Single Nucleotide Polymorphism in Cell Adhesion Molecule L1 Affects Learning and Memory in a Mouse Model of Traumatic Brain Injury.

The L1 cell adhesion molecule (L1) has demonstrated a range of beneficial effects in animal models of spinal cord injury, neurodegenerative disease, and ischemia; however, the role of L1 in TBI has not been fully examined. Mutations in the L1 gene affecting the extracellular domain of this type 1 transmembrane glycoprotein have been identified in patients with L1 syndrome. These patients suffer from hydrocephalus, MASA (mental retardation, adducted thumbs, shuffling gait, aphasia) symptoms, and corpus callosum agenesis. Clinicians have observed that recovery post-traumatic brain injury (TBI) varies among the population. This variability may be explained by the genetic differences present in the general population. In this study, we utilized a novel mouse model of L1 syndrome with a mutation at aspartic acid position 201 in the extracellular domain of L1 (L1-201). We assessed the impact of this specific single nucleotide polymorphism (SNP) localized to the X-chromosome L1 gene on recovery outcomes following TBI by comparing the L1-201 mouse mutants with their wild-type littermates. We demonstrate that male L1-201 mice exhibit significantly worse learning and memory outcomes in the Morris water maze after lateral fluid percussion (LFP) injury compared to male wild-type mice and a trend to worse motor function on the rotarod. However, no significant changes were observed in markers for inflammatory responses or apoptosis after TBI.

Lateral fluid percussion injury: A rat model of experimental traumatic brain injury.

Traumatic brain injury (TBI) represents one of the leading causes of disability and death worldwide. The annual economic impact of TBI-including direct and indirect costs-is high, particularly impacting low- and middle-income countries. Despite extensive research, a comprehensive understanding of the primary and secondary TBI pathophysiology, followed by the development of promising therapeutic approaches, remains limited. These fundamental caveats in knowledge have motivated the development of various experimental models to explore the molecular mechanisms underpinning the pathogenesis of TBI. In this context, the Lateral Fluid Percussion Injury (LFPI) model produces a brain injury that mimics most of the neurological and systemic aspects observed in human TBI. Moreover, its high reproducibility makes the LFPI model one of the most widely used rodent-based TBI models. In this chapter, we provide a detailed surgical protocol of the LFPI model used to induce TBI in adult Wistar rats. We further highlight the neuroscore test as a valuable tool for the evaluation of TBI-induced sensorimotor consequences and their severity in rats. Lastly, we briefly summarize the current knowledge on the pathological aspects and functional outcomes observed in the LFPI-induced TBI model in rodents.

Successful harmonization in EpiBioS4Rx biomarker study on post-traumatic epilepsy paves the way towards powered preclinical multicenter studies.

OBJECTIVE: Project 1 of the Preclinical Multicenter Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) consortium aims to identify preclinical biomarkers for antiepileptogenic therapies following traumatic brain injury (TBI). The international participating centers in Finland, Australia, and the United States have made a concerted effort to ensure protocol harmonization. Here, we evaluate the success of harmonization process by assessing the timing, coverage, and performance between the study sites. METHOD: We collected data on animal housing conditions, lateral fluid-percussion injury model production, postoperative care, mortality, post-TBI physiological monitoring, timing of blood sampling and quality, MR imaging timing and protocols, and duration of video-electroencephalography (EEG) follow-up using common data elements. Learning effect in harmonization was assessed by comparing procedural accuracy between the early and late stages of the project. RESULTS: The animal housing conditions were comparable between the study sites but the postoperative care procedures varied. Impact pressure, duration of apnea, righting reflex, and acute mortality differed between the study sites (p < 0.001). The severity of TBI on D2 post TBI assessed using the composite neuroscore test was similar between the sites, but recovery of acute somato-motor deficits varied (p < 0.001). A total of 99% of rats included in the final cohort in UEF, 100% in Monash, and 79% in UCLA had blood samples taken at all time points. The timing of sampling differed on day (D)2 (p < 0.05) but not D9 (p > 0.05). Plasma quality was poor in 4% of the samples in UEF, 1% in Monash and 14% in UCLA. More than 97% of the final cohort were MR imaged at all timepoints in all study sites. The timing of imaging did not differ on D2 and D9 (p > 0.05), but varied at D30, 5 months, and ex vivo timepoints (p < 0.001). The percentage of rats that completed the monthly high-density video-EEG follow-up and the duration of video-EEG recording on the 7th post-injury month used for seizure detection for diagnosis of post-traumatic epilepsy differed between the sites (p < 0.001), yet the prevalence of PTE (UEF 21%, Monash 22%, UCLA 23%) was comparable between the sites (p > 0.05). A decrease in acute mortality and increase in plasma quality across time reflected a learning effect in the TBI production and blood sampling protocols. SIGNIFICANCE: Our study is the first demonstration of the feasibility of protocol harmonization for performing powered preclinical multi-center trials for biomarker and therapy discovery of post-traumatic epilepsy.

Divergent Spatial Learning, Enhanced Neuronal Transcription, and Blood-Brain Barrier Disruption Develop During Recovery from Post-Injury Sleep Fragmentation.

Traumatic brain injury (TBI) causes pathophysiology that may significantly decrease quality of life over time. A major propagator of this response is chronic, maladaptive neuroinflammation, which can be exacerbated by stressors such as sleep fragmentation (SF). This study determined whether post-TBI SF had lasting behavioral and inflammatory effects even with a period of recovery. To test this, male and female mice received a moderate lateral fluid percussion TBI or sham surgery. Half the mice were left undisturbed, and half were exposed to daily SF for 30 days. All mice were then undisturbed between 30 and 60 days post-injury (DPI), allowing mice to recover from SF (SF-R). SF-R did not impair global Barnes maze performance. Nonetheless, TBI SF-R mice displayed retrogression in latency to reach the goal box within testing days. These nuanced behavioral changes in TBI SF-R mice were associated with enhanced expression of neuronal processing/signaling genes and indicators of blood-brain barrier (BBB) dysfunction. Aquaporin-4 (AQP4) expression, a marker of BBB integrity, was differentially altered by TBI and TBI SF-R. For example, TBI enhanced cortical AQP4 whereas TBI SF-R mice had the lowest cortical expression of perivascular AQP4, dysregulated AQP4 polarization, and the highest number of CD45+ cells in the ipsilateral cortex. Altogether, post-TBI SF caused lasting, divergent behavioral responses associated with enhanced expression of neuronal transcription and BBB disruption even after a period of recovery from SF. Understanding lasting impacts from post-TBI stressors can better inform both acute and chronic post-injury care to improve long-term outcome post-TBI.

Sleep Disturbance and Severe Hydrocephalus in a Normally Behaving Wistar Rat With Traumatic Brain Injury.

We report on a case study of a Wistar rat that was investigated in detail because it exhibited no N3 sleep in electroencephalography (EEG) after lateral fluid-percussion injury (FPI)-induced traumatic brain injury (TBI). The rat (#112) belonged to a cohort of 28 adult Wistar rats exposed to lateral FPI. Rats were monitored by continuous video EEG for 30 days to follow-up on the evolution of sleep disturbances. The beam walking test was used to measure post-TBI functional recovery. Severity of the cortical lesion area, total brain volume, and cortical volume were measured from histological brain sections. Rat #112 had a normal body and skull appearance. Its baseline body weight did not differ from that of the rest of the cohort. At baseline, rat #112 crossed the beam in 6.3 sec (score range for the rest of the cohort, 4.7-44.3) and showed no evident slipping of the paws, scoring a 5.3 (score range for the rest of cohort, 4.3-6.0). On day 30 post-TBI, however, rat #112 was the only rat with a score of 0 on the beam. Histological analysis at 30 days post-TBI revealed a small 0.6-mm2 post-TBI lesion in the somatosensory cortex (lesion size range for the rest of the cohort, 1.2-10.9). The brain volume of rat #112 was 2-fold larger than the mean volume of the rest of the cohort (1592 vs. 758 mm3), the ventricles were remarkably enlarged, and the layered cerebral cortex was very thin. Analysis of the sleep EEG revealed that rat #112 had rapid eye movement sleep and wakefulness, but no N3 sleep, during the 72-h EEG epoch analyzed. This case report demonstrates that brain abnormalities presumably unrelated to the impact-induced cortical lesion, such as presumed pre-existing hydrocephalus, may worsen TBI-induced behavioral and electrographical outcome measures and complicate the assessment of the cause of the abnormalities.

Early preclinical plasma protein biomarkers of brain trauma are influenced by early seizures and levetiracetam.

OBJECTIVE: We used the lateral fluid percussion injury (LFPI) model of moderate-to-severe traumatic brain injury (TBI) to identify early plasma biomarkers predicting injury, early post-traumatic seizures or neuromotor functional recovery (neuroscores), considering the effect of levetiracetam, which is commonly given after severe TBI. METHODS: Adult male Sprague-Dawley rats underwent left parietal LFPI, received levetiracetam (200 mg/kg bolus, 200 mg/kg/day subcutaneously for 7 days [7d]) or vehicle post-LFPI, and were continuously video-EEG recorded (n = 14/group). Sham (craniotomy only, n = 6), and naïve controls (n = 10) were also used. Neuroscores and plasma collection were done at 2d or 7d post-LFPI or equivalent timepoints in sham/naïve. Plasma protein biomarker levels were determined by reverse phase protein microarray and classified according to injury severity (LFPI vs. sham/control), levetiracetam treatment, early seizures, and 2d-to-7d neuroscore recovery, using machine learning. RESULTS: Low 2d plasma levels of Thr231 -phosphorylated tau protein (pTAU-Thr231 ) and S100B combined (ROC AUC = 0.7790) predicted prior craniotomy surgery (diagnostic biomarker). Levetiracetam-treated LFPI rats were differentiated from vehicle treated by the 2d-HMGB1, 2d-pTAU-Thr231 , and 2d-UCHL1 plasma levels combined (ROC AUC = 0.9394) (pharmacodynamic biomarker). Levetiracetam prevented the seizure effects on two biomarkers that predicted early seizures only among vehicle-treated LFPI rats: pTAU-Thr231 (ROC AUC = 1) and UCHL1 (ROC AUC = 0.8333) (prognostic biomarker of early seizures among vehicle-treated LFPI rats). Levetiracetam-resistant early seizures were predicted by high 2d-IFNγ plasma levels (ROC AUC = 0.8750) (response biomarker). 2d-to-7d neuroscore recovery was best predicted by higher 2d-S100B, lower 2d-HMGB1, and 2d-to-7d increase in HMGB1 or decrease in TNF (P < 0.05) (prognostic biomarkers). SIGNIFICANCE: Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Moderate traumatic brain injury triggers long-term risks for the development of peripheral pain sensitivity and depressive-like behavior in mice.

As traumatic brain injury (TBI) is one of the major causes of permanent disability, there is increasing interest in the long-term outcome of TBI. While motor deficits, cognitive impairment and longer-term risks of neurodegenerative disease are well-established consequences in animal models of TBI, pain is discussed less often despite its high prevalence. The current study addresses the need to characterize the extent of chronic pain and long-term behavioral impairments induced by moderate lateral fluid percussion injury (latFPI) in mice up to 12 months post-TBI and evaluates the validity of the model. Adult male BALB/c mice were subjected to latFPI, and the results were compared with outcomes in sham-operated mice. Mouse behavior was assessed at 1 and 7 days and 1, 3, 6, 9, and 12 months post-injury using sensory-motor (neurological severity score, NSS), cold (acetone) and mechanical sensitivity (von Frey), depressive-like behavior (tail suspension), locomotor (open field), motor coordination (rotarod) and cognitive (Morris water maze, y-maze, passive avoidance) tests. Animals with TBI demonstrated significantly higher NSS than the sham-operated group for up to 9 months after the injury. Cold sensitization was significantly increased in the contralateral hind paw in the TBI group compared to that of the sham group at 3, 6, and 9 months after TBI. In the von Frey test, the withdrawal threshold of the contralateral and ipsilateral hind paws was reduced at 6 months after TBI and lasted for up to 12 months post-injury. latFPI induced progressive depressive-like behavior starting at 6 months post-injury. No significant deficits were observed in memory, motor coordination or locomotion over the 12-month assessment period. The present study demonstrates that moderate TBI in mice elicits long-lasting impairment of sensory-motor function, results in progressive depression and potentiates peripheral pain. Hence, the latFPI model provides a relevant preclinical setting for the study of the link between brain injury and chronic sequelae such as depression and peripheral pain.

Seizure Susceptibility and Sleep Disturbance as Biomarkers of Epileptogenesis after Experimental TBI.

Objectives: We investigated whether seizure susceptibility increases over weeks−months after experimental traumatic brain injury (TBI), and whether seizure susceptibility in rats predicts the development of post-traumatic epilepsy (PTE) or epileptiform activity. We further investigated whether rats develop chronic sleep disturbance after TBI, and whether sleep disturbance parameters­alone or in combination with pentylenetetrazol (PTZ) test parameters­could serve as novel biomarkers for the development of post-traumatic epileptogenesis. Methods: TBI was induced in adult male Sprague-Dawley rats with lateral fluid-percussion injury. Sham-operated experimental controls underwent craniectomy without exposure to an impact force. Seizure susceptibility was tested with a PTZ test (30 mg/kg, intraperitoneally) on day (D) 30, D60, D90, and D180 after TBI (n = 28) or sham operation (n = 16) under video electroencephalogram (vEEG). In the 7th post-injury month, rats underwent continuous vEEG monitoring to detect spontaneous seizures and assess sleep disturbances. At the end of the experiments, rats were perfused for brain histology. Results: In the TBI group, the percentage of rats with PTZ-induced seizures increased over time (adjusted p < 0.05 compared with D30). Combinations of three PTZ test parameters (latency to the first epileptiform discharge (ED), number of EDs, and number of PTZ-induced seizures) survived the leave-one-out validation for differentiating rats with or without epileptiform activity, indicating an area under the receiver operating curve (AUC) of 0.743 (95% CI 0.472−0.992, p = 0.05) with a misclassification rate of 36% on D90, and an AUC of 0.752 (95% CI 0.483−0.929, p < 0.05) with a misclassification rate of 32% on D180. Sleep analysis revealed that the number of transitions to N3 or rapid eye movement (REM) sleep, along with the total number of transitions, was increased in the TBI group during the lights-on period (all p < 0.05). The sleep fragmentation index during the lights-on period was greater in the TBI rats than in sham-operated rats (p < 0.05). A combination of sleep parameters showed promise as diagnostic biomarkers of prior TBI, with an AUC of 0.792 (95% CI 0.549−0.934, p < 0.01) and a misclassification rate of 28%. Rats with epilepsy or any epileptiform activity had more transitions from N3 to the awake stage (p < 0.05), and the number of N3−awake transitions differentiated rats with or without epileptiform activity, with an AUC of 0.857 (95% CI 0.651−1.063, p < 0.01). Combining sleep parameters with PTZ parameters did not improve the biomarker performance. Significance: This is the first attempt to monitor the evolution of seizure susceptibility over months in a well-described rat model of PTE. Our data suggest that assessment of seizure susceptibility and sleep disturbance can provide diagnostic biomarkers of prior TBI and prognostic biomarkers of post-traumatic epileptogenesis.

Brain Injury Effects on Neuronal Activation and Synaptic Transmission in the Basolateral Amygdala of Adult Male and Female Wistar Rats.

Traumatic brain injury (TBI) is defined as brain damage produced by an external mechanical force that leads to behavioral, cognitive, and psychiatric sequelae. The basolateral amygdala (BLA) is involved in emotional regulation, and its function and morphology are altered following TBI. Little is known about potential sex-specific effects of TBI on BLA neuronal function, but it is critical for the field to identify potential sex differences in TBI effects on brain and behavior. Here, we hypothesized that TBI would produce sex-specific acute (1 h) effects on BLA neuronal activation, excitability, and synaptic transmission in adult male and female rats. Forty-nine Wistar rats (n = 23 males and 26 females) were randomized to TBI (using lateral fluid percussion) or Sham groups in two separate studies. Study 1 used in situ hybridization (i.e., RNAscope) to measure BLA expression of c-fos (a marker of cell activation), vGlut, and vGat (markers of glutamatergic and GABAergic neurons, respectively) messenger RNA (mRNA). Study 2 used slice electrophysiology to measure intrinsic excitability and excitatory/inhibitory synaptic transmission in putative pyramidal neurons in the BLA. Physiological measures of injury severity were collected from all animals. Our results show that females exhibit increased apnea duration and reduced respiratory rate post-TBI relative to males. In male and female rats, TBI increased c-fos expression in BLA glutamatergic cells but not in BLA GABAergic cells, and TBI increased firing rate in BLA pyramidal neurons. Further, TBI increased spontaneous excitatory and inhibitory postsynaptic current (sEPSC and sIPSC) amplitude in BLA neurons of females relative to all other groups. TBI increased sEPSC frequency in BLA neurons of females relative to males but did not alter sIPSC frequency. In summary, lateral fluid percussion produced different physiological responses in male and female rats, as well as sex-specific alterations in BLA neuronal activation, excitability, and synaptic transmission 1 h after injury.

Reduced GFAP Expression in Bergmann Glial Cells in the Cerebellum of Sigma-1 Receptor Knockout Mice Determines the Neurobehavioral Outcomes after Traumatic Brain Injury.

Neuroprotective effects of Sigma-1 receptor (S1R) ligands have been observed in multiple animal models of neurodegenerative diseases. Traumatic brain injury (TBI)-related neurodegeneration can induce long-lasting physical, cognitive, and behavioral disabilities. The aim of our study was to evaluate the role of S1R in the development of neurological deficits after TBI. Adult male wild-type CD-1 (WT) and S1R knockout (S1R-/-) mice were subjected to lateral fluid percussion injury, and behavioral and histological outcomes were assessed for up to 12 months postinjury. Neurological deficits and motor coordination impairment were less pronounced in S1R-/- mice with TBI than in WT mice with TBI 24 h after injury. TBI-induced short-term memory impairments were present in WT but not S1R-/- mice 7 months after injury. Compared to WT animals, S1R-/- mice exhibited better motor coordination and less pronounced despair behavior for up to 12 months postinjury. TBI induced astrocyte activation in the cortex of WT but not S1R-/- mice. S1R-/- mice presented a significantly reduced GFAP expression in Bergmann glial cells in the molecular layer of the cerebellum compared to WT mice. Our findings suggest that S1R deficiency reduces TBI-induced motor coordination impairments by reducing GFAP expression in Bergmann glial cells in the cerebellum.

Targeting Oxidative Stress with Antioxidant Duotherapy after Experimental Traumatic Brain Injury.

We assessed the effect of antioxidant therapy using the Food and Drug Administration-approved respiratory drug N-acetylcysteine (NAC) or sulforaphane (SFN) as monotherapies or duotherapy in vitro in neuron-BV2 microglial co-cultures and validated the results in a lateral fluid-percussion model of TBI in rats. As in vitro measures, we assessed neuronal viability by microtubule-associated-protein 2 immunostaining, neuroinflammation by monitoring tumor necrosis factor (TNF) levels, and neurotoxicity by measuring nitrite levels. In vitro, duotherapy with NAC and SFN reduced nitrite levels to 40% (p < 0.001) and neuroinflammation to -29% (p < 0.001) compared with untreated culture. The treatment also improved neuronal viability up to 72% of that in a positive control (p < 0.001). The effect of NAC was negligible, however, compared with SFN. In vivo, antioxidant duotherapy slightly improved performance in the beam walking test. Interestingly, duotherapy treatment decreased the plasma interleukin-6 and TNF levels in sham-operated controls (p < 0.05). After TBI, no treatment effect on HMGB1 or plasma cytokine levels was detected. Also, no treatment effects on the composite neuroscore or cortical lesion area were detected. The robust favorable effect of duotherapy on neuroprotection, neuroinflammation, and oxidative stress in neuron-BV2 microglial co-cultures translated to modest favorable in vivo effects in a severe TBI model.

Reorganization of Thalamic Inputs to Lesioned Cortex Following Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) disrupts thalamic and cortical integrity. The effect of post-injury reorganization and plasticity in thalamocortical pathways on the functional outcome remains unclear. We evaluated whether TBI causes structural changes in the thalamocortical axonal projection terminals in the primary somatosensory cortex (S1) that lead to hyperexcitability. TBI was induced in adult male Sprague Dawley rats with lateral fluid-percussion injury. A virus carrying the fluorescent-tagged opsin channel rhodopsin 2 transgene was injected into the ventroposterior thalamus. We then traced the thalamocortical pathways and analyzed the reorganization of their axonal terminals in S1. Next, we optogenetically stimulated the thalamocortical relays from the ventral posterior lateral and medial nuclei to assess the post-TBI functionality of the pathway. Immunohistochemical analysis revealed that TBI did not alter the spatial distribution or lamina-specific targeting of projection terminals in S1. TBI reduced the axon terminal density in the motor cortex by 44% and in S1 by 30%. A nematic tensor-based analysis revealed that in control rats, the axon terminals in layer V were orientated perpendicular to the pial surface (60.3°). In TBI rats their orientation was more parallel to the pial surface (5.43°, difference between the groups p < 0.05). Moreover, the level of anisotropy of the axon terminals was high in controls (0.063) compared with TBI rats (0.045, p < 0.05). Optical stimulation of the sensory thalamus increased alpha activity in electroencephalography by 312% in controls (p > 0.05) and 237% (p > 0.05) in TBI rats compared with the baseline. However, only TBI rats showed increased beta activity (33%) with harmonics at 5 Hz. Our findings indicate that TBI induces reorganization of thalamocortical axonal terminals in the perilesional cortex, which alters responses to thalamic stimulation.

An In Vivo Study of a Rat Fluid-Percussion-Induced Traumatic Brain Injury Model with [11C]PBR28 and [18F]flumazenil PET Imaging.

Traumatic brain injury (TBI) modelled by lateral fluid percussion-induction (LFPI) in rats is a widely used experimental rodent model to explore and understand the underlying cellular and molecular alterations in the brain caused by TBI in humans. Current improvements in imaging with positron emission tomography (PET) have made it possible to map certain features of TBI-induced cellular and molecular changes equally in humans and animals. The PET imaging technique is an apt supplement to nanotheranostic-based treatment alternatives that are emerging to tackle TBI. The present study aims to investigate whether the two radioligands, [11C]PBR28 and [18F]flumazenil, are able to accurately quantify in vivo molecular-cellular changes in a rodent TBI-model for two different biochemical targets of the processes. In addition, it serves to observe any palpable variations associated with primary and secondary injury sites, and in the affected versus the contralateral hemispheres. As [11C]PBR28 is a radioligand of the 18 kD translocator protein, the up-regulation of which is coupled to the level of neuroinflammation in the brain, and [18F]flumazenil is a radioligand for GABAA-benzodiazepine receptors, whose level mirrors interneuronal activity and eventually cell death, the use of the two radioligands may reveal two critical features of TBI. An up-regulation in the [11C]PBR28 uptake triggered by the LFP in the injured (right) hemisphere was noted on day 14, while the uptake of [18F]flumazenil was down-regulated on day 14. When comparing the left (contralateral) and right (LFPI) hemispheres, the differences between the two in neuroinflammation were obvious. Our results demonstrate a potential way to measure the molecular alterations in a rodent-based TBI model using PET imaging with [11C]PBR28 and [18F]flumazenil. These radioligands are promising options that can be eventually used in exploring the complex in vivo pharmacokinetics and delivery mechanisms of nanoparticles in TBI treatment.

Plau/Plaur double-deficiency did not worsen lesion severity or vascular integrity after traumatic brain injury.

Binding of urokinase-type plasminogen activator receptor (uPAR) to its ligand uPA or to its plasma membrane partner, platelet-derived growth factor receptor ß (PDGFRß), promotes neuroprotection, cell proliferation, and angiogenesis. Following injury, single deficiency in uPA or uPAR leads in increased tissue loss and compromised vascular remodeling. We hypothesized that double-deficiency of uPAR (Plaur) and uPA (Plau) would result in increased lesion area and poor vascular integrity after traumatic brain injury (TBI). TBI was induced by lateral fluid-percussion injury in Plau/Plaur double-knockout (dKO) and wild-type (Wt) mice. The cortical lesion area was quantified in unfolded cortical maps prepared from thionin-stained sections at 4 d or 30 d post-TBI. The density of PDGFRß+ pericytes and blood vessels was calculated from immunostained sections. Blood-brain barrier leakage was analyzed using ImageJ® from IgG-immunostained sections. Genotype had no effect on the total area of the cortical lesion at 4 d or 30 d post-TBI (p > 0.05) or its progression as the overall lesion area was comparable at 4 d and 30 d post-TBI in both genotypes (p > 0.05). Subfield analysis, however, indicated that damage to the visual cortex at 4 d post-TBI in dKO-TBI mice was 53 % of that in Wt-TBI mice (p < 0.05). Both genotypes had a higher density of PDGFRß-positive pericytes at 4 d than at 30 d post-TBI (p < 0.05), but no genotype effect was detected between these time-points (p > 0.05). TBI-induced increase in the density of PDGFRß+ blood vessels at the region adjacent to the lesion core was comparable in both genotypes (p > 0.05). Genotype had no effect on TBI-induced IgG leakage into the perilesional cortical parenchyma (p > 0.05). Contrary to our expectations, Plau/Plaur double-deficiency did not aggravate TBI-related structural outcome.

Early Increase in Cortical T2 Relaxation Is a Prognostic Biomarker for the Evolution of Severe Cortical Damage, but Not for Epileptogenesis, after Experimental Traumatic Brain Injury.

Prognostic biomarkers for post-injury outcome are necessary for the development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI). We hypothesized that T2 relaxation magnetic resonance imaging (MRI) predicts the progression of perilesional cortical pathology and epileptogenesis. The EPITARGET animal cohort used for MRI analysis included 120 adult male Sprague-Dawley rats with TBI induced by lateral fluid-percussion injury and 24 sham-operated controls. T2 MRI was performed at days 2, 7, and 21 post-TBI. The lesioned cortex was outlined, and the T2 value of each imaging voxel within the lesion area was scored using a five-grade pathology classification. Analysis of 1-month video-electroencephalography recordings initiated 5 months post-TBI indicated that 27% (31 of 114) of the animals with TBI developed epilepsy. Multiple linear regression analysis indicated that T2-based classification of lesion volume at day 2 and day 7 post-TBI explained the necrotic lesion volume with greatly increased T2 (>102 ms) at day 21 post-TBI (F(13,103) = 52.5; p < 0.001; R2 = 0.87; adjusted R2 = 0.85). The volume of moderately increased (78-102 ms) T2 at day 7 post-TBI predicted the evolution of large (>12 mm3) cortical lesions (area under the curve, 0.92; p < 0.001; cutoff, 1.9 mm3; false positive rate, 0.10; true positive rate, 0.62). Logistic regression analysis, however, showed that the different severities of T2 lesion volumes at days 2, 7, and 21 post-TBI did not explain the development of epilepsy (χ2(18,95) = 18.4; p = 0.427). In addition, the location of the T2 abnormality within the cortex did not correlate with epileptogenesis. A single measurement of T2 relaxation MRI in the acute post-TBI phase is useful for identifying post-TBI subjects at highest risk of developing large cortical lesions, and thus, in the greatest need of neuroprotective therapies after TBI, but not the development of post-traumatic epilepsy.

Acute Downregulation of Novel Hypothalamic Protein Sushi Repeat-Containing Protein X-Linked 2 after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) causes damage to the hypothalamo-hypophyseal axis, leading to endocrine dysregulation in up to 40% of TBI patients. Hence, there is an urgent need to identify non-invasive biomarkers for TBI-associated hypothalamo-hypophyseal pathology. Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel hypothalamic protein expressed in both rat and human brain. Our objective was to investigate the effect of acquired brain injury on plasma SRPX2 protein levels and SRPX2 expression in the brain. We induced severe lateral fluid-percussion injury in adult male rats and investigated changes in SRPX2 expression at 2 h, 6 h, 24 h, 48 h, 72 h, 5 days, 7 days, 14 days, 1 month, and 3 months post-injury. The plasma SRPX2 level was assessed by Western blot analysis. Hypothalamic SRPX2-immunoreactive neuronal numbers were estimated from immunostained preparations. At 2 h post-TBI, plasma SRPX2 levels were markedly decreased compared with the naïve group (area under the curve = 1.00, p < 0.05). Severe TBI caused a reduction in the number of hypothalamic SRPX2-immunoreactive neurons bilaterally at 2 h post-TBI compared with naïve group (5032 ± 527 vs. 9440 ± 351, p < 0.05). At 1 month after severe TBI, however, the brain and plasma SRPX2 levels were comparable between the TBI and naïve groups (p > 0.05). Unsupervised hierarchical clustering using SRPX2 expression differentiated animals into injured and uninjured clusters. Our findings indicate that TBI leads to an acute reduction in SRPX2 protein expression and reduced plasma SRPX2 level may serve as a candidate biomarker of hypothalamic injury.

BDNF Val66Met Genetic Polymorphism Results in Poor Recovery Following Repeated Mild Traumatic Brain Injury in a Mouse Model and Treatment With AAV-BDNF Improves Outcomes.

Clinicians have long noticed that some Traumatic Brain Injury (TBI) patients have worse symptoms and take a longer time to recover than others, for reasons unexplained by known factors. Identifying what makes some individuals more susceptible is critical to understanding the underlying mechanisms through which TBI causes deleterious effects. We have sought to determine the effect of a single nucleotide polymorphism (SNP) in Brain-derived neurotrophic factor (BDNF) at amino acid 66 (rs6265) on recovery after TBI. There is controversy from human studies as to whether the BDNF Val66Val or Val66Met allele is the risk factor for worse outcomes after brain trauma. We therefore investigated cellular and behavioral outcomes in genetically engineered mice following repeated mild TBI (rmTBI) using a lateral fluid percussion (LFP) injury model. We found that relative to injured Val66Val carriers, injured Val66Met carriers had a larger inflammation volume and increased levels of neurodegeneration, apoptosis, p-tau, activated microglia, and gliosis in the cortex and/or hippocampus at 1 and/or 21 days post-injury (DPI). We therefore concluded that the Val66Met genetic polymorphism is a risk factor for poor outcomes after rmTBI. In order to determine the mechanism for these differences, we investigated levels of the apoptotic-inducing pro BDNF and survival-inducing mature BDNF isoforms and found that Met carriers had less total BDNF in the cortex and a higher pro/mature ratio of BDNF in the hippocampus. We then developed a personalized approach to treating genetically susceptible individuals by overexpressing wildtype BDNF in injured Val66Met mice using an AAV-BDNF virus. This intervention improved cellular, motor, and cognitive behavior outcomes at 21 DPI and increased levels of mature BDNF and phosphorylation of mature BDNF's receptor trkB. This study lays the groundwork for further investigation into the genetics that play a role in the extent of injury after rmTBI and highlights how personalized therapeutics may be targeted for recovery in susceptible individuals.

Pioglitazone improves working memory performance when administered in chronic TBI.

Traumatic brain injury (TBI) is a leading cause of long-term disability in the United States. Even in comparatively mild injuries, cognitive and behavioral symptoms can persist for years, and there are currently no established strategies for mitigating symptoms in chronic injury. A key feature of TBI-induced damage in acute and chronic injury is disruption of metabolic pathways. As neurotransmission, and therefore cognition, are highly dependent on the supply of energy, we hypothesized that modulating metabolic activity could help restore behavioral performance even when treatment was initiated weeks after TBI. We treated rats with pioglitazone, a FDA-approved drug for diabetes, beginning 46 days after lateral fluid percussion injury and tested working memory performance in the radial arm maze (RAM) after 14 days of treatment. Pioglitazone treated TBI rats performed significantly better in the RAM test than untreated TBI rats, and similarly to control animals. While hexokinase activity in hippocampus was increased by pioglitazone treatment, there was no upregulation of either the neuronal glucose transporter or hexokinase enzyme expression. Expression of glial markers GFAP and Iba-1 were also not influenced by pioglitazone treatment. These studies suggest that targeting brain metabolism, in particular hippocampal metabolism, may be effective in alleviating cognitive symptoms in chronic TBI.

Inhibition of EphA/Ephrin-A signaling using genetic and pharmacologic approaches improves recovery following traumatic brain injury in mice.

Primary Objective: Eph/Ephrin signaling is inhibitory for developing axons and blocking Eph pathways enhances regeneration after spinal cord injury. It was hypothesized that inhibition of Eph signaling promotes cellular and behavioral recovery after traumatic brain injury (TBI). Research design: Lateral fluid percussion (LFP) injury was performed on wildtype (WT) and EphA6 knockout (KO) mice. EphA6-Fc, Ephrin-A5-Fc fusion proteins, and sodium orthovanadate were used to alter the signaling pathway. Immunohistochemistry and tissue explants revealed cellular changes. Rotarod tests demonstrated vestibulomotor function. Outcomes: The EphA6 receptor expression is upregulated following LFP. Uninjured EphA6 KO mice exhibit greater neurite density and clustered Ephrin-A5-Fc causes growth cone collapse in vitro. After LFP, EphA6 KO mice demonstrate longer neurites and decreased neuronal cell death and astrocytosis compared to WT mice. Blocking EphA signaling by soluble EphA6-Fc fusion protein reduces cell death and improves motor function following LFP whereas clustered Ephrin-A5-Fc exacerbates cell death and neurodegeneration. Sodium orthovanadate rescues growth cone collapse in vitro as well as cell death and neurodegeneration in vivo. Conclusions: Eph/Ephrin signaling plays an inhibitory role following TBI. Targeting the Eph signaling pathway with Fc fusion proteins and pharmacological agents can be a novel strategy to counter the damaging effects of TBI. Abbreviations: LFP: lateral fluid percussion; TBI: traumatic brain injury; KO: knockout; WT: wildtype; PTP2: protein phosphotyrosine phosphatase 2; Tg: transgenic; YFP: yellow fluorescent protein; ATM: atmospheres; RT-qPCR: Real-time-quantitative PCR; dpi: days post injury; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4',6-diamidino-2-phenylindole; PBS: phosphate buffered saline; GFAP: glial fibrillary acidic protein; FLJC: fluorojade C; CA: cornu ammonis; SEM: standard error of the mean; ANOVA: analysis of variance; PLSD: posthoc least significant difference.