All experimental procedures in this study were approved by Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Mice and treatment
For behavior testing, corticospinal tract tracing and immunohistochemical studies, a total of 40 male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) at the age of 8–10 weeks were randomly divided into four groups: a sham control group, a TBI-vehicle control group, a TBI-SCF + G-CSF-single treatment group, and a TBI-SCF + G-CSF-repeated treatment group (n = 10/group). Twenty-three weeks after the final treatment, half of the mice in each of the four groups (i.e. n = 5/group) were injected with biotinylated dextran amine (BDA) in the contralateral cortex for the TBI mice or in the left cortex for the sham mice to trace the corticospinal tracts. The treatments were initiated 3 months after TBI (i.e. in the chronic phase of TBI). Recombinant mouse SCF (PeproTech, 200 μg/kg/day, dissolved in 0.9% saline) and recombinant human G-CSF (Amgen, 50 μg/kg/day, dissolved in 5% dextrose) were subcutaneously injected for 7 consecutive days. For sham and vehicle control groups, an equal volume of vehicle solution (i.e. 0.9% saline and 5% dextrose) was subcutaneously injected for 7 days. For SCF + G-CSF-repeated treatments, a total of three 7-day- treatment courses with a 4-week interval between each therapeutic course were performed. For SCF + G-CSF-single treatment, a 7-day injection of SCF + G-CSF was subcutaneously performed in the first course of therapy followed by vehicle solution injections in the rest of two courses of therapy. All mice were euthanized at the end of the experiment (i.e. 25 weeks after the final treatment) (Fig. 1a). Due to health concerns, three mice in the sham group and one mouse in the vehicle control group were euthanized before the end of experiment and excluded from this study.
For analysis of the engulfment of synapses by microglia in vivo, an additional nine TBI mice were randomly divided into two groups: a vehicle control group (TBI, n = 4) and an SCF + G-CSF (TBI, n = 5) treatment group. Three months after TBI, mice were given a 7-day treatment as described above. The mice were then sacrificed 24 h after the final injection (Fig. 8a).
All mice were housed under a reversed 12:12 h light–dark cycle (Dark hours: 6:30 am to 6:30 pm. Light hours: 6:30 pm to 6:30am) with ad libitum access to water and a standard laboratory diet.
Controlled cortical impact model of TBI
A controlled cortical impact model of TBI was performed to make a reproducible and severe TBI as previously described [58]. Briefly, after being anesthetized with Avertin (400 mg/kg, Sigma- Aldrich, St. Louis, MO, USA), mice were immobilized on a stereotaxic instrument (Leica Biosystems Inc., Wetzlar, Germany). A 4-mm-diameter circular craniotomy (centered at 0.0 mm to the bregma and 2.0 mm lateral to the midline) was made on the right side of the skull. The cortex in the right hemisphere was impacted by an electromagnetically driven impactor with a 3-mm diameter flat impact tip (Impact One stereotaxic impactor, Leica Biosystems Inc., Wetzlar, Germany) at a 4° angle to the vertical line with a 1.5 m/s strike speed, 2 mm impact depth from the surface of the dura and 8500 ms dwell time. After surgery, mice were allowed to fully recover on a homeothermic blanket set at 37 °C before transferring to their home cages. Sustained-release buprenorphine (0.6 mg/kg) was subcutaneously injected to alleviate pain after surgery.
Neurobehavioral tests
Mice were brought to the testing room (using red light for illumination) at least 30 min prior to testing to allow acclimation to the new environment. The behavior tests were started about 8:30 a.m. and ended before 2:00 p.m. The ANY-Maze Video Tracking System (Stoelting Co.) was used for recording mouse performance during the tests.
To test spatial learning, the Morris water maze test was performed 3 weeks after the final repeated SCF + G-CSF treatments as previously described [58]. In brief, mice were tested in a water tank (1.2 m in diameter) filled with water (room temperature) mixed with nontoxic white paint to cover the platform (12 cm in diameter, 1 cm beneath the water) which was set in the center of one of four imaginary quadrants in the tank. Mice were tested for five consecutive days. Each day, mice were examined in four trials (1 min/trail) with a random start from one of the four quadrants. On the first day, mice were held on the platform for 15 s after each trial for training. The average latency to find the platform each day was analyzed.
To evaluate somatosensory-motor deficits, the adhesive tape removal test as described elsewhere [58] was performed. Briefly, on day 1, mice were habituated in a testing beaker for three trials (2 min/trial). On day 2, mice were tested in the same beaker, and two 6-mm circular stickers were pasted onto the palm of each forepaw. The latency to take off each sticker from the forepaws by their mouth was recorded. Each mouse was tested for three trials with a 15 min rest period between trials.
The Rotarod test was used to evaluate motor learning and coordination using a rotarod apparatus (Coulbourn Instrument, Holliston, MA, USA). Mice were placed on the rod at 0 rotation per minute (rpm). The rod was then started at 4 rpm with a linear acceleration (4 rpm every 30 s) until it reached 40 rpm. Each trial was ended when the mouse spun around or fell off the rod. Each mouse was evaluated for five consecutive days. Each day three trials with a 15-min rest break between the trials were performed. The average fall latency of the three trials was analyzed.
Corticospinal tract tracing
To trace the corticospinal tract from the contralateral somatosensory-motor cortex, BDA (biotinylated, 10,000 MW, Invitrogen, Carlsbad, CA, USA) was injected into the left sensorimotor cortex 2 weeks before mice were euthanized. Mice were anesthetized with Avertin and placed in a stereotaxic instrument. The dura was exposed, and 10% BDA was stereotaxically injected into two sites (0.75 μL per site) using a Micro4 microsyringe pump controller with a 10 μL Hamilton microsyringe (150 nL/min). The coordinates of the two injection sites were 0.75 mm and − 0.75 mm to the bregma, 1.5 mm lateral to the midline and 1.0 mm deep from the dura. After the completion of injection, the needle was kept in place for another 5 min before retracting. The skin was sutured, and mice were kept on a homeothermic blanket set at 37 °C before transferring to the home cage.
Tissue processing and immunohistochemistry
Mice were transcardially perfused with 0.01 M phosphate buffered saline (PBS) with 10 U/mL heparin, followed by 10% neutral formalin solution (Sigma Aldrich, St. Louis, MO, USA). Brains and spinal cords were removed and post-fixed in 10% neutral formalin solution overnight at 4 °C. The tissues were dehydrated in 30% sucrose solution in PBS and sectioned (coronal sections, in 30 μm thickness) using a cryostat (Leica Biosystems Inc., Wetzlar, Germany). The sections were stored in a − 30 °C freezer with cryo-protectant solution (0.1 M PB with 30% Ethylene glycol and 30% Glycerol) until used.
For free-floating immunofluorescence staining, sections were rinsed with PBS followed by blocking nonspecific binding using 10% normal donkey serum diluted with 1% IgG-free bovine serum albumin (BSA) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and 0.3% Triton X-100 (Sigma- Aldrich, St. Louis, MO, USA) in PBS for one hour at room temperature. A Mouse on Mouse Immunodetection Kit (Vector Laboratories, Burlingame, CA, USA) was used for staining with mouse monoclonal antibodies to block endogenous mouse IgG. Subsequently, the sections were incubated with primary antibodies: rabbit anti-myelin basic protein (MBP, 1:500, Abcam, Cambridge, UK), mouse anti-postsynaptic density protein 95 (PSD-95, 1:500, Novus Biologicals, Centennial, CO, USA), rabbit anti-synaptophysin (SYN, 1:600, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-Gephyrin (1:500, Thermo Fisher Scientific, Waltham, MA USA), sheep anti-Gephyrin (1:600, Novus Biologicals, Centennial, CO, USA), goat anti-Ionized calcium binding adaptor molecule 1 (Iba1, 1:600, Novus Biologicals, Centennial, CO, USA), and rabbit anti-Purinergic Receptor P2Y12 (P2RY12, 1:1000, Brigham and women’s hospital, Boston, MA, USA). In negative control sections, the primary antibodies were omitted. The primary antibodies were diluted in 1% IgG-free BSA and 0.3% Triton X-100 in PBS and incubated overnight at 4 °C with gentle shaking. After rinsing with PBS, the sections were incubated in the corresponding secondary antibodies for two hours in the dark at room temperature. The secondary antibodies were donkey anti-mouse IgG Alexa Fluor 488/594, donkey anti-rabbit IgG Alexa Fluor 488/594, donkey anti-goat IgG Alexa Fluor 647, and donkey anti-sheep IgG Alexa Fluor 488 (all secondary antibodies, 1:500 dilution, Invitrogen, Carlsbad, CA, USA). Finally, the sections were rinsed with PBS and mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA USA) with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). To visualize BDA-labeled dendrites or axons, Cy3-conjugated avidin (1:300, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used during the secondary antibody incubation as described above. In addition, the spinal cord sections were also stained using an ImmunoCruz® ABC Kit (Santa Cruz Biotechnology, Dallas, TX, USA) to detect BDA-labeled axons according to the manufacturer’s instructions.
Quantitative image analysis
BDA-labeled corticospinal tract at 5–7 segments of the cervical spinal cord were visualized using immunohistochemistry or immunofluorescence staining. To analyze the number of axons crossing the midline of the coronal spinal cord section, three spinal cord sections from each segment of the spinal cord were chosen, and the number of BDA-labeled sprouting axons were manually counted under a 40 × objective lens of the microscope (Zeiss, Oberkochen, Germany). The average number of sprouting axons per segment was statistically analyzed. For Sholl analysis, the spinal cord sections were imaged using the “Tile scan” mode of a Zeiss LSM 780 confocal microscope (Zeiss, Oberkochen, Germany). The sprouted BDA-labeled axons were traced using the plugin “neuronJ” in Fiji (NIH software) followed by Sholl analysis (a plugin in Fiji) with 10 μm step size started from the central canal of the spinal cord [20, 47, 64].
To quantify the different types of dendritic spines, three brain sections from bregma − 0.5 mm to 0.5 mm (avoided the injection sites) were chosen from each mouse. After staining with Cy3-conjugated avidin, the dendrites from layer 2/3 neurons of the contralateral cortex were imaged using the Zeiss LSM 780 confocal microscope with z-stack scanning (0.3 μm interval, 40 × objective lens with a resolution of 1024 × 1024 pixels). After acquiring the z-stack image, maximum intensity projection (a plugin in Fiji) was used to produce a projection image for analysis. The number of different types of dendritic spines was manually counted. At least 10 dendrites were analyzed for each mouse. The types of dendritic spines were distinguished according to the geometric characteristics of the dendritic spines [60].
To quantitate the relative PSD-95, Gephyrin and Iba1 expression, two fields in the stratum radiatum of hippocampal CA1 and cortex (layer 2–5) were randomly selected and imaged under a 40 × objective lens using the z-stacks scanning mode (1-μm interval, total 18 μm). The background of the images was subtracted from all fluorescent channels (rolling ball radius: 50 pixels), split channels and thresholded using Fiji software. Subsequently, the volumes of PSD-95+ and Gephyrin+ puncta and Iba1+ cells were calculated using the “3D RoiManager” plugin in Fiji [54]. The volumes of PSD-95+ and Gephyrin+ puncta and Iba1+ cells were then normalized to the sham group.
To analyze the morphological changes of microglial cells, brain sections were stained with a microglial cell marker, Iba1. Two fields in both the contralateral and ipsilateral stratum radiatum of hippocampal CA1 and cortex (layer 2–5) were imaged with the Zeiss LSM780 confocal microscope under a 40 × objective lens. Z-stack imaging with a 1-μm z-step was performed (total 18 μm). The acquired z-stack images were projected into a single image using the maximum intensity z-projection. The Iba1+ cells with an integral cell body were randomly selected from each field and performed threshold process to generate binarized images. The binarized image was filtered by particle size to remove image noise. The filtered images were analyzed using Sholl analysis from the center of the cell soma with a 2-μm step size using Fiji software. To count Iba1 positive cells, the z-projected images were used. The Iba1 positive cells with integrated cell soma and counterstained with DAPI were quantified using Fiji software.
In vivo engulfment of synapse analysis
Two brain sections from bregma − 1.46 to − 2.18 mm were chosen for triple immunostaining with PSD-95, Gephyrin, Iba1 or P2RY12. After immunostaining, two fields in the stratum radiatum of hippocampal CA1 per brain section were acquired using the Zeiss LSM780 confocal microscope (40 × objective lens, 1024 × 1024 pixels of the resolution and 1-μm intervals in z-stacks). To analyze the engulfment of synapses, the volumes of Iba1+PSD-95+ and Iba1+Gephyrin+ puncta or P2RY12+PSD-95+ and P2RY12+ Gephyrin+ puncta were calculated using the plugin “3D RoiManager” in Fiji software. The engulfing index was presented using the following formulas: (Iba1+PSD-95+ or Iba1+Gephyrin+ volume)/(Iba1+ volume) or (P2RY12+PSD-95+ or P2RY12+Gephyrin+ volume)/(P2RY12+ volume).
Synaptosome preparation
Synaptosomes were purified from adult mouse brain according to a well-established method published elsewhere [19]. Briefly, the whole brain without the cerebellum was homogenized in 10% (w/v) homogenizing buffer (0.32 M sucrose, 1 mM EDTA, 5 nM Tris and 0.25 mM DTT, pH 7.4). The homogenate was centrifuged at 1000g for 10 min at 4 °C. The supernatant was diluted and layered over top of homogenizing buffer containing 3%, 10%, 15% and 23% (vol/vol) Percoll, respectively. After a 5 min centrifuge at 31,000 g (4 °C), the fraction between the 15% and 23% Percoll gradient solution was collected, diluted to four times in homogenizing buffer and centrifuged at 20,000g (4 °C) for 30 min. The pellet was resuspended in isotonic PBS. The protein concentration was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. A Vybrant™ Multicolor Cell-Labeling Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to label the synaptosomes based on the manufacturer’s instructions. One microliter DiO was added to 200 µL synaptosome suspension and incubated at 37 °C for 1 min, followed by centrifuging at 20,000g (4 °C) for 30 min. The DiO-labeled pellet was resuspended in PBS to be used for testing uptake of synaptosomes by microglia in vitro.
Primary microglial cell culture
Primary microglial cell cultures were prepared from dissecting cerebral cortex of 1-day-old neonatal C57BL/6 mice based on the protocol developed by Saura and coworkers [61]. Briefly, the cortex was dissected and digested using 0.25% trypsin–EDTA at 37 °C for 30 min. Digested tissue was resuspended in DMEM/F12 with 10% fetal bovine serum (FBS), pipetted into single cells and filtered through a 70-μm cell strainer (Thermo Fisher Scientific, Waltham, MA, USA). The filtered cell suspension was centrifuged at 300g for 10 min. Pellets were resuspended in DMEM/F12 with 10% FBS and plated on Poly-d-Lysine (PDL, 100 μg/mL in sterile distilled water, Sigma- Aldrich, St. Louis, MO, USA) coated T75 cell culture flasks. The culture medium was replaced every 4 days until achieving confluency. The mixed glial cultures were then incubated in 0.05% trypsin–EDTA (0.25% trypsin–EDTA diluted in DMEM/F12) for 30–60 min. The detached cells were discarded, and the undetached cells were collected for further study.
In vitro engulfment of synaptosome assay
For the flow cytometry assay, isolated primary microglial cells were grown on a PDL-coated 24-well plate overnight and then treated with or without SCF + G-CSF (20 ng/mL each) for 48 h. The DiO-labeled synaptosomes were added to each well of cultured microglial cells (final concentration: 1 μg/mL). After a 6-h incubation with synaptosomes, cells were washed with warm PBS (37 °C) three times and detached with 0.25% trypsin–EDTA. After centrifugation, cells were resuspended in ice-cold flow cytometry buffer (2% FBS, 2 mM EDTA in PBS) and directly went through a BD LSRFortessa™ cell analyzer. Data were analyzed using FlowJo software. The DiO positive (DiO+) cells and median fluorescence intensity (MFI) were analyzed.
For the immunocytochemistry assay, microglia cells were grown on 12-mm diameter coverslips coated with PDL. The cell concentration was 50,000 cells per coverslip. Cells were then treated with or without SCF + G-CSF (20 ng/mL each) for 48 h. After incubating with DiO-labeled synaptosomes (1 μg/mL) for 6 h, cells were washed with warm PBS (37 °C) and fixed using 10% neutral formalin solution for 10 min (Sigma-Aldrich, St. Louis, MO, USA). After rinsing with PBS, cells were incubated with PE-conjugated CX3CR1 antibody (1:100, BioLegend, San Diego, CA, USA) for 2 h and then washed with PBS three times. Considering Triton-X100 could reduce fluorescence of DiO-labeled synaptosome membrane, the cell membrane receptor marker CX3CR1 was used instead of Iba1 to label microglia.
For the western blot assay, microglial cells were grown on PDL coated six-well plates. Cells were treated with or without SCF + G-CSF (20 ng/mL each) for 48 h. Microglial cells were then incubated with unlabeled synaptosomes (1 μg/mL) for 6 h, rinsed with PBS and lysed in ice-cold lysis buffer (20 mM sodium phosphate, 150 mM sodium chloride, 50 mM sodium fluoride, 5 mM EDTA and 1% Triton X-100 with proteinase inhibitor cocktail) for 30 min. The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected. The protein concentration was then quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The quantified samples were boiled in the loading buffer, electrophoresed in 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane (0.45 μm pore size, Amersham Biosciences GE, Little Chalfont, UK). Protein blots were blocked with 5% non-fat milk for 1 h (room temperature, RT) and probed overnight at 4 °C with rabbit anti-beta-Actin (β-Actin, 1:5000, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-PSD-95 (1:1000, Sigma-Aldrich, St. Louis, MO, USA), and rabbit anti-SYN (1:1000, Sigma-Aldrich, St. Louis, MO, USA) in 5% IgG-free BSA (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted with Tris-buffered saline (TBS). After rinsing with TBS containing Tween-20 (TBS-T, 0.5% Tween-20) three times, the blots were incubated with the corresponding alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:10,000, Sigma-Aldrich, St. Louis, MO, USA) for 2 h (RT). The membranes were then washed with TBS-T, incubated with ECF substrate (Sigma-Aldrich, St. Louis, MO, USA) and visualized using the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Proteins were extracted from microglial cells in four independent experiments. The levels of protein expression were quantified using Fiji (ImageJ, NIH software).
Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). All data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). The data of water maze and rotarod tests were examined by repeated two-way analysis of variance (ANOVA) followed by Fisher’s LSD tests. Sholl analysis data were examined with two-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests. Other data were analyzed using one-way ANOVA followed by Fisher’s LSD tests for multiple group comparisons and Student’s t test for two group comparisons. The Kolmogorov–Smirnov test was used for the comparison of cumulative frequency. Two-tailed statistical significance tests were used throughout, and p < 0.05 was considered statistically significant.