All experiments were strictly conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center.
Drug Preparation and Properties: RGFP966 (a selective HDAC3 inhibitor) was purchased from Biorbyt (Cambridge, UK). RGFP966 is an N-(o-aminophenyl)carboxamide HDAC inhibitor [25–27]. With systemic administration, the distribution of RGFP966 to the CNS is relatively efficient, with a brain: plasma ratio of 0.45 [28]. A substrate-dependent biochemical assay using recombinant human HDACs found that RGFP966 is a specific inhibitor for HDAC3, with an IC50 of 0.08 µM and no effective inhibition of any other HDACs at concentrations up to 15 µM [28]. With systemic administration of 10 mg/kg RGFP966, the maximum drug concentration (Cmax) in the brain is 3.15 µM; thus, RGFP966 at the dose used in this study is a specific inhibitor of HDAC3 in vivo [28].
Animal Middle Cerebral Artery Occlusion (MCAO) Model
As previously described [29], adult male Wistar rats (270–300 g, 2–3 months) were anesthetized with isoflurane, administered via a precision vaporizer in oxygen (3.5-5% for induction, followed by 1.5% for maintenance). The analgesic Buprenorphine SR 1.2 mg/kg was administrated subcutaneously before surgery. Body temperature was maintained at 37 ± 0.5°C throughout the surgical procedure using a heating pad and a feedback-regulated water heating system. A 4 − 0 nylon suture with its tip rounded by heating near a flame was inserted into the external carotid artery (ECA) through a small puncture. The nylon suture, whose length was determined by the animal's weight, was gently advanced from the ECA into the lumen of the internal carotid artery (ICA) until the suture blocked the origin of the middle cerebral artery (MCA). The nylon suture was retained inside the ICA for 2 hours and the neck incision was closed. The animals were moved to their cage to awaken. Animals were re-anesthetized with isoflurane after 2 hours, and restoration of blood flow was performed by the withdrawal of the suture until the tip cleared the lumen of the ECA. The incision was then closed.
Experimental Groups
To examine the effects of HDAC3 inhibition on cerebral edema and BBB leakage, adult male Wistar rats was subjected to 2-h MCAO, and randomly selected animals were treated i.p. with either vehicle (1% Tween 80) or a selective HDAC3 inhibitor (RGFP966, 10 mg/kg) at 2 and 24 h after MCAO. The dose of RGFP966 used in this study (10 mg/kg) was selected based on our previous study [24] and other studies demonstrating proper concentrations in the brain [28, 30]. Before each administration, RGFP966 was freshly dissolved in 1% Tween 80. Rats were sacrificed 3 days after 2-h MCAO for histological and immunohistochemistry analysis (n = 6/group) and at 48 hours for EBD and Western blot assays (n = 4/group).
Modified Neurological Severity Score (mNSS) and Inclusion and Exclusion Criteria: mNSS is a composite of motor, sensory, balance, and reflex tests. mNSS is graded on a scale of 0 to 18 (normal score: 0; maximal deficit score: 18). One point is awarded for the inability to perform the test or for the lack of a tested reflex; thus, the higher the score, the more severe is the injury [31, 32].
mNSS was employed as a prespecified severity inclusion and exclusion criteria [33]. Specifically, if the animal’s score was mNSS 5 or more after MCAO surgery, it was included in the randomization. Animals that scored less than 5 were excluded. For each experimental animal, mNSS was performed before MCAO, at 2 hours, 1 day, and 3 days after MCAO.
Tissue Preparation for Immunohistochemistry
Animals were anesthetized with ketamine (80 mg/kg) and xylazine (13 mg/kg) via i.p. injection. The animals were then subjected to cardiac puncture and perfused with saline followed by 4% paraformaldehyde (4% PFA) via a needle inserted into the left ventricle of the heart. The brains were removed, fixed in 4% PFA overnight, and then embedded in 20% sucrose for 2 days. Using a rat brain matrix (Braintree Scientific, MA), each forebrain was cut into 2 mm thick coronal blocks for a total of seven blocks per animal.
Quantification of Cerebral Edema
A series of 10 µm thick sections was cut from each block and stained with hematoxylin and eosin (H&E) for calculation of the cerebral edema for each group, as previously described [33, 34]. Each H&E–stained coronal section was digitized under 2.5 x objective of Celestron Digital Microscope Pro. and analyzed using NIH ImageJ software. Cerebral edema was calculated by subtracting the volume (mm3) of the contralateral hemisphere from the ipsilateral hemisphere [33, 34].
Immunohistochemistry: A series of coronal sections (10 µm thick) were obtained from the center of the lesion (Bregma − 1 mm to + 1 mm) and mounted on slides for analysis. For immunohistochemistry, the following primary antibodies were employed: anti-HDAC3 (Abcam, ab32369, 1:500), anti-GFAP (Millipore, MAB3026, 1:600), anti–Iba-1 (Novus Biologicals, NB100-1028ss, 1:500), anti-albumin (Invitrogen, MAB1455, 1:400), anti-AQP4 (Milipore Sigma, AB3594, 1:200), anti–claudin-5 (Santa Cruz, sc-374221, 1:400) and anti-ZO-1 (Invitrogen, 61-7300, 1:400). Negative controls were performed by omitting the primary antibody. Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI).
Image Acquisition and Quantitation
For quantitative measurements, immunostained coronal sections were digitized using a 20–40 x objective epifluorescence (Nikon Eclipse E600) microscope. Four-six fields of view were acquired from the peri-infarct cortex and the total number of immunoreactive cells were counted using NIH ImageJ software. The total number of positive cells per mm2 area is presented.
Evans Blue Dye (EBD) Assay
As previously described [35], 2% solution of EBD (Sigma, St. Louis, MO) was prepared in 0.9% saline and injected to the tail vein. After 2–4 hours of EBD injection, the rats were sacrificed and perfused transcardially with 0.9% of saline. The cerebral hemispheres were separated and homogenized in N,N-dimethylformamide (Sigma, MO) and then incubated for 72 h in a water bath at 55°C. The samples were centrifuged at 1,500 g for 20 min. The extracted EBD in the supernatant was quantified by absorbance at 620 nm. Results were expressed as µg of Evans blue per gram of brain hemisphere by comparison with solution standards and reported as fold change compared to the contralateral hemisphere.
Western Blot: Animals were sacrificed and brain tissues were harvested and then snap-frozen in liquid nitrogen and stored at − 80°C. Tissues were thawed, washed in ice-cold PBS, and lysed in RIPA buffer containing protease inhibitors (Sigma). Samples were then sonicated, incubated on ice for 30 minutes, and centrifuged at 10,000 g for 20 min at 4°C. Protein concentration in the supernatant was determined by Pierce BCA Protein Assay Kit (Life Technologies). Equal amounts of protein (20 µg) were combined with loading buffer, boiled for 5 min, and loaded onto 4–20% precast polyacrylamide gel (Bio-Rad Laboratories). Separated proteins were transferred onto nitrocellulose membranes, blocked with casein-based blocking reagent (I-Block, Life Technologies) for 60 minutes at room temperature, and then incubated overnight at 4°C with the following primary antibodies: MMP-9 (Cell signaling, #13667, 1:1000) and acetyl NF-kappa( Cell signaling, S2S3J, 1:1000). Secondary antibodies used were HRP-linked specific for rabbit (1:2000, Cell Signaling) and mouse (1:2000, Cell Signaling). After incubation, membranes were washed with PBS-T and exposed to the appropriate horseradish peroxidase-linked secondary antibody. Blots were developed with Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected using a BioRad ChemiDoc Touch Imaging System (BioRad Laboratories). Data were analyzed using ImageJ software. Total abundance of target protein was normalized to appropriate endogenous control and reported as fold change.
Blinded Assessment of Outcomes
All measurements (mNSS testing, cerebral edema calculation, and immunohistochemical measurements) were performed by an investigator who had no knowledge of the experimental groups and to which an animal belongs in line with STAIR criteria [36, 37].
Statistical Analysis
An unpaired student t-test was used to test differences in histological measures among the treatment groups. Repeated measures two-way ANOVAs were performed for functional tests. Spearman or Pearson correlation coefficients were calculated among the immunostaining evaluation measurements and their correlation with functional outcome and cerebral edema. Statistical significance was set at p-value < 0.05.