Shumilov et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03158-9(2024) 21:171

Journal of Neuroinflammation

Open Access

RESEARCH

Early depletion of gut microbiota shapeoligodendrocyte response after traumaticbrain injury
Kirill Shumilov1, Allen Ni2, Maria Garcia-Bonilla1, Marta Celorrio2 and Stuart H. Friess2,3*
Abstract
White matter injury (WMI) is thought to be a major contributor to long-term cognitive dysfunctions after traumaticbrain injury (TBI). This damage occurs partly due to apoptotic death of oligodendrocyte lineage cells (OLCs) after
the injury, triggered directly by the trauma or in response to degenerating axons. Recent research suggests that the
gut microbiota modulates the inflammatory response through the regulation of peripheral immune cell infiltration
after TBI. Additionally, T-cells directly impact OLCs differentiation and proliferation. Therefore, we hypothesized thatthe gut microbiota plays a critical role in regulating the OLC response to WMI influencing T-cells differentiation
and activation. Gut microbial depletion early after TBI chronically reduced re-myelination, acutely decreased OLCs
proliferation, and was associated with increased myelin debris accumulation. Surprisingly, the absence of T-cells ingut microbiota depleted mice restored OLC proliferation and remyelination after TBI. OLCs co-cultured with T-cells
derived from gut microbiota depleted mice resulted in impaired proliferation and increased expression of MHCII compared with T cells from control-injured mice. Furthermore, MHC-II expression in OLCs appears to be linked
to impaired proliferation under gut microbiota depletion and TBI conditions. Collectively our data indicates that
depletion of the gut microbiota after TBI impaired remyelination, reduced OLCs proliferation with concomitantly
increased OLC MHCII expression, and required the presence of T cells. This data suggests that T cells are an
important mechanistic link by which the gut microbiota modulate the oligodendrocyte response and white matterrecovery after TBI.
Keywords Traumatic brain injury, White matter injury, Gut microbiota, T cells, Oligodendrocytes, Neuroinflammation,
Remyelination*Correspondence:
Stuart H. Friess
friess@wustl.edu
1
Department of Neurosurgery, Virginia Commonwealth University,
Richmond, VA, USA
2
Department of Pediatrics, Washington University in St. Louis School of
Medicine, St. Louis, MO, USA
3
Division of Critical Care Medicine, Department of Pediatrics, Washington
University in St. Louis School of Medicine and St. Louis Children’s Hospital,
Campus Box 8028, 3rd Fl MPRB 660 S. Euclid Avenue, St. Louis, MO
63110, USA© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

Introduction
In the United States, approximately 1.7 million people
experience traumatic brain injury (TBI) each year, andover 5 million face TBI-related disabilities [1]. The overall health cost attributable to non-fatal TBI in the UnitedStates has been estimated to be over $40 billion per year[2]. Traumatic white matter injury (WMI) is thought tobe a major contributor to long-term cognitive dysfunction in TBI survivors [3–5]. Oligodendrocytes, which
provide metabolic support to axons and are the producers of myelin in the central nervous system (CNS),
undergo apoptosis after TBI, triggered by direct injury
or in response to axonal degeneration [6, 7]. Mature oligodendrocytes present in the brain at the time of injury
have limited, if any, capability to contribute to remyelination, with the bulk of CNS remyelination attributed
to new oligodendrocyte production by oligodendrocyte lineage cells (OLCs) [8–10]. OLC-mediated remyelination involves a three-phase process of proliferation,
recruitment, and differentiation, which can be adversely
impacted by inflammation after TBI [11, 12].
Research over the past few years has revealed that the
gut microbiota influence neurogenesis, myelination andsubsequently functional and behavior outcomes [13, 14].
This complex interplay is known as the gut-brain axis, abidirectional communication that includes neuro-endocrine-immunological activity [15]. Early-life gut microbial depletion has been shown to have long-term effects
on maturation of myelinating oligodendrocytes leading
to altered cognition and anxiety-like behavior [16]. The
gut microbiota has been shown to modulate the inflammatory response after acute CNS injury impacting injury
severity and recovery [17–19]. Recently, we showed thatgut microbiota depletion after TBI alters the innate andadaptive immune response [17]. The innate immune
response plays an important role in myelin debris clearance which influences OLC proliferation and migration[20]. Activated T-cells have been found to have a trophic
role by promoting OLC proliferation in vitro [21], while
effector T-cells inhibit OLCs differentiation in an inflammatory demyelinating mouse model [22]. However,the possible T cell-OLC crosstalk and the mechanismof cell-cell communication after WMI hasn’t yet been
elucidated.
Gut microbiota perturbations are common after TBI[22, 23], but their potential impact on remyelination after
traumatic WMI is currently unknown. Therefore, wehypothesized that the gut microbiota plays a critical role
regulating the OLC response to traumatic WMI influencing T-cells differentiation and activation. We evaluatedwhether an intact enteric microbiome early after TBI
is critical for OLC proliferation and remyelination. We
further investigated if the presence of T cells is requiredfor gut microbial modulation of the OLC response andPage 2 of 18

subsequent remyelination following TBI. Finally, using
an in vitro model system we further explored OLC/T-cellinteractions and the role of the gut microbiome.

Materials and methods
Animals

All procedures were approved by the Washington University Animal Studies Committee (Protocol 19–0864)
and are consistent with the National Institutes of
Health guidelines for the care and use of animals. Animals were housed 5/cage and had free access to waterand food with a 12-hour light/dark cycle. C57BL/6J(RRID: IMSR_ORNL: C57BL/6J-A/A) and B6.129P2Tcrbtm1MomTcrdtm1Mom/JJ (TCRβ−/−TCRδ−/−) (RRID:IMSR_JAX:002122) 8 week-old male and female mice
(Jackson Laboratory, Bar Harbor, ME) weighing 20–25 g(g) were used for in vivo studies. C57BL/6J male andfemale neonatal P2-P5 pups were used for in vitro
studies.
Controlled cortical impact with gut microbial depletion

Controlled cortical impact (CCI) was performed using apreviously described protocol [17]. Mice were anesthetized with 5% isoflurane and maintained at 2% isoflurane
throughout the procedure. Buprenorphine sustainedrelease (0.5 mg/kg) was administered subcutaneously,prior to scalp incision. Ear bars were positioned to secure
the head within the stereotaxic frame (MyNeurolab, St.
Louis, MO). A 5 mm craniectomy was performed using
an electric drill, centered 2.7 mm lateral to the midline
and 3 mm anterior to lambda. Animals were randomly
assigned to either CCI or sham group after craniectomy
using a computer-generated randomization algorithm.
The electronic impactor (Leica Biosystems, Richmond,VA) equipped with a 3 mm tip was aligned with the craniectomy site using the following coordinates: 1.2 mm
lateral to the midline and 1.5 mm anterior to lambda[17]. The impact was delivered at a depth of 2 mm witha velocity of 5 m/s and a dwell time of 100 ms. A loosefitting 7 mm plastic cap was secured over the craniectomy site using Vetbond (3 M, St. Paul, MN). The skinincision was closed with interrupted sutures and treatedwith antibiotic ointment. Animals were then placed on a
warming pad for recovery.
For gut microbiota depletion, broad-spectrum antibiotics were administered for 7 consecutive days in thedrinking water consisting of 250 mg vancomycin, 500 mg
neomycin sulfate, 500 mg ampicillin, 500 mg metronidazole (VNAM), and 10 g grape-flavored Kool-Aid (Kraft
Heinz, IL, Chicago) in 500 mL of sterile-filtered water[25]. Control animals received only Kool-Aid in drinking
water to reduce the bitter flavor of the antibiotics.

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

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We performed two fecal microbiota transplantations(FMTs) of the gut microbiota from VNAM- or KoolAid-treated uninjured animals into germ-free (GF) miceas previously described [24]. Briefly at Day 17 and Day
10 prior to injury, GF mice received FMTs from animals with 7 days of VNAM or Kool-Aid treatment. For
each extraction, fecal pellets were collected 7 days after
initiation of VNAM or Kool-Aid treatment. The pellets
were mixed with phosphate-buffered saline (PBS) andafter 5 min, the sample was vortexed to break up the
fecal matter and allowed to sit for ∼5 min for debris tosettle. The supernatant was then removed with an uncut
P1000 pipette tip into a sterile 5 mL tube to avoid material clogging the gavage needle. Mice were gavaged witha sample volume of 250 µL. Ten days after the last FMT,
we performed CCI in all the mice and they remained insealed cages in the animal facility for 7 days before being
euthanized.

performed on four 50-µm thick slices spaced 300 μm
apart with the most rostral slice being the first appearance of the dorsal hippocampus. Free-floating slices wererinsed 3 times with tris-buffered saline (TBS) for 5 min atroom temperature (RT), and then incubated for 12 min at
60 °C in pre-warmed BGII solution (0.3% in 0.9% NaCl),followed by 2 washes in distilled water at RT. Next, slices
were incubated in pre-heated sodium thiosulfate (1% in
distilled water) at 60 °C for 3 min. After 3 washes in TBS,tissue was mounted on charged slides and dried overnight. Slides were dehydrated using a serial of gradedalcohols (50%, 70%, 95%, and twice with 100%) and coverslipped with DPX (Sigma-Aldrich, St. Louis, MO).
Images were generated using 20X objective with a Brightfield Zeiss Axio Scan Z1 microscope (Zeiss, White Plains,NY). Percent of myelinated area of the CC was quantified
using ImageJ software [25]. The CC region of interest was
defined as the area between the mid CC and the cingulum unless it was truncated by the injury.

CD3-cpecific monoclonal antibody injection

Fluorescence immunohistochemistry

For in vivo depletion of CD3 expressing cells, mice were
injected intraperitoneally (i.p.) with 200 µg of InVivoPlus anti-mouse CD3ε, clone 145-2C11 (1 µg/µl, Cat#
BP0001-1, Bio X Cell, Lebanon, New Hampshire), starting 6 days before injury, followed by 100 µg injections
every four days for 1 month, to ensure T-cells depletion.
The control group received the same injection regime
with InVivoPlus polyclonal Armenian hamster IgG (Cat#BP0091 Bio X Cell, Lebanon, New Hampshire).
To detect cell proliferation in the pericontusional corpus
callosum (CC), animals received intraperitoneal injections of 5-bromo-2’-deoxyuridine (BrdU, Sigma-Aldrich,
St. Louis, MO) 50 mg/kg i.p. daily for 4 consecutive days
starting 3 days post-injury.

Fluorescence immunohistochemical staining was performed on free-floating sections. Tissue was incubatedwith pre-heated HCl 1 N (Sigma-Aldrich) for 30 min at
45 °C to increase the antigen exposure for BrdU detection. After the three washes with PBS, 20% normal donkey serum, 3% bovine serum albumin, and 0.3% triton
X-100 in PBS were used to block nonspecific staining for
all antibodies. Sections with a thickness of 50 μm were
stained with the primary antibodies (Table 1) at 4 °C
overnight. The next day, antibody binding was detectedby incubating sections with Alexa Fluor secondary antibody (Table 1) for 2 h in PBS with 0.3% triton X-100.
Sections were mounted on glass slides in PBS, dried, andcoverslipped with mounting medium for fluorescencewith 4’,6-Diamidino-2-Phenylindole, (DAPI, Thermo
Fisher Scientific, Waltham, MA).

Tissue processing

Quantitative fluorescent immunohistochemistry

Mice were euthanized under isoflurane anesthesia, bytranscardial perfusion with cold 0.3% heparin in PBS followed by 4% paraformaldehyde solution in PBS (PFA,
Sigma-Aldrich, St. Louis, MO). Brains were post-fixed
in 4% PFA for 24 h at 4 °C followed by equilibration in
30% sucrose for 48 h before sectioning. Using a freezing
microtome, brains were cut, and four 50-µm thick cryosections with complete CC spaced 300 μm apart wereused for the subsequent analysis.

Fluorescent images were obtained with a Zeiss Axio
Imager Z2 with ApoTome 2 fluorescence microscopewith a 20X objective. 20-µm z stacks with an interval of
1 μm were obtained of the ipsilateral CC. Quantification of OLC proliferation was performed by counting
the number of cells that co-localized with nuclear and/or cytoplasmic Olig1 immunolabeling and BrdU staining in 4 slices spaced 300 μm apart by a blinded observer.
Degraded myelin basic protein (dMBP) fluorescentimmunostaining was performed on adjacent sections.
Images were generated using 20X objective with a fluorescence slide scanner Zeiss Axio Scan 7 microscope(Zeiss, White Plains, NY). dMBP percent area of the CC
was quantified using ImageJ software [25].

Fecal microbiota transplantation

5-bromo-2’-deoxyuridine (BrdU) treatment

Myelin Black Gold II staining

Myelin Black Gold II (BGII, Histo-Chem, Jefferson, AR)
staining was performed as previously described [26, 27].
Briefly, to visualize individual myelin fibers in the CC
and quantify myelinated percent area, BGII staining wasShumilov et al. Journal of Neuroinflammation(2024) 21:171

Table 1 Overview of the antibodies for immunohistochemistry
and flow cytometry used in this study
Antibody

Fluorophore

CD45

BV425

CD3ɛ

AF700

CD4

BUV395

CD8a

BV785

CD11b

BV510

MHC-II

TNFa

APC

IL-4

PerCP-Cy5.5

IL-17

PE-Cy7

Olig1
BrdU

βAPP

dMBP
Secondary AF594
antibody
Secondary AF647
antibody
Secondary AF488
antibody
Secondaryantibody

Clone

Species

Source Productnumber30-F11 Rat
BioLe- 103,134
monoclo- gendnal
500-A2 Armenian BioLe- 100,320
Hamster gend
monoclonal
GK1.5
Rat
BD
565,974
monoclo- Bioscinalences
53 − 6.7 Mouse
BioLe- 100,750
monoclo- gendnal
M1/70 Rat
BioLe- 101,263
monoclo- gend
nal
AF6MouseeBio46-5320120.1
monoclo- science 80
nal
MP6Mouse
BioLe- 506,307
XT22
monoclo- gendnal
11B11 Rat
BioLe- 504,123
monoclo- gend
nal
TC11Mouse
BioLe- 206,921
18H10.1 monoclo- gendnal
RabbitMilAB15620
polyclonal lipore
BU1/75 Rat
Abcam Ab6326
monoclonal
Rat
Abcam Ab32136
monoclonal
Rabbit
MilAb5864
polyclonal lipore
DonkeyThermo A-21,209
anti-rat
FisherDonkeyThermo A-31,571
antiFisher
mouse
DonkeyThermo A-21,206
anti-rabbit Fisher
Biotinyl- Vector BAated goat Labo- 1000-1.5
anti-rabbit ratories

Primary oligodendrocyte lineage cell culture

Primary OLC culture and isolation was performed aspreviously described with a modified protocol [26, 27].
Briefly, brains from postnatal day P2-5 mice pups wereisolated under a dissection microscope. Then, the cortical tissue were dissociated with a digestion cocktail
containing papain (1.5 mg/ml, Worthington, Lakewood,

Page 4 of 18OH) in a tissue culture incubator at 37 °C and 5% CO2
for 55 min. After a centrifugation at 100 g for 7 min,cells were resuspended in proliferation medium with
DEMEM (Gibco, Waltham, MA), glutamax (1%, Gibco,
Waltham, MA), penicillin-streptomycin (1%, Gibco,
Waltham, MA), and fetal bovine serum (10%, Gemini Biproducts, Sacramento, CA) and plated in T-25 culture
flask pre-coated with poly-L-lysine (100 µg/ml, SigmaAldrich, St. Louis, MO), the dissociated cortical tissue
where plated at a concentration of 2,5 brains/flask. The
mixed glia culture was incubated at 37 °C and 5% CO2.
When confluence reached 90–100% (day 3–4), flasks
were shaken at 330 RPM overnight, to dislodge OLCs
and microglia from astrocytes which strongly attach tothe flask. The supernatant containing OLCs and microglia were collected and added to a 100 mm tissue culture
dish. Petri dishes were incubated at 37 °C and 5% CO2 for
30 min, to allow microglia to adhere. Medium containing
enriched OLCs was harvested and centrifuged at 300 g
for 7 min. Cells were then seeded at a density of 1 × 104
cells/well on a 96-well plate or 1 × 105 cells/well on a 6
well plate coated with poly-L-lysine and grown at 37 °C
and 5% CO2 for 2 days in OLC basal medium. OLC basal
medium containing DEMEM/F12 (MilliporeSigma, Burlington, MA) was supplemented with N2 (1%, ThermoFisher), B27 (1%, ThermoFisher), penicillin-streptomycin(1%, Gibco, Waltham, MA), BSA (0.3%, Sigma-Aldrich,
St Louis, MO), bEGF (10 ng/ml, PeproTech, Waltham,MA), FGF (10ng/ml, PeproTech) and PDGFaa (10 ng/ml
PeproTech).
T-cells isolation from spleen

Spleens were collected from injured animals treatedwith VNAM or Kool-Aid as described above. Splenocytes were obtained by mechanical shredding and filteredthrough a 70-µm cell strainer, and centrifuged at 500 g
for 10 min. The resulting cell suspensions were incubatedwith red blood lysis buffer (Roche Diagnostics Gmbh,
Mannheim, Germany) for 5 min at 4°C, centrifuged andfiltered through 40-µm cell strainer. T-cells were thenisolated following manufacturers protocol by negative
selection using pan T-cell isolation kit II (Miltenyi biotec). This isolation kit is based on a cocktail of biotinconjugated antibodies against CD11b, CD11c, CD19,
CD49b, CD105, Anti-MHC-class II, and Ter-119. Cells
were counted and co-cultured with OLCs at a density of
3 × 104 cells/well on a 96-well plate or 3 × 105 cells/well on
a 6-well plate at 37 °C and 5% CO2 for 24 h.
Immunocytochemistry

Cells were permeabilized using 0.3% TX-100 in PBS for
10 min. Then cells were incubated with HCl 1 N (SigmaAldrich, St. Louis, MO) for 30 min at 45 °C to increase
the antigen exposure for BrdU detection. Non-specificShumilov et al. Journal of Neuroinflammation(2024) 21:171

antibody interactions were blocked using 20% NDS in
PBS for 1 h. Primary antibodies (Table 1) were diluted in
the same blocking solution and incubated overnight at
4 °C. The cells were washed with PBS and secondary antibodies (Table 1) diluted in PBS were incubated for 2 h.
For nuclei detection, cells were incubated for 10 min withDAPI (1:5000, Life Technology, Carlsbad, CA) and stored
at 4 °C in PBS. The immunofluorescent images were takenusing Zeiss Celldiscover 7 (Zeiss, White Plains, NY) with
10x objective, 4 tiles per well were automatically scanned.
We used ImageJ (NIH public software) particle analysis
plugin with macro instructions [27] to quantify the number of cells and percent of area immunostained. To analyze the co-localization of BrdU and DAPI positive cells
quantitative analysis of Mander’s coefficient were performed using the JACoP plugin for ImageJ.
Flow cytometry analysis

After 24 h of co-culture with OPC, T-cells were stimulated for intracellular cytokines expression in vitro.
Supernatant from the co-culture were collected centrifuged at 300 g for 1 min and resuspended in RPMI 1640
with glutamax, 10% FCS, 12.5 mM of Hepes (Gibco), 1%
of Pen/Strep (Gibco, Waltham, MA), 50µM of B-mercaptoetanol (Sigma-Aldrich, St. Louis, MO) and 10 µg/
ml of gentamycine (Sigma-Aldrich) with 100 ng/mlof phorbol 12-myristate 13-acetate (PMA), 1 µg/ml ofionomycine (Sigma-Aldrich) and 1x brefeldin (BioLegend, San Diego, CA) at 37 °C and 5% CO2 for 4 h. OPCs
were detached from the surface of the culture plate byrepetitive resuspensions in FACS buffer and collected
in 1 ml tubes, followed by centrifugation at 300 g for
7 min. Next, cells were incubated for 5 min with Zombie
NIR Dye (BioLegend, San Diego, CA). Then, cells were
washed with FACS buffer, stained with their respective
antibody mix (Table 1) for 30 min at RT, and analyzed
on a BD LSRFortessa flow cytometer (BD Biosciences,Franklin Lakes, NJ) using the Software v10.6.1 (BD Biosciences, Franklin Lakes, NJ). T-cells were defined as
CD45highCD11b−CD3+.
Intracellular cytokine staining and analysis

Cells were incubated for 5 min at RT with Zombie NIR
Dye (BioLegend, San Diego, CA) to assess their viability. The Zombie NIR Dye was quenched, and cells were
washed with cytometry buffer and blocked with FcRblocking reagent (1:50, Miltenyi Biotec, Bergisch Gladbach, Germany). Then, the samples were washed withcytometry buffer, stained with antibodies (Table 1)
for 15 min at RT, Microglial cells were defined as
CD45lowCD11b + and T cells as CD45hiCD11b − CD3+.
For the intracellular staining, cells were first stained withsurface markers as indicated above, stimulated, fixedand permeabilized by using FoxP3/transcription factor

Page 5 of 18

staining buffer set (eBiosciences) following the manufacturer’s instructions. Briefly, cells were stimulated for 4 hwith 0.2 µg/mL phorbol 12-myristate 13-acetate (PMA)/
2 µg/mL ionomycin/ 1x brefeldin A to characterize T cellsubset changes associated with GMD after TBI. Next
cells were fixed for 7 min at 4 °C, washed, permeabilized and stained with intracellular markers (Table 1) for
30 min at 4 °C. We measured the percentages of IL-17 + T
cells. Then, cells were washed and analyzed on a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes,
NJ) using the Software v10.6.1 (BD Biosciences, Franklin
Lakes, NJ). Fluorescence minus one (FMO) and isotype
control antibodies were used as negative controls foreach marker.
Statistical analysis

Blinding of investigators to experimental groups wasmaintained until data were fully analyzed. Data wereassessed for normal distribution with the Shapiro-Wilk
test and expressed as mean ± SEM. Two-tailed Student’s
t-test was used when comparing two conditions. For
more than two conditions, ANOVA and Tukey’s multiple
comparison post-hoc test were employed. All analysis
was performed with GraphPad Prism v10.1.0 (GraphPad
software. Boston, MA).

Results
Gut microbiota depletion impedes white matter repair
three months after TBI

Our previous work has demonstrated that gut microbial
depletion for 1 week significantly impacts neuroinflammation and fear memory 3 months after TBI [17]. Since,
chronic white matter degeneration is influenced by prolongated neuroinflammation [28], we decided to investigate the impact of gut microbiota depletion on whitematter remyelination 3 months after TBI. Gut microbiota
was depleted by administering broad-spectrum antibiotics orally to mice immediately after CCI for 1 week
(Fig. 1a). Three months post-TBI, white matter remyelination was assessed by staining the peri-contusional CC
with BGII and measuring the percentage of myelinatedarea adjacent to the lesion site (Fig. 1b and c). Our findings revealed that early gut microbiota depletion after
injury significantly reduced the percentage of myelinatedarea in the CC compared with Kool-Aid-treated animals
3 months post-TBI (Fig. 1c). However, the underlying
mechanisms by which gut microbiota influences postTBI recovery remain to be elucidated. Therefore, we next
performed a more in-depth analysis of gut microbiota
depletion impact on acute WMI.

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 1 Gut microbial depletion impairs remyelination after TBI. a Experimental design: one week of VNAM treatment after TBI; animals were sacrificed
3 months after injury. b Representative images of the CC (black dashed lines) areas stained with BGII. c Quantification of percentage of the myelinatedarea of CC interaction of injury and gut microbiome F(1,21) = 16.29 p = 0.0006 interaction of VNAM and CCI. Mean values are plotted ± SEM. ***p = 0.0006;****p < 0.0001. Two-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine statistical differences; n = 6–7 mice pergroup. Scale bar = 200 μm and 50 μm in the inserts. Abbreviations: CC: corpus callosum; CCI: controlled cortical impact; CX: cortex; BGII: myelin black gold
II; VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

Depletion of gut microbiota decreases oligodendrocyte
lineage cell proliferation and increases myelin debris one
week after TBI

Next, we wanted to further explore the effect of gut
microbiota depletion on acute WMI and remyelination
7-day post-TBI (Fig. 2a). One critical inhibitor of remyelination and oligodendrocytes proliferation is myelin
debris accumulation [29]. After WMI, myelin debris isaccumulated specially in CC due to glial dysfunction andcan be associated with axon regeneration impairment
and neuroinflammation [30]. We found an increase in
myelin debris accumulation in the CC of VNAM-treatedinjured mice compared with injured controls (Fig. 2b-c).
Additionally, gut microbiota depletion suppressed OLC

proliferation, a crucial step in remyelination, as indicatedby a significant reduction in BrdU/Olig1 double-positive
cells (Fig. 2d-e). The density of β-APP swelling, indicative of axonal injury, did not differ between VNAM and
Kool-Aid treated mice one week after TBI (Fig. 2f-g).
Furthermore, to determine if the impact of gut microbiota depletion on the white matter remyelination after
TBI was mediated directly by antibiotics or indirectly
via modulation of the gut microbiota, we utilized a fecal
microbiota transplantation (FMT) approach (Fig. 2h).
Consistent with the results described above, GF micethat received FMT from VNAM-treated animals exhibited significantly higher percent area of myelin debris
accumulation compared to those receiving Kool-AidShumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 2 Gut microbial depletion reduces oligodendrocyte lineage cell proliferation after TBI. a Experimental design: one week of VNAM treatment
after TBI; animals were sacrificed one week after injury. Representative images of CC (white dashed lines) stained with b dMBP, c Olig1+/BrdU+, and dbAPP. e Quantification of percentage of the dMBP-stained area of the CC. f Quantification of Olig1+/BrdU+ cells in the ipsilateral CC. g Quantification ofβAPP axonal swelling density of the CC. h Experimental design: Germ free (GF) mice were gavaged with two fecal microbiota transplants (FMT) of the gut
microbiota from mice treated with VNAM or Kool-Aid uninjured animals on Day − 7 and day − 17 prior to injury. i Representative fluorescent images ofCC (white dashed lines) stained with dMBP. j Quantification of percentage of the dMBP-stained area of the CC. Mean values are plotted ± SEM. *p < 0.05.
Unpaired t tests was used to determine statistical differences; n = 5–9 mice per group. Scale bar = 200 μm and 50 μm in the inserts. Abbreviations: CC:
corpus callosum; CCI: controlled cortical impact; CX: cortex; dMBP: degraded myelin basic protein; FMT: fecal matter transplant; GF: germ free; SPF: specificpathogens free; VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

FMT (Fig. 2i-j). Taken together, these results support our
hypothesis that the gut microbiota plays a pivotal role inmodulating post-traumatic myelin debris clearance and
OLC proliferation.
Pharmacological depletion or genetic deletion of T-cells
rescues OLC proliferation and remyelination impaired by
gut microbial depletion

Previously, we have demonstrated that gut microbiota
depletion after TBI impairs hippocampal neurogenesis,
promotes pro-inflammatory microglia phenotype andsurprisingly reduced T cell infiltration into the brain up
to one month after injury [17] but then normalizes by 3months after injury (Supplemental Fig. 1). Building uponthese findings, we wanted to further investigate the roleof the T-cells in gut-brain communication in the context
of WMI. To achieve the depletion of T-cells, we employedpharmacological treatment with anti-CD3 IgG for one
month, to be able to address changes in myelin density(Fig. 3a). Peripheral blood analysis following T-cell depletion revealed a nearly complete absence of CD4+ (Fig. 3b)
and CD8+ (Fig. 3c) lymphocytes. Surprisingly, the percentof myelinated area (Fig. 3d and f ) and impaired OLC proliferation (Fig. 3e and g), effect of gut microbial disruption, was restored after the depletion of T-cells compared
to VNAM-treated mice with control IgG injections. Furthermore, TCRβ−/−TCRδ−/− mice (absence of alpha beta
T-cell receptor and any gamma delta T-cell receptor)
exposed to VNAM for one week (Fig. 4a) showed similarfindings. The density of oligodendrocyte lineage proliferative cells (Fig. 4b-c), and myelin debris accumulation(Fig. 4d-e) remained unaffected by microbial depletionin TCRβ−/−TCRδ−/− mice. Taken together, these findings
suggest that T-cells have a crucial role in gut-brain communication and the modulation of remyelination following traumatic WMI.
In vitro co-culture of T-cells derived from injured mice withgut microbiota depletion reduced proliferation of OLCs

OLCs are susceptible to damage from inflammatoryenvironments triggered by T-cell cytokine production(Larochelle et al., 2021). This exposure leads to a significant shift in their gene expression profile (Falcao et al.,2018). Cytokines, such as IL17 further modulate OLC
functions, leading to apoptotic death of oligodendrocytes[31]. Since, the absence of T-cells mitigated the impact
of gut microbiota depletion on traumatic WMI repair,
we decided to investigate the mechanism of interaction
between OLCs and T-cells in vitro (Fig. 5a). T-cells isolated from spleens of injured animals with and without
microbiota depletion 7 days after injury, were co-cultured with OLCs. We found decreased Olig1+ (Fig. 5c),and BrdU+ (Fig. 5d) cell density when OLCs were cocultured with T-cells from injured mice with microbiota

Page 8 of 18

depletion. Analysis using Mander’s coefficient revealed
co-localization of 96% of BrdU staining with Olig1(0.9606 ± 0.02), indicating that the vast majority of proliferating cells were OLCs. Additionally, morphologicalanalysis of OLCs showed an increase of circularity coefficient, indicative of a decreased maturation [32], when
T-cells derived from injured animals with gut microbiota
depletion were added to the medium (Fig. 5e). To further
explore this cellular interaction, medium from T-cell and
OLCs culture where exchanged (Sup. Figure 1a), however, no changes of Olig1 or BrdU density was detected
after 24 h of culture (Sup. Figure 1b-c).
Next, we characterized the T-cell differentiation fromthe in vitro co-culture using flow cytometry (Fig. 6a). No
significant differences were observed in the total numberof CD3+ lymphocytes (Fig. 6b). However, the percentage
of CD8+ (Fig. 6c) were significantly lower in both co-culture groups, and CD4+ (Fig. 6e) showed a decrease onlyin the Kool-Aid group. The percentage of CD4+ T-cells
expressing IL17 (Fig. 6f ) cytokines was significantly
higher in lymphocytes derived from spleens of injured
mice with depleted gut microbiota compared withinjured controls. No significative changes were detected
when IL4 (Fig. 6g) expression was analyzed. Furthermore,when OPCs medium was added to T-cells culture a similar increase IL17 (Sup. Figure 1 g) was detected in T cells
derived from gut microbiota depleted mice. Collectively,
our findings provide evidence that differentiation ofT-cells towards more pro-inflammatory phenotype could
impair white matter repair after TBI through the modulation of OLC proliferation.
To further elucidate the nature of T-cell and OLC interactions in vitro, we employed a trans-well co-culture
system (Fig. 7a). The permeable barrier allowed media
exchange while preventing direct cell contact. Interestingly, Olig1+ (Fig. 7c), and BrdU+ (Fig. 7d) presented atrend towards a decreased OLCs density. However, CD4+lymphocytes derived from spleens of VNAM treated andinjured animals exhibited significantly elevated expression of IL17 (Fig. 7f ) cytokine compared to Kool-Aid
controls. This finding suggests that, while modulation of
OLC proliferation could have a greater impact when contact with T-cells is direct, CD4+ lymphocytes differentiation occurs indirectly through extracellular signaling.
Our findings provide additional evidences of the immunomodulatory role of OLCs, highlighting their complex
bidirectional interplay with T-cells.
Gut microbiota depletion increase T-cell-induced MHCII
expression in OLCs after TBI

There is a growing body of evidence suggesting that
OLCs can express immunomodulatory factors such as
cytokines/chemokines and their receptors [33, 34] et al.,2014). T-cells/OLCs bidirectional communication wasShumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 3 Pharmacological depletion and genetical deletion of T-cells restore OLCs proliferation after TBI and gut microbial depletion. a Experimental design: six days before injury animals received the first injection of CD3 IgG/control IgG then every four days; one week of VNAM treatment after
TBI; animals were sacrificed one month after injury. Quantification of cell absolute numbers in the blood for b CD4 T-cells (CD11b−CD3+CD4+) and c CD8
T-cells (CD11b−CD3+CD8+). d Representative images of the CC (black dashed lines) areas stained with BGII. e Representative fluorescent images of CC(white dashed lines) stained with Olig1/BrdU. f Quantification of percentage of the myelinated area of CC F(1,30) = 1.572 p = 0.2195 interaction of VNAM andCD3 depletion. g Quantification of Olig1+/BrdU+ proliferative cell density of the CC F(1,35) = 7.986 p = 0.0077 interaction of VNAM and CD3 depletion. Meanvalues are plotted ± SEM. ****p < 0.0001; ***p < 0.0006; **p < 0.005. Two-way ANOVA followed by Tukey multiple comparison post hoc test was used todetermine statistical differences; n = 6–7 mice per group. Scale bar = 200 μm and 50 μm in the inserts. Abbreviations: CC: corpus callosum; CCI: controlled
cortical impact; CX: cortex; BGII: myelin black gold II; VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 4 Gut microbial depletion after TBI does not impact OLC proliferation and dMBP accumulation in TCRβ-/-TCRδ-/-mice. a Experimental design: one week of VNAM treatment after TBI; animals were sacrificed one week after injury. b Representative fluorescent images of CC (white dashed lines)
stained with Olig1 and BrdU. c Quantification of Olig1+/BrdU+ proliferative cell density of the CC. d Representative images of CC (white dashed lines)
stained with dMBP. e Quantification of percentage of the myelinated area of CC. Mean values are plotted ± SEM. Unpaired t tests was used to determine
statistical differences; n = 4–5 mice per group. Scale bar = 200 μm. Abbreviations: CC: corpus callosum; CCI: controlled cortical impact; CX: cortex; dMBP:
degraded myelin basic protein; VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

described in a previous study, where OLCs increased
expression of immunoprotective genes suggesting a
potential mechanism of immune functions in the context of multiple sclerosis (MS) disease [35]. To further
explore the mechanism of bidirectional communication
between T-cells and OLCs, we decided to analyze major
histocompatibility complex II (MHC-II) expression. The

presence of genes associated with MHC class I and II
was found in OLCs in response to interferon exposure[35, 36]. OLCs MHC-II intensity values was analyzed invitro, co-cultured with direct T-cells contact (Fig. 8a),and with trans-wells cell culture (Fig. 8b). Additionally,
Olig1+/MHC-II+ cell density was analyzed in the pericontusional CC (Fig. 8c). We found that OLCs exposedShumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 5 Co-culture of T-cells derived from injured animals with gut microbiota depletion impairs OLCs proliferationin vitro. a Experimental design: Oligodendrocyte lineage cells were isolated form P5 mice brains and cultured for five days. b Representative fluorescent images stained Olig1 and
BrdU. c Quantification of Olig1 cell density F(1.99,17.99) = 14.85 p = 0.0002 VNAM treatment interaction. d Quantification of BrdU proliferative cell density
F(1.215,10.95) = 14.32 p = 0.0022 VNAM treatment interaction. e Quantification of cell circularity coefficient F(1.582,9.495) = 22.96 p = 0.0004 VNAM treatment
interaction. Mean values are plotted ± SEM. ***p < 0.0005; **p < 0.005. One-way ANOVA followed by Tukey multiple comparison post hoc test was used todetermine statistical differences; n = 10 wells per group. Scale bar = 20 μm. Abbreviations: OLCs: oligodendrocyte lineage cells; PFA: paraformaldehyde;
VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

to T-cell derived from microbiota depleted animals hada significantly higher MHC-II intensity mean value compared with control (Fig. 8d). Surprisingly, this difference
vanished under in vitro trans-well co-culture conditions (Fig. 8e), suggesting that cell-to-cell contact might
be necessary for T-cell-mediated MHC-II expression
on OLCs. Furthermore, Olig1 and MHC-II co-localization significantly increased in peri-contusional CC of
injured and VNAM-exposed animals compared withinjured controls (Fig. 8f ), indicating potential activationof immunomodulatory functions in OLCs. This data
supports the potential immunomodulatory functions of
OLCs under gut-microbial depleted context and the pivotal role of T cells in gut-brain communication.

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 6 Co-culture of OLCs increased IL17 CD4+T-cells derived from injured animals with gut microbiota depletion. a Experimental design: cells
were isolated form P5 mice brains and cultured for five days; after overnight shaking supernatant cells were co-cultured in plates with T-cells for 24 h. b
Gating strategy. c Quantification of CD3+ cells. d Quantification of the % of CD8+ cells from CD3+ T-cells F(1,18) = 5.24. e Quantification of the % of CD4+
cells from CD3+ T-cells F(1,18) = 163.8. f Quantification of the % of IL17+ cells from CD4+ T-cells F(1,18) = 34.76. g Quantification of the % of IL4+ cells from
CD4+ T-cells. Mean values are plotted ± SEM. ****p < 0.0001. Two-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine
statistical differences; n = 6–9 wells per group. Abbreviations: VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

Discussion
This report provides evidence that the gut-brain axis
influences white matter remyelination after TBI. Depletion of the gut microbiota after TBI impaired OLC proliferation and resulted in long-term reductions in whitematter remyelination. Furthermore, our data suggeststhat T-cells play an important mechanistic link in gutbrain communication in regards to OLC proliferation
and remyelination. Consistent with observations of MS
related disease, where MHC-II expressing OLCs could
activate effector T-cells [35], we show that T-cells froma gut microbial depleted host could differentiate into

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 7 Trans-well co-culture of T-cells derived from injured and microbiota depleted animals with OLCs partially reduced OLCs proliferationand increased CD4+IL17 expression. a Experimental design: cells were isolated form P5 mice brains and cultured for five days; after overnight shaking supernatant cells were cultured in plates with trans-well co-cultured T-cells for 24 h. b Representative fluorescent images stained Olig1/BrdU cells.
c Quantification of Olig1 cell density. d Quantification of BrdU proliferative cell density. e-f Gating and quantification of the % of IL17+ cells from CD4+
T-cells. Mean values are plotted ± SEM. ***p < 0.0003 **p < 0.066; *p < 0.046. Unpaired t test was used to determine statistical differences; n = 7 wells pergroup. Scale bar = 20 μm. Abbreviations: OLCs: oligodendrocyte lineage cells; PFA: paraformaldehyde; VNAM: vancomycin, neomycin-sulfate, ampicillin,
and metronidazole

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

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Fig. 8 Gut microbial depletion after TBI increase MHCII expression in OLCs through direct T-cell contact. a Representative fluorescent images of
Olig1+/MHCII+ staining of OLCs 24 h after T-cell co-culture. b Representative fluorescent images of Olig1/MHCII staining of OLCs 24 h after T-cell transwell co-culture. c Representative fluorescent images of CC (white dashed lines) stained with Olig1/MHCII. d Quantification of MHCII intensity mean valueof OLCs 24 h after T-cell co-culture F(1.078,6.469) = 22.95 p = 0.0014 VNAM treatment interaction. e Quantification of MHCII intensity mean value of OLCs
24 h after T-cell trans-well co-culture. f Quantification of Olig1+/MHCII+ cell density of the CC. Mean values are plotted ± SEM. ***p < 0.0003 **p < 0.0026;*p < 0.0449 One-way ANOVA followed by Tukey multiple comparison post hoc test and Unpaired t tests was used to determine statistical differences; n = 7
wells per group in d; n = 5 wells per group in e; n = 6–7 mice per group in f. Scale bar = 20 μm in a and b. Scale bar = 200 μm in c. Abbreviations: CC: corpus
callosum; CX: cortex; VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole

Shumilov et al. Journal of Neuroinflammation(2024) 21:171

IL17 CD4+ in presence of OLCs in vitro. While MHCII expression in OLCs appears to be linked to impaired
proliferation. Collectively, our findings suggest that oligodendrocytes are not passive in the neuroinflammatoryand degenerative environment caused by brain trauma
but instead could exert an active role in the modulationof immune response.
Extensive research has established the microbiome’s
influence on inflammatory responses and immunology[37–39]. Notably, its role in shaping brain development
and regulating central nervous system (CNS) functions
is becoming increasingly evident [40]. Disruption of gut
microbial composition and diversity has been linked tovarious neurodevelopmental disorders, including autism
spectrum disorders, depression, and schizophrenia [38,41, 42]. Furthermore, the gut microbiome has the potential to modulate white matter structural integrity in a
diet-dependent manner [43]. Our investigation into TBI
and gut microbial depletion reveals a profound impact
on WMI recovery. We observed impaired white matter
remyelination, characterized by decreased OLC proliferation and myelin debris accumulation, following TBI in
animals with depleted microbiota. This correlation was
further demonstrated through microbial transplantationin GF mice, showing a direct impact of the gut microbiota on WMI. This evidence emphasizes the gut microbiota’s potential as a modulator of post-TBI recovery.
TBI triggers complex immune responses, including therecruitment of lymphocytes to the injured site. WhileT-cell infiltration was traditionally linked to worsenedoutcomes and exacerbated brain damage [44], otherresearch suggests that specific T-cell populations might
actually play a protective role in brain injury recovery [45, 46]. Gut microbiota are known to regulate theimmune cell response after TBI. In our previous work
injured mice with gut microbial depletion were found tohave altered microglial morphology and increased neurodegeneration associated with impaired T-cell infiltration[17]. In this manuscript, we found that in the absenceof T-cells, gut-microbiota depletion had a significantly
reduced impact on myelin repair and OLC proliferation
following injury. This effect was observed both in thepharmacological deletion of CD3+ lymphocytes and in
genetic absence of T-cells in TCRβ-/-TCRδ-/- mice. Thesefindings provide evidence for a critical role of T-cells in
gut-brain communication in the setting of TBI. Further
elucidating the mechanistic and regulatory interactions
between the gut microbiota, the immune response, andthe brain in the setting of TBI can provide foundational
knowledge for the development novel therapeutic strategies to enhance white matter repair.
While the role of resident immune cells in the CNS is
well-established, the involvement of non-immune glial
cells like astrocytes and OLCs in neuroinflammation has

Page 15 of 18

only recently emerged as a critical area of research. In thecontext of neurological disorders, OLCs can transition
to disease-specific cell states [47]. Disease-specific OLCs
are characterized by the expression of immune specificgenes allowing the direct cross-tale between immune celland therefore modulating immune response [35]. Furthermore, defective perivascular migration of OLCs not
only impairs their recruitment to the lesion site but canalso disrupt the blood brain barrier, making it more permeable to infiltrating CD3+ lymphocytes [48]. To further
understand the role of T-cells in the modulation of OLCs
we performed an in vitro co-culture with both cell types.
We found that OLCs exposed to T-cells from gut-microbiota-depleted and injured mice exhibited stunted proliferation. This suppression was partially mediated by direct
cell contact. Analysis of T-cells population revealed anincrease in CD4+ lymphocytes expressing IL17 in gutmicrobiota-depleted mice. This finding aligns with existing evidence suggesting that immune cells modulate OLC
proliferation through IL17 [31, 49]. Furthermore, CD4+
T-cells of the Th1 and Th17 lineage play a pivotal role in
MS perpetuation and establishment [50, 51]. Emerging
evidences point to a role of Th17 cells in a wide variety ofcognitive, neurovascular, and neurodegenerative diseases[52]. However, additional research is required to uncover
the precise mechanisms behind the gut microbial influence on white matter repair after injury.
The overall immunomodulatory role of OLCs in CNS
diseases and disorders still needs to be investigated.
Emerging evidence suggests that OLCs could modulate
immune cells activation through the increased expression of gene modules associated with interferon response
and MHC class I and II [35, 36]. These studies establisheda novel role of OLCs in antigen presentation in vivo [35,36, 53]. This novel OLCs function was further confirmedby single-nuclei RNA sequencing analysis in corticalgray matter and subcortical white matter [54]. This study
revealed an increased expression of MHC genes as signature of stressed oligodendrocyte in MS lesions [54].
We then wondered whether T-cells isolated from injured
and gut microbiota depleted animals could influence
MHC class II expression in vitro. Our data revealed that
MHC-II was upregulated in OLCs in cell contact dependent manner. Furthermore, increased colocalization of
MHC-II and Olig1 was observed in the peri-contusional
CC after TBI with gut microbial depletion. These findings suggest a potential mechanism by which T-cells
might directly interact with OLCs through MHC-II,
modifying their proliferation and amplifying their immunomodulatory role. Further research is crucial to determine whether OLCs immunomodulatory functions areprominent or merely limited to a fine-tuning effect. Ourin vivo and in vitro data implicate T helper cells (CD4+)
as the critical T cell subset interacting with OLCs. FutureShumilov et al. Journal of Neuroinflammation(2024) 21:171

studies with specific antibody depletion of CD4+ T cells
are planned.
There are several limitations in our investigation.
Our in vivo studies included young adult mice, but the
response to injury/recovery and gut dysbiosis may be
influenced by sex and age [55, 56]. Furthermore, gut
dysbiosis and systemic inflammation can be influenced
directly by age-related dysregulation of bile acid homeostasis [57, 58]. Future research should prioritize exploring this complex interplay in the context of white matter
repair. The level of antibiotics remaining in the feces of
SPF mice, transplanted to GF mice, wasn’t analyzed. It is
possible that the GF mice had some exposure to VNAM
during FMT. However, the antibiotics used are not systemically absorbed (except for metronidazole) and thesecond FMT was, performed 10 days before injury. We
used a focal injury model which limited our ability toevaluate other white matter regions affect in human TBI.
Future studies using animal models of diffuse TBI are
warranted. Another limitation of our study is the absenceof sham control in the analysis of OLCs proliferation,
myelin debris accumulation and axonal swelling in both
WT and TCRβ-/-TCRδ-/- mice. Nevertheless, the proliferation of OLCs in the CC of adult uninjured mice are
inherently rare under homeostatic conditions as well as
the accumulation of myelin debris and axonal swelling.
The absence of in vitro model of myelination and OLCs
differentiation could be considered as a limitation to ourstudy. Previous studies have identified accelerated remyelination in brain slice cultures with regulatory T-cells,
that directly promoted OPCs differentiation [51, 59]. Costaining with a marker of mature oligodendrocytes, suchas proteolipid protein, or OPC specific marker PDGFr-α
would have allowed to evaluate OLCs differentiation. The
discrete area of analysis and the focal nature of the injury
model are limitations to our study. Further investigation
should include mild and diffuse injury models to improve
the translatability to human cases of TBI. Also, microglia could have a critical role in gut microbial modulation of myelination, future studies should include in vivo
microglia depletion and in vitro co-culture with OLs toaddress this mechanism.
In summary, depletion of the gut microbiota after
TBI impaired OLC proliferation, increased OLC MHCII expression, and reduced white matter remyelination.
Absence of T-cells protected injured mice from the detrimental effects of gut microbial depletion on WM providing evidence for T-cells as a cellular mechanistic link
for gut microbiota-brain communication in the setting of
TBI. Future studies should address the molecular mechanisms of gut microbial regulation of the T-cell response(such as bacterial metabolites) and its role in regulating
OLC differentiation and maturation in the setting of TBI.

Page 16 of 18

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03158-9.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Author contributions
K.S.: Conceptualization, Investigation, Formal analysis, Writing – original draft.
A.N.: Investigation. M.G.B: Investigation, Writing- Review and Editing. M.C. :Investigation, Formal analysis, Writing- Review and Editing. S.H.F.: Supervision,
Methodology, Formal analysis, Writing- Review and Editing, Project
Administration, Funding acquisition.
Funding
NINDS R01NS097721.
Data availability
The datasets generated and analyzed during the current study are available
from the corresponding author on reasonable request.

Declarations
Competing interests
The authors declare no competing interests.
Ethics approval
All authors have adhered to the standards of the National Research Council’s
Guide for the care and use of laboratory animals and the ARRIVE guidelines. All
protocols were approved by Washington University in St. Louis Animal Studies
Committee.
Consent for publication
Not applicable.
Received: 18 April 2024 / Accepted: 19 June 2024

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