A total of 50 Wistar rats (male; body weight, 270–330 g, age: 10 weeks-12 weeks) were obtained from the Animal Center of Shandong University (Jinan, China). All rats were maintained at 25±1°C, with 12 h light/12 h dark cycle of housing, food and water available. All animal experiment protocols were approved by the Institutional Animal Care Committee of Shandong University (Jinan, China), and were performed in strict consistence with its guidelines.

After 1 week of accommodation, the rats were subjected to middle cerebral artery occlusion (MCAO) surgery as previously described (10,11). Briefly, following anesthetization with 10% chloral hydrate (350 mg/kg; administered intraperitoneally) the left common carotid artery (CCA) was revealed and clipped using an artery clamp. During surgery, body temperature was maintained at 36.5–37.0°C using a heating pad on the surgical table. The incision region was disinfected with povidone-iodine solution. The external carotid artery (ECA) was separated and ligatured. A nylon suture with a blunted tip (0.35 mm diameter) was drawn into the ECA and then into internal carotid artery (ICA). The middle cerebral artery was occluded by the suture 18 mm distal from the carotid bifurcation. Ischemia reperfusion injury was executed by removing the suture after 2 h of occlusion. Following closure of the incision, the rats were returned to cages with food and water available after the incision was closed.

Rb1 was dissolved in saline and intravenously injected following initiation of ischemia. The animals were randomized distributed into 5 groups according to the random number table. Firstly, the rats were numbered by body weight. Second, we chose the any row in the random number table and copy 50 random numbers. Sort random numbers from small to large. Specify the first 10 numbers as the first group from the sorted numbers, followed by analogy: i) sham control, the ECA was surgically prepared for insertion of the filament as described above, but the filament was not inserted and saline was received intravenously; ii) MCAO group, subjected to MCAO and saline was received intravenously and iii) Rb1 group, subjected to MCAO and 50, 100 or 200 mg/kg of Rb1 was received intravenously. We conducted a pre-experiment to investigate the effect of Rb1 100 mg/kg on the focal cerebral ischemic reperfusion rats. The results manifested that Rb1 100 mg/kg remarkably decreased the ischemic injury. Therefore, we chose Rb1 50, 100 and 200 mg/kg as the dose of Rb1.

Neurological examination was performed blindly 24 h after reperfusion, according to Zea Longa's method (10). The scores of the neurological tests were categorized according to 5 grades: 0, no neurological deficit; 1, unable to extend right forepaw fully upon lifting of the whole body by the tail; 2, circling to the right; 3, falling to the right, and 4, unable to walk spontaneously and reduced levels of consciousness.

Coronal brain sections (2-mm thickness) were incubated with 2% TTC at 37°C for 30 min with gentle agitation, then fixed with 10% formalin in PBS. Pale unstained sections were considered to be indicative of infarct regions, whereas red-stained sections were indicative of normal tissue. The slices were photographed from each side, and the infarct regions and were detected both hemispheres using a morphological image-analysis system (Jie Da software, China). Infarct volume was calculated as a percentage of the contralateral hemisphere volume using an ‘indirect method’ (area of intact contralateral hemisphere-area of intact regions of the ipsilateral hemisphere) to compensate for edema formation in the ipsilateral hemisphere. The volume of infarction was obtained according to the following formula, and expressed as percentage of infarction in the ipsilateral hemisphere (11):

V, volume of fraction; Ai, infarct area of each slice, and h, slice thickness.

After being anesthetized with 10% chloral hydrate (350 mg/kg; injected intraperitoneally) the rats were sacrificed by cardiac perfusion, the brains were immediately removed and the bregma-3~3.8 mm areas were immobilized in 4% neutral buffered formalin and embedded in paraffin. No peritonitis was observed in the rats during the entire experimental protocol. The areas were mounted onto slides, deparaffinized with xylene, rehydrated using a graded alcohol series, stained with hematoxylin and eosin and analyzed under a light microscope at magnification, ×100. The brains were sliced into 10-µm thick coronal sections at the level of the bregma. TUNEL staining was performed using an in situ apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. TUNEL staining was detected under a fluorescence microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan). A total of 3 sections from each animal were analyzed by 2 investigators, blinded to the origin of the sections. For each section, TUNEL-positive cells were counted in 5 non-overlapping high-power fields at magnification, ×200.

Protein samples were prepared as previously described (12) and the protein concentration was determined using the Bradford method. The protein samples were heated at 95°C for 5 min, loaded at 30 µg per lane, separated using 10% SDS-PAGE, and electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies for cleaved caspase-3 (cat. no., 9664; Cell Signaling Technology, Inc., Danvers, MA, USA), cleaved caspase-9 (cat. no., 9507; Cell Signaling Technology, Inc.), with β-actin functioning as a loading control (cat. no., ab6276; Abcam, Cambridge, UK), overnight at 4°C. Following washing with Tris-buffered saline with Tween (TBS-T), the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, then washed again with TBS-T. The antibodies were then visualized by enhanced chemiluminescence and the density of the protein bands was analyzed using an AlphaEaseFC system (ProteinSimple, San Jose, CA, USA).

Cortex samples were homogenized in 1 ml homogenization buffer and centrifuged at 14,000 × g for 10 min at 4°C. ELISA kits were used to verify the levels of high-mobility group box 1 (HMGB1) and NF-κB p65, TNF-α, iNOS, NO and IL-6, according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

All data are expressed as the mean ± standard deviation and analyzed using one-way analysis of variance followed by the Least Significant Difference test. All the statistics analyses were performed using SPSS software (v.18; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Neurological scores were determined 24 h after I/R injury. No neurological deficitobserved in sham animals, whereas MCAO animals suffered from I/R injury, displayed all the characteristics of neuron damage and had relatively high neurological deficit scores (2.07±0.24; Table I). The results also show that Rb1 treatment significantly improved the neurological deficits of MCAO mice, and the deficit score in animals treated with 50, 100 and 200 mg/kg Rb1 were decreased to 1.71±0.43, 1.25±0.72 and 1.05±0.36, respectively.

Infarct area of brain tissues from the animals measured 24 h after I/R injury by TTC staining are presented in Fig. 1. No infarct was observed in sham animals, whereas in the MCAO group, the infarct area reached 31.56% the whole brain. However, as shown in Fig. 1B, Rb1-treatment decreased infarct volumes in MCAO rats in a dose-dependent manner: 50, 100 and 200 mg/kg Rb1 treatment reduced the infarct volume to 25.89% (P<0.05 vs. MCAO animals), 18.35% (P<0.01 vs. MCAO animals) and 10.13% (P<0.01 vs. MCAO animals), respectively.

Hematoxylin and eosin staining was applied to examine the histopathological abnormalities following focal cerebral I/R (Fig. 2). No histopathological damage was detected in the corext or pyramidal neurons in the hippocampus CA1 region of sham animals (Fig. 2A and B). In the MCAO rat model, the majority of the neurons in the infarct core were atrophied and/or reduced in size, exhibiting a eosinophilic cytoplasm and triangulated pycnotic nucleus compared with the intact and well-arranged neurons with eumorphism in the sham group. However, the number of pyramidal neurons in the MCAO model was significantly decreased compared with the sham group, and large necrotic neurons surrounding the infarct core and in the peri-infarct zone were noted, exhibiting pycnotic shape and condensed nuclear material. The necrotic tissue was notablyremarkably diminished following Rb1 treatment. This suggests a reduction in nerve injury, characterized by the decreased number of cells with obvious historical change, such as liquefaction necrosis, pycnosis, nucleoli abolition and nuclear fragmentation.

The level of HMGB1 in the peri-infarct zones of ischemic cortex samples from each group was measured. A significantly increased level of HMGB1 was identified in brain tissue subjected to focal cerebral ischemia reperfusion. Furthermore, an increased level of HMGB1 was observed in MCAO rats compared with the sham group (P<0.01; Fig. 3A), which was notably decreased by Rb1-treatment.

High levels of TNF-α, iNOS, NO and IL-6 were also identified in MCAO rats (P<0.05, sham vs. MACO; Fig. 3), which was significantly attenuated in Rb1-treated animals.

Neuronal cell apoptosis was measured by TUNEL staining in the rats of each group (Fig. 4). The number of TUNEL-positive neurons in the cortex region was significantly increased in MCAO group after I/R injury compared with the sham group (P<0.01). Following Rb1 treatment, the number of TUNEL-positive neurons in the cortex region was significantly reduced compared with the sham group (P<0.05; Fig. 4). A higher proportion of apoptotic neurons was detected in MCAO group, compared with the sham group (P<0.01). Rb1 treatment at 50, 100 or 200 mg/kg reduced the TUNEL-positive staining by 30.2, 19.6 and 9.2%, respectively (P<0.01 vs. MCAO).

The expression levels of cleaved caspase-3 and caspase-9 was also investigated. High expression of apoptosis-related proteins was exhibited by brain tissues from the MCAO group compared with the sham group (P<0.05; Fig. 4D). The elevated levels of cleaved caspase-3 and caspase-9 were significantly attenuated with Rb1 treatment (Fig. 4C and D).