Development of a Modified Surgical Technique for Simulating Ischemic Cerebral Cortex Injury in Rats

Materials and Methods

Animals. All animal experiments were approved by the Institutional Animal Care and Utilization Committee (IACUC) of National Yang-Ming University, Taipei, Taiwan (approval ID: 1050315). Ten-week-old male Wistar Kyoto (WKY) rats weighing 200-300 g each were purchased from BioLASCO (Taipei, Taiwan). The rats were housed in the Laboratory Animal Center in National Yang-Ming University with a 12-h light/dark cycle at a constant temperature (25±2°C), and with unrestricted access to water and food.

Modified surgical method of MCAO. In order to improve the efficiency of MCAO, a modified surgical method was developed. Rats were anesthetized with an intraperitoneal injection of Zoletil (30 mg/kg) and Xylazine (8 mg/kg). A vertical incision was made on the midline of the neck, and the bilateral common carotid arteries (CCAs) were identified. After retracting the temporal muscle, a craniotomy was performed on the right temporal bone using a cutting burr (Figure 1a and b). During this period, the inferior edge of the hole was lowered as much as possible, until interrupted by the zygomatic arch. Then Dumont forceps (Roboz, Gaithersburg, MD, USA) were used to hold the residual bone on the inferior edge of the hole, with one tip between the dura and inner surface of cranial bone, and another tip on the outer surface of cranial bone (Figure 1c). After holding it with about 1mm contact depth, the bone was pulled out piecemeal with a lateral and superior direction until the olfactory tract was exposed (Figure 1d). Then the dura matter was removed, and the MCA was permanently occluded by suture ligation at the level of the olfactory tract. Subsequently, bilateral CCA occlusion was performed by vessel clamp temporarily for 90 min.

Another rat group was subjected to MCAO for purposes of comparison, using the conventional method. In this group, rats underwent the above procedure, but forceps were not used to remove the residual bone on the inferior edge of the hole. Thus, MCA was exposed to the level above the inferior cerebral vein and the distal MCA was permanently occluded by suture ligation above the inferior cerebral vein. Bilateral CCA occlusion was also performed temporarily for 90 min. The sham group received identical anesthesia and surgical procedure without MCA and CCA occlusion. Rats that died during or after surgery were excluded for further experiment (n=7).

Animal Behavioral Test. The rotarod test was utilized to evaluate motor coordination and balance alterations. Before MCAO was applied, rats were trained for running and balancing on the rotating drum (Rotamex-5, Columbus Instruments, Columbus, OH, USA) for 3 days. The rats deemed stable and that performed well on rotarod were assigned to groups that randomly received modified and conventional MCAO. At 1 (n=13/group) and 3 (n=7/group) days after MCAO, the rotarod was accelerated from 4 rpm to 40 rpm, with an acceleration of 1 rpm/10 sec. The latency before falling off the drum was recorded. Every rat received three trials for each training and testing day and the interval between each trial was 10 min. Successful trials after MCAO were defined as less than 75% running time on rod at 1 day after surgery, compared with preoperative performance.

Quantification of Infarct Volume. At 1 (n=8/group) and 3 (n=4/group) days after MCAO, rats were anesthetized by intraperitoneal injection of Zoletil and Xylazine and transcardially perfused with phosphate-buffered saline (PBS). Their brains were expeditiously removed from the skull to determine infarction size, and then sliced into 2-mm-thick coronal sections. Then, they were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, St. Louis, MO, USA) solution for 15 min at room temperature without light, and then fixed with 4% paraformaldehyde. The unstained area represented the infarcted region, and the non-infarcted region revealed as red color on TTC-stained sections. To eliminate the effect of brain edema, the infarcted region was determined by subtracting the non-infarcted region in the ipsilateral hemisphere from that in the contralateral hemisphere. Infarct volumes were then calculated as the sum of the infarcted areas on each brain slice by using Image J software (National Institutes of Health, Bethesda, MD, USA).

Measurement of inflammatory cytokines. At 1 (n=4/group) day after MCAO, rats were anesthetized by intraperitoneal injection of Zoletil and Xylazine and transcardially perfused with PBS. Their brains were removed and stored in −80°C ultra-low temperature freezer. Then, the cerebral cortex of MCAO rats and sham group were homogenized and the levels of inflammatory cytokines (IL-1β and IL-6) were measured using commercially available ELISA kits (R&D, Minneapolis, MN, USA).

Statistical analysis. Data are expressed as mean±the standard error of the mean (SEM). Categorical data were compared using chi-square test. Independent samples t-test was used to analyze the difference between two groups. All statistical analyses were performed using the SPSS 24.0 software package (SPSS, Inc, Chicago, IL, USA). Differences were considered significant at p<0.05.

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Figure 1.

The procedures of the modified surgical method for middle cerebral artery occlusion (MCAO). (A) After skin incision and temporal muscle retraction, craniotomy contour was created using a burr tool. (B) Craniotomy and exposure of the underlying brain tissue. (C) Removal of the residual bone on the inferior edge of the craniotomy (arrow) using fine forceps. (D) Exposure of the middle cerebral artery at the level of olfactory branch (arrow) and preparation for MCAO.