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. Author manuscript; available in PMC: 2023 Jun 12.
Published in final edited form as: Brain Res. 2023 Mar 24;1808:148338. doi: 10.1016/j.brainres.2023.148338

Immunocal® limits gliosis in mouse models of repetitive mild-moderate traumatic brain injury

Lilia A Koza a,b, Claudia Pena a,b, Madison Russell a,b, Alec C Smith a,b, Jacob Molnar a,b, Maeve Devine a,b, Natalie J Serkova c, Daniel A Linseman a,b,*
PMCID: PMC10258892  NIHMSID: NIHMS1889436  PMID: 36966959

Abstract

Successive traumatic brain injuries (TBIs) exacerbate neuroinflammation and oxidative stress. No therapeutics exist for populations at high risk of repetitive mild TBIs (rmTBIs). We explored the preventative therapeutic effects of Immunocal®, a cysteine-rich whey protein supplement and glutathione (GSH) precursor, following rmTBI and repetitive mild-moderate TBI (rmmTBI). Populations that suffer rmTBIs largely go undiagnosed and untreated; therefore, we first examined the potential therapeutic effect of Immunocal® long-term following rmTBI. Mice were treated with Immunocal® prior to, during, and following rmTBI induced by controlled cortical impact until analysis at 2 weeks, 2 months, and 6 months following the last rmTBI. Astrogliosis and microgliosis were measured in cortex at each time point and edema and macrophage infiltration by MRI were analyzed at 2 months post-rmTBI. Immunocal® significantly reduced astrogliosis at 2 weeks and 2 months post-rmTBI. Macrophage activation was observed at 2 months post-rmTBI but Immunocal® had no significant effect on this endpoint. We did not observe significant microgliosis or edema after rmTBI. The dosing regimen was repeated in mice subjected to rmmTBI; however, using this experimental paradigm, we examined the preventative therapeutic effects of Immunocal® at a much earlier timepoint because populations that suffer more severe rmmTBIs are more likely to receive acute diagnosis and treatment. Increases in astrogliosis, microgliosis, and serum neurofilament light (NfL), as well as reductions in the GSH:GSSG ratio, were observed 72 h post-rmmTBI. Immunocal® only significantly reduced microgliosis after rmmTBI. In summary, we report that astrogliosis persists for 2 months post-rmTBI and that inflammation, neuronal damage, and altered redox homeostasis present acutely following rmmTBI. Immunocal® significantly limited gliosis in these models; however, its neuroprotection was partially overwhelmed by repetitive injury. Treatments that modulate distinct aspects of TBI pathophysiology, used in combination with GSH precursors like Immunocal®, may show more protection in these repetitive TBI models.

Keywords: Traumatic brain injury, Secondary injury, Neuroinflammation, Oxidative stress, Glutathione, Neuroprotection

1. Introduction

Each year, approximately 69 million individuals worldwide sustain a traumatic brain injury (TBI), defined as an injury resulting from an impact to the head (Dewan et al., 2018). In the United States, 1.5 million individuals suffer a TBI of which 80,000 experience long-term disability (Flanagan, 2015). In 2010, the lifetime economic burden of TBI for the United States was estimated to be $76.5 billion (Seifert, 2007). Clearly, TBI has severe emotional, physical, and economic consequences.

Primary injury from TBI occurs immediately and results from mechanical forces from the impact (Harish et al., 2015). Secondary injury is progressive and includes neuroinflammation, blood brain barrier (BBB) disruption, oxidative stress, and excitotoxicity (Jassam et al., 2017) (Hiebert et al., 2015; Pearn et al., 2017). These processes contribute to symptoms and cognitive and motor deficits (Honan et al., 2015; Yin et al., 2016; Decker et al., 2018; Wang et al., 2018; Voormolen et al., 2019). Approximately 80 % of TBIs sustained worldwide are mild in severity (McCrory et al., 2013). Repetitive TBIs (rTBIs), even of mild severity, can prolong and worsen TBI secondary injury (Fujita et al., 2012; Bailes et al., 2013; Fehily and Fitzgerald, 2017; Mouzon et al., 2018; Dhillon et al., 2020). Continual treatment of secondary injury is necessary for patients that suffer rmTBI as they may have an increased risk for neurodegeneration (Faden and Loane, 2015; Edwards et al., 2017; Gao et al., 2017).

Currently, there are no FDA approved preventative treatments for high-risk individuals who sustain rmTBI. The current study investigated Immunocal® treatment in mouse models of rmTBI or repetitive mild-moderate TBI (rmmTBI) induced by controlled cortical impact (CCI), adapted from a well-established model (Mouzon et al., 2018; Mouzon et al., 2014; Mouzon et al., 2012). Immunocal® is a non-denatured whey protein supplement that contains a large amount of cystine and gluta-mylcysteine, which can be converted to cysteine after crossing the BBB (Baruchel and Viau, 1996; Shih et al., 2006; Ross et al., 2012). Cysteine is the limiting substrate in the production of glutathione (GSH), a key endogenous antioxidant which has been shown to play significant roles in mitigating oxidative stress from neurodegeneration and neurotrauma (Bannai and Tateishi, 1986; Mazzetti et al., 2015; Koza and Linseman, 2019). We have previously shown that Immunocal® enhances the synthesis of GSH in neurons and provides neuroprotection in vitro and in vivo (Ross et al., 2012; Ross et al., 2014; Winter et al., 2017). We previously studied Immunocal® in a mouse model of single moderate TBI induced by CCI and found that supplementation with Immunocal® for 28 days prior to TBI preserved the ratio of GSH:glutathione disulfide (GSSG). Immunocal® also offered significant neuroprotection (Ignowski et al., 2018).

We first analyzed the potential beneficial effects of Immunocal® long-term (out to 6 months) in response to rmTBI. High-risk individuals, such as athletes in contact sports, may sustain rmTBIs that go undiagnosed and untreated. For example, data has shown that athletes in contact sports who have no clinically identified history of rmTBI exhibit neurodegenerative pathology post-mortem (Bailes et al., 2013). These findings indicate that continual long-term treatment for populations at high-risk for rmTBI is necessary. We proposed Immunocal® as a long-term treatment as it can be easily administered as a daily dietary supplement. Secondly, we analyzed the therapeutic potential of Immunocal® acutely in response to more severe, rmmTBI. High-risk populations that sustain more severe rmmTBI are likely to be diagnosed acutely; therefore, assessment of the anti-inflammatory and neuroprotective effects of Immunocal® in response to this scenario was also performed.

Mice were treated with Immunocal® 28 days prior to, during, and following 5 rmTBIs or 3 rmmTBIs induced by CCI and the effects of Immunocal® treatment were assessed on recovery, edema, macrophage infiltration, astrogliosis, microgliosis, serum neurofilament light (NfL), and brain GSH:GSSG, using ex vivo and in vivo (magnetic resonance imaging, MRI) techniques.

2. Methods

2.1. Animal care and treatment

Animal work was performed under approved protocols to study rmTBI (#1011533; approved: 2/7/2017; expired: 2/6/2020) or rmmTBI (#1638687; approved: 8/27/2020; expires: 8/26/2023) by the Institutional Animal Care and Use Committee at the University of Denver. Male CD1-Elite mice (aged 35 days) were purchased from Charles River Laboratories (Hollister, CA) and maintained at the University of Denver animal facility on a 12 h light/dark cycle with food and water provided ad libitum. Mice acclimated for two weeks prior to the study.

2.2. Mice subjected to rmTBI for analysis at 2 weeks, 2 months, and 6 months post-TBI

Mice were divided into three groups with 23 mice in each group: Sham, untreated rmTBI, or Immunocal®-treated rmTBI. Immunocal®-treated rmTBI mice were dosed with a 3 % w/v solution in sterile drinking water provided ad libitum for 5 days/week over 28 days prior to, during the rmTBIs, and until euthanasia. A similar dosing regimen yielded therapeutic effects in mice subjected to single moderate TBI (Ignowski et al., 2018). Body weights were obtained weekly. An MRI session was performed on 11 (n = 3–4 per group) of the mice analyzed at 2 months post-rmTBI. Immunohistochemical staining of cortex with glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba-1) to measure astrogliosis and microgliosis, respectively, from mice analyzed at 2 weeks, (n = 9 per group), 2 months (n = 9 per group), and 6 months (n = 5 per group) post-rmTBI. N values vary due to unviable tissue or high/low outliers resulting in exclusion.

2.3. Repetitive mTBI procedure

Five rmTBIs were performed with an inter-concussion interval of 48 h over 10 consecutive days. Following the 28-day Immunocal® dosing, the first mTBI was induced by CCI using the Leica Impact One system (Leica Biosystems, Buffalo Grove, IL). Briefly, mice were anesthetized using an isoflurane vaporizer (VetEquip, Inc., Livermore, CA), their head was shaved, and they were placed into a stereotaxic frame (Braintree Scientific Inc., Braintree, MA) to prevent movement during impact. The impactor probe (5 mm diameter) was directly centered along the midline at bregma. A mTBI was administered with an impact depth of 1 mm at a velocity of 5 m/s (dwell time 200msec) as previously described (Mouzon et al., 2018; Ross et al., 2014; Winter et al., 2017). Untreated and Immunocal®-treated rmTBI mice were subjected to rmTBI (Supplementary Table 1). Apnea times >3 s were not observed or recorded. Sham were not subjected to impact but were otherwise treated identically to rmTBI mice. Mice recovered from anesthesia on a thermal pad and their righting reflex times were measured. Righting reflex was the point at which the animal was able to maintain a sternal position. Mice returned to their home cage once ambulatory.

2.4. Mice subjected to rmmTBI and analysis at 72 h post-TBI

Mice were divided into three groups with 7 mice in each group: Sham, untreated rmmTBI, or Immunocal®-treated rmmTBI. Two Immunocal®-treated rmmTBI and 1 untreated rmmTBI mouse were euthanized prior to study conclusion due to adverse effects from the rmmTBI (e.g., difficulties with ambulation, seizure activity). The dosing regimen and body weight assessment was performed as described above. During and following the rmmTBIs until euthanasia, mice were monitored for distress. Mice were euthanized at 72 h post-rmmTBI. Immunohistochemical staining of cortex for GFAP to measure astrogliosis and Iba-1 to measure microgliosis, brain GSH and GSSG, and serum NfL were performed for all mice subjected to rmmTBI. However, n values vary slightly due to unviable tissue or high/low outliers resulting in exclusion.

2.5. Repetitive mmTBI procedure

Three mmTBIs were performed with an inter-concussion interval of 48 h over 5 consecutive days. Repetitive mmTBIs were performed as described above with the following modifications. A concave 3 mm metallic disk was affixed to the shaved head along the midline at bregma using tissue adhesive. Mice were in a stereotaxic frame and the impactor probe administered an impact depth of 2 mm at a velocity of 5.5 m/s (dwell time 200msec) to induce mmTBI (Supplementary Table 1). Untreated and Immunocal®-treated rmmTBI mice underwent mmTBI and were monitored for TBI-induced apnea (discontinuation of normal respiration). Sham animals were not subjected to impact. Mice recovered and righting reflex times were measured as previously described.

2.6. MRI analysis

All mouse brain MRI acquisitions were performed in the Colorado Animal Imaging Shared Resource (University of Colorado Anschutz Medical Campus) under an approved animal IACUC protocol #00596 (expired 11/7/2022). Briefly, the animals were anesthetized with 1.5–2 % Isoflurane, positioned inside a warmed animal holder and inserted into the Bruker 9.4 Tesla/ 20 cm BioSpec MRI scanner with a mouse head phase-array RF coil. A multi-parametric high-resolution MR protocol was applied: “T2w-turboRARE MRI” (structural MRI, 48 μm in-plane resolution) → “T2wMRI-MSME mapping with 16 echoes” (with/ without iron oxide nanoparticles Ferumoxytol for macrophage imaging) → “T1wMRI-MSME” (with/ without gadolinium contrast MultiHance for BBB breakdown) → EPS DWI with 6-gradients (for restricted diffusion/ brain edema) (Frey et al., 2014; Pierce et al., 2019; Dahl et al., 2020; Serkova et al., 2010).

2.6.1. Quantitative image analysis

All injured areas were assessed by placing region of interest (ROIs) on T2wMRI axial and sagittal slices. The changes in T2-relaxation times were assessed in the injured and control cortex from the T2w-maps in pre- and post-ferumxytol scans (24 h apart) to assess iron accumulation of macrophages. Increased T1-signal intensities in T1W post-gadolinium MRI scans were used to assess the areas of BBB leakage. Increased apparent diffusion coefficient (ADC) values from DWI for the left and right hemisphere were taken in two brain slices, anterior and posterior to bregma, were used to characterize brain edema. Change in T2-weighted values for the left and right hemisphere were also averaged for each mouse. All analysis was performed using Bruker ParaVision NEO360 v2.0 software.

2.6.2. Euthanasia and sample collection

Mice were euthanized with an overdose of isoflurane and decapitated at respective study endpoints. For rmmTBI mice, trunk blood was collected. Whole brain was removed, washed in 1X phosphate buffered saline (PBS, pH = 7.4) and placed into a 1 mm cut stainless steel brain matrix (Plastics One, Roanoke, VA) submerged in ice cold Gey’s balanced salt solution (Sigma Aldrich, St. Louis, MO). Using a razor blade, each brain was sectioned and used as shown in Supplementary Fig. 1.

2.7. Immunohistochemical staining of GFAP and Iba-1 in cortex

2.7.1. Tissue processing

The brain section was washed with ice cold Grey’s solution and placed in 4 % paraformaldehyde at 4 °C overnight. Each section was washed with cold 1× PBS and placed in 30 % sucrose in 1× PBS for cryopreservation. Sections were frozen in optimal cutting temperature (OCT) compound with liquid nitrogen and stored at −80 °C. Prior to sectioning, tissue was allowed to acclimate in the microtome cryostat for 30 min. Brain was cut in 20 μm sections, collecting every viable tissue section onto the surface of Fisherbrand Superfrost Colorfrost Plus coated slides (Fisher Scientific, Pittsburgh, PA) and stored at −20 °C.

2.7.2. Immunohistochemical staining

Slides were equilibrated at room temperature for 30 min. Sections were outlined with Liquid Blocker Super PAP Pen (Daido Sangyo Co., Tokyo, Japan) and washed twice with 1X PBS to remove residual OCT. Tissue underwent a standard staining protocol using primary antibodies against GFAP and Iba-1, Cy3- or Alexa Fluor 488-conjugated secondary antibodies, and Hoechst nuclear stain, as previously described (Ignowski et al., 2018). Two slides with 6 sections of cortex per mouse were stained for Iba-1 and GFAP.

2.8. Immunofluorescence microscopy of GFAP and Iba-1-stained cortex

Three 20× images per section were captured by a blinded researcher using a Zeiss Axio Observer epi fluorescence microscope (locations shown in Supplementary Fig. 2). Iba-1 and GFAP images were captured on the Alexa Fluor 488 and Cy3 channel, respectively.

2.9. Quantification of astrocyte and microglia number in cortex

Iba-1 and GFAP images were quantified by two blinded researchers. Using Adobe Photoshop CC 19.1.7 (Adobe Inc., San Jose, CA), background staining was removed by setting a black point for the image in an area of high background staining. The count tool was then used to label positively stained GFAP and Hoechst cells, counted as astrocytes, or positively stained Iba-1 and Hoechst cells, counted as microglia. An average of the total number of astrocytes or microglia counted for each mouse was obtained from 18 to 36 images per mouse.

2.10. Measurement of serum NfL by ELISA

2.10.1. Blood processing

Trunk blood was collected into 200 μL serum capillary top sample tubes (SAI Infusion Technologies, Lake Villa, IL) and clotted for 1 h at room temperature prior to centrifugation at 4000 rpm for 10 min at 4 °C. Supernatant was collected and serum was isolated and stored at −80 °C.

2.10.2. ELISA

Manufacturer’s protocols were followed for mouse Neurofilament Light ELISA assay kit (Aviva Systems Biology, San Diego, CA). Samples were diluted 1:2 with provided standard diluent and run in duplicate. Optical density (O.D.) absorbance was read at 450 nm using a spectrophotometer and relative O.D. with corrected absorbance was calculated for standards and samples. Values for NfL concentration were interpolated from the standard curve and an average NfL concentration was calculated for each mouse.

2.11. Colorimetric detection of brain GSH and GSSG

2.11.1. Tissue processing

Brain sections (Supplementary Fig. 1) were frozen in liquid nitrogen and stored at −80 °C. Brain sections were homogenized in 250 μL of ice cold 100 mM phosphate buffer (pH = 7) using a dounce homogenizer. The homogenate was centrifuged at 14,000 rpm for 10 min at 4 °C and supernatant was removed. An aliquot was taken for protein assay determination. One volume of cold 5 % 5-sulfo-salicylic acid (Sigma-Aldrich, St/ Louis, MO) was added to remaining samples which were incubated for 10 min at 4 °C. Samples were centrifuged at 14,000 rpm for 10 min at 4 °C and supernatant was analyzed.

2.11.2. Colorimetric detection assay

Manufacturer’s protocols were followed for the Glutathione Colorimetric Detection Kit (Invitrogen, Waltham, MA). Samples were diluted by adding 1.5 volumes of provided 1X assay buffer and run in duplicate. O.D. absorbance was read at 405 nm using a spectrophotometer and the relative O.D. with corrected absorbance was calculated for standards and samples. Values for GSH and GSSG were interpolated from standard curves and normalized based on protein concentration. Average values for GSH and GSSG were calculated for each mouse and the GSH:GSSG ratio was calculated.

2.12. Statistical analysis

Data were statistically analyzed and graphs were created using GraphPad Prism 5.01 for Windows (GraphPad Software, San Diego, CA). Statistical differences between groups were evaluated using a one-way ANOVA with post-hoc Tukey’s test at each time point for body weight, righting reflex, ADC and T-2 weighted values, quantification of astrocytes and microglia, NfL levels, and GSH, GSSG, and GSH:GSSG measurements. A post-hoc Dunnett’s Multiple Comparison test was also used for GSH. Statistical differences in righting reflex between and within groups were analyzed by a paired t-test. Statistical differences between groups were analyzed by an unpaired t-test for apnea values. Differences were statistically significant when p < 0.05 for all analyses.

3. Results

3.1. Immunocal®-treatment effects on body weight and righting reflex times after rmTBI

Sham, untreated rmTBI, and Immunocal®-treated rmTBI mice were analyzed at 2 weeks (n = 9 per group), 2 months (n = 9 per group), and 6 months (n = 5 per group) post-rmTBI. Body weights were monitored weekly (Fig. 1A-C). There were no significantly sustained changes in percentage of peak body weight between groups.

Fig. 1.

Fig. 1.

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Percent of peak body weight and righting reflex times of mice subjected to sham surgery (Sham) or untreated (TBI) and Immunocal®-treated (TBI + ICAL) mice subjected to repetitive mild TBI (rmTBI). Body weight of untreated TBI, TBI + ICAL, and sham mice was assessed once per week beginning with Immunocal® treatment at 4 weeks (28 days) prior to the first mTBI, during the mTBI’s, and continuing until euthanasia for analysis at (A) 2 weeks (n = 9 mice per group), (B) 2 months (n = 9 mice per group), and (C) 6 months (n = 5 mice per group) post-last rmTBI or sham surgery. Body weight is expressed as the percentage of peak body weight at each time point. No significant differences in percent of peak body weight were observed at any time points for mice analyzed at (A) 2 weeks post-rmTBI. For mice analyzed at (B) 2 months post-rmTBI, untreated TBI mice had significantly lower percent of peak body weight only at 1-week post-last mTBI when compared to sham mice (p = 0.031). No significant differences in percent of peak body weight were observed between TBI + ICAL mice and sham or untreated TBI mice at any time points for mice analyzed at 2 months post-last TBI. For mice analyzed at (C) 6 months post-rmTBI, untreated TBI mice had a significantly lower percent of peak body weight compared to sham mice only at 3 weeks post-rmTBI (p = 0.017). TBI + ICAL mice had a significantly lower percent of peak body weight compared to TBI mice only at 21 weeks post-rmTBI (p = 0.019). No significant differences in percent of peak body weight were observed between TBI + ICAL and sham mice at any time points. All data in (A) - (C) are displayed as the mean ± SEM and a one-way ANOVA with post-hoc Tukey’s test was used to analyze data at each time point (* indicates p < 0.05 for untreated TBI versus sham mice and # indicates p < 0.05 for TBI + ICAL versus TBI mice). Righting reflex times for sham, TBI, and TBI + ICAL mice analyzed at (D) all time points (2 weeks, 2 months, and 6 months post- last rmTBI; n = 23 per group) were measured immediately following each mTBI or sham surgery after anesthesia was discontinued. Untreated TBI mice had significantly increased righting reflex times compared to sham mice following all mTBIs, however, TBI + ICAL mice only exhibited significantly increased righting reflex times compared to sham mice following the first three TBI’s. All data in (D) are displayed as the mean ± SEM and righting reflex times between groups are analyzed by a one-way ANOVA with post-hoc Tukey’s test for each TBI day number (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and NS indicates p > 0.05 or nonsignificant).

Righting reflex times, indicative of TBI and impairment, were measured immediately following each rmTBI or sham surgery and combined for mice analyzed at all time points post-rmTBI (Grin’kina et al., 2016). Righting reflex times for untreated and Immunocal®-treated rmTBI mice did not significantly differ but were significantly increased versus sham mice (Fig. 1D).

3.2. MRI analysis of Immunocal®-treatment effects on macrophage activation after rmTBI

Groups were analyzed by MRI at 2 months post-rmTBI. Gadolinium contrast was used to determine edema. Since the mTBIs were performed at midline at bregma, the ADC values for the left and right hemispheres at bregma were averaged. No significant differences were observed between sham, untreated rmTBI, and Immunocal®-treated rmTBI mice (p = 0.742; Fig. 2A). It seems that rmTBI did not induce significant edema 2 months post-rmTBI.

Fig. 2.

Fig. 2.

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Repetitive mild traumatic brain injury (rmTBI) did not result in edema but did cause an increase in inflammation as analyzed by magnetic resonance imaging (MRI). Iron oxide T2-weighted brain MRI analysis was performed on untreated (TBI) and Immunocal® pre- and post-treated (TBI + ICAL) mice subjected to rmTBI, or mice subjected to sham surgery (Sham) at 2 months post-last mTBI or sham surgery (n = 3–4 per group). Averaged apparent diffusion coefficient (ADC) values for the left and right hemisphere (A), which indicate edema when increased, were not significantly different between groups (p = 0.742). The average combined left and right hemisphere change (Δ) in T2-weighted values (B) are significantly decreased in untreated TBI mice when compared to sham mice which is indicative of the accumulation of the iron nanoparticle contrast in activated macrophages (p = 0.025). No significant differences in the average change in T2-weighted values were observed between TBI + ICAL and sham mice or untreated TBI mice. All MRI data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test (* indicates p < 0.05 and NS indicates p > 0.05 or non-significant).

Iron-oxide T2-weighted contrast was used to measure macrophage infiltration and is indicative of increased brain inflammation. Change in T2 values were averaged for the left and right hemisphere. Untreated rmTBI mice had significantly increased average T2 values versus sham mice, indicating accumulation of iron-oxide nanoparticles in macrophages (p = 0.025). Immunocal® treatment did not protect against this as Immunocal®-treated rmTBI mice did not have significantly different values versus untreated rmTBI mice (Fig. 2B).

3.3. Immunocal®-treatment significantly reduces astrogliosis at 2 weeks and 2 months after rmTBI

Neuroinflammation has been reported in vivo in response to closed skull rmTBI (Hoogenboom et al., 2019). The rmTBI model used to model our rmTBI model showed significant GFAP and Iba-1 immunoreactivity in brain at 24 h and 6 months post-rmTBI versus sham mice (Ross et al., 2014; Winter et al., 2017). We anticipated astrogliosis and microgliosis around the injury site out to 6 months post-rmTBI. Cortices of mice were stained for GFAP, Iba-1, and Hoechst to analyze astrogliosis, microgliosis, and nuclei, respectively (Figs. 3A and 4A). Astrocytes or microglia, counted as GFAP or Iba-1 (and Hoechst) positive cells, respectively, were quantified and averaged for groups analyzed at 2 weeks, 2 months, and 6 months post-TBI (Figs. 3B and 4B).

Fig. 3.

Fig. 3.

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Immunocal®-treatment pre- and post-repetitive mild traumatic brain injury (rmTBI) significantly reduced astrogliosis in the cortex of mice subjected to rmTBI when analyzed at 2 weeks and 2 months, but not at 6 months post-last rmTBI. Representative images of cortex stained for GFAP (green) and DAPI (blue), to label astrocytes and nuclei, respectively, from mice subjected to sham surgery (SHAM) and Immunocal® pre- and post-treated mice (TBI + ICAL) and untreated mice (TBI) subjected to rmTBI analyzed at (A) 2 months post-rmTBI. White arrows indicate GFAP and DAPI positive cells, counted as astrocytes. Scale bar = 20 μm. Quantification of the average number (#) of astrocytes in cortex stained with GFAP and DAPI, as described in (A) is shown for sham, untreated TBI, and TBI + ICAL mice analyzed at (B) 2 weeks (n = 6–7 mice per group), 2 months (n = 7–9 mice per group), and 6 months (4–5 mice per group) post-rmTBI. The average number of astrocytes in the cortex of untreated TBI mice was significantly increased at 2 weeks (p = 0.011) and 2 months (p = 0.002) post-rmTBI compared to sham mice. Immunocal®-treatment pre- and post-rmTBI significantly reduced the average number of astrocytes in the cortex in mice subjected to rmTBI at 2 weeks (p = 0.011) and 2 months (p = 0.002) post-rmTBI when compared to untreated TBI mice and showed no significant difference compared to sham mice. No significant differences in the average number of astrocytes in the cortex were observed between groups analyzed at 6 months post-rmTBI (p = 0.216). All astrocyte quantification data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test at each time point (* indicates p < 0.05, ** indicates p < 0.01, and NS indicates p > 0.05 or non-significant).

Fig. 4.

Fig. 4.

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Microgliosis was not observed in the cortex of mice subjected to repetitive mild traumatic brain injury (rmTBI) when analyzed at 2 weeks, 2 months, or 6 months post-last mTBI. Representative images of cortex stained for Iba-1 (green) and DAPI (blue), to label microglia and nuclei, respectively, from mice subjected to sham surgery (SHAM) and untreated mice (TBI) and Immunocal®-treated pre- and post-rmTBI mice (TBI + ICAL) subjected to rmTBI analyzed at (A) 2 weeks post-rmTBI. White arrows indicate Iba-1 and DAPI positive cells, counted as microglia. Scale bar = 20 μm. Quantification of the average number (#) of microglia in cortex stained with Iba-1 and DAPI, as described in (A) is shown for sham, untreated TBI, and TBI + ICAL mice analyzed at (B) 2 weeks (n = 7–9 mice per group), 2 months (n = 8–9 mice per group), and 6 months (n = 4 mice per group) post-rmTBI. No significant differences of the average number of microglia in the cortex were observed between sham, TBI, and TBI + ICAL mice at 2 weeks (p = 0.229), 2 months (p = 0.450), or 6 months (p = 0.291) post-rmTBI. All microglia quantification data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test at each time point.

Untreated rmTBI mice had significantly increased average number of astrocytes in cortex versus sham mice when analyzed at 2 weeks (p = 0.011) and 2 months (p = 0.002) post-rmTBI (Fig. 3B). Immunocal®-treatment essentially prevented astrogliosis evidenced by significantly decreased average number of astrocytes in cortex versus untreated rmTBI mice and no significant differences versus sham mice at 2 weeks (p = 0.011) and 2 months post-rmTBI (p = 0.002). No significant differences were observed between groups 6 months post-rmTBI (p = 0.216; Fig. 3B).

No significant differences in the number of microglia were observed between groups at 2 weeks (p = 0.229), 2 months (p = 0.450), or 6 months (p = 0.291) post-rmTBI (Fig. 4B). It seems that rmTBI induced sustained astrogliosis at 2 weeks and 2 months, but not 6 months, post-rmTBI. Immunocal® prevented the increase in astrogliosis, however, microgliosis was not observed in this mouse model of rmTBI.

3.4. Immunocal®-treatment effects on body weight, apnea, and righting reflex times after rmmTBI

We next performed an analysis to observe the effects of Immunocal® acutely on recovery and pathology in a mouse model of rmmTBI. Mice were divided into sham, untreated rmmTBI, and Immunocal®-treated rmmTBI mice. Mice were analyzed 72 h post-rmmTBI (n = 5–7 per group).

Body weight was assessed weekly. Percentage of peak body weight for Immunocal®-treated rmmTBI mice was significantly increased day 28 prior to the first TBI versus sham and untreated rmmTBI mice (p = 0.023). There were no significant differences between untreated rmmTBI and sham mice at any time point. Immunocal®-treated rmmTBI mice displayed a small but significantly decreased percentage of peak body weight versus sham and untreated rmmTBI mice 3 days post-rmmTBI (p = 0.000; Fig. 5A).

Fig. 5.

Fig. 5.

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Percent of peak bodyweight, apnea, and righting reflex times of mice subjected to sham surgery (Sham) or untreated (TBI) and Immunocal®-treated (TBI + ICAL) mice subjected to repetitive mild-moderate traumatic brain injury (rmmTBI) analyzed at 72 h post-last TBI. Bodyweight of sham, untreated TBI, and TBI + ICAL mice was assessed once per week beginning with Immunocal® treatment at 4 weeks (28 days) prior to the first mmTBI, during the mmTBI’s, and continuing until euthanasia for analysis at (A) 72 h (3 days) post-rmmTBI or sham surgery (n = 5–7 mice per group). Bodyweight is expressed as the percentage of peak body weight at each time point. Data are displayed as the mean ± SEM. Bodyweight for TBI + ICAL mice was significantly increased at day 28 pre-TBI (p = 0.023) and significantly decreased at 72 h post-TBI (p = 0.000) when compared to both sham and untreated TBI mice. Bodyweight data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test at each time point (* indicates p < 0.05 and ** indicates p < 0.01 for TBI + ICAL versus untreated TBI mice. # indicates p < 0.05 and ### indicates p < 0.001 for TBI + ICAL versus sham mice). Apnea times were recorded immediately after each TBI impact or sham surgery for mice analyzed at (B) 72 h post-rmmTBI or sham surgery (n = 5–7 mice per group). Mice subjected to sham surgery did not display apnea and are not shown. Both untreated TBI and TBI + ICAL mice experienced apnea but no significant differences were observed between these groups following TBI 1 (p = 0.383), TBI 2 (p = 0.822), or TBI 3 (p = 0.261). Data are displayed as the mean ± SEM and are analyzed by an unpaired t-test for each TBI number. Righting reflex times for mice analyzed at (C) 72 h post-rmmTBI were measured immediately following each mmTBI or sham surgery after anesthesia was discontinued (n = 5–7 mice per group). Untreated TBI and TBI + ICAL mice had significantly increased righting reflex times compared to sham mice following TBI 1 (p < 0.000), TBI 2 (p < 0.000), and TBI 3 (p = 0.001). No significant differences in righting reflex times were observed between untreated TBI and TBI + ICAL following any TBI. All righting reflex data are displayed as the mean ± SEM and are analyzed one-way ANOVA with post-hoc Tukey’s test for each TBI number (** indicates p < 0.01, *** indicates p < 0.001, and NS indicates p > 0.05 or nonsignificant).

Untreated and Immunocal®-treated rmmTBI mice experienced apnea which suggests this model is increased in severity. Sham mice did not experience apnea and are not shown. No significant differences in average apnea times between groups were found following the mmTBI procedures (Fig. 5B).

Righting reflex times for all groups were measured immediately following each mmTBI or sham surgery (Fig. 5C). Righting reflex times for untreated and Immunocal®-treated rmTBI mice did not significantly differ but were significantly increased versus sham mice (Fig. 5C).

3.5. Analysis of Immunocal®-treatment effects on increases in serum NfL induced by rmmTBI

Neurofilament light chain (NfL) is a subunit of neurofilament proteins, scaffolding proteins that are highly expressed in myelinated axons. Increased NfL in serum is an indicator of neuroaxonal injury resulting from neurodegeneration and rmTBI (Khalil et al., 2018; Ojo et al., 2015; Pham et al., 2021).

We evaluated if rmmTBI induced increased levels of serum NfL and if Immunocal®-treatment would prevent this. Serum was analyzed for NfL by ELISA in all groups 72 h post-rmmTBI. Untreated rmmTBI mice exhibited a significant increase of NfL versus sham mice (p = 0.035). Immunocal®-treated rmmTBI mice did not significantly differ from untreated rmmTBI or sham mice (Fig. 6). These data indicate that rmmTBI induced neuronal damage, however, Immunocal®-treatment did not significantly decrease this effect.

Fig. 6.

Fig. 6.

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Repetitive mild-moderate traumatic brain injury (rmmTBI) results in acute neuronal damage in mice analyzed at 72 h post-last mmTBI. Quantification of neurofilament light (NfL) levels, an indication of acute neuronal damage, in serum detected by enzyme-linked immunosorbent assay from mice subjected to sham surgery (SHAM) and untreated mice (TBI) and Immunocal®-treated pre- and post-rmmTBI mice (TBI + ICAL) subjected to rmmTBI analyzed at 72 h post-rmmTBI or sham surgery (n = 4–5 mice per group). NfL levels were significantly elevated in untreated TBI mice when compared to sham mice (p = 0.035), however, no differences were observed between untreated TBI and TBI + ICAL mice or TBI + ICAL and sham mice. Neurofilament light data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test (* indicates p < 0.05 and NS indicates p > 0.05 or nonsignificant).

3.6. Analysis of Immunocal®-treatment effects on changes in brain GSH and GSSG induced by rmmTBI

Glutathione, an endogenous antioxidant, can protect against reactive oxygen (ROS) and nitrogen species (RNS) and maintain redox homeostasis. Upon reacting with ROS/RNS, GSH oxidizes to GSSG. The ratio of GSH:GSSG is indicative of redox status (Dwivedi et al., 2020). Immunocal® contains cystine which can be converted to cysteine, the limiting substrate in GSH synthesis (Ross et al., 2012; Bannai and Tateishi, 1986). We have previously shown that Immunocal® enhances GSH synthesis in neurons and that a similar dosing regimen preserves GSH: GSSG in a mouse model of single moderate TBI (Ross et al., 2012; Ross et al., 2014; Winter et al., 2017; Ignowski et al., 2018).

Therefore, GSH and GSSG were measured in brain from all groups analyzed 72 h post-rmmTBI. A significant difference was found in GSH levels between all groups and, although a Tukey’s post-hoc analysis did not identify differences, a Dunnett’s Multiple Comparison test identified a significant GSH decrease in untreated rmmTBI versus sham mice (p = 0.046; Fig. 7A). Brain GSSG was significantly increased in untreated rmmTBI mice versus sham mice (p = 0.022; Fig. 7B). Immunocal®-treated rmmTBI mice showed no significant differences in GSH or GSSG versus untreated rmmTBI or sham mice. No significant in differences in GSH:GSSG were observed between untreated and Immunocal®-treated rmmTBI mice although both groups showed a significant decrease versus sham mice (p = 0.003; Fig. 7C). It seems that rmmTBI decreases GSH and significantly increases GSSG, resulting in a decreased brain GSH:GSSG ratio, that Immunocal®-treatment did not alter.

Fig. 7.

Fig. 7.

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Repetitive mild-moderate traumatic brain injury (rmmTBI) reduced glutathione (GSH) and significantly increased glutathione disulfide (GSSG) in brain of mice analyzed at 72 h post-last mmTBI. Quantification of GSH measured in brain homogenate isolated from mice subjected to sham surgery (SHAM) and untreated mice (TBI) and Immunocal®-treated pre- and post-rmmTBI mice (TBI + ICAL) exposed to rmmTBI analyzed at (A) 72 h post-rmmTBI or sham surgery (n = 5–6 mice per group). GSH levels were significantly different between groups (p = 0.046). A post-hoc Tukey’s test was unable to identify which groups were significantly different but a post-hoc Dunnett’s Multiple Comparison test identified a significant difference between untreated TBI and sham mice. All GSH data are displayed as mean ± SEM. Quantification of GSSG as measured in brain homogenate of sham, untreated TBI, and TBI + ICAL analyzed at (B) 72 h post-rmmTBI or sham surgery (n = 5–6 mice per group). Untreated TBI mice had significantly elevated levels of GSSG when compared to sham mice (p = 0.022). No significant differences in GSSG levels were observed in TBI + ICAL mice when compared to untreated TBI mice or sham mice. All GSSG data are displayed as mean ± SEM and analyzed by a one-way ANOVA with post-hoc Tukey’s test (* indicates p < 0.05 and NS indicates p > 0.05 or nonsignificant). The ratio of GSH:GSSG as measured in brain homogenate of sham, untreated TBI, and TBI + ICAL analyzed at (C) 72 h post-TBI or sham surgery (n = 5–6 mice per group). Both untreated TBI and TBI + ICAL mice had a significantly reduced ratio of GSH: GSSG when compared to sham mice (p = 0.003). No significant differences were observed between untreated TBI mice and TBI + ICAL mice. GSH: GSSG data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test (* indicates p < 0.05, ** indicates p < 0.01, and NS indicates p > 0.05 or nonsignificant).

3.7. Immunocal®-treatment significantly reduces microgliosis at 72 h after rmmTBI

Astrogliosis, and prevention of astrogliosis with Immunocal® treatment were observed at 2 weeks and 2 months following rmTBI. We hypothesized that we would observe astrogliosis at a much earlier time point, 72 h post-rmmTBI and evaluated if Immunocal®-treatment would attenuate this effect. Although microgliosis was not observed in the rmTBI model, we analyzed microgliosis to see if it was induced by a more severe rmmTBI model and if Immunocal®-treatment would mitigate this effect. Cortices from all groups were stained for GFAP, Iba-1, and Hoechst to analyze astrogliosis, microgliosis, and nuclei, respectively 72 h post-rmmTBI (Figs. 8A and 9A). Astrocytes or microglia, counted as GFAP or Iba-1 (and Hoechst) positive cells, respectively, were quantified and averaged for all groups (Figs. 8B and 9B).

Fig. 8.

Fig. 8.

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Significant astrogliosis present in the cortex of mice subjected to repetitive mild-moderate-traumatic brain injury (rmmTBI) when analyzed at 72 h post-last mmTBI. Representative images of cortex stained for GFAP (green) and DAPI (blue), to label astrocytes and nuclei, respectively, from mice subjected to sham surgery (SHAM) and untreated mice (TBI) and Immunocal®-treated pre- and post-rmmTBI mice (TBI + ICAL) exposed to rmmTBI analyzed at (A) 72 h post-rmmTBI. White arrows indicate GFAP and DAPI positive cells, counted as astrocytes. Scale bar = 20 μm. Quantification of the average number of astrocytes in cortex stained with GFAP and DAPI, as described in (A) is shown for sham, TBI + ICAL, and untreated TBI mice analyzed at (B) 72 h post-rmmTBI (5–7 mice per group). The average number of astrocytes in the cortex of untreated TBI mice was significantly increased compared to sham mice (p = 0.033). No significant differences were observed between TBI + ICAL mice versus sham or untreated TBI mice. Astrocyte quantification data are displayed as the mean ± SEM and are analyzed by a one-way ANOVA with post-hoc Tukey’s test (* indicates p < 0.05 and NS indicates p > 0.05 or nonsignificant).

Fig. 9.

Fig. 9.

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Immunocal®-treatment pre- and post-repetitive mild-moderate TBI (rmmTBI) significantly reduced microgliosis in the cortex of mice when analyzed at 72 h post-last mmTBI. Representative images of cortex stained for Iba-1 (green) and DAPI (blue), to label microglia and nuclei, respectively, from mice subjected to sham surgery (SHAM) and untreated mice (TBI) and Immunocal®-treated pre- and post-rmmTBI mice (TBI + ICAL) exposed to rmmTBI analyzed at (A) 72 h post-rmmTBI. White arrows indicate Iba-1 and DAPI positive cells, counted as microglia. Scale bar = 20 μm. Quantification of the average number of microglia in cortex stained with Iba-1 and DAPI, as described in (A) is shown for sham, TBI, and TBI + ICAL mice analyzed at (B) 72 h post-rmmTBI (n = 3 mice per group). The average number of microglia in cortex of untreated TBI mice was significantly increased compared to sham mice (p = 0.021). Immunocal®-treatment pre- and post-rmmTBI significantly reduced the average number of microglia in the cortex at this time point when compared to untreated TBI mice (p = 0.021). No significant differences in the average number of microglia were observed between TBI + ICAL mice versus sham mice. Microglia quantification data are displayed as the mean ± SEM and are analyzed as a one-way ANOVA with post-hoc Tukey’s test (* indicates p < 0.05 and NS indicates p > 0.05 or nonsignificant).

A significant increase in the average number of astrocytes in cortex of untreated rmmTBI mice was observed versus sham mice at 72 h post-rmmTBI (p = 0.033, Fig. 8B). However, Immunocal®-treatment did not significantly reduce the average number of astrocytes versus untreated rmmTBI mice at this time point.

Interestingly, a significant increase in the average number of microglia in the cortex of untreated rmmTBI mice was observed versus sham mice at 72 h post-rmmTBI (p = 0.021; Fig. 9B). Immunocal®-treated rmmTBI mice showed a significantly decreased average number of microglia versus untreated rmmTBI mice and no difference from sham mice (p = 0.021). Both astrogliosis and microgliosis are present in the cortex of mice 72 h post-rmmTBI, however, Immunocal® only prevents microgliosis at this early time point.

4. Discussion

There is a need for preventative and restorative long-term treatments for individuals at high-risk for rTBI. Repetitive mTBI can cause cumulative injury to the brain, resulting in an enhanced susceptibility to neurodegeneration (Faden and Loane, 2015; Edwards et al., 2017; Gao et al., 2017; Bramlett and Dietrich, 2015). We investigated Immunocal®, a whey protein supplement shown to preserve brain GSH and provide neuroprotection in a mouse model of single moderate TBI, as a preventative and restorative treatment in models of rmTBI and rmmTBI (Ross et al., 2012; Winter et al., 2017; Ignowski et al., 2018). Since individuals at high-risk for rmTBI, such as contact sport athletes, likely sustain rmTBIs that go undiagnosed and, as a result, may not receive treatment, we assessed the therapeutic benefit of Immunocal® long-term (at 2 weeks, 2 months, and 6 months) in a mouse model of rmTBI. On the other hand, we analyzed Immunocal® treatment acutely (72 h) following more severe rmmTBI as more severe rmmTBIs are more likely to be clinically recognized and treated (Bailes et al., 2013).

We observed significant astrogliosis, but not microgliosis, in cortex around the injury site at 2 weeks and 2 months post-rmTBI which Immunocal® significantly reduced. Mouzon et al. (Mouzon et al., 2014) also did not report microgliosis in the cortex at 6 and 12 months post-rmTBI, however, microgliosis in corpus callosum was observed at these timepoints (Ross et al., 2014). Microgliosis in cortex in rmTBI mouse models is not observed chronically (Hoogenboom et al., 2019; Fidan et al., 2016). The rmTBI model may not be severe enough to elicit prolonged microgliosis in cortex. Microglia display heterogeneity in brain and increased numbers are observed in the substantia nigra, dentate gyrus, basil ganglia, and olfactory bulb in mice (Tan et al., 2020; Lawson et al., 1992). Microgliosis could have been present in sensitive areas or may subside before 2 weeks following rmTBI. Macrophage infiltration was observed 2 months post-rmTBI by MRI although no significant edema was observed. Furthermore, we quantified Iba-1 positive cells as microglia, so the observed inflammation could be due to supraependymal, epiplexus, or pericyte cells (Jordan and Thomas, 1988).

We observed a more robust injury response to rmmTBI. Clinically, NfL in serum is elevated following rTBI (Al Nimer et al., 2015; Shahim et al., 2017; Hossain et al., 2019; Shahim et al., 2020). We’ve shown that rmmTBI increases NfL in serum from mice 72 h post-rmmTBI, although Immunocal®-treatment had no effect. We also observed increased astrogliosis and microgliosis in cortex in response to rmmTBI. Similarly, astrogliosis and microgliosis in cortex 1–7 days post-TBI is reported following more severe rTBI (Petraglia et al., 2014; Chen et al., 2017). Interestingly, Immunocal®-treatment only prevented microgliosis in this model.

Lastly, we measured a decreased GSH:GSSG ratio at 72 h post-rmmTBI. Increased oxidative stress in brain results from TBI and leads to antioxidant depletion and cell death (Cornelius et al., 2013; Morris et al., 2019). Glutathione is an endogenous antioxidant that maintains redox homeostasis in brain. We did not observe GSH preservation or an increased GSH:GSSG ratio in Immunocal®-treated rmmTBI mice. We have previously shown that Immunocal® pre-treatment prevented the reduction in brain GSH:GSSG in a single moderate TBI mouse model (Ignowski et al., 2018). The repetitive nature of the model used here may cause sustained oxidative stress. Therefore, Immunocal® was unable to keep up with the increased oxidative burden and sustain brain GSH.

Immunocal®-treatment significantly limited astrogliosis in the rmTBI model and microgliosis in the rmmTBI model. Immunocal® offers its therapeutic effects through enhancing GSH synthesis. Lindenau et al. (1998) reported increased GPx localization and activity was only measured in activated astrocytes following excitotoxicity but not in resting astrocytes (Lindenau et al., 1998). However, GPx localizes to resting microglia and more activity was observed in activated microglia versus astrocytes (Lindenau et al., 1998). Higher GPx activity is beneficial in protecting microglia from ROS produced intrinsically or by nearby microglia (Lindenau et al., 1998). Astrocytes produce GSH to support neurons and increase GSH in neurons when cultured together (Dringen et al., 1999). Microglia synthesis of GSH is coupled to glutamate uptake which may be abundant following injury. Persson et al. (2006) reported that, in response to toxins, GSH synthesis and release was protective to microglia (Persson et al., 2006). Microglia may become resilient through GSH synthesis whereas astrocytes synthesize GSH to protect neurons. Furthermore, microglia have a higher cytosolic concentration of GSH in culture than either astrocytes or neurons (Chatterjee et al., 1999; Hirrlinger et al., 2000). In the more severe model of rmmTBI, a decrease in microgliosis and not astrogliosis may result from Immunocal® supplementation because microglia synthesize GSH for their own protection.

The present study only used male mice; however, further preclinical study should use both sexes. Furthermore, this study should be followed up with a more thorough analysis of oxidative stress and inflammatory biomarkers in brain taken at regular intervals in both the rmTBI and rmmTBI models. This would help broaden our understanding of the timeline of pathology induced by these models and further elucidate the effects that Immunocal® supplementation has on lipid peroxidation, DNA damage, and pro-inflammatory cytokine production.

In summary, rmTBI can induce lasting gliosis out to 2 months post-rmTBI. Furthermore, inflammation, neuronal damage, and altered redox homeostasis present acutely following rmmTBI. These findings support the use of easily administered preventative and restorative treatments, such as Immunocal®, for patients at high-risk for rTBI. Immunocal® supplementation for rmTBI and rmmTBI significantly limited gliosis. However, we believe that the repetitive nature of these models induces pathology that cannot be ameliorated by a single agent. Additional treatments (e.g., anti-glutamatergic compounds, lipid-based free radical scavengers, and mitochondrial protective agents), used in combination with a GSH precursor such as Immunocal®, may produce a synergistic therapeutic effect in more severe models of rTBI. The data presented here are important in that they show the limits of GSH-based neuroprotection in repetitive models of TBI.

Supplementary Material

MMC1
Fx1
Fx2

Acknowledgements

Funding: This study was supported in large part by funding from Immunotec Inc. (Quebec, Canada), which is the manufacturer of Immunocal®. The University of Colorado Animal Imaging Shared Resources are supported by the University of Colorado Cancer Center (NCI P30 CA046934) and the Colorado Clinical and Translational Sciences Institute (NIH/NCATS UL1 TR001082).

The authors would like to dedicate this work to the memory of Ms. Madison Russell (deceased).

Footnotes

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have received funding from Immunotec Inc. (Quebec, Canada) to support research on the therapeutic effects of Immunocal® in pre-clinical mouse models of TBI. DAL is currently a member of the Scientific Advisory Board for Immunotec Inc.

CRediT authorship contribution statement

Lilia A. Koza: Investigation, Resources, Validation, Formal analysis, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration. Claudia Pena: Investigation, Resources, Validation. Madison Russell: Investigation, Validation. Alec C. Smith: Investigation, Validation. Jacob Molnar: Investigation, Validation. Maeve Devine: Investigation, Validation. Natalie J. Serkova: Methodology, Investigation, Resources, Validation, Supervision, Writing – review & editing. Daniel A. Linseman: Conceptualization, Methodology, Investigation, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.brainres.2023.148338.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MMC1
NIHMS1889436-supplement-MMC1.docx (205.4KB, docx)
Fx1
NIHMS1889436-supplement-Fx1.jpg (215.8KB, jpg)
Fx2
NIHMS1889436-supplement-Fx2.jpg (121.4KB, jpg)

Data Availability Statement

Data will be made available on request.

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