All animal studies were approved by the Ethic Committee of Beijing Tiantan Hospital.
Main materials and instruments
C57BL/6 N mice (male, 7–8 weeks, 23 ± 2 g) were obtained from Charles River (Beijing, China). The mice had free access to food and water and were housed at 25 ± 1 °C under a 12 h light/12 h dark cycle. A laser speckle imaging system ((RFLSI III, RWD Life Science, China) and sutures for mice weighing 20–25 g (RWD Life Science, China) were used.
Establishment of the mouse CIRI model
Before the experiment, the mice were fasted for 12 h and provided free access to water. The CIRI mouse model was induced by middle cerebral artery occlusion (MCAO) according to a previous study [16]. Briefly, the mice were intraperitoneally anesthetized with 2% pentobarbital sodium (45 mg/kg). A silicon-coated suture was then inserted from the distal left common carotid artery and advanced into the internal carotid artery to reach and obstruct the middle cerebral artery (MCA) for 1 h, and then the silicon-coated suture was removed out for reperfusion.
The neurological function of the mice was scored according to the 5-point Longa scoring method [11], and the scoring criteria were as follows: a score of 0 means no neurologic deficit, a score of 1 means failure to extend the contralateral forepaw fully, a score of 2 means circling to the contralateral side, a score of 3 means falling to the contralateral side and a score of 4 means not walking spontaneously and having a depressed level of consciousness. Mice with a score of 2–3 were included in the subsequent experiment.
Grouping
The mice that met the scoring criteria were divided into a good outcome group and a poor outcome group according to observations within 7 days. Mice that died within 7 days were considered to have a poor outcome and mice that survived more than 7 days were considered to have a good outcome. In addition, five mice underwent only incision of the neck skin and vascular separation and were designated as the sham group.
Cerebral cortical blood flow monitoring by LSCI
After anesthesia, the mice were randomly fixed in the prone position on a thermostatic mouse plate to receive the cerebral cortical blood flow monitoring on both sides by LSCI. The operators were blinded to the group allocation. The skin and mucous membrane of the head were separated to keep the skull fully exposed. The LSCI system was focused on the mouse skull to obtain a clear color map. Normal saline was instilled on the skull to maintain a wet condition. The cortical blood flow of the both cerebral hemispheres supplied by the MCA was monitored at baseline (before modeling), 1 h after ischemia, immediately after reperfusion and 24 h after reperfusion. Keeping the mice under anesthesia to obtain stable breathing and heart rates was necessary during the whole monitoring process. Continuous monitoring was performed for 15–30 s. The region of interest (ROI) was set, and the value of the ROI represents the blood flow in the selected region.
HE staining
Mice were anesthetized by 1% isoflurane (R510-22, RWD, China) using a gas anesthesia machine (R540IP, RWD, China) and subsequently perfused by injecting 4% paraformaldehyde (60 ml) through the heart. The brain was removed and fixed with 4% paraformaldehyde. The left damaged brain tissue was dissected to embed in paraffin and then sectioned using a microtome (RM2016, Leica, Germany) with a thickness of 5 μm. The sections were baked in an oven at 60 °C and dewaxed, and then stained with Harris hematoxylin for 3–8 min. After this, the sections were stained with eosin for 1–3 min after washing twice with tap water. Finally, the sections were dehydrated twice with 95% alcohol (5 min each) and washed three times with xylene (5 min each), and then fixed with natural gum. All sections were scanned with a digital pathology scanner system (Pannoramic 250, 3D HISTECH Ltd., Hungary), and 5 fields in injured areas were randomly selected for analysis. The output digital pathological images were 400x enlarged.
Statistical analysis
SPSS version 21.0 (IBM Corp., Armonk, NY, US) was used for statistical analysis, and the data are expressed as the mean ± standard deviation. One-way ANOVA was used to compare differences between groups, and p < 0.05 indicated that the difference was statistically significant. The formal sample size has not been decided due to insufficient data. However, based on previous studies [17,18,19], 3–5 mice per group could meet the statistical requirements.