Allicin (purity, >98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A tumor necrosis factor (TNF)-α ELISA kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). A myeloperoxidase (MPO) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2,3,5-triphenyltet-razolium chloride (TTC) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

A total of 60 adult male Sprague-Dawley rats (250–300 g; 6–8 weeks-old) were purchased from the Experimental Animal Center of Harbin Medical University (Harbin, China). All animals were maintained under a 12-h light/dark regime with ad libitum access to food and water. The rats were housed at 22°C with 50% relative humidity. The animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States' National Institutes of Health (publication no. 85–23, revised 1996; Bethesda, MD, USA). The protocol was approved by the Committee of Experimental Animals of Harbin Medical University (Harbin, China).

Rats were randomly assigned to three groups: Sham-surgery group (n=20), MCAO group (n=20) and MCAO + allicin group (n=20). The rats were anesthetized intraperitoneally (i.p.) with chloral hydrate (350 mg/kg; Beyotime Institute of Biotechnology). Subsequently, the rat was fixed in a supine position and a midline skin incision was made to expose the right common carotid artery, the internal carotid artery (ICA) and the external carotid artery (ECA). The MCAO was performed as described previously (13). Briefly, a 4-0 nylon monofilament (Sunbio Biotech, Beijing, China) with a head-rounded tip was inserted into the ECA and gently advanced into the ICA for ~20 mm to obstruct the blood flow of the right middle cerebral artery. After a 90-minute occlusion, the filament was withdrawn, allowing for the reperfusion for 24 h. The rectal temperature of the rat was maintained at 37°C±0.5°C using a heating pad. Rats in the sham group were subjected to the same procedure, with the exception of the MCAO. Allicin was administered at a dose of 50 mg/kg i.p. 3 h after reperfusion daily for five consecutive days. Allicin was dissolved in 2% dimethyl sulfoxide (DMSO; Sigma-Aldrich). Rats in the sham and MCAO groups were injected with an equal volume of DMSO.

The neurological score was assessed by an observer blinded to the animal groups after 24 h reperfusion according to previously described methods (14). The neurological deficits were evaluated on a five-point scale as follows: No neurological deficits = 0; failure to fully extend left paw = 1; circling to the left = 2; falling to the left = 3; no spontaneous walking and depressed levels of consciousness = 4.

At 24 h after MCAO, the brain water content was detected according to a previously described method (15). The rats were sacrificed by deep anesthesia with chloral hydrate (350 mg/kg, i.p.) and brains were immediately removed and placed on a frozen plate. Tissue samples were collected from infarct areas of ischemic rats and from the corresponding areas in the sham-surgery rats. The samples were weighed to determine the wet weight. Subsequently, samples were dried in a desiccating oven at 110°C for 24 h and then weighed to determine the dry weight. The brain water content was calculated using the following formula: brain water content (%) = (wet weight − dry weight) ×100%/wet weight.

The animals were sacrificed 24 h after reperfusion (n=6 in each group) by deep anesthesia with chloral hydrate (350 mg/kg, i.p.). The brains were rapidly removed and cut into five coronal sections (2 mm). The sections were incubated in a solution of 2% TTC at 37°C for 30 min. The infarct sizes were measured using ImageJ software version 1.6 (National Institutes of Health).

At 24 h after reperfusion, the rats were anesthetized and transcardially perfused with saline, followed by 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in phosphate-buffered saline. Subsequently, the samples were dehydrated in a graded series of ethanol and embedded in paraffin (Leica Biosystems, Wetzlar, Germany). Following epitope retrieval at 120°C for 5–10 min using citrate buffer (10 mM citric acid, 0.05% Tween 20; pH 6.0), the sections were incubated with 3% H₂O₂ for 15 min and then with 5% bovine serum albumin (Beyotime Institute of Biotechnology) for 1 h. The sections were then incubated with primary mouse monoclonal neuronal nuclei (NeuN) antibody (1:100; cat. no. MAB377; EMD Millipore, Billerica, MD, USA) overnight at 4°C, followed by incubation with goat anti-mouse secondary antibody (1:1,000; P044701; Dako, Carpinteria, CA, USA) for 30 min at 37°C. The immunoreactivity was visualized using a Dako Envision system-horseradish peroxidase kit (Dako). Finally, counterstaining was performed using hematoxylin (Beyotime Institute of Biotechnology). NeuN-positive staining in the cortex was identified by the presence of deep brown staining of cells specifically localized to the nucleus (TI-S; Nikon Corporation, Tokyo, Japan). The images were analyzed using the Image-Pro plus 6.0 image analysis system (Media Cybernetics, Inc., Rockville, MD, USA).

Total proteins of the cortex were extracted and quantified according to the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The protein samples were subjected to 8% SDS-PAGE (Beijing Solarbio Science & Technology Co., Ltd.) followed by electrotransfer onto polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). Subsequently, the membranes were blocked with 5% non-fat milk for 2 h at room temperature. The membranes were then incubated overnight at 4°C with the rabbit anti-cleaved caspase-3 polyclonal antibody (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc., Danvers, MA, USA) and with rabbit anti-β-actin polyclonal antibody (1:3,000; sc-7210; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), followed by three washes with Tris-buffered saline with Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, the membranes were incubated with the secondary antibody (1:5,000; Sc-2004; goat anti-rabbit immunoglobulin G-horseradish peroxidase; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature, followed by three washes with TBST. Finally, the bands were detected via chemiluminescence with the ECL Plus western blotting detection kit (EMD Millipore). Blots were analyzed using the Bio-Rad Imaging software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and Quantity One software package version 4.6.2 (Bio-Rad Laboratories).

At 24 h after the MCAO, blood samples (1 ml) were harvested from the femoral vein. Following centrifugation at 1,000 × g for 15 min, the supernatant was collected and stored at −80°C. Serum TNF-α was assayed using a TNF-α ELISA kit according to the manufacturer's instructions.

Following reperfusion, brain tissue from the penumbral cortex was immediately harvested and stored at −80°C. The brain tissue was homogenized in ice-cold phosphate-buffered saline. Subsequently, the homogenate was centrifuged at 1,000 × g for 15 min. MPO was detected using an MPO kit according to manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute).

Values are expressed as the mean ± standard deviation. Statistical analyses were performed using the SPSS 16.0 software package (SPSS, Inc., Chicago, IL, USA). Data analysis was performed by one-way analysis of variance, followed by a least significant difference-t-test for inter-group comparisons. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, the rats in the sham group did not exhibit any neurological deficits, while the MCAO rats exhibited severe neurological deficits according the neurological scoring system. Allicin treatment markedly ameliorated the neurological deficits compared with those in the MCAO group (P<0.05).

At 24 h after MCAO, the brain water content of the MCAO rats was markedly higher than that of the sham-surgery rats. Treatment with allicin significantly decreased brain edema compared with those in the MCAO group (P<0.05; Fig. 2).

The rats in the sham group did not exhibit any infarct area in the brain, while in the MCAO group, a clear infarction was observed. Following treatment with allicin, the infarct volume in the MCAO + allicin group was significantly lower compared with that in the MCAO group (P<0.05; Fig. 3).

Compared with the sham group, the number of NeuN-positive neurons was significantly decreased in the MCAO group. The decrease of NeuN-positive neurons was attenuated by allicin treatment (P<0.05; Fig. 4).

To identify whether the neuroprotective effect of allicin was associated with its anti-apoptotic activity, the levels of cleaved caspase-3 were assessed. Compared with those in the sham group, the levels of cleaved caspase-3 were significantly increased in the MCAO group. Of note, allicin markedly decreased the levels of cleaved caspase-3 (P<0.05; Fig. 5).

A cerebral ichemia/reperfusion (I/R) injury causes the production of TNF-α (16). Thus, the levels of serum TNF-α were assessed. Fig. 6 shows that, compared with the MCAO group, allicin significantly decreased the serum levels of TNF-α (P<0.05).

As indicated in Fig. 7, the MPO activity in the sham group was at a normal level, while that in the MCAO group was markedly elevated (P<0.05). Of note, treatment with allicin significantly lowered cerebral MPO activity (P<0.05).