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.


| INTRODUCTION
2][3][4][5] Depending on the nature of the impact and injury, TBI varies from a mild concussion to a severe, disabling, and long-lasting condition. 6Being a heterogeneous neurological disorder, TBI induces symptoms that range from brief unconsciousness and amnesia to coma and prolonged central nervous system (CNS) dysfunction.8][9] TBI, including mild repetitive TBI, is one of the most significant non-genetic and non-agerelated risk factors for neurodegenerative diseases, such as chronic traumatic encephalopathy (CTE), 10,11 Alzheimer's disease (AD), AD-like dementias, 5,[12][13][14][15] and even brain cancer. 168][19] Due to the complex nature of the pathology, which involves neurodegeneration, formation of lesions, disruption of blood-brain barrier (BBB), gliosis, and demyelination, among many processes, and because of the poor regenerative capacity of the brain, TBI treatments need to target multiple cellular and molecular pathways and processes. 20][23][24][25][26][27] Some of these peptides found in blood and CSF, including neuron-specific enolase (NSE), S100 calciumbinding protein B (S100B), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP), also serve as promising biomarkers of TBI.Circulating IgG antibodies against neuronal and glial antigens may also indicate neurodegenerative disease, nervous system injury, and inflammatory response, where the damage to the BBB, combined with the breakdown of cells and tissues containing neuronal and glial peptides, contributes to poor recovery from the injury and exacerbates neurological sequelae.This is particularly relevant to repetitive mild traumatic brain injury (mTBI) and consequent CTE because the immune system is activated continuously due to repeated BBB disruption and cascades of cytotoxic events. 10,11,28,294][35] Hence, the presence of serum IgG antibodies against food-derived antigens is considered a marker of immune tolerance associated with the production and activation of regulatory T cells (Treg) and not a marker of food sensitivity or intolerance. 36utritional support is considered a plausible approach toward ameliorating, and possibly preventing, the long-term consequences of head injury. 37,38TBI patients who do not meet the daily recommended allowance for key nutrients have a higher risk of mortality, longer hospital stays, and poor neurological scores. 37,39,40The current consensus is that advanced nutritional planning for patients with TBI needs to start promptly after the injury, as it improves their survival and recovery rates. 41][51][52][53][54] In this study, we sought to evaluate the impacts of nutritional supplements derived from bovine glandular tissues on TBI recovery efficiency.6][57][58][59][60] We hypothesized that the concentrated material extracted from thymus-nutrients, peptides, and other bioactive molecules combined-could facilitate faster TBI recovery.To this end, we tested three types of supplements derived from bovine thymus-the desiccated form (TD), cytosolic extract (TCF), and the nuclear extract (TNF), for their capacity to improve recovery following a parasagittal controlled cortical impact (CCI) in rats-an established model of TBI. 61We report that the functional recovery following CCI, as assessed by balance and memory tests, is significantly improved if the animals were supplemented with TNF, but not with other thymus extracts.We report that multiple molecular signaling pathways are differentially regulated in the hippocampus following TBI and with TNF supplementation.We further demonstrate that the induction of autoantibodies against neuronal and glial antigens by TNF through a possible oral tolerance mechanism may be beneficial during TBI recovery period.Together, our data support the need for optimal nutritional supplementation during TBI recovery phase.

| Study 1: CCI model
This study was conducted at PsychoGenics, Inc. (Paramus, NJ, USA), in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (IACUC number: PsychoGenics-242-0514, approved on July 14, 2021).Adult, male, Sprague-Dawley rats (6-7 weeks of age) were purchased from Charles River Laboratories (Wilmington, MA, USA).Animals were received at PsychoGenics Inc. and assigned unique identification numbers (PGI ID) and tail marks.Animals were housed in pairs in ventilated cages and were acclimated for 7 days prior to study enrollment.All rats were examined, handled, and weighed prior to the study initiation to assure adequate health and suitability.During the study, 12/12 h light/dark cycles were maintained.The room temperature was maintained between 20 and 23°C with a relative humidity around 50%.Chow (Laboratory Rodent Diet 5001; LabDiet, St. Louis, MO, USA) and water were provided ad libitum for the duration of the study.Animals were randomly assigned across treatment groups.Body weights were taken twice a week during the study.All animal procedures were reviewed by the DHMRI IACUC (protocol approved on August 20, 2020).Adult, male, Sprague-Dawley rats (6-7 weeks of age, Charles River Laboratories) were paired in ventilated cages and acclimated for 7 days prior to study enrollment.All rats were examined, handled, and weighed prior to the study initiation to assure adequate health and suitability.During the study, 12/12 h light/dark cycles were maintained.The room temperature was maintained between 20 and 23°C with a relative humidity around 50%.Chow (Teklad Rodent Diet 8604; West Lafayette, IN) and water were provided ad libitum for the duration of the study.Animals were randomly assigned across treatment groups.Body weights were taken twice a week during the study.Glandular extracts were administered orally (oral gavage, PO) starting on day 1 and ending on day 28.Rats were humanely euthanized on day 29.

| Supplements
Desiccated thymus and thymus gland extracts-cytoplasmic (Thymex™) and nuclear (Thymus PMG™)-were provided by Standard Process Inc. (Palmyra, WI, USA).Cytoplasmic and nuclear extracts were made from frozen raw bovine glands and dried on-site using Standard Process Inc. proprietary processes.All test products are in powder form and are suitable for consumption by animals and humans.These extracts have been commercially available as dietary supplements in the United States since the early 1950s.

| Study 1
The test articles (TD, TCF, and TNF) were homogeneously dispersed in sterile water and administered orally (oral gavage, PO) twice a day at a dose of 175 mg/kg (350 mg/kg/ day) and a dose volume of 5 mL/kg.Considering a 6.2 conversion factor between rat and human, the human equivalent dose would be approximately 3.4 grams per day. 62he treatment started 28 days before the CCI lesion (Day −28) and continued until the day before tissue collection.Fifteen rats were used in each of the following treatment groups: (1) CCI-Thymus Desiccated (TD; bovine thymus substance powder); (2) CCI-Thymus Extract-Cytosolic Fraction (TCF); (3) CCI-Thymus Extract-Nuclear Fraction (TNF); (4) CCI-Vehicle; and (5) Sham-Vehicle.

| Study 2
Test articles TD, TCF, and TNF and nuclear extracts of bovine heart, adrenal, liver, pancreas, and kidney glands were dissolved in sterile water and administered orally (oral gavage, PO) twice a day at a dose of 175 mg/kg (350 mg/kg/day) and a dose volume of 5 mL/kg.

| Parasagittal controlled cortical impact (CCI)
CCI to the parasagittal cortex was produced by an electronic cortical contusion device (Custom Design & Fabrication, Inc [CDF], Richmond, VA, USA) that creates a reliable contusion injury to the exposed area of the brain with a brass-tipped impounder.Rats were anesthetized with isoflurane (4%-5% for induction, 2%-3% for maintenance) (Novaplus™; Irving, TX, USA) at O2 flow of 300 cm 3 /min and were mounted on a stereotaxic frame.Under aseptic conditions, a sagittal incision was made on the scalp and the fascia was retracted to expose the cranium.A 6-mm-diameter trephine drill was used to open the skull centered approximately 4 mm lateral to the sagittal suture, mid-way between bregma and lambda.CCI brain injuries were produced with a 5-mm-diameter rounded brass impactor attached to a computer-controlled piston, propelled electronically with under the following parameters: velocity = 2.5 m/s; depth = 3 mm; and duration = 100 ms.After CCI, any cortical surface hemorrhaging was controlled, and the scalp was closed with surgical staples.The CCI was performed in the left-brain hemisphere.The animals were allowed to recover in a warm recovery chamber, and appropriate post-operative care was provided, including (1) administration of fluid by subcutaneous (SC) injection of 6 mL of Lactate Ringer's solution, (2) administration of analgesics by SC injection of buprenorphine at 0.05 mg/kg (1 dose immediately after injury and 1 dose the day following injury), and (3) daily monitoring for 3 days after surgery.Animals in the Sham group were subjected to the same anesthesia, craniectomy, and post-operative care and medication as CCI animals except for brain lesion.

| Beam balance test
The beam balance test examines vestibulomotor function of the animals. 63,64Animals were trained prior to surgery to maintain their balance on the beam for 60 s.The rats were tested during the first week following CCI injury at days 3, 5, and 7.The animals were gently placed on a suspended narrow beam 1.5 cm wide with a rough surface, and the duration that they remained on the beam was measured with a maximum cutoff time of 60 s.Each animal received three trials per test day, and the trials were averaged to obtain a beam balance time.Cushioned pads were placed under the beam to prevent injury in the event the animal fell.

| Morris water maze test
Two weeks after injury, rats were tested in the Morris water maze (MWM)-a well-characterized test of spatial learning and memory in rodents. 65,66The MWM test was conducted beginning two weeks after the injury (day 14 to day 20).Rats were acclimated to the test room at least 1 h prior to testing.The MWM consists of a circular pool (160 cm diameter × 50 cm height) containing water that was made opaque with non-toxic black paint to hide the escape platform.The water temperature was kept at 25 ± 1°C.The water surface was 15 cm from the rim of the pool, and the inner wall was always carefully wiped to eliminate any local cues.The pool was in a large room with several extra-maze visual cues, including highly visible geometric images (squares, triangles, circles, etc.) hung on the wall, diffused lighting, and curtains to hide the experimenter and the awaiting rats.Behavior was tracked by a video camera hanging above the pool.Video tracking software (ANY-maze; Wood Dale, IL, USA) recorded and analyzed the behavior of the animal in the pool.After training, the rats were gently dried with clean paper towels and placed in a warmed holding cage for the rats to dry before being returned to their home cage.

| MWM: Training
During training, rats were placed into the water facing the wall of the pool and allowed to search for the platform.If the rats found the platform within 60 s, the trials were stopped, and animals were allowed to stay on the platform for 30 s before being moved.If the platform was not found within 60 s, rats were placed on the platform for 30 s to allow them time to learn the position of the platform in relation to the room's visual cues.Rats were given 4 trials a day for 5 days, with a 30-s interval between trials.The starting point was randomly rotated for every rat in each trial.

| MWM: Probe trial
On the sixth day, the rats received a probe trial.The probe test consisted of placing the animals in the pool for 60 s without the platform and monitoring the time spent in the four quadrants of the pool.An animal that has learned the position of the platform will spend more time searching for that target quadrant of the pool.

| Interim serum collection
Blood from the tail vein (study 1) or jugular vein (study 2) (~1 mL) was collected the day before the first test article dosing (day −28 for study 1 and day 0 for study 2) and before CCI (day −1) for study 1.Whole blood was collected into tubes without any coagulant and allowed to clot by leaving it undisturbed at room temperature for 30 min.Samples were then placed in a refrigerated centrifuge at 4°C and were spun at 2000× g for 10 min.Following centrifugation, serum was transferred into a clean polypropylene tube.The samples were maintained at 2-8°C while handling.The samples were aliquoted into small tubes (2 aliquots/rat) and stored at −80°C.

| Terminal blood and tissue collections
Animals were anesthetized with isoflurane and monitored for loss of reflexes.Blood (approximately 2 mL) was collected via cardiac puncture into tubes without any coagulant and allowed to clot by leaving it undisturbed at room temperature for 30 min.Samples were then placed in a refrigerated centrifuge at 4°C and were spun at 2000× g for 10 min.Following centrifugation, serum was transferred into clean polypropylene tubes.The samples were maintained at 2-8°C while handling.The samples were aliquoted into small tubes (2 aliquots/rat) and stored at −80°C.In study 1, brain was extracted, the hippocampi dissected from both hemispheres, weighed, and placed in individual tubes.All tissue samples were frozen on dry ice and stored at −80°C.

| RNA extraction
A total of 25 RNA extractions (representing 5 rats per treatment group) from brain hippocampi (right and left hippocampi combined) were performed using the RNeasy Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's guidelines.RNA libraries were constructed from 600 ng of total RNA input using the Universal Plus mRNA-Seq kit (Tecan Trading AG, Switzerland), according to the manufacturer's instructions.Completed libraries were quantitated using qPCR, and their sizes were visualized on the Bioanalyzer (Agilent, Santa Clara, CA, USA).All libraries passed QC, showing neither the presence of adapter dimer contamination nor excess amplification, and produced an average size peak of 400-500 base pairs. 2.9 | RNA sequencing (RNA-Seq) All 25 libraries were combined in equimolar proportions into one pool and loaded, along with 1% PhiX spike-in, in a single NovaSeq S2 flow cell for clustering, with two lanes per sample.The sequencing was performed on a NovaSeq 6000 instrument to generate 150 bp reads.The run metrics showed low phasing (<0.1%) and error (0.25%) rates, indicating that the loading and the chemistry performance were optimum.Most clusters (83.6%) passed filters, which resulted in an overall yield of 1.51 terabase pairs with 92.7% of reads ≥Q30 (99.9% base accuracy).

| Read alignment and data analysis
Raw reads were archived locally and assessed for quality via FastQC (https:// www.bioin forma tics.babra ham.ac.uk/ proje cts/ fastqc/ ) and filtered using Trimmomatic v0.39 with a minimum per base PHRED score cutoff of 20 and read length of 150. 67Unpaired reads after filtration were discarded.Matching read pairs passing quality filtering were aligned to the Rattus norvegicus mRatBN7.2reference genome obtained from NCBI using STAR v2.7.3a (Figure S1A). 68Following alignment, we quantified transcript abundance using fea-tureCounts, version 2.0.0. 69Differential gene expression was inferred between experiment groups (Vehicle-CCI, TD-CCI, TCF-CCI, and TNF-CCI) versus the Vehicle-Sham group using DESeq2, version 1.26.0. 70Genes were considered differentially expressed if the FDR-adjusted p-value was less than .05.Gene expression overlap and exclusivity for the experiments were defined using UpSetR, 71 which is an R implementation of UpSet. 72hanges in canonical signaling pathways were assessed using Ingenuity Pathway Analysis (IPA) (QIAGEN Inc.; https:// digit alins ights.qiagen.com/ IPA). 73

| Western blot
Total protein was extracted from the left (injured) and right (contralateral) hippocampi, and protein concentration was determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher, Rockford, IL, USA).A 12% SDS-polyacrylamide gel was loaded with 50 μg of protein in each well and run at 200 V for 32 min; protein was electrotransferred onto an Amersham Protran nitrocellulose membrane (GE Healthcare, Boston, MA) at 10 V for 30 min.The membrane was then blocked with SuperBlock T20 blocking buffer (Thermo Fisher) for 1 h at room temperature with gentle shaking.Primary antibodies against Beclin1 (MBL Life Science; PD017MS), LC3 (MBL Life Science; M186-3), beta-actin (Thermo Fisher; MA-15739), and GAPDH (Thermo Fisher; MA5-15738) were diluted at 1:1000 with PBS-T and applied to the membrane overnight at 4°C.The membranes were washed with PBS-T and incubated with a 1:10 000 dilution of either goat anti-mouse IgG (H + L) HRP (Thermo Fisher; 31430) or goat antirabbit IgG (H + L) HRP (Thermo Fisher; 65-6120) secondary antibodies at 1:10 000 for 1 h at room temperature with gentle shaking.Following the secondary antibody incubation, the membranes were washed with PBS-T three times and then washed two times with 1XPBS.Immediately after washing, Pierce 1-Step Ultra TMB Blotting Solution (Thermo Fisher) was added to the membranes for visualization and then imaged using a ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA).Western blots were quantified using the National Institutes of Health ImageJ 1.53 software (Bethesda, MD, USA).

| Thymus antigen extraction and analysis
Antigens were extracted from TD, TCF, and TNF supplement samples essentially as described by Vojdani et al  (2009). 74Each sample was mixed on a multitube shaker for 2 h at room temperature in either coco buffer (0.5% NaHCO3, 1% NaCl), 10X PBS (pH 7.4), 70% ethanol, or cold acetone, respectively.After shaking, samples were centrifuged at room temperature at 2000× g for 15 min.The supernatant was removed and dialyzed against 0.01X PBS overnight in 23-cm-long snakeskin tubing.After dialysis, extracted antigens were combined, and protein concentration was determined using a Pierce BCA Assay Kit (Waltham, MA, USA).

| Antigen detection by enzyme-linked immunosorbent assay (ELISA)
Extracted antigens were diluted to a 2.5 ng/μL concentration with a 0.1 M carbonate/bicarbonate (pH 9.6) coating buffer.Then, 100 μL of the antigen solution was added to a polystyrene flat-bottom ELISA plate with a final well concentration of 250 ng/well.Plates were incubated overnight at 4°C to allow proteins to adhere to the wells and then washed with 300 μL of 1X PBS (pH 7.4) and 0.05% Tween-20 wash buffer.After washing, the wells were coated with 300 μL of 1% BSA and 0.05% Tween-20 in 1X PBS-blocking buffer at room temperature for 2 h.After incubation, the 1% BSA blocking buffer was decanted, and the plates were washed with wash buffer.Afterward, 300 μL of Superblock T20 (Thermo Fisher, PI37516) was added to all wells and then incubated on a shaker at room temperature for 2 h.Primary antibodies against neuron-specific enolase (NSE; Thermo Fisher, LF-MA0064), S100 calcium-binding protein B (S100B; Thermo Fisher, MA1-25005), myelin basic protein (MBP; Thermo Fisher, MA1-10837), glial fibrillary acidic protein (GFAP; Thermo Fisher, 13-0300), intercellular adhesion molecule 5 (ICAM-5; Thermo Fisher, PA5-42734), and brain-derived neurotrophic factor (BDNF; Thermo Fisher, PA1-18357) were diluted 1:250 in 1% BSA and 0.05% Tween-20 in 1X PBS-blocking buffer.In triplicate, 100 μL of diluted primary antibodies were added to their respective wells, with the negative controls receiving primary antibody diluent.The plates were then incubated on a shaker at room temperature for 2 h.The appropriate secondary antibodies were diluted to 1:15 000 in Milli-Q water and kept on ice.After primary antibody incubation, plates were washed just as before, and then 100 μL of the diluted secondary antibodies were added to all wells, including the negative controls.Plates were again incubated on a shaker for 2 h at room temperature and then washed.Subsequently, 100 μL of TMB Ultra Substrate (Fisher, PI34028) was added to all wells and incubated for 30 min at 37°C.Immediately after incubation, 50 μL of 2 M sulfuric acid stop solution was added to all wells to terminate the reaction and mixed on a shaker for 5 min.Optical density (OD) was read at 450 nm using a BioTek Synergy H1 spectrophotometer.All samples were run in triplicate.

| IgG detection in serum of animals from studies 1 and 2 using ELISA
Antigens used in this study included neuron-specific enolase (NSE; Sigma, SRP6108), S100 calcium-binding protein B (S100B; Sigma, APREST73328), and myelin basic protein (MBP; Sigma, APREST78641) along with cardiac proteins troponin (TNNC1; Sigma, APREST91012), myoglobin (MB; Sigma, APREST73560), and myosin heavy chain-7 (MYH7; Sigma, APREST70519).Other glandular tissue-specific antigens included adrenal antigens of CYP21A2 (Sigma, APREST83903), CYP17A1 (Sigma, APREST83903), and CYP11A1 (Sigma, APREST73008); hepatic antigens of CYP2D6 (Sigma, APREST83904), SepSecs (Sigma, APREST92927), UGT1A6 (Sigma, APREST88381), and ASGR2 (Sigma, APREST72295); pancreatic antigen PRSS1 (Atlas Antibodies, APREST89201); and renal antigen UGT1A6 (Sigma, APREST88381).Antigens were diluted to a concentration of 2.5 ng/μL with a 0.1 M carbonate/bicarbonate (pH 9.6) coating buffer.One hundred μL of each protein was added to its respective wells in duplicate with a final well concentration of 250 ng/well.The plates were incubated overnight at 4°C to allow the proteins to adhere and coat the wells.The plates were washed with 300 μL of 1X PBS (pH 7.4) and 0.05% Tween-20 wash buffer throughout the assay.The protein-coated plate was washed, and then, 300 μL of 1% BSA and 0.05% Tween-20 in 1X PBS-blocking buffer was added to all wells with incubation at 37°C for 2 h and then washed.Afterward, 300 μL of Superblock T20 (Fisher, PI37516) was added to all wells, incubated at 37°C for 2 h, and then washed.Serum samples were diluted 1:10 with 1% BSA and 0.05% Tween-20 in 1X PBS-blocking buffer.100 μL of each sample was added to the appropriate wells with incubation at 37°C for 2 h and then washed.After that, 100 μL of a 1:15 000 dilution of goat anti-rat IgG-HRP secondary antibodies (Fisher, 31470) was added to all wells with incubation at 37°C for 2 h and then washed.After incubation, 100 μL of TMB Ultra Substrate (Fisher, PI34028) was added to all wells, followed by incubation at 37°C for 30 min.Immediately after incubation, 50 μL of 2 M sulfuric acid stop solution was added to all wells, and the absorbance was measured at 450 nm with a BioTek Synergy H1 spectrophotometer.All samples were run in duplicates.

| Statistical analysis
A non-parametric Kruskal-Wallis one-way analysis of variance was performed for rats 5 days after CCI and 7 days after CCI individually to examine the latency to fall from beam balance test, followed by the Conover-Iman post-hoc test with p-value adjusted by the Benjamini-Hochberg method.
MWM test was analyzed with a Welch one-way analysis of variance to examine the treatment effect on target quadrant in the probe test (TQ), followed by a Benjamini-Hochberg post-hoc test.MWM probe trial results were analyzed with Dirichlet distribution according to Maugard et al. 75 , to determine whether the fraction of time spent in the quadrants was uniformly distributed.Bonferroni's method was used to adjust the p-values.In the case of a divergence from uniformity, a one-tailed single-sample ttest was applied to assess what were the quadrants responsible for this divergence.
A non-parametric Kruskal-Wallis one-way analysis of variance was performed to examine the ELISA IgG antibody response to targeted neuronal proteins.The Conover-Iman post-hoc multiple comparison test was conducted to further examine the difference between groups with no adjustment of p-values (Fisher's method).Fisher's exact test was performed to compare treatment effect on serum IgG levels.
Statistical significance was set at p < .05.Significant differences from Vehicle-Sham in figures were marked with asterisk (*), while lack of significant differences from Vehicle-CCI in figures was marked with (Ϯ).All statistical evaluations except for Dirichlet tests were performed using R statistical software package version 4.2.1 (R Core Team, 2022) for Microsoft Windows.Dirichlet tests on MWM probe trial results were programmed using Python in Jupyter Notebook with the Dirichlet package from Eric Suh and modified by Maugard et al (2019) 75 (https:// github.com/ xuod/ diric hlet).
Western blot data were analyzed using one-way ANOVA and Bonferroni's correction method.All data are reported as mean ± standard error of the means (SEM).

| Supplementation with TNF improves the vestibulomotor and memory functions following CCI
To begin investigating whether thymus-derived supplements would improve recovery following CCI, we developed an experimental protocol, where animals were pre-treated with TD, TCF, or TNF for 28 days prior to injury and continued to receive supplementation for 20 days following CCI (Table 1).Animals were divided into 5 groups (n = 15 per group).Vehicle-Sham group served as surgical control, and animals who experienced CCI but did not receive any supplementation served as untreated controls-Vehicle-CCI group.Treatment groups included TD-CCI, TCF-CCI, and TNF-CCI.
Animal weights were monitored throughout the study, and no significant main effect of treatments, or any treatment versus time interactions, on body weights among any of the treatment groups was observed (Figure S2).This finding demonstrates that neither the CCI procedure itself nor thymus glandular extracts had adverse effects on body weights during the study.
Next, we evaluated whether TD, TCF, or TNF treatments would enhance animal vestibulomotor function following CCI compared to vehicle-treated CCI animals.We used the beam balance test and measured the latency of animals to fall off the beam before and after CCI and with or without supplementation.At baseline, all animals were able to maintain their balance on the beam for at least 60 s (Figure 1A).All CCI-lesioned animals showed decline in latency to fall off the beam at days 3, 5, and 7 post-injury.Sham-Vehicle animals were able to maintain their balance on the beam on average for approximately 50 s at days 3, 5, and 7 following CCI, indicating minimal impact of the sham surgery on the vestibulomotor function of these animals.Among all 4 groups that underwent CCI, TNF-CCI animals stayed on the beam significantly longer on days 5 and 7 following the injury (Figure 1A).Notably, animals supplemented with TD or TCF extracts did not show significant improvement when compared to the Vehicle-CCI group.TBI is known to disrupt memory function, both short-and long-term. 76,77o test whether thymus extracts can mediate restoration of spatial learning and reference memory after brain injury, we tested animals using MWM on days 14-20 after CCI.Following a 5-day training protocol, animals were tested on day 20 (probe trial) after the injury and time spent in the target quadrant of the pool was recorded.We found that rats supplemented with TNF spent significantly longer time in the target quadrant of the pool compared to Vehicle-CCI, TD-CCI, and TCF-CCI groups (Figure 1B).We verified this observation using Dirichlet distribution analyses and found that both-the Vehicle-Sham and TNF-CCI groups-spent significantly more time in the target quadrant (over 25% of expected time between four quadrants) and significantly less than 25% of time in the opposite quadrant (Figure 1C).TNFsupplemented animals spent significantly less than 25% of time in the opposite, non-target quadrant (p = .00004655);their time spent in the target quadrant was significantly larger than 25% (p = .01871).The fraction of time spent in the adjacent quadrants did not differ from 25% for TNF-CCI group (p = .1079and .5783).Similarly, Vehicle-Sham group animals spent significantly more time (>25%) in the target quadrant (p = .00008542)and significantly less time (<25%) in the opposite quadrant (p = .0009534),whereas the fraction of time spent in the adjacent quadrants did not differ from 25% (p = .4091and .03557).Together, these data show that TNF group animals displayed a pronounced bias toward the target MWM quadrant with a distribution comparable to Vehicle-Sham group, indicating a significant recovery in cognitive function following CCI.

| RNA-Seq reveals a unique impact of TNF supplementation on hippocampal gene expression pattern
To begin dissecting the molecular pathways that may underlie TNF-mediated improvement in animal recovery following CCI, we used RNA-Seq approach to compare hippocampal gene expression profiles between all 5 groups of animals who underwent CCI versus the Vehicle-Sham group.In this study, we randomly selected 5 animals from each treatment group and combined the left and right hippocampi from each animal to perform analyses of global hippocampal gene expression changes following injury using RNA-Seq.Data quality assessment showed that over 98% of obtained sequences aligned to Rattus norvegicus genome (Figure S1A).We find that among the three thymus extracts, TNF administration elicits the largest change in the number of genes, including the unique genes, that are differentially expressed compared to the Vehicle-Sham group (Table S1).
Next, we assessed the similarities and the differences in gene expression patterns between all groups using UpSetR-a novel visualization technique that enables investigation of overlaps and uniqueness in set-based data. 71,72The largest number of genes differentially expressed compared to the Vehicle-Sham group was observed in TNF-CCI animals, while TNF-CCI and TD-CCI groups display the largest number of common differentially expressed genes (Figure 2A).In addition, TNF supplementation alone elicited the largest impact on expression of genes within individual canonical signaling pathways compared to other experimental groups (Figure 2B), and TNF-CCI and TD-CCI groups together accounted for nearly 100% of signaling pathways that are modulated by thymus extract supplementation at gene expression level (Figure 2B).
We further examined the expression patterns of 50 most upregulated and 50 most downregulated genes in each treatment condition compared to Vehicle-Sham group.We found that TNF-CCI group induced changes in gene expression levels that were not observed with other supplements and in Vehicle-CCI animals, while expression patterns were most different between the TNF-CCI versus TCF-CCI or Vehicle-CCI groups (Figure 3).Differences in hippocampal gene expression patterns between the animal groups were also observed using volcano plots (Figure S1B-E).These results suggest that the nuclear fraction of bovine thymus, particularly its concentrated TNF form, may carry unique bioactive components with significant impact on gene regulation that could explain, in part, the observed enhancement of functional recovery following TBI.

| Signaling pathways implicated in TBI pathology and recovery are modulated by thymus extract supplementation
Brain injury induces changes in many cellular and molecular pathways that respond to tissue damage and support healing and recovery. 78We therefore used Ingenuity Pathway Analysis (IPA) to evaluate which canonical signaling pathways are affected by CCI alone and can be modulated by thymus extract supplementation and the extent to which each of the thymus extracts impacts individual pathways, in relation to the Vehicle-Sham group.We find that the number of signaling pathways significantly altered in thymus extract-supplemented animals differs dramatically compared to the Vehicle-CCI group (compare Figure S3A-C vs. S3D).Importantly, the changes in signaling pathways that are affected by CCI and thymus extract treatments are similar between TNF-CCI and TD-CCI groups, reflecting fewer differentially expressed genes compared to Vehicle-Sham group (Figure S3A vs. S3B; Table S1).
0][81][82][83] Together, these data show that extracts containing the thymus nuclear fraction exert pronounced effects on hippocampal gene expression patterns following CCI.Yet, only TNF supplementation significantly improves animal recovery.

| Autophagy marker expression in the hippocampus is modulated by TNF supplementation
5][86] While Beclin1 is a protein important for induction of autophagy, microtubule-associated protein 1A/1B-light chain 3 (LC3) plays a key role in the accumulation of autophagosomes. 87Reduction in the hippocampal expression of Beclin1 and LC3 proteins by nutritional supplementation with resveratrol, however, was found to be beneficial in the context of recovery from TBI. 88 Considering that phagosome formation is one of the top signaling pathways impacted by TNF supplementation in our study, we examined the expression of both autophagy markers in the hippocampi of tested animals 21 days following CCI at the protein level.We find that Beclin1 levels in the left hippocampi, the side of CCI, are moderately but not significantly reduced with thymus extract supplementation compared to the Vehicle-CCI group (Figure 5A,B,E).Importantly, the levels of LC3 in the left hippocampi are significantly reduced in TNF-supplemented animals compared to the Vehicle-CCI group (Figure 5C,E; p = .035).No significant changes in Beclin1 and LC3 expression were detected in the right hippocampi (uninjured side).These results suggest that the process of autophagy is modulated by TNF supplementation.

I G U R E 3
Unique patterns of most up-and downregulated DEGs are observed with thymus extract supplementation.DEGs were compared between thymus extract-supplemented and the Vehicle-Sham groups.Unique DEGs per treatment group included 151, 65, 22, and 31 genes for TNF-CCI, TD-CCI, TCF-CCI, and Vehicle-CCI groups, respectively.A total of 167 unique DEGs were detected across all groups with an adjusted p-value <.05.Log2 expression fold changes for each gene are shown.

| Extracts derived from bovine thymus contain antigens found within the nervous system, and their oral supplementation increases serum levels of IgG antibodies against those antigens
0][91][92][93] However, when autoantibody production is elicited by antigens delivered in food, the immune response can be suppressed through an oral tolerance mechanism, which involves initial production of IgM antibodies against given antigens and later transition to production of IgG antibodies against the same antigens, which can confer long-term suppression of the immune response. 94,95When antigens delivered in food are identical or highly homologous to those that are normally not present in circulation at high levels and are released from the brain tissues upon injury, the circulating IgG antibodies induced through ingestion of food would prevent the immune system attack on cells that carry those antigens, thus reducing inflammation.6][57][58][59][60] We therefore tested whether TD, TCF, and TNF extracts contain antigens found in the nervous system and whether antibodies against those antigens could be induced in rats after 28 days of oral supplementation.We find that TD, TCF, and TNF extracts contain multiple antigens commonly implicated in neurodegenerative diseases-GFAP, MBP, NSE, and S100B-whereas BDNF and ICAM-5 are not detected in thymus extracts at appreciable levels (Figure 6A).Among the three extracts, TD contains the highest level of neuronal and glial antigens.
We next examined whether supplementation with thymus extracts or nuclear extracts of other bovine glandular tissues could induce antibodies against neuronal and glial antigens.To evaluate serum IgG antibody induction, animals were supplemented with TD, TCF, and TNF, as well as with nuclear fraction extracts from bovine heart, adrenal gland, liver, pancreas, and kidney, for 28 days and serum IgG levels against GFAP, MBP, NSE, and S100B were measured using ELISA (Figure 6B,C).We find that the strongest induction of serum IgG directed against the selected neuronal and glial antigens occurs with TCF, TNF, and heart nuclear fraction supplementation (Figure 6B).Interestingly, supplementation with TD, which we found to contain the largest amounts of nervous system antigens, does not elicit strong induction of IgGs against those same antigens, similarly to all other glandular tissue nuclear extracts we tested (Figure 6B,C).
The observation that the nuclear fraction of bovine heart could elicit production of IgG antibodies against nervous system proteins, NSE and MBP, similarly to TCF and TNF (Figure 6B,C), led us to further investigate the possible relationship between the thymus and the heart tissue antigenicity and whether induction of IgGs against tissuespecific antigens is a common feature among glandular tissue extracts.We first measured the induction of serum IgG antibodies against heart tissue markers-MYH7, myoglobin, and troponin, in rats supplemented with TD, TCF, and TNF and with nuclear extracts of bovine heart, adrenal gland, liver, pancreas, and kidney.A large induction of IgG antibodies against myoglobin and moderate induction of IgGs against troponin were observed with heart-nuclear fraction supplementation, while serum levels of IgGs against MYH7 remained largely unchanged (Figure S4B).TCF and TNF extract supplementation increases the serum levels of IgGs against myoglobin and troponin, while TD extract increases the levels of IgGs against MYH7 (Figure S4B).
We next tested whether bovine glandular tissue extracts, namely nuclear fraction extracts from bovine F I G U R E 5 Levels of autophagy marker, LC3 protein, in the hippocampus are reduced by TNF supplementation.(A, B) Beclin1 protein levels were measured by western blot in left and right hippocampi separately, with left hippocampus being in proximity to the site of CCI.No statistically significant differences in Beclin1 levels were detected among all tested conditions.(C, D) LC3 protein levels are significantly reduced in the left hippocampus of TNF-supplemented animals (injured side).(E) Representative western blot images showing Beclin1, LC3, and actin protein bands.*p = .035;n = 5 animals per group.heart, adrenal gland, liver, pancreas, and kidney, could also induce IgG antibodies against their respective tissue antigens.We find that all tested extracts can induce production of IgG antibodies against some of the antigens specific to their respective tissues of origin, with heart and adrenal gland extracts being most potent (Figure S4A,C).
Together, these data demonstrate that induction of IgG antibodies against antigens delivered through oral route, potentially without eliciting an inflammatory response, is possibly one of the mechanisms whereby bovine tissue extract supplementation could confer organ and tissue protection in diverse pathological conditions that are exacerbated by immune-mediated inflammatory damage.

| TNF supplementation promotes induction of IgG antibodies against glial and neuronal antigens in animals that underwent CCI
To evaluate the possibility that the benefit of oral TNF supplementation in enhancing the recovery from TBI F I G U R E 6 Thymus extracts contain antigens found in the nervous system and induce production of IgG antibodies in rats supplemented for 28 days.(A) BDNF, GFAP, ICAM, MBP, NSE, and S100B antigens were detected in TD, TCF, and TNF extracts using ELISA.TD supplement contains the largest amounts of GFAP, NSE, and S100B antigens compared to the other two types of extracts.N = 4 samples per extract.(B, C) Serum levels of IgG antibodies against GFAP, MBP, NSE, and S100B were measured using ELISA following 28 days of supplementation with either thymus extracts or nuclear extracts from other bovine glandular tissues-heart, adrenal gland, liver, pancreas, and kidney.A pronounced induction of serum IgG antibodies against GFAP, MBP, and NSE was observed in animals supplemented with TCF, TNF, and heart-nuclear fraction extracts.Among thymus extract-supplemented animals, IgG induction was higher in TCF and TNF groups (p = .03and .051,respectively, n = 5 per group).IgG levels in TCF-supplemented animals were higher compared to all groups besides TNF-and heart-nuclear fraction-supplemented animals (p < .05)(C).
stems from its capacity to induce IgG antibodies toward neuronal and glial antigens, in the context of oral tolerance, we first evaluated whether supplementation with TNF, TCF, and TD increases serum IgG antibodies against GFAP, MBP, NSE, and S100B following CCI.We find that while IgG induction against GFAP, MBP, and NSE is generally increasing with TNF and TCF supplementation (Figure 7A-C), combined IgG levels against all tested antigens are significantly higher in TNF-supplemented animals on day 21 following CCI compared to all other groups (Figure 7E,F; p < .05).These data show that continuous TNF supplementation prior to and following brain injury increases serum levels of IgG antibodies against nervous system antigens.

| Serum IgG levels correlate with vestibulomotor function recovery
The notable increase in the levels of IgG antibodies against nervous system antigens observed with TNF supplementation led us to further examine the possible relationship F I G U R E 7 Increase in IgG antibodies against neuronal and glial antigens is induced by TNF supplementation.(A-D) IgG antibodies against GFAP (A), MBP (B), NSE (C), and S100B (D) were measured in serum samples prior to CCI injury (day 0) and on day 21 following CCI using ELISA.(E, F) Combined IgG levels against tested antigens show significant increase in TNF-supplemented animals at day 21 following CCI.*p < .05;n = 13-15 per group.
between the induction of IgG antibodies and functional recovery from TBI.To this end, we performed simple linear regression analyses to determine the correlation between combined IgG levels in individual animals and performance on behavioral tests for each experimental group.We find that the induction of serum IgG levels between day −28 and day 0, prior to CCI, positively correlates with performance on beam balance test at day 5 following injury in TNF-supplemented animals (Figure 8A).Conversely, increase in IgG levels in Vehicle-CCI animals negatively correlates with the latency to fall off the beam at day 5 (Figure 8D).No notable correlations between serum IgG levels and beam balance test performance were found in TD-CCI and TCF-CCI animals (Figure 8D).Likewise, no significant correlations were found between serum combined IgG levels and latency to fall off the beam at day 7 among all groups, except for Vehicle-CCI animals, whose IgG levels were negatively correlated with balance beam performance (data not shown).Likewise, no significant correlations were found between serum IgG levels and performance on MWM test of spatial memory across all groups (data not shown).These results demonstrate that the increase in IgG antibodies in TNF-supplemented animals, but not in other supplemented CCI groups, is associated with better vestibulomotor function recovery following CCI injury, while high IgG antibody levels against neuronal and glial antigens in Vehicle-CCI group are correlated with poor balance function recovery.Together with reduction in autophagy marker expression, our data suggest that TNF supplementation may be beneficial for faster cellular and functional recovery from TBI and can be possibly assisted by immune system tolerance toward nervous system antigens, induced through an oral tolerance mechanism.
F I G U R E 8 Correlation between serum IgG levels against nervous system antigens and performance on beam balance test.(A-D) Correlation between combined IgG levels against GFAP, MBP, NSE, and S100B and latency to fall off the beam on day 5 following CCI was examined in individual animals using simple linear regression analyses (n = 13-15 per group).Higher IgG levels in TNF-supplemented animals (A) correlated with higher latency to fall off the beam.In Vehicle-CCI animals (D), higher combined IgG levels correlated with shorter latency to fall off the beam.Despite the high prevalence and long-lasting consequences of TBI, effective treatments for this condition are still lacking, while timely nutritional support and intervention are considered essential for reducing the risk of death and poor neurologic outcomes. 3,20,41In this study, we assessed the potential benefit of bovine thymus gland extract supplementation in a well-characterized model of acute head injury, CCI, which recapitulates many common TBI symptoms observed in humans and is validated for experimental reliability by numerous research groups. 96,97This work reveals possible cellular and molecular mechanisms that may underlie nutrition-mediated improvement and acceleration of the recovery from TBI.
Previous studies have demonstrated that TBI significantly impairs vestibulomotor function in the early days following injury and this effect diminishes or disappears completely over time. 98In a rat model, this was shown using the balance beam test, where balance impairment appears most pronounced during the first week following TBI. 77onsistent with this, in our study we show that while latency to fall off the beam is significantly lengthened in TNF-supplemented animals at days 5 and 7 following injury, it remains low in the remaining experimental groups that underwent CCI (Figure 1A).Consistent with previous reports of the prolonged cognitive and memory impairment following TBI compared to sensory/motor dysfunction, 98,99 we find that performance on MWM is poor among the animals in Vehicle-CCI, TD-CCI, and TCF-CCI groups.This effect of TBI is significantly ameliorated in TNF-treated animals, who were comparable to Vehicle-Sham animals in performance.Behavioral studies together demonstrate that TNF supplementation prior to and during CCI recovery phase elicits a pronounced improvement in both the early and long-lasting symptoms of TBI that involve vestibulomotor and cognitive functions, respectively (Figure 1B,C).These findings suggest that nuclear fraction of bovine thymus, which may have a unique composition compared to other thymus extracts we examined, contains bioactive components that may be beneficial for faster recovery from TBI.
0][81][82][83] While not many changes in gene expression were reported between days 14 and 30 after TBI, large numbers of genes appear to remain either up-or downregulated long-term. 79,81In our study, we find that CCI induces persistent changes in gene expression patterns in Vehicle-CCI animals, with the majority of differentially expressed genes involved in inflammation and tissue repair (Figure 3).Conversely, we find that thymus extract supplementation and TNF supplementation, in particular, induce notably larger changes in hippocampal gene expression patterns compared to Vehicle-CCI group (797 vs. 70 differentially expressed genes for TNF-CCI versus Vehicle-CCI groups, respectively) (Table S1).These data suggest that beyond the expected normalization of hippocampal gene expression pattern that may accompany recovery from TBI, TNF affects the expression of many additional genes, which are likely to collectively influence the efficiency and the time course of functional recovery from brain injury.This finding is consistent with a known capacity of dietary components to change gene expression in various tissues through a variety of mechanisms. 100Notably, the patterns of activation versus suppression of the signaling pathways most affected by thymus gland extract supplementation appear similar between supplemented groups, in particular between TNF-and TD-supplemented animals (Figures 4 and S3), suggesting that the material contained in the nuclear fraction of bovine thymus is similar in composition between these two materials, albeit differentially concentrated, which can explain the observed overlap in affected signaling pathways within the hippocampus.
9][80][81][82][83] Notably, phagosome formation and autophagy, specifically, are among the most significantly affected signaling pathways in thymus extract-supplemented animals (Figure 4).2][103][104] On the one hand, increased autophagy following TBI can be beneficial for converting damaged or dysfunctional molecules and cellular organelles into their recyclable components.On the other hand, prolonged autophagy can also eliminate functional proteins, lipids, nucleic acids, and cellular organelles.Evaluation of Beclin1 and LC3 levels in the hippocampi of injured animals in our study revealed a reduction in their expression at day 21 following CCI in response to TNF supplementation (Figure 5).Interestingly, LC3 expression in Vehicle-CCI animals remained increased compared to supplemented animals.These results demonstrate that downregulation of key players in autophagosome formation coincides with better recovery from TBI, suggesting one of three possible explanations: (1) the process of autophagy is no longer highly necessary, and tissue repair has been largely completed in TNF-supplemented animals; (2) prolonged, damaging autophagy has been attenuated by TNF supplementation, leading to faster tissue recovery; or (3) TNF and other thymus extract supplementation may have a direct impact on Beclin1 and LC3 expression levels, unrelated to TBI recovery efficiency.Further studies are needed to discern between these possibilities.
6][107][108][109][110][111][112] Increased levels of circulating autoantibodies against antigens of neuronal and glial origin are found in many neurologic diseases and may be an important contributing factor toward disease pathogenesis and progression. 112,113In fact, a proposed mechanism for the development of CTE following repeated head trauma involves repeated BBB permeabilization events and release of neuronal and glial proteins from the injured tissue into the blood, resulting in induction of the immune response to these proteins, evidenced by higher titers of autoantibodies against antigens of neuronal and glial origin. 29ogether, these events lead to persistent neuroinflammation and autoimmune processes, including loss of self-tolerance, often observed in athletes and military personnel who experience mild, repetitive concussions. 28,29,114,115Harnessing the immune system function, on the other hand, is one of the promising approaches toward the treatment of many pathological conditions, including TBI. 33,116 Here, we demonstrate that bovine tissue extracts are capable of eliciting production of IgG antibodies against antigens specific to their tissues of origin (Figure S4).Notably, the nature of the extract (i.e., nuclear vs. cytoplasmic) may determine what antigens are concentrated, their bioavailability and antigenicity.Consistent with this, we find that supplementation with heart-nuclear fraction extract elicits production of IgGs against myoglobin and troponin, but not MYH7.This may be due to low concentration of MYH7 in the nuclear fraction of bovine heart tissue or to its low bioavailability, among other possibilities.Yet, heart tissue extract is potent at inducing IgG antibodies against neuronal and glial antigens, NSE in particular, which also serves as a predictive biomarker for patient outcomes following cardiac arrest 117 (Figure 6B).Similarly, we find that thymus extracts increase serum levels of IgGs against proteins present in the heart tissues consistent with the expression of cardiac markers, such as myoglobin, in thymus tissues. 118ur results also demonstrate that serum levels of circulating IgG autoantibodies against nervous system antigens are elevated in TNF-supplemented animals on day 21 post-injury and that the increased levels of IgG antibodies correlate with better behavioral recovery from CCI; the vestibulomotor function and spatial memory are both improved with TNF supplementation.The variation in the extent of IgG induction observed with groups of supplemented healthy control animals (Figure 6B,C) compared to those undergoing supplementation prior to CCI (Figure 7) can be explained by the dissimilarities in the supplementation duration.Healthy control animals were supplemented with TNF for 28 days, while those who underwent TBI for 21 days.
Thus, the time necessary for the switch between the production of IgM-versus IgG-class antibodies, which is a key feature of a primary immune response, was different between these two studies. 112,119While IgM antibody levels in rodents generally peak between 10 and 15 days following the initial antigen exposure and then rapidly decline, IgG levels toward the same antigen are detected later in the course of the immune response, peak between 15 and 30 days following antigen introduction, and display a wide time window of expression, depending on antigen exposure continuity.In addition, different environments and diets, combined with varying daily regimens, which included behavioral test training for the CCI group, could contribute to the discrepancy in the observed increase in IgG antibodies against nervous system antigens between the two experiments.Nevertheless, neither significant increases in IgG antibody levels nor behavioral improvements are observed in TD-CCI, TCF-CCI, and Vehicle-CCI groups at timepoints evaluated in this study (Figures 6B,C and 7).When each animal is examined separately, however, few individual TD-, TCF-, or Vehicle-treated rats do display an increase in serum levels of IgGs against nervous system antigens, albeit contrary to TNF-supplemented animals; this increase corresponds to poor performance on beam balance test, with significant negative correlation between IgG levels and latency to fall off the beam detected for Vehicle-CCI animals (Figure 8).This observation suggests that the rise in serum levels of IgG antibodies against nervous system antigens in TNFsupplemented animals is likely a part of the immune tolerance mechanism, whereby the inflammatory response of the immune system against antigens delivered orally is suppressed through expansion and activation of Treg cells, whereas for other groups, the increase in IgG levels could signify neuroinflammation caused by the stress of daily handling and oral gavage, which was reported by others. 120Neuroinflammation, neuronal silencing, and even ablation of cells within the nervous system are major contributors to the clinical presentation of TBI and CTE and are elicited by the circulating antigens released from the injured brain tissues. 29,114,115herefore, our finding that TNF supplementation can induce oral tolerance toward self-antigens could be directly translated into therapies for human patients suffering from TBI-related pathologies and to individuals who are at risk of sustaining TBI.Further studies are necessary to determine whether the induction of IgG antibodies through oral supplementation with bovine tissue extracts suppresses the immune system response to the antigens present in those extracts and whether the progression of diseases that stem from the elevated autoimmune response could be delayed via nutrient supplementation and oral tolerance mechanism.
In this study, we demonstrate that a dietary supplement derived from animal glandular material is potent at improving the recovery from TBI when administered prior to and following the injury.2][123] Compared to individual dietary compounds and nutrients, however, thymus extracts are derived from the complex matrices of thymus tissue and are likely to contain a variety of bioactive molecules, including peptides, lipids, and nucleic acids, which together can influence an array of cellular and molecular processes.In the context of brain injury, where etiology is complex and involves many distinct processes and pathways, nutritional supplementation that offers multiple beneficial bioactive compounds within a naturally occurring matrix can possibly deliver a multifaceted neuroprotective effect that is required to effectively support TBI patients.
3][134][135][136][137][138][139][140][141] These observations suggest that diets low in organ meats may not deliver the optimal amounts of nutrients that facilitate neuroprotection, support cognitive function, or facilitate organ recovery from injuries.Nutritional supplements derived from animal glandular tissues should be considered in the design of dietary plans for patients suffering from medical conditions and healthy individuals alike.
In summary, we demonstrate that TNF supplementation enhances the functional recovery from TBI in a rat model; both the balance function and the spatial memory improve when the animals are receiving TNF by oral gavage prior to and following the injury.We further show that TNF supplementation induces dramatic changes in gene expression patterns in the hippocampus of animals who experienced TBI and validate that the process of autophagy is modulated in supplemented animals.Induction of IgG antibodies against neuronal and glial antigens in the sera of TNF-supplemented animals correlates with their better performance on the beam balance test shortly after the injury.Together, our data demonstrate, for the first time, that nutritional supplement containing bovine thymus nuclear fraction is potent at ameliorating the functional consequences of TBI and may aid in the regulation of the immune system, possibly through an oral tolerance mechanism.The specific mechanisms whereby thymus nuclear extract exerts its beneficial effects on brain health following injury remain to be elucidated.

F I G U R E 1
TNF supplementation improves vestibulomotor function and spatial memory following CCI.(A) Latency to fall off the beam is increased with TNF supplementation at days 5 and 7 following CCI compared to other conditions.(B) CCI induces significant reduction in the time spent in the target quadrant of the pool during MWM test, while TNF supplementation improves time spent in the target quadrant compared to other conditions.*Significant difference from Vehicle-Sham group; Ϯ Significant difference from Vehicle-CCI group; p < .05.Data are presented as mean ± SEM. (C) Dirichlet distribution analysis shows randomized presence of animals across four MWM pool quadrants in Vehicle-CCI, TCF-CCI, and TD-CCI groups, indicating persistent impairment in cognitive function 21 days after CCI.Vehicle-Sham and TNF-CCI animals showed bias toward the target quadrant, suggesting better cognitive function.(−) = not significant, ***p < .001;n = 11-15 per group.

F I G U R E 2
UpSetR plots for IPA pathways in each experiment.(A) UpSetR plot of differentially expressed genes (DEGs): The bar on the bottom left shows the number of DEGs for each experiment.The internal bar shows the number of DEGs unique and shared by each of the experimental conditions.Overlap was determined using R package UpSetR.The TNF-CCI group (maroon bar, bottom left) shows the greatest number of unique DEGs expressed, followed by TCF-CCI.All 3 treatment groups show a greater number of DEGs when compared to the Vehicle-CCI condition.The greatest overlap between conditions was observed with TNF-CCI and TD-CCI groups (217 genes exclusively).(B) UpSetR plot at the gene pathway level: Gene pathways are most active in TNF-CCI and TD-CCI groups with much overlap.No unique pathways were detected in the Vehicle-CCI group.

F I G U R E 4 Z
-scores of the top 50 signaling pathways changed with TNF-CCI supplementation (first colored column).TD-CCI shows the highest degree of pathway similarities with TNF-CCI group (red boxes), while Vehicle-CCI animals display the least similarities.Numbers represent the Z-scores generated using IPA.Pathways without predictable Z-scores are shown in gray.
2.1.2| Study 2: Induction of IgG antibodies following oral administration of various glandular extracts in healthy adult rats This study was conducted at the David H. Murdock Research Institute (DHMRI) (Kannapolis, NC, USA) in a facility accredited by AAALAC.