Vascular impairment as a pathological mechanism underlying long-lasting cognitive dysfunction after pediatric traumatic brain injury

Accepted Manuscript Vascular impairment as a pathological mechanism underlying long-lasting cognitive dysfunction after pediatric traumatic brain injury Ichkova Aleksandra, Beatriz Rodriguez-Grande, Claire Bar, Frederic Villega, Jan Pieter Konsman, Jerome Badaut PII:

S0197-0186(17)30181-X

DOI:

10.1016/j.neuint.2017.03.022

Reference:

NCI 4041

To appear in:

Neurochemistry International

Received Date: 20 November 2016 Revised Date:

29 March 2017

Accepted Date: 31 March 2017

Please cite this article as: Aleksandra, I., Rodriguez-Grande, B., Bar, C., Villega, F., Konsman, J.P., Badaut, J., Vascular impairment as a pathological mechanism underlying long-lasting cognitive dysfunction after pediatric traumatic brain injury, Neurochemistry International (2017), doi: 10.1016/ j.neuint.2017.03.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Vascular impairment as a pathological mechanism underlying longlasting cognitive dysfunction after pediatric traumatic brain injury

Aleksandra Ichkova1, Beatriz Rodriguez-Grande1, Claire Bar1,2, Frederic

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Villega2, Jan Pieter Konsman1 and Jerome Badaut1, 3, 4,§

CNRS UMR 5287, INCIA, University of Bordeaux, France

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Department of Pediatric Neurology, University Children’s Hospital of

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Bordeaux, France.

Department of Basic Sciences, Loma Linda University School of Medicine,

§

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Loma Linda, CA, USA

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Corresponding author:

Jerome Badaut, PhD

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CNRS UMR 5287, Bordeaux University 146 rue Léo Saignat

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33076 Bordeaux cedex, France E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Traumatic brain injury (TBI) is the leading cause of death and disability in children. Indeed, the acute mechanical injury often evolves to a chronic brain disorder with long-term cognitive, emotional and social dysfunction even in the

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case of mild TBI. Contrary to the commonly held idea that children show better recovery from injuries than adults, pediatric TBI patients actually have worse outcome than adults for the same degree of injury severity. Acute

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trauma to the young brain likely interferes with the fine-tuned developmental processes and may give rise to long-lasting consequences on brain’s function.

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This review will focus on cerebrovascular dysfunction as an important early event TBI that may lead to long-term phenotypic changes in the brain after pediatric TBI. These, in turn may be associated with accelerated brain aging and cognitive dysfunction. Finally, since there no effective treatments are

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currently available, understanding the unique pathophysiological mechanisms

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of pediatric TBI is crucial for the development of new therapeutic options.

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ACCEPTED MANUSCRIPT Highlights: •

Traumatic brain injury is the leading cause of death and disability among children and young adults Outcome after pediatric TBI is worse than after adult TBI

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Cerebrovascular dysfunction after pediatric TBI can lead to accelerated

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brain aging and cognitive dysfunction

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•

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dysfunction, neurovascular unit

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Keywords: traumatic brain injury, pediatric, development, cerebrovascular

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ACCEPTED MANUSCRIPT 1. Introduction Traumatic brain injury (TBI) occurs as the consequence of a direct or indirect mechanical force applied to the head. The mechanical force can be a rapid acceleration or deceleration, direct impact or penetration by a projectile,

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and it is followed by a post-injury altered consciousness with various severities (Maas et al., 2008). The clinical classification of TBI severity is done according to the Glasgow Coma Scale (GCS) for assessment of neurological

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function according which the injury can be considered as mild, moderate or severe.

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TBI is the leading cause of death and disability in pediatrics with infants (<2 years) and adolescents (15-18) being the most commonly affected (Thurman, 2016, Dewan, 2016). Every year around three million pediatric patients worldwide are admitted to hospitals as a result of a TBI (Dewan et al.,

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2016) but this number is likely to be an underestimation as most of the mild TBI cases are not even reported to the health services. However, regardless of whether the injury is mild or not, the initial brain injury can evolve into a

It has been estimated that at least 20% of the pediatric patients

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2015).

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chronic brain condition and reduce quality of life of patients (Babikian et al.,

hospitalized after TBI will have lifelong physical disabilities (Thurman, 2016). Even in the absence of physical disabilities, pediatric TBI patients often display cognitive impairments with persistent attention, memory and concentration deficits as well as emotional disorders such as anxiety and depression, which, in turn, greatly influence academic and work performance and cause profound social dysfunction later in life (Babikian et al., 2015; Ryan et al., 2016). It is therefore becoming clear that the costs for rehabilitation,

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ACCEPTED MANUSCRIPT hospitalization and educational support after pediatric TBI represent important financial burden for the society. Indeed, in the USA alone, around 1 billion dollars per year are charged only for hospital visits of pediatric TBI patients (Schneier et al., 2006).

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The idea that the developing brain would recover faster and better from an injury driven by the assumption that being “plastic is fantastic” needs to be revised (Giza et al., 2009; Giza and Prins, 2006). In fact, the outcome after

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pediatric TBI is even worse compared to adult TBI for the same degree of severity, of the initial injury with more profound edema formation, more severe

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dysregulation of cerebral blood flow (CBF), higher mortality risk and more behavioral sequelae (Giza and Prins, 2006; Pop and Badaut, 2011). Human brain maturation includes of synaptogenesis, synaptic pruning, myelination, reorganization of neuronal networks, changes in basal CBF and metabolism

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and continues up to the twenties (Giza et al., 2009; Toga et al., 2006). Any injury during the brain maturation has the potential to impact these finely tuned processes with long-lasting consequences on the brain’s function.

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However, little is known of the pathophysiological mechanisms underlying the

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long-term consequences of pediatric TBI. As stated above, TBI concerns an external mechanical primary injury,

which results in shear stress being exerted on the brain tissue, which may lead to hemorrhages and cell death, followed by secondary injuries. These latter include metabolic, inflammatory and vascular changes, edema formation and excitotoxicity, all of which have the potential to aggravate outcome, for example through increased cell death. Some of these secondary injuries including reduced cerebral blood flow, impaired cerebral autoregulation (CA),

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ACCEPTED MANUSCRIPT disruption of the blood brain barrier (BBB) and edema formation are hallmarks of pediatric TBI (Figure 1). These vascular changes have long-lasting consequences and have been suggested as potential mechanisms to explain long-term cognitive and emotional dysfunction (Figure 1; (Jullienne et al.,

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2016; Jullienne et al., 2014; Keightley et al., 2014; Pop and Badaut, 2011; Pop et al., 2013). Cerebral blood vessels are part of a physiological entity called the neurovascular unit (NVU) which includes glial cells, pericytes and

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neurons (Zhang et al., 2012). The present review will address the relationship between vascular function and cognition and will focus on the changes in the

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NVU after TBI and how its dysfunction can be relevant for the increased vulnerability to long-term cognitive damage after pediatric TBI. We believe that studying TBI-associated changes in the NVU are critical for a better understanding of the mechanisms underlying poor outcome after pediatric

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TBI.

Even though pediatric TBI has a high incidence rate and devastating long-term consequences, there is so far no effective treatment. Moreover, TBI

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is receiving relatively limited support in terms of basic research funding

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compared to other brain disorders such as neurodegenerative diseases. This basic research is however required to better understand the pathophysiology of pediatric TBI and develop new treatments.

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ACCEPTED MANUSCRIPT 2. Developmental aspects after traumatic brain injury: clinical studies 2.1 TBI interferes with ongoing brain maturation and cognitive development Brain development begins very early in prenatal life and continues well into

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young adulthood with significant structural changes in the gray and the white matter structure. Post-natal brain development starts with growth of short cortico-cortical connections, followed by rapid synaptogenesis and dendritic

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development, and finally by myelination and development of local circuitry. Magnetic resonance imaging (MRI), and in particular diffusion tensor imaging

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(DTI), has provided new insights into of maturation progression within the brain microstructure with a decrease of the overall diffusion of water molecules in the tissue and increase in fractional anisotropy (FA) which may reflect progressive myelination. In fact, increases in FA between structurally

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connected regions have been shown to be correlated with specific skill acquisition throughout childhood and adolescence, including reading performance, visuospatial abilities, language response inhibition, and

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memory. The corpus callosum and visual nerve tracts reach the earliest

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plateau of FA during brain development compared to “cortical-associativetracts” that are supposed to underlie the most complex executive and attention functions (Jernigan et al., 2011; Lebel et al., 2008). This maturation process is genetically programmed, but also very sensitive to environmental stimuli and experience through dynamic modifications of structural and functional neuronal networks. While this neuroplasticity is considered beneficial in healthy development, recent research work indicates that children aged <5 years are highly vulnerable to long-term deficits after TBI

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ACCEPTED MANUSCRIPT (Anderson et al., 2011). In general, pediatric TBI is frequently associated with poor outcomes including impairment of processing speed, attention, memory and executive functions with a direct relationship between the severity of the injury and the importance of the deficits (Anderson et al., 2013; Babikian and

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Asarnow, 2009; Garcia et al., 2015). Furthermore, age at the time of the injury is relevant to the severity of lasting dysfunctions. In fact, traumatic injuries early in brain development have worse outcomes than later ones because TBI

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disrupts recently established skills and also potentially interferes with the course of acquisition and consolidation of later skills (Anderson et al., 2005;

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Catroppa et al., 2008). Therefore, neuroplasticity may turn into vulnerability depending on injury-related factors and environmental influences during certain critical periods of brain development (Anderson et al., 2011). The acute injury processes after TBI may give rise to chronic disease

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symptoms with cognitive deficits emerging several years post-insult (Anderson et al., 2012; Babikian et al., 2015). For example, 10 years after mild to severe TBI, children exhibit impairment in intellectual abilities,

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particularly in processing speed, leading to long-term consequences on

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school performance (Anderson et al., 2012). Moreover, when the level of exigency increases with later grades in school and requires more complex and efficient cognitive processing abilities, the gap between these children and their healthy counterparts appears more evident (Babikian et al., 2015; Chevignard et al., 2016; Ryan et al., 2015; Ryan et al., 2016). In contrast, TBI in adulthood mostly affects the learning repertoire and acquisition of new knowledge at long-term.

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ACCEPTED MANUSCRIPT 2.2 Cerebrovascular function and cognition Brain activity is highly dependent on adequate blood supply and even a short interruption of blood flow can lead to irreversible cellular damage. Vascular dysfunction and neuronal damage are associated with cognitive

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impairments in many neurodegenerative diseases as well as in healthy aging (Iadecola, 2004; Raz et al., 2016). Cerebrovascular dysfunction is a landmark of the secondary injuries in pediatric TBI and the resulting changes in blood

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supply may have direct consequences for neuronal functioning (Jullienne et al., 2016; Pop and Badaut, 2011). Decreased CBF after TBI can prompt

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neuronal death and damage by not meeting the metabolic demands of neuronal activity. Moreover, edema formation and changes in BBB permeability can disturb the brain homeostasis and contribute to neuronal dysfunction with a cascade of secondary injury events including excitoxicity

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(glutamate release and resulting higher metabolic demand). Disruption of the microvasculature and the BBB together with neuronal injury have recently been proposed as pathological mechanisms for cognitive dysfunction after

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TBI (Wang and Li, 2016).

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Various clinical studies have shown a significant alteration of the vascular reactivity during the acute period after pediatric TBI (see below). Interestingly, studies employing transcranial doppler (TCD) ultrasonography have shown that major hyperemia but also hypoperfusion are positively correlated to poorer cognitive outcomes in children with Sickle cells disease and preterm infants, respectively (Bakker et al., 2014). Similarly, bloodoxygen level-dependent (BOLD) functional MRI studies point to some vascular alterations in pediatric populations that are at risk of cognitive

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ACCEPTED MANUSCRIPT impairments, such as preterm infants or children already presenting cognitive impairments like autistic children (Taylor et al., 2013). There is some evidence from adult TBI clinical studies that cognitive dysfunction could be associated with vascular modifications. For example, chronic traumatic encephalopathy

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dementia

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cognitive

impairments,

is

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(CTE), a disease associated with repetitive brain trauma with symptoms such characterized

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hyperphosphorylated tau deposits mainly around the small vessels in the

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cortex (McKee et al., 2015). Moreover, it seems that treatments for adult TBI that improve cerebral perfusion and decrease the intracranial pressure such

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as hypothermia lead to improved cognitive outcome (Adelson et al., 2005). All together, these clinical data support the hypothesis that cerebrovascular impairment is associated with poor cognitive outcome. Similarly, the link between cerebrovascular dysfunction and cognitive dysfunction is also more

accepted

for

several

brain

disorders

including

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neurodegenerative diseases such as Alzheimer disease. Indeed, reduced CBF and breakdown of the BBB likely precede cognitive impairments in

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Alzheimer’s disease (Bell and Zlokovic, 2009). Similarly, the long-term

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cognitive impairments after early life TBI may represent consequences of vascular dysfunction and ensuing gray and white matter damage (Figure 1, (Jullienne et al., 2016; Keightley et al., 2014; Pop and Badaut, 2011).

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ACCEPTED MANUSCRIPT 3. Cerebrovascular physiology in healthy children and development of the BBB 3.1 Cerebral blood flow and autoregulation Referent values for CBF and limits of autoregulation obtained in healthy

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children are scarce. One of the ways to measure cerebral blood flow velocity (CBFV) is by using noninvasive TCD ultrasonography. These measurements show that CBFV increases with age, peaks at 6-9 years old and decreases

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after 10 years (Udomphorn et al., 2008). Similarly, MRI arterial spin labeling (ASL) demonstrated that CBF values are highest in children aged 4-12 (97+/-

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5 mL/100 g/minute in gray matter and 26+/-1 mL/100 g/minute in white matter) to drop in adolescence (age 13-19) until they reach adult values (gray matter 58+/-4 mL/100 g/minute, white matter 20+/-1 mL/100 g/minute) around the twenties (Biagi et al., 2007). The higher CBF values in childhood are likely

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related to the higher metabolic demand of the developing brain. Cerebral vasoreactivity (CVR) is the capacity of the brain vasculature to respond by vasodilation to brain activity and can be assessed by inducing

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changes in blood CO2. CVR values are also higher in children compared to

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adults (Philip et al., 2009). A recent study with healthy subjects aged 9-30 used blood-oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) to assess changes in CVR in response to computer-controlled CO2 stimuli (Leung et al., 2016). The results of this study show that CVR increases with age in both gray and white matter until the age of 14.7 years and then decreases with age (Leung et al., 2016). Cerebral autoregulation (CA) is a crucial physiological principle by which the cerebral blood vessels dilate and constrict to maintain CBF constant under

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ACCEPTED MANUSCRIPT a range of cerebral perfusion pressure (CPP) values of 50 to 150 mm Hg. Outside of this range CBF is significantly impaired; low CPP values lead to hypoperfusion and ischemia, while high CPP values contribute to hyperemia. The few studies that have investigated cerebral autoregulation in healthy

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children (6 months – 14 years) report no differences in the lower limit of CA among the different ages and when compared to adult values (Vavilala et al., 2003). However, a study comparing autoregulatory capacity between healthy

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adults and adolescents has found that the autoregulatory index, that describes how fast the blood flow velocity returns to baseline levels after

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transiently decreasing the mean arterial pressure (MAP), is lower in adolescents (age 12-17) when compared to adults (Vavilala, 2002). Altogether, the findings indicate that properties of the brain vasculature may

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differ between children and adults during brain development.

3.2 BBB and brain development

The maintenance of BBB properties is important for brain homeostasis and

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neuronal functioning. At the level of the endothelial cells the BBB can be

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subdivided into three elemental features (Abbott et al., 2006): 1) the “physical barrier”, formed by junctional complexes of tight junction proteins (e.g. occludin, claudin-5 and zona occludens-1) between endothelial cells that prevent paracellular diffusion, thus “forcing” most substances to face the endothelial cell barrier; 2) the “transport barrier”, composed by specific transport proteins in endothelial cells that control the crossing of nutrients, ions and toxins between the blood stream and brain (Abbott et al., 2010; Abbott et al., 2006); 3) the “metabolic barrier” formed by a combination of

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ACCEPTED MANUSCRIPT intracellular and extracellular enzymes that inactivate molecules capable of penetrating cerebral endothelial cells. It is important, however, to point out that certain solutes of large molecular weight are able to cross the “physically intact” BBB by transcytosis, that is they are internalized by vesicular caveolae

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on one side of the endothelial cell, routed across the cytoplasm and finally exocytosed at the opposite site (for review De Bock et al., 2016). These structural and functional aspects of the BBB are remarkably conserved among

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species (Bundgaard and Abbott, 2008; Cserr and Bundgaard, 1984).

Maturation of the BBB starts soon after angiogenesis. Physical and

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transport barrier phenotypes are observed as early as embryonic day (E) 12 in rodents, when tight junction proteins and some influx transporters start to be expressed (Daneman et al, 2010). An important concept is that the BBB does not “switch” to a tight barrier at a specific time during angiogenesis.

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Instead, the tightness of the barrier gradually increases in a region-dependent manner while angiogenesis is progressing (Robertson et al., 1985). Interestingly, pericyte recruitment to the CNS correlates temporally with

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maturation of BBB (Daneman et al., 2010; Winkler et al., 2010, 2011).

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Endothelial cells later become covered with astrocytic end-feet that will contribute to the final maturation of the BBB and induce increased tight junction protein and specific transporter expression (Abbott et al., 2006). Paracellular diffusion diminishes during these latter stages of endothelial cellastrocytic end-feet complex formation and basement membrane development as part of perivascular maturation process. In parallel, the levels of certain transporters, e.g., monocarboxylate transporters (MCT), decline with cerebral maturity in brain and vasculature of humans and rodents (Abbott et al., 2006).

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ACCEPTED MANUSCRIPT Hormones are also important regulators of barrier maturation (Stonestreet et al., 2000). Interestingly, during the perinatal period increases in endogenous corticosteroids serve to protect the fetal and neonatal brain by reducing its own permeability (Stonestreet et al., 2000; Sysyn et al., 2001). However, in a

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more mature brain, increased glucocorticoids can result in increased BBB permeability (Stonestreet et al., 2000; Sysyn et al., 2001). Moreover, the BBB in the developing brain shows higher inward transport of glucose and amino

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acids, as well as different expression of some efflux transporters and signaling pathways (Daneman et al., 2010; Saunders et al., 2012) compared to adults

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indicating functional differences between the BBB in the developing and the adult brain. Therefore, it is critical to bear in mind that the vascular response to injury changes depending on developmental stage (Fernández-López et al., 2012). It was shown that the BBB is more vulnerable to damage in response

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to hypoxic-ischemic insult in younger (post-natal day 7) compared to older (post-natal day 21) rat pups (Muramatsu et al., 1997). Furthermore, the BBB transporter activity differed between juvenile post-natal day 21 and adult rats

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under neuroinflammatory conditions (Harati et al., 2012).

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Since BBB disruption is a major cause of mortality and long-term neurological deficits in moderate to severe TBI adult patients (Shlosberg et al., 2010) and is also present early after mild-TBI (Readnower et al., 2010), there is a need to study BBB changes in pediatric TBI at early and long term.

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ACCEPTED MANUSCRIPT 4. Cerebrovascular dysfunction after pediatric TBI 4.1 Cerebral blood flow and autoregulation Cerebrovascular dysfunction is an important hallmark of TBI and can be associated with accelerated brain aging and cognitive dysfunction (Jullienne

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et al., 2016). Contrary to the adult brain, the intensive metabolic, cellular and vascular changes render the young brain more sensitive to changes in blood supply (Obermeier et al., 2013; Toga et al., 2006). In accordance with this

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idea, an important mismatch between the metabolic demand and blood supply has been reported after pediatric TBI. Pediatric patients exhibit more profound

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reductions in CBF after TBI than adults (for review Pop and Badaut, 2011). The decrease of CBF puts the brain at risk of ischemia, which is a significant predictor of poor outcome after pediatric TBI (Adelson et al., 2011; Philip et al., 2009).

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Cerebral autoregulation (CA) is also impaired after TBI and negatively influences long-term outcome. In fact, impaired CA has been associated with unfavorable outcome up to 12 months post TBI (Nagel et al.,

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2016; Vavilala et al., 2006; Vavilala et al., 2008) with younger age at the time

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of injury being a risk factor for impaired CA (Freeman et al., 2008). Clinical studies have demonstrated impaired cerebral autoregulation in around 40% of children with moderate and severe TBI and in 17% of children with mild TBI (Vavilala et al., 2004; Vavilala et al., 2008). The loss of CA means that the regulation of CBF becomes dependent on the CPP with a low CPP resulting in low CBF thus exposing the brain to ischemia and ensuing secondary injuries. Additionally, CO2 vasoreactivty is also impaired after pediatric TBI such that changes of less than 2% are associated with poor outcome

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ACCEPTED MANUSCRIPT comparable to that observed after impairment of CA (Adelson et al., 1997; Maa et al., 2016). Reductions of CBF, impaired cerebrovascular reactivity and CA have been reported in various experimental pediatric TBI models. Decreased

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CBF and greater constrictions in pial arteries have been observed in an experimental TBI model in the pig with greater CBF and pial arteries diameter reductions occurring in the newborn (1-5 days old) compared to the juvenile

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pigs (3-4 weeks old) (Armstead and Kurth, 1994). The 1-5 days old pigs correspond to newborn to infant in human age and the juvenile pigs to a child

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5-8 years of age. Moreover, CA was also impaired in this model and again with greater extent in the newborn compared to the juvenile pig (Armstead, 1999b). Interestingly, the CBF reductions and impaired CA could be a result of increased endothelin (ET)-1 production and secretion, as high ET-1 levels

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have been observed in the cerebrospinal fluid (CSF) in both animal studies using pediatric TBI models (Armstead, 1999a; Armstead and Kreipke, 2011) and in clinical pediatric studies (Salonia et al., 2010). Moreover, the increase

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in CSF ET-1 was of greater magnitude and duration in the newborn compared

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to the juvenile pig, which can explain the greater impairment of the CA in the newborn pigs (Armstead, 1999). In accordance with these data, pre-treatment with an ET-1 antagonist partially restored the impaired CA response after TBI in the newborn and juvenile pigs and attenuated decreased CBF (Armstead, 1999a; Armstead and Raghupathi, 2011). In an experimental study using rat TBI model to compare the outcome between juvenile (post-natal day 17), preadolescent rats (post-natal day 28) and adult rats, more prolonged hypotension has been observed in the developing brain compared to the adult

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ACCEPTED MANUSCRIPT brain, which may have contributed to the increased mortality during development (Prins et al., 1996). The cerebral vasodilation to N-methyl-Daspartate receptor agonist (NMDA) in pial arteries is reversed to vasoconstriction after TBI in newborn and juvenile piglets and again with

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greater extent in the newborn (Armstead, 2004; Armstead et al., 2011a). The activation of the intracellular signaling involving the c-Jun N-terminal kinase (JNK) contributes to this impaired response (Armstead et al., 2011b).

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Interestingly, inhibition of the JNK pathway increases CBF and improves behavioral outcome in a post-natal day 17-rat model of TBI (personal

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communication, unpublished data). These findings point to both ET-1 and JNK as a potential therapeutic targets in pediatric TBI. Taken together, both clinical and experimental studies show important changes in CBF, CA and vascular reactivity after pediatric TBI that might be associated with poor

4.2 Edema

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outcome on the long-term.

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One consequence of trauma-associated damage is edema formation.

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Children develop diffuse edema more often and earlier after TBI compared to adults (Aldrich et al., 1992; Bauer and Fritz, 2004; Lang et al., 1994). This difference with the adult can be due to weaker cervical support combined with higher water content, relatively larger heads and softer skulls in younger children, which alter the biomechanics of the injury and can lead to a different type of damage, even with the same TBI parameters (Ommaya et al., 2002). Mortality in children with diffuse edema is more than twice as frequent as in children without it (Aldrich et al., 1992) and, for survivors, edema formation

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ACCEPTED MANUSCRIPT correlates with poorer long-term outcome (Feickert et al., 1999). Animal studies have shown that, within the juvenile population, post-natal day 21 mice have more edema and for a longer time than post-natal day 24 mice (Rossi, 2015). Altogether these data suggest that younger children may be

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more vulnerable to TBI than adult, in large part due to more important edema formation.

Edema formation occurs first at a cellular level, involving cell swelling

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due to ionic imbalance and osmotic gradients, and then at a vascular level, involving BBB dysfunctions (Jullienne et al., 2016; Unterberg et al., 2004). In

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addition to the post-TBI changes in transcytosis and tight junction expression discussed above, water channels are also key elements in edema formation. The expression of the water channel aquaporin 4 (AQP4), present in astrocyte end-feet, is increased 3 days after TBI in post-natal day 17 rats and

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contributes to the peak of edema formation (Fukuda et al., 2013; Fukuda et al., 2012). Later on, AQP4 also seems to be involved in edema resolution, however this has not yet been studied in detail in models of pediatric TBI

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(Badaut et al., 2014).

4.3 Blood brain barrier and perivascular changes Changes in the NVU, including BBB dysfunction as a result of a

physical “opening/leakage” of the barrier have been described during the first week after injury both in adult and pediatric TBI. The mechanisms behind the “opening/leakage” of BBB are still part of active research. At early time-points after experimental pediatric TBI, BBB opening can be visualized with stains like Evans blue or immunoglobulin G (IgG) detection

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ACCEPTED MANUSCRIPT post mortem (Badaut et al., 2015). In pediatric TBI models the BBB “opening/leakage” occurs within the first day after injury as shown by the presence of the MRI contrast agent gadolinium. This contributes to vasogenic edema formation, which may underlie increased contrast in T2-weighted MRI

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(Beaumont et al., 2006; Fukuda et al., 2012). In this case, it is associated with a decrease of claudin-5 expression, a protein involved in tight junction formation (Badaut et al., 2015).

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However, BBB “leakage” can occur early on without a “physical rupture” of the BBB or changes in claudin-5 expression. Instead, changes in

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the rate of caveolin-1-mediated trancytosis may play a role in this early BBB permeability, as shown in an adult experimental stroke model (Knowland et al., 2014). Experimental studies have shown that caveolin-1 is also involved in the stabilization of the tight junction proteins such as claudin-5 within the lipid

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raft domain and of the efflux pump P-glycoprotein (P-gp) (Jodoin et al., 2003; McCaffrey et al., 2007; McCaffrey et al., 2012). P-gp is an endothelial efflux pump known to expel several proteins from endothelial cells to the

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extracellular space, mostly into the blood compartment. In post-natal day 17

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rats, a bi-phasic increase of caveolin-1 expression in the endothelial cells at 1 and 7 days post TBI suggests that the early increase may contribute to increased transcytosis and the second to the stabilization of tight junction proteins and P-gp (Badaut et al., 2015). Until now, the exact role of caveolin-1 in brain injury and especially in pediatric TBI is still unclear (Badaut et al., 2015). Monocarboxylate transporters (MCTs), which are expressed on the endothelial cells membrane, are affected after TBI. MCT expression in the

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ACCEPTED MANUSCRIPT brain and the vasculature declines with normal cerebral maturation in humans and rodents (Abbott et al., 2006). However, MCT levels in brain lysates are increased during the first 24 hours after controlled cortical impact (CCI) in adolescent post-natal day 35 rats and post-natal day 75 rats (Prins and Giza,

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2006). Sham post-natal day 35 rats had higher baseline MCT levels than adult sham rats but both age groups had a 26-28% increase of MCT expression at 6 hours after injury and 16% increase at 24 hours after injury. MCTs are

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known to facilitate the transport of the ketone bodies (Pierre and Pellerin, 2005). In a comparable study, the potential benefit of ketogenic diet on TBI

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outcome has been tested. However, only post-natal day 35 rats had improvements in motor function and cognition and showed preserved cortical tissue after consuming a ketogenic diet that increased MCT levels after injury, whereas no therapeutic effect of the diet was observed in post-natal day 75

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rats (Appelberg et al., 2009). This overall improvement after ketogenic diet has been recently explained by the improvement of the mitochondrial respiratory complex activity after injury in post-natal day 35 rats (Greco et al.,

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2016). Interestingly, clinical studies have demonstrated that intravenous

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injection of hypertonic lactate is beneficial in recovery after TBI in adults (Bouzat et al., 2014; Quintard et al., 2016). In our studies, we showed that despite the return to normal T2 values

and the absence of IgG extravasation at 7 days after TBI in post-natal day 17 rats, long-term phenotypic changes occurred in the brain endothelium (Pop et al., 2013). These changes were associated with a sustained increase of caveolin-1 and claudin-5 expressions and decreased P-gp expression (Figure 2, 3) up to 2 months after injury (Badaut and Bix, 2014; Pop et al., 2013). As

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ACCEPTED MANUSCRIPT discussed above, changes in claudin-5 and P-gp expression could be due to the alteration of the stability of these proteins by the modification of caveolin-1 expression in endothelial cells (Badaut et al., 2015). P-gp has been proposed by several research groups to be implicated in amyloid-beta (Aβ) clearance

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from the brain tissue into the blood circulation (Cirrito et al., 2005). Interestingly, decreased levels of P-gp occur in parallel with Aβ accumulation in the brain 2 and 6 months after injury in our experimental model of pediatric

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TBI (Jullienne et al., 2014; Pop et al., 2013). We observed persistent behavioral and motor deficits up to 6 months post injury, primarily in spatial

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memory measured with Morris water maze (Kamper et al., 2013). The phenotypic changes in the brain endothelial cells were found to be associated with a decrease of capillary diameter (Figure 2) and structural changes in the perivascular matrix with an increase of perlecan and fibronectin expression at

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2 and 6 months post injury (Figure 2) (Jullienne et al., 2014). Both perlecan and fibronectin have been shown to be implicated in impaired drainage of Aβ (Figure 3, (Hawkes et al., 2013; Hawkes et al., 2014) by accelerating its

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aggregation and providing a higher stability of Aβ in the basement membrane

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(Castillo et al., 1997; Cotman et al., 2000). Altogether, these long-term vascular changes may contribute to decreased Aβ clearance and result in its accumulation of Aβ (Figure 3) due to a decrease of its clearance and in a way similar to what has been proposed to occur in Alzheimer’s disease (Jullienne et al., 2014; Pop et al., 2013). These new insights into the long-term pathophysiology of TBI may give rise to new therapeutical targets beyond the acute period in pediatric TBI patients.

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ACCEPTED MANUSCRIPT 5. Treatments Hypothermia, hyperosmolar therapy and a wide range of antiinflammatory drugs have been tested with more or less success in adult TBI after having shown beneficial effects in animal models, and most of the

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current studies that explore the mechanisms of action of these therapies are carried out in experimental models of adult TBI. However, very few therapies have actually been evaluated in pediatric TBI whether in experimental studies

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or in the clinic. Given the different characteristics of the developing brain, it is

compared to adulthood.

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likely that those same treatments would have different effects in early life

Since high intracranial pressure and edema are common complications after pediatric TBI, several therapies targeting these have been tested in experimental models. Hypothermia has been known for a long time to reduce

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edema after TBI (Jiang et al., 1992). A recent study has shown that pharmacological hypothermia, which can be achieved by targeting specific receptors or channels involved in thermoregulation, can reduce brain damage

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and improve behavior also in post-natal 14 rats (Gu et al., 2015).

The

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beneficial effect of hypothermia is thought to occur partly through a reduction of inflammation, metabolic rate and excitotoxicity (Sahuquillo and Vilalta, 2007). However, the efficacy of hypothermia in pediatric TBI remains unclear with some conflicting results among clinical studies (Zhang et al., 2015). In clinical practice, neuroprotective strategies after pediatric TBI concern essentially in monitoring of intracranial pressure to sustain CPP using hyperosmolar therapy, CSF drainage and decompressive craniotomy (Kochanek et al., 2012). While hyperosmolar therapy has moderate benefits

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ACCEPTED MANUSCRIPT (Roumeliotis et al., 2016), decompressive surgery improves outcome in survivors of severe pediatric TBI, even though it does not directly reduce edema (Figaji et al., 2003; Mhanna et al., 2015). On a molecular level, studies targeting water channels and molecular

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pathways linked to vascular dysfunction have been evaluated in juvenile animals. After TBI in post-natal day 17 rats, silencing of AQP4 reduced edema and improved motor and cognitive function (Fukuda et al., 2013) while

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inhibition of the JNK pathway increased CBF and improved behavioral outcome (personal communication, unpublished data). Moreover, the

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combination of these strategies (inhibition of JNK and silencing of AQP4) has been shown to have synergistic effects at early, but not at later, time-points (Ajao et al., 2011; Ajao et al., 2012; Fukuda et al., 2013; Margulies et al., 2016). Reduction of post-TBI vasoconstriction using an inhibitor/antagonist of

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ET-1 production/action has also given good results in neonatal and juvenile piglets as it resulted in less neurodegeneration (Kasemsri and Armstead, 1997).

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Anti-inflammatory treatments, progesterone or implantation of iPSC-

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derived neural progenitor cells have also been tested in different experimental models of pediatric TBI with most of them leading to improved outcome (Hanlon et al., 2016; Mannix et al., 2014; Robertson and Saraswati, 2015; Wei et al., 2016), however their impact on the vasculature has not been evaluated.

6. Conclusions In this review, we focused on the vascular dysfunction after pediatric TBI. From the clinical data, our experiments and those of others, we believe

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ACCEPTED MANUSCRIPT that the vascular dysfunction observed just after primary injury continues for several months and years. In addition to the primary neuronal damages, the vascular dysfunction is very likely one of the mechanisms contributing to the

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long-term cognitive dysfunction.

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Acknowledgements:

The authors wish to acknowledge the following funding: Fondation des

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gueules cassées (JB), eraNET neuron CNSaflame (JB), ANR-TRAIL (JB) and CNRS support.

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ACCEPTED MANUSCRIPT Figure Legends

Figure 1: Summary time-line of pathophysiological cascades following juvenile TBI. Preclinical and clinical data agree on a consistent description of

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the course of secondary events within days and weeks after TBI (solid lines) compared to the control level in the time (dashed center line). Behavior (purple line), from motor dysfunctions to memory, is impaired within minutes

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of most pediatric TBI injuries and partial recovery occurs in the weeks and months following injury. Certain behavior repertoire exhibits continued

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impairment years later. Potential underlying mechanisms for acute and longterm behavioral impairment may be: decrease of the cerebral blood flow, autoregulation impairment, hypometabolism (red line); increased edema and brain swelling (brown line) during the first week; increase of BBB dysfunction

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with BBB disruption as well as phenotype changes at long-term (Figure 2 and 3); associated with increased neuropathology accumulation of proteins such as Aβ (blue line and see Figure 3). Some of the changes after pediatric TBI

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the BBB permeability (green line) returns to normal after a week, however long-term BBB modifications are observed (see Figure 2 and 3). All this evidence indicates increased neuropathology long time after injury, even for mild TBI. Since changes in the NVU after TBI are not transient, there is an opportunity to develop new drugs targeting the NVU that can be useful for treating this type of injuries both in the short- and long-term. (Adapted with permission from Pop and Badaut, 2011; TBI: traumatic brain injury; BBB: blood-brain barrier; IgG: immunoglobulin G; Aβ: beta-amyloid)

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Figure 2:

Longterm neurovascular phenotypic transformation in

pediatric TBI. (A, B) Tomato-lectin (t-lectin) histochemistry stained the cerebral blood vessels showing significant morphological changes in TBI

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animals (B) compared to naive animals (A) at 2 months post-injury. Capillary diameter is significantly decreased in TBI group (B) (modified with permission from Jullienne et al., 2014). (C, D) P-gp immunostaining is specific for

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endothelial cells (green) and not present in the astrocyte end-feet of GFAPpositive astrocytes in both naive and TBI (red) P-gp expression is significantly

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decreased in TBI (D) compared to the control group (C) (modified from Pop et al., 2013). (E, F) The perivascular protein fibronectin is almost not visible in adult naive animals (E) but it was observed (red) in TBI group (F) around cerebral blood vessels 2 months after the injury (modified with permission

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from Jullienne et al., 2014).

Figure 3: Accumulation of rodent-Aβ deposition 2 months after pediatric

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TBI caused by decrease of the clearance system. (A, B) Rodent-Aβ

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staining (A) is observed in several brain regions remote from the site of the impact (B, red arrowheads). Rodent-Aβ stains a cluster of several extracellular diffuse deposits (white arrows) and vascular labeling (white arrowheads) in the frontal cortex (bregma +2mm). The distribution of the presence of Aβ is observed around the cavity after the lesion (B, black arrowheads) and at distance in frontal and occipital in the contralateral side (red arrowheads). Scale bar A=100 µm. (Modified with permission from Pop et al., 2013) (C) A schematic representation of the changes in the neurovascular

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P-gp, an efflux transporter proposed to contribute to the clearance of Aβ. In addition to the modifications of the BBB properties, the diameter of the capillaries is significantly decreased with possible consequences on the

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cerebral blood flow. The changes in the protein of the perivascular matrix fibronectin and perlecan/ domain V may also contribute to the decrease of the

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Aβ clearance. Collectively, these changes within the NVU may contribute to the decreased Aβ clearance observed after TBI and accelerate the neurodegenerative process (Schematic drawing modified and adapted with

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permission from Pop et al., 2013 and Jullienne et al., 2014).

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