DEX was purchased from Jiangsu Singch Pharm. Co., Ltd. (Jiangsu, China) and the α2-AR antagonist yohimbine (YOH) was purchased from Tocris Bioscience (Bristol, UK) 2,3,5-Triphenyltetrazolium chloride (TTC) and pentobarbital sodium were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primary antibodies against AMPK and ELISA kits were purchased from R&D Systems Europe, Ltd., (Abingdon, UK). Rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from R&D Systems Europe, Ltd.

A total of 126 male Sprague-Dawley (SD) rats (8 weeks, 220–250 g) were purchased from the Experimental Animal Center of Shanghai Jiaotong University (Shanghai, China) and housed in a controlled environment with a 12-h light/dark cycle, 60±5% humidity and 22±2°C with access to water and food ad libitum. All procedures were conducted in accordance with The National Institute of Health Guide for the Care and Use of Laboratory Animals and this study was approved by the Renji Hospital Laboratory Animal Ethics Committee (reference no. 20170723-006).

As illustrated in Fig. 1, SD rats were divided to seven experimental groups (18 rats/group): Sham surgery [treated with intraperitoneal (i.p.) saline]; middle cerebral artery occlusion (MCAO) surgery (90 min); DEX10 [10 µg/kg i.p. injection 30 min prior to MCAO]; DEX50 (50 µg/kg i.p. 30 min prior to MCAO); DEX100 (100 µg/kg i.p. 30 min prior to MCAO); DEX50+Yohimbine [YOH; 5 mg/kg 10 min prior to DEX (50 µg/kg i.p.) administration and MCAO] and YOH (5 mg/kg 40 min prior to MCAO).

The MCAO model was established as previously described (22). Briefly, rats were anesthetized with an i.p injection of 3% pentobarbital sodium (50 mg/kg; Sigma-Aldrich; Merck KGaA), and the middle cerebral artery (MCA) was occluded by threading a monofilament sterile nylon suture with a heat-rounded tip through the internal carotid artery, which was advanced until it blocked the origin of the MCA. At 90 min following ischemia induction, reperfusion was initiated by withdrawal of the monofilament. In the Sham surgery group, the MCA was separated only and MCAO was not performed. During all surgical procedures rats were maintained at 37°C using a heating blanket and a heat lamp.

DEX was dissolved in normal saline and administered by i.p injection at three single doses (10, 50 100 µg/kg; 0.25 ml administration) 30 min prior to MCAO surgery. YOH (5 mg/kg; 0.25 ml) was administered by i.p 10 min prior to the dexmedetomidine (50 µg/kg). The concentrations of DEX and YOH were selected according to the previous reports (19,23–26). The vehicle control was normal saline, which was administrated by i.p injection in 0.25 ml 30 min prior to ischemia induction (Fig. 1). SD rats were divided to seven experimental groups (18 rats/group): Sham surgery; middle cerebral artery occlusion (MCAO) surgery (90 min); DEX10 [10 µg/kg i.p injection 30 min prior to MCAO]; DEX50 (50 µg/kg i.p 30 min prior to MCAO); DEX100 (100 µg/kg i.p 30 min prior to MCAO); DEX50+Yohimbine [YOH; 5 mg/kg 10 min prior to DEX (50 µg/kg i.p) administration and MCAO] and YOH (5 mg/kg 40 min prior to MCAO).

Neurological deficit scores were evaluated as reported previously (26): 0, no motor deficits (normal); 1, forelimb weakness and torso turning to the ipsilateral side when held by tail (slight); 2, circling to the contralateral side, with normal posture at rest (moderate); 3, unable to bear weight on the affected side at rest (severe); 4, no spontaneous locomotor activity or barrel rolling (serious).

Infarct volume was evaluated by TTC staining at 24 h following I/R. A total of 6 rats/group were euthanized and the brains were quickly removed. The brains were sliced into five coronal sections (2 mm) and stained with 1% solution of TTC at 37°C for 20 min, followed by fixation in 4% paraformaldehyde at room temperature for 30 min. Images of TTC-stained sections were captured by light microscopy (×1; DSC-HX9V, SONY Corporation, Tokyo, Japan) and the digital images were analyzed using Image-Pro Plus v6.0 (Media Cybernetics, Inc., Rockville, MD, USA) image analysis software. Lesion volumes were calculated by multiplying the area by the thickness of slices. The percentage of hemisphere lesion volume (%HLV) was calculated by the following formula (27): %HLV = {[total infarct volume-(the volume of intact ipsilateral hemisphere-the volume of intact contralateral hemisphere)]/contralateral hemisphere volume} ×100.

At 24 h post-I/R, 6 rats/group were sacrificed and the brain cortex tissue were obtained from the infarcted cerebral hemisphere and homogenized at 4°C in lysis buffer for ELISA. The production levels of TNF-α and IL-1β were measured in brain tissue homogenates using specific ELISA kits (TNF-α: cat. no. MTA00B; IL-1β: cat. no. MLB00C; R&D Systems Europe, Ltd.), according to the manufacturer's protocol; results were expressed as pg/ml.

At 24 h following I/R, 6 rats/group were euthanized, rats were anesthetized with 4% chloral hydrate and intracardially perfused first with 250 ml saline and then fixed with 250 ml 4% paraformaldehyde (at 4°C for 30 min). Then the fixed brains were processed by embedding in paraffin blocks, and the brains (2-µm) were sectioned. Apoptotic cells in the cortex were determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. TUNEL staining was performed 6 times according to the manufacturer's protocol (R&D Germany); TUNEL-positive cells emitted a green fluorescent color and were quantified using fluorescence microscopy (Olympus BX53; Olympus corporation) at magnification, ×40, and 5 fields for each section were examined from the ischemic cortex. The average percent of TUNEL-positive cells out of the total number of cells were determined. Nuclei were stained with DAPI.

At 24 h post-I/R, the cortex of 6 rats/group were obtained and homogenized. Total protein extracted from cortex homogenates with a ProteinExt^™ Mammalian Total Protein Extraction kit (TransGenBiotech, Beijing, China) was used to analyze protein expression by western blotting. Following that, protein concentrations were measured using a BCA Protein Quantification kit (Yeasen, Shanghai, China) with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (40 µg/lane) were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were placed in QuickBlock Blocking Buffer for Western Blot (Beyotime Institute of Biotechnology, Beijing, China) for 30 min to block non-specific binding sites at 4°C prior to incubating with mouse primary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) against AMPKα (1:1,000; cat. no. 2532), phosphorylated (p)-AMPK (1:1,000; cat. no. 4186) and β-actin (1:2,000; Abcam; cat. no. ab8226) at 4°C overnight. The membranes were subsequently washed with TBST (0.05% Tween-20), incubated with the appropriate secondary antibodies (horseradish peroxidase-conjugated anti-mouse IgG secondary antibody; R&D Systems Europe, Ltd.; cat. no. HAF007, 1:2,000) at room temperature for 2 h and washed with TBST. This experiment was repeated 6 times. The protein bands were detected using a Bio-Rad Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and quantified using the Quantity One software package (version 2, Bio-Rad Laboratories, Inc.). The expression of p-AMPK was normalized to β-actin.

A total of 6 rats/group were subjected to neuromotor tests (screen clinging, horizontal bar and prehensile traction) at days 1, 2 and 5 post-I/R, as previously described (28), with a score of 9 being the best possible score. The rats were placed on a screen (29×30 cm), which was rotated in the vertical plane. The time that the rat was able to hold onto the vertical screen was recorded for a maximum of 15 seconds (screen clinging). In addition, the rats were placed at the center of a horizontal wooden rod (diameter, 2.5 cm) and the time that the rat was able to remain balanced on the rod was recorded for a maximum of 30 sec (horizontal bar). Finally, the time that the animal was able to cling to a horizontal rope was recorded for a maximum of 5 sec (prehensile traction). The tests were performed by a researcher who was blinded to animal group assignments.

Data were analyzed by one-way analysis of variance followed by Tukey's post hoc test except total motor scores using GraphPad Prism 5.0 (Graphpad, Inc., La Jolla, CA, USA). Neuromotor scores using the Tukey's Multiple Comparison Test data were expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated six times.

Compared with rats in the MCAO group, the neurological scores and infarct volumes were significantly decreased in rats in each of the three DEX pre-treatment groups (P<0.01; Fig. 2A and B). No significant differences were identified for neurological scores and infarct volume in the YOH group compared with the MCAO group. Compared with the DEX10 group, the neurological scores and infarct volumes were significantly decreased in DEX50 and DEX100 groups (P<0.05); However, no significant differences were identified between the DEX50 and the DEX100 group (P>0.05). The neurological scores and infarct volumes were significantly increased in rats in the DEX50+YOH group (P<0.001).

Compared with the Sham group, the percent of TUNEL-positive cells and infarct size in the cortex of ischemic hemisphere in MCAO group increased (P<0.001; Fig. 2C and D). Compared with the MCAO group, the percent of TUNEL-positive cells was significantly decreased in the DEX treatment groups (P<0.05 or P<0.01); however, no significant difference in apoptotic cells was identified in the YOH group compared with the MCAO group. Compared with the DEX10 group, the percent of TUNEL-positive cells was significantly decreased in the DEX50 and DEX100 groups (Fig. 2D; P<0.01). Compared with the DEX50 group, there was a significant increase in apoptotic cells in the DEX50+YOH group (P<0.01).

The production levels of TNF-α and IL-1β in the brain tissues were examined to elucidate the effects of DEX on MCAO-induced inflammation (Fig. 3A and B, respectively). Compared with the Sham group, the levels of TNF-α and IL-1β were significantly increased in the MCAO group (P<0.001). Compared with the MCAO group, the levels of TNF-α and IL-1β were significantly decreased in the three DEX treatment groups (P<0.01). YOH had no observable effects on the production of TNF-α and IL-1β in the cortex compared with the MCAO group. Compared to DEX50 group, the levels of TNF-α and IL-1β were higher in the DEX50+YOH-treatment group (P<0.01).

The expression levels of p-AMPK were also examined in the ischemic brain cortex. The levels of p-AMPK were elevated in ischemic cortex (Fig. 3C and D). Compared with the MCAO group, pretreatment with DEX (10, 50 and 100 µg/kg) resulted in a significant increase in the expression levels of p-AMPK (P<0.01). p-AMPK expression levels were increased in DEX50 and DEX100 groups compared with the DEX10 group (P<0.05). Compared with the DEX50 group, p-AMPK expression was significantly lower in the DEX50+YOH group (P<0.01). Furthermore, the ratio of p-AMPK/AMPK as illustrated in Fig. 4.

Focal cerebral ischemia resulted in reduced motor function scores in MCAO rats compared with Sham group rats at day 1 following I/R (Fig. 5A), and the motor functions remained low at days 2 and 5 (Fig. 5B and C, respectively). The decline of motor function was partially improved by pre-treatment with DEX, and the higher doses (50 and 100 µg/kg) improved the motor function scores more significantly compared with the lower dose (10 µg/kg; P<0.05). No significant differences in motor functions were identified in rats in the YOH groups compared with the MCAO group; However, compared with the DEX50 group, pretreatment of YOH resulted in a significant reduction in the motor score (P<0.01).