Dexmedetomidine inhibits neuronal apoptosis by inducing Sigma-1 receptor signaling in cerebral ischemia-reperfusion injury

Dexmedetomidine alleviated neurological dysfunction after CIRI

To study the effects of dexmedetomidine on CIRI, we established four groups of rats: a sham operation group, a dexmedetomidine treatment group, a middle cerebral artery occlusion (MCAO) model group and an MCAO + dexmedetomidine group. Twenty-four hours after inducing cerebral ischemia and reperfusion, we assessed the neurological deficits in each group by calculating the neurological function score (Figure 1). The preoperative neurological scores of all the groups were in the normal range. The neurological deficit scores in the dexmedetomidine group did not differ significantly from those in the sham group (P>0.05). However, the neurological deficit scores were significantly higher in the MCAO group than in the sham group (P<0.05). The neurological deficit scores in the MCAO + dexmedetomidine group were significantly lower than those in the MCAO group (P<0.05). These results demonstrated that dexmedetomidine had no obvious toxicity in normal rats, and ameliorated the neurological dysfunction caused by 24-hour CIRI.

Figure 1. Dexmedetomidine improved neurological function after CIRI. Neurological deficit scores. Data are shown as the mean ± standard error of the mean (SEM), n = 12 per group. *P < 0.05 compared with the sham group; ^#P < 0.05 compared with the MCAO group. Sham operation group, Sham group; dexmedetomidine administration group, Dex group; MCAO model control group, MCAO group; MCAO + dexmedetomidine administration group, MCAO + Dex group.

Dexmedetomidine attenuated the neuronal apoptosis induced by CIRI

To investigate the molecular mechanism whereby dexmedetomidine protected against neurological dysfunction following CIRI, we used terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to detect apoptosis in slices of the cerebral ischemic penumbra from each group (Figure 2). The number of apoptotic cells was significantly greater in the MCAO group than in the sham group, but this increase was attenuated in the MCAO + dexmedetomidine group (P<0.01). There was no significant difference in the rate of apoptosis between the dexmedetomidine group and the sham group (P>0.05). These results indicated that 24-hour CIRI significantly increased the rate of neuronal apoptosis, while dexmedetomidine exerted neuroprotective effects by inhibiting this neuronal apoptosis.

Figure 2. Dexmedetomidine attenuated the neuronal apoptosis induced by CIRI. (A) TUNEL staining was performed on slices from the cerebral ischemic penumbra. a. Sham operation (Sham) group, b. dexmedetomidine administration (Dex) group, c. MCAO model control (MCAO) group, d. MCAO + dexmedetomidine administration (MCAO + Dex) group. (B) Ratio of positive cells. Magnification is 400x. n = 6. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 compared with the sham group; ^#P < 0.05, ^##P < 0.01 compared with the MCAO group.

Dexmedetomidine upregulated the expression of Sig-1R after CIRI

Sig-1R is widely distributed on the MAM and is an important contributor to the ERS response [16, 17].

ERS promotes the dissociation of Sig-1R from GRP78, freeing these proteins to inhibit ERS-induced apoptosis [18]. To investigate the involvement of Sig-1R in our CIRI model, we performed Western blotting to detect Sig-1R protein expression in ER-rich brain tissue lysates from each group. First, we examined the expression of Sig-1R after 0, 3, 6, 12 and 24 hours of ischemia-reperfusion in the MCAO model (Figure 3A). Sig-1R protein expression increased after 3 hours of reperfusion, increased significantly after 6 hours of reperfusion, and then decreased with increasing reperfusion times (P<0.05, Figure 3B). After 24 hours, the expression of Sig-1R was significantly lower than it was at 0 hours (P<0.05, Figure 3B). These results suggested that CIRI upregulated Sig-1R expression on the ER, and that Sig-1R was redistributed within cells as the reperfusion time increased.

Figure 3. Sig-1R and GRP78 protein expression on the ER after CIRI. (A) The protein levels of Sig-1R and GRP78 were determined by Western blotting. (B) Sig-1R protein levels with increasing reperfusion times. (C) GRP78 protein levels with increasing reperfusion times. The results were normalized to the percentage of Calnexin expression. Data are shown as the mean ± SEM, n = 6 per group. ^#P < 0.05, ^##P < 0.01 compared with the MCAO 0-hour group.

Subsequently, we examined the expression changes of Sig-1R in brain tissues from the four groups of rats after 24 hours of ischemia-reperfusion. Sig-1R expression was significantly lower in the MCAO group than in the sham group, but this decrease was significantly attenuated in the MCAO + dexmedetomidine group (P<0.05, Figure 4A, 4C). These results suggested that dexmedetomidine upregulated the expression of Sig-1R in brain tissues after 24-hour CIRI.

Figure 4. The effects of dexmedetomidine on the expression of Sig-1R and ERS-induced apoptotic proteins after CIRI. (A) The protein levels of Sig-1R in the cerebral ischemic penumbra were determined by Western blotting. (B) The levels of ERS-induced apoptotic proteins (CHOP, p-JNK/JNK, Caspase-3) in the cerebral ischemic penumbra were determined by Western blotting. (C) Sig-1R protein expression. (D) CHOP protein expression. (E) p-JNK/JNK protein expression. (F) Caspase-3 protein expression. The results were normalized to the percentage of Calnexin or β-actin expression. Data are shown as the mean ± SEM, n = 6 per group. *P < 0.05, **P < 0.01 compared with the sham group; ^#P < 0.05, ^##P < 0.01 compared with the MCAO group. Sham operation group, Sham group; dexmedetomidine administration group, Dex group; MCAO model control group, MCAO group; MCAO + dexmedetomidine administration group, MCAO + Dex group.

The upregulation of Sig-1R by dexmedetomidine may have inhibited apoptotic signaling in the ER

To determine the effects of Sig-1R activation on the downstream ERS signaling pathway after CIRI, we examined the expression of the ERS marker protein GRP78 in ER-rich brain tissue lysates at different ischemia-reperfusion time points by Western blotting (Figure 3A). GRP78 protein expression increased slightly after 3 hours of reperfusion, increased significantly after 6 hours of reperfusion, reached a peak at 12 hours, and then decreased at 24 hours (P<0.05, Figure 3C). Thus, the protein expression trend of GRP78 was basically consistent with that of Sig-1R.

To determine the effects of dexmedetomidine on the ERS signaling pathway involving Sig-1R after CIRI, we detected the expression of ERS marker proteins (C/EBP homologous protein [CHOP], c-Jun N-terminal kinase [JNK] and Caspase-3) in brain tissues by Western blotting (Figure 4B). CHOP and Caspase-3 levels were significantly higher in the MCAO group than in the sham group, as was the ratio of phosphorylated JNK to total JNK (P<0.01, Figure 4D–4F). These increases were significantly attenuated in the MCAO + dexmedetomidine group (P<0.01, Figure 4D–4F). These results suggested that 24-hour CIRI activated ERS and promoted neuronal apoptosis, while dexmedetomidine inhibited neuronal apoptosis through this pathway.

To confirm the effects of dexmedetomidine on the Sig-1R-associated ERS signaling pathway, we treated rats with a Sig-1R-specific inhibitor (BD1063) 30 minutes before performing the MCAO operation, and subsequently detected the changes in GRP78 expression in ER-rich brain tissue lysates (Figure 5A). Compared with the MCAO group, the expression of GRP78 on the ER was significantly reduced in the MCAO + BD1063 group, while it was significantly elevated in the MCAO + dexmedetomidine group (P<0.05, Figure 5C). GRP78 expression was significantly lower in the MCAO + BD1063 + dexmedetomidine group than in the MCAO + dexmedetomidine group (P<0.05, Figure 5C). These results suggested that the Sig-1R-specific inhibitor significantly reduced GRP78 expression in brain tissues after 24-hour CIRI, and also prevented the upregulation of GRP78 by dexmedetomidine to a certain extent.

Figure 5. The effects of dexmedetomidine on the ERS signaling pathway involving Sig-1R after CIRI. (A) GRP78 protein levels on the ER were determined by Western blotting. (B) The ERS-induced apoptotic proteins (CHOP, Caspase-3) from the cerebral ischemic penumbra were determined by Western blotting. (C) GRP78 protein levels. (D) CHOP protein levels. (E) Caspase-3 protein levels. The results were normalized to the percentage of Calnexin or β-actin expression. Data are shown as the mean ± SEM, n = 6 per group. ^#P < 0.05, ^##P < 0.01 compared with the MCAO group; ^☆P < 0.05, ^☆☆P < 0.01 compared with the MCAO + dexmedetomidine group. MCAO + BD1063 group, MCAO + BD group; MCAO + dexmedetomidine group, MCAO + Dex group; MCAO + BD1063 + dexmedetomidine group, MCAO + BD + Dex group.

We also examined the protein levels of the ERS-induced apoptotic markers CHOP and Caspase-3 in brain tissues, and found that they were significantly higher in the MCAO + BD1063 group than in the MCAO group, but were significantly lower in the MCAO + dexmedetomidine group (P<0.05, Figure 5B, 5D, 5E). CHOP and Caspase-3 levels were significantly higher in the MCAO + BD1063 + dexmedetomidine group than in the MCAO + dexmedetomidine group (P<0.05, Figure 5B, 5D, 5E). These results suggested that dexmedetomidine may at least partially inhibit ERS-induced apoptosis by activating Sig-1R in brain tissues, thus attenuating brain damage after 24 hours of ischemia-reperfusion.

Experimental animals

In total, 102 healthy specific-pathogen-free adult male Sprague Dawley rats (age: 8-12 weeks, weight: 220-260 g) were provided by Beijing HFK Bioscience Co., Ltd. (Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences). The rats were acclimated to the laboratory for one week before the experiment. The animals were housed four per cage and were free to ingest standard feed and water. The room temperature was set to 22 ± 2 °C, the relative humidity was set to 50-60%, and a 12-hour light-dark cycle was used. The room was regularly ventilated, the water and feed were exchanged and the cushions were replaced to keep the animals healthy. All the animal experiments were approved by the Experimental Animal Ethics Committee of the Tianjin Central Hospital of Gynecology Obstetrics and were performed in strict accordance with the guidelines of the National Laboratory Animal Ethics Regulations.

Animal model preparation

The rats were fasted for 12 hours before the operation, but were allowed access to water. The MCAO model in rats was established by thread embolization according to the Zea-Longa method [43]. The rats were anesthetized by intraperitoneal injection of a 10% chloral hydrate solution (300 mg/kg) and fixed in the supine position. The total neck and internal and external carotid arteries along the midline of the neck were carefully separated. The proximal common end of the common carotid artery and the distal end of the external carotid artery were ligated. Then, a prepared nylon thread bolt was slowly inserted into the internal carotid artery, stopped advancing when encountering slight resistance, and the bolt was fixed with a reserved line. After the blood flow was successfully blocked for two hours, the bolt was pulled out and reperfusion was allowed to proceed for 24 hours. The wound was stitched layer-by-layer at the end of the operation. During the procedure, the ambient temperature was maintained at 37 ± 0.5 °C, and the rectal temperature, respiration rate and heart rate were monitored. After the rats were awakened, they were returned to the animal house for feeding and observation.

Grouping and treatment of the experimental animals

Our experiments were divided into three parts. Firstly, to elucidate the possible protective mechanism of dexmedetomidine in a model of CIRI, we randomly divided the rats into four groups: the sham operation group, the dexmedetomidine administration group (dexmedetomidine was purchased from Jiangsu Enhua Pharmaceutical Co., Ltd., National Drug Standard H20110086), the MCAO model control group, and the MCAO + dexmedetomidine administration group. TUNEL staining and Western blotting were performed after the neurological deficit score was determined. Secondly, to observe the effects of the ERS pathway involving Sig-1R on CIRI, we subjected rats to ischemia for two hours and randomly sampled their brain tissues at five time points of reperfusion (0, 3, 6, 12 and 24 hours) for Western blotting. Finally, to verify that the ERS pathway involving Sig-1R was responsible for the protective effects of dexmedetomidine in CIRI, we obtained a specific inhibitor of Sig-1R (BD1063, MedChemExpress; cat. no. HY-18101A) and randomly divided the rats into four groups: the MCAO group, the MCAO + BD1063 group, the MCAO + dexmedetomidine group, and the MCAO + BD1063 + dexmedetomidine group. Western blotting was performed after the neurological deficit score was determined.

In the above groupings, the sham group underwent the same anesthesia procedure and operation as the MCAO group, but a thread was not inserted into the middle cerebral artery. For the dexmedetomidine group, dexmedetomidine was intravenously administered at a loading dose of 1 μg/kg at the very beginning of the surgery, and was then administered at 0.05 μg/kg/min for the next two hours [26, 44]. The other operations in the dexmedetomidine group were the same as in the sham group. In the MCAO model control group, the middle cerebral artery thread occlusion method was used for two hours. Then, the thread thrombus was pulled out and reperfusion was allowed to proceed for 24 hours. In the MCAO + dexmedetomidine group, the dexmedetomidine treatment was the same as in the dexmedetomidine group, and the rest of the operation was the same as in the MCAO group. In the MCAO + BD1063 group, BD1063 (3 mg/kg) was injected subcutaneously 30 minutes before the operation, and the rest of the operation was the same as in the MCAO group [45]. In the MCAO + BD1063 + dexmedetomidine group, BD1063 was injected subcutaneously 30 minutes before the operation, dexmedetomidine was intravenously administered at the beginning of the surgery, and the rest of the operation was the same as in the MCAO group. The sham and MCAO groups were administered the same amount of normal saline. The rats were placed in a clean environment after the surgery, and were free to eat and drink under the same conditions previously described.

Neurological deficit score

The neurobehavioral score was scored by a researcher who was blinded to the experimental groups. After the rats were subjected to 24 hours of CIRI, the neurological function of each rat was scored according to Zea-Longa’s five-point scoring system [43]: 0 points, the rat had no neurological symptoms; 1 point, the rat could not fully extend its contralateral forepaw when lifting its tail; 2 points, the rat turned to the opposite side of the operation while walking; 4 points, the rat could not walk on its own or lost consciousness. A neurological deficit score of 2 to 3 was used as the success criterion for establishing the MCAO model. The rats that did not meet this criterion were excluded, along with those that underwent subarachnoid hemorrhaging or death within 24 hours. Additional rats were randomly sampled under the same conditions and were subjected to the same experimental procedures to ensure that each group contained an adequate number of rats.

Material and specimen processing

After 24 hours of CIRI, six rats in each group were randomly selected for the TUNEL assay or Western blotting. The rats were intraperitoneally anesthetized with a 10% chloral hydrate solution (300 mg/kg) and fixed on the operating table. The anterior cardiac region was fully exposed, the left ventricle was punctured with a perfusion needle from the apex, and the needle was fixed. The right auricle was cut and the tissue was continuously perfused with 0.9% saline until no bloody liquid was observed. Then, the perfusion solution was replaced with 4% paraformaldehyde until the limbs of the rats were stiff. The cerebral cortex on the side of the CIRI was separated. The penumbra of the cerebral cortex on the side of the CIRI was isolated according to previously published methods [26, 46]. We initially cut about 2 mm from the outside of the sagittal suture through the right hemisphere longitudinally (from top to bottom). Lateral diagonal cuts were then made at approximately the “2 o’clock” position, and the penumbra was separated from the core for further experimentation. The isolated penumbra of the cerebral cortex was fixed at 4 °C for the TUNEL experiment. After cardiac perfusion with ice-cold saline, the brain was removed and the penumbra of the cerebral cortex on the side of the CIRI was separated on ice. The penumbra of the cerebral cortex was stored in a liquid nitrogen tank until Western blotting.

Detection of apoptosis by TUNEL staining

The fixed samples of the cerebral ischemic penumbra were embedded in paraffin and sliced into 5-micron sections for TUNEL staining. The experiment was carried out strictly according to the instructions of the TUNEL staining kit (Roche Diagnostics Corp., Indianapolis, IN, USA; Cat. No. 11684817910). Under 400x fluorescence microscopy, four non-overlapping visual fields were randomly selected, and the percentage of the apoptotic cell index (ACI%, the number of apoptotic cells/the total number of apoptotic cells × 100%) was calculated.

Western blotting experiment

Protein isolation and Western blotting were performed as previously described [18]. In short, the cryopreserved rat brain tissue samples from the CIRI side were homogenized with lysis buffer on ice, and the lysate was left on ice until full lysis was achieved. The tissue homogenate was centrifuged at 12,000 x g for 30 minutes at 4 °C. The supernatant was the total cell lysate, and an appropriate amount was collected for Western blotting. The remaining ER-rich cerebral ischemic penumbra was centrifuged (800 × g) for 10 minutes at 4 °C, and the resulting supernatant was centrifuged (10,000 × g) for 20 minutes at 4 °C. The new supernatant was collected at 4 °C and centrifuged at 100,000 × g for 60 minutes, yielding an ER-rich precipitate. The precipitate was added to a lysis buffer containing 1% Triton X-100, and an appropriate amount was collected for Western blot analysis.

The protein concentration was determined by the bicinchoninic acid method. A sodium dodecyl sulfate polyacrylamide gel was prepared, and 30 μg of protein was loaded per well. The proteins were electrophoretically separated and transferred to a polyvinylidene fluoride membrane. The membrane was washed thoroughly and blocked with 5% skim milk powder for two hours. The membrane was cut into strips and incubated overnight at 4 °C with one of the following primary antibodies: Sig-1R (1:1000; Proteintech Group, Wuhan, China; cat. no. 15168-1-AP), GRP78 (1:3000; Abcam, Cambridge, UK; cat. no. ab108613), p-JNK (1:5000; Abcam, Cambridge, UK; cat. no. ab124956), JNK (1:1000; Abcam, Cambridge, UK; cat. no. ab179461), CHOP (1:1000; Abcam, Cambridge, UK; cat. no. ab11419), Caspase-3 (1:2000; Abcam, Cambridge, UK; cat. no. ab184787). β-actin (1:3000; Proteintech Group, Wuhan, China; cat. no. 20536-1-AP) and Calnexin (1:1000; Abcam, Cambridge, UK; cat. no. ab133615) were used as internal references. After the membrane was thoroughly washed, a horseradish peroxidase-labeled secondary antibody (1:1000; Solarbio, Beijing, China; cat. no. SE134 or SE131) was applied for two hours at room temperature prior to enhanced chemiluminescence development. Image J software was used for image analysis. The relative expression of the target strip was expressed as the ratio of the gray value of the strip to the gray value of the internal reference strip.

Statistical analysis

The Data were expressed as mean ± standard deviation (SD). The statistical significance of differences between groups was obtained using one-way analysis of variance (ANOVA) for multiple comparisons. The statistical analyses were conducted with GraphPad Prism 5.0 software. P < 0.05 was considered statistically significant.