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 water and food with a 12-hour light/dark cycle. C57BL/6J (RRID: IMSR_ORNL:C57BL/6J-A/A) and B6.129P2-Tcrbtm1MomTcrdtm1Mom/JJ (TCRb-/-TCRd-/-) (RRID:IMSR_JAX:002122) 8 week-old male and female mice (Jackson Laboratory, Bar Harbor, ME) weighing 20-25 grams (g) were used for in vivo studies. C57BL/6J male and female neonatal P2-P5 pups were used for in vitro studies.
Controlled cortical impact with gut microbial depletion
Controlled cortical impact (CCI) was performed using a previously described protocol (17). Mice were anesthetized with 5% isoflurane and maintained at 2% isoflurane throughout the procedure. Buprenorphine sustained-release (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 (Celorrio et al., 2021). The impact was delivered at a depth of 2 mm with a velocity of 5 m/s and a dwell time of 100 ms. A loose-fitting 7 mm plastic cap was secured over the craniectomy site using Vetbond (3M, St. Paul, MN). The skin incision was closed with interrupted sutures and treated with 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 the drinking 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 (Steed et al., 2017). Control animals received only Kool-Aid in drinking water to reduce the bitter flavor of the antibiotics.
Fecal microbiota transplantation
We performed two fecal microbiota transplantations (FMTs) of the gut microbiota from VNAM- or Kool-Aid-treated uninjured animals into germ-free (GF) mice as 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) and after 5 min, the sample was vortexed to break up the fecal matter and allowed to sit for ∼5 min to allow for debris to settle. 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 with a sample volume of 250 μL. Ten days after the last FMT, we performed CCI in all the mice and they remained in sealed cages in the animal facility for 7 days before being euthanized.
CD3-cpecific monoclonal antibody injection
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).
5-bromo-2'-deoxyuridine (BrdU) treatment
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.
Tissue Processing
Mice were euthanized under isoflurane anesthesia, by transcardial 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 were used for the subsequent analysis.
Myelin Black Gold II staining
Myelin Black Gold II (BGII, Histo-Chem, Jefferson, AR) staining was performed as previously described (Celorrio et al., 2022; Shumilov et al., 2023). Briefly, to visualize individual myelin fibers in the CC and quantify myelinated percent area, BGII staining was 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 were rinsed 3 times with tris-buffered saline (TBS) for 5 min at room 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 graded alcohols (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.
Fluorescence Immunohistochemistry
Fluorescence immunohistochemical staining was performed on free-floating sections. Tissue was incubated with pre-heated HCl 1N (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 detected by incubating sections with Alexa Fluor secondary antibody (Table 1) for 2 hours in PBS with 0.3% triton X-100. Sections were mounted on glass slides in PBS, dried, and coverslipped with mounting medium for fluorescence with 4',6-Diamidino-2-Phenylindole, (DAPI, Thermo Fisher Scientific, Waltham, MA).
Quantitative fluorescent immunohistochemistry
Fluorescent images were obtained with a Zeiss Axio Imager Z2 with ApoTome 2 fluorescence microscope with 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) fluorescent immunostaining 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).
Primary oligodendrocyte lineage cell culture
Primary OLC culture and isolation was performed as previously described with a modified protocol (26, 27). Briefly, brains from postnatal day P2-5 mice pups were isolated under a dissection microscope. Then, the cortical tissue were dissociated with a digestion cocktail containing papain (1.5 mg/ml, Worthington, Lakewood, OH) 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 Bi-products, Sacramento, CA) and plated in T-25 culture flask pre-coated with poly-L-lysine (100 μg/ml, Sigma-Aldrich, 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 to the 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 1x104 cells/well on a 96-well plate or 1x105 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, MO), FGF (10ng/ml, PeproTech) and PDGFaa (10 ng/ml PeproTech).
T-cells isolation from spleen
Spleens were collected from injured animals treated with VNAM or Kool-Aid as described above. Splenocytes were obtained by mechanical shredding and filtered through a 70-µm cell strainer, and centrifuged at 500g for 10 min. The resulting cell suspensions were incubated with red blood lysis buffer (Roche Diagnostics Gmbh, Mannheim, Germany) for 5 min at 4°C, centrifuged and filtered through 40-µm cell strainer. T-cells were then isolated following manufacturers protocol by negative selection using pan T-cell isolation kit II (Miltenyi biotec). This isolation kit is based on a cocktail of biotin-conjugated antibodies against CD11b, CD11c, CD19, CD45, CD49b, CD105, Anti-MHC-class II, and Ter-119. Cells were counted and co-cultured with OLCs at a density of 3x104 cells/well on a 96-well plate or 3x105 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 1N (Sigma-Aldrich, St. Louis, MO) for 30 min at 45°C to increase the antigen exposure for BrdU detection. Non-specific 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 with DAPI (1:5000, Life Technology, Carlsbad, CA) and stored at 4°C in PBS. The immunofluorescent images were taken using 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 10µg/ml of gentamycin (Sigma-Aldrich) with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA), 1 µg/ml of ionomycin (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 by repetitive 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+.
Statistical analysis
Blinding of investigators to experimental groups was maintained until data were fully analyzed. Data were assessed 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).