A study of optimal concentration range and time window of sevoflurane preconditioning for brain protection in MCAO rats

Male Sprague-Dawley (SD) rats (3 months of age, weighing 330–370 g) (Beijing VitalRiver Laboratory Animial Technology Co.China) were used for experiments in this study. Rats were bred and maintained under standardized housing connditions with food and water ad libitum. The experimental protocol was approved by the Peking University Biomedical Ethics Committee Experimental Animal Ethics Branch (Approval No. LA 2016300).

The MCAO model was established by using the suture method, and induced by sevoflurane inhalation. After anesthesia, a mask inhalation of 1 MAC sevoflurane + 60% oxygen was performed. During operation, the rectal temperature of the rats was maintained at 36–37 °C. After disinfecting neck, a median of 2 cm incision and separation were made, the distal right external carotid artery was ligated with a 4–0 surgical suture, and then a proximal slipknot ligation was performed. The common carotid artery and internal carotid artery were blocked with bulldog clamp. An oblique incision was made at the middle segment of the two ligation areas of external carotid artery, followed by the insertion of a thread (ZL400, Guangzhou Xinzan Biotechnology Co., Ltd.), and loosening of the bulldog clamp of the internal carotid artery. The thread was then inserted into the internal carotid artery, pushed to the initial segment of the anterior cerebral artery, and stopped until a resistance was reached, and it was generally not more than 20 mm. The blood supply to the middle cerebral artery was blocked, and the fixed thread for the suture was tightened, a bulldog clamp of the internal carotid artery was loosened, and then a suture was made to the wound. After 120 min, the thread that was exposed outside of the neck skin was gently pulled out and stopped until there was resistance. The excess parts were cut off to recirculate the blood, forming an ischemia-reperfusion model [18].

A total of 140 male SD rats were randomly assigned to 14 groups (10 in each group), which were as follows: 0.5 MAC (1%) sevoflurane 6 h, 12 h, 24 h, and 48 h group; 1.0 MAC (2%) sevoflurane 6 h, 12 h, 24 h, and 48 h group; 1.3 MAC (2.6%) sevoflurane 6 h, 12 h, 24 h, and 48 h group, MCAO group and sham group. Rats were preconditioned with sevoflurane for 3 h. MCAO and sham groups underwent no preconditioning with sevoflurane. The respiratory box was pre-filled with corresponding concentration of sevoflurane (sevoflurane + 60% oxygen) for 15 min. The rats were then placed in an anaesthetic respiratory box to maintain spontaneous breathing. The corresponding concentration of sevoflurane was continuously blown from the air inlet end, and the air outlet was connected to the atmosphere and the bypass end-expiratory sevoflurane concentration was detected to ensure that the MAC value remains unchanged at 1.3 MAC, 1.0 MAC, and 0.5 MAC. Rats in the sham and MCAO groups just inhaled a mixture of 60% oxygen and air for 3 h. Physiological parameters of the rats were monitored during preconditioning. The respiratory box was pre-filled with 60% oxygen, and the rats were placed in a respiratory box for 3 h. The respiratory box was evenly padded with a 37 °C insulation blanket to keep the rats warm. The MCAO model was established at different time periods (6 h/12 h/24 h/48 h) after preconditioning. There were 10 rats in each group (5 for measuring cerebral infarct volume and 5 for determining brain water content).

Except for these 140 rats, we took another 5 rats in each group of MCAO, Sham, 0.5 MAC, 1.0 MAC and 1.3 MAC respectively to determine whether sevflurane anesthesia caused physiologic side effects.

The neurological severity score was performed 24 h after MCAO operation in each group by blinding method (scoring was separately performed by two people and averaging). The NSS scoring standard was used with 18 points in total.

The rats were induced by sevoflurane inhalation. The respiratory box was pre-filled with 2% sevoflurane (sevoflurane + 60% oxygen) for 15 min. The rats were then placed in an anaesthetic respiratory box. The rats were immediately decapitated to take the brains When they were anesthetized and lost consciousness.

After reperfusion for 24 h, the anesthetized rats with sevoflurane lost consciousness and were decapitated to take the brains, and the olfactory bulb, cerebellum and low brain stem were removed to make into sections. These sections were then placed in 20 ml of 2% triphenyltetrazolium chloride (TTC, Sigma) staining solution, followed by placing them in a water bath at 37 °C in dark place, and taken out after 10 min. Normal brain tissues appeared red and the infarct areas remained white. The brain sections were immediately kept in 4% paraformaldehyde for fixation for 24 h, and pictures were taken with a digital camera. The infarct volume of each section was calculated by using image analysis system Image ProPlus version 6.0. The percentage of cerebral infarct volume was calculated as the (total infarct volume of brain section /total area of​brain section) × 100%.

Rats were anesthetized with sevoflurane, and immediately decapitated to remove the cerebellum and the lower brain stem. The micro-precision balance was immediately used to weigh the wet weight of the brain, placed in an oven, baked at 100 °C for 72 h until constant weight was reached. After weighing the dry weight, the brain water content was calculated by using the formula, water content (%) = (wet weight - dry weight)/wet weight × 100%.

Sevoflurane is a new type inhalation anesthetic with quick inspiration, rapid induction and fine controllability. Generally associated with stable hemodynamics, dose dependent vasodilatation, and cardiac depression. And could also provide cardioprotection through pharmacologic preconditioning. Sevoflurane was administered through the lung and primarily eliminated by the lungs.

We used Power and Sample Size 14.0 and superiority test method to calculate the sample size. Take α = 0.05, β = 0.9, according to the values in the literature, the sample size of each group was calculated to be 10.

Measurement data were expressed as mean ± standard deviation (x ± s). Statistical analysis was performed by using SPSS software, version 17.0. One-way analysis of variance was used for between-group comparison of data with normal distribution. The tukey was used for the post hoc analysis. The between-group comparison of the measurement data with skewed distribution was performed by using Kruskal and wallis method rank sum test, and the bonferroni adjusted method was used to modify the p value. Two-sided P < 0.05 was considered to be statistically significant.

After sevoflurane inhalation anesthesia, the righting reflex of the rats was disappeared within a few minutes, and the skin color of the nasolabial and toe ends of each group of rats appeared ruddy, with no significant fluctuations in SaO₂, heart rate and rectal temperature. After anesthesia, the rats were awakened after 5–15 min.

To determine whether sevflurane anesthesia caused physiologic side effects, such as hypoxia, hypercapnia, or hypoglycemia, 5 rats were choosed in MCAO, Sham, 0.5 MAC, 1.0 MAC and 1.3 MAC respectively. MCAO and sham groups underwent no preconditioning with sevoflurane. 2 mL blood was withdrawn by cardiac puncture at the end of the sevflurane or oxygen exposure. In the MCAO group, 2 mL blood was withdrawn by cardiac puncture after the establishment of MCAO. Arterial blood gas (ABG) and blood glucose measurements were performed using a portable blood gas analyzer (OPTI Medical Systems, Georgia, USA) and One Touch Ultra blood glucose monitoring system (Life Scan Inc., California, USA) respectively. Those rats were not used for any other part of the study.

The results showed no statistical differences in pH, PaO₂, PaCO₂, and blood glucose for each group. No adverse reactions, such as hypoxemia, hypercapnia, and hypoglycemia were observed in each group. These results suggested that the effects of these adverse reactions on behavioral outcomes can be excluded (Table. 1).

The behavioral score for 0.5 MAC sevoflurane preconditioning was 10.0 (8.0–13.0) for the 6 h group, 10.0 (9.0–11.0) for the 12 h group, 10.0 (10.0–12.0) for the 24 h group, and 9.5 (7.0–13.0) for the 48 h group. There was no statistical difference between the MCAO group and the 6 h, 12 h, 24 h, and 48 h groups (P = 0.809, 0.513, 0.809, 0.295), (Fig. 1).

The cerebral infarct volume was 251.22 ± 57.32 mm³ in the MCAO group, 202.69 ± 99.34 mm³ in the 1.3 MAC sevoflurane preconditioning 6 h group, 237.87 ± 68.01 mm³ in the 12 h group, 123.11 ± 33.29 mm³ in the 24 h group, and 247.18 ± 107.91 mm³ in the 48 h group. There were statistical differences in the 24 h group (P = 0.002) when compared with MCAO group, while showed no statistical differences with 6 h, 12 h, and 48 h groups (P = 0.245, 0.747, 0.927). The cerebral infarct volume was 264.63 ± 73.52 mm³ in the 1.0 MAC sevoflurane preconditioning 6 h group, 226.17 ± 37.66 mm³ in the 12 h group, 117.68 ± 15.36 mm³ in the 24 h group, and 274.40 ± 73.49 mm³ in the 48 h group. When compared with MCAO group, the 24 h group (P = 0.004) showed statistical differences, while no statistical differences were observed with the 6 h, 12 h, and 48 h groups (P = 0.746, 0.605, 0.600). The cerebral infarct volume was 191.38 ± 64.58 mm³ in the 0.5 MAC sevoflurane preconditioning 6 h group, 245.36 ± 62.12 mm³ in the 12 h group, 228.95 ± 72.92 mm³ in the 24 h group, and 184.42 ± 61.02 mm³ in the 48 h group. There was no statistical difference with the 6 h, 12 h, 24 h, and 48 h groups as compared to the MCAO group (P = 0.180, 0.894, 0.591, 0.135), (Figs. 2 and 3).

The percentage of infarct volume was 37.89 ± 7.66% in the MCAO group, 29.15 ± 15.68% in the 1.3 MAC sevoflurane preconditioning 6 h group, 33.29 ± 9.33% in the 12 h group, 17.01 ± 5.12% in the 24 h group, and 28.63 ± 10.73% in the 48 h group. Compared with the MCAO group, a statistically significant difference was observed with the 24 h group (P = 0.001), while no statistical difference was observed with the 6 h, 12 h, and 48 h groups (P = 0.152, 0.447, 0.154). The percentage of infarct volume was 35.4 ± 6.47% in the 1.0 MAC sevoflurane preconditioning 6 h group, 34.0 ± 3.61% in the 12 h group, 17.5 ± 2.08% in the 24 h group, and 40.5 ± 11.39% in the 48 h group. A statistically significant difference was observed in the 24 h group (P = 0.003), while no statistical difference in the 6 h, 12 h, and 48 h groups when compared with the MCAO group (P = 0.680, 0.581, 0.685). The percentage of infarct volume was 25.8 ± 8.73% in the 0.5 MAC sevoflurane preconditioning 6 h group, 35.3 ± 11.96% in the 12 h group, 36.0 ± 14.66% in the 24 h group, and 27.3 ± 9.95% in the 48 h group. Comparison with MCAO group showed no statistical difference with the 6 h, 12 h, 24 h, and 48 h groups (P = 0.064, 0.682, 0.754, 0.103), (Fig. 4).

The brain water content was 82.61 ± 0.48% in the sham group and 83.29 ± 0.21% in the MCAO group, and the difference was statistically significant (P = 0.014). The brain water content was 83.32 ± 0.27% in the 1.3 MAC sevoflurane preconditioning 6 h group, 83.49 ± 0.43% in the 12 h group, 82.29 ± 0.68% in the 24 h group, and 83.39 ± 0.79% in the 48 h group. Comparison with MCAO group showed a statistically significant difference with the 24 h group (P = 0.000), while no statistical significance was observed with the 6 h, 12 h, and 48 h groups (P = 0.905, 0.456, 0.911). The brain water content was 83.34 ± 0.21% in the 1.0 MAC sevoflurane preconditioning 6 h group, 83.24 ± 0.35% in the 12 h group, 82.68 ± 0.62% in the 24 h group, and 83.28 ± 0.11% in the 48 h group. Compared with the MCAO group, a statistically significant difference with the 24 h group (P = 0.027), and no statistical significance with the 6 h, 12 h, and 48 h groups (P = 0.870, 0.849, 0.977) were observed. The brain water content was 83.36 ± 0.21% in the 0.5 MAC sevoflurane preconditioning 6 h group, 82.88 ± 0.43% in the 12 h group, 82.91 ± 0.38% in the 24 h group, and 82.81 ± 0.57% in the 48 h group. There was no statistically significant difference with the 6 h, 12 h, 24 h, and 48 h groups when compared with the MCAO group (P = 0.812, 0.134, 0.160, 0.099), (Fig. 5).