–2179. Guntinas-Lichius, O., Irintchev, A., Streppel, M., Lenzen, M., Grosheva, M., Wewetzer, K., Neiss, W., Angelov, D., 2005b. Factors limiting motor recovery after facial nerve transection in the rat: combined structural and functional analyses. Eur. J. Neurosci. 21, 391–402. Guntinas-Lichius, O., Straesser, A., Streppel, M., 2007. Quality of life after facial nerve repair. Laryngoscope 117, 421–426. Guseva, D., Angelov, D., Irintchev, A., Schachner, M., 2009. Ablation of adhesion molecule L1 in mice favours Schwann cell proliferation and functional recovery after peripheral nerve injury. Brain 132, 2180–2195. Haftel, V.K., Bichler, E.K., Wang, Q.B., Prather, J.F., Pinter, M.J., Cope, T.C., 2005. Central suppression of regenerated proprioceptive afferents. J. Neurosci. 25, 4733–4742. Hamilton, S.K., Hinkle, M.L., Nicolini, J., Rambo, L.N., Rexwinkle, A.M., Rose, S.J., Sabatier, M.J., Backus, D., English, A.W., 2011. Misdirection of regenerating axons and functional recovery following sciatic nerve injury in rats. J. Comp. Neurol. 519, 21–33. Herbison, G.J., Jaweed, M.M., Ditunno, J.F., 1981. Contractile properties of reinnervating skeletal muscle in the rat. Arch. Phys. Med. Rehabil. 62, 35–39. Hildebrand, C., Kocsis, J.D., Berglund, S., Waxman, S.G., 1985. Myelin sheath remodelling in regenerated rat sciatic nerve. Brain Res. 358, 163–170. Höke, A., 2006. Mechanisms of disease: what factors limit the success of peripheral nerve regeneration in humans? Nat. Clin. Pract. Neurol. 2, 448–454. Ijkema-Paassen, J., Meek, M.F., Gramsbergen, A., 2002. Reinnervation of muscles after transection of the sciatic nerve in adult rats. Muscle Nerve 25, 891–897. Irintchev, A., Carmody, J., Wernig, A., 1991. Effects on recovery of soleus and extensor digitorum longus muscles of prolonged wheel running during a period of repeated nerve damage. Neuroscience 44, 515–519. Irintchev, A., Draguhn, A., Wernig, A., 1990. Reinnervation and recovery of mouse soleus muscle after long-term denervation. Neuroscience 39, 231–243. Irintchev, A., Simova, O., Eberhardt, K., Morellini, F., Schachner, M., 2005. Impacts of lesion severity and tyrosine kinase receptor B deficiency on functional outcome of femoral nerve injury assessed by a novel single-frame motion analysis in mice. Eur. J. Neurosci. 22, 802–808. Isokawa-Akesson, M., Komisaruk, B.R., 1987. Difference in projections to the lateral and medial facial nucleus: anatomically separate pathways for rhythmical vibrissa movement in rats. Exp. Brain Res. 65, 385–398. Jakovcevski, I., Wu, J., Karl, N., Leshchyns’ka, I., Sytnyk, V., Chen, J., Irintchev, A., Schachner, M., 2007. Glial scar expression of CHL1, the close homolog of the

adhesion molecule L1, limits recovery after spinal cord injury. J. Neurosci. 27, 7222–7233. Jaquet, J., Luijsterburg, A., Kalmijn, S., Kuypers, P., Hofman, A., Hovius, S., 2001. Median, ulnar, and combined median-ulnar nerve injuries: functional outcome and return to productivity. J. Trauma 51, 687–692. Kanaya, F., Firrell, J.C., Breidenbach, W.C., 1996. Sciatic function index, nerve conduction tests, muscle contraction, and axon morphometry as indicators of regeneration. Plast. Reconstr. Surg. 98, 1264–1271, discussion 1272–1264. Kiryakova, S., Söhnchen, J., Grosheva, M., Schuetz, U., Marinova, T., Dzhupanova, R., Sinis, N., Hübbers, C.U., Skouras, E., Ankerne, J., Fries, J.W., Irintchev, A., Dunlop, S.A., Angelov, D.N., 2010. Recovery of whisking function promoted by manual stimulation of the vibrissal muscles after facial nerve injury requires insulin-like growth factor 1 (IGF-1). Exp. Neurol. 222, 226–234. Kiryu-Seo, S., Gamo, K., Tachibana, T., Tanaka, K., Kiyama, H., 2006. Unique antiapoptotic activity of EAAC1 in injured motor neurons. EMBO J. 25, 3411–3421. Kiryu-Seo, S., Hirayama, T., Kato, R., Kiyama, H., 2005. Noxa is a critical mediator of p53-dependent motor neuron death after nerve injury in adult mouse. J. Neurosci. 25, 1442–1447. Kleene, R., Schachner, M., 2004. Glycans and neural cell interactions. Nat. Rev. Neurosci. 5, 195–208. Kocsis, J.D., Waxman, S.G., 1983. Long-term regenerated nerve fibres retain sensitivity to potassium channel blocking agents. Nature 304, 640–642. Koerber, H.R., Seymour, A.W., Mendell, L.M., 1989. Mismatches between peripheral receptor type and central projections after peripheral nerve regeneration. Neurosci. Lett. 99, 67–72. Krarup, C., Archibald, S., Madison, R., 2002. Factors that influence peripheral nerve regeneration: an electrophysiological study of the monkey median nerve. Ann. Neurol. 51, 69–81. Kreiger, N., Kelsey, J., Harris, C., Pastides, H., 1981. Injuries to the upper extremity: patterns of occurrence. Clin. Plast. Surg. 8, 13–19. Lago, N., Navarro, X., 2006. Correlation between target reinnervation and distribution of motor axons in the injured rat sciatic nerve. J. Neurotrauma 23, 227–240. Lee, H., Jakovcevski, I., Radonjic, N., Hoelters, L., Schachner, M., Irintchev, A., 2009. Better functional outcome of compression spinal cord injury in mice is associated with enhanced H-reflex responses. Exp. Neurol. 216, 365–374. Lee, S., Wolfe, S., 2000. Peripheral nerve injury and repair. J. Am. Acad. Orthop. Surg. 8, 243–252. Lindsay, R.W., Heaton, J.T., Edwards, C., Smitson, C., Vakharia, K., Hadlock, T.A., 2010. Daily facial stimulation to improve recovery after facial nerve repair in rats. Arch. Facial Plast. Surg. 12, 180–185. Lundborg, G., 2003. Richard P. Bunge memorial lecture. Nerve injury and repair – a challenge to the plastic brain. J. Peripher. Nerv. Syst. 8, 209–226. Lundborg, G., Rosén, B., 2007. Hand function after nerve repair. Acta Physiol. (Oxf.) 189, 207–217. Madison, R., Archibald, S., Brushart, T., 1996. Reinnervation accuracy of the rat femoral nerve by motor and sensory neurons. J. Neurosci. 16, 5698–5703. Madison, R.D., Robinson, G.A., Chadaram, S.R., 2007. The specificity of motor neurone regeneration (preferential reinnervation). Acta Physiol. (Oxf.) 189, 201–206. Madison, R.D., Sofroniew, M.V., Robinson, G.A., 2009. Schwann cell influence on motor neuron regeneration accuracy. Neuroscience 163, 213–221. Malin, D., Sonnenberg-Riethmacher, E., Guseva, D., Wagener, R., Aszódi, A., Irintchev, A., Riethmacher, D., 2009. The extracellular-matrix protein matrilin 2 participates in peripheral nerve regeneration. J. Cell Sci. 122, 995–1004. Martin, L.J., Chen, K., Liu, Z., 2005. Adult motor neuron apoptosis is mediated by nitric oxide and Fas death receptor linked by DNA damage and p53 activation. J. Neurosci. 25, 6449–6459. Martini, R., Schachner, M., 1986. Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve. J. Cell Biol. 103, 2439–2448. Martini, R., Schachner, M., Brushart, T.M., 1994. The L2/HNK-1 carbohydrate is preferentially expressed by previously motor axon-associated Schwann cells in reinnervated peripheral nerves. J. Neurosci. 14, 7180–7191. Martini, R., Xin, Y., Schmitz, B., Schachner, M., 1992. The L2/HNK-1 carbohydrate epitope is involved in the preferential outgrowth of motor neurons on ventral roots and motor nerves. Eur. J. Neurosci. 4, 628–639. Martins, R.S., Siqueira, M.G., da Silva, C.F., Plese, J.P., 2006. Correlation between parameters of electrophysiological, histomorphometric and sciatic functional index evaluations after rat sciatic nerve repair. Arq. Neuropsiquiatr. 64, 750–756. McComas, A.J., 1998. Oro-facial muscles: internal structure, function and ageing. Gerodontology 15, 3–14. Mears, S., Schachner, M., Brushart, T.M., 2003. Antibodies to myelin-associated glycoprotein accelerate preferential motor reinnervation. J. Peripher. Nerv. Syst. 8, 91–99. Mehanna, A., Jakovcevski, I., Acar, A., Xiao, M., Loers, G., Rougon, G., Irintchev, A., Schachner, M., 2010. Polysialic acid glycomimetic promotes functional recovery and plasticity after spinal cord injury in mice. Mol. Ther. 18, 34–43. Mehanna, A., Mishra, B., Kurschat, N., Schulze, C., Bian, S., Loers, G., Irintchev, A., Schachner, M., 2009. Polysialic acid glycomimetics promote myelination and functional recovery after peripheral nerve injury in mice. Brain 132, 1449–1462. Mert, T., Gunay, I., Daglioglu, Y.K., 2004. Role of potassium channels in the frequencydependent activity of regenerating nerves. Pharmacology 72, 157–166. Moran, L.B., Graeber, M.B., 2004. The facial nerve axotomy model. Brain Res. Brain Res. Rev. 44, 154–178.

A. Irintchev / Annals of Anatomy 193 (2011) 276–285 Muir, D., 2010. The potentiation of peripheral nerve sheaths in regeneration and repair. Exp. Neurol. 223, 102–111. Muir, G.D., Webb, A.A., 2000. Mini-review: assessment of behavioural recovery following spinal cord injury in rats. Eur. J. Neurosci. 12, 3079–3086. Munro, C., Szalai, J., Mackinnon, S., Midha, R., 1998. Lack of association between outcome measures of nerve regeneration. Muscle Nerve 21, 1095– 1097. Navarro, X., Vivó, M., Valero-Cabré, A., 2007. Neural plasticity after peripheral nerve injury and regeneration. Prog. Neurobiol. 82, 163–201. Nguyen, Q., Kleinfeld, D., 2005. Positive feedback in a brainstem tactile sensorimotor loop. Neuron 45, 447–457. Nichols, C.M., Myckatyn, T.M., Rickman, S.R., Fox, I.K., Hadlock, T., Mackinnon, S.E., 2005. Choosing the correct functional assay: a comprehensive assessment of functional tests in the rat. Behav. Brain Res. 163, 143–158. Noble, J., Munro, C., Prasad, V., Midha, R., 1998. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J. Trauma 45, 116–122. Rice, F.L., Fundin, B.T., Arvidsson, J., Aldskogius, H., Johansson, O., 1997. Comprehensive immunofluorescence and lectin binding analysis of vibrissal follicle sinus complex innervation in the mystacial pad of the rat. J. Comp. Neurol. 385, 149–184. Robinson, G.A., Madison, R.D., 2003. Preferential motor reinnervation in the mouse: comparison of femoral nerve repair using a fibrin sealant or suture. Muscle Nerve 28, 227–231. Robinson, G.A., Madison, R.D., 2004. Motor neurons can preferentially reinnervate cutaneous pathways. Exp. Neurol. 190, 407–413. Robinson, G.A., Madison, R.D., 2005. Manipulations of the mouse femoral nerve influence the accuracy of pathway reinnervation by motor neurons. Exp. Neurol. 192, 39–45. Robinson, G.A., Madison, R.D., 2006. Developmentally regulated changes in femoral nerve regeneration in the mouse and rat. Exp. Neurol. 197, 341–346. Robinson, L., 2000. Traumatic injury to peripheral nerves. Muscle Nerve 23, 863– 873. Rutishauser, U., 2008. Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nat. Rev. Neurosci. 9, 26–35. Sanes, J.N., Suner, S., Donoghue, J.P., 1990. Dynamic organization of primary motor cortex output to target muscles in adult rats. I. Long-term patterns of reorganization following motor or mixed peripheral nerve lesions. Exp. Brain Res. 79, 479–491. Santos, P., Williams, S., Thomas, S., 1995. Neuromuscular evaluation using rat gait analysis. J. Neurosci. Methods 61, 79–84. Schiaveto de Souza, A., da Silva, C., Del Bel, E., 2004. Methodological evaluation to analyze functional recovery after sciatic nerve injury. J. Neurotrauma 21, 627–635. Scholz, T., Krichevsky, A., Sumarto, A., Jaffurs, D., Wirth, G.A., Paydar, K., Evans, G.R., 2009. Peripheral nerve injuries: an international survey of current treatments and future perspectives. J. Reconstr. Microsurg. 25, 339–344. Shenaq, J., Shenaq, S., Spira, M., 1989. Reliability of sciatic function index in assessing nerve regeneration across a 1 cm gap. Microsurgery 10, 214–219. Siemionow, M., Brzezicki, G., 2009. Chapter 8: current techniques and concepts in peripheral nerve repair. Int. Rev. Neurobiol. 87, 141–172. Simova, O., Irintchev, A., Mehanna, A., Liu, J., Dihné, M., Bächle, D., Sewald, N., Loers, G., Schachner, M., 2006. Carbohydrate mimics promote functional recovery after peripheral nerve repair. Ann. Neurol. 60, 430–437. Sinis, N., Guntinas-Lichius, O., Irintchev, A., Skouras, E., Kuerten, S., Pavlov, S., Schaller, H., Dunlop, S., Angelov, D., 2008. Manual stimulation of forearm muscles does not improve recovery of motor function after injury to a mixed peripheral nerve. Exp. Brain Res. 185, 469–483. Sumner, A., 1990. Aberrant reinnervation. Muscle Nerve 13, 801–803. Sunderland, S., 1991. Nerve Injuries and their Repair: A Critical Appraisal. Churchill Livingstone, Edinburgh, New York.

285

Svensson, M., Törnqvist, E., Aldskogius, H., Cova, J.L., 1991. Synaptic detachment from hypoglossal neurons after different types of nerve injury in the cat. J. Hirnforsch. 32, 547–552. Taylor, K.S., Anastakis, D.J., Davis, K.D., 2009. Cutting your nerve changes your brain. Brain 132, 3122–3133. Tomita, K., Kubo, T., Matsuda, K., Yano, K., Tohyama, M., Hosokawa, K., 2007. Myelinassociated glycoprotein reduces axonal branching and enhances functional recovery after sciatic nerve transection in rats. Glia 55, 1498–1507. Tomov, T., Guntinas-Lichius, O., Grosheva, M., Streppel, M., Schraermeyer, U., Neiss, W., Angelov, D., 2002. An example of neural plasticity evoked by putative behavioral demand and early use of vibrissal hairs after facial nerve transection. Exp. Neurol. 178, 207–218. Ugolini, G., Raoul, C., Ferri, A., Haenggeli, C., Yamamoto, Y., Salaün, D., Henderson, C.E., Kato, A.C., Pettmann, B., Hueber, A.O., 2003. Fas/tumor necrosis factor receptor death signaling is required for axotomy-induced death of motoneurons in vivo. J. Neurosci. 23, 8526–8531. Urbanchek, M., Chung, K., Asato, H., Washington, L., Kuzon, W.J., 1999. Rat walking tracks do not reflect maximal muscle force capacity. J. Reconstr. Microsurg. 15, 143–149. Valero-Cabré, A., Navarro, X., 2002. Functional impact of axonal misdirection after peripheral nerve injuries followed by graft or tube repair. J. Neurotrauma 19, 1475–1485. Varejão, A., Cabrita, A., Geuna, S., Melo-Pinto, P., Filipe, V., Gramsbergen, A., Meek, M., 2003a. Toe out angle: a functional index for the evaluation of sciatic nerve recovery in the rat model. Exp. Neurol. 183, 695–699. Varejão, A., Cabrita, A., Meek, M., Bulas-Cruz, J., Filipe, V., Gabriel, R., Ferreira, A., Geuna, S., Winter, D., 2003b. Ankle kinematics to evaluate functional recovery in crushed rat sciatic nerve. Muscle Nerve 27, 706–714. Varejão, A., Melo-Pinto, P., Meek, M., Filipe, V., Bulas-Cruz, J., 2004. Methods for the experimental functional assessment of rat sciatic nerve regeneration. Neurol. Res. 26, 186–194. Vleggeert-Lankamp, C.L., 2007. The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: a systematic review. Laboratory investigation. J. Neurosurg. 107, 1168–1189. Vogelaar, C.F., Vrinten, D.H., Hoekman, M.F., Brakkee, J.H., Burbach, J.P., Hamers, F.P., 2004. Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome. Brain Res. 1027, 67–72. Walker, J., Evans, J., Meade, P., Resig, P., Sisken, B., 1994. Gait-stance duration as a measure of injury and recovery in the rat sciatic nerve model. J. Neurosci. Methods 52, 47–52. Wall, P.D., Devor, M., Inbal, R., Scadding, J.W., Schonfeld, D., Seltzer, Z., Tomkiewicz, M.M., 1979. Autotomy following peripheral nerve lesions: experimental anaesthesia dolorosa. Pain 7, 103–111. Welt, C., Abbs, J.H., 1990. Musculotopic organization of the facial motor nucleus in Macaca fascicularis: a morphometric and retrograde tracing study with cholera toxin B-HRP. J. Comp. Neurol. 291, 621–636. Whitehead, J., Keller-Peck, C., Kucera, J., Tourtellotte, W.G., 2005. Glial cell-line derived neurotrophic factor-dependent fusimotor neuron survival during development. Mech. Dev. 122, 27–41. Wolthers, M., Moldovan, M., Binderup, T., Schmalbruch, H., Krarup, C., 2005. Comparative electrophysiological, functional, and histological studies of nerve lesions in rats. Microsurgery 25, 508–519. Xu, Q.G., Forden, J., Walsh, S.K., Gordon, T., Midha, R., 2010. Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat. J. Neurosurg. 112, 890–899. Yeagle, S.P., Mayer, R.F., Max, S.R., 1983. Contractile properties of rat fast-twitch skeletal muscle during reinnervation: effects of testosterone and castration. Exp. Neurol. 82, 344–357. Yu, P., Matloub, H., Sanger, J., Narini, P., 2001. Gait analysis in rats with peripheral nerve injury. Muscle Nerve 24, 231–239.