Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury

Wildtype C57Bl/6 male mice were purchased from Jackson Laboratories (Bar Harbor, ME) and were 3–4 months old at the time of injury. Male human APOE targeted replacement mice with the human APOE3 or APOE4 inserted at the endogenous murine APOE promoter on a C57BL/6 J background were 3–4 months old at the time of injury. A limitation of the work in this study is that it only included male mice. Further research will be needed to determine if these results translate to female mice.

Animals were group housed under a 12 h light/dark cycle with ad libitum access to food and water. All mice were euthanized via carbon dioxide inhalation followed by saline perfusion. All procedures were conducted in accordance with protocols approved by the Georgetown University Animal Care and Use Committee. Experiments adhered to guidelines from the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services.

CCI was conducted as previously described [8, 19]. Briefly, surgical anesthesia was induced using 4% isoflurane with maintenance in 2%, at flow rate of 1–1.5 L/min in freely breathing oxygen. The anesthetized animal was mounted in a stereotaxic frame and the surgical site clipped and aseptically sterilized. A 10 mm midline incision was made over the skull, the skin and fascia reflected and craniotomy performed (4 mm) on the central aspect of the left parietal bone. The impounder tip of a Leica StereoOne Impactor was sterilized, positioned to the surface of the exposed dura, and set to impact the cortical surface at 5.25-m/s velocity, 1.5 mm tissue deformation as previously described [23]. Sham animals received isoflurane anesthesia, skin incision, and reflection, but no impact. After injury, the incision was closed with wound clips, anesthesia discontinued, 1 ml saline administered by intraperitoneal (i.p) injection and the mouse placed in a heated cage to maintain normothermia for a 45 min recovery period. All animals monitored carefully for 4 h after surgery and then daily.

For cyclosporine A (CsA) experiments, wildtype mice were randomly assigned in a blinded manner to receive either cyclosporine A (Sigma-Aldrich, St Louis, MO) or vehicle (5% ethanol, 25% cremaphor oil, and 70% saline), at 15 min post-injury. CsA was administered i.p. at 20 mg/kg in 10 mL/kg initially, with repeat doses administered at 10 mg/kg at 10 mL/kg, i.p. at 24 h intervals. The safety and efficacy of this dosing regimen in rodents has previously been established [24].

Evans blue (EB) extravasation assessed BBB permeability following CCI. Two hours before euthanasia, animals were administered 200 μL of EB dye (2% w/v, i.p., Sigma-Aldrich, St Louis, MO) in saline. After euthanasia, animals were immediately transcardially perfused with ice-cold PBS, brains extracted and micro-dissected to obtain the cortex and thalamus/striatum in both the ipsilateral and contralateral hemispheres. Samples were weighed and placed in ice-cold trichloroacetic acid (50% w/v in distilled water), before homogenization by sonication. Homogenates were incubated at 4 °C for 30 min, followed by centrifugation at 16,000×g for 30 min at 4 °C. The supernatant of each sample was removed and absorbance measured at an excitation of 540 nm and an emission of 680 nm. Absorbance measurements were adjusted for tissue weight and subsequent levels of EB in each sample was calculated relative to a standard curve of known EB concentrations.

Subcellular fractions containing soluble and membrane bound proteins were separated by sequential centrifugation. Mice were perfused with ice-cold PBS; brain removed and a 5 mm-diameter region of tissue surrounding the cortex lesion, or tissue corresponding to the area of lesion in sham mice, was excised by punch extraction. Ipsilateral cortex punches were homogenized in PBS with protease and phosphatase inhibitors using a glass dounce (88668, Pierce, Waltham, MA), then centrifuged at 135,000 x g for 45 min at 4 °C. Following centrifugation, the supernatant containing PBS soluble proteins was removed and stored at − 80 °C. The remaining pellet was resuspended in ice-cold RIPA buffer containing protease and phosphatase inhibitors (20–188, Millipore, Billerica, MA), sonicated and centrifuged at 14,000 x g for 15 min at 4 °C. The subsequent RIPA supernatant containing membrane bound proteins was removed and stored at − 80 °C.

Protein concentrations were measured using the BCA protein assay kit (23227, Pierce), with 20 μg of protein added to a 1:1 v/v of reducing buffer (2× Laemmli sample buffer (1610737, BioRad, Hercules, CA), 5% v/v β-mercaptoethanol (1610710, BioRad)) and heated at 95 °C for 5 min. Proteins were separated using SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes. Following transfer, membranes were blocked in 5% w/v skim milk powder in PBS-T (PBS, 0.05% v/v Tween-20, pH 7.6) for 1 h. Blots were then incubated with primary antibodies in 2% w/v skim milk/TBS-T, overnight at 4 °C. Primary antibodies used were PDGFRβ (1:1000, #3169, Cell Signaling, Danvers, MA), apoE (1:1000, ab1907, Abcam, Cambridge, MA), MMP-9 (1:800, 38898, Abcam) and β-Actin (1:5000, A5441, Sigma-Aldrich). Following primary antibody incubation, membranes were washed for 3 × 10 min in PBS-T at RT, before incubation with HRP-conjugated goat anti-rabbit, goat anti-mouse or rabbit anti-goat (Jackson ImmunoResearch, Westgrove, PA, 1:1000 in 2% w/v skim milk powder in PBS-T) secondary antibodies, for 90 min at RT. Membranes then washed for 3 × 10 min in PBS-T before detection using SuperSignal West Pico Chemiluminescent Substrate detection kit (34580, ThermoScientific, Waltham, MA) and visualization with Amersham 600 imaging machine (GE healthcare, Chicago, IL). Raw pixel intensities of bands from Western blots were quantified by densitometry using Image J software (Version 1.47, NIH, Bethesda, MD).

Free-floating parallel brain sections (20 μm thickness) from wildtype, APOE3, and APOE4 mice were cut using a microtome (Microm HM 430, Thermo Fisher Scientific, Tusin, CA), and washed three times with PBS, before staining and incubated with blocking buffer (5% normal goat serum (NGS) in PBS) and 0.3% Triton X-100 (PBST) for 2 h at room temperature. Brain sections were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal PDGFRβ antibody for pericytes (1:200, #3169, Cell Signaling), dylight 488 labeled Lycopersicon Esculentum (tomato lectin) (1:1000, DL-1174, Vector Laboratories, Burlingame, CA) for blood vessels, rabbit anti-CD31 (1:500, ab28364, Abcam) for microvessels, mouse monoclonal anti-Claudin-5 antibody (1:500, 35–2500, Invitrogen, Carlsbad, CA) and rabbit anti-Zonula Occludens 1 (1:500, 21,773–1-AP, Proteintech, Rosemont, IL) for tight junctions in the epithelial cells. After three 5 min washes in PBS, sections were incubated for 2 h with a corresponding anti-rabbit Alexa Fluor 568-conjugated or anti-mouse Alexa Fluor 594-conjugated IgG secondary antibodies (1:1000, S11227, Invitrogen) for 2 h at room temperature. As negative controls, sections were incubated without primary antibody. Sections were rinsed with PBS and distilled water and coverslipped with Fluoro-Gel with Tris Buffer mounting medium (Electron Microscopy Sciences, Hatfield, PA). Quantitative image analysis of the PDGFRβ Claudin-5, Zonula Occludens-1 and CD31 immunoreactive cortical regions were performed on five fields (× 20, 151.894 mm²), and three cortical sections per animal through the level of impact site using the same densitometric analysis method as previously described [23]. Image J software was used for analysis as previously described [23].

Following euthanasia and transcardial perfusion with ice-cold PBS, brains were harvested and flash frozen on dry ice. Brains were cryosectioned into 20 μm sections in series that encompass the ipsilateral lesion, as well as the contralateral hemisphere. Fluorescein conjugated, dye-quenched gelatin (DQ-gelatin, 1 mg/ml, D12054, Molecular Probes, Invitrogen) was prepared in reaction buffer (50 mM Tris HCl, Tris Base NH₂C(CH₂OH)₃, 150 mM NaCl, 5 mM CaCl₂, 0.2 mM NaN₃, pH 7.6) and stored at 4 °C. Sections were incubated in DQ-gelatin reaction buffer in the dark for 1 h at 37 °C. Following incubation, reaction buffer was removed and coverslips placed over the tissue using vectashield. Fluorescence visualized using an Olympus BX51 microscope (Olympus American, Center Valley, PA) equipped with Olympus DP72 camera, and cell Sens Standard software.

Coronal brain sections (20 μm thickness) were washed three times in PBS before mounting on gelatin-coated glass slides (Superfrost Plus, Thermo Fisher Scientific) and stored at − 80 °C until use. Tissue was allowed to dry at room temperature (RT) and then stored at -20 °C until use. Fluorescent in situ hybridization (FISH) was performed using RNAscope® Technology 2.0 (Advanced Cell Diagnostics (ACD), Hayward, CA), as previously described [23, 25]. In short, mounted tissue sections were serially dehydrated in 50%, 70%, 95%, 100% and 100% ethanol for 5 min each. In between all pretreatment steps, tissue sections were briefly washed with ultra-pure water. The pretreat solution 1 (hydrogen peroxide reagent) was applied for 10 min at RT, and then the tissue sections were boiled in pretreat solution 2 (target retrieval reagent) for 15 min. Mounted slices were treated with pretreat solution 3 (protease reagent) for 30 min at 40 °C on the HybEz™ hybridization system (ACD). Mouse Apoe probe (Cat. No. 313278, ACD) targets the region 83–1245 (Accession number: NM_009696.3) of the Apoe sequence with 19 pairs of ZZ-target probes. In addition, the negative (Cat. No. 310043, ACD) and positive (Cat. No. 313911, ACD) control probes were applied and let hybridized for 2 h at 40 °C. The amplification steps were performed according to manufacturer’s directions. In between every amplification step, sections were washed with 1× wash buffer. Detection was performed using a mixture ratio of the Red-A to the Red-B solution of 1:60. Sections were incubated for 10 min at RT and rinsed with ultra-pure water. Following in situ hybridization, the sections were processed for immunohistochemistry. Briefly, following the blocking step with 5% NGS in PBS for 1 h at RT, post-hybridized slides were incubated with an antibody against anti-mouse GFAP (glial fibrillary acidic protein, 1:1000, EMD Millipore, Temecula, CA) or anti-rabbit PDGFRβ in the presence of 2% NGS in PBS overnight at 4 °C. After three washes with PBS, the slides were incubated with corresponding anti-mouse or anti-rabbit Alexa Fluor® 488 secondary antibodies (1:500; Molecular probes®, ThermoFisher Scientific, Grand Island, NY) for 2 h at RT. Brain sections were rinsed with PBS three times and incubated for 5 min in PBS with DAPI solution (1: 50,000, Sigma-Aldrich) for counterstained nuclei. Fluorescent images were acquired on an Olympus XB51 microscope equipped with Olympus DP72 camera, and cell Sens Standard software.

Microvessels were isolated as previously described [26]. Briefly, sham and CCI mice were euthanized and ipsilateral and contralateral brain tissue punch excised and rinsed in sucrose buffer (S.B, 0.32 mol/L sucrose, 3 mmol/L HEPES, pH 7.4). Samples were homogenized in a glass dounce homogenizer containing 2 ml S.B., then centrifuged for 10 min × 1000 g at 4 °C. Following centrifugation, the supernatant (containing the visible white upper myelin layer) was discarded and remaining pellet resuspended in 2 ml S.B., re-homogenized using glass dounce and centrifuged again for 10 min × 1000 g at 4 °C. Supernatant was discarded and the sediment pellet was resuspended in a further 2 ml S.B. and centrifuged (30s × 100 g, 4 °C). Following centrifugation, supernatant was removed and kept, and the resulting pellet underwent a second resuspension (in 2 ml S.B) and centrifuged (30s × 100 g, 4 °C). The supernatant was removed and pooled together, before a final centrifugation for 2 min × 200 g at 4 °C. The resulting pellet was resuspended (25 μl, 0.1% BSA in PBS) and microvessels confirmed by Toluidin Blue staining and brightfield microscopy, before being prepared for RNA extraction.

RNA was extracted from microvessels using TRIzol® reagent (15596026, Invitrogen) as per manufacturer’s guidelines. Concentration and purity of the RNA was assessed using the NanoDrop 1000 spectrophotometer (ThermoScientific). One microgram of RNA was reverse transcribed into cDNA using a High-Capacity RNA-to-cDNA Reverse Transcription Kit (4368814, Applied Biosystems, Foster City, CA) in accordance with manufacturer’s guidelines. The resulting cDNA was diluted 1:3 in diethylpyrocarbonate (DEPC) treated H₂O for use in RT-QPCR. Samples were stored at − 80 °C until use.

RT-QPCR was conducted as described previously [27, 28]. Briefly, RT-QPCR was performed in triplicate in standard 384-well plates using the Prism 7900HT fast sequence detection system (Applied Biosystems). Taqman probes were used to analyze GAPDH (Mm99999915_m1), apoe (Mm01307193_g1), APOE (Hs00171168_m1), Mmp-9 (Mm01317678_m1), Claudin-5 (Mm00727012_s1), Occludin (Mm00500912_m1), Zonula Occludens-1 (Mm00493699_m1) and Pdgfrβ (Mm00435546_m1) under the following cycle conditions, 50 °C for 2 min, 95 °C for 20 s, (95 °C for 1 s, 60 °C for 20 s) × 40 repeats. The SDS 2.4 software (Applied Biosystems) was used to generate threshold cycle (Ct) values, and fold change in mRNA expression calculated using the ΔΔCt method (2-ΔΔCt) as previously described [29].

It has been well established that BBB dysfunction occurs following TBI, contributing to secondary injury processes and the overall degree of injury severity [30]. To characterize the time course of BBB permeability following primary impact, wildtype mice were subject to TBI before BBB permeability assessed by EB analysis. Levels of EB extravasation were increased by 411% in the ipsilateral cortex at day 1 (P < 0.001) and remained elevated by 201% at day 3 post-injury (P < 0.05) compared to sham animals (Fig. 1a). Full closure of the BBB to EB permeability was observed approximately 7–10 days post-injury. No change in BBB permeability was observed in the contralateral cortex (Fig. 1b) or the ipsilateral or contralateral thalamus/striatum at any timepoint post-TBI (Fig. 1c-d).

We also assessed BBB permeability using GdDTPA enhanced MRI. Consistent with EB observations, T1-weighted MRI with Gadolinium-enhanced contrast displayed increased leakage in the pericontusional area of the ipsilateral cortex that extended approximately 5 mm from the rostral to the caudal aspect of the lesion (Fig. 1e). We assessed GdDTPA leakage into the mouse brain at multiple timepoints up to 21 days post-TBI and observed GdDTPA signal at 1, 3, and 7 days post-injury compared to pre-injection scans, but not at 14 or 21 days post-injury (Fig. 1f). We quantified this signal and confirmed that the BBB was fully closed to GdDTPA leakage at 14d post-TBI (Fig. 1g).

The apoE protein has been demonstrated to modulate cerebrovascular integrity at the NVU via an apoE-LRP1 mediated suppression of MMP-9 [9]. To characterize the apoE and MMP-9 response following TBI stimulus, wildtype mice were subject to TBI before a micropunch of the ipsilateral cortex was extracted and analyzed at 1, 3 and 7 days post-injury. TBI induced a biphasic response in PBS-soluble apoE protein (Fig. 2a), with protein levels significantly reduced by 73% at day 1 (P < 0.001) and by 21% at day 3 (P < 0.01) post-injury compared to sham (Fig. 2a). At 7 days post-injury, apoE protein levels were significantly increased by 22% compared to sham levels (P < 0.05; Fig. 2a). Apoe mRNA expression in cortical punch was elevated by 3.5-fold in the ipsilateral cortex at 7 days post-injury (P < 0.05) (Fig. 2b). To explore the source of apoe mRNA after TBI, we used apoe mRNA FISH alongside markers of astrocytes and pericytes. We found widespread expression of apoe in our mice, with the majority of expression found in GFAP-positive astrocytes (Fig. 2c). Fig. 2C1–4 show apoe mRNA and GFAP immunofluorescence surrounding a cortical microvessel. The astrocytic projections and endfeet surrounding the microvessel are clear, however it is also apparent that cells associated with the microvessel are also producing apoe mRNA. To investigate this further, we repeated our apoe mRNA FISH with PDGFRβ immunofluorescence. We found colocalization of the PDGFRβ positive cells with apoe mRNA (Fig. 2d, D1–4).

To determine if microvessels were locally producing MMP, we used in-situ zymography. We report that MMP activity was greatly increased in the ipsilateral cortex at 1 days post-TBI, and that microvessel-related MMP activity was clearly visible in the ipsilateral cortex using this technique (Fig. 2e, arrows indicate microvessel-associated activity). At 3 days post-injury MMP activity was still visible, but there were noticeably less microvessel-associated activity compared to 1 day post-TBI. At 7 days post-injury there was still ongoing microvessel associated activity, but this was spatially limited to the area directly adjacent to the lesion (Fig. 2e). We next isolated microvessels from micropunches of the ipsilateral and contralateral cortex of TBI and sham mice and analyzed these vessels for the presence of MMP-9 protein. MMP-9 protein was detectable in the ipsilateral cortex microvessels from TBI mice, but not from the contralateral microvessels, or from sham microvessels (Fig. 2f). We further performed gel zymography on isolated microvessels from the ipsilateral cortex at 1, 3, and 7 days post-TBI, and found that while TBI substantially increased microvessel MMP-9 activity at 1 day post-TBI, but that this activity was below detection threshold at 3 and 7 days post-injury (Fig. 2g-h).

The NVU is a crucial structure that regulates BBB vascular integrity, stability and maintenance. To further elucidate the specific TBI mediated BBB response at the NVU, wildtype mice were subject to TBI before ipsilateral cortex microvessels were isolated and analyzed by RT-QPCR. Baseline characterization of microvessel preparations from sham mice revealed elevated Glut1 and PDGFRβ mRNA expression, indicative of enriched endothelial and pericyte populations compared to whole cortex tissue (Fig. 3a). Furthermore, these microvessels contain minimal astrocytic presence, with reduced levels GFAP and S100β observed compared to whole cortex fractions (Fig. 3a). Following TBI, apoe mRNA expression was significantly increased at 3 and 7 days post-injury compared to sham (P < 0.05; Fig. 3b). Mmp-9 expression was significantly increased 1 day post-injury (P < 0.001; Fig. 3c), but returned towards baseline by 3 days post-injury. There was a time-dependent decrease in Pdgfrβ mRNA, a pericyte marker, until 3 days post-TBI (P < 0.05), before recovering at day 7 post-injury (Fig. 3d). At the tight-junction protein level, we observed significant decreases in mRNA for Zonula Occludens-1 and Occludin at 1 and 3 days post-injury (P < 0.05; Fig. 3e, f), with a similar trend in the mRNA for Claudin-5 (Fig. 3g). All tight-junction protein mRNA returned to baseline expression by 7 days post-injury. We confirmed that PDGFRβ is found on vessels by examining protein co-localization with vessel specific lectin and examining vessel size and morphology. PDGFRβ colocalizes with lectin, and was found primarily on vessels with a diameter of 5–20μM (Fig. 3h).

To investigate the role of CypA–MMP-9 in BBB stabilization and repair, wildtype mice were subject to TBI, followed by administration of either CsA or vehicle. We assessed acute BBB permeability at 1 day to establish if CypA was involved in the acute response to trauma, and at 5 days post-injury to assess if inhibition of CypA could induce faster BBB stabilization than the 7–10 day spontaneous closure window we observed in untreated mice. At 1 day following TBI, EB extravasation from the ipsilateral cortex was significantly elevated in both vehicle and CsA treated mice (P < 0.001). At 5 days post-injury, BBB permeability remained significantly elevated by 135% in vehicle treated mice (P < 0.01 vs vehicle sham), but had returned to sham levels in CsA treated animals (P < 0.05 vs. CsA sham mice, Fig. 4a). In a separate cohort of mice, the effect of CsA on NVU responses was investigated by isolating microvessels from the ipsilateral cortex of CsA and vehicle treated wildtype mice after TBI. RT-QPCR analysis showed Mmp-9 expression was increased in the vehicle group at 1 and 5 days post-injury, a response that is significantly attenuated by CsA administration (P < 0.01; Fig. 4b). Pericyte expression was unchanged, with Pdgfrβ mRNA reduced in both groups at 1 day post-TBI (P < 0.001, Fig. 4c), before rebounding by day 5 post-injury. At the tight junction level, we observed significant decreases in mRNA for Zonula Occludens-1 and Occludin in both CsA and vehicle groups at 1 day post-TBI (P < 0.001; Fig. 4d, e). However, at 5 days post injury, CsA treatment resulted in a 20% increase in Zonula Occludens-1 (P < 0.001; Fig. 4d), whilst Occludin levels were unchanged to that observed in sham animals.

The human apoE4 protein is known to be functionally deficient compared to the apoE3 protein, and we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI. APOE3 and APOE4 mice were subject to TBI and their BBB permeability assessed using the EB method. 3 days following TBI, EB extravasation from the ipsilateral cortex was increased by 202% in APOE3 mice (P < 0.0001 vs APOE3 sham) and by 174% in APOE4 mice (P < 0.001 vs APOE4 sham) (Fig. 5a). Similar to wildtype mice, BBB permeability in APOE3 TBI mice had returned to within 5% of APOE3 sham levels by 10 days post-TBI, and was significantly reduced compared to APOE3 mice at 3 days post-TBI (P < 0.001). In contrast, BBB closure to EB was not achieved in APOE4 mice at 10 days post-TBI. At 10 days post-injury, BBB permeability in APOE4 mice remained significantly elevated by 94% compared to APOE4 sham mice (P < 0.05), and was not significantly decreased compared to APOE4 3 day mice (Fig. 5a). Previously we have demonstrated reduced levels of soluble apoE in the ipsilateral cortex of APOE4 mice compared to APOE3, up to 7 days post-TBI [8]. To investigate the NVU specific apoe response, ipsilateral microvessels were isolated and assessed after TBI. APOE3 microvessels displayed significantly elevated apoe mRNA expression at 3 and 10 days post-injury compared to APOE4 counterparts (P < 0.05; Fig. 5b). Given the role of brain pericytes in maintaining and regulating the integrity of the BBB [31, 32], we investigated the pericyte response to TBI in APOE3 and APOE4 mice. Western blot analysis of the ipsilateral cortex revealed that both APOE3 and APOE4 exhibit acute pericytes loss, with a 47% loss of PDGFRβ protein in APOE3 mice at 1 day post-TBI, and a 49% loss in APOE4 mice at the same timepoint (P < 0.05 vs genotype sham; Fig. 5c). PDGFRβ expression recovers by 3 days post-TBI in APOE3 mice, and is elevated by 44% at 7d post-TBI. In contrast, PDGFRβ remains significantly depressed at 3 days in APOE4 mice (P < 0.05 vs APOE4 sham), and remains 47% below APOE3 PDGFRβ levels at 7 days post-TBI (Fig. 5c, P < 0.01). In support of this finding, we found that immunohistochemistry for PDGFRβ was reduced in the pericontusional cortex of APOE4 mice compared to APOE3 mice at 7 days post injury (Fig. 5d-e). Furthermore, RT-QPCR analysis of the pericyte marker Pdgfrβ in isolated microvessels, revealed APOE4 animals exhibit decreased expression at 7 (P < 0.001, Fig. 5f) and 10 days post-injury (P < 0.05, Fig. 5f) in comparison to APOE3 mice.

To investigate the genotype specific effects of APOE on BBB dynamics, APOE3 and APOE4 mice were subject to TBI before cerebrovascular integrity assessed. We used lectin and CD31 to determine if the number of blood vessels was impacted in the perilesion area 7 days post-TBI. In sham mice, we found no effect of genotype using either lectin or CD31. However we found that in TBI mice, lectin is no longer specific for blood vessels, with widespread cellular staining through the glial scar that had the morphological characteristics of glial cells (Fig. 6a). CD31 immunohistochemistry remained specific for blood vessels, and we found that neither APOE genotype nor TBI caused a significant change in the area of blood vessel coverage in the pericontusional area (Fig. 6a). At the tight junction level, immunohistochemistry analysis showed APOE4 mice display reduced levels of Claudin-5 and Zonula Occludens-1 at 7 days post-TBI compared to APOE3 mice (Fig. 6b, c). In support of our protein analysis, APOE4 microvessels isolated from a micropunch of the ipsilateral cortex display significantly reduced mRNA expression of Claudin-5 (P < 0.05, Fig. 6d), Zonula Occludens-1 (Day 3 and 10, P < 0.05, Fig. 6e) and Occludin (P < 0.05, Fig. 6f) after TBI compared to APOE3 counterparts. Furthermore, increased levels of Mmp-9 is observed in APOE4 microvessels at day 1 (P < 0.01, Fig. 6g) and day 3 (P < 0.05, Fig. 6g) post-TBI compared to APOE3 microvessels. In addition, Mmp-9 expression remained elevated in APOE4 microvessels at day 7 and day 10 (P < 0.05, Fig. 6g) post-injury, relative to genotype control.