Lateral Fluid Percussion Injury Research Articles

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.

Brain abscess – A rare confounding factor for diagnosis of post-traumatic epilepsy after lateral fluid-percussion injury

ObjectiveTo assess the prevalence of brain abscesses as a confounding factor for the diagnosis of post-traumatic epilepsy (PTE) in a rat model of lateral fluid-percussion-induced (FPI) traumatic brain injury (TBI). MethodsThis retrospective study included 583 rats from 3 study cohorts collected over 2009–2022 in a single laboratory. The rats had undergone sham-operation or TBI using lateral FPI. Rats were implanted with epidural and/or intracerebral electrodes for electroencephalogram recordings. Brains were processed for histology to screen for abscess(es). In abscess cases, (a) unfolded cortical maps were constructed to assess the cortical location and area of the abscess, (b) the abscess tissue was Gram stained to determine the presence of gram-positive and gram-negative bacteria, and (c) immunostaining was performed to detect infiltrating neutrophils, T-lymphocytes, and glial cells as tissue biomarkers of inflammation. In vivo and/or ex vivo magnetic resonance images available from a subcohort of animals were reviewed to evaluate the presence of abscesses. Plasma samples available from a subcohort of rats were used for enzyme-linked immunosorbent assays to determine the levels of lipopolysaccharide (LPS) as a circulating biomarker for gram-negative bacteria. ResultsBrain abscesses were detected in 2.6% (15/583) of the rats (6 sham, 9 TBI). In histology, brain abscesses were characterized as vascularized encapsulated lesions filled with neutrophils and surrounded by microglia/macrophages and astrocytes. The abscesses were mainly located under the screw electrodes, support screws, or craniectomy. Epilepsy was diagnosed in 60% (9/15) of rats with an abscess (4 sham, 5 TBI). Of these, 67% (6/9) had seizure clusters. The average seizure frequency in abscess cases was 0.436 ± 0.281 seizures/d. Plasma LPS levels were comparable between rats with and without abscesses (p > 0.05). SignificanceAlthough rare, a brain abscess is a potential confounding factor for epilepsy diagnosis in animal models of structural epilepsies following brain surgery and electrode implantation, particularly if seizures occur in sham-operated experimental controls and/or in clusters.

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

ObjectiveProject 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. MethodWe 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. ResultsThe 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. SignificanceOur 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.

Sex-Specific and Traumatic Brain Injury Effects on Dopamine Receptor Expression in the Hippocampus.

Traumatic brain injury (TBI) is a major health concern. Each year, over 50 million individuals worldwide suffer from TBI, and this leads to a number of acute and chronic health issues. These include affective and cognitive impairment, as well as an increased risk of alcohol and drug use. The dopaminergic system, a key component of reward circuitry, has been linked to alcohol and other substance use disorders, and previous research indicates that TBI can induce plasticity within this system. Understanding how TBI modifies the dopaminergic system may offer insights into the heightened substance use and reward-seeking behavior following TBI. The hippocampus, a critical component of the reward circuit, is responsible for encoding and integrating the spatial and salient aspects of rewarding stimuli. This study explored TBI-related changes in neuronal D2 receptor expression within the hippocampus, examining the hypothesis that sex differences exist in both baseline hippocampal D2 receptor expression and its response to TBI. Utilizing D2-expressing tdTomato transgenic male and female mice, we implemented either a sham injury or the lateral fluid percussion injury (FPI) model of TBI and subsequently performed a region-specific quantification of D2 expression in the hippocampus. The results show that male mice exhibit higher baseline hippocampal D2 expression compared to female mice. Additionally, there was a significant interaction effect between sex and injury on the expression of D2 in the hippocampus, particularly in regions of the dentate gyrus. Furthermore, TBI led to significant reductions in hippocampal D2 expression in male mice, while female mice remained mostly unaffected. These results suggest that hippocampal D2 expression varies between male and female mice, with the female dopaminergic system demonstrating less susceptibility to TBI-induced plasticity.

Early deficits in dentate circuit and behavioral pattern separation after concussive brain injury

Traumatic brain injury leads to cellular and circuit changes in the dentate gyrus, a gateway to hippocampal information processing. Intrinsic granule cell firing properties and strong feedback inhibition in the dentate are proposed as critical to its ability to generate unique representation of similar inputs by a process known as pattern separation. Here we evaluate the impact of brain injury on cellular decorrelation of temporally patterned inputs in slices and behavioral discrimination of spatial locations in vivo one week after concussive lateral fluid percussion injury (FPI) in mice. Despite posttraumatic increases in perforant path evoked excitatory drive to granule cells and enhanced ΔFosB labeling, indicating sustained increase in excitability, the reliability of granule cell spiking was not compromised after FPI. Although granule cells continued to effectively decorrelate output spike trains recorded in response to similar temporally patterned input sets after FPI, their ability to decorrelate highly similar input patterns was reduced. In parallel, encoding of similar spatial locations in a novel object location task that involves the dentate inhibitory circuits was impaired one week after FPI. Injury induced changes in pattern separation were accompanied by loss of somatostatin expressing inhibitory neurons in the hilus. Together, these data suggest that the early posttraumatic changes in the dentate circuit undermine dentate circuit decorrelation of temporal input patterns as well as behavioral discrimination of similar spatial locations, both of which could contribute to deficits in episodic memory.

Mild Traumatic Brain Injury Affects Orexin/Hypocretin Physiology Differently in Male and Female Mice.

Traumatic brain injury (TBI) is known to affect the physiology of neural circuits in several brain regions, which can contribute to behavioral changes after injury. Disordered sleep is a behavior that is often seen after TBI, but there is little research into how injury affects the circuitry that contributes to disrupted sleep regulation. Orexin/hypocretin neurons (hereafter referred to as orexin neurons) located in the lateral hypothalamus normally stabilize wakefulness in healthy animals and have been suggested as a source of dysregulated sleep behavior. Despite this, few studies have examined how TBI affects orexin neuron circuitry. Further, almost no animal studies of orexin neurons after TBI have included female animals. Here, we address these gaps by studying changes to orexin physiology using ex vivo acute brain slices and whole-cell patch clamp recording. We hypothesized that orexin neurons would have reduced afferent excitatory activity after injury. Ultimately, this hypothesis was supported but there were additional physiological changes that occurred that we did not originally hypothesize. We studied physiological properties in orexin neurons approximately 1 week after mild traumatic brain injury (mTBI) in 6-8-week-old male and female mice. mTBI was performed with a lateral fluid percussion injury between 1.4 and 1.6 atmospheres. Mild TBI increased the size of action potential afterhyperpolarization in orexin neurons from female mice, but not male mice and reduced the action potential threshold in male mice, but not in female mice. Mild TBI reduced afferent excitatory activity and increased afferent inhibitory activity onto orexin neurons. Alterations in afferent excitatory activity occurred in different parameters in male and female animals. The increased afferent inhibitory activity after injury is more pronounced in recordings from female animals. Our results indicate that mTBI changes the physiology of orexin neuron circuitry and that these changes are not the same in male and female animals.

Selective neuroimmune modulation by type I interferon drives neuropathology and neurologic dysfunction following traumatic brain injury.

Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/β receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Molecular alteration of reactive microglia also occurred with diminished expression of genes needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.

HDAC inhibitor attenuates rat traumatic brain injury induced neurological impairments

Oxidative stress plays an important role in the secondary neuronal damage after traumatic brain injury (TBI). Inhibition of histone deacetylases (HDACs) has been shown to reduce reactive oxygen species (ROS) production and NADPH oxidases (Nox) transcription. Vorinostat is an HDAC inhibitor. This study investigated the influence of vorinostat on neurological impairments in a rat model of TBI induced by lateral fluid percussion injury (LFPI). Different concentrations of vorinostat (5, 25, and 50 mg/kg) were administered via intraperitoneal injection. Neurological deficits were evaluated by modified neurological severity scoring (mNSS). Evans blue extravasation was performed to assess blood–brain barrier (BBB) permeability. Morris water maze assay was performed to evaluate cognitive impairments. Protein levels were evaluated through ELISA and Western blot. Vorinostat was found to attenuate TBI induced brain edema and BBB permeability in rats. Vorinostat also alleviated TBI-induced neurological impairments and anxiety-like behavior in rats. Vorinostat attenuated TBI induced apoptosis and oxidative stresses in ipsilateral injury cortical tissue. Vorinostat inhibited HDAC1, HDAC3, and Nox4 while activated AMPK signaling in ipsilateral injury cortical tissue. In conclusion, administration of vorinostat alleviates the secondary damage of TBI in rat model. The oxidative stress in the ipsilateral injury cortical tissues is decreased by the inhibition of Nox4 expression and the activation of AMPK.

Neuroprotective Effects of a Serotonin Receptor Peptide Following Sham vs. Mild Traumatic Brain Injury in the Zucker Rat.

Accelerated cognitive decline frequently complicates traumatic brain injury. Obesity and type 2 diabetes mellitus drive peripheral inflammation which may accelerate traumatic brain injury-associated neurodegeneration. The Zucker rat harbors G-protein coupled receptor agonist IgG autoantibodies and in vitro neurotoxicity caused by these autoantibodies was prevented by a novel synthetic fragment of the serotonin 2A receptor. The aim of the present study was to test whether genetic obesity manifested in Zucker diabetic fatty rat is associated with greater spatial memory impairment before and after mild traumatic brain injury compared to Zucker lean rats. Furthermore, we investigated whether these neurodegenerative complications can be lessened by administration of a novel putative neuroprotective peptide comprised of a fragment of the second extracellular loop of the serotonin 2A receptor. Age-matched lean and fatty diabetic Zucker rats were tested in the Morris water maze (spatial memory) prior to receiving a sham-injury or lateral fluid percussion (LFP) mild traumatic brain injury. Behavioral testing was repeated at 1-week, 1-month, and 3-month intervals following injury. A synthetic peptide consisting of a portion of the 5-hydroxytryptamine (serotonin) 2A receptor (2 mg/kg) (vehicle, or an inactive scrambled version of the peptide (2 mg/kg)) was administered via intraperitoneal route every other day for 7 days after sham or LFP injury to lean rats or 7 days before and after sham or LFP injury to fatty rats. Mild traumatic brain injury impaired recall of spatial memory in fatty and lean rats. Zucker fatty rats subjected to sham-injury or mild TBI experienced a significantly greater longitudinal decline in recall of spatial memory compared to lean Zucker rats. A synthetic peptide fragment of the 5-hydroxytryptamine 2A receptor significantly enhanced acquisition of spatial learning and it appeared to strengthen recall of spatial learning (one-week) after sham injury in Zucker rats. These data suggest that the Zucker diabetic fatty rat is a suitable animal model to investigate the role of metabolic factor(s) in accelerated cognitive decline. A novel synthetic peptide comprised of a fragment of the second extracellular loop of the human serotonin 2A receptor appeared to have neuroprotective effects on both acquisition and recall of spatial memory in subsets of Zucker rats, with relatively greater benefit in sham-injured, lean Zucker rats.

Optogenetic Stimulation of CA1 Pyramidal Neurons at Theta Enhances Recognition Memory in Brain Injured Animals.

Abstract The hippocampus plays a prominent role in learning and memory formation. The functional integrity of this structure is often compromised after traumatic brain injury (TBI), resulting in lasting cognitive dysfunction. The activity of hippocampal neurons, particularly place cells, is coordinated by local theta oscillations. Previous studies aimed at examining hippocampal theta oscillations after experimental TBI have reported disparate findings. Using a diffuse brain injury model, the lateral fluid percussion injury (FPI; 2.0 atm), we report a significant reduction in hippocampal theta power that persists for at least three weeks after injury. We questioned whether the behavioral deficit associated with this reduction of theta power can be overcome by optogenetically stimulating CA1 neurons at theta in brain injured rats. Our results show that memory impairments in brain injured animals could be reversed by optogenetically stimulating CA1 pyramidal neurons expressing channelrhodopsin (ChR2) during learning. In contrast, injured animals receiving a control virus (lacking ChR2) did not benefit from optostimulation. These results suggest that direct stimulation of CA1 pyramidal neurons at theta may be a viable option for enhancing memory after TBI.

Levetiracetam Pharmacokinetics and Brain Uptake in a Lateral Fluid Percussion Injury Rat Model.

Post-traumatic epilepsy (PTE) occurs in some patients after moderate/severe traumatic brain injury (TBI). Although there are no approved therapies to prevent epileptogenesis, levetiracetam (LEV) is commonly given for seizure prophylaxis due to its good safety profile. This led us to study LEV as part of the Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) Project. The objective of this work is to characterize the pharmacokinetics (PK) and brain uptake of LEV in naïve control rats and in the lateral fluid percussion injury (LFPI) rat model of TBI after either single intraperitoneal doses or a loading dose followed by a 7-day subcutaneous infusion. Sprague-Dawley rats were used as controls and for the LFPI model induced at the left parietal region using injury parameters optimized for moderate/severe TBI. Naïve and LFPI rats received either a bolus injection (intraperitoneal) or a bolus injection followed by subcutaneous infusion over 7 days. Blood and parietal cortical samples were collected at specified time points throughout the study. LEV concentrations in plasma and brain were measured using validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods. Noncompartmental analysis and a naive-pooled compartmental PK modeling approach were used. Brain-to-plasma ratios ranged from 0.54 to 1.4 to 1. LEV concentrations were well fit by one-compartment, first-order absorption PK models with a clearance of 112 ml/h per kg and volume of distribution of 293 ml/kg. The single-dose pharmacokinetic data were used to guide dose selection for the longer-term studies, and target drug exposures were confirmed. Obtaining LEV PK information early in the screening phase allowed us to guide optimal treatment protocols in EpiBioS4Rx. SIGNIFICANCE STATEMENT: The characterization of levetiracetam pharmacokinetics and brain uptake in an animal model of post-traumatic epilepsy is essential to identify target concentrations and guide optimal treatment for future studies.

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.

Mature and Myelinating Oligodendrocytes Are Specifically Vulnerable to Mild Fluid Percussion Injury in Mice.

Myelin loss and oligodendrocyte death are well documented in patients with traumatic brain injury (TBI), as well as in experimental animal models after moderate-to-severe TBI. In comparison, mild TBI (mTBI) does not necessarily result in myelin loss or oligodendrocyte death, but causes structural alterations in the myelin. To gain more insight into the impact of mTBI on oligodendrocyte lineage in the adult brain, we subjected mice to mild lateral fluid percussion injury (mFPI) and characterized the early impact (1 and 3 days post-injury) on oligodendrocytes in the corpus callosum using multiple oligodendrocyte lineage markers (platelet-derived growth factor receptor [PDGFR]-α, glutathione S-transferase [GST]-π, CC1, breast carcinoma-amplified sequence 1 [BCAS1], myelin basic protein [MBP], myelin-associated glycoprotein [MAG], proteolipid protein [PLP], and FluoroMyelin™). Two regions of the corpus callosum in relation to the impact site were analyzed: areas near (focal) and anterior (distal) to the impact site. mFPI did not result in oligodendrocyte death in either the focal or distal corpus callosum, nor impact on oligodendrocyte precursors (PDGFR-α+) and GST-π+ oligodendrocyte numbers. In the focal but not distal corpus callosum, mFPI caused a decrease in CC1+ as well as BCAS1+ actively myelinating oligodendrocytes and reduced FluoroMyelin intensity without altering myelin protein expression (MBP, PLP, and MAG). Disruption in node-paranode organization and loss of Nav1.6+ nodes were observed in both the focal and distal regions, even in areas without obvious axonal damage. Altogether, our study shows regional differences in mature and myelinating oligodendrocyte in response to mFPI. Further, mFPI elicits a widespread impact on node-paranode organization that affects regions both close to and remotely located from the site of injury.

Inherent Susceptibility to Acquired Epilepsy in Selectively Bred Rats Influences the Acute Response to Traumatic Brain Injury.

Traumatic brain injury (TBI) often causes seizures associated with a neuroinflammatory response and neurodegeneration. TBI responses may be influenced by differences between individuals at a genetic level, yet this concept remains understudied. Here, we asked whether inherent differences in one's vulnerability to acquired epilepsy would determine acute physiological and neuroinflammatory responses acutely after experimental TBI, by comparing selectively bred "seizure-prone" (FAST) rats with "seizure-resistant" (SLOW) rats, as well as control parental strains (Long Evans and Wistar rats). Eleven-week-old male rats received a moderate-to-severe lateral fluid percussion injury (LFPI) or sham surgery. Rats were assessed for acute injury indicators and neuromotor performance, and blood was serially collected. At 7 days post-injury, brains were collected for quantification of tissue atrophy by cresyl violet (CV) histology, and immunofluorescent staining of activated inflammatory cells. FAST rats showed an exacerbated physiological response acutely post-injury, with a 100% seizure rate and mortality within 24 h. Conversely, SLOW rats showed no acute seizures and a more rapid neuromotor recovery compared with controls. Brains from SLOW rats also showed only modest immunoreactivity for microglia/macrophages and astrocytes in the injured hemisphere compared with controls. Further, group differences were apparent between the control strains, with greater neuromotor deficits observed in Long Evans rats compared with Wistars post-TBI. Brain-injured Long Evans rats also showed the most pronounced inflammatory response to TBI across multiple brain regions, whereas Wistar rats showed the greatest extent of regional brain atrophy. These findings indicate that differential genetic predisposition to develop acquired epilepsy (i.e., FAST vs. SLOW rat strains) determines acute responses after experimental TBI. Differences in the neuropathological response to TBI between commonly used control rat strains is also a novel finding, and an important consideration for future study design. Our results support further investigation into whether genetic predisposition to acute seizures predicts the chronic outcomes after TBI, including the development of post-traumatic epilepsy.

Tau Phosphorylation Patterns in the Rat Cerebral Cortex After Traumatic Brain Injury and Sodium Selenate Effects: An Epibios4rx Project 2 Study.

Sodium selenate (SS) activates protein phosphatase 2 (PP2A) and reduces phosphorylated tau (pTAU) and late post-traumatic seizures after lateral fluid percussion injury (LFPI). In EpiBioS4Rx Project 2, a multi-center international study for post-traumatic targets, biomarkers, and treatments, we tested the target relevance and modification by SS of pTAU forms and PP2A and in the LFPI model, at two sites: Einstein and Melbourne. In Experiment 1, adult male rats were assigned to LFPI and sham (both sites) and naïve controls (Einstein). Motor function was monitored by neuroscores. Brains were studied with immunohistochemistry (IHC), Western blots (WBs), or PP2A activity assay, from 2 days to 8 weeks post-operatively. In Experiment 2, LFPI rats received SS for 7 days (SS0.33: 0.33 mg/kg/day; SS1: 1 mg/kg/day, subcutaneously) or vehicle (Veh) post-LFPI and pTAU, PR55 expression, or PP2A activity were studied at 2 days and 1 week (on treatment), or 2 weeks (1 week off treatment). Plasma selenium and SS levels were measured. In Experiment 1 IHC, LFPI rats had higher cortical pTAU-Ser202/Thr205-immunoreactivity (AT8-ir) and pTAU-Ser199/202-ir at 2 days, and pTAU-Thr231-ir (AT180-ir) at 2 days, 2 weeks, and 8 weeks, ipsilaterally to LFPI, than controls. LFPI-2d rats also had higher AT8/total-TAU5-ir in cortical extracts ipsilateral to the lesion (WB). PP2A (PR55-ir) showed time- and region-dependent changes in IHC, but not in WB. PP2A activity was lower in LFPI-1wk than in sham rats. In Experiment 2, SS did not affect neuroscores or cellular AT8-ir, AT180-ir, or PR55-ir in IHC. In WB, total cortical AT8/total-TAU-ir was lower in SS0.33 and SS1 LFPI rats than in Veh rats (2 days, 1 week); total cortical PR55-ir (WB) and PP2A activity were higher in SS1 than Veh rats (2 days). SS dose dependently increased plasma selenium and SS levels. Concordant across-sites data confirm time and pTAU form-specific cortical increases ipsilateral to LFPI. The discordant SS effects may either suggest SS-induced reduction in the numbers of cells with increased pTAU-ir, need for longer treatment, or the involvement of other mechanisms of action.

Proteomics of Deep Cervical Lymph Nodes After Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) damages the glymphatic-lymphatic system. We hypothesized that brain injury associated with trauma results in the enrichment of brain-relevant proteins in deep cervical lymph nodes (DCLNs), the end station of meningeal lymphatic vessels, and that some of these proteins will present mechanistic tissue biomarkers for TBI. Proteomes of rat DCLNs were investigated in the left DCLN (ipsilateral to injury) and right DCLN at 6.5 months after severe TBI induced by lateral fluid percussion injury or after sham operation. DCLN proteomes were identified using sequential window acquisition of all theoretical mass spectra. Group comparisons, together with functional protein annotation analyses, were used to identify regulated protein candidates for further validation and pathway analyses. Validation of a selected candidate was assessed using enzyme-linked immunosorbent assay. Analysis comparing post-TBI animals with sham-operated controls revealed 25 upregulated and 16 downregulated proteins in the ipsilateral DCLN and 20 upregulated and 28 downregulated proteins in the contralateral DCLN of post-TBI animals. Protein class and function analyses highlighted the dysregulation of enzymes and binding proteins. Pathway analysis indicated an increase in autophagy. Biomarker analysis suggested that a subgroup of post-TBI animals had an increase in zonula occludens-1 coexpressed with proteins linked to molecular transport and amyloid precursor protein. We propose here that, after TBI, a subgroup of animals exhibit dysregulation of the TBI-relevant protein interactome in DCLNs, and that DCLNs might thus serve as an interesting biomarker source in future studies aiming to elucidate pathological brain functioning.

Posttraumatic and Idiopathic Spike-Wave Discharges in Rats: Discrimination by Morphology and Thalamus Involvement.

The possibility of epileptiform activity generation by the thalamocortical neuronal network after focal brain injuries, including traumatic brain injury (TBI), is actively debated. Presumably, posttraumatic spike-wave discharges (SWDs) involve a cortico-thalamocortical neuronal network. Differentiation of posttraumatic and idiopathic (i.e., spontaneously generated) SWDs is imperative for understanding posttraumatic epileptogenic mechanisms. Experiments were performed on male Sprague-Dawley rats with electrodes implanted into the somatosensory cortex and the thalamic ventral posterolateral nucleus. Local field potentials were recorded for 7 days before and 7 days after TBI (lateral fluid percussion injury, 2.5 atm). The morphology of 365 SWDs (89 idiopathic before craniotomy, and 262 posttraumatic that appeared only after TBI) and their appearance in the thalamus were analyzed. The occurrence of SWDs in the thalamus determined their spike-wave form and bilateral lateralization in the neocortex. Posttraumatic discharges were characterized by more "mature" characteristics as compared to spontaneously generated discharges: higher proportions of bilateral spreading, well-defined spike-wave form, and thalamus involvement. Based on SWD parameters, the etiology could be established with an accuracy of 75% (AUC 0.79). Our results support the hypothesis that the formation of posttraumatic SWDs involves a cortico-thalamocortical neuronal network. The results form a basis for further research of mechanisms associated with posttraumatic epileptiform activity and epileptogenesis.

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

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. 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. 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). Antiseizure medications and early seizures need to be considered in the interpretation of early post-traumatic biomarkers.

Maintenance of a Lateral Fluid Percussion Injury Device.

Traumatic brain injury (TBI) accounts for roughly 2.5 million emergency room visits and hospitalizations annually and is a leading cause of death and disability in children and young adults. TBI is caused by a sudden force applied to the head and, to better understand human TBI and its underlying mechanisms, experimental injury models are necessary. Lateral fluid percussion injury (LFPI) is a commonly used injury model due to similarities in the pathological changes found in human TBI compared to LFPI, including hemorrhages, vascular disruption, neurological deficits, and neuron loss. LFPI employs a pendulum and a fluid-filled cylinder, the latter having a moveable piston at one end, and a Luer lock connection to stiff, fluid-filled tubing at the other end. Preparation of the animal involves performing a craniectomy and attaching a Luer hub over the site. The next day, the tubing from the injury device is connected to the Luer hub on the animal's skull and the pendulum is raised to a specified height and released. The impact of the pendulum with the piston generates a pressure pulse which is transmitted to the intact dura mater of the animal via the tubing and produces the experimental TBI. Proper care and maintenance are essential for the LFPI device to function reliably, as the character and severity of the injury can vary greatly depending on the condition of the device. Here, we demonstrate how to properly clean, fill, and assemble the LFPI device, and ensure that it is adequately maintained for optimal results.