Animals and experimental setup:
All of the studies performed were approved by the United States Veterans Affairs Animal Component of Research Protocol (ACORP). Additionally, Lexington VA Vivarium is accredited by the Association for the Assessment and Accreditation for Laboratory Animal Care, International (AAALAC, International) and all experiments were performed with its guidelines. All animal experiments were compliant with ARRIVE guidelines and experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Male (~ 260g average weight) and female (~ 220g average weight) Sprague Dawley rats (Charles River) were used at 7–8 weeks.
Animals were randomly assigned to groups, using random number generators. Researchers were blinded to treatment groups during outcome assessment and data analysis. The animals were housed 2–3 per cage (NexGen™ Rat 1800, Allentown Inc.) and maintained in a 12h light/12h dark cycle. Confounding factors were minimized by including various treatment groups in the same cage, ensuring all experimental groups are operated on/analyzed at the same time (especially if the assay required multiple cohorts of animals), and all animals were housed in the same room. All animals were fed a balanced diet ad libitum and water was reverse osmosis generated. The exact number of animals per study are reported within the figure legends. For additional details on common data elements used in this study, see Table 1.
Blast injury model: The McMillan Blast Device (MBD) consists of a cylindrical steel tube, 12-inch internal diameter, separated into a 20-ft. expansion chamber and a 2.5-ft compression chamber. We used compressed helium in the compression chamber and 10-mil-thick (0.254 mm) polyethylene terephthalate (Mylar®) membrane to separate the two chambers (Mylar A; Tekra Corp., New Berlin, WI). Industrial grade compressed helium (American Welding & Gas, Lexington, KY), was filled to approximately 33 psi and then manually ruptured by an 8-point blade affixed to a pneumatic cylinder.
The blast over(under)pressure wave was recorded by a pitot probe (face-on/reflected and side-on) pressure (custom-built; Stumptown Research & Development, LLC, Black Mountain, NC; XTL-190S-100A, Kulite Semiconductor Products, Inc., Leonia, NJ), and piezo-resistive side-on sensors (Model #XTEL-100-190S-100A; Kulite). For the exact sensor locations, see Fig. 1C. Three side-on/wall sensors were positioned equi-distanced (6 inches) around the animal position. Data from each sensor was routed directly to the TMX-18 (AstroNova, Inc., West Warwick, RI). Data were analyzed using AstroView software (AstroNova, Inc.). Data were graphed using FlexPro software.
Animal blast exposure: Sprague-Dawley rats were transported to the blasting site. The rats were temporarily housed in a climate-controlled room enclosed away from the blast tests and had access to food and water ad libitum throughout the course of the transport. Immediately prior to injury, the rats were anesthetized with isoflurane using SomnoSuite Low-Flow Anesthesia System (Kent Scientific Corporation, Torrington, CT). Rats received 900 mL/min flow at 4.5% isoflurane until fully anesthetized and then placed on a nose cone (400 mL/min at 3.0% isoflurane) for physiologic recordings. Rats were placed into a mesh netting support (Industrial Netting, Minneapolis, MN) and secured into the MBD (two feet from open end of tube) laterally with the left side facing the blast[37]. Once loaded into the MBD, the rat's body was protected from direct forces by a steel tube that surrounded the body but left the head completely exposed to the blast. The rats were subjected to compressed helium-driven blasts at 11 psi peak static overpressure (Fig. 1; blast parameters for each group detailed in Table 2) to model mbTBI. Physiological recordings were taken using SomnoSuite technology (MouseSTAT) five minutes before and five minutes after mbTBI procedure. Rats were monitored and recovered before transportation back to the Lexington VA Vivarium.
Open field test: Open field (OF) testing was performed in the morning hours three days prior to, and two days following low level blast exposure to assess anxiety-like behavior in male and female rats [25]. Rats were placed in a 32” x 32” x 12” dimly lit box for 10 minutes and their exploration was recorded using Ethovision software. The box was divided into two zones, with the inner zone being half the size and centered within the outer zone. The software tracked the nose point of the rats and recorded the number of entrances and time spent in each zone. The box was cleaned using 70% EtOH between each test.
Elevated maze plus: Rats were placed in the Elevated Plus Maze (EPM; Med Associates Inc. Fairfax, VT, USA) 7 days following low level blast exposure to assess to assess anxiety in male and female rats [33]. The maze is designed with two open arms, 20” x 4”, and two closed arms, 20” x 4” x 15.94”, with like arms across from each other and an open junction in the middle, 4” x 4”. The plus maze has no roof, and raised off the ground 29.31”. Rats were placed in the junction between the open and closed arms and allowed to explore for five minutes. The number of entrances into and time spent in either the closed or open arms was recorded using IR beam detection by MedPC software. The lights were dimmed and the box was cleaned between each trial using 70% EtOH.
Tissue Processing: Cohorts of animals were euthanized at 6 hours, 24 hours, 7 days and 14 days following mbTBI. Rats received intraperitoneal injection of Fatal Plus (Vortech Pharmaceuticals, Dearborn, MI) before transcardial perfusion with cold, sterile saline. After perfusion, rats were decapitated and the brains were then removed from the skull. The left hemisphere of the brains was rapidly dissected to isolate the amygdala and immediately frozen on dry ice. The right hemisphere of the brains was fixed with 4% paraformaldehyde (PFA) for 24 hrs. Following post-fixation, tissue was placed into 30% sucrose in PBS buffer solution for at least 48h for cryoprotection. The brain was then flash frozen in -25 to -35°C isopentane before being cut into 40µm thick coronal sections using a sliding microtome (Microm HM 450, Thermo Fisher). Tissue sections were stored at -20°C in cryoprotectant (30% glycerol, 30% ethylene glycol in 1X TBS).
Western blot: Western blot analysis was performed for tight junction proteins (zonula occludens-1 (ZO-1), Occludin and Claudin-5) and glial fibrillary acidic protein (GFAP) from amygdala brain tissue homogenates. Lysates were made using RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0), centrifuged at 16,100 x G for 30-minutes and total protein levels were estimated from supernatant using a BCA kit (23225, Thermofisher). Western blot samples were made using XT sample buffer (1610791, Biorad) with DTT and boiled at 95℃ for 10 minutes. Samples were resolved in duplicate 4–12% BIS-TRIS gels (3450125, Biorad) under reducing condition and transferred to PVDF membrane. Probing was done against ZO-1 (1:1000; ZO1-1A12, Thermofisher), Occludin (1:1000; OC-3F10, Thermofisher), Claudin-5 (4C3C2, Thermofisher), GFAP (1:1000; G3893, Sigma) and beta-actin (1:5000; 8H10D10, Cell Signaling). Signals were detected using chemiluminescence substrate (34075, Thermofisher) with anti-mouse IgG-HRP (1:10,000; GENA93, Millipore Sigma), anti-rabbit IgG-HRP (1:20,000; GENA934, Millipore Sigma) or with fluorescence signals using IRDye 68RD goat anti-mouse (1: 10,000; 926-68070, Li-Cor) and IRDye 800 CW goat anti-rabbit (1:10,000; 926-32211, Li-Cor). Protein levels were quantified by densitometric analysis using ImageJ software.
Immunohistochemistry: PFA fixed, 40µm thick coronal brain sections were immunostained to examine GFAP expression. Briefly, tissue sections were washed from cryoprotectant and endogenous peroxide activity was blocked with H2O2 (10% H2O2 in methanol) for 30 min. Following three time washing with TBS (Tris buffered saline), sections were blocked with blocking buffer contains 5% normal horse serum in TBST (0.1% tween-20 in TBS) and incubated with rabbit anti-GFAP antibody (1: 500; G3893, Sigma) in blocking buffer overnight at 4℃. The next day, sections were washed and incubated with biotin donkey anti-mouse IgG (1: 250; 715-065-15, Jackson Immuno Research) in blocking buffer at room temperature (RT) for 1 hr. After rinsing the secondary antibody, sections were incubated with avidin-biotin complex (PK-4000; Vector Laboratories) for 1 hr at RT followed by DAB treatment for 2 minutes as per manufacturers direction. After washing, sections were mounted on glass slides, dried overnight, dehydrated with ethanol and xylene before final permount mounting. Slides were scanned on the Zeiss Axio Scan Z.1 and amygdala regions were traced in HALO for quantification.
Immunofluorescence: Randomly selected (n = 3–6/group) above mentioned brain sections were double immuno-stained for GFAP (1:250; G9269, Sigma) and SMI-71 (1: 250; 836804, Biolegend) or SMI-71 alone to quantify the astrocyte coverage around the brain vasculature and BBB integrity respectively. Briefly, brain sections were permeabilized in 0.2% Trion X-100 in TBST for 15 mints followed by blocking in blocking buffer (1%BSA + 10% normal horse serum + 0.1% Triton X-100 in TBST) at RT for 1 hr. Then sections were incubated with mixture of rabbit anti-GFAP and mouse anti-SMI-71 or SMI-71 primary antibody in blocking buffer overnight at 4℃. Following day, sections were washed and incubated with mixture of Alexa flour 488 donkey anti-rabbit (1:500; A212206, Invitrogen) and Alexa flour 594 donkey anti-mouse (1: 500; A212203, Invitrogen) or Alexa flour 594 donkey anti-mouse alone as secondary antibody in blocking buffer at room temperature (RT) for 1 hr. After rinsing, samples were mounted on glass slides using prolong-glass antifade mount with Nucblue (P36981; Invitrogen). GFAP and SMI-71 double stained slides were scanned and astrocytic end-feet coverage around the blood vessel were analyzed using Nikon confocal microscope (20X; 100X with oil) with NIS-Elements version 5.30.05. Randomly 25 vessels were selected (red channel; SMI 71) in amygdala region from each brain sections (n = 3/group) and measured GFAP mean intensity (green channel) after subtracting the background fluorescence. SMI-71 stained slides were scanned using BioTek-Cytation-5 with Gen5 Image + 3.11 software. Vascular integrity was quantified as vascular density by SMI-71 using ImageJ with vascular density macro.
Statistical Analysis: Power analysis was conducted (using G*Power statistical software; version 3.0.10) for all experimental data and based on previous published literature from our group. Analysis was completed based on the ANOVA statistical tests and output of F score. A priori analysis was performed and effect size was calculated based on expected mean ± SD within each group. Sample size was calculated for behavioral experiments using the following parameters: α = 0.05, 1 − β = 0.8, and standard deviation 20% of mean for experimental groups. Primary outcomes for sample size determination were time in closed arms and tight junction expression. Based on deviation and detectable differences, it was determined that only a subset of the animals was needed to measure IHC and fluorescent histological markers.
Statistical analysis was performed using Graph Pad Prism (GraphPad Software, CA, USA) or JMP 12 (SAS, NC, USA). For all analyses, a significant difference among groups was defined as p < 0.05. For each measure, data were measured using interval/ratio scales. The Brown-Forsythe and Bartlett’s tests were performed to ensure homogeneity of variance. Furthermore, the Shapiro-Wilk test was completed to ensure normality. As these criteria were met for all experimental data, parametric statistics were employed for all analyses. Two-way ANOVA test were completed and the Bonferroni post-hoc test was utilized to examine injury effect within sex, where appropriate. Additionally, two-tailed, unpaired t test was also used.