Lateral Fluid Percussion Model Research Articles

Sleep fragmentation after traumatic brain injury impairs behavior and conveys long-lasting impacts on neuroinflammation

Traumatic brain injury (TBI) causes a prolonged inflammatory response in the central nervous system (CNS) driven by microglia. Microglial reactivity is exacerbated by stress, which often provokes sleep disturbances. We have previously shown that sleep fragmentation (SF) stress after experimental TBI increases microglial reactivity and impairs hippocampal function 30 days post-injury (DPI). The neuroimmune response is highly dynamic the first few weeks after TBI, which is also when injury induced sleep-wake deficits are detected. Therefore, we hypothesized that even a few weeks of TBI SF stress would synergize with injury induced sleep-wake deficits to promote neuroinflammation and impair outcome. Here, we investigated the effects of environmental SF in a lateral fluid percussion model of mouse TBI. Half of the mice were undisturbed, and half were exposed to 5 h of SF around the onset of the light cycle, daily, for 14 days. All mice were then undisturbed 15–30 DPI, providing a period for SF stress recovery (SF-R). Mice exposed to SF stress slept more than those in control housing 7–14 DPI and engaged in more total daily sleep bouts during the dark period. However, SF stress did not exacerbate post-TBI sleep deficits. Testing in the Morris water maze revealed sex dependent differences in spatial reference memory 9–14 DPI with males performing worse than females. Post-TBI SF stress suppressed neurogenesis-related gene expression and increased inflammatory signaling in the cortex at 14 DPI. No differences in sleep behavior were detected between groups during the SF stress recovery period 15–30 DPI. Microscopy revealed cortical and hippocampal IBA1 and CD68 percent-area increased in TBI SF-R mice 30 DPI. Additionally, neuroinflammatory gene expression was increased, and synaptogenesis-related gene expression was suppressed in TBI-SF mice 30 DPI. Finally, IPA canonical pathway analysis showed post-TBI SF impaired and delayed activation of synapse-related pathways between 14 and 30 DPI. These data show that transient SF stress after TBI impairs recovery and conveys long-lasting impacts on neuroimmune function independent of continuous sleep deficits. Together, these finding support that even limited exposure to post-TBI SF stress can have lasting impacts on cognitive recovery and regulation of the immune response to trauma.

Designer Receptor Exclusively Activated by Designer Drug (DREADD)-Mediated Activation of the Periaqueductal Gray Restores Nociceptive Descending Inhibition After Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) patients frequently experience chronic pain that can enhance their suffering and significantly impair rehabilitative efforts. Clinical studies suggest that damage to the periaqueductal gray matter (PAG) following TBI, a principal center involved in endogenous pain control, may underlie the development of chronic pain. We hypothesized that TBI would diminish the usual pain control functions of the PAG, but that directly stimulating this center using a chemogenetic approach would restore descending pain modulation. We used a well-characterized lateral fluid percussion model (1.3 ± 0.1 atm) of TBI in male rats (n = 271) and measured hindpaw mechanical nociceptive withdrawal thresholds using von Frey filaments. To investigate the role of the PAG in pain both before and after TBI, we activated the neurons of the PAG using a Designer Receptor Exclusively Activated by Designer Drug (DREADD) viral construct. Immunohistochemical analysis of brain tissue was used to assess the location and confirm the appropriate expression of the viral constructs in the PAG. Activation of the PAG DREADD using clozapine N-oxide (CNO) caused hindpaw analgesia that could be blocked using opioid receptor antagonist, naloxone, in uninjured but not TBI rats. Due to the importance of descending serotonergic signaling in modulating nociception, we ablated spinal serotonin signaling using 5,7-DHT. This treatment strongly reduced CNO-mediated anti-nociceptive effects in TBI but not uninjured rats. To define the serotonergic receptor(s) required for the CNO-stimulated effects in TBI rats, we administered 5-HT7 (SB-269970) and 5-HT1A (WAY-100635) receptor antagonists but observed no effects. The selective 5-HT2A receptor antagonist ketanserin, however, blocked CNO's effects in the DREADD expressing TBI but not DREADD expressing sham TBI animals. Blockade of alpha-1 adrenergic receptors with prazosin also had no effect after TBI. Descending pain control originating in the PAG is mediated through opioid receptors in uninjured rats. TBI, however, fundamentally alters the descending nociceptive control circuitry such that serotonergic influences predominate, and those are mediated by the 5-HT2A receptor. These results provide further evidence that the PAG is a key target for anti-nociception after TBI.

Epilepsy phenotype and its reproducibility after lateral fluid percussion-induced traumatic brain injury in rats: Multicenter EpiBioS4Rx study project 1.

This study was undertaken to assess reproducibility of the epilepsy outcome and phenotype in a lateral fluid percussion model of posttraumatic epilepsy (PTE) across three study sites. A total of 525 adult male Sprague Dawley rats were randomized to lateral fluid percussion-induced brain injury (FPI) or sham operation. Of these, 264 were assigned to magnetic resonance imaging (MRI cohort, 43 sham, 221 traumatic brain injury [TBI]) and 261 to electrophysiological follow-up (EEG cohort, 41 sham, 220 TBI). A major effort was made to harmonize the rats, materials, equipment, procedures, and monitoring systems. On the 7th post-TBI month, rats were video-EEG monitored for epilepsy diagnosis. A total of 245 rats were video-EEG phenotyped for epilepsy on the 7th postinjury month (121 in MRI cohort, 124 in EEG cohort). In the whole cohort (n = 245), the prevalence of PTE in rats with TBI was 22%, being 27% in the MRI and 18% in the EEG cohort (p > .05). Prevalence of PTE did not differ between the three study sites (p > .05). The average seizure frequency was .317 ± .725 seizures/day at University of Eastern Finland (UEF; Finland), .085 ± .067 at Monash University (Monash; Australia), and .299 ± .266 at University of California, Los Angeles (UCLA; USA; p < .01 as compared to Monash). The average seizure duration did not differ between UEF (104 ± 48 s), Monash (90 ± 33 s), and UCLA (105 ± 473 s; p > .05). Of the 219 seizures, 53% occurred as part of a seizure cluster (≥3 seizures/24 h; p >.05 between the study sites). Of the 209 seizures, 56% occurred during lights-on period and 44% during lights-off period (p > .05 between the study sites). The PTE phenotype induced by lateral FPI is reproducible in a multicenter design. Our study supports the feasibility of performing preclinical multicenter trials in PTE to increase statistical power and experimental rigor to produce clinically translatable data to combat epileptogenesis after TBI.

Repeated Mild Traumatic Brain Injury and JZL184 Produce Sex-Specific Increases in Anxiety-Like Behavior and Alcohol Consumption in Wistar Rats.

Alcohol use disorder (AUD) is highly comorbid with traumatic brain injury (TBI). Previously, using a lateral fluid percussion model (LFP) (an open-head injury model) to generate a single mild to moderate traumatic brain injury (TBI) we showed that TBI produces escalation in alcohol drinking, that alcohol exposure negatively impacts TBI outcomes, and that the endocannabinoid degradation inhibitor (JZL184) confers significant protection from behavioral and neuropathological outcomes in male rodents. In the present study, we used a weight drop model (a closed-head injury model) to produce repeated mild TBI (rmTBI; three TBIs separated by 24 hours) in male and female rats to examine the sex-specific effects on anxiety-like behavior and alcohol consumption, and whether systemic treatment with JZL184 would reverse TBI effects on those behaviors. In two separate studies, adult male and female Wistar rats were subjected to rmTBI or sham procedure using the weight drop model. Physiological measures of injury severity were collected from all animals. Animals in both studies were allowed to consume alcohol using an intermittent 2-bottle choice procedure (12 pre-TBI sessions and 12 post-TBI sessions). Neurological severity and neurobehavioral scores (NSS and NBS, respectively) were tested 24 hours after the final injury. Anxiety-like behavior was tested at 37-38 days post-injury in Study 1-, and 6-8-days post-injury in Study 2. Our results show that females exhibited reduced respiratory rates relative to males with no significant differences between Sham and rmTBI, no effect of rmTBI or sex on righting reflex, and increased neurological deficits in rmTBI groups in both studies. In Study 1, rmTBI increased alcohol consumption in female but not male rats. Male rats consistently exhibited higher levels of anxiety-like behavior than females. The rmTBI did not affect anxiety-like behavior 37-38 days post-injury. In Study 2, rmTBI once again increased alcohol consumption in female but not male rats, and repeated systemic treatment with JZL184 did not affect alcohol consumption. Also in Study 2, rmTBI increased anxiety-like behavior in males but not females and repeated systemic treatment with JZL184 produced an unexpected increase in anxiety-like behavior 6-8 days post-injury. In summary, rmTBI increased alcohol consumption in female rats, systemic JZL184 treatment did not alter alcohol consumption, and both rmTBI and systemic JZL184 treatment increased anxiety-like behavior 6-8 days post-injury in males but not females, highlighting robust sex differences in rmTBI effects.

Early Measures of TBI Severity Poorly Predict Later Individual Impairment in a Rat Fluid Percussion Model.

Multiple measures of injury severity are suggested as common data elements in preclinical traumatic brain injury (TBI) research. The robustness of these measures in characterizing injury severity is unclear. In particular, it is not known how reliably they predict individual outcomes after experimental TBI. We assessed several commonly used measures of initial injury severity for their ability to predict chronic cognitive outcomes in a rat lateral fluid percussion (LFPI) model of TBI. At the time of injury, we assessed reflex righting time, neurologic severity scores, and 24 h weight loss. Sixty days after LFPI, we evaluated working memory using a spontaneous alternation T-maze task. We found that righting time and weight loss had no correlation to chronic T-maze performance, while neurologic severity score correlated weakly. Taken together, our results indicate that commonly used early measures of injury severity do not robustly predict longer-term outcomes. This finding parallels the uncertainty in predicting individual outcomes in TBI clinical populations.

Hippocampal position and orientation as prognostic biomarkers for posttraumatic epileptogenesis: An experimental study in a rat lateral fluid percussion model.

ObjectiveThis study was undertaken to identify prognostic biomarkers for posttraumatic epileptogenesis derived from parameters related to the hippocampal position and orientation.MethodsData were derived from two preclinical magnetic resonance imaging (MRI) follow‐up studies: EPITARGET (156 rats) and Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx; University of Eastern Finland cohort, 43 rats). Epileptogenesis was induced with lateral fluid percussion‐induced traumatic brain injury (TBI) in adult male Sprague Dawley rats. In the EPITARGET cohort, T2∗‐weighted MRI was performed at 2, 7, and 21 days and in the EpiBioS4Rx cohort at 2, 9, and 30 days and 5 months post‐TBI. Both hippocampi were segmented using convolutional neural networks. The extracted segmentation mask was used for a geometric construction, extracting 39 parameters that described the position and orientation of the left and right hippocampus. In each cohort, we assessed the parameters as prognostic biomarkers for posttraumatic epilepsy (PTE) both individually, using repeated measures analysis of variance, and in combination, using random forest classifiers.ResultsThe extracted parameters were highly effective in discriminating between sham‐operated and TBI rats in both the EPITARGET and EpiBioS4Rx cohorts at all timepoints (t; balanced accuracy > .9). The most discriminating parameter was the inclination of the hippocampus ipsilateral to the lesion at t = 2 days and the volumes at t ≥ 7 days after TBI. Furthermore, in the EpiBioS4Rx cohort, we could effectively discriminate epileptogenic from nonepileptogenic animals with a longer MRI follow‐up, at t = 150 days (area under the curve = .78, balanced accuracy = .80, p = .0050), based on the orientation of both hippocampi. We found that the ipsilateral hippocampus rotated outward on the horizontal plane, whereas the contralateral hippocampus rotated away from the vertical direction.SignificanceWe demonstrate that assessment of TBI‐induced hippocampal deformation by clinically translatable MRI methodologies detects subjects with prior TBI as well as those at high risk of PTE, paving the way toward subject stratification for antiepileptogenesis studies.

The Effects of Mild Traumatic Brain Injury (mTBI) on Serotonergic Gene Expression

Traumatic brain injury (TBI) is a risk factor for suicide. Approximately 1.7 million TBIs occur each year and new TBI cases in the U.S. military have more than doubled in the last 5 years. The majority of military TBIs are mild in severity and can lead to chronic symptoms including, impulsivity, emotional instability, and impaired inhibition. Impulsivity is one of the most frequent behavioral changes seen following mTBI and involves poor response inhibition and/or poor decision making. Also, impulsivity is a key risk factor for suicide. The serotonergic system is an important modulator of impulsivity and, thus, a molecular target for understanding the behavioral changes in impulsivity that occur following mTBI. One major source of serotonin in the brain is the raphe nuclei in the pons. The serotonergic neurons in the raphe nuclei project to the prefrontal cortex, frontal cortex and hippocampus and are involved in many cognitive processes related to impulsivity and suicidality. In addition, both animal and human research indicates that the brainstem is affected in mTBI, particularly, in the lateral fluid percussion model utilized in this study. This provides additional support for the notion that mTBI may lead to dysfunction of the serotonin producing raphe nuclei located in the brain stem. In this study, changes in serotonergic gene expression in the raphe nuclei and target regions including the prefrontal cortex, frontal cortex and hippocampus were assessed using qRT‐PCR. Rats sustained a single mTBI induced using the lateral fluid percussion model and were sacrificed three months following injury. Brains were extracted, flash frozen, sectioned at 300um and tissue for the raphe nuclei and target regions was collected using a 1.5 mm tissue punch. RNA was extracted from the tissue punches, reverse transcribed into cDNA and qRT‐PCR was performed. The hypothesis states that mTBI leads to altered expression of serotonin related genes in the raphe nuclei and/or raphe nuclei target regions including the prefrontal cortex, frontal cortex, and hippocampus. Results show altered expression levels of tryptophan hydroxylase and serotonin transporter genes in both the medial raphe and dorsal raphe nuclei in mTBI rats compared to SHAM control rats. Changes in expression levels of the 5HT1A and 5HT2A receptor genes were also seen in the frontal cortex, prefrontal cortex, and ventral hippocampus. The current study serves to provide evidence for a link between TBI and altered serotonin levels to better understand the molecular mechanisms underlying impulsivity and suicidality in humans following TBI.

Activation of the Locus Coeruleus Mediated by Designer Receptor Exclusively Activated by Designer Drug Restores Descending Nociceptive Inhibition after Traumatic Brain Injury in Rats.

Disruption of endogenous pain control mechanisms including descending pain inhibition has been linked to several forms of pain including chronic pain after traumatic brain injury (TBI). The locus coeruleus (LC) is the principal noradrenergic (NA) nucleus participating in descending pain inhibition. We therefore hypothesized that selectively stimulating LC neurons would reduce nociception after TBI. All experiments used a well-characterized rat lateral fluid percussion model of TBI. NA neurons were stimulated by administering clozapine N-oxide (CNO) to rats selectively expressing a designer receptor exclusively activated by designer drug (DREADD) viral construct in their LC's. Mechanical nociceptive thresholds were measured using von Frey fibers. The efficacy of diffuse noxious inhibitory control (DNIC), a critical endogenous pain control mechanism, was assessed using the hindpaw administration of capsaicin. Immunohistochemical analyses demonstrated the selective expression of the DREADD construct in LC neurons after stereotactic injection. During the 1st week after TBI, when rats demonstrated hindlimb (HL) nociceptive sensitization, CNO administration provided transient anti-allodynia in DREADD-expressing rats but not in rats injected with control virus. Seven weeks after TBI we observed a complete loss of DNIC in response to capsaicin. However, CNO administration largely restored DNIC in TBI DREADD-expressing rats but not those injected with control virus. Unexpectedly, the effects of LC activation in the DREADD-expressing rats were blocked by the α-1 adrenergic receptor antagonist prazosin, but not the α-2 adrenergic receptor antagonist atipamezole. These results suggest that directly stimulating the LC after TBI can reduce both early and late manifestations of dysfunctional endogenous pain regulation. Clinical approaches to activating descending pain circuits may reduce suffering in those with pain after TBI.

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.

Diacylglycerol Lipase-β Knockout Mice Display a Sex-Dependent Attenuation of Traumatic Brain Injury-Induced Mortality with No Impact on Memory or Other Functional Consequences.

Background: The endogenous cannabinoid system modulates inflammatory signaling in a variety of pathological states, including traumatic brain injury (TBI). The selective expression of diacylglycerol lipase-β (DAGL-β), the 2-arachidonylglycerol biosynthetic enzyme, on resident immune cells of the brain (microglia) and the role of this pathway in neuroinflammation, suggest that this enzyme may contribute to TBI-induced neuroinflammation. Accordingly, we tested whether DAGL-β-/- mice would show a protective phenotype from the deleterious consequences of TBI on cognitive and neurological motor functions. Materials and Methods: DAGL-β-/- and -β+/+ mice were subjected to the lateral fluid percussion model of TBI and assessed for learning and memory in the Morris water maze (MWM) Fixed Platform (reference memory) and Reversal (cognitive flexibility) tasks, as well as in a cued MWM task to infer potential sensorimotor/motivational deficits. In addition, subjects were assessed for motor behavior (Rotarod and the Neurological Severity Score assays) and in the light/dark box and the elevated plus maze to infer whether these manipulations affected anxiety-like behavior. Finally, we also examined whether brain injury disrupts the ceramide/sphingolipid lipid signaling system and if DAGL-β deletion offers protection. Results: TBI disrupted all measures of neurological motor function and reduced body weight, but did not affect body temperature or performance in common assays used to infer anxiety. TBI also impaired performance in MWM Fixed Platform and Reversal tasks, but did not affect cued MWM performance. Although no differences were found between DAGL-β-/- and -β+/+ mice in any of these measures, male DAGL-β-/- mice displayed an unexpected survival-protective phenotype, which persisted at increased injury severities. In contrast, TBI did not elicit mortality in female mice regardless of genotype. TBI also produced significant changes in sphingolipid profiles (a family of lipids, members of which have been linked to both apoptotic and antiapoptotic pathways), in which DAGL-β deletion modestly altered levels of select species. Conclusions: These findings indicate that although DAGL-β does not play a necessary role in TBI-induced cognitive and neurological function, it appears to contribute to the increased vulnerability of male mice to TBI-induced mortality, whereas female mice show high survival rates irrespective of DAGL-β expression.

Traumatic Brain Injury and Alcohol Drinking Alter Basolateral Amygdala Endocannabinoids in Female Rats.

Traumatic brain injury (TBI) affects approximately 3 million Americans yearly and increases vulnerability to developing psychiatric comorbidities. Alcohol use disorder (AUD) is the most prevalent psychiatric diagnosis preceding injury and TBI may increase subsequent alcohol use. The basolateral amygdala (BLA) is a limbic structure commonly affected by TBI that is implicated in anxiety and AUD. Endocannabinoids (eCBs) regulate synaptic activity in the BLA, and BLA eCB modulation alters anxiety-like behavior and stress reactivity. Previous work from our laboratories showed that systemic eCB degradation inhibition ameliorates TBI-induced increases in anxiety-like behavior and motivation to respond for alcohol in male rats. Here, we used a lateral fluid percussion model to test moderate TBI effects on anxiety-like behavior, alcohol drinking, and eCB levels and cell signaling in BLA, as well as the effect of alcohol drinking on anxiety-like behavior and the BLA eCB system, in female rats. Our results show that TBI does not promote escalation of operant alcohol self-administration or increase anxiety-like behavior in female rats. In the BLA, TBI and alcohol drinking alter tissue amounts of 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA) 1 h post-injury, and 2-AG levels remain low 11 days post-injury. Eleven days after injury, BLA pyramidal neurons were hyperexcitable, but measures of synaptic transmission and eCB signaling were unchanged. These data show that TBI impacts BLA 2-AG tissue levels, that this effect is modified by alcohol drinking, and also that TBI increases BLA cell excitability.

Loss of diffuse noxious inhibitory control after traumatic brain injury in rats: A chronic issue

Chronic pain is one of the most challenging and debilitating symptoms to manage after traumatic brain injury (TBI), yet the underlying mechanisms remain elusive. The disruption of normal endogenous pain control mechanisms has been linked to several forms of chronic pain and may play a role in pain after TBI. We hypothesized therefore that dysfunctional descending noradrenergic and serotonergic pain control circuits may contribute to the loss of diffuse noxious inhibitory control (DNIC), a critical endogenous pain control mechanism, weeks to months after TBI. For these studies, the rat lateral fluid percussion model of mild TBI was used along with a DNIC paradigm involving a capsaicin-conditioning stimulus. We observed sustained failure of the DNIC response up to 180-days post injury. We confirmed, that descending α2 adrenoceptor-mediated noradrenergic signaling was critical for endogenous pain inhibition in uninjured rats. However, augmenting descending noradrenergic signaling using reboxetine, a selective noradrenaline reuptake inhibitor, failed to restore DNIC after TBI. Furthermore, blocking serotonin-mediated descending signaling using selective spinal serotonergic fiber depletion with 5, 7-dihydroxytryptamine was also unsuccessful at restoring endogenous pain modulation after TBI. Unexpectedly, increasing descending serotonergic signaling using the selective serotonin reuptake inhibitor escitalopram and the serotonin-norepinephrine reuptake inhibitor duloxetine restored the DNIC response in TBI rats at both 49- and 180- days post injury. Consistent with these observations, spinal serotonergic fiber depletion with 5, 7-dihydroxytryptamine eliminated the effects of escitalopram. Intact α2 adrenoceptor signaling, however, was not required for the serotonin-mediated restoration of DNIC after TBI. These results suggest that TBI causes maladaptation of descending nociceptive signaling mechanisms and changes in the function of both adrenergic and serotonergic circuits. Such changes could predispose those with TBI to chronic pain.

Propylparaben Reduces the Long-Term Consequences in Hippocampus Induced by Traumatic Brain Injury in Rats: Its Implications as Therapeutic Strategy to Prevent Neurodegenerative Diseases.

Severe traumatic brain injury (TBI), an important risk factor for Alzheimer's disease, induces long-term hippocampal damage and hyperexcitability. On the other hand, studies support that propylparaben (PPB) induces hippocampal neuroprotection in neurodegenerative diseases. Experiments were designed to evaluate the effects of subchronic treatment with PPB on TBI-induced changes in the hippocampus of rats. Severe TBI was induced using the lateral fluid percussion model. Subsequently, rats received subchronic administration with PPB (178 mg/kg, TBI+PPB) or vehicle (TBI+PEG) daily for 5 days. The following changes were examined during the experimental procedure: sensorimotor dysfunction, changes in hippocampal excitability, as well as neuronal damage and volume. TBI+PEG group showed sensorimotor dysfunction (p < 0.001), hyperexcitability (64.2%, p < 0.001), and low neuronal preservation ipsi- and contralateral to the trauma. Magnetic resonance imaging (MRI) analysis revealed lower volume (17.2%; p < 0.01) and great damage to the ipsilateral hippocampus. TBI+PPB group showed sensorimotor dysfunction that was partially reversed 30 days after trauma. This group showed hippocampal excitability and neuronal preservation similar to the control group. However, MRI analysis revealed lower hippocampal volume (p < 0.05) when compared with the control group. The present study confirms that post-TBI subchronic administration with PPB reduces the long-term consequences of trauma in the hippocampus. Implications of PPB as a neuroprotective strategy to prevent the development of Alzheimer's disease as consequence of TBI are discussed.

In Vitro and In Vivo Pipeline for Validation of Disease-Modifying Effects of Systems Biology-Derived Network Treatments for Traumatic Brain Injury-Lessons Learned.

We developed a pipeline for the discovery of transcriptomics-derived disease-modifying therapies and used it to validate treatments in vitro and in vivo that could be repurposed for TBI treatment. Desmethylclomipramine, ionomycin, sirolimus and trimipramine, identified by in silico LINCS analysis as candidate treatments modulating the TBI-induced transcriptomics networks, were tested in neuron-BV2 microglial co-cultures, using tumour necrosis factor α as a monitoring biomarker for neuroinflammation, nitrite for nitric oxide-mediated neurotoxicity and microtubule associated protein 2-based immunostaining for neuronal survival. Based on (a) therapeutic time window in silico, (b) blood-brain barrier penetration and water solubility, (c) anti-inflammatory and neuroprotective effects in vitro (p < 0.05) and (d) target engagement of Nrf2 target genes (p < 0.05), desmethylclomipramine was validated in a lateral fluid-percussion model of TBI in rats. Despite the favourable in silico and in vitro outcomes, in vivo assessment of clomipramine, which metabolizes to desmethylclomipramine, failed to demonstrate favourable effects on motor and memory tests. In fact, clomipramine treatment worsened the composite neuroscore (p < 0.05). Weight loss (p < 0.05) and prolonged upregulation of plasma cytokines (p < 0.05) may have contributed to the worsened somatomotor outcome. Our pipeline provides a rational stepwise procedure for evaluating favourable and unfavourable effects of systems-biology discovered compounds that modulate post-TBI transcriptomics.

Harmonization of the pipeline for seizure detection to phenotype post-traumatic epilepsy in a preclinical multicenter study on post-traumatic epileptogenesis

RationaleThe Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) Centre without walls is an NIH funded multicenter consortium. One of EpiBioS4Rx projects is a preclinical post-traumatic epileptogenesis biomarker study that involves three study sites: The University of Eastern Finland, Monash University (Melbourne) and the University of California Los Angeles. Our objective is to create a platform for evaluating biomarkers and testing new antiepileptogenic treatments for post-traumatic epilepsy (PTE) using the lateral fluid percussion injury (FPI) model in rats. As only 30–50% of rats with severe lateral FPI develop PTE by 6 months post-injury, prolonged video-EEG monitoring is crucial to identify animals with PTE. Our objective is to harmonize the surgical and data collection procedures, equipment, and data analysis for chronic EEG recording in order to phenotype PTE in this rat model across the three study sites. MethodsTraumatic brain injury (TBI) was induced using lateral FPI in adult male Sprague-Dawley rats aged 11–12 weeks. Animals were divided into two cohorts: a) the long-term video-EEG follow-up cohort (Specific Aim 1), which was implanted with EEG electrodes within 24 h after the injury; and b) the magnetic resonance imaging (MRI) follow-up cohort (Specific Aim 2), at 5 months after lateral FPI. Four cortical epidural screw electrodes (2 ipsilateral, 2 contralateral) and three intracerebral bipolar electrodes were implanted (septal CA1 and the dentate gyrus, layers II and VI of the perilesional cortex both anterior and posterior to the injury site). During the 7th post-TBI month, animals underwent 4 weeks of continuous video-EEG recordings to diagnose of PTE. ResultsAll centers harmonized the induction of TBI and surgical procedures for the implantation of EEG recordings, utilizing 4 or more EEG recording channels to cover areas ipsilateral and contralateral to the brain injury, perilesional cortex and the hippocampus and dentate gyrus. Ground and reference screw electrodes were implanted. At all sites the minimum sampling rate was 512 Hz, utilizing a finite impulse response (FIR) and impedance below 10 KΩ through the entire recording. As part of the quality control criteria we avoided electrical noise, and monitoring changes in impedance over time and the appearance of noise on the recordings. To reduce electrical noise, we regularly checked the integrity of the cables, stability of the EEG recording cap and the appropriate connection of the electrodes with the cables. Following the pipeline presented in this article and after applying the quality control criteria to our EEG recordings all of the sites were successful to phenotype seizure in chronic EEG recordings of animals after TBI. DiscussionDespite differences in video-EEG acquisition equipment used, the three centers were able to consistently phenotype seizures in the lateral fluid-percussion model applying the pipeline presented here. The harmonization of methodology will help to improve the rigor of preclinical research, improving reproducibility of pre-clinical research in the search of biomarkers and therapies to prevent antiepileptogenesis.

Chronic Dysregulation of Cortical and Subcortical Metabolism After Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. Although chronic disability is common after TBI, effective treatments remain elusive and chronic TBI pathophysiology is not well understood. Early after TBI, brain metabolism is disrupted due to unregulated ion release, mitochondrial damage, and interruption of molecular trafficking. This metabolic disruption causes at least part of the TBI pathology. However, it is not clear how persistent or pervasive metabolic injury is at later stages of injury. Using untargeted 1H-NMR metabolomics, we examined ex vivo hippocampus, striatum, thalamus, frontal cortex, and brainstem tissue in a rat lateral fluid percussion model of chronic brain injury. We found altered tissue concentrations of metabolites in the hippocampus and thalamus consistent with dysregulation of energy metabolism and excitatory neurotransmission. Furthermore, differential correlation analysis provided additional evidence of metabolic dysregulation, most notably in brainstem and frontal cortex, suggesting that metabolic consequences of injury are persistent and widespread. Interestingly, the patterns of network changes were region-specific. The individual metabolic signatures after injury in different structures of the brain at rest may reflect different compensatory mechanisms engaged to meet variable metabolic demands across brain regions.

Traumatic Brain Injury Disrupts Pain Signaling in the Brainstem and Spinal Cord.

Chronic pain is a common consequence of traumatic brain injury (TBI) that can increase the suffering of a patient and pose a significant challenge to rehabilitative efforts. Unfortunately, the mechanisms linking TBI to pain are poorly understood, and specific treatments for TBI-related pain are still lacking. Our laboratory has shown that TBI causes pain sensitization in areas distant to the site of primary injury, and that changes in spinal gene expression may underlie this sensitization. The aim of this study was to examine the roles that pain modulatory pathways descending from the brainstem play in pain after TBI. Deficiencies in one type of descending inhibition, diffuse noxious inhibitory control (DNIC), have been suggested to be responsible for the development of chronic pain by allowing excess and uncontrolled afferent nociceptive inputs. Here we expand our knowledge of pain after TBI in two ways: (1) by outlining the neuropathology in pain-related centers of the brain and spinal cord involved in DNIC using the rat lateral fluid percussion (LFP) model of TBI, and (2) by evaluating the effects of a potent histone acetyl transferase inhibitor, anacardic acid (AA), on LFP-induced pain behaviors and neuropathology when administered for several days after TBI. The results revealed that TBI induces transient mechanical allodynia and a chronic persistent loss of DNIC. Further, while short-term AA treatment can block acute nociceptive sensitization and some early neuropathological changes, this treatment neither prevented the loss of DNIC nor did it alter long-term neuropathological changes in the brain or spinal cord.

A Lateral Fluid Percussion Injury Model for Studying Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) diagnoses have increased in frequency during the past decade, becoming a silent epidemic. The pathophysiology of TBI involves pathophysiological processes affecting the brain, induced by traumatic biomechanical forces resulting in temporary impairment of neurological function. Preclinical models have been generated to recapitulate the mechanical, neuroinflammatory, and behavioral outcomes observed in the clinical setting. The lateral fluid percussion (LFP) model is the most extensively used and well-characterized model of nonpenetrating and nonischemic TBI. The model is reproducible and can be adjusted to produce a mild to moderate and severe injury, as reflected by mortality and return of reflexes, by adjusting the amount of force applied. The histopathological changes achieved with this model reproduce that seen in human TBI including focal contusion in the cortex, with accompanying intraparenchymal punctate hemorrhage, followed by inflammation and neuronal degeneration. This chapter describes the LFP model, which produces a mixed model of focal and diffuse brain injury that progresses over time affecting predominantly the cortical parenchyma.

Intranasal Oxytocin Attenuates Reactive and Ongoing, Chronic Pain in a Model of Mild Traumatic Brain Injury.

Approximately 1.7 million Americans sustain a traumatic brain injury (TBI) each year and chronic pain is a common complication. We studied the effects of intranasally administered oxytocin as a potential treatment for chronic pain in an animal model of mild TBI. The lateral fluid percussion model of mild TBI was chosen for this purpose and after exposure to mild TBI the rats (n = 12) developed hind paw and facial allodynia compared to sham animals (n = 6). Oxytocin or a vehicle was afterwards administered intranasally and reactive pain was assessed by hind paw and facial von Frey testing. Some animals received the oxytocin receptor antagonist, atosiban, in addition to oxytocin/vehicle treatment (n = 12). The effect of oxytocin on ongoing and spontaneous pain was examined through conditioned place preference testing. To determine whether the effects of intranasal oxytocin could be attributed to delivery via the peripheral blood stream, some TBI animals received an intravenous injection of the same oxytocin dose that was given intranasally. ELISA immunoassays were carried out (n = 6) to measure concentrations of oxytocin in the trigeminal ganglia, pons, spinal cord, and olfactory bulb after intranasal administration and evaluate the most likely route of entry. These studies confirmed that the fluid percussion model can be used to study post-TBI facial allodynia. Oxytocin attenuated both reactive and spontaneous, ongoing non-reactive pain following mild TBI for at least 3-4 hours after intranasal administration by binding to OT or VA1-receptors most likely by a peri-trigeminal nerve mediated uptake. Intranasal oxytocin attenuates measures of reactive and non-reactive pain in a model of mild TBI and may represent a novel treatment for chronic pain in TBI patients.

Post-injury administration of a combination of memantine and 17β-estradiol is protective in a rat model of traumatic brain injury

Traumatic brain injury (TBI) often leads to substantial adverse cognitive and health outcomes, including permanent disability and death. Preventing these outcomes requires attenuation of the secondary biochemical damage that follows the initial biomechanical insult, but a clinically proven pharmacotherapeutic capable of such has not been identified. In fact, the heterogeneous nature of TBI and the complexity of secondary injury cascades suggest a polytherapeutic approach that targets multiple pathways might be necessary. We and others have reported that 17β-estradiol (E2) is neuroprotective in models of central nervous system injury. Although E2 is neuroprotective and favorably modulates several key components of secondary injury, it does not effectively block the destructive excitotoxic cascade. Thus, administering E2 in combination with a second drug that targets excitotoxicity, such as the FDA-approved uncompetitive NMDA receptor antagonist memantine hydrochloride, may provide additional benefits. Here, we assessed the neuroprotective potential of an acutely administered intravenous bolus dose of a combination of memantine and E2 after induction of experimental TBI in the clinically relevant lateral fluid percussion model. Our results indicate that the combination of these drugs conferred neuroprotection by increasing neuronal survival and decreasing neuronal degeneration in the hippocampus and cortex ipsilateral to injury. Furthermore, administration of this combination improved vestibulomotor deficits and modestly reduced anxiety. We conclude that further investigation of the neuroprotective potential of memantine administered with E2 is warranted.