A total of 90 male SD rats (age, 8–9 weeks; weight, 250±10 g) were purchased from Jinan Pengyue Experimental Animal Breeding, Co., Ltd. All animals were given water and food ad libitum, and were maintained on a 12-h light/dark cycle in a temperature-controlled room at 24–26°C with humidity of 50–65%. All rats acclimated for 1 week before experimental procedures. All animal experiments were approved by the Animal Ethics Committee of Jining Medical University, and performed were in accordance with the National Experimental Animal Feeding Guidelines (19). The rats were sacrificed with decapitation after deep anesthesia causing rapid and unconscious death without pain.

SD rats were fasted for 12 h before the experiment. Rats were anesthetized using 10% chloral hydrate (300 mg/kg) via intraperitoneal injection for up to 2 h after reperfusion. After intraperitoneal injection of 10% chloral hydrate, the rats did not show symptoms of peritonitis. After a midline incision was introduced in the neck, the right common carotid artery, right external carotid artery and internal carotid artery (ICA) were exposed and separated. A 2.5 nylon mono-filament (0.265 mm in diameter) was inserted through the common carotid artery into the lumen of the ICA and advanced 18–22 mm from the bifurcation until it blocked the origin of the right middle cerebral artery. In the drug and model groups, the filament remained in the lumen for 2 h, and was subsequently withdrawn to allow reperfusion for 3, 6, 12, 24 and 48 h. The sham-operated group was treated the same as the model group except for the occlusion of the middle cerebral arteries after the neck incision. A total of 30 rats were randomly divided into 6 groups: sham operation group (Sham), MCAO model group and reperfusion for 3 h (CIRI 3h), 6 h (CIRI 6h), 12 h (CIRI 12h), 24 h (CIRI 24h) and 48 h (CIRI 48h). Based on the expression of ERS-related proteins, CIRI 24h was selected for OXA intervention. Therefore, 20 rats were randomly divided into the sham operation group (Sham), the MCAO model group and reperfusion for 24 h (CIRI), CIRI with intracerebroventricular (ICV) injection of normal saline (NS; CIRI + NS) and CIRI with intracerebroventricular (ICV) injection of OXA (CIRI + OXA). Another 40 rats were used for TTC and TUNEL staining. In addition, there was ~1/3 mortality and model failure rate.

At the beginning of reperfusion, the rats were anesthetized via intraperitoneal injection with 10% chloral hydrate (300 mg/kg). A burr hole for ICV administration was carefully made in the skull at 0.8-mm dorsal and 1.6-mm lateral to the right from Bregma using a Dremel drill. A total of 10 µl OXA (30 µg/kg) (20) from Phoenix Pharmaceuticals, Inc. in 0.9% NaCl or 10 µl NS (0.9% NaCl) were injected at 2 µl/min for 5 min.

After reperfusion for 24 h, neurological function of all rats was evaluated according to the Longa five-point scale (21): 0, no neurological deficit; 1, failed to fully extend their left forepaw; 2, circling to the left when walking; 3, falling to the left when walking; and 4, failure to walk spontaneously or stroke-related mortality. Only rats scoring 1, 2 or 3 were selected for use in subsequent experiments. The test was performed in a blinded manner.

2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining. Rats were anesthetized with 10% chloral hydrate (300 mg/kg), and brains were collected and incubated on ice for 30 min. Coronal thin slices (2 mm) of brain were stained with 1% TTC (Sigma-Aldrich; Merck KGaA) and incubated at 37°C in the dark for 15–20 min. Viable tissues were stained deep red, whereas infarcts were pale white. TTC-stained brains were then fixed with 4% formaldehyde for 24 h at room temperature and imaged with a digital camera. The infarct areas were measured using the ImageJ 2 software (National Institutes of Health).

TUNEL staining was performed according to the manufacturer's instructions (Promega Corporation). Briefly, coronal slices (30 µm) were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. After equilibration for 10 min, the slices were incubated with rTdT reaction buffer for 60 min at a constant temperature of 37°C and then incubated with 2X SSC for 15 min to stop the rTdT reaction at a constant temperature of 37°C. Finally, the nuclei were counterstained with DAPI (100 ng/ml) for 30 min at room temperature, and then the anti-fluorescence quencher was added dropwise for mounting. Images of three fields of view were randomly capture under an optical microscope with a magnification of ×200 (Olympus IX 71; Olympus Corporation) and the percentages of TUNEL-positive cells vs. total cells were calculated

Brain tissues of rats were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with PMSF (Beyotime Institute of Biotechnology), and protein concentrations were quantified using the BCA method (Beyotime Institute of Biotechnology). Equal amounts of protein (25 µg) from each sample were separated via 10% SDS-PAGE and transferred to PVDF membranes at 4°C. The membranes were blocked with 5% non-fat milk powder in TBS-Tween-20 (0.1%) for 2 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: Anti-cleaved caspase-3 (cat. no. 9661S), anti-GRP78 (cat. no. 3177S), anti-IRE1α (cat. no. 3294S), anti-PERK (cat. no. 5683S), anti-p-PERK (cat. no. 3179S), anti-CHOP (cat. no. 2895S), anti-eIF2α (cat. no. 5324S), anti-p-eIF2α (cat. no. 9721S), anti-JNK (cat. no. 9252T), anti-p-JNK (cat. no. 9255S), anti-cleaved caspase-12 (cat. no. 35965S) (all 1:1,000; Cell Signaling Technology, Inc.), anti-p-IRE1α (cat. no. ab48187; 1:1000; Abcam) and anti-β-actin (cat. no. TA-09; 1:1,000; OriGene Technologies, Inc.). Next, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (1:5,000; OriGene Technologies, Inc.), and visualized using an ECL system (MultiSciences Biotech Co,. Ltd. The grayscale intensities of protein bands were quantified using ImageJ 2 software (National Institutes of Health).

All data are representative of ≥3 independent experiments and were analyzed using the GraphPad Prism 5 software (GraphPad Software, Inc.). Data are presented as the mean ± SEM. One-way ANOVA followed by Tukey's test were performed. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the neuroprotective effect of OXA in the CIRI model, infarct volume and neurological deficit score were measured, respectively. Healthy cerebral tissues were stained red, whereas infarcted areas were stained white (Fig. 1A). No infarction areas were detected in the sham group, whereas the CIRI group had a significantly higher rate of infarction (40±2%) compared with the sham group (P<0.001), demonstrating that CIRI-induced cerebral infarct had been successfully introduced. However, the OXA group had a significantly lower rate of infarction (24.5±1.5%) compared with the CIRI group (P<0.05), demonstrating that intervention with OXA significantly decreased the infarct area.

As presented in Fig. 1B, the average neurological deficit scores of the CIRI model rats was 3 compared with the sham group, according to the Longa score scale (P<0.001). After OXA intervention, neurological deficit scores were significantly decreased (P<0.01), indicating that OXA has the potential ability to improve neurological deficit scores. Thus, OXA exhibited a notable neuroprotective effect on infarct volume and neurological deficit score in CIRI model rats.

To determine whether CIRI was accompanied by ERS, western blot analysis was conducted to evaluate the expression levels of ERS-related proteins in the cerebral cortex on the ischemic side at different times post-reperfusion. The expression patterns of five proteins exhibited significant differences at various times during the post-reperfusion period (Fig. 2). After IR, the expression level of HSPA5 was increased to a peak at 6 h and then declined (P<0.01 and P<0.001). The phosphorylation level of PERK was elevated at 3 and 6 h, then decreased before increasing again from 24 h (P<0.01 and P<0.001). The expression of p-eIF2α was highest at 6 h post-reperfusion, and then gradually decreased (P<0.05 and P<0.01). p-IRE1α reached its highest levels at 3 h post-reperfusion, and then decreased (P<0.01 and P<0.001). The expression of p-JNK remained at a high level for 48 h post-reperfusion (P<0.05 and P<0.01). These observations demonstrate that the patterns of gene expression are complex and diverse, increasing the challenge of understanding the molecular pathology of CIRI. Taken together, however, the present findings suggested that these proteins were activated in CIRI, indicating that ERS is accompanied by CIRI.

To investigate the effect of OXA on the expression of ERS-related proteins, CIR was simulated by 2 h MCAO followed by 24 h of reperfusion, and OXA was administered via ICV injection during reperfusion. Next, the expression levels of ERS-related protein were detected via western blotting (Fig. 3A). Compared with Sham group, the fold changes of the expression levels of HSPA5, p-PERK, p-eIF2α, p-IRE1α, and p-JNK in CIRI group and CIRI + OA group were uneven. GRP78 expression was significantly higher in the CIRI group (22.6-fold) compared with the Sham model (Fig. 3B), but significantly lower in the OXA model compared with the CIRI group (P<0.01). No statistically significant difference was observed between CIRI and CIRI + NS models. The phosphorylation levels of PERK, eIF2α, IRE1α and JNK were upregulated in the CIRI group compared with the Sham group (P<0.05, P<0.01 and P<0.001), but all were downregulated following intervention with OXA (P<0.05, P<0.01 and P<0.001). In addition, only the phosphorylation levels of eIF2α and JNK were statistically significant between the Sham and OXA models (P<0.05 and P<0.01). In brief, these results indicated that CIRI activated ERS-related proteins in the rat cortex, and that OXA could decrease their activities.

The caspase-12/caspase-9/caspase-3 or CHOP apoptotic pathways are mediated by ERS (22). Moreover, caspase-12 is regarded as a representative molecule in ERS-mediated apoptosis, and caspase-3 serves a key role in regulating apoptosis, which can directly lead to cell death (22,23). Therefore, the expression levels of CHOP, cleaved caspase-12 and cleaved caspase-3 were measured using western blotting (Fig. 4). CIRI upregulated the expression of all three proteins (all P<0.05). Moreover, treatment with OXA significantly decreased the expression of CIRI-induced CHOP, cleaved caspase-12 and cleaved caspase-3 (all P<0.05). However, the expression levels of these proteins did not differ significantly between the CIRI + NS and CIRI groups. These data suggested that OXA exerted an anti-apoptotic effect on CIRI by inhibiting the expression of apoptosis-related genes.

To determine the effect of OXA on apoptosis in the CIRI model, apoptotic cells in brain tissues were detected using TUNEL staining. TUNEL-positive cells were barely visible in the Sham group, whereas substantial levels of TUNEL-positive neurons (47.33%) were detected in the CIRI group (Fig. 5). However, the number of TUNEL-positive cells in the CIRI + OXA group was significantly decreased (28.33%) compared with the CIRI group (P<0.01). These results demonstrated that OXA exerted an anti-apoptotic effect in the cortex following CIR.