Animal's care and experimental protocols were approved by the Animal Care and Use Committee of Anhui Medical University (Hefei, China) and conformed to the Guide for the Care and Use of Laboratory Animals as outlined by the National Institutes of Health. New Zealand white rabbits used in the present study were purchased from Animal Central of TaiHu Hospital (cat. no. SYXK2012-0033; Wuxi, China). Rabbits were raised in a comfortable room with normal atmospheric moisture and fed with a standard diet every 3–4 h at the Animal Center of Taihu Hospital for 10 days prior to the start of the experiments. The temperature of the feeding and operation rooms was maintained at 22°C. Atorvastatin (20 mg/kg/d; Pfizer, Inc., Wuxi, China) was administered by gastric gavage once daily for 3 days prior to SAH operation and 22 h post-SAH to maintain drug levels (14). Neurological deficits were assessed 24 h following SAH operation and rabbits were euthanized immediately following evaluation. The data of blood pressure, injected arterial blood gas data, body weight and blood pressure were used for monitoring only, and were not shown in the results of the present study.

A total of 48 adult male New Zealand white rabbits (weight, 2.5–3.2 kg) were randomly assigned to 3 groups (n=16/group): i) Sham group, which received an injection of saline into the cisterna magna (2 ml); ii) SAH group; and iii) SAH + atorvastatin group. As 3 rabbits died in the SAH group and 4 rabbits died in the SAH + atorvastatin group, 3 additional rabbits were added for the SAH group and 4 additional rabbits were added for the SAH + atorvastatin group. Atorvastatin (20 mg/kg^/d) was administered when the SAH model was established and continued every 24 for 72 h, as previously described (10). A total of 8 rabbits from each group were sacrificed on day 3 with the fixation-perfusion method in 10% formaldehyde. The hippocampus and basilar artery (BA) were removed for terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL), hematoxylin and eosin (H&E) and for immunohistochemical staining analyses. The remaining eight rabbits were exsanguinated and decollated, and the artery was removed and frozen in deep cryogenic freezer for further biochemical studies. Prior to sacrifice, serum samples were collected from each rabbit to evaluate the protein expression levels of endothelin-1 (ET-1) by ELISA.

SAH was induced according to a previously described two-hemorrhage rabbit model (10,14). Briefly, rabbits were anesthetized with an auricular marginal vein injection of 10% chloral hydrate (2.5 ml/kg). Vital signs were kept stable and a 23-gauge butterfly needle was inserted into the cisterna magna. When cerebrospinal fluid was observed from the butterfly needle, then 2 ml non-heparinized fresh autologous auricular arteries blood was injected into the cisterna magna from the butterfly needle with strict aseptic technique and the injection lasted 1 min. To maintain the blood flow from cisterna magna to the basilar cistern, all rabbits were held at a 30-degree head-down position for 30 min. Rabbits were returned to the feeding room following recovery from anesthesia. The second injection was administered after 48 h in a similar manner.

Neurological scores were recorded by the same independent observer who was blinded to the study. A previously modified scoring table was used to evaluate the neurological function every day (14,15). The Neurological study consisted of the following three tests: i) Appetite. Scores indicate the following: 2, Scarcely ate; 1, left meal unfinished; 0, finished meal. ii) Activity. Scores indicate the following: 2, Almost always lying down; 1, lying down, will stand and walk with some stimulation; 0, active, alert or standing. iii) Deficits. Scores indicate the following: 2, Impossible to walk and stand due to ataxia and paresis; 1, unable walk due to ataxia or paresis; 0, no deficits.

A total of 8 rabbits from each group were anesthetized with an auricular marginal vein injection of 10% chloral hydrate (4 ml/kg). The chest was quickly cut open for cannula intubation in the left ventricle and the right atrium was cut open. Perfusion started with 1,500 ml of physiological phosphate buffered saline (PBS; pH 7.3) at 37°C, followed by 1,000 ml buffered formaldehyde (10%) under 120 cm H₂O perfusion pressure. Following fixation-perfusion, whole brain tissues were removed and stored in 10% formalin at 22°C.

This tissue was then post-fixed in 4% paraformaldehyde at 4°C overnight, dehydrated and embedded in paraffin, and cut into sections (4 µm). To avoid the arterial branches, the BAs were transected at the same middle position each time (2 mm). Every fourth section along the coronal plane was stained with H&E and observed under a microscope. The diameter, perimeter and cross-sectional area of the BA were measured by an independent investigator, blinded to the study, using an Olympus light microscope (Olympus Corporation, Tokyo, Japan) and Image-Pro Plus 6.0 Software (Media Cybernetics, Inc., Rockville, MD, USA). The diameter, perimeter and cross-sectional area of BA were calculated as previously described (16). T-score indicates the BA perimeter/BA thickness.

Brain tissues (brainstem) were isolated from brain tissues before storing in formalin, and were routinely fixed, embedded and cut into sections (4 µm) for immunohistochemistry. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min at room temperature. Sections were incubated with anti-von Willebrand factor (vWF; cat no. ab778; dilution 1:30; Abcam, Cambridge, UK) and anti-thrombomodulin (TM; cat no. ab6980; 1:1,000; Abcam) primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies (goat serum; cat no. ab138478; Abcam) for 1 h at room temperature. Following washing, sections were developed in 3,3′-diaminobenzidine (DAB) solution and counterstained with hematoxylin for 5 min at room temperature. Negative controls were included by omitting the primary antibody. A light Olympus microscope (Olympus Corporation) and Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) were used for analysis.

The TUNEL Staining kit (Roche Diagnostics, Basel, Switzerland) was used for hippocampus staining, and the TUNEL-positive cells were analyzed by fluorescein-dUTP with dNTP or POD with DAB (manufacturer's protocol for in situ apoptosis detection kit (Roche Diagnostics) according to the methods described previously (17). A negative control was used by eliminating the TUNEL reaction mixture. Cells exhibiting nuclear condensation/fragmentation and apoptotic bodies in the absence of cytoplasmic TUNEL reactivity, brown staining of nuclei were considered as apoptotic cells. Apoptotic cells were confirmed with the help of a pathologist blinded to the grouping. The number of TUNEL-positive cells in each region (number/mm²) were counted in a high-powered field (magnification, ×400) by an investigator who was blinded to the study. A total of 8 rabbits from each group were used. A total of 5 fields were analyzed, and the experiment was repeated three times.

Western blot analysis was performed as described previously for evaluating the levels of Caspase-3 proteins (18). The samples (20 µg protein), as determined by using a bicinchoninic acid assay (Abcam), were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with the following primary antibodies: Rabbit anti-Caspase-3 (cat no. ab4051; 1:500; Abcam) antibody. GAPDH (cat no. G5262; 1:6,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used as a loading control. Following incubation with the primary antibodies for 1 h at room temperature, membranes were washed with TBS + 5% Tween-20 (TBST) and incubated with appropriate horseradish peroxidase-labeled secondary antibodies (cat no. sc2357; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature in 1% nonfat milk in TBST for 1 h at room temperature. Following two rinses and four washes with PBST, membranes were incubated with Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Healthcare Life Sciences, Shanghai, China) for 60 sec and exposed to autoradiography film for visualization of the bands. Results were quantified by Quantity One version 4.5 software (Bio-Rad Laboratories, Hercules, CA, USA). A total of 8 rabbits from each group were used.

At day 3 following surgical intervention, blood samples were collected from anesthetized animals (n=8/group) and analyzed for ET-1 expression levels using a rabbit ET-1 ELISA kit (cat no. F2003; Westang Bio-Tech Co., Ltd., Shanghai, China) specific for rabbits. Plasma was separated from the blood by centrifugation at 3,000 × g for 15 min, and the supernatant was assayed for the protein concentrations of ET-1, according to the manufacturer's protocol. ET-1 concentrations (pg/ml) were determined based on a standard curve, prepared using a known set of serial dilutions of standard proteins. The experiment was repeated three times.

Total RNA was extracted from hippocampus brain samples (n=8/group) using TRIzol Reagent (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), following the manufacture's protocol. β-actin was used as an internal control. First-strand cDNA was synthesized from the total RNA as previously described by using a kit from Abcam (cat no. 185916) (10,19). vWF and TM mRNA levels in each sample were determined by qPCR using SYBR Green Master Mix (Toyobo Co., Ltd., Osaka, Japan). The qPCR thermocycling conditions were as follows: 45°C (2 min) and 95°C (10 min), followed by 40 cycles of denaturation 95°C (15 sec); annealing 60°C (1 min); extension 72°C (1 min). All samples were analyzed in triplicate. The primers were as follows: TM, forward CTC TAG CAC CTA CAA TAC CCC ATT, reverse CCC GAG TCC AGT GTC CCT CT (146); vWF, forward TTT TCT TAT GTT CTC CAC GAA GGG, reverse CAT TGA TGA GGC AGG GGT TCT (151); β-actin, forward CCC ATC TAT GAG GGT TAC GC, reverse TTT AAT GTC ACG CAC GAT TTC (150) (20).

All data were presented as the mean ± standard deviation. SPSS 14.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Differences between the two groups were analyzed using a two-tailed unpaired Student's t-test. The differences among multiple groups were assessed using a one-way analysis of variance followed by Tukey's post hoc test. Ranked data between the two groups were evaluated using rank sum test. P<0.05 was considered to indicate a statistically significant difference.

No significant differences were identified in blood pressure, injected arterial blood gas data, body weight and blood pressure (data not shown). The mortality of SAH group was 18.75% (3 of 16), 25% (4 of 16) in SAH + atorvastatin group and 0% in the Sham group. No significant difference was identified for the mortality of SAH + atorvastatin treated group compared with the SAH group (P>0.05). During the process of model establishment, the dead and not eligible rabbits were excluded from the present study and were replaced with new rabbits to maintain the number of animals in each group.

The neurological scores of rabbits in SAH + atorvastatin group were significantly lower than the scores in the SAH group at 72 h following SAH induction (P<0.05; Fig. 1; Table I). In addition, treatment with atorvastatin was demonstrated to improve neurological functional post-SAH in experimental rabbits.

Notable differences were observed in BA morphology among the three groups on day 3 post-SAH (Fig. 2A): The inner perimeter, diameter and cross-sectional area of BA in the SAH group was smaller, and the BA wall was thicker compared with the Sham group. To avoid the individual differences, the relatively fixed values of T (T=inner perimeter/wall thickness) were calculated (Fig. 2B). Compared with the SAH group, the T value of the BA in the SAH + atorvastatin treatment group exhibited a significant increase (115.4±11.0 vs. 89.6±9.11; P<0.01).

Immunohistochemical analysis revealed only a few vWF- and TM-positive regions located in the vessels in the Sham group (Fig. 3A). However, vWF and TM expression levels were upregulated in the SAH and SAH + atorvastatin treatment groups. The SAH group and the SAH + atorvastatin treatment groups exhibited a significant increase in the number of vWF- and TM-positive immunostained regions compared with the Sham group (P<0.01; Fig 3B). Immunohistochemical analysis also indicated a significant decrease in the expression vWF and TM in the SAH + atorvastatin treatment group compared with the SAH group (P<0.01).

Few TUNEL-positive cells were detected in the hippocampus of rabbits in the Sham group. However, TUNEL-positive cells were significantly increased in SAH-induced rabbits at 72 h, and TUNEL-positive cells were significantly decreased in the SAH + atorvastatin treatment group compared with untreated SAH rabbits (P<0.05; Fig. 4).

ELISA was performed to examine the alterations in protein concentration of the vasoconstrictor ET-1 to determine the effects of atorvastatin on cerebral edema post-SAH. Compared with the Sham group, ET-1 expression was significantly upregulated in the SAH rabbits (85.24+6.25 vs. 39.72+4.67 pg/ml; P<0.01; Fig. 5). Following atorvastatin treatment, the elevation of plasma ET-1 concentration was significantly lower compared with the SAH group (62.92+7.27 vs. 85.24+6.25 pg/ml; P<0.01; Fig. 5).

vWF and TM mRNA expression levels of were detected by RT-qPCR: vWF and TM mRNAs were expressed at low levels in the brain tissue in the Sham group, whereas the levels of vWF and TM mRNA expression were significantly increased in the SAH and SAH + atorvastatin treatment groups. Compared with the SAH group, the vWF and TM mRNA expression levels were reduced in the SAH + atorvastatin treatment group. (P<0.01; Fig. 6).

Caspase-3 protein expression in the hippocampus was detected by western blot to observe neuronal apoptosis at 72 h following SAH. SAH induced an evident increase of Caspase-3 expression levels in the hippocampus compared with the Sham group, whereas the level of Caspase-3 was markedly decreased in the SAH + atorvastatin group (P<0.05 vs. SAH; Fig. 7).