Probiotic treatment causes sex-specific neuroprotection after traumatic brain injury in mice

Background Recent studies have shed light on the potential role of gut dysbiosis in shaping traumatic brain injury (TBI) outcomes. Changes in the levels and types of Lactobacillus bacteria present might impact the immune system disturbances, neuroinflammatory responses, anxiety and depressive-like behaviors, and compromised neuroprotection mechanisms triggered by TBI. Objective This study aimed to investigate the effects of a daily pan-probiotic (PP) mixture in drinking water containing strains of Lactobacillus plantarum, L. reuteri, L. helveticus, L. fermentum, L. rhamnosus, L. gasseri, and L. casei, administered for either two or seven weeks before inducing TBI on both male and female mice. Methods Mice were subjected to controlled cortical impact (CCI) injury. Short-chain fatty acids (SCFAs) analysis was performed for metabolite measurements. The taxonomic profiles of murine fecal samples were evaluated using 16S rRNA V1-V3 sequencing analysis. Histological analyses were used to assess neuroinflammation and gut changes post-TBI, while behavioral tests were conducted to evaluate sensorimotor and cognitive functions. Results Our findings suggest that PP administration modulates the diversity and composition of the microbiome and increases the levels of SCFAs in a sex-dependent manner. We also observed a reduction of lesion volume, cell death, and microglial and macrophage activation after PP treatment following TBI in male mice. Furthermore, PP-treated mice show motor function improvements and decreases in anxiety and depressive-like behaviors. Conclusion Our findings suggest that PP administration can mitigate neuroinflammation and ameliorate motor and anxiety and depressive-like behavior deficits following TBI. These results underscore the potential of probiotic interventions as a viable therapeutic strategy to address TBI-induced impairments, emphasizing the need for gender-specific treatment approaches.


BACKGROUND
Traumatic brain injury (TBI) remains a signi cant cause of death and disability worldwide [1].The longterm effects of TBI are associated with an increased risk of neurodegenerative and neuropsychiatric disorders, such as Alzheimer's and Parkinson's disease [2,3].TBI leads to severe outcomes, with no existing treatments for neuroprotection or regeneration.The pathophysiology of TBI is closely linked to neuroin ammation, predominantly driven by microglial cells [4], and in ltrated macrophages, neutrophils, and leukocytes that contribute to secondary brain damage post-TBI [5].The in ammatory responses within the central nervous system (CNS) can lead to alterations in peripheral organs.For instance, in ammation in the intestinal lining can trigger an imbalance in the gut microbiome, known as gut dysbiosis [6].
Recent research suggests that many symbiotic bacteria living in our intestines may substantially in uence how the brain responds to chronic in ammation and disease [7].The microbiome-gut-brain axis represents a bidirectional connection between the gut, microbial communities, and brain.This connection is facilitated through the bloodstream or vagus nerve [8], affecting neurological health [9] and the pathogenesis of neurological disorders [10].The microbiome plays a critical role in essential neurological processes and structures, such as maintaining the integrity of the blood-brain barrier (BBB) [11], myelin formation [12], and microglial activation [13].
Interestingly, survivors of TBI often report gastrointestinal complaints, and animal studies have demonstrated gastrointestinal dysfunction and dysbiosis following TBI [10].Our previous studies have established a link between TBI and changes in gut microbiota, particularly a disruption known as bacterial dysbiosis [14].Imbalances in the gut microbiota are associated with worsened outcomes of TBI by leading to neurodegeneration [15] and negative impacts on brain recovery [16].Alterations in the gut microbiome extend beyond immediate local impacts, affecting essential bacterial populations such as the bene cial Lactobacillaceae family, which are vital for maintaining gut health and are prevalent within the human gastrointestinal system [17].A decrease or lack of these advantageous microbes can amplify neuroin ammation and exacerbate neurocognitive impairments linked to TBI.
Furthermore, TBI has also been associated with decreased levels of microbial metabolites, such as shortchain fatty acids (SCFAs), negatively in uencing the healing process [18].Recent research in animal models indicates that altering the gut microbiome via fecal microbiota transplants can improve recovery outcomes from TBI [19], and stroke [20].These bene ts are thought to be associated with the production of SCFAs, including butyrate, propionate, and acetate, generated through the bacterial breakdown of dietary bers [21,22].SCFAs are key in diminishing in ammation and mitigating oxidative stress [23], impacting BBB integrity, stimulating neurogenesis, reducing neuroin ammation, and promoting microglial maturation [24,25].Furthermore, probiotic treatments, which involve administering live bene cial microorganisms like Lactobacillus strains, have proven effective in boosting SCFA levels [26,27] thus potentially offering signi cant health advantages to the host [28].
Despite these previous ndings, there has been no investigation into the effects of probiotics comprising Lactobacillus strains on the modulation of microglia and motor and cognitive functions by altering the gut microbiota composition in a mouse model of TBI.We tested the hypothesis that administrating a mixture of probiotics from the Lactobacillus strains following TBI in mice would reduce gut dysbiosis and improve motor function by attenuating the neuroin ammatory response.Our ndings support the idea that Lactobacillus spp.treatment could represent a promising therapeutic approach to managing TBI, which would be easy to translate into a clinical setting, considering probiotics' well-documented safety and tolerability.

MATERIALS AND METHODS
Animals, traumatic brain injury model, and probiotic administration.
For our experiments, we utilized male and female C57BL/6 mice, aged 9-12 weeks and weighing 20-26 g, obtained from (Jackson Laboratories, Bar Harbor, ME, US).The mice were housed in an environment with a 14-h light/10-h dark cycle at a stable temperature of 23°C ± 2°C and had continuous access to food and water.After arriving, the mice were given a minimum of three days to adjust to their new surroundings before participating in any experimental procedures.Both adult male and randomly cycling female C57BL/6 mice were divided into groups for the study.We included only males for the germ-free (GF) mice because males have a higher in ammatory response after TBI than females [29].C57BL/6-GF male mice (12 weeks) were obtained from the Baylor College of Medicine Gnotobiotic Rodent Facility (Houston, TX).An internal standard was serially diluted to assess the "germ-free" status of the mice upon arrival, and the copy number of the 16S rRNA gene in feces from each transfer crate was analyzed using quantitative PCR (qPCR).16S rRNA gene was not detected in feces from transfer crates, con rming that the mice were GF.
Anesthesia was induced with 4-5% iso urane, then maintained at 1.5-2% with a 1-1.5 L/min oxygen ow rate.Each mouse, under anesthesia, was secured in a stereotaxic frame for the surgery.A moderate to severe TBI was in icted on the left side of the brain, targeting the primary motor and somatosensory cortices, using an electromagnetic device (Leica StereoOne Impactor) driven controlled cortical impact (CCI) injury.Throughout the surgical procedure, the body temperature of the mouse was maintained at 37°C using a heating pad.A midline incision approximately 10 mm long was made on the skull.The skin and fascia were pulled back to expose the skull, where a 4 mm craniotomy was then performed on the left parietal bone.The sterilized tip of 1.5 mm diameter was placed onto the surface of the exposed dura and set to deliver an impact to the cortical surface with a velocity of 3.25 m/s and a tissue deformation of 1.5 mm.Mice in the sham-operated group underwent identical procedures, including iso urane anesthesia, but without the CCI injury.Post-TBI, the incision was sutured, and the mouse was placed in a warmed recovery chamber to maintain average body temperature.All animals were closely monitored for 4 h post-surgery and then checked daily.The mice were distributed into four distinct groups, each consisting of 4 to10 mice: (i) a sham group (sham-operated control, vehicle (VH)-treated), (ii) a sham group (sham-operated with pan-probiotic (PP) treatment), (iii) a CCI group (VH-treated), and (iv) a CCI group (PP treatment) (Fig. 1).
Pan-probiotic (PP) mixture in drinking water containing strains of Lactobacillus plantarum, L. reuteri, L. helveticus, L. fermentum, L. rhamnosus, L. gasseri, and L. casei, administered for either two or seven weeks before inducing TBI on both male and female mice.Mice were treated with PP in their drinking water for 2 weeks before TBI and continued for 5 more weeks until euthanasia for tissue collection at 35 days post-injury (dpi).A separate set of mice was treated with PP for 7 weeks, injured and then euthanized at 3 dpi to assess the acute effects.All mice in the four groups were given a standard diet and either PP-treated or regular drinking water for VH groups.For euthanasia, the mice were deeply anesthetized using iso urane at either 3 dpi (acute injury) or 35 dpi (chronic injury) and underwent transcardiac perfusion with cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde.The GF mice were removed from the isolator in the gnotobiotic facility and transferred to the laminar ow with high e ciency particulate air (HEPA) lter to perform the CCI injury model.After surgery, GF mice were transferred to autoclaved cages, provided with autoclave chow and water, and maintained in a conventional animal holding room for 3 days until euthanasia.Brain tissues were then harvested for immunohistochemical analysis, and serum samples were collected via centrifugation of blood and stored at − 80°C for SCFAs analysis.Samples of intestinal tissue were promptly collected and immediately frozen in dry ice.This study was performed under the National Institute of Health guidelines, and the Institutional Animal Care approved all experiments and Use Committees at Baylor College of Medicine and Houston Methodist Research Institute (Houston, TX).

Preparation of bacterial cell suspension for oral feeding
The PP mixture comprised various Lactobacillus strains supplied by the American Type Culture Collection (ATCC) (Manassas, VA, USA).The strains included Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC BAA-793), Lactobacillus reuteri (ATCC 23272), Lactobacillus helveticus (ATCC BAA-2840), Lactobacillus fermentum (ATCC 23271), Lactobacillus rhamnosus (ATCC BAA-2836), and Lactobacillus casei (ATCC BAA-2843).Following the supplier's protocol, we cultured the strains in MRS broth (Beckton Dickinson, Sparks, MD), and preserved them as glycerol stocks at -80°C.To initiate culture growth, we prepared 5 mL of pre-warmed MRS broth with the glycerol stocks and then incubated these starter cultures at 37°C in a 5% CO 2 environment for 2 h.The bacteria were cultured overnight in 1 L of MRS broth under identical conditions until they reached the logarithmic growth phase, con rmed by measuring optical density at 600 nm (OD600).After growth, the bacterial cells were collected through several centrifugation steps at 3000xg and 4°C, followed by a wash in cold PBS.We then resuspended the bacterial pellets in 10% glycerol in PBS and promptly froze them in 1 mL aliquots for future administration to mice.The viability and concentration of the bacteria were veri ed by serial dilution and colony-forming unit (CFU) enumeration on agar plates.
Brain Tissue Preparation and Quanti cation of Lesion Volume.
The brains were extracted and further xed in 4% paraformaldehyde overnight.After 24 h of xation, the brains underwent cryopreservation in a 30% sucrose solution.Coronal brain sections measuring 16 µm in thickness were cut using a cryostat (Epredia Cryostar NX50, Fisher Scienti c, Waltham, MA).The sectioning process was done in intervals of 0.5 mm throughout the brain, beginning 1.56 mm anterior from the bregma landmark to ensure a consistent and thorough examination of the affected areas for subsequent histochemical analysis.An average of 10-12 brain sections, equally spaced from 0 to -2.70 mm relative to the bregma, were selected for cresyl violet staining to target the area affected by injury.
Sections were placed on gelatin-coated glass slides (SuperFrost Plus, Thermo Fisher Scienti c, IL) and submerged in 0.5% cresyl violet solution (Sigma-Aldrich, St. Louis, MO), prepared in distilled water and ltered.Following staining, the slides underwent a dehydration process in graded ethanol solutions (100%, 95%, 70%, and 50%) for 2 min each and were then cleared in xylene twice, each for 2 min.The sections were then covered and slipped using a Permount mounting medium (Thermo Fisher Scienti c) for preservation.The lesion area was quanti ed by examining every 16th section throughout the full range of the lesion, using a methodology outlined in our previous publications [29][30][31][32].The area of the ipsilateral hemisphere was also measured for each corresponding section.The lesion volume was calculated by multiplying the aggregate of lesion areas by the interval between sections.To determine the percent lesion volume, we divided the lesion area by the area of the total ipsilateral hemisphere.
Serum SCFA analysis.SCFAs were separated using mobile phases 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B).Separation of metabolites was performed on Acquity UPLC HSS T3 1.8 um (2.1×100mM).The SCFA were measured in ESI negative mode using a 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to an HPLC system (Agilent Technologies, Santa Clara, CA) with multiple reaction monitoring (MRM).The acquired data was analyzed using Agilent Mass Hunter quantitative software.(Agilent Technologies, Santa Clara, CA).
Immuno uorescence analysis and cell death assay.
We prepared serial free-oating brain sections, each 16 µm thick, for our immunohistochemical studies, at the dorsal hippocampus level.These sections underwent immunohistochemistry processing, which included 3 successive 5-min washes in PBS enhanced with 0.5% Triton X-100 (PBS-T).Sections were then treated with 5% normal goat serum (NGS) in PBS-T to block nonspeci c binding for 1 h at room temperature.Overnight incubation at 4°C followed, using 3% NGS in PBS-T with primary antibodies targeting anti-rabbit Iba-1 (Wako, #019-19741) at a dilution of 1:500 for labeling microglia/macrophages, and anti-rat F4/80 (R&D Systems, #MAB5580) at a dilution of 1:200.The following day the sections were washed 3 times for 5 min each in PBS-T and incubated with the corresponding secondary antibodies (all 1:1000, Invitrogen), for 2 h at room temperature.The sections were then rinsed with PBS three times for 5 min each, and incubated in PBS with DAPI solution (1:50,000, Sigma-Aldrich, St. Louis, MO) for counterstained nuclei.The sections were rinsed with distilled water and covered with Fluoro-Gel with Tris Buffer mounting medium (Electron Microscopy Sciences, Hat eld, PA).Semiquantitative image analysis of the staining's in the cortical regions was performed using Image J software as previously described [29].To assess cell death, brain sections were processed for DNA strand breaks using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) from Fluorescence In Situ Cell Death Detection kit (Roche Diagnostic, Indianapolis, IN) according to the manufacturer's instructions.
Image Acquisition and Sholl Analysis of Microglia Morphology.
To analyze the microglia morphology in the injured brains after 3 dpi, we used confocal microscopy to collect full thickness 40X magni cation z-stack images using a Leica confocal microscope at the injured cortex from 16 um-thick brain sections that were immunostained for Iba-1 (n = 4 per group).Z-stacks were collected at 2048 X 2048 resolution with 3 frame averages for each color channel and were exported as .TIF les.After importing the Z-stacks, we performed Sholl analysis of microglial morphology using the Neurolucida Explorer (MBF Bioscience) setting increments of 2 µm with a starting radius of 2 µm, and the branches were detected using the user-guided tree tracing.When examining microglial processes in detail using Sholl analysis, the number of intersections and the average length of processes were quanti ed across different distances from the cell body.We combined intersection data from each microglia in a region into single pro le plots, enabling comparison of the overall morphology between cortical microglia in the injured brains treated with VH or PP.
Histological Analysis of the Gut.
To assess gut in ammation, we evaluated intestinal villus-to-crypt ratios in the jejunal region of the intestine.The intestines harvested from mice treated with VH or PP after 3 or 35 dpi were initially xed in 4% paraformaldehyde for 48 h and transferred to 70% ethanol.Tissue processing was carried out using a Shandon Excelsior ES Tissue Processor, and embedding took place on a Shandon HistoCentre Embedding System, following the manufacturer's standard protocols for processing and embedding.
Subsequently, slides were sectioned to a thickness of 5 µm.The para n sections were then subjected to depara nization and rehydration.Hematoxylin solution was applied for 6 h at a temperature ranging from 60 to 70°C, followed by rinsing with tap water until the water became colorless.The tissue was differentiated using 0.3% acid alcohol in water, with 2 min treatments.After rinsing with tap water, the sections were stained with eosin for 2 min.Alcian blue staining was employed to visualize mucins produced by the goblet cells in the intestine.Depara nized and rehydrated sections were immersed in Alcian blue solution for 30 minutes and then counterstained with 0.1% Nuclear Fast Red for 5 minutes.Finally, the intestine sections were dehydrated and mounted using permanent mounting media.
Quantitative Analysis.
For quantitative analysis of immunolabeled sections, we implemented unbiased, standardized sampling techniques to measure tissue areas corresponding to the injured cortex showing positive immunoreactivity, as we previously described [29,31].For proportional area measurements, the microglia/macrophages Iba-1-immunoreactivity was reported as the proportional area of tissue occupied by immunohistochemical stained cellular pro les within a de ned target area.Thresholded images converted to 8-bit grayscale were made using ImageJ (NIH, Bethesda, MD).The thresholding function was then used to set a black-and-white threshold corresponding to the imaged eld, with the averaged background subtracted.Once a threshold was set, the "Analyze Particles" function was used to sum up the total area of positive staining and calculate the fraction of the total positive area for the stain as previously described [29].To quantify the number of TUNEL, Iba-1, and F4/80 + cells in the injured cortex, an average of four coronal sections from the lesion epicenter (− 1.34 to − 2.30 mm from Bregma) were counted and imaged for each animal, n = 8/group.
Collection and Extraction of Fecal Samples for DNA Analysis.
Fresh stool pellets were aseptically collected and placed in sterile tubes, immediately snap-frozen, and subsequently stored at − 80°C for preservation.Genomic bacterial DNA was extracted from these frozen stool samples utilizing the QIAamp PowerFecal Pro DNA Kit (Qiagen, Germantown, MD).To facilitate DNA extraction, bead beating was carried out in three cycles, each lasting one min, at a speed of 6.5 m/s.There was a 5 min rest period between each cycle.This mechanical disruption was performed using a FastPrep-24 system (MP Biomedicals, Irvine, CA).The DNA isolation proceeded per the manufacturer's instructions, following the bead-beating process.The concentration of the extracted genomic DNA was subsequently quanti ed using a DS-11 Series Spectrophotometer/Fluorometer (DeNovix, Wilmington, DE).
The speci c gene sequences employed were designed to target the 16S ribosomal RNA gene region V1 to V3.The primers used for ampli cation contain adapters for MiSeq sequencing and single-index barcodes so that the PCR products may be pooled and sequenced directly, targeting at least 10,000 reads per sample [33].Primers used for the 16S V1-V3 ampli cation were 27F (AGAGTTTGATYMTGGCTCAG, where Y = C (90%) or T (10%); M = A (30%) or C (70%)) and 534R (ATTACCGCGGCKGCTGG, where K = G (10%) or T (90%)) [34].Amplicons were generated using primers corresponding to the variable regions, and the PCR products were puri ed.Subsequently, sequencing libraries for the V1-V3 target were constructed following the instructions provided by the Illumina MiSeq system with end products of 300 bp paired-end libraries.

Amplicon sequence analysis pipeline
Raw data les in binary base call (BCL) format were converted into FASTQs and demultiplexed based on the single-index barcodes using the Illumina 'bcl2fastq' software.Demultiplexed read pairs underwent an initial quality ltering using bbduk.sh(BBMap version 38.82), removing Illumina adapters, PhiX reads and reads with a Phred quality score below 15 and length below 100 bp after trimming.16S V1-V3 qualitycontrolled reads were then merged using bbmerge.sh(BBMap version 38.82), with merge parameters optimized for the 16S V1-V3 amplicon type (vstrict = t qtrim = t trimq = 15).Further processing was performed using nf-core/ampliseq version 2.8.0 of the nf-core collection of work ows, utilizing reproducible software environments from the Bioconda and Biocontainers projects [35][36][37][38].Data quality was evaluated with FastQC (version 0.12.1) and summarized with MultiQC (version 1.18) [39].Sequences were processed sample-wise (independent) with DADA2 (version 1.28) to eliminate PhiX contamination, trim reads (forward reads at 275 bp and reverse reads at 265 bp; reads shorter than this were discarded), discard reads with > 2 expected errors, to correct errors, to merge read pairs, and to remove PCR chimeras.

Western Blot Analysis
Serum was collected by allowing trunk blood to clot at room temperature for 30 min, followed by centrifugation at 4000 revolutions per minute (rpm) and 4°C for 20 min.The serum was then stored at − 80°C.For analysis, serum samples were diluted 1:5 with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) and heated at 100°C for 10 min.The samples were then loaded onto 12% Mini-PROTEAN TGX Stain-Free gels (Bio-Rad Laboratories, Hercules, CA) for protein separation.Following electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA).The membranes underwent blocking with 5% w/v skim milk powder in PBS-Tween 20 (PBS-Tw) for 1 h at room temperature.They were then incubated overnight at 4°C with a goat anti-SAA primary antibody (1:500; AF2948, R&D Systems, Minneapolis, MN).This was followed by a 1 h room temperature incubation with a horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (1:3000; Thermo Fisher Scienti c, Waltham, MA).The membranes were developed using Clarity Western ECL (Bio-Rad Laboratories, Hercules, CA) and imaged with a ChemiDoc MP system (Bio-Rad Laboratories, Hercules, CA).Densitometry quanti cation and chemiluminescence imaging were carried out using ImageLab 6.0.1 software (Bio-Rad Laboratories, Hercules, CA).
Behavioral tests.
The rotarod test is a method employed to assess sensorimotor function in mice [45].For this evaluation, we used the Rotamex 5 system from Columbus Instruments (Columbus, OH) and followed the protocol as we have previously described [30,46].Each mouse underwent a training regimen spanning 2 days, with 3 daily trials.The procedure began by initially placing the mouse on a stationary rod, allowing the animal to explore for 30 sec.Then, the rod began to rotate, with the drum's speed gradually increasing from 4 to 40 rpm.Each trial concluded when the mouse fell off the rotarod, or a 5-min time span had elapsed, and the latency to fall was recorded.The mean value of these measurements served as the baseline assessment for each mouse.The testing sessions were conducted at 1, 3, 7, 14, and 35 dpi, with two investigators blinded to the animal groups overseeing the assessments.
Elevated Plus Maze Test.

Statistical Analysis
For the SCFA analysis, differentially expressed metabolites were detected using the unequal variance twosided Student's t-test followed by Benjamini-Hochberg correction, with signi cance achieved at an FDRadjusted p < 0.25.A one-way analysis of variance (ANOVA) with post-hoc Tukey's multiple comparison test was employed for immunohistochemistry data.We adhered to a signi cance threshold (alpha) 0.05 across all datasets.For the sholl analysis the area under the curve was calculated, and an unpaired Student's t-test was utilized to analyze mean uorescence intensity and cell count.We used two-way ANOVA for behavioral analysis followed by post-hoc Tukey's multiple comparison.Analysis of the rotarod test and histochemical/immuno uorescence utilized a one-way ANOVA post-hoc Tukey's multiple comparison to compare the time after injury and sex as the independent variables.All mice were randomized to experimental conditions, and experimenters were blind to the treatment groups throughout the study.Data was represented as the mean and standard error of the mean (± SEM), and *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered statistically signi cant.GraphPad Prism 8 Software (GraphPad; San Diego, CA, US) was used for statistical analysis.

RESULTS
Pan-probiotics increase the relative abundance of SCFAs in serum.
At 3 dpi, there were no signi cant differences in the levels of acetate, propionate, butyrate, isobutyrate, 2methyl-butyrate, isovalerate, valerate, and caproate between VH and PP treatment groups across both male and female mice (Fig. 2a-h).However, there was a general trend for an increase in the butyrate, isobutyrate, valerate, and caproate groups in the PP-treated males compared to females (Fig. 2c, d, g, h).
At 35 dpi, notable differences were observed, isobutyrate, 2-methyl-butyrate, isovalerate, valerate, and caproate levels demonstrated signi cant increases in the PP-treated males compared to VH, with 2methyl-butyrate and caproate also showing signi cant increases in PP-treated males versus females (**p < 0.01, *p < 0.05, respectively) (Fig. 2l-p).This indicates that the PP treatment successfully replenishes certain SCFAs, typically diminished following TBI, after 7 weeks of treatment.However, it is worth noting that there was a reduction in the relative abundance of acetate in the female PP mice when compared to their VH counterparts (Fig. 2i).These ndings suggest that the PP treatment's effect on SCFA pro les varies by sex in the setting of TBI, potentially in uencing metabolic and in ammatory responses.
The effect of pan-probiotic administration on gut microbiota composition.
Next, we evaluated whether PP treatment could affect the fecal microbiota composition and abundance based on the number of OTUs in the microbiome.The alpha diversity of the microbiome compositions revealed statistically signi cant differences between the VH and PP groups in the sham group over a simulated 7-week period, as indicated by the Shannon index (Fig. 3a).There was a statistically signi cant distinction between the VH and PP treatment groups in the TBI mice, according to the Simpson index (Fig. 3b), which adjusts for biases in sample uniformity, leading to a different result compared to the Shannon diversity of the same dataset.The introduction of PP should increase the microbial counts in the gut [49].The beta diversity analysis of the same study groups shows that there is a statistically signi cant variation between the VH and PP treatments after 2 weeks (Pr(> F) = 0.014), (R 2 = 0.063), (Fig. 3e-g).The relationship between the gut microbiomes of the VH and PP mice was not highly altered at the 2-week timepoint when observing the phylum level, as revealed by the stacked bar representation for the different groups, with a statistical signi cance between the sham-VH group mice.The Firmicutes to Bacteroidetes ratio showed a signi cant reduction in the VH group throughout treatment from 2 weeks to the 7 weeks sham-VH group (Fig. 3h), while a signi cant change was also observed between the sham and TBI groups in females (*p < 0.05, **p < 0.01, ***p < 0.001) (Fig. 3i).No signi cant differences were detected between the sham and the TBI PP-treated groups, indicating a return towards normal Firmicutes and Bacteroidota levels.
The temporal dynamics of microbiota genera following VH and PP treatments in mice revealed sexdependent modi cations in the microbiota composition.At 2-week of treatment, the relative abundance of the top 20 microbial genera in mice treated with VH exhibited a striking sex-dependent pattern.In male mice, there was a pronounced presence of Dubosiella, contrasting with the female mice, who showed a higher abundance of Faecalibaculum, particularly within those subjected to TBI-VH treatment (Fig. 4a).In the PP-treated groups, sham male mice demonstrated a higher frequency of Lactobacillus, while an increased presence of Faecalibaculum characterized their female counterparts.In TBI-PP mice, a notable dominance of Dubosiella was observed in males, many of which also harbored Faecalibaculum.In contrast, female mice predominantly displayed an abundance of Faecalibaculum (Fig. 4b).Lactobacillus was identi ed as the most prevalent genus throughout these subsets, indicating its signi cant role in the gut microbiota composition under these experimental conditions.By 7-week of treatment there was a notable balance in the microbial composition between male and female VH-treated mice, regardless of whether they were subjected to sham or TBI interventions (Fig. 4c).Conversely, in the PP-treated groups, sex-dependent variations were more pronounced.Sham-treated male mice predominantly hosted Ileibacterium, a genus not previously highlighted at an earlier time point.TBI-exposed female mice, however, showed a higher abundance of Lactobacillus relative to their male counterparts, underscoring the ongoing sex-speci c in uence of PP treatment in the context of TBI (Fig. 4d).
Germ-free mice show a higher lesion level than wild-type mice after TBI, and pan-probiotics treatment reduces the lesion and cell death following TBI in males.
GF male mice showed around 33% more lesion volume than wild-type male mice at 3 dpi (****p < 0.0001) (Fig. 6a), indicating the critical role of microbiota in brain damage repair.In our examination of the effects of a PP treatment on TBI, we observed that lesion volume showed a signi cant reduction in male subjects treated with PP compared to VH (*p < 0.05).At the same time, females did not exhibit a signi cant change (Fig. 6b).Representative cresyl-violet brain sections highlight the areas of the lesion (Fig. 6c).TUNEL assay results indicated a signi cant decrease in dying cells in males receiving PP treatment compared to VH (*p < 0.05), with females showing a less pronounced lesion volume compared to male counterparts in the VH group (*p < 0.05) (Fig. 6d, d1-4).These ndings suggest that the PP treatment may confer neuroprotective effects in a sex-speci c manner post-TBI.
Administering pan-probiotics diminishes the activation of microglia/macrophages in males three days following traumatic brain injury.
To evaluate the effects of PP treatment on the anti-in ammatory activity after acute TBI, we analyzed the activation of the microglia and macrophage population using the Iba-1 marker.The quantitative analysis of Iba-1 + cells revealed a signi cant decrease in cell density in male and female mice treated with PP compared to the VH group 3 dpi (Fig. 7a, c-f).The Iba-1 + area within the observed elds also showed a decrease, with the male PP group demonstrating a signi cant enlargement in comparison to the male VH group (***p < 0.001) (Fig. 7a, c-f).The Iba-1 + area within the observed elds also showed an increase, with the male VH group demonstrating a signi cant enlargement in comparison to the male PP group (**p < 0.01) (Fig. 7b, c-f).High-magni cation images of Iba-1 + cells (Fig. 7j-m) highlighted the morphological differences, with the VH-treated microglia appearing to have a more activated phenotype.
The Sholl analysis was performed, and data indicated a reduced complexity in the dendritic architecture in PP-treated males (Fig. 7g-I), as shown by the decreased number of intersections (Fig. 7n) and the shorter average length of processes (Fig. 7p) at distances greater than 10 µm from the cell body compared to the VH-treated mice.In addition, F4/80 + macrophages analysis revealed a signi cant elevation in cell density in VH-treated males in comparison to PP male mice (**p < 0.01) (Fig. 7r), aligning with the observed alterations in microglia morphology and suggesting a potential modulatory effect of PP on microglia activation.The females were not statistically signi cant when comparing PP and VH treatments (Fig. 7o, q).Representative images of F4/80 + staining corroborate the quanti ed data, with a noticeable reduction in signal intensity in the PP-treated groups compared to the VH-treated groups (Fig. 7s-w).Overall, the ndings suggest that post-TBI microglial activation and morphological changes are reduced by PP treatment, with more pronounced effects observed in males.
Pan-probiotic treatment improves motor and partiality and reduces anxiety-related behaviors associated with TBI.
In assessing behavioral outcomes following TBI, male and female mice exhibited distinct responses after PP treatment across various tests.In the rotarod test, males demonstrated a signi cant decrease in latency to fall post-TBI compared to sham-operated controls, indicating a motor de cit as previously established in our mouse CCI model.This effect was most pronounced in TBI-VH treated males at early time points (1, 3, and 7 dpi, ###p < 0.001), with gradual recovery over 35 dpi, yet still signi cantly lower than sham-VH groups (##p < 0.01 at 14 dpi and not signi cantly different at 35 dpi).Females showed a similar motor impairment, with TBI-VH groups exhibiting reduced latency to fall compared to sham-VH groups (###p < 0.001 at 1 and 3 dpi, ##p < 0.01 at 7 dpi) (Fig. 8a, b).PP treatment signi cantly improved the motor de cits induced by TBI in both male and female mice (Fig. 8a, b) (*p < 0.05 at 1 and 3 dpi for males and at 1 and 7 dpi for females).In the EPM, both male and female sham-PP and TBI-PP groups spent more time in the open arms entries, showing a non-signi cant trend towards increased compared to VH groups (p = 0.11 and p = 0.06, respectively) (Fig. 8c).
The FST revealed a signi cant reduction in immobility time observed between females sham-VH and TBI-VH groups (##p < 0.01).Additionally, female TBI-PP mice spent signi cantly less time immobile than TBI-VH groups (**p < 0.01), suggesting an effect of the PP treatment in improving the depressive-like behavior induced by TBI.In contrast, male TBI-PP subjects did not show signi cant differences in immobility time compared to TBI-VH groups, although a trend toward decreased immobility was noted (Fig. 8d).Shamoperated controls did not exhibit any signi cant changes across treatments.
Pan-probiotics did not alter the gut permeability and systemic in ammation.
In a histological examination of the intestines following TBI, both male and female subjects displayed changes in gut morphology at different time points post-injury.At 3 dpi, villi length in the VH-treated males and females appeared similar to sham controls; however, PP treatment resulted in a reduction in villi length in males that was not observed in females (Fig. 9a, a1-4, b).By 35 dpi, villi length in the PPtreated males signi cantly recovered.In contrast, PP-treated females showed a nonsigni cant trend towards increased villi length compared to VH-treated counterparts (**p < 0.01) (Fig. 9c, c1-4, d).Mucin + area analysis revealed no signi cant differences between the groups at 3 dpi (Fig. 9e, e1-4, f), and mucin + goblet cell count did not differ signi cantly (Fig. 9g).However, at 35 dpi, mucin + goblet cell numbers in PP-treated males were slightly increased compared to VH-treated males, though not reaching statistical signi cance (Fig. 9h, h1-4, i, j).As a systemic in ammatory marker, SAA levels showed no signi cant differences between groups.However, the PP treatment maintains a trend of reduction in SAA levels compared to the VH groups, suggesting that PP treatment reduces the acute phase response at 3 dpi, mainly in males (Fig. 9k).Additionally, body weight measurements for 35 days indicated no signi cant differences between groups, with all mice showing a typical growth curve regardless of sex or injury (Fig. 9l).Collectively, these results suggest that while PP treatment reduced villi length in females, the overall absence of signi cant changes in mucin production, goblet cell number, systemic in ammation (as measured by SAA levels), or body weight suggests that the observed histological alterations in the gut may be localized responses to TBI and treatment, rather than systemic effects.

DISCUSSION
In recent years, research has demonstrated the pivotal role of the gut-microbiome-brain axis in maintaining neurological health [51].The gut microbiota can affect CNS functions in both healthy physiological conditions, encompassing neurotransmission, neurogenesis, and glial activation [52].In the context of neuroin ammatory diseases, there is a signi cant alteration in the composition and diversity of the microbiome, which in uences the progression of neuropathological conditions [53].This phenomenon is observed in individuals who have suffered brain damage, who demonstrate increased systemic in ammation, changes in the composition of gut microbiota, disrupted intestinal motility, and neuroin ammation [54][55][56].
The temporal dynamics of microbial communities elucidated in this study provide valuable insights into the sex-dependent effects of probiotic treatment in the context of brain injury.Our study showed that gut microbiota dysbiosis following TBI can be reversed by administering PP, which concurrently exhibits neuroprotective effects within the injured brain.Our previous work showed the impact of TBI on bacterial dysbiosis corresponding to relative abundance and the diversity of the Lactobacillaceae family [14], which is one of the ubiquitously seen healthy bacteria in the human gut.Lactobacillus, a genus of bacteria belonging to the phylum Firmicutes [57], have been shown to have neuroprotective effects [58].A probiotic regimen consisting of Bi dobacterium, Lactobacillus, and Enterococcus faecalis markedly reduced levels of pro-in ammatory cytokines post-treatment in severe TBI patients [59].In our study, we observed a dramatic decrease in Lactobacillus gasseri at the species level at 24 h post-TBI [14].We hypothesized that the administration of a probiotic mixture consisting of various Lactobacilli strains, , and also enhances the microbial community's diversity [70].While the administration of probiotics does not correlate with their ability to colonize the gut permanently, research has shown that even if probiotics do not establish long-term residence in the intestines, their introduction can still modify the existing intestinal microbiota composition [71].
The positive impacts of probiotics are driven by their metabolite pro les, like SCFAs, regardless of whether the probiotic strain establishes long-term colonization in the intestine [66,71].SCFAs are recognized for their neuroprotective [72], neuronal survival, plasticity [73], and anti-in ammatory properties [24].They also modulate microglial activation [74], maturation and function [75] across various conditions [76].Lactobacilli produce SCFAs such as acetate, propionate, and butyrate [77].Our ndings indicate that PP treatment for 7 weeks increases the relative abundance of SCFAs in serum in a favorable metabolic environment for TBI recovery.Interestingly, SCFA levels did not show signi cant changes at 3 dpi.This might indicate that the acute neuroin ammatory response to TBI abolishes the production of SCFAs by selected PP.However, PP administration increases SCFA levels once the in ammation is restored, evidenced at 35 dpi.We have not observed differences between male and female mice, yet other studies have documented sex-based differences in SCFA pro les [78,79].These variations are likely attributable to the temporal course of hormonal disparities following TBI [29].
There were increases in microbial counts and signi cant variations in beta diversity, especially pronounced in TBI mice for 7 weeks of PP administration.Alpha diversity analysis also indicates an augmentation in the sham-treated male mice receiving PP compared to those treated with VH, with no similar increase observed in females, underscoring the gender-speci c microbial response.Our results suggest that the PP administration corresponded with changes in phyla like Firmicutes and Bacteroidetes and alterations in the gut microbiota that were speci c to each sex.Our ndings suggest a signi cant increase in the abundance of genera such as Lactiplantibacillus, Lactobacillus, and Limosilactobacillus, as well as the species L. gasseri in male mice, which could be indicative of the PP targeted effect on male microbiota.We found speci c shifts in microbial taxa in the PP treated mice, such as the increased abundance of bene cial bacteria like Lactobacillaceae, Limnosilactobacillus and Lactiplantibacillus populations, but a decline in L. helveticus, suggest that PP treatment could correct dysbiosis associated with TBI.Previous reports associated Limnosilactobacillus and Lactiplantibacillus with anti-in ammatory properties and gut health [80].Females in the TBI-PP group exhibited more complex taxonomic changes post-TBI with notable increases in taxa such as Faecalibaculum, Lactiplantibacillus, Lactobacillaceae, and Lachnospiraceae, along with Limosilactobacillus and L. gasseri.The increase of Clostridium taxa could suggest a nuanced interaction between the administered probiotic and host physiology in the injury context [81].We found an increase in Faecalibaculum in the TBI-PP-treated group.This bacterium is likely associated with the observed reduction in depression and anxiety [82].Surprisingly, the decline of L. helveticus in both sham and TBI-PP mice may re ect a rebalancing of the gut ecosystem, possibly correcting the associated dysbiosis providing an opportunity for other bacteria to compete more effectively for resources than L. helveticus.Also, different strains may produce substances that inhibit the growth of potentially bene cial bacteria [83].
A limitation of our current study is that the characterization of gut dysbiosis through short-read 16S sequencing is constrained by the small amplicon size, leading to possible taxonomic misidenti cation [84].16S amplicon sequencing is much more affordable and scalable than shotgun metagenomics.
However, the inability to con dently classify bacteria at the species and strain levels and the lack of accurate functional annotations that can be performed are major detriments when attempting to understand the gut microbiome changes [85].One promising area that spans the gap between short-read amplicon sequencing and shotgun metagenomics is nanopore full-length amplicon sequencing, which has been gaining popularity in microbiome research through its ability to identify bacteria taxa at the species level [86] which we will implement in future studies.
Our study observed a tendency for PP treatment to diminish the levels of SAA in serum, a protein associated with acute-phase peripheral in ammation, aligning with documented evidence that probiotic supplementation can lessen the occurrence of infections [87].Our ndings indicate no changes in the intestinal epithelium, implying that the PP-administered treatment does not adversely in uence gut integrity.Concerning neuroprotective outcomes, our data indicates that PP exerts a sex-speci c impact, predominantly evidenced by decreased lesion volume and cell death in male mice aligning with the observed neuroprotective outcomes, a nding that is consistent with outcomes reported in our prior research [29].Probiotic supplementation has been shown to mitigate apoptosis and enhance in ammatory states in animal models.For instance, the administration of C. butyricum diminished neuronal cell death triggered by bilateral common carotid artery occlusion in diabetic mice [88].Another investigation using broiler chickens as the model found that L. plantarum supplementation decreased apoptosis and intestinal in ammation, an outcome attributed to enhanced SCFA production and alterations in the microbiota composition [89].Our results show how animals lacking microbiota, GF mice, respond to TBI with extensive brain injury.Previous studies have indicated that supplementing GF mice with SCFAs ameliorates abnormalities in microglia [75].
The host-microbiota plays a role in microglial maturation and activation [90], and as the resident immune cells of the CNS, play a pivotal role in the brain's response to injury, shifting from a 'resting' state to an 'activated' state [91], and releasing proin ammatory cytokines [4].Interestingly, our ndings indicate that PP treatment exerts a differential effect on the microglial activation state, as evidenced by a signi cant decrease in Iba-1 + cell density and alterations in cell morphology, particularly in male mice.Moreover, the observed reduction in F4/80 + macrophages in male mice receiving PP treatment may also in uence peripheral immune cells in ltrating the brain after injury.The more marked effects seen in male, relative to female mice, align with an increasing body of evidence suggesting that male and female brains may exhibit distinct responses to TBI and subsequent treatments, a phenomenon we have previously demonstrated [29,92,93].Lactobacillus supplementation has been linked to reductions in anxiety and depression [67], [68].In our study, PP treatment tends to mitigate anxiety-related behaviors, as evidenced by the performance in the EPM and the FST in males, and an improvement in motor functions.A study in neonatal mice shows how infection with C. rodentium determined the role of L. rhamnosus and L. helveticus in mitigating corticosterone-induced colitis and stress-induced cognitive dysfunctions [94].Moreover, a novel composite probiotic mixture (VSL#3) containing diverse strains of Bi dobacteria and Lactobacilli prevented the diet-induced memory de cits, however, no differences were observed in anxietylike behavior [95].In a clinical study, participants who received a 4-week multispecies probiotics intervention showed a signi cantly reduced overall cognitive reactivity to sad mood [96].Consequently, in our research, employing PP appears to ameliorate the anxiety and depressive behaviors triggered by TBI, a nding that aligns with trends observed in other animal studies.

CONCLUSIONS
In conclusion, our study provides compelling evidence of the potential bene ts of PP supplementation in TBI.The modulation of the gut microbiome, metabolic pro les, and in ammatory processes, coupled with the neuroprotective effects and behavioral improvements observed, underscore the therapeutic promise of probiotics in a sex-dependent manner.The insights provided by these ndings emphasize the potential of targeting the gut microbiota and its metabolic pathways as a novel therapeutic strategy for TBI.These outcomes provide a promising avenue for developing probiotic-based interventions in TBI.However, further research is needed to fully elucidate the mechanisms underlying these effects and their long-term implications.These results collectively contribute to a burgeoning discourse on the gut-brain axis, emphasizing the potential of probiotic treatments in modulating the gut microbiome post-TBI, with implications for therapeutic interventions and recovery.
Taxonomic Pro le dynamics in the gut microbiome following pan-probiotic treatment.(a-d) The Shannon and Simpson indices in males and females indicate that fecal microbiota diversity was higher following a 2-week pan-probiotic (PP, in red) treatment when compared to a vehicle (VH, in blue) treatment in both the sham groups (n=10) and the traumatic brain injury (TBI) groups (n=29-30).This pattern persisted into the   2 weeks of treatment.Taxa names are listed on the y-axis, and the log fold change is plotted on the xaxis.In the sham treated with VH, both male (a) and female (b) groups showed no notable differences in microbial taxa.In the sham mice treated with PP, males (c) exhibited a signi cant rise in the abundance of Lactiplantibacillus, Lactobacillus, Limosilactobacillus, and L. gasseri.Females in the sham-PP group signi cantly increased, speci cally in Lactiplantibacillus (d).In the TBI mice, neither males (e) nor females (f) in the VH group demonstrated signi cant changes in taxa.However, males (g) in the TBI-PP group presented an increased abundance of Lactobacillaceae and Lactiplantibacillus, alongside decreased L. helveticus.Females (h) in the TBI-PP group revealed taxonomic alterations characterized by increased levels of Faecalibaculum, Lactiplantibacillus, Lactobacillaceae, Lachnospiraceae, and Limosilactobacillus, as well as L. gasseri, with a concurrent reduction in the abundance of the taxa L. helveticus and Clostridium.Error bars denote the standard error of the mean (SEM).A log-fold-change minimum threshold of 0.5 was used within the ANCOMBC2 visualization.Statistical signi cance was determined using the resulting q-value from the ANCOMBC2 analysis with (*q<0.05,**q<0.01,***q<0.001).Probiotic treatment restored gut morphology and slightly enhanced mucin production after traumatic brain injury (TBI) in male mice.Histological assessment of the intestinal morphology and mucin production was conducted at 3-and 35-days post-injury (dpi) in both male and female mice, using either a vehicle (VH) or a pan-probiotic treatment (PP).At 3 days post-TBI, there was no signi cant change in length of the villi in the colon across all groups (a, a1-4, b).At 35 dpi, male PP-treated mice showed a signi cant increase in villus length compared to their VH counterparts, highlighting the PP restorative potential (c, c1-4, d).When assessing mucin production in the colon, no signi cant differences were noted at 3 dpi regarding the mucin+ area or the count of mucin+ goblet cells across any group (e, f).The number of mucin+ goblet cells followed a similar trend (g).At 35 dpi, although not statistically signi cant, an increase in both mucin area and goblet cell numbers in the PP-treated groups hinted at a delayed bene cial effect of the probiotics (h, i).Serum amyloid A (SAA) levels, a marker of in ammation, remained unchanged across treatments and sexes.However, the PP group tends to show a more uniform reduction in SAA levels than VH (k).The study also observed body weight changes, indicating that TBI in uenced weight gain.PP-treated mice showed a trend towards quicker weight recovery than those given VH across both sexes (j).VH versus PP (**p<0.01).Scale bars (a1-4, c1-4, e1-4, h1-4) represent 50 µm.

Figure 9
Figure 9 The Elevated Plus Maze Test (EPM) is a neurobehavioral paradigm that examines the con ict between an mouse's innate exploratory drive and its fear of open areas.The EPM is designed to measure anxietyrelated behaviors.It consists of a plus-shaped apparatus with 2 open and 2 closed arms.Mice typically avoid open spaces due to the increased risk of exposure to predators.Therefore, animals that spend more time in the open arms are considered to exhibit less anxiety-like behavior.In our study, we utilized an EPM The Forced Swimming Test (FST) is a widely recognized animal model utilized to study despair behavior and is highly sensitive for evaluating the effects of pharmacological interventions on depression-like behaviors[48].The FST measures the propensity of the animal to give up when placed in a cylinder lled with water from which they cannot escape.Rodents will initially try to escape, but over time, they will start to oat more and struggle less, which is interpreted as a state of behavioral despair akin to depressivelike behavior.In this context, more time spent immobile ( oating without trying to escape) is considered an indicator of greater depressive-like behavior.At 22 dpi, mice were placed inside glass cylinders The cumulative time spent by the mice in this immobile state served as an indicator of behavioral despair.Additionally, any time the mice spent climbing or swimming was considered part of the total swimming time.
[47]ratus based on the design introduced by Pellow et al.[47].The plus-shaped maze is custom-made using white acrylic material.It comprises 2 open arms (50×10 cm) and 2 closed arms (50×10×40 cm), positioned in such a way that the 2 arms of each type are situated opposite to each other and connected by a central platform (5×5 cm).The maze is elevated to a height of 75 cm above the oor.At 21 dpi, each mouse was gently placed at the central platform of the maze, with its head oriented toward one of the open arms.Subsequently, we recorded data pertaining to the time spent in the open arms, over a 5-min duration.These measurements were expressed as percentages.After each trial, the maze's platform was cleaned with 70% isopropanol.The behavioral assessments of the mice were conducted during the light phase of the light-dark cycle.measuring30 cm in height and 20 cm in diameter, lled with water at a temperature of 25 ± 1°C, reaching a depth of 15 cm.The test duration was established to be 6 min.During the nal 4 min of the test, a trained observer, who was blinded to the treatment groups, recorded the behavioral responses of the mice.In this context, despair behavior in animals was quanti ed in terms of total swimming and immobility.Mouse immobility was passive oating with minimal movement, just enough to keep the animal's head above the water.
speci cally L. plantarum, L. reuteri, L. helveticus, L. fermentum, L. rhamnosus, L. gasseri, and L. casei.mighthave a neuroprotective action during the acute and chronic phases in a mouse model of TBI.Our selection was supported by literature documenting the bene cial effects of the Lactobacillus strains in various animal model studies, where modulation of the in ammatory response was a fundamental aspect of the disease process [60-63].Different strains of Lactobacillus have been shown to decrease the production of in ammatory cytokines such as TNF-α and IL-6 [64, 65], downregulate pro-apoptotic neurons, and activate in ammatory mediators[66].The administration of Lactobacillus has also been shown to mitigate locomotor dysfunction, anxiety, and depression after brain insults[67, 68].The probiotic L. acidophilus has been shown to help repair the intestinal mucosal barrier and improve the terminal ileum villus morphology and GI motility[69] 7-week treatment for both the sham (n=10) and TBI (n=19) groups under VH or PP treatments.Statistical signi cance was assessed using Bonferroni's multiple comparisons test (*p<0.033,**p<0.002,***p<0.001).(e-g) Principal Coordinate Analysis (PCoA) ordination plots were based on Weighted UniFrac distances and PERMANOVA assessed group dissimilarities.Beta diversity did not show signi cant differences between the VH and PP groups after 2 weeks of treatment nor between sham groups after 7 weeks of treatment.However, a signi cant variation in beta diversity was observed between VH and PP groups in TBI mice at the 7-week treatment (g).Firmicutes/Bacteroidetes ratio decreased in the VH group after two weeks compared to the VH from sham mice after 7 weeks of treatment in males (h) and females (i).In addition, the TBI group had an increased ration compared to the Sham mice at 7 weeks of treatment.No differences were observed in this ratio within the PP groups.Statistical signi cance was assessed using Bonferroni's multiple comparisons test (*p<0.033,**p<0.002,***p<0.001).