Adult male Sprague-Dawley rats (300 ± 30 g) were cared for according to the Guide for the Care and Use of Laboratory Animals. The committee for experimental animals of Second Military Medical University approved all surgical procedures. Rats were anesthetized with chloral hydrate (350 mg/kg intraperitoneally; IP) and subjected to MCAO as described previously [6]. In brief, we exposed the right common carotid artery, internal carotid artery, and external carotid artery surgically. A 4–0 monofilament nylon suture (Beijing Sunbio Biotech Co. Ltd.) with a rounded tip was inserted into the internal carotid artery through the external carotid artery stump and gently advanced to occlude the MCA. After 60 min of MCAO, the suture was removed to restore blood flow (reperfusion confirmed by laser Doppler). Sham-operated rats were manipulated in the same way; the monofilament was inserted to a depth of 1.0–2.0 mm with the MCA not occluded. Core body temperatures were monitored with a rectal probe and maintained at 37 °C during the whole procedure. All surgical procedures were performed under an operating stereomicroscope.

In one set, 120 rats were randomly divided into sham group, reperfusion 6 h group, reperfusion 24 h group, reperfusion 48 h group, and reperfusion 72 h group (24 rats per group). In another set, 72 rats were randomly divided into sham group, reperfusion 24 h group, and ROCK inhibitor (fasudil) treated group (24 rats per group) and were IP administered with saline or fasudil (15 mg/kg) once before operation and once 12 h after operation.

The ischemic core and penumbra of the cortex were dissected following the protocols described previously [7]. Briefly, each hemisphere was cut longitudinally, from dorsal to ventral at 1.5 mm from the midline to exclude medial brain structures that were supplied primarily by the anterior cerebral artery. A transverse diagonal incision at approximately the “2 o’clock” position separated the core from the penumbra.

Evans Blue (EB, 2% in saline, 4 mL/kg) was injected intravenously 24 h after the onset of MCAO. The chest was subsequently opened under halothane anesthesia 1 h later. Rats were perfused with saline through the left ventricle at 110 mm Hg pressure until colorless perfusion fluid was obtained from the right atrium. After decapitation, the brain was blocked into 2 segments that included the levels bregma +2.7 and −0.3 mm. Coronal blocks were next divided into right and left hemispheres for local measurement of EB dye. Samples were weighed and placed in 50% trichloroacetic acid solution. Following homogenization and centrifugation, the extracted dye was diluted with ethanol (1:3), and its fluorescence was determined at 620 nm. Calculations were based on external standards in the same solvent (100–500 ng/mL). The tissue content of EB was quantified from a linear standard curve derived from known amounts of the dye and was expressed per gram of tissue.

For tissue harvesting, the rats were anesthetized with a lethal dose of sodium pentobarbital (80 mg/kg body weight). After decapitation, the brain was washed in 20% phosphate-buffered sucrose, then embedded and mounted in Tissue-Tek OCT compound. Serial longitudinal frozen sections (4 μm) were cut, fixed, and washed with phosphate-buffered saline (PBS) for 5 min, 3 times. After incubation with 5% normal goat serum for 1 h at RT, the sections were incubated with Laminin primary antibody (rabbit anti rat, Abcam) in a humid chamber at 32 °C for 1 h. The immunoreaction was visualized by treatment with CY3-conjugated goat anti-rabbit secondary antibody (Jackson) at 32 °C for 30 min in the dark. Immunostained sections were mounted and analyzed using a Leica epifluorescence microscope (Leica Microsystems Wetzlar GmbH, Germany). Three fields were randomly selected and semi-quantitation of the positive staining was performed using Leica-Qwin software with the florescence value calculated as Ga (positive gray value)–GA (background gray value).

Total RNA was isolated from right cortical samples by Trizol reagent (Invitrogen), and RT was performed with TOYOBO RT kit (TOYOBO) according to the manufacturer’s instructions. PCR was performed with primers for ROCK (forward: 5′-CTGCTGAAGTCGTGCTTGCA-3′;

Reverse: 5′-AGCATGTTATCGGGCTTCACA-3′, amplicon: 79 bp);

MMP9 (forward: 5′-ACGAGGACTCCCCTCTGCAT-3′;

Reverse: 5′-AGGCCTTGGGTCAGGTTTAGA-3′, amplicon: 83 bp);

GAPDH was used as an internal standard (forward: 5′-CAGTGCCAGCCTCGTCTCAT-3′;

Reverse: 5′-TGGTAACCAGGCGTCCGATA-3′, amplicon: 79 bp).

The PCR products were quantified by ABI Prism^® 7900HT Sequence Detection System.

All data are expressed as χ̄ ± s. All data were analyzed with SPSS11.0 statistical software. One-way ANOVA, Bonferroni, Kruskal-Wallis H test and Pearson correlated analysis were performed. P < 0.05 was considered as statistically significant.

By real-time quantitative PCR, we observed the changes of mRNA level of ROCK at different time points after MCAO (Table 1). The expression level of ROCK increased 6 h after reperfusion and the increase was significant compared with a sham group (P < 0.01). The expression of ROCK kept increasing 6–24 h after reperfusion, and then decreased gradually 48 h after perfusion. The different time points group all showed significant difference in ROCK expression compared with the sham group (P < 0.01), while there was no significant difference between 24 h after reperfusion group and 6 h after reperfusion group (P > 0.05).

By EB method, we measured EB contents at different time points after MCAO (Table 1). Clearly, EB content increased 6 h after perfusion, with significant difference from sham group (P < 0.01). EB content gradually increased 6–48 h after perfusion, suggesting that BBB permeability increased gradually and microvascular structure was gradually damaged. However, EB content began to decrease 48 h after perfusion. The difference in EB content was significant when each individual group was compared with sham group (P < 0.01) or compared with each other (P < 0.01).

By immunofluorescence staining, we detected the expression of Laminin in the microvascular basal membrane in rat ischemic penumbra. As shown in Figure 1, we observed different staining patterns for Laminin at different time points after MCAO. While strong positive staining for Laminin was detected in the sham group, slightly weaker staining was observed 6 h after perfusion. Moreover, 24 h after perfusion, the staining for Laminin was significantly weaker with both the positive areas and the staining intensity decreased. We could also observe basal membrane damage such as breakdown. The weakest staining for Laminin was observed at 48 h after perfusion, when both the positive areas and staining intensity were lowest. The staining was blurry and the shape was irregular, demonstrating that substantial basal membrane damage occurred. In comparison, the staining for Laminin was recovered and microvascular regeneration could be observed 72 h after perfusion.

To obtain a quantitative comparison of Laminin expression, we performed semi-quantitative analysis with Leica-Qwin software. The results demonstrated that Laminin expression decreased gradually 6–48 h after perfusion, was lowest at 48 h after perfusion, and then gradually increased (Table 1). At the different time points, all groups showed significant difference in Laminin expression compared with the sham group (P < 0.01). In addition, although there was no significant difference between the 24 h after reperfusion group and 72 h after reperfusion group (P > 0.05), there was a significant difference when any other two groups were compared (P < 0.05).

Since we observed similar changes in ROCK mRNA level, brain EB content and Laminin expression after MCAO, next we attempted to address their association. Based on statistical analysis, the correlation coefficient r between ROCK expression and brain EB content was 0.925, and it was statistically significant (P = 0.025 < 0.05). The correlation coefficient r between ROCK expression and Laminin expression was −0.955, and it was also statistically significant (P = 0.011 < 0.05). Thus, these data suggest that ROCK expression is positively correlated with BBB permeability and negatively correlated with Laminin expression.

Since ROCK plays a role in the pathology of stroke, we decided to use the ROCK inhibitor fasudil to evaluate its effects on microvascular damage after MCAO. First, we examined the impact of fasudil on BBB permeability by measuring brain EB content. As shown in Table 2, compared with the sham group, brain EB content was significantly higher in both the 24 h reperfusion group and 24 h reperfusion plus fasudil group (P < 0.05). However, brain EB content was significantly lower in the 24 h reperfusion plus fasudil group than in the 24 h reperfusion group (P < 0.05), suggesting that the ROCK inhibitor decreases BBB permeability.

Next we examined the effect of fasudil on basal membrane by immunofluorescence staining for Laminin. As shown in Figure 2, compared with the sham group, 24 h after perfusion, the staining for Laminin was much weaker and basal membrane breakdown was obvious. Interestingly, fasudil partially rescued positive staining for Laminin and promoted microvascular regeneration.

Further semi-quantitative analysis demonstrated that Laminin expression was significantly lower in both the 24 h reperfusion group and 24 h reperfusion plus fasudil group compared with the sham group (P < 0.05). Nevertheless, Laminin expression was significantly higher in the 24 h reperfusion plus fasudil group compared with the 24 h reperfusion group (P < 0.05) (Table 2). Collectively, these data suggest that the ROCK inhibitor could antagonize basal lamina damage and the downregulation of Laminin following brain ischemia reperfusion.

Finally, we examined the effect of fasudil on MMP9 expression after MCAO. Real-time quantitative PCR experiments demonstrated that the mRNA level of MMP9 was significantly higher in both the 24 h reperfusion group and the 24 h reperfusion plus fasudil group, compared with the sham group (P < 0.05). However, the mRNA level of MMP9 was significantly lower in the 24 h reperfusion plus fasudil group compared with 24 h reperfusion group (P < 0.05) (Table 2). Thus, these results indicate that ROCK inhibitor inhibits MMP9 expression following brain ischemia reperfusion.