ComBating inter-site differences in field strength: harmonizing preclinical traumatic brain injury MRI data.

Integrating datasets from multiple sites and scanners can increase statistical power for neuroimaging studies but can also introduce significant inter-site confounds. We evaluated the effectiveness of ComBat, an empirical Bayes approach, to combine longitudinal preclinical MRI data acquired at 4.7 or 9.4 T at two different sites in Australia. Male Sprague Dawley rats underwent MRI on Days 2, 9, 28, and 150 following moderate/severe traumatic brain injury (TBI) or sham injury as part of Project 1 of the NIH/NINDS-funded Centre Without Walls EpiBioS4Rx project. Diffusion-weighted and multiple-gradient-echo images were acquired, and outcomes included QSM, FA, and ADC. Acute injury measures including apnea and self-righting reflex were consistent between sites. Mixed-effect analysis of ipsilateral and contralateral corpus callosum (CC) summary values revealed a significant effect of site on FA and ADC values, which was removed following ComBat harmonization. Bland-Altman plots for each metric showed reduced variability across sites following ComBat harmonization, including for QSM, despite appearing to be largely unaffected by inter-site differences and no effect of site observed. Following harmonization, the combined inter-site data revealed significant differences in the imaging metrics consistent with previously reported outcomes. TBI resulted in significantly reduced FA and increased susceptibility in the ipsilateral CC, and significantly reduced FA in the contralateral CC compared with sham-injured rats. Additionally, TBI rats also exhibited a reversal in ipsilateral CC ADC values over time with significantly reduced ADC at Day 9, followed by increased ADC 150 days after injury. Our findings demonstrate the need for harmonizing multi-site preclinical MRI data and show that this can be successfully achieved using ComBat while preserving phenotypical changes due to TBI.

The waiting game: investigating the neurobiological transition from acute to persistent pain in adolescent rats.

Persistent postsurgical pain affects 20% of youth undergoing a surgical procedure, with females exhibiting increased prevalence of chronic pain compared with males. This study sought to examine the sexually-dimorphic neurobiological changes underlying the transition from acute to persistent pain following surgery in adolescence. Male and female Sprague Dawley rats were randomly allocated to a sham or injury (plantar-incision surgery) condition and assessed for pain sensitivity while also undergoing magnetic resonance imaging at both an acute and chronic timepoint within adolescence. We found that injury resulted in persistent pain in both sexes, with females displaying most significant sensitivity. Injury resulted in significant gray matter density increases in brain areas including the cerebellum, caudate putamen/insula, and amygdala and decreases in the hippocampus, hypothalamus, nucleus accumbens, and lateral septal nucleus. Gray matter density changes in the hippocampus and lateral septal nucleus were driven by male rats whereas changes in the amygdala and caudate putamen/insula were driven by female rats. Overall, our results indicate persistent behavioral and neurobiological changes following surgery in adolescence, with sexually-dimorphic and age-specific outcomes, highlighting the importance of studying both sexes and adolescents, rather than extrapolating from male adult literature.

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.

A high-fat high-sugar diet and adversity early in life modulate pain outcomes at the behavioural and molecular level in adolescent rats: The role of sex.

Given that adolescence is a significant period of brain plasticity and development, early life factors have the potential to alter long term outcomes. For instance, adversities such as consumption of a high-fat high-sugar (HFHS) diet and adverse childhood experiences (ACEs; e.g., neglect), and their resulting inflammation and microglial activation can influence pain outcomes by priming the neuroimmune system to overrespond to stressors. Chronic pain is highly prevalent amongst the adolescent population, with the prevalence and manifestation being sexually dimorphic. Although clinical studies show that females are twice as likely to report pain problems compared to males, the majority of pre-clinical work uses male rodents. Therefore, our aim was to examine the effects of sex, a HFHS diet, and an ACE on chronic pain outcomes following a stressor in adolescence. Rat dams were randomly assigned to a Standard or HFHS diet, with pups maintained on their respective diets then randomly allocated to a No Stress or ACE paradigm, and a Sham or Injury condition as a stressor. Results showed that early life adversities increased nociceptive sensitivity, inflammation, and microglial activation systemically and within the brain. Behaviourally, pain outcomes were more prominent in females, however the neuroimmune response was exacerbated in males. These results demonstrate the sexual dimorphism of chronic pain outcomes following early life adversities and provide insight into the mechanisms driving these changes, which will inform more targeted and effective treatment strategies for youth living with chronic pain.

E2730, an uncompetitive γ-aminobutyric acid transporter-1 inhibitor, suppresses epileptic seizures in a rat model of chronic mesial temporal lobe epilepsy.

OBJECTIVE: More than one third of mesial temporal lobe epilepsy (MTLE) patients are resistant to current antiseizure medications (ASMs), and half experience mild-to-moderate adverse effects of ASMs. There is therefore a strong need to develop and test novel ASMs. The objective of this work is to evaluate the pharmacokinetics and neurological toxicity of E2730, a novel uncompetitive inhibitor of γ-aminobutyric acid transporter-1, and to test its seizure suppression effects in a rat model of chronic MTLE. METHODS: We first examined plasma levels and adverse neurological effects of E2730 in healthy Wistar rats. Adult male rats were implanted with osmotic pumps delivering either 10, 20, or 100 mg/kg/day of E2730 subcutaneously for 1 week. Blood sampling and behavioral assessments were performed at several timepoints. We next examined whether E2730 suppressed seizures in rats with chronic MTLE. These rats were exposed to kainic acid-induced status epilepticus, and 9 weeks later, when chronic epilepsy was established, were assigned to receive one of the three doses of E2730 or vehicle for 1 week in a randomized crossover design. Continuous video-electroencephalographic monitoring was acquired during the treatment period to evaluate epileptic seizures. RESULTS: Plasma levels following continuous infusion of E2730 showed a clear dose-related increase in concentration. The drug was well tolerated at all doses, and any sedation or neuromotor impairment was mild and transient, resolving within 48 h of treatment initiation. Remarkably, E2730 treatment in chronically epileptic rats led to seizure suppression in a dose-dependent manner, with 65% of rats becoming seizure-free at the highest dose tested. Mean seizure class did not differ between the treatment groups. SIGNIFICANCE: This study shows that continuous subcutaneous infusion of E2730 over 7 days results in a marked, dose-dependent suppression of spontaneous recurrent seizures, with minimal adverse neurological effects, in a rat model of chronic MTLE. E2730 shows strong promise as an effective new ASM to be translated into clinical trials.

Repeat mild traumatic brain injuries (RmTBI) modify nociception and disrupt orexinergic connectivity within the descending pain pathway.

Repeat mild traumatic brain injuries (RmTBI) result in substantial burden to the public health system given their association with chronic post-injury pathologies, such as chronic pain and post-traumatic headache. Although this may relate to dysfunctional descending pain modulation (DPM), it is uncertain what mechanisms drive changes within this pathway. One possibility is altered orexinergic system functioning, as orexin is a potent anti-nociceptive neuromodulator. Orexin is exclusively produced by the lateral hypothalamus (LH) and receives excitatory innervation from the lateral parabrachial nucleus (lPBN). Therefore, we used neuronal tract-tracing to investigate the relationship between RmTBI and connectivity between lPBN and the LH, as well as orexinergic projections to a key site within the DPM, the periaqueductal gray (PAG). Prior to injury induction, retrograde and anterograde tract-tracing surgery was performed on 70 young-adult male Sprague Dawley rats, targeting the lPBN and PAG. Rodents were then randomly assigned to receive RmTBIs or sham injuries before undergoing testing for anxiety-like behaviour and nociceptive sensitivity. Immunohistochemical analysis identified distinct and co-localized orexin and tract-tracing cell bodies and projections within the LH. The RmTBI group exhibited altered nociception and reduced anxiety as well as a loss of orexin cell bodies and a reduction of hypothalamic projections to the ventrolateral nucleus of the PAG. However, there was no significant effect of injury on neuronal connectivity between the lPBN and orexinergic cell bodies within the LH. Our identification of structural losses and the resulting physiological changes in the orexinergic system following RmTBI begins to clarify acute post-injury mechanistic changes that drive may drive the development of post-traumatic headache and the chronification of pain.

White and Gray Matter Abnormalities in Australian Footballers With a History of Sports-Related Concussion: An MRI Study.

Sports-related concussion (SRC) is a form of mild traumatic brain injury that has been linked to long-term neurological abnormalities. Australian rules football is a collision sport with wide national participation and is growing in popularity worldwide. However, the chronic neurological consequences of SRC in Australian footballers remain poorly understood. This study investigated the presence of brain abnormalities in Australian footballers with a history of sports-related concussion (HoC) using multimodal MRI. Male Australian footballers with HoC (n = 26), as well as noncollision sport athletes with no HoC (n = 27), were recruited to the study. None of the footballers had sustained a concussion in the preceding 6 months, and all players were asymptomatic. Data were acquired using a 3T MRI scanner. White matter integrity was assessed using diffusion tensor imaging. Cortical thickness, subcortical volumes, and cavum septum pellucidum (CSP) were analyzed using structural MRI. Australian footballers had evidence of widespread microstructural white matter damage and cortical thinning. No significant differences were found regarding subcortical volumes or CSP. These novel findings provide evidence of persisting white and gray matter abnormalities in Australian footballers with HoC, and raise concerns related to the long-term neurological health of these athletes.

Caffeine consumption during development alters spine density and recovery from repetitive mild traumatic brain injury in young adult rats.

Caffeine is the most commonly used psychostimulant throughout the world, with its consumption being especially prevalent among adolescents and young adults, as over 75% of this group consumes caffeine daily. Similarly, the adolescent and young adult age group exhibit the highest incidence of traumatic brain injury (TBI). Given that both caffeine consumption and mild TBI (mTBI) are more prevalent among the late adolescent/young adult age group and that changes in dendritic spine morphology during this developmental period are poorly understood, this study sought to examine the effects of caffeine consumption during late adolescence/early adulthood on recovery from repetitive mTBI (RmTBI). The study specifically focused on changes to neuronal dendritic morphology as synaptic changes likely underlie long-term behavioral outcomes. The results demonstrate that during young adulthood caffeine consumption differentially affects the RmTBI outcomes of males and females, where the effects of caffeine and RmTBI were often additive in males while being equally detrimental, but rarely additive, in females. In general, caffeine and RmTBI induced the greatest impairments in males on cognitive and motor tasks whereas in females the most significant detriments were on pain-related tasks. Both caffeine and RmTBI increased spine density in the Cg3 (medial prefrontal cortex [mPFC]), AID (orbitofrontal cortex [OFC]), and nucleus accumbens (NAc), which is proposed to reflect an impairment in the normal pruning processes. Overall, despite caffeine's neuroprotective abilities among other age groups, this study offers concerning results regarding the detrimental effects of caffeine and RmTBI, in isolation, and especially in combination, in this susceptible population.

The cholinergic forebrain arousal system acts directly on the circadian pacemaker.

Sleep and wake states are regulated by a variety of mechanisms. One such important system is the circadian clock, which provides temporal structure to sleep and wake. Conversely, changes in behavioral state, such as sleep deprivation (SD) or arousal, can phase shift the circadian clock. Here we demonstrate that the level of wakefulness is critical for this arousal resetting of the circadian clock. Specifically, drowsy animals with significant power in the 7- to 9-Hz band of their EEGs do not exhibit phase shifts in response to a mild SD procedure. We then show that treatments that both produce arousal and reset the phase of circadian clock activate (i.e., induce Fos expression in) the basal forebrain. Many of the activated cells are cholinergic. Using retrograde tract tracing, we demonstrate that cholinergic cells activated by these arousal procedures project to the circadian clock in the suprachiasmatic nuclei (SCN). We then demonstrate that arousal-induced phase shifts are blocked when animals are pretreated with atropine injections to the SCN, demonstrating that cholinergic activity at the SCN is necessary for arousal-induced phase shifting. Finally, we demonstrate that electrical stimulation of the substantia innominata of the basal forebrain phase shifts the circadian clock in a manner similar to that of our arousal procedures and that these shifts are also blocked by infusions of atropine to the SCN. These results establish a functional link between the major forebrain arousal center and the circadian system.

Serotonin 1A receptors alter expression of movement representations.

Serotonin has a myriad of central functions involving mood, appetite, sleep, and memory and while its release within the spinal cord is particularly important for generating movement, the corresponding role on cortical movement representations (motor maps) is unknown. Using adult rats we determined that pharmacological depletion of serotonin (5-HT) via intracerebroventricular administration of 5,7 dihydroxytryptamine resulted in altered movements of the forelimb in a skilled reaching task as well as higher movement thresholds and smaller maps derived using high-resolution intracortical microstimulation (ICMS). We ruled out the possibility that reduced spinal cord excitability could account for the serotonin depletion-induced changes as we observed an enhanced Hoffman reflex (H-reflex), indicating a hyperexcitable spinal cord. Motor maps derived in 5-HT1A receptor knock-out mice also showed higher movement thresholds and smaller maps compared with wild-type controls. Direct cortical application of the 5-HT1A/7 agonist 8-OH-DPAT lowered movement thresholds in vivo and increased map size in 5-HT-depleted rats. In rats, electrical stimulation of the dorsal raphe lowered movement thresholds and this effect could be blocked by direct cortical application of the 5-HT1A antagonist WAY-100135, indicating that serotonin is primarily acting through the 5-HT1A receptor. Next we developed a novel in vitro ICMS preparation that allowed us to track layer V pyramidal cell excitability. Bath application of WAY-100135 raised the ICMS current intensity to induce action potential firing whereas the agonist 8-OH-DPAT had the opposite effect. Together our results demonstrate that serotonin, acting through 5-HT1A receptors, plays an excitatory role in forelimb motor map expression.

Microbiome depletion prior to repeat mild TBI differentially alters social deficits and prefrontal cortex plasticity in adolescent and adult rats.

Although aging, repeat mild traumatic brain injury (RmTBI), and microbiome modifications independently change social behavior, there has been no investigation into their cumulative effects on social behavior and neuroplasticity within the prefrontal cortex. Therefore, we examined how microbiome depletion prior to RmTBI affected social behavior and neuroplasticity in adolescent and adult rats. Play, temperament analysis, elevated plus maze, and the hot/cold plate assessed socio-emotional function. Analyses of perineuronal nets (PNNs) and parvalbumin (PV) interneurons was completed. Social-emotional deficits were more pronounced in adults, with microbiome depletion attenuating social behavior deficits associated with RmTBI in both age groups. Microbiome depletion increased branch length and PNN arborization within the PFC but decreased the overall number of PNNs. Adults and males were more vulnerable to RmTBI. Interestingly, microbiome depletion may have attenuated the changes to neuroplasticity and subsequent social deficits, suggesting that the microbiome is a viable, but age-specific, target for RmTBI therapeutics.

Treatment with the vascular endothelial growth factor-A antibody, bevacizumab, has sex-specific effects in a rat model of mild traumatic brain injury.

Mild traumatic brain injury (mTBI) involves damage to the cerebrovascular system. Vascular endothelial growth factor-A (VEGF-A) is an important modulator of vascular health and VEGF-A promotes the brain's ability to recover after more severe forms of brain injury; however, the role of VEGF-A in mTBI remains poorly understood. Bevacizumab (BEV) is a monoclonal antibody that binds to VEGF-A and neutralises its actions. To better understand the role of VEGF-A in mTBI recovery, this study examined how BEV treatment affected outcomes in rats given a mTBI. Adult Sprague-Dawley rats were assigned to sham-injury + vehicle treatment (VEH), sham-injury + BEV treatment, mTBI + VEH treatment, mTBI + BEV treatment groups. Treatment was administered intracerebroventricularly via a cannula beginning at the time of injury and continuing until the end of the study. Rats underwent behavioral testing after injury and were euthanized on day 11. In both females and males, BEV had a negative impact on cognitive function. mTBI and BEV treatment increased the expression of inflammatory markers in females. In males, BEV treatment altered markers related to hypoxia and vascular health. These novel findings of sex-specific responses to BEV and mTBI provide important insights into the role of VEGF-A in mTBI.

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.

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.

Critical Windows: Exploring the Association Between Perinatal Trauma, Epigenetics, and Chronic Pain.

Chronic pain is highly prevalent and burdensome, affecting millions of people worldwide. Although it emerges at any point in life, it often manifests in adolescence. Given that adolescence is a unique developmental period, additional strains associated with persistent and often idiopathic pain lead to significant long-term consequences. While there is no singular cause for the chronification of pain, epigenetic modifications that lead to neural reorganization may underpin central sensitization and subsequent manifestation of pain hypersensitivity. Epigenetic processes are particularly active during the prenatal and early postnatal years. We demonstrate how exposure to various traumas, such as intimate partner violence while in utero or adverse childhood experiences, can significantly influence epigenetic regulation within the brain and in turn modify pain-related processes. We provide compelling evidence that the burden of chronic pain is likely initiated early in life, often being transmitted from mother to offspring. We also highlight two promising prophylactic strategies, oxytocin administration and probiotic use, that have the potential to attenuate the epigenetic consequences of early adversity. Overall, we advance understanding of the causal relationship between trauma and adolescent chronic pain by highlighting epigenetic mechanisms that underlie this transmission of risk, ultimately informing how to prevent this rising epidemic.

Repetitive mild traumatic brain injury alters central and peripheral clock gene expression in the adolescent rat.

Mild traumatic brain injury (mTBI) or concussion is a common injury worldwide leading to substantial medical costs and a high burden on society. In adolescents, falls and sports related trauma are often the causes of mTBI. Importantly, critical brain growth and development occurs during this sensitive period making the prospect of a brain injury a worrying phenomenon. Upwards of 70% of patients report circadian disruption following these injuries and this has been shown to impede recovery. Therefore, we sought to determine if core circadian clock gene expression was disrupted in rat model of repetitive mTBI (RmTBI). Male and female adolescent rats (n = 129) received sham or RmTBI. The animals were then euthanized at different times throughout the day and night. Tissue from the hypothalamus, cerebellum, hippocampus, liver, and small intestine were evaluated for the expression of per1, per2, cry1, clock, bmal1 and rev-erb-α. We found most clock genes varied across the day/night indicating circadian expression patterns. In the hypothalamus we found RmTBI altered the expression of cry1 and bmal1 in addition to sex differences in per2, cry1, clock, bmal1 and rev-erb- α. In the cerebellum, per1, per2, cry1, clock, bmal1 and rev-erb-α rhythms were all knocked out by RmTBI in addition to sex differences in cry1, clock and bmal1 expression. We also detected a significant decrease in overall expression of all clock genes in males in the middle of the night. In the hippocampus we found that RmTBI changed the rhythm of rev-erb-α expression in addition to sex differences in bmal1 expression. In the liver we detected strong rhythms in all genes examined, however only per2 expression was knocked out by RmTBI, in addition we also detected sex differences in per2 and cry1. We also detected an overall decrease in female clock gene expression in the early night. In the small intestine, RmTBI altered cry1 expression and there were sex differences in rev-erb-α. These results indicate that RmTBI alters core circadian clock gene expression in the central and peripheral nervous system in a time, tissue and sex dependent manner. This may be disrupting important phase relationships between the brain and peripheral nervous system and contributing to post-injury symptomology and also highlights the importance for time and sex dependent assessment of injury outcomes.

Gene expression changes in the cerebellum are associated with persistent post-injury pain in adolescent rats exposed to early life stress.

Chronic pain develops following injury in approximately 20% of adolescents, at twice the rate in females than males. Adverse childhood experiences also increase the risk for poor health outcomes, such as chronic pain. Emerging literature suggests the cerebellum to be involved in pain processing, however detailed explorations into how the cerebellum contributes to pain are lacking. Therefore, this study aimed to characterise chronic pain outcomes and cerebellar gene expression changes following early life stress and injury in both sexes. The adverse childhood experience of neglect was modelled using a maternal separation (MS) paradigm, which was combined with a subsequent injury (mild traumatic brain injury (mTBI) or plantar incision surgery) in adolescent male and female Sprague-Dawley rats. We measured behavioural nociceptive sensitivity, systemic modulators of pain such as calcitonin gene-related protein (CGRP) and Substance P, as well as gene expression of IL1ß, GFAP, GR, MR, GABRA1, CNR1, MAOA, and DAT1 in the cerebellum to examine associations between pain and neuroinflammation, the stress response, inhibitory neurotransmission, and monoaminergic function. We found increases in mechanical nociceptive sensitivity following plantar incision surgery. Sex differences were observed in anxiety-like behaviour and neuroinflammation, whereas systemic pain modulators showed cumulative effects with the addition of stressors. Most interestingly however, the increases in nociceptive sensitivity were associated with the suppressed expression of cerebellar genes that regulate stress, inhibition, cannabinoid function, and dopaminergic function, alongside sex-dependent distinctions for genes involved in inflammation and injury. This study highlights a novel link between nociception and molecular function in the cerebellum. Further investigation into how the cerebellum contributes to pain in males and females will facilitate novel therapeutic insights and opportunities.

Gut instinct: Sex differences in the gut microbiome are associated with changes in adolescent nociception following maternal separation in rats.

Adolescent chronic pain is a growing public health epidemic. Our understanding of its etiology is limited; however, several factors can increase susceptibility, often developing in response to an acute pain trigger such as a surgical procedure or mild traumatic brain injury (mTBI), or an adverse childhood experience (ACE). Additionally, the prevalence and manifestation of chronic pain is sexually dimorphic, with double the rates in females than males. Despite this, the majority of pre-clinical pain research focuses on males, leaving a gap in mechanistic understanding for females. Given that emerging evidence has linked the gut microbiome and the brain-gut-immune axis to various pain disorders, we aimed to investigate sex-dependent changes in taxonomic and functional gut microbiome features following an ACE and acute injury as chronic pain triggers. Male and female Sprague Dawley rat pups were randomly assigned to either a maternal separation (MS) or no stress paradigm, then further into a sham, mTBI, or surgery condition. Chronically, the von Frey test was used to measure mechanical nociception, and fecal samples were collected for 16S rRNA sequencing. Animals in the surgery group had an increase in pain sensitivity when compared to mTBI and sham groups, and this was complemented by changes to the gut microbiome. In addition, significant sex differences were identified in gut microbiome composition, which were exacerbated in response to MS. Overall, we provide preliminary evidence for sex differences and ACE-induced changes in bacterial composition that, when combined, may be contributing to heterogeneity in pain outcomes.

Diurnal circadian clock gene expression is altered in models of genetic and acquired epilepsy.

OBJECTIVES: Growing evidence demonstrates a relationship between epilepsy and the circadian system. However, relatively little is known about circadian function in disease states, such as epilepsy. This study aimed to characterize brain and peripheral core circadian clock gene expression in rat models of genetic and acquired epilepsy. METHODS: For the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) study, we used 40 GAERS and 40 non-epileptic control (NEC) rats. For the kainic acid status epilepticus (KASE) study, we used 40 KASE and 40 sham rats. Rats were housed in a 7 am:7 pm light-dark cycle. Hypothalamus, hippocampus, liver, and small intestine samples were collected every 3 h throughout the light period. We then assessed core diurnal clock gene expression of per1, cry1, clock, and bmal1. RESULTS: In the GAERS rats, all tissues exhibited significant changes in clock gene expression (P < 0.05) when compared to NEC. In the KASE rats, there were fewer effects of the epileptic condition in the hypothalamus, hippocampus, or small intestine (P > 0.05) compared with shams. SIGNIFICANCE: These results indicate marked diurnal disruption to core circadian clock gene expression in rats with both generalized and focal chronic epilepsy. This could contribute to epileptic symptomology and implicate the circadian system as a viable target for future treatments.

Serum neurofilament light as a biomarker of vulnerability to a second mild traumatic brain injury.

A second mild traumatic brain injury (mTBI) sustained prior to neuropathological recovery can lead to exacerbated effects. Without objective indicators of this neuropathology, individuals may return to activities at risk of mTBI when their brain is still vulnerable. With axonal injury recognized as a neuropathological hallmark of mTBI, we hypothesized that serum levels of neurofilament light (NfL), a highly sensitive biomarker of axonal injury, may be predictive of vulnerability to worse outcomes in the event of a second mTBI. Given this hypothesis is difficult to test clinically, we used a two-hit model of mTBI in rats and staggered inter-injury intervals by 1-, 3-, 7-, or 14-days. Repeat-mTBI rats were dichotomized into NfLhigh (NfL>median at the time of re-injury) and NfLlow (NfL