Animals
The study was conducted on male adult Sprague–Dawley rats weighing 200–250 g obtained from the Animal House of Shiraz University of Medical Sciences. All procedures were in accordance with the Institutional Animal Ethics Committee of Shiraz University of Medical Sciences and followed the NIH Guidelines for care and use of animals (NIH Publication No. 85–23, revised in 1996). The animals were kept under controlled temperature (22°C–24°C), humidity (40%–60%), and artificial 12‑hour light/dark cycle, and were fed a regular diet (Pars Dam, Iran) and water ad libitum.
Experimental Protocol and Groups
Animals were randomly divided into one of two groups: hypothyroid (HPO) and control normal (CN). In the HPO group, the hypothyroid status was induced over a 4‑week period by the oral administration of methimazole (Iran Hormone Company, Iran) in drinking water at a concentration of 0.025% w/v. In the CN group, the rats were treated in the same manner as the HPO group, except that they were given tap water for the 4‑week period. At the end of the treatment period, the animals of each group were randomly divided for surgery as follows:
Ischemic CN (ICN, n=14) group: Surgery was accomplished at the neck region, and the right MCA was occluded for 60 minutes, followed by reopening and 24 hours of reperfusion.
Sham CN (SCN, n=8) group: All procedures were done like the ICN group, with the exception of MCA occlusion.
Ischemic HPO (IHPO, n=14) group: Experiments were performed like the ICN group.
Sham HPO (SHPO, n=8) group: All procedures were accomplished like the SCN group.
Subsequently, the rats of each group were further divided into 2 subgroups for the evaluation of: 1) cerebral infarct size and 2) BBB integrity. The overall success rate of the induction of brain ischemia was 60%, and the mortality rate of ischemia during the 24‑hour period of reperfusion was 10% in all groups. The data presented in this study were obtained from animals that tolerated 60 minutes of brain ischemia and survived 24 hours after reperfusion.
Parameters Measured during the First Period of the Experiment
In both HPO and CN groups, the water intake was measured daily during the first week and every other day afterward. Body weight and systolic blood pressure (with a tail‑cuff plethysmography and a Power Lab system) were measured once a week. The levels of thyroid hormones and core body temperature were checked at the beginning and the end of the treatment period. The blood sample was obtained by slightly anesthetizing the animal with isoflurane and collecting 1 mL of blood from the tip of the snipped tail. Afterwards, the samples were centrifuged (4,000 × g), and the separated serum was stored in a freezer (‑70 ⁰C) until further processing. Plasma T3 and T4 quantitative measurements were performed with ELISA (DiaPlus, Canada). In addition, the achieved hypothyroidism was confirmed by various clinical signs such as decreased body temperature, systolic blood pressure, bradycardia, and decreased body weight.
Surgical Procedures
The animals were fasting overnight prior to surgery, but they had free access to water. Anesthesia was induced through a facemask with 3% isoflurane in 30% oxygen and 70% nitrous oxide and maintained by 1.5% isoflurane in the same gas mixture. Continuous recording of blood pressure was done during surgery via cannulation of the right femoral artery. Similarly, core temperature was continuously recorded by means of rectal temperature probe and maintained at 37±0.5⁰C with a heating pad and a lamp.
Measurement of Regional Blood Flow
A laser Doppler flowmeter (AD Instrument, model: ML191, Australia) was used to measure the regional cerebral blood flow (rCBF) of the right hemisphere that receives its blood flow from the right MCA, according to the method described by Lecrux et al(Lecrux, Nicole et al. 2007). MCA occlusion was considered successful if there was a 75%–85% decrease in rCBF and a swift return to the pre‑occluded level after reopening (Figure 1).
Induction of Focal Cerebral Ischemia
Transient focal cerebral ischemia was induced by using the intraluminal threated technique of Longa et al. (Longa, Weinstein et al. 1989). Cerebral ischemia was started by advancing prepared silicon-coated nylon thread through the exposed right carotid artery up to the cranium and circle of Willis until the origin of MCA was occluded. Occlusion was confirmed by the observation of a sharp decline in rCBF trace. Reperfusion of the ischemic hemisphere, starting after pulling out the thread, was confirmed by restored rCBF. Finally, all instruments were removed, incisions were sutured, and the animals were allowed to recover from anesthesia and returned to a warm cage for recuperation during a 24‑hour reperfusion period.
Evaluation of Neurological Disability
Evaluation of neurological motor function was carried out blindly on every animal 24 hours after surgery or ischemia by using a 5‑point neurological deficit score (NDS) described previously. Then, the animals were scarified and their brain was prepared for the evaluation of ischemia infarct volume or BBB disruption.
Measurements of Cerebral Infarct Volume
Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride staining method according to Swanson et al. (Swanson, Morton et al. 1990). After deep anesthesia with sodium thiopental, the animals were beheaded and their brains were removed, cleaned, and sliced. Staining was achieved with 2% TTC (2, 3, 5‑triphenyltetrazolium chloride, Sigma), as previously described. The images of the slices were digitized by using a Cannon camera. The visible infarct zones were quantified using image analysis software. Finally, cerebral infarct volume was calculated, as described previously. Finally, cerebral infarct volume was calculated as described previously.
Quantitative Measurement of the Blood-Brain Barrier Permeability
The integrity of the brain vessels was quantitatively assessed using Evans Blue (EB) extravasation. After preparation, the optical density values of the prepared samples were measured at 620 nm with a Microplate reader (BioTek model: Gen 5, Germany). The tissue contents of EB were calculated according to the standard curves of dyes described, and the results are expressed as μg/g wet tissue weight.
Statistical Analysis
All values are presented as means±SEMs. The analyses were performed using SPSS, version 21. ANOVA with repeated measures was used to evaluate the changes that had occurred during the 4‑week treatment period in the body weight, daily drinking water, heart rate, blood pressure, and core temperature. One‑way ANOVA with the Tukey post hoc test was used to evaluate variations in the rCBF, infarct volume, and BBB permeability. The Kruskal-Wallis test was also utilized to evaluate the NDS. Values of P<0.05 were considered statistically significant.