A Stable Focal Cerebral Ischemia Injury Model in Adult Mice: Assessment Using 7T MR Imaging

Animals

All interventions and animal care procedures were performed in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Animal Surgery provided by our university, and were approved by the institutional Animal Use and Care Committee. One hundred twenty adult male C57Bl/6J mice, 6 weeks old and between 20–25 g, were obtained from the Comparative Medicine Center of Yangzhou University, Yangzhou, China. All animals were housed in a standard animal facility with a 12-hr light-dark cycle and allowed standard food and water ad libitum.

One hundred twenty animals were randomly assigned to 10 groups of 12 each. The first 5 groups (groups I–V) underwent the intraluminal suture occlusion, with suture insertion depths of 0.8 cm–0.9 cm, 1.0 cm, 1.2 cm, 1.4 cm, and maximum (no longer advanceable), respectively. The sixth group (group VI) underwent thromboembolic occlusion. The last 4 groups (groups VII–X) received HI injury with hypoxia exposure times of 30, 60, 90, and 120 minutes, respectively.

Intraluminal Suture Model

The intraluminal suture model was produced according to the previously reported method.⁸ Briefly, after anesthesia with peritoneal injection of 1% pentobarbital sodium (6 mL/Kg), a middle cervical incision was made and the right submandibular gland was separated and the carotid sheath was exposed by a blunt splitting technique. The right CCA was carefully dissected from its bifurcation to the skull base. The thyroid artery and occipital artery branches of the ECA and PPA were ligated with a 5–0 nylon suture. The ECA was dissected and ligated proximally. Microvascular clips were placed on the ICA and CCA to block blood flow. A nylon poly-l-lysine-coated 2-cm suture with a blunt head (Model 1418; Sunbio Biotech, Beijing, China) was inserted via the proximal ECA into the ICA. The insertion lengths of the sutures were 0.8–0.9 cm, 1.0 cm, 1.2 cm, 1.4 cm, and maximum (until no longer advanceable). After 20 minutes occlusion, the clips were removed and the suture was withdrawn to restore blood supply to the MCA territory. The wounds were closed in layers and the animals were returned and housed separately.

Thromboembolic Model

The thromboembolic occlusion was established as described.¹² In brief, fresh heterologous arterial blood (0.2 mL) was rapidly mixed with 0.04 mL thrombin (1 mg/mL, Sigma, St. Louis, Missouri) in an EP tube (Eppendorf, Hamburg, Germany) and then immediately transferred to a 30-cm microcatheter (PE-0402, ID = 0.2 mm, OD = 0.38 mm; Anlai, Ningbo, China). The microcatheter was placed at ambient pressure for 2 hours at 37°C to allow clot formation, followed by 48 hours clot stabilization at 4°C. Five-cm microcatheters were cut, and the clots were rinsed out and immersed in saline for 4 hours at 4°C to remove the remaining red blood cells. The fibrin-rich segments were dedicatedly selected under microscopy inspection, cut into small cylindrical pieces of 1–2 mm in length, suspended in PBS containing 1 mg/mL serum albumin, and again transferred into individual microcatheters. After animals were anesthetized with pentobarbital sodium (6 mL/Kg), the right CCA was exposed and the root of ECA, thyroid and occipital arteries, and PPA were ligated. A microvascular clip was placed on the CCA. The emboli-contained microcatheter was inserted through the incision of the ECA and was fixed with a suture. The clot was injected over a period of 5 seconds, and then the microvascular clip was removed and the microcatheter was withdrawn.

HI Model

The HI model was produced according to the previously reported method,⁵ with minor modification. Briefly, after the mice were anesthetized with pentobarbital sodium (6 mL/Kg), the carotid sheath was exposed and the right CCA was isolated and ligated with a 5–0 nylon suture. After animals recovered from the anesthesia, they were transferred to a chamber ventilated with a hypoxic gas mixture (8% O₂ + 92% N₂) at 1 L/min airflow. After 30-, 60-, 90-, or 120-minute hypoxia exposure, animals were retrieved from the hypoxia chamber and housed separately in a standard animal facility.

Neurologic Impairment Scoring

Twenty-four hours after surgery, neurologic impairment was assessed as described.¹ The neurologic findings were scored on a 5-point scale: a score of 0 indicated no neurologic deficit, a score of 1 (failure to extend left forepaw fully) indicated a mild focal neurologic deficit, a score of 2 (circling to the left) indicated a moderate focal neurologic deficit, and a score of 3 (falling to the left) indicated a severe focal deficit; mice with a score of 4 could not walk spontaneously and had a depressed level of consciousness. Scores between 1 and 4 were considered modeling success.

MR Imaging

After neurologic assessment, MR imaging was performed on a 7T animal scanner (PharmaScan, Bruker, Germany) using a 23-mm mouse brain coil. Anesthesia was induced by 1.5% halothane and maintained with 1% halothane in 70% N₂O and 30% O₂. Animals were positioned in a plastic holder with a stereotaxic headframe, with heart rate and respiratory monitoring. Coronal and axial brain images were obtained with a rapid acquisition with relaxation enhancement T2-weighted imaging acquired with the following parameters: TR = 5000 ms, TE = 60 ms, section thickness = 0.5 mm, section gap = 0 mm, FOV = 35 mm × 35 mm, matrix size = 256 × 256, flip angle = 180°, number of signal intensity acquisitions = 6.

Imaging Evaluation

On T2-weighted images, the infarct volume was measured and the infarct location was observed. For infarct volume measurement, the obtained coronal MR images in DICOM format were processed in ImageJ software (National Institutes of Health, Bethesda, Maryland). Appropriate contrast enhancement of the images was completed to maximize the signal intensity from the hyperintense cerebral infarct region. Manual planimetry was performed. For each section, the hyperintense cerebral infarct region and total brain area were manually outlined. Section hyperintense areas were then summated to generate infarct volume as a percentage of total brain volume, according to the following formula¹³: infarct percentage = infarct volume / total brain volume * 100% = (A₁ + A₂ + A₃ +. A_n) t / (V₁ + V₂ + V₃ +. V_n) t, where A represents the infarct area, V represents the total brain area on each section, and t is the section thickness.

Survival Rate

At 1 day and 7 days postsurgery, the number of living animals in each group was recorded and survival rates were calculated.

Histologic Evaluation

At 1 day after MR imaging, 2 successfully modeled animals in each group were randomly sacrificed by overdose of anesthesia for TTC. The whole brain was removed and preserved at −80°C for 20 minutes and then cut into 6 coronal sections of 2-mm thickness. All sections were incubated with 2% TTC in PBS for 30 minutes at room temperature, then fixed in 4% paraformaldehyde for 5–6 hours. Images of the sections were acquired using a stereomicroscope equipped with a digital camera system (SEZ 300; Shenzhen Finial Technology, Shenzhen, China). TTC staining was evaluated as described.¹⁴ The infarct location and size were observed.

Statistical Analysis

The ordinal data were presented as means ± SD. The infarct volume was compared using ANOVA, followed by a post hoc Student-Newman-Keuls test. The sample size (n = 12 in each group) was determined as previously described,¹⁵ and a statistical power of 0.90 could be achieved using the ANOVA test. The survival rates and infarct locations were compared using Fisher exact test. A P value < .05 was considered statistically significant. All statistical analyses were performed with SPSS 13.0 software (SPSS, Chicago, Illinois).