Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury

Studies were performed using C57BL/6 adult male mice (10–12 weeks old, 22–26 g). The mice were housed in the Animal Care facility at the University of Maryland School of Medicine under a 12-h light-dark cycle with ad libitum access to food and water. All surgical procedures were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine.

Our custom-designed controlled cortical impact (CCI) TBI device consists of a microprocessor-controlled pneumatic impactor with a 3.5-mm diameter tip. Mice were anesthetized with isoflurane evaporated in a gas mixture containing 70% N₂O and 30% O₂ and administered through a nose mask (induction at 4% and maintenance at 2%). Depth of anesthesia was assessed by monitoring respiration rate and pedal withdrawal reflexes. Mice were placed on a heated pad, and core body temperature was maintained at 37 °C. The head was mounted in a stereotaxic frame, and the surgical site was clipped and cleaned with Nolvasan and ethanol scrubs. A 10-mm midline incision was made over the skull, the skin and fascia were reflected, and a 5-mm craniotomy was made on the central aspect of the left parietal bone. The impounder tip of the injury device was then extended to its full stroke distance (44 mm), positioned to the surface of the exposed dura, and reset to impact the cortical surface. Moderate-level TBI was induced using an impactor velocity of 6 m/s and deformation depth of 2 mm as previously described [22]. After injury, the incision was closed with interrupted 6-0 silk sutures, anesthesia was terminated, and the animal was placed into a heated cage to maintain normal core temperature for 45 min post-injury. Sham animals underwent the same procedure as TBI mice except for the impact.

Mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.), and blood was collected in heparinized syringes by aortic puncture. Blood was immediately combined with fixative (100 μl/ml Caltag Reagent A fixation medium, Invitrogen, Carlsbad, CA) to diminish ex vivo microparticle (MP) aggregation. All reagents and solutions used for MP analysis were sterile and filtered (0.1-μm filter). Heparinized blood was centrifuged for 5 min at 1500×g, and supernatant was centrifuged at 15,000×g for 30 min to pellet platelets and other cell debris [23]. Blood supernatants were used to collect total blood MP (~250 μl) that were purified by adding PBS (4 ml) and centrifuged at 100,000×g for 60 min at 4 °C as previously described [23‐30]. In each experiment, flow cytometry in combination with microbead standards ranging in size from 300 to 3000 nm was used to characterize MP size. MP were distinguished from larger (apoptotic body; >1000 nm) and smaller (exosomes; <100 nm) vesicles based on size (SSC), and their phenotype was confirmed using the unique MP surface marker, annexin V (details below; see Fig. 1a). In parallel experiments, equal numbers of circulating MP from sham and TBI mice (8 × 10⁵ MP) were resuspended in 100 μl serum-free DMEM media and co-cultured with BV2 microglia (2 × 10⁴ cells/well) for 24 h at 37 °C and at 5% CO₂ prior to cell extraction for markers of microglial activation. The enriched MP population obtained using this protocol may also contain other types of extracellular vesicles such as exosomes.

Total and cell-derived MP were identified by flow cytometry using an eight-color, triple-laser MACSQuant Analyzer (Miltenyi Biotec, Auburn, CA). MACSQuant was calibrated every other day with calibration beads (Miltenyi Biotec, Auburn, CA), and forward and side scatters were set at logarithmic gain. Photomultiplier tube voltage and triggers were optimized to detect submicron-sized particles. Microbeads of different diameters (0.3 μm (Sigma; LB3), 1.09 μm (Spherotech, Lake Forest, IL; BCP-10-5), and 3.0 μm (Spherotech, Lake Forest, IL; BP-30-5)) were used to set initial parameters and to confirm MP characteristics in each experiment. To reduce small particle contaminants, all reagents and solutions used for MP analysis were sterile and filtered (0.1-μm filter; EMD Millipore, Billerica, MA) before use. The expression of phosphatidylserine (PS) on MP was detected using an anti-annexin V (FITC or APC) (1:50, BD Biosciences PharMingen, catalog no: 556419) in annexin buffer (5 mM KCl, 1 mM MgCl₂, 136 mM NaCl, 2 mM CaCl₂, 1% BSA; pH 7.4). MP were incubated with antibodies for 30 min at room temperature (RT) in the dark. Annexin buffer (150 μl) was added to each sample prior to MACSQuant analysis. True-negative controls were established by a fluorescence-minus-one analysis and using isotype-matched irrelevant antibodies at the same concentration and under the same conditions. Annexin V-positive particles with diameters from 300 to 1000 nm were defined as MP. Blood MP were further characterized by double labeling with specific cell-specific antibodies: leukocytes (anti-CD18 (PE); 1:50, Biolegend; catalog no: 101408) and monocyte/macrophages (anti-F4/80 (APC); 1:50, Thermo Fisher, catalog no: MF48005). Microglial staining was performed using anti-P2Y12 (1:1000, AnaSpec Inc., Fremont, CA) and anti-CD45-PerCP (1:10, Miltenyi Biotec, Auburn, CA) as follows: MP were incubated with anti-P2Y12 in annexin buffer for 1 h at RT, washed and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibodies (1:500; Life Technologies) for 30 min, washed and further incubated with pre-conjugated anti-CD45 for 30 min at RT, and washed in annexin buffer prior to analysis by flow cytometry. FlowJo software (Vx; Tree Star, Inc., Ashland, OR) was used for analysis, and data are presented as percent of annexin V-positive MP in the MP gate as set by microbeads, unless otherwise specified.

BV2 microglia (murine microglial cell line) were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal equine serum (HyClone, Logan, UT), and 1% penicillin and streptomycin (Invitrogen) at 37 °C with 5% CO₂. DMEM media was filtered using 0.1-μm filters. BV2 microglia were seeded at a density of 0.6 × 10⁶ in 60-mm dishes and stimulated with lipopolysaccharide (LPS, 20 ng/ml; Sigma-Aldrich) or control media for 24 h. MP released into conditioned BV2 microglial media were characterized using a MACSQuant flow cytometer (Miltenyi Biotec, San Diego, CA) analysis as described before. In parallel experiments, MP in conditioned media were pelleted by centrifugation as described before and MP-enriched pellets were either resuspended in RIPA buffer (Teknova, Hollister, CA) for Western blotting or extracted using TRIzol reagent (Invitrogen) for RNA analysis. In another experiment, BV2 microglia cells were stained with 1 μM calcein AM (Invitrogen) in DMEM for 30 min at 37 °C and then stimulated with LPS (20 ng/ml) for 24 h. Calcein AM-labeled MP were isolated from conditioned media by centrifugation at 100,000×g for 60 min at 4 °C [23‐30] and were stained with anti-annexin V (APC) (1:50, BD Biosciences PharMingen, catalog no: 550474) for MP characterization by flow cytometry as described before.

Levels of endotoxin (LPS) in pelleted MP from control and LPS-stimulated BV2 microglia were determined using a LAL Chromogenic Endotoxin assay (Thermo Fisher Scientific, MA, USA) as per manufacturer’s instructions. A standard curve was used to determine the concentration of endotoxin in each sample. Endotoxin levels are expressed as endotoxin unit per milliliter (EU/ml).

BV2 microglia were seeded at a density of 0.6 × 10⁶ in 60-mm dishes and stimulated with LPS (20 ng/ml) or control media for 24 h. MP released into conditioned BV2 microglial media were isolated by centrifugation as described before. Control and LPS-stimulated MP (total 8 × 10⁵ MP) were co-cultured with BV2 microglia (2 × 10⁴/96 well) or primary microglia (1 × 10⁵/96 well) that were obtained from postnatal day 1 C57BL/6 mouse pups as previously described [31] for 24 h at 37 °C and at 5% CO₂ prior to cell extraction using TRIzol reagent (Invitrogen) to assess markers of microglial activation.

Magnetic bead-conjugated anti-CD11b and MACS separation technology (Miltenyi Biotec, Auburn, CA) was used to isolate microglia/macrophages from sham and TBI brain tissue of C57BL/6J mice at 7 days post-injury (n = 5/group; study 2 above). Briefly, ipsilateral cortical and hippocampal tissues were rapidly microdissected, and a single cell suspension was prepared using enzymatic digestion (Neural Tissue Dissociation Kit; Miltenyi Biotec) in combination with a gentle MACS dissociator. Myelin was removed using Myelin Removal Beads II and LS columns (Miltenyi Biotec), and the cells were incubated with anti-CD11b microbeads (Miltenyi Biotec) and loaded onto MS columns (Miltenyi Biotec) placed in the magnetic field of a MACS separator. The negative fraction (flow through) was collected, and the column was washed three times with MACS buffer (Miltenyi Biotech). CD11b-positive cells were eluted by removing the magnetic field, resulting in the isolation of approximately 93% viable CD11b-positive cells from sham and TBI mice [32]. Expression level of CD11b-positive selected cells (MFI) was quantified by flow cytometry using anti-CD11b (APC) (1:50; Miltenyi Biotech, catalog no: 130-091-241). CD11b-positive cells were subsequently seeded at 1 × 10⁵ cells/well in a 96-well plate in DMEM-F12 containing 10% fetal calf serum and incubated at 37 °C under 5% CO₂ for 24 h. Ex vivo-secreted MP from sham and TBI CD11b-positive cells were collected from condition media by ultra centrifugation as described before.

Total RNA including miR was extracted from snap-frozen tissue, cells, or enriched MP, using a miRNeasy mini isolation kit (Qiagen, Valencia, CA). Complementary DNA (cDNA) synthesis was performed on 1 μg of total RNA using a Verso cDNA RT kit (Thermo Scientific, Pittsburg, PA), as per manufacturer’s instructions. For messenger RNA (mRNA) analysis, real-time PCR was performed using TaqMan gene expression assays on an ABI 7900 HT FAST Real-Time PCR machine (Applied Biosystems). Gene expression was calculated relative to the endogenous control sample (GAPDH) to determine relative expression values (2^−∆∆Ct, where Ct is the threshold cycle). For miR analysis, a total of 10 ng of total RNA was reverse transcribed using TaqMan miRNA Reverse Transcription Kit (Applied Biosystems) with miR-specific primer of miR-155 and control U6. Reverse transcription reaction products (1.5 μl) were used for qPCR as described above. Following real-time PCR, miR expression was calculated relative to the endogenous control sample (U6) to determine relative expression values (2^−∆∆Ct, where Ct is the threshold cycle).

Proteins were extracted using RIPA buffer (Teknova, Hollister, CA), equalized, and loaded equally onto 5–20% gradient gels for SDS PAGE (Bio-Rad; Hercules, CA). Proteins were transferred onto nitrocellulose membranes and blocked overnight in 5% milk in 1x PBS containing 0.01% Tween-20 (PBS-T). The membrane was incubated in rabbit anti-interleukin-1β (anti-IL-1β) (1:1000; Cell Signaling, catalog no: sc-7884) and mouse anti-β-actin (1:20,000; Sigma, catalog no: A1978) overnight at 4 °C, then washed three times in PBS-T for 5 min, and incubated in appropriate HRP-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA) for 1 h at RT. Membranes were washed three times in PBS-T, and proteins were visualized using Super Signal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL). Chemiluminescence was captured using ChemiDoc TM XRS+ System (Bio-Rad; Hercules, CA), and protein bands were quantified by densitometric analysis using Image J (NIH, Bethesda, MD). The data reflects the intensity of the target protein band normalized based on the intensity of the endogenous control for each sample (expressed in arbitrary units). Protein from cells was normalized to β-actin, and protein from MP was normalized to Ponceau-S.

Twenty-micrometer coronal brain sections from −1.70 mm from the bregma were selected, and standard immunostaining techniques were employed. Briefly, 20-μm sections were washed three times with 1x PBS, blocked for 1 h in goat serum containing 0.4% Triton X-100, and incubated overnight at 4 °C with primary antibody (rabbit anti-P2Y12 (1:1000, AnaSpec Inc., Fremont, CA; catalog no: AS-55043A), rabbit anti-Iba-1 (1:200; Wako Chemicals, Richmond, VA; catalog no: 019-197)). Sections were washed three times with 1x PBS and incubated with appropriate Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 2 h at RT. Sections were washed three times with 1x PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml, Sigma), and mounted with glass cover slips using Hydromount solution (National Diagnostics, Atlanta, GA). Images were acquired using a fluorescent Nikon Ti-E inverted microscope, at ×10 (Plan Apo 10× NA 0.45) or ×20 (Plan APO 20× NA 0.75) magnification. Exposure times were kept constant for all sections in each experiment. All images were quantified using Nikon ND-Elements Software (AR 4.20.01). 6000–10,000 positive areas were quantified per mouse per experiment, and expression levels were expressed as binary area per region of interest (ROI) × 10⁶.

Neurolucida software (MBF Biosciences, Williston, VT) was used to quantify cell body area and ramification length of P2Y12-positive microglia in the cortex, hippocampus, and thalamus of enriched MP intracortical injected mice as previously described [33]. Immunostained microglia were outlined using the live image setting so that the width of the ramified branches could be traced while focusing on the section. Cell bodies were outlined using the contour tool followed by tracing of the individual ramification using the dendrite line tool. Microglial ramification length is expressed in micrometers,, and cell body area is expressed in square millimeters.

Randomization and blinding protocols were employed, and individuals performing in vitro and in vivo analysis were blinded to isolated MP groups. Quantitative data were expressed as mean ± standard errors of the mean (S.E.M.). qPCR and flow cytometry data were analyzed by one-way ANOVA, followed by post hoc adjustments using a Student-Newman-Keuls test. Remaining data were analyzed using Student’s t test. Statistical analyses were performed using GraphPad Prism Program, Version 3.02 for Windows (GraphPad Software, San Diego, CA, USA). A p < 0.05 was considered statistically significant.

C57BL/6 male mice were subjected to TBI (moderate-level CCI) or sham surgery, and 24 h later, animals were euthanized and blood was collected for MP isolation and characterization. Ipsilateral cortical tissue was also collected to assess markers of brain inflammation. MP in blood were measured by flow cytometry for annexin V, which binds the externalized phosphatidylserine (PS) present on the surface of MP. To exclude the presence of annexin V-positive apoptotic bodies (>1000 nm) and smaller exosomes (<100 nm) in the sample, microbead standards (300, 1000, and 3000 nm) were used to gate on MP (MP gate set between 300 and 1000 nm) (Fig. 1a). There was a significant increase in annexin V-positive MP in the blood of TBI mice when compared to sham-injured controls (p < 0.05; Fig. 1a).

We next evaluated the origin of the circulating MP based on unique markers derived from parental cells. P2Y12/CD45-positive MP (microglial derived) were significantly increased in the circulation when compared to sham-injured control levels (p < 0.05; Fig. 1b). The CD18-positive MP (leukocyte derived) increase after TBI did not reach statistical significance, and TBI did not increase levels of F4/80-positive MP (monocyte derived) in the circulation at 24 h post-injury. These data indicate that microglial-derived MP are released into the circulation after moderate-level TBI.

Assessment of injured cortical tissue revealed a robust neuroinflammatory response to TBI, with increased mRNA expression of markers of microglial activation and the induction of pro-inflammatory cytokines and chemokines. Specifically, TBI resulted in a significant increase in mRNA for CD11b (p < 0.01), nitric oxide synthase 2 (NOS2, p < 0.01), interleukin-1β (IL-1β, p < 0.05), tumor necrosis factor-alpha (TNF-α, p < 0.001), C-C motif chemokine ligand 2 (CCL2, p < 0.01), interleukin-6 (IL-6, p < 0.001), pro-inflammatory microRNA-155 (miR-155, p < 0.05), and the purinergic receptor P2X7 (p < 0.05) when compared to the sham-injured group (Fig. 2a). Histological assessment of P2Y12 expression on microglia in the ipsilateral cortex revealed that the following moderate TBI P2Y12-positive microglia transform from a ramified morphological state to an activated state that withdrew branched processes to form thick bundles around highly enlarged cell bodies (Fig. 2b). Further, Neurolucida reconstruction analysis of P2Y12-positive cells demonstrated that ramification length was significantly decreased (p < 0.01) and cell body area was significantly increased (p < 0.01) in microglia in the TBI cortex as compared to sham-injured cortex (Fig. 2c).

To examine if circulating MP released after TBI could promote inflammation, we co-cultured BV2 microglia in the presence or absence of enriched MP from the blood of sham-injured and TBI mice (Fig. 3). Equal number of circulating enriched MP from sham and TBI blood (total 8 × 10⁵) were incubated with naive BV2 microglia for 24 h, and subsequent activation was assessed by mRNA expression of selected microglial activation markers. When compared to BV2 microglia treated with sham circulating enriched MP, there was a significant increase in IL-1β (p < 0.01) and CCL2 (p < 0.05) in recipient microglia co-cultured with TBI circulating enriched MP (Fig. 3) and a trend towards increased in IL-6 and NOS2 expression in this group. There was also a significant increase in P2X7 (p < 0.05) in recipient microglia treated with TBI circulating enriched MP when compared to naïve microglia. There was no difference in expression of other pro-inflammatory molecules such as TNF-α and miR-155.

We performed a MP release assay in BV2 microglia following LPS stimulation (20 ng/ml) for 24 h. MP release was quantified by flow cytometry using Calcein AM-stained BV2 microglia that were co-stained with the MP marker annexin V. When compared to MP levels in control BV2 microglia, LPS stimulation significantly increased the number of annexin V/calcein AM-positive MP (p < 0.001 vs control; Fig. 4a). We next measured IL-1β and miR-155 in BV2 microglial cells and isolated enriched MP from the conditioned media to determine the relative expression of pro-inflammatory mediators in MP vs cellular compartments. Following LPS stimulation, IL-1β protein was not detected in the cells, but it was significantly increased in LPS-stimulated enriched MP (p < 0.001 vs LPS-stimulated cells; Fig. 4b). In addition, following LPS stimulation, miR-155 was significantly increased in BV2 microglial cells (p < 0.05 vs control cells; Fig. 4c). Notably, miR-155 was even more highly enriched in LPS-stimulated MP (p < 0.01 vs LPS-stimulated cells), indicating that enriched MP contain elevated concentrations of pro-inflammatory molecules IL-1β and miR-155 following activation.

To test the hypothesis that enriched MP from activated microglia can seed neuroinflammation, we isolated MP from control and LPS-stimulated BV2 microglia and co-cultured them with naïve BV2 or primary microglia for 24 h prior to assessing cellular markers of activation by qPCR. When compared to control enriched-MP-treated recipient BV2 microglia, there was a significant increase in IL-1β (p < 0.001), TNF-α (p < 0.001), CCL2 (p < 0.001), IL-6 (p < 0.01), and NOS2 (p < 0.001) mRNA in recipient BV2 microglia treated with LPS enriched MP (Fig. 5a). There was also a significant increase in miR-155 expression in BV2 microglia (p < 0.001) treated with LPS enriched MP when compared to cells treated with control MP (Fig. 5a).

To demonstrate the specific activity of microglial-derived enriched MP in activating microglia in these experiments, we neutralized MP by co-incubating them with polyethylene glycol telomere B (PEG-TB), a surfactant that depletes MP without activating blood immune cells [25]. To establish the dose of PEG-TB required to neutralize microglial-derived MP, we performed a dose response study in control and LPS-stimulated enriched MP derived from BV2 microglia and incubated them with increasing concentrations of PEG-TB for 1 h prior to MP characterization by flow cytometry. We determined that 6 μl PEG-TB/100 μl significantly depleted MP levels under both conditions (Fig. 5b), and this concentration was employed to neutralize microglial-derived MP activity in in vitro studies. Next, we determined that when LPS enriched MP were neutralized by PEG-TB and co-cultured with naïve BV2 microglia for 24 h, markers of microglial activation were significantly reduced. Specifically, recipient BV2 microglia incubated with LPS MP + PEG-TB had significantly reduced expression of IL-1β (p < 0.001) and TNF-α (p < 0.001) when compared to the LPS MP group (Fig. 5c). We confirmed the effects of LPS-stimulated enriched MP on primary cortical microglia. There was a significant increase in IL-1β (p < 0.01), TNF-α (p < 0.01), and IL-6 (p < 0.05) mRNA in recipient primary microglia treated with LPS enriched MP when compared to control MP-treated cells (Fig. 6). There was also a significant increase in miR-155 expression in primary microglia (p < 0.01) treated with LPS enriched MP when compared to cells treated with control MP (Fig. 6).

Finally, we confirmed that the effects of LPS enriched MP on microglial activation were not due to LPS crossover. We measured LPS levels in the LPS enriched MP samples using a limulus amebocyte lysate (LAL) assay and determined that the LPS concentration was negligible (0.081 ± 0.009 EU/ml (<0.1 ng)) and well below LPS concentrations previously shown to transfer LPS activity in enriched MP [34].

To determine whether microglial-derived enriched MP could seed neuroinflammation in the uninjured brain, we isolated enriched MP from control and LPS-stimulated BV2 microglia and stereotactically injected them into the cortex of adult male C57BL/6 mice. As a control to demonstrate the specific activity of microglial-derived enriched MP in promoting brain inflammation, we neutralized enriched MP by co-incubating them with PEG-TB as described before. After 24 h, cortical tissue was collected and markers of brain inflammation were assessed. When LPS enriched MP were injected into the uninjured cortex, they produced a robust neuroinflammatory response resulting in a significant increase in IL-1β (p < 0.01), NOS2 (p < 0.01), TNF-α (p < 0.001), IL-6 (p < 0.05), and miR-155 (p < 0.01) when compared to the control MP-injected group (Fig. 7). Notably, when LPS enriched MP were neutralized by PEG-TB and injected into the uninjured cortex, the cortical neuroinflammatory response was attenuated, such that the LPS enriched MP + PEG-TB group had significantly reduced expression of IL-1β (p < 0.001), NOS2 (p < 0.001), TNF-α (p < 0.001), IL-6 (p < 0.05), and miR-155 (p < 0.01) when compared to the LPS enriched MP group (Fig. 7). Intracortical injection with control MP, control MP + PEG-TB, or PEG-TB alone did not promote a neuroinflammatory response in the cortex.

In a separate study, control and LPS enriched MP were stereotactically injected into the cortex of uninjured C57BL/6 mice. At 7 days post-surgery, mice were euthanized and brain tissue was saline perfused and fixed for immunohistochemical analysis of microglial activation in the cortex, hippocampus, and thalamus using Iba-1 and P2Y12 immunostaining. There was a significant increase in Iba-1 staining in the ipsilateral cortex, hippocampus, and thalamus of C57BL/6 injected with LPS enriched MP (p < 0.001) when compared to mice injected with control MP or sham mice (Fig. 8a–c). Further morphological analysis of P2Y12-positive microglia using Neurolucida reconstruction software revealed that C57BL/6 mice injected with LPS enriched MP resulted in increased microglial activation. Specifically, microglial ramifications were significantly reduced in length in the cortex and hippocampus of the LPS enriched-MP-injected cortex (p < 0.01 for both vs control MP), and the microglial cell body area was significantly increased in the cortex (p < 0.001), hippocampus (p < 0.01), and thalamus (p < 0.01 vs control MP; Fig. 9a–c). These data indicate that enriched MP derived from LPS-stimulated microglia produce a robust neuroinflammatory response when injected into the cortex of uninjured mice.

To relate MP-mediated seeding of neuroinflammation to secondary injury mechanisms in the TBI brain, we subjected adult male C57BL/6 mice to sham or moderate-level TBI and isolated microglia/macrophages from the ipsilateral cortex at 7 days post-injury using MACS CD11b magnetic beads. We then cultured CD11b-positive cells for 24 h and collected microglia/macrophage-derived enriched MP from sham and TBI samples. Equal numbers of enriched MP (total 8 × 10³ MP) were stereotactically injected into the cortex of uninjured adult male C57BL/6 mice, and cortical tissue was collected 24 h later to assess markers of neuroinflammation. Injection of sham MP significantly increased IL-1β (p < 0.001) and TNF-α (p < 0.001) expression, but not miR-155 expression, when compared to the naïve control group (Fig. 10). Furthermore, injection of TBI enriched MP significantly increased IL-1β (p < 0.001), TNF-α (p < 0.05), and miR-155 (p < 0.01) expression in the cortex when compared to the sham MP-injected group. Although there was a modest increase in IL-6 and NOS2 expression in sham MP and TBI enriched MP-injected groups when compared to the naïve control group, these changes did not reach statistical significance. These data indicate that enriched MP derived from the microglia/macrophages isolated from the TBI brain produce a robust neuroinflammatory response when injected into the uninjured cortex.