Cortical Impact Research Articles

Pulmonary Effects of Traumatic Brain Injury in Mice: A Gene Set Enrichment Analysis.

Acute lung injury occurs in 20-25% of cases following traumatic brain injury (TBI). We investigated changes in lung transcriptome expression post-TBI using animal models and bioinformatics. Employing unilateral controlled cortical impact for TBI, we conducted microarray analysis after lung acquisition, followed by gene set enrichment analysis of differentially expressed genes. Our findings indicate significant upregulation of inflammation-related genes and downregulation of nervous system genes. There was enhanced infiltration of adaptive immune cells, evidenced by positive enrichment in Lung-Th1, CD4, and CD8 T cells. Analysis using the Tabula Sapiens database revealed enrichment in lung-adventitial cells, pericytes, myofibroblasts, and fibroblasts, indicating potential effects on lung vasculature and fibrosis. Gene set enrichment analysis linked TBI to lung diseases, notably idiopathic pulmonary hypertension. A Venn diagram overlap analysis identified a common set of 20 genes, with FOSL2 showing the most significant fold change. Additionally, we observed a significant increase in ADRA1A→IL6 production post-TBI using the L1000 library. Our study highlights the impact of brain trauma on lung injury, revealing crucial gene expression changes related to immune cell infiltration, cytokine production, and potential alterations in lung vasculature and fibrosis, along with a specific spectrum of disease influence.

Dose-Dependent Tranexamic Acid Blunting of Penumbral Leukocyte Mobilization and Blood-Brain Barrier Permeability Following Traumatic Brain Injury: An In Vivo Murine Study.

Early posttraumatic brain injury (TBI) tranexamic acid (TXA) may reduce blood-brain barrier (BBB) permeability, but it is unclear if this effect is fixed regardless of dose. We hypothesized that post-TBI TXA demonstrates a dose-dependent reduction of in vivo penumbral leukocyte mobilization, BBB microvascular permeability, and enhancement of neuroclinical recovery. CD1 male mice (n = 40) were randomly assigned to TBI by controlled cortical impact (injury [I]) or sham TBI (S), followed by intravenous bolus of either saline (placebo [P]) or TXA (15, 30, or 60mg/kg). At 48h, in vivo pial intravital microscopy visualized live penumbral BBB microvascular leukocytes and albumin leakage. Neuroclinical recovery was assessed by Garcia NeurologicalTest scores and animal weight changes at 24h and 48h after injury. I + TXA60 reduced live penumbral leukocyte rolling compared with I + P (p < 0.001) and both lower TXA doses (p = 0.017 vs. I + TXA15, p = 0.012 vs. I + TXA30). Leukocyte adhesion was infrequent and similar across groups. Only I + TXA60 significantly reduced BBB permeability compared with that in the I + P (p = 0.004) group. All TXA doses improved Garcia Test scores relative to I + P at both 24h and 48h (p < 0.001 vs. I + P for all at both time points). Mean 24-h body weight loss was greatest in the I + P (- 8.7 ± 1.3%) group and lowest in the I +TXA15 (- 4.4 ± 1.0%, p = 0.051 vs. I + P) group. Only higher TXA dosing definitively abrogates penumbral leukocyte mobilization, preserving BBB integrity post TBI. Some neuroclinical recovery is observed, even with lower TXA dosing. Better outcomes with higher dose TXA after TBI may occur secondary to blunting of leukocyte-mediated penumbral cerebrovascular inflammation.

White Matter Integrity and Motor Function Disruption Due to Traumatic Brain Injury in Piglets: Impacts on Motor-Related Brain Fibers.

Pediatric traumatic brain injury (TBI) often induces significant disability in patients, including long-term motor deficits. Early detection of injury severity is key in determining a prognosis and creating appropriate intervention and rehabilitation plans. However, conventional magnetic resonance imaging (MRI) scans, such as T2 Weighted (T2W) sequences, do not reliably assess the extent of microstructural white matter injury. Diffusion tensor imaging (DTI) tractography enables three-dimensional reconstruction of specific white matter tracts throughout the brain in order to detect white matter injury based on anisotropic diffusion. The objective of this study was to employ DTI tractography to detect acute changes to white matter integrity within the intersecting fibers of key motor-related brain regions following TBI. Piglets were assigned to either the sham craniectomy group (sham; n = 6) or the controlled cortical impact TBI group (TBI; n = 6). Gait and MRI were collected at seven days post-surgery (DPS). T2W sequences confirmed a localized injury predominately in the ipsilateral hemisphere in TBI animals. TBI animals, relative to sham animals, showed an increased apparent diffusion coefficient (ADC) and decreased fractional anisotropy (FA) in fiber bundles associated with key brain regions involved in motor function. TBI animals exhibited gait deficits, including stride and step length, compared to sham animals. Together these data demonstrate acute reductions in the white matter integrity, measured by DTI tractography, of fibers intersecting key brain regions that strongly corresponded with acute motor deficits in a pediatric piglet TBI model. These results provide the foundation for the further development of DTI-based biomarkers to evaluate motor outcomes following TBI.

Fingolimod improves diffuse brain injury by promoting AQP4 polarization and functional recovery of the glymphatic system.

Diffuse brain injury (DBI) models are characterized by intense global brain inflammation and edema, which characterize the most severe form of TBI. In a previous experiment, we found that fingolimod promoted recovery after controlled cortical impact injury (CCI) by modulating inflammation around brain lesions. However, it remains unclear whether fingolimod can also attenuate DBI because of its different injury mechanisms. Furthermore, whether fingolimod has additional underlying effects on repairing DBI is unknown. The impact acceleration model of DBI was established in adult Sprague-Dawley rats. Fingolimod (0.5 mg/kg) was administered 0.5, 24, and 48 h after injury for 3 consecutive days. Immunohistochemistry, immunofluorescence analysis, cytokine array, and western blotting were used to evaluate inflammatory cells, inflammatory factors, AQP4 polarization, apoptosis in brain cells, and the accumulation of APP after DBI in rats. To evaluate the function of the glymphatic system (GS), a fluorescent tracer was injected into the cistern. The neural function of rats with DBI was evaluated using various tests, including the modified neurological severity score (mNSS), horizontal ladder-crossing test, beam walking test, and tape sensing and removal test. Brain water content was also measured. Fingolimod administration for 3 consecutive days could reduce the levels of inflammatory cytokines, neutrophil recruitment, microglia, and astrocyte activation in the brain following DBI. Moreover, fingolimod reduced apoptotic protein expression, brain cell apoptosis, brain edema, and APP accumulation. Additionally, fingolimod inhibited the loss of AQP4 polarization, improved lymphatic system function, and reduced damage to nervous system function. Notably, inhibiting the GS weakened the therapeutic effect of fingolimod on the neurological function of rats with DBI and increased the accumulation of APP in the brain. In brief, these findings suggest that fingolimod alleviates whole-brain inflammation and GS system damage after DBI and that inhibiting the GS could weaken the positive effect of fingolimod on nerve function in rats with DBI. Thus, inhibiting inflammation and regulating the GS may be critical for the therapeutic effect of fingolimod on DBI.

Identification and neuroprotective properties of NA-184, a calpain-2 inhibitor.

Our laboratory has shown that calpain-2 activation in the brain following acute injury is directly related to neuronal damage and the long-term functional consequences of the injury, while calpain-1 activation is generally neuroprotective and calpain-1 deletion exacerbates neuronal injury. We have also shown that a relatively selective calpain-2 inhibitor, referred to as C2I, enhanced long-term potentiation and learning and memory, and provided neuroprotection in the controlled cortical impact (CCI) model of traumatic brain injury (TBI) in mice. Using molecular dynamic simulation and Site Identification by Ligand Competitive Saturation (SILCS) software, we generated about 130 analogs of C2I and tested them in a number of invitro and invivo assays. These led to the identification of two interesting compounds, NA-112 and NA-184. Further analyses indicated that NA-184, (S)-2-(3-benzylureido)-N-((R,S)-1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide, selectively and dose-dependent inhibited calpain-2 activity without evident inhibition of calpain-1 at the tested concentrations in mouse brain tissues and human cell lines. Like NA-112, NA-184 inhibited TBI-induced calpain-2 activation and cell death in mice and rats, both male and females. Pharmacokinetic and pharmacodynamic analyses indicated that NA-184 exhibited properties, including stability in plasma and liver and blood-brain barrier permeability, that make it a good clinical candidate for the treatment of TBI.

Systemic treatment with ubiquitin carboxy terminal hydrolase L1 TAT protein ameliorates axonal injury and reduces functional deficits after traumatic brain injury in mice

Traumatic brain injury (TBI) is often associated with axonal injury that leads to significant motor and cognitive deficits. Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in neurons and loss of its activity plays an important role in the pathogenesis of TBI. Fusion protein was constructed containing wild type (WT) UCHL1 and the HIV trans-activator of transcription capsid protein transduction domain (TAT-UCHL1) that facilitates transport of the protein into neurons after systemic administration. Additional mutant proteins bearing cysteine to alanine UCHL1 mutations at cysteine 152 (C152A TAT-UCHL1) that prevents nitric oxide and reactive lipid binding of C152, and at cysteine 220 (C220A TAT-UCHL1) that inhibits farnesylation of the C220 site were also constructed. WT, C152A, and C220A TAT-UCHL1 proteins administered to mice systemically after controlled cortical impact (CCI) were detectable in brain at 1 h, 4 h and 24 h after CCI by immunoblot. Mice treated with C152A or WT TAT-UCHL1 decreased axonal injury detected by NF200 immunohistochemistry 24 h after CCI, but C220A TAT-UCHL1 treatment had no significant effect. Further study indicated that WT TAT-UCHL1 treatment administered 24 h after CCI alleviated axonal injury as detected by SMI32 immunoreactivity 7 d after CCI, improved motor and cognitive deficits, reduced accumulation of total and K48-linked poly-Ub proteins, and attenuated the increase of the autophagy marker Beclin-1. These results suggest that UCHL1 activity contributes to the pathogenesis of white matter injury, and that restoration of UCHL1 activity by systemic treatment with WT TAT-UCHL1 after CCI may improve motor and cognitive deficits. These results also suggest that farnesylation of the C220 site may be required for the protective effects of UCHL1.

Rapid volume pulsations of the extracellular space accompany epileptiform activity in trauma-injured neocortex and depend on the sodium-bicarbonate cotransporter NBCe1

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.

RACK1 promotes autophagy via the PERK signaling pathway to protect against traumatic brain injury in rats.

Neuronal cell death is a primary factor that determines the outcome after traumatic brain injury (TBI). We previously revealed the importance of receptor for activated C kinase (RACK1), a multifunctional scaffold protein, in maintaining neuronal survival after TBI, but the specific mechanism remains unclear. The aim of this study was to explore the mechanism underlying RACK1-mediated neuroprotection in TBI. TBI model was established using controlled cortical impact injury in Sprague-Dawley rats. Genetic intervention and pharmacological inhibition of RACK1 and PERK-autophagy signaling were administrated by intracerebroventricular injection. Western blotting, coimmunoprecipitation, transmission electron microscopy, real-time PCR, immunofluorescence, TUNEL staining, Nissl staining, neurobehavioral tests, and contusion volume assessment were performed. Endogenous RACK1 was upregulated and correlated with autophagy induction after TBI. RACK1 knockdown markedly inhibited TBI-induced autophagy, whereas RACK1 overexpression exerted the opposite effects. Moreover, RACK1 overexpression ameliorated neuronal apoptosis, neurological deficits, and cortical tissue loss after TBI, and these effects were abrogated by the autophagy inhibitor 3-methyladenine or siRNAs targeting Beclin1 and Atg5. Mechanistically, RACK1 interacted with PERK and activated PERK signaling. Pharmacological and genetic inhibition of the PERK pathway abolished RACK1-induced autophagy after TBI. Our findings indicate that RACK1 protected against TBI-induced neuronal damage partly through autophagy induction by regulating the PERK signaling pathway.

Cognitive and Motor Function Effects of Antipsychotics in Traumatic Brain Injury: A Systematic Review of Pre-Clinical Studies.

Traumatic brain injury (TBI) survivors often suffer from agitated behaviors and will most likely receive pharmacological treatments. Choosing an optimal and safe treatment that will not interfere with neurological recovery remains controversial. By interfering with dopaminergic circuits, antipsychotics may impede processes important to cognitive recovery. Despite their frequent use, there have been no large randomized controlled studies of antipsychotics for the management of agitated behaviors during the acute TBI recovery period. We conducted a systematic review and meta-analysis of pre-clinical studies evaluating the effects of antipsychotics post-TBI on both cognitive and motor recovery. MEDLINE and Embase databases were searched up to August 2, 2023. Pre-clinical studies evaluating the effects of antipsychotics on cognitive and motor functions post-TBI were considered. Risk of bias was evaluated with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool. We identified 15 studies including a total of 1188 rodents, mostly conducted in male Sprague-Dawley rats using cortical impact injury. The analysis revealed no consistent effect of haloperidol on motor functions, but risperidone was associated with a significant impairment in motor function on day 5 post-injury (7.05 sec; 95% confidence interval [CI]: 1.47, 12.62; I2 = 92%). Other atypical antipsychotics did not result in impaired motor function. When evaluating cognitive function, haloperidol- (23.00 sec; 95% CI: 17.42-28.59; I2 = 7%) and risperidone-treated rats (24.27 sec; 95% CI: 16.18-32.36; I2 = 0%) were consistently impaired when compared to controls. In studies evaluating atypical antipsychotics, no impairments were observed. Clinicians should avoid the regular use of haloperidol and risperidone, and future human studies should be conducted with atypical antipsychotics.

Piezo2 Contributes to Traumatic Brain Injury by Activating the RhoA/ROCK1 Pathways

Traumatic brain injury (TBI) can lead to short-term and long-term physical and cognitive impairments, which have significant impacts on patients, families, and society. Currently, treatment outcomes for this disease are often unsatisfactory, due at least in part to the fact that the molecular mechanisms underlying the development of TBI are largely unknown. Here, we observed significant upregulation of Piezo2, a key mechanosensitive ion channel protein, in the injured brain tissue of a mouse model of TBI induced by controlled cortical impact. Pharmacological inhibition and genetic knockdown of Piezo2 after TBI attenuated neuronal death, brain edema, brain tissue necrosis, and deficits in neural function and cognitive function. Mechanistically, the increase in Piezo2 expression contributed to TBI-induced neuronal death and subsequent production of TNF-α and IL-1β, likely through activation of the RhoA/ROCK1 pathways in the central nervous system. Our findings suggest that Piezo2 is a key player in and a potential therapeutic target for TBI.

Engineered Dual Antioxidant Enzyme Complexes Targeting ICAM-1 on Brain Endothelium Reduce Brain Injury-Associated Neuroinflammation.

The neuroinflammatory cascade triggered by traumatic brain injury (TBI) represents a clinically important point for therapeutic intervention. Neuroinflammation generates oxidative stress in the form of high-energy reactive oxygen and nitrogen species, which are key mediators of TBI pathology. The role of the blood-brain barrier (BBB) is essential for proper neuronal function and is vulnerable to oxidative stress. Results herein explore the notion that attenuating oxidative stress at the vasculature after TBI may result in improved BBB integrity and neuroprotection. Utilizing amino-chemistry, a biological construct (designated "dual conjugate" for short) was generated by covalently binding two antioxidant enzymes (superoxide dismutase 1 (SOD-1) and catalase (CAT)) to antibodies specific for ICAM-1. Bioengineering of the conjugate preserved its targeting and enzymatic functions, as evaluated by real-time bioenergetic measurements (via the Seahorse-XF platform), in brain endothelial cells exposed to increasing concentrations of hydrogen peroxide or a superoxide anion donor. Results showed that the dual conjugate effectively mitigated the mitochondrial stress due to oxidative damage. Furthermore, dual conjugate administration also improved BBB and endothelial protection under oxidative insult in an in vitro model of TBI utilizing a software-controlled stretching device that induces a 20% in mechanical strain on the endothelial cells. Additionally, the dual conjugate was also effective in reducing indices of neuroinflammation in a controlled cortical impact (CCI)-TBI animal model. Thus, these studies provide proof of concept that targeted dual antioxidant biologicals may offer a means to regulate oxidative stress-associated cellular damage during neurotrauma.

Temporal-Specific Sex and Injury-Dependent Changes on Neurogranin-Associated Synaptic Signaling After Controlled Cortical Impact in Rats.

Extensive effort has been made to study the role of synaptic deficits in cognitive impairment after traumatic brain injury (TBI). Neurogranin (Ng) is a calcium-sensitive calmodulin (CaM)-binding protein essential for Ca2+/CaM-dependent kinase II (CaMKII) autophosphorylation which subsequently modulates synaptic plasticity. Given the loss of Ng expression after injury, additional research is warranted to discern changes in hippocampal post-synaptic signaling after TBI. Under isoflurane anesthesia, adult, male and female Sprague-Dawley rats received a sham/control or controlled cortical impact (CCI) injury. Ipsilateral hippocampal synaptosomes were isolated at 24h and 1, 2, and 4weeks post-injury, and western blot was used to evaluate protein expression of Ng-associated signaling proteins. Non-parametric Mann-Whitney tests were used to determine significance of injury for each sex at each time point. There were significant changes in the hippocampal synaptic expression of Ng and associated synaptic proteins such as phosphorylated Ng, CaMKII, and CaM up to 4weeks post-CCI, demonstrating TBI alters hippocampal post-synaptic signaling. This study furthers our understanding of mechanisms of cognitive dysfunction within the synapse sub-acutely after TBI.

Dopamine enhances recovery after traumatic brain injury through ubiquitylation and autophagic degradation of RIPK1

BackgroundAlthough the neurotransmitter dopamine (DA) plays a crucial pathophysiologic role after traumatic brain injury (TBI), its function and specific underlying mechanisms of action remain unclear.MethodsAdult male mice underwent controlled cortical impact (CCI). We administered DA intraperitoneally to mice for 14 consecutive days, starting 8 h before CCI. On day 3 after brain injury, cortical lesion volume and brain water content were measured. On days 7–13, behavioral tests were performed.ResultsHerein we report that DA inhibits neural death after injury, which is mediated via the dopamine D1 receptor (DRD1). Our results also showed that DRD1 signaling promotes RIPK1 ubiquitination via the E3 ubiquitin ligase Chip and then degradation through autophagy. Importantly, in vivo data revealed that DRD1 signaling prevented neural death, suppressed neuroinflammation, and restored many TBI-related functional sequelae.ConclusionsThese data reveal a novel mechanism involving dopamine, and suggest that DRD1 activation positively regulates Chip-mediated ubiquitylation of RIPK1—leading to its autophagic degradation.

STING-Dependent Signaling in Microglia or Peripheral Immune Cells Orchestrates the Early Inflammatory Response and Influences Brain Injury Outcome.

While originally identified as an antiviral pathway, recent work has implicated that cyclic GMP-AMP-synthase-Stimulator of Interferon Genes (cGAS-STING) signaling is playing a critical role in the neuroinflammatory response to traumatic brain injury (TBI). STING activation results in a robust inflammatory response characterized by the production of inflammatory cytokines called interferons, as well as hundreds of interferon stimulated genes (ISGs). Global knock-out (KO) mice inhibiting this pathway display neuroprotection with evidence that this pathway is active days after injury; yet, the early neuroinflammatory events stimulated by STING signaling remain understudied. Furthermore, the source of STING signaling during brain injury is unknown. Using a murine controlled cortical impact (CCI) model of TBI, we investigated the peripheral immune and microglial response to injury utilizing male chimeric and conditional STING KO animals, respectively. We demonstrate that peripheral and microglial STING signaling contribute to negative outcomes in cortical lesion volume, cell death, and functional outcomes postinjury. A reduction in overall peripheral immune cell and neutrophil infiltration at the injury site is STING dependent in these models at 24 h. Transcriptomic analysis at 2 h, when STING is active, reveals that microglia drive an early, distinct transcriptional program to elicit proinflammatory genes including interleukin 1-β (IL-1β), which is lost in conditional knock-out mice. The upregulation of alternative innate immune pathways also occurs after injury in these animals, which supports a complex relationship between brain-resident and peripheral immune cells to coordinate the proinflammatory response and immune cell influx to damaged tissue after injury.

Traumatic Brain Injury Alters the Trajectory of Age-Related Mitochondrial Change.

Some epidemiologic studies associate traumatic brain injury (TBI) with Alzheimer's disease (AD). To test whether a TBI-induced acceleration of age-related mitochondrial change could potentially mediate the reported TBI-AD association. We administered unilateral controlled cortical impact (CCI) or sham injuries to 5-month-old C57BL/6J and tau transgenic rTg4510 mice. In the non-transgenics, we assessed behavior (1-5 days, 1 month, and 15 months), lesion size (1 and 15 months), respiratory chain enzymes (1 and 15 months), and mitochondrial DNA copy number (mtDNAcn) (1 and 15 months) after CCI/sham. In the transgenics we quantified post-injury mtDNAcn and tangle burden. In the non-transgenics CCI caused acute behavioral deficits that improved or resolved by 1-month post-injury. Protein-normalized complex I and cytochrome oxidase activities were not significantly altered at 1 or 15 months, although complex I activity in the CCI ipsilesional cortex declined during that period. Hippocampal mtDNAcn was not altered by injury at 1 month, increased with age, and rose to the greatest extent in the CCI contralesional hippocampus. In the injured then aged transgenics, the ipsilesional hippocampus contained less mtDNA and fewer tangles than the contralesional hippocampus; mtDNAcn and tangle counts did not correlate. As mice age their brains increase mtDNAcn as part of a compensatory response that preserves mitochondrial function, and TBI enhances this response. TBI may, therefore, increase the amount of compensation required to preserve late-life mitochondrial function. If TBI does modify AD risk, altering the trajectory or biology of aging-related mitochondrial changes could mediate the effect.

Fecal microbiota transplantation unveils sex-specific differences in a controlled cortical impact injury mouse model.

Contusion type of traumatic brain injury (TBI) is a major cause of locomotor disability and mortality worldwide. While post-TBI deleterious consequences are influenced by gender and gut dysbiosis, the sex-specific importance of commensal gut microbiota is underexplored after TBI. In this study, we investigated the impact of controlled cortical impact (CCI) injury on gut microbiota signature in a sex-specific manner in mice. We depleted the gut microflora of male and female C57BL/6 mice using antibiotic treatment. Thereafter, male mice were colonized by the gut microbiota of female mice and vice versa, employing the fecal microbiota transplantation (FMT) method. CCI surgery was executed using a stereotaxic impactor (Impact One™). For the 16S rRNA gene amplicon study, fecal boli of mice were collected at 3 days post-CCI (dpi). CCI-operated male and female mice exhibited a significant alteration in the genera of Akkermansia, Alistipes, Bacteroides, Clostridium, Lactobacillus, Prevotella, and Ruminococcus. At the species level, less abundance of Lactobacillus helveticus and Lactobacillus hamsteri was observed in female mice, implicating the importance of sex-specific bacteriotherapy in CCI-induced neurological deficits. FMT from female donor mice to male mice displayed an increase in genera of Alistipes, Lactobacillus, and Ruminococcus and species of Bacteroides acidifaciens and Ruminococcus gnavus. Female FMT-recipient mice from male donors showed an upsurge in the genus Lactobacillus and species of Lactobacillus helveticus, Lactobacillus hamsteri, and Prevotella copri. These results suggest that the post-CCI neurological complications may be influenced by the differential gut microbiota perturbation in male and female mice.

Functional recovery following traumatic brain injury in rats is enhanced by oral supplementation with bovine thymus extract.

Traumatic brain injury (TBI) is one of the leading causes of death worldwide. There are currently no effective treatments for TBI, and trauma survivors suffer from a variety of long-lasting health consequences. With nutritional support recently emerging as a vital step in improving TBI patients' outcomes, we sought to evaluate the potential therapeutic benefits of nutritional supplements derived from bovine thymus gland, which can deliver a variety of nutrients and bioactive molecules. In a rat model of controlled cortical impact (CCI), we determined that animals supplemented with a nuclear fraction of bovine thymus (TNF) display greatly improved performance on beam balance and spatial memory tests following CCI. Using RNA-Seq, we identified an array of signaling pathways that are modulated by TNF supplementation in rat hippocampus, including those involved in the process of autophagy. We further show that bovine thymus-derived extracts contain antigens found in neural tissues and that supplementation of rats with thymus extracts induces production of serum IgG antibodies against neuronal and glial antigens, which may explain the enhanced animal recovery following CCI through possible oral tolerance mechanism. Collectively, our data demonstrate, for the first time, the potency of a nutritional supplement containing nuclear fraction of bovine thymus in enhancing the functional recovery from TBI.

The cellular senescence response and neuroinflammation in juvenile mice following controlled cortical impact and repetitive mild traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of disability and increases the risk of developing neurodegenerative diseases. The mechanisms linking TBI to neurodegeneration remain to be defined. It has been proposed that the induction of cellular senescence after injury could amplify neuroinflammation and induce long-term tissue changes. The induction of a senescence response post-injury in the immature brain has yet to be characterised. We carried out two types of brain injury in juvenile CD1 mice: invasive TBI using controlled cortical impact (CCI) and repetitive mild TBI (rmTBI) using weight drop injury. The analysis of senescence-related signals showed an increase in γH2AX-53BP1 nuclear foci, p53, p19ARF, and p16INK4a expression in the CCI group, 5 days post-injury (dpi). At 35 days, the difference was no longer statistically significant. Gene expression showed the activation of different senescence pathways in the ipsilateral and contralateral hemispheres in the injured mice. CCI-injured mice showed a neuroinflammatory early phase after injury (increased Iba1 and GFAP expression), which persisted for GFAP. After CCI, there was an increase at 5 days in p16INK4, whereas in rmTBI, a significant increase was seen at 35 dpi. Both injuries caused a decrease in p21 at 35 dpi. In rmTBI, other markers showed no significant change. The PCR array data predicted the activation of pathways connected to senescence after rmTBI. These results indicate the induction of a complex cellular senescence and glial reaction in the immature mouse brain, with clear differences between an invasive brain injury and a repetitive mild injury.

Acute gastrointestinal permeability after traumatic brain injury in mice precedes a bloom in Akkermansia muciniphila supported by intestinal hypoxia

Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI.

Sex-dependent improvement in traumatic brain injury outcomes after liposomal delivery of dexamethasone in mice.

Traumatic brain injury (TBI) can have long-lasting physical, emotional, and cognitive consequences due to the neurodegeneration caused by its robust inflammatory response. Despite advances in rehabilitation care, effective neuroprotective treatments for TBI patients are lacking. Furthermore, current drug delivery methods for TBI treatment are inefficient in targeting inflamed brain areas. To address this issue, we have developed a liposomal nanocarrier (Lipo) encapsulating dexamethasone (Dex), an agonist for the glucocorticoid receptor utilized to alleviate inflammation and swelling in various conditions. In vitro studies show that Lipo-Dex were well tolerated in human and murine neural cells. Lipo-Dex showed significant suppression of inflammatory cytokines, IL-6 and TNF-α, release after induction of neural inflammation with lipopolysaccharide. Further, the Lipo-Dex were administered to young adult male and female C57BL/6 mice immediately after controlled cortical impact injury (a TBI model). Our findings demonstrate that Lipo-Dex can selectively target the injured brain, thereby reducing lesion volume, cell death, astrogliosis, the release of pro-inflammatory cytokines, and microglial activation compared to Lipo-treated mice in a sex-dependent manner, showing a major impact only in male mice. This highlights the importance of considering sex as a crucial variable in developing and evaluating new nano-therapies for brain injury. These results suggest that Lipo-Dex administration may effectively treat acute TBI.