Adult male Sprague-Dawley rats, 10–12 weeks old, weighing 250–280 g, were purchased from the Animal Center of Southern Medical University (Guangzhou, China). All protocols were approved by the animal care committee of Southern Medical University and undertaken according to the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD, USA). The rats were kept under constant laboratory conditions (20–25°C, 60±5% humidity) and a 12 h light/dark cycle. They were allowed free access to food and water up until 12 h prior to surgery at which point only water was available.

LA powder (Shanghai Modern Pharmaceutical Co., Ltd., Shanghai, China) was mixed with sterile saline. Subsequently, 1 M NaOH was added until the suspension dissolved. The pH was lowered to 7.4 using hydrochloric acid (14). Rats were administered with LA or saline via subcutaneous injection 2 h prior to MCAO (15).

In preliminary experiments, different dosages of LA were administered to determine the optimal dosage. Rats were randomly assigned to the following groups (n=6): i) Sham group; ii) I/R group, wherein the animals received I/R and a vehicle treatment; iii) LA group, in which the rats received I/R and LA (50 mg/kg); iv) LA group, in which the rats received I/R and LA (70 mg/kg); v) LA group, in which the rats received I/R and LA (100 mg/kg). Following determining 100 mg/kg as the optimal dosage, rats were randomly divided into the following groups (n=9): i) Sham group; ii) I/R group, in which the rats received I/R and a vehicle treatment; iii) LA group, wherein the rats received I/R and LA (100 mg/kg).

Rats fasted overnight, but were allowed free access to water prior to the MCAO procedure. MCAO was induced as described previously (16). Briefly, rats were anesthetized with 10% chloral hydrate (3.5 ml/kg) by intraperitoneal injection. The skin and surrounding fur were disinfected with 75% ethyl alcohol. Following incision to the skin, the left common carotid artery (CCA) was exposed and carefully separated from the vagal nerves. The left external carotid artery (ECA) and the left internal carotid artery (ICA) were carefully isolated and the ECA was ligated. In addition, the CCA was temporally occluded with 3-0 silk thread. The left MCA was occluded for 90 min with a paraffin-coated nylon filament (17), which was introduced into the ICA for 18–20 mm until resistance was detected. After 90 min of occlusion, the ECA was permanently occluded. The silk thread occluding the CCA was removed for reperfusion for 24 h. A heating lamp was used to maintain body temperature at 37°C during surgery.

After 90 min of ischemia followed by 24 h of reperfusion animals were anesthetized with 10% chloral hydrate (3.5 ml/kg) and decapitated. The brains were quickly removed and frozen at −20°C for 20 min, dissected into 2 mm coronal slices and immediately incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC; Mbchem, Shanghai, China) at 37°C for 10 min as described previously (16). Following TTC staining, the normal brain tissue was dark red, whereas infarcted tissue was unstained. Following TTC staining, the tissues were then fixed in 4% paraformaldehyde (Mbchem) for 24 h and scanned with a digital camera (Canon, Inc., Tokyo, Japan). Infarct size was calculated using Image J software (National Institutes of Health, Bethesda, MD, USA) by an individual blinded to the identity of the experimental groups and the result was expressed as a percentage of infarct area to total brain area.

Neurological deficit evaluations were performed at the end of the experiment by an observer masked to the identity of experimental groups using the following criteria as previously described (16): 0, no neurologic deficit or normal function; 1, failure to extend right forepaw fully; 2, circling to right; 3, leaning to right; 4, absence of spontaneous motor activity. Therefore, a higher score was associated with poorer neurological function.

Brain water content was measured as described previously (18). Briefly, after 24 h of reperfusion animals were anesthetized with 10% chloral hydrate (3.5 ml/kg) and decapitated. The brains were rinsed with saline and separated into ischemic and non-ischemic hemispheres, then immediately weighed to gain the wet weight (WW). The brains were placed in an oven at 100°C for 24 h and weighed to obtain the dry weight (DW). The brain water content (%) was measured using the following formula: (WW − DW)/WW × 100%.

After 24 h of reperfusion, animals were anesthetized with 10% chloral hydrate (3.5 ml/kg) and decapitated. The rat brains were quickly removed and detached, then rinsed with cold saline. The brains were then homogenized in cold saline (1:10, wt/vol) and centrifuged at 3,000 × g for 10 min to prepare tissue homogenate. The levels of MDA, NO and the activities of T-AOC and SOD were determined using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Animals were anesthetized with 10% chloral hydrate (3.5 ml/kg) and decapitated 24 h after reperfusion. The rat brains were quickly removed and detached, then rinsed with cold saline. Tissue samples were lysed in ice-cold RIPA buffer (Nanjing Keygen Biotech, Co., Ltd., Nanjing, China) containing 1% phenylmethylsulfonyl fluoride, 1% phosphatase inhibitors and 0.1% protease inhibitor with a glass homogenizer on ice, repeated for 5 min and incubated on ice for 10 min, then centrifuged at 13,000 × g for 20 min at 4°C. The supernatant was aliquoted and stored at −80°C. Protein concentration was measured using the BCA kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The protein (30 μg) was then separated on 8–15% polyacrylamide SDS gels and transferred onto a polyvinylidene difluoride membrane. The membranes were treated with blocking solution (5% non-fat dry milk or 5% bovine serum albumin in Tris-buffered saline with Tween^® 20 and incubated at room temperature for 2 h. Following this, the membranes were incubated with a rabbit anti-rat polyclonal cleaved caspase-3 antibody (1:1,000; Cell Signaling Technology, Inc., Beverly, MA, USA), a rabbit anti-rat polyclonal BDNF antibody (1:1,000; Chemicon, Temecula, CA, USA), a rabbit anti-rat polyclonal PI3K antibody (1:1,000; Cell Signaling Technology, Inc.), a rabbit anti-rat polyclonal p-Akt antibody (1:2,000; Cell Signaling Technology, Inc.) and a rabbit anti-rat polyclonal p-ERK1/2 antibody (1:2,000; Cell Signaling Technology, Inc.) at 4°C overnight, followed by incubation with the corresponding goat anti-rabbit secondary antibodies (Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) at room temperature for 1 h. GADPH was used as a control to ensure equal protein loading. The blots were visualized with ECL-Plus reagent (Pierce Biotechnology, Inc.) and the exposures were transferred to radiographic films. The radiographic films were scanned by a cannon scanner (Canon, Inc.) and analyzed using Image J software (National Institutes of Health).

All data were analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and are expressed as the mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance and P<0.05 was considered to indicate a statistically significant difference.

The experimental procedure is shown in Fig. 1A. In order to investigate the effect of LA on infarct size, the brain infarct area was analyzed by TCC staining after 24 h of reperfusion. There was no infarct area in the sham rat brain (Fig. 1B). Pretreatment with 100 mg/kg LA significantly reduced total infarct size by 51.3% compared with ischemic rats with vehicle treatment from 26.9±1.7% to 13.8±1.4% (P<0.05), while 50 mg/kg and 70 mg/kg did not reveal a significant effect (26.9±2.1% and 24.1±2.3%, respectively; Fig. 1C). Therefore, 100 mg/kg LA was selected as the optimum dosage for further investigation.

As shown in Fig. 2A, 24 h after reperfusion, the neurological score was 3.17±0.41 in the I/R group, while this significantly decreased following treatment with LA (100 mg/kg) to 1.83±0.41 (P<0.05), however, 50 and 70 mg/kg LA did not have a significant effect (3.0±0.63 and 2.5±0.55, respectively).

Brain water content was quantified using the dry-wet weight method and the result is shown in Fig. 2B. The brain water content in the ischemic area of the I/R group was significantly higher than that of the sham group. Pretreatment of rats with 100 mg/kg LA significantly reduced brain edema in the rats that underwent I/R (82.1±0.5% vs. 80.3±0.6%; P<0.05).

As shown in Fig. 3, the content of MDA and NO significantly increased in the I/R group (P<0.05) compared with that in the sham group and decreased in the LA-pretreated group (P<0.05). The activities of T-AOC and SOD were significantly higher in the LA-treated group (P<0.05) than in the I/R group.

Cleaved caspase-3, activated from caspase-3, has been identified as a key mediator of apoptosis and is considered to be one of the final steps in cell apoptosis (19,20). As shown in Fig. 4, LA significantly suppressed the level of cleaved caspase-3 compared with the I/R group (P<0.05).

The expression of BDNF, PI3K, p-Akt and p-ERK1/2 were examined using western blotting (Fig. 5A). As shown in Fig. 5B, compared with the I/R group, LA significantly increased the level of BDNF and p-Akt (P<0.05). The results demonstrated that the levels of PI3K and p-ERK1/2 were significantly downregulated following cerebral I/R, however, pretreatment with LA was able to reverse this effect and increase the expression of these proteins (Fig. 5C–E; P<0.05).