BYNWD (Guangdong Provincial Traditional Chinese Medical Hospital, Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China) is composed of Radix Astragali (Gansu, China), Radix Angelicae Sinensis (Gansu, China), Radix Paeonia Rubra (Sichuan, China), Rhizoma Chuanxiong (Sichuan, China), Semen Persicae (Hebei, China), Flos Carthami (Sichuan, China) and Lumbricus (Guangxi, China). The components were mixed with a ratio of 120:6:4.5:3:3:3:3 (dry weight) (17). To maintain the consistency of the herbal chemical ingredients, all the herbal components were originally obtained from the standard naive sources as stated above with good agriculture practice grade and the drugs were extracted with standard methods according to Chinese Pharmacopoeia (China Pharmacopoeia and Committee, 2000). BYHWD was prepared with distilled water at 100°C for 30 min twice and the drug solution was concentrated to the final concentration of 2 g/ml (equivalent to dry weight of raw materials).

Adult male Sprague-Dawley rats weighing 220–250 g were purchased from Guangzhou University of Chinese Medicine Experimental Animal Center (Guangzhou, China). All the experimental procedures were carried out strictly in compliance with the NIH guidelines for the Care and Use of Laboratory Animals. Focal ischemia was induced by middle cerebral artery occlusion (MCAO) as previously reported (18). Briefly, rats were anesthetized with 350 mg/kg chloral hydrate via intraperitoneal injection. The right carotid bifurcation was exposed, and the external carotid artery was coagulated distal to the bifurcation. A 4-0 nylon monofilament suture with a rounded tip was introduced into the internal carotid artery through the stump of the external carotid artery and gently advanced for ~20 mm. After 2 h of occlusion, the filament was gently withdrawn, and the incision was closed. During the whole process, the rat rectal temperature was maintained at 37°C by placing the animals on a heating bed. In the sham-operated rats, the common carotid was exposed without ligation. Two hours after surgery, the rats were intragastrically administered with BYHWD every 12 h at the dose that was equivalent to 36 g raw materials/(kg/day) (1). The other rats were administered the same volume of distilled water. In some experiments, the rats were injected intravenously with the PACAP receptor antagonist PACAP-(6–38) (Tocris Bioscience, Ellisville, MO, USA) every 48 h (19).

The study was approved by the Ethics Committee of the Guangzhou University of Chinese Medicine (no. ds20130001).

Following the experiments, the animals were anesthetized with an overdose of 10% chloral hydrate and perfused through the ascending aorta with 0.9% saline and 4% buffered paraformaldehyde. Brains were quickly removed, post-fixed in the same fixative for 2 h and subsequently transferred into a graded series of phosphate-buffered sucrose solutions (10–30%) at 4°C. Coronal sections (15 µm) were embeded in optimum cutting temperature compound, and cut at the levels of hippocampus (22.8–23.8 mm from the bregma) on a cryostat and mounted on polylysine-coated slides. Sections were used to stain individually for the following primary antibodies: Anti-GLT-1 (1:400; cat. no. AB1783; Merch Millipore, Billerica, MA, USA), anti-GS (1:400;cat. no. ab73593; Abcam, Cambridge, MA, USA) and anti-GFAP (1:800; cat. no. MAB360; Merck Millipore) overnight at 4°C. For immunohistochemistry, the sections were incubated with biotinylated immunoglobulin G and further processed with the avidin-biotin complex method (Vector, San Diego, CA, USA). For immunofluorescence, the sections were incubated with the corresponding secondary antibodies for 2 h at room temperature and captured after rinsing. For double immunofluorescence, sections were incubated with a mixture of antibodies, anti-GLT-1, anti-GS and anti-GFAP (astrocyte marker), followed by a mixture of fluorescein isothiocyanate- and CY3-conjugated secondary antibodies (Chemicon, Temecula, CA, USA). Images were captured using an Olympus microscope (Olympus, Tokyo, Japan). To quantify GLT-1, GS and GFAP immunoreactivity (IR), measurement was performed with a computerized image analysis system (Image-Pro-Plus; Media Cybernetics, Silver Spring, MD, USA) according to a previously described method (18). Briefly, a density threshold was first set above the background level to identify the positively stained structure and the area occupied by these structures was measured as the positive area. An average percentage of the immunoreactive area to the total outlined area was obtained. Five to six animals were included in each group for quantification of immunohistochemistry.

PACAP-38 was measured by the PACAP-38 enzyme-linked immunosorbent assay (ELISA) kit (TSZ Bioscience, North Brunswick, NJ, USA). Briefly, rats were decapitated and their brains were quickly removed. Hippocampi were weighed and immersed in 10-fold (volume/hippocampus weight) 0.2 mol/l Tris-HCl buffer (pH 7.3) containing 20 mmol/EDTA, and subsequently boiled and centrifuged as previously described (20). Relevant reagents in the kit were added according to the manufacturer's instructions. The absorbance of the product was measured at 450 nm using a microplate ELISA reader (Bio-Rad, Hercules, CA, USA). The concentration of PACAP-38 in the samples was estimated from the standard curve that was constructed in each assay. Samples were assayed in triplicate.

Observers blinded to experimental conditions evaluated the outcome measures. All the data were presented as mean ± standard error of the mean, and significant differences between groups were analyzed with one-way analysis of variance. The Student's t-test was used to compare the means of two samples. All the analysis was carried out using the Statistical Package for Social Sciences (SPSS, version 13.0 for Windows; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Expression of GLT-1 and GS was examined after ischemia for 2 h and reperfusion for 1, 3, 5 and 7 days, respectively, to observe their changes over time. Compared with the control group, ischemia significantly decreased GLT-1 expression. Among the ischemia group, GLT-1 expression in the hippocampus CA1 area on day 3 was higher than that on day 1, and it subsequently decreased on days 5 and 7 (F=123.014, P<0.001). Additionally, BYHWD significantly enhanced GLT-1 expression compared with the ischemia group, with the distinct difference on day 3 (F=171.564, P<0.001) (Fig. 1A).

Similar with GLT-1, BYHWD markedly upregulated the expression of GS in the hippocampal CA1 region from day 1 and peaked on day 3 compared with the ischemia group. The level of GS was gradually decreased, but a significant difference remained between the BYHWD and ischemia groups on days 5 and 7 (F=104.439, P<0.001) (Fig. 1B).

As BYHWD upregulated the expression of GLT-1 and GS expression on day 3, the following experiments were carried out on day 3. As shown in Fig. 2, focal ischemia for 2 h followed by 72 h of reperfusion resulted in less PACAP-38 expression (t=3.987, P=0.017), but administration of BYHWD significantly increased the expression (t=3.999, P=0.016) (Fig. 2A). Combined with the fact that PACAP-38 regulated glial glutamate transportation and metabolism, whether PACAP-38 was involved in the process of BYHWD protecting ischemia injury by enhancing GLT-1 and GS. As shown in Fig. 2, focal ischemia for 2 h followed by 72 h of reperfusion resulted in decreased GLT-1 (t=20.222, P<0.001) and GS (t=14.596, P<0.001). BYHWD enhanced GLT-1 (t=10.654, P<0.001) and GS (t=11.794, P<0.001) expression compared with the ischemia group, while co-administration of PACAP-(6–38) (a non-specific PACAP receptor antagonist) significantly reduced the expression of GLT-1 (t=9.798, P<0.001) and GS (t=5.368, P=0.001) (Fig. 2B–D).

As shown in Fig. 3, GLT-1 and GS were mainly colocalized with GFAP (an indicator of astrocytes) detected by double immunofluorescent staining (Fig. 3A and B), indicating that they were mainly expressed in astrocytes. Subsequently, whether BYHWD affected GFAP immunostaining was investigated. Immunofluorescent staining showed that GFAP expression was enhanced in the ischemia group compared with the control group (t=10.579, P<0.001). However, BYHWD significantly attenuated the expression (t=6.481, P<0.001). Additionally, co-administration of PACAP-(6–38) reversed the effect of BYHWD on decreasing GFAP expression (t=5.418, P=0.001) (Fig. 3C and D). Representative images showed that astrocytes in hippocampal CA1 appeared to exhibit hypertrophic morphology in the ischemia group, while BYHWD attenuated the astrogliosis, and PACAP-(6–38) inhibited the effect of BYHWD (Fig. 3C).