Three-month-old C57BL/6J male mice (Jackson Laboratory) were used for this study. The mice were housed on a 12-h light–dark cycle with ad libitum access to food and water. All animal experiment procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. The animals were randomly distributed into four experimental groups. All experiments and analysis for this study were conducted in a blinded, randomized, and controlled design to group assignment and treatment. All efforts were made to minimize the animal number and animal suffering. The number of animals for each experiment was calculated based on the power analysis and is stated in each figure or figure legend .
Transient focal cerebral ischemia model
Transient focal cerebral ischemia model was induced by 35-min reversible middle cerebral artery occlusion (tMCAO) with a silicone-coated filament followed by reperfusion, as our group described previously [22, 23]. This transient unilateral cerebral ischemia model generates a reproducible ischemic lesion in the ipsilateral hemisphere. Briefly, each mouse was anesthetized with isoflurane (3% for induction and 1.5–2% for maintenance) in an oxygen/air mixture during surgery. Artificial tear ointment was applied to the eyes for protection and lubrication. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully exposed. The CCA was ligated with a 6–0 silk suture at the proximal portion. A 12-mm length of a 6–0 silicone-coated nylon filament (Doccol, Cat. No. 602123) was gently inserted into the CCA and then advanced into the ICA ~ 9–10 mm from the internal carotid bifurcation until mild resistance was felt and cerebral blood flow (CBF) exhibited a dramatic reduction (> 75% of the baseline value), assessed by a laser Doppler flowmetry (Moor Instruments Ltd). Reperfusion was performed by gently retracting the filament after 35 min of MCA occlusion. The body temperature was maintained at 37 °C by a thermostatically controlled heating pad during the whole surgical procedure. Post-surgery mice were allowed to recover in a temperature- and humidity-controlled chamber. The sham-surgery (sham) mice were subjected to the same surgical procedures except for the MCA occlusion.
Treatment with BET degrader dBET1
The proteolysis-targeting chimera (PROTAC) dBET1 is a BET protein degrader comprising BET bromodomain antagonist ( +)-JQ1 conjugated to a cereblon E3 ubiquitin ligase ligand . dBET1 (Chemietek, Indianapolis, IN, USA; Cat. #CTDBET1; > 99.5% purity; CAS #1,799,711–21-9) was dissolved in dry dimethyl sulfoxide (75 mg/ml) and stored at − 20 °C as we previous described . The freshly prepared dBET1 working solution or the same volume of vehicle was injected intraperitoneally at 4 and 24 h after the onset of reperfusion. dBET1 was given at a dose of 30 mg/kg. For a 30-g mouse, 12 µl dBET1 stock (or 12 µl dimethyl sulfoxide for vehicle) and 12 µl of 25% Tween 80 in sterile water were mixed and vortexed for 1 min. 476 µl of 11.2% of Captisol® (β-cyclodextrin sulfobutyl ether sodium; Ligand, San Diego, CA, USA) in sterile water was added and thoroughly vortexed again before injection.
Measurement of infarct volume
The infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 48 h after stroke . Mice were euthanized, transcardially perfused with ice-cold saline, and the brains were harvested. Mouse brains were sliced into six 1-mm-thick coronal sections with a brain matrix. The fourth section, starting from the rostral side, was dissected and immediately frozen in liquid nitrogen and stored in the -80 °C freezer until further molecular biology analyses. The other sections were incubated in a 2% TTC solution (Sigma-Aldrich) at room temperature for 30 min, followed by fixation with 4% paraformaldehyde (PFA, pH 7.4). All rostral sides of the stained sections and the caudal side of the third section (corresponds to the rostral side of the fourth coronal section) were scanned at 600 dpi using an HP Scanjet 8300 scanner (Palo Alto, CA) and saved as a JPEG file. The infarcted area of each section was delineated and determined using Adobe Photoshop CS5, and the overall infarct volume was calculated. The details are described in our previous reports [19, 26].
A battery of behavioral tests, including neurological deficit score, open field locomotor activity, and vertical grid test, were performed to assess the neurological performance of mice at indicated timepoints. Investigators performing the tests and analyses were blinded to group assignment and treatment.
Neurological deficit score
The neurological deficits scoring can assess the overall neurological severity and multiple deficits in animal studies of stroke [27, 28]. At 48 h after stroke, mice’s neurological deficit score (NDS) was evaluated using six individual tests, including body symmetry, gait, circling behavior, front limb symmetry, compulsory circling, and climbing, as we previously described [29, 30]. Scoring for each test was performed independently by two trained investigators using a 4-point scoring system (0, no deficits; 4, severe deficits). The average score of each mouse from two investigators was used for statistical analysis.
Vertical grid test
Vertical grid test is a sensitive method to assess neuromuscular strength and motor coordination in rodents [31, 32]. The vertical grid apparatus is an open frame (55 cm height × 8 cm wide × 5 cm depth) with a wire mesh (0.8 cm × 0.8 cm aperture) on the backside. It is vertically placed in a cage filled with bedding material. Within 1 week prior to and 48 h after tMCAO, each mouse was placed on the top of wire mesh, facing downward, and was allowed to climb down to the cage. The total time to climb down was recorded. If the mouse failed while descending or could not descend down into the cage within 60 s, the performance was expressed as the maximum duration of 60 s. Each mouse performed 3 trials with at least 5-s intervals.
Open field test
The open field test is used to assess the locomotor activity of mice. Within 1 week prior to and at 48 h after tMCAO, the spontaneous locomotor activity of mice was measured in an open field paradigm with an automated video tracking system (Anymaze software, Stoelting, Wood Dale, IL) as we described previously . Mice were individually placed in an open field chamber (40 × 40 × 40 cm) with grey sidewalls and allowed to explore for 10 min. The total traveled distance was used as the indices of motor/exploratory behavior of each mouse. The open field arena was cleaned with 70% ethanol between tests.
Immunohistochemistry and immunofluorescence
Each mouse was anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde (pH 7.4) in PBS. The brain was removed, post-fixed, and cryoprotected in 30% sucrose (w/v) and sliced into 30-μm-thick coronal sections on a semi-automatic vibrating microtome (Compresstome® VF310-0Z; Precisionary Instruments, Natick, MA). Immunohistochemistry and immunofluorescence stainings were performed using standard protocols [33, 34]. The primary antibodies were rabbit polyclonal ionized calcium-binding adapter protein 1 (Iba1; 1:5000; catalog #019–19,741, Wako Bioproducts, Richmond, VA), rabbit polyclonal glial fibrillary acidic protein (GFAP; 1:3000; catalog #Z0334, DAKO, Carpinteria, CA), horse anti-mouse IgG (biotinylated; 1:200; catalog #BA-2000, Vector Laboratories, Burlingame, CA), purified rat anti-mouse Ly-6G (1:200; catalog #127,602, Biolegend, San Diego, CA), and goat anti-mouse intercellular adhesion molecule-1 (ICAM-1/CD54; 1:1000; catalog #AF796, R&D Systems, Minneapolis, MN). Sections were washed and incubated with Elite ABC-HRP Kit for Ly-6G and IgG stainings (catalog #PK-6100, Vector Laboratories, Burlingame, CA) or appropriate secondary antibodies for other stainings on the following day. The following secondary antibodies were used: goat anti-rabbit (1:2000; catalog #5450–0010, SeraCare, Gaithersburg, MD) and donkey anti-goat Alexa Fluor Plus-594 (1:500; catalog #A32758, Life Technologies, Grand Island, NY). For immunohistochemistry, the immunoreaction was visualized using a 3,3-diaminobenzidine chromogen solution (DAB substrate kit; Vector Laboratories). The brightfield images were captured by ScanScope CS and analyzed using ImageScope software (Aperio Technologies, Vista, CA) or ImageJ software. Immunofluorescence images were captured by an automated Axio Scan.Z1 slide scanner and analyzed using Zen 2.3 software (Zeiss, Berlin, Germany) or ImageJ software. The immunostaining signals were quantified in a 100 × 100 µm square applied at indicated brain regions in the figures. Three consecutive coronal sections were quantitatively analyzed and provided an average value. We counted Iba1-positive cells area (%), GFAP-positive astrocytes area (%), IgG immunointensity, Ly-6G positive cells per field (i.e., 200 µm × 200 µm square), and ICAM-1 immunointensity in indicated brain regions.
Protein extraction and western blot
Protein extraction and western blot were performed as previously described . The deeply anesthetized mice were transcardially perfused with ice-cold saline. Harvested brain tissues (peri-infarct/contralateral cortex or hemispheres) were collected and stored in the − 80 °C freezer. Tissues were homogenized in lysis buffer, and total protein concentration was determined using the PierceTM BCA assay kit (catalog #23,227, Thermo Scientific, Rockford, IL). Samples were aliquoted and stored at − 80 °C freezer until further analysis. An equal amount of protein was separated using 4–20% polyacrylamide gradient gels (BioRad, Hercules, CA) and transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were incubated overnight with rabbit anti-BRD4 (1:2000; Catalog #A301-985A50; Bethyl Laboratories, Montgomery, TX, USA), rabbit anti-zona occludens 1 (ZO-1; 1:1000; catalog #61–7300, Life Technologies, Grand Island, NY), rabbit anti-Occludin (1:1000; catalog #ab167161, Abcam, Cambridge, MA), rabbit anti-MMP-9 (1:500; catalog #sc-6841-R, Santa Cruz Biotechnology, Dallas, Texas), mouse-4-hydroxynonenal (4-HNE; 1:2000, catalog #MAB3249, R&N systems, Minneapolis, MN), mouse anti-gp91phox (1: 500; catalog #611,414, BD Biosciences, San Jose, California), rabbit anti-glutathione peroxidase 1 (GPX1; 1:1000; catalog #ab22604, Abcam, Cambridge, MA), rabbit anti-Superoxide dismutase 2 (SOD2; 1:2,000, catalog #ab13534, Abcam, Cambridge, MA), and mouse anti-β-actin (1:10,000; catalog #A1978, Sigma-Aldrich, St. Louis, MO). The membranes were then washed with TBST three times at 5 min intervals, incubated with goat anti-rabbit IRDye 800CW (1:30,000; Li-Cor, Lincoln, NE) or donkey anti-mouse IRDye 680LT (1:40,000; Li-Cor) secondary antibodies for 1 h at room temperature. Membranes were scanned with an Odyssey infrared scanning system (Li-Cor), and quantification analyses of immunoreactive bands were performed using ImageJ software. Unedited immunoblots are provided in Additional file 1: Figure S1. For gp91phox, we optimized the immunoblotting conditions and found a better signal when gels are run under non-reducing conditions (Additional file 2: Figure S2).
RNA isolation and quantitative real-time PCR
Mice were euthanized and transcardially perfused with ice-cold saline, and the brains were collected and sliced into six 1-mm-thick coronal sections with a brain matrix. The cortical tissue was dissected from the fourth section and was stored at − 80 °C until RNA extraction. Total RNA from the cortical tissue was isolated using a modified method of acid guanidinium thiocyanate–phenol–chloroform extraction [19, 35]. RNA concentration and purity were determined by a Take3 Micro-Volume Plate Reader (Biotek Instruments, Winooski, VT). Quantitative real-time PCR was performed in a total reaction volume of 10 μL using Luna® Universal One-Step RT-qPCR Kit (catalog # E3005, New England BioLabs, Ipswich, MA) according to the manufacturer’s protocol. Reactions were run in a BioRad CFX96 Touch Real-Time PCR Detection System. The primer sequences were included in Table 1. Each reaction was performed in triplicate, and the relative expression value for each target gene was calculated using the ΔΔCt method after normalization to the housekeeping gene Ywhaz.
PRISM software (GraphPad V.6) was used to perform the statistical analyses. D’Agostino–Pearson normality test was utilized to confirm Gaussian distribution before performing parametric statistical tests. An independent unpaired Student’s t test was performed for comparison of two groups. Multiple comparisons were made using one-way or two-way ANOVA followed by Bonferroni’s post hoc test. All data were expressed as group mean ± SEM, and P ≤ 0.05 was considered statistically significant. The number of samples for each experiment is stated in each figure or figure legend. All studies were analyzed with investigators blinded to treatments and groups.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.