The Protection Of Nasopharyngeal Cooling Against Globle Cerebral Ischemia-reperfusion In Rats

Objective:By using the method of modified Pulsinelli's four-vessel occlusion to establish the model of global cerebral ischemia-reperfusion injury in rats, to observe the speed of the whole body cooling and nasopharyngeal cooling, to detect the concentration of extracellular glutamate ([Glu]e)in hippocampal CA1 field and the expression of Bcl-2 and Bax during global cerebral ischemia-reperfusion injury.Method:Eighteen male Wistar rats weighing 200±50g were randomly divided into 3 groups(n=6): Normal temperature group/group A; Whole body cooling group/group B; Nasopharyngeal cooling group/group C.Anaesthesia was induced by an intraperitoneal injection of 10% chloral hydrate (0.3ml/100g). The trachea was cannulated via endotracheal intubation. Electroencephalogram (EEG) and electrocardiogram (ECG) were continuously monitored. A cannula was placed through the right femoral vein into the vena cava for local anesthetic and sodium lactated Ringer's solution infusion. The global cerebral ischemic model was established by Pulsinelli's four-vessel occlusion: The bilateral vertebral arteries were electrocauterized permanent, after 24h, the bilateral common carotid arteries were occlused for 20 min. After placement in a stereotaxic apparatus, A microdialysis probe was implanted in the left hippocampal CA1 region (2 mm to the left and 3.6 mm posterior to the bregma and 2 mm below the cortical surface). The microdialysis probe was perfused with Ringer's solution at a rate of 2.5μL/min using an infusion pump, and the dialysates were collected every 10 mins for 2h after onset of reperfusion. The right hippocampal temperature was measured by placing a small thermocouple in the CA1 region.Normal temperature group/group A: both the rectal temperature and the hippocampal temperature were maintained at 37±0.5℃by using a heated water blanket and an infrared lamp during the experiment. The whole body cooling group/group B: the hippocampal temperature was reduced to 33℃and maintained it for one hour by using a fan and a ice bag. Nasopharyngeal cooling group/group C: gel silica pipes were inserted into both nasal cavities, and cold physiologic saline (5℃) was infused at a rate of 100 mL·min-1·kg-1 until the hippocampal temperature decreased to 33℃. A cotton ball was placed in the lower pharynx and a suction device was placed for drainage, so as not to cause aspiration of saline. The rectal temperature was maintained at 37.0±0.5℃using a heated water blanket and an infrared lamp. Arteria carotis communes were clamped for 20min when the temperature was to 33℃in group B and group C, and the objective temperature was maintained for 60 mins, and then rewarming was initiated with the use of heated water blanket and an infrared lamp.After reperfusion for 8h, all rats were anesthetized again, fixed with 4% PFA perfused via ascending aorta. Then, the rats'brain were carefully sampled and stored in 4℃. What's more, specimans of the brain were used in immunohistochemical test to observe the expression of Bcl-2 and Bax. The dialysates were stored under -70℃and be used to determine the concentration of glutamate by high performance capillary electrophoresis(HPCE).Result:1. There were no statistical differences of the rats'weight in the three groups.2. The comparison of the cooling speed of hippocampal temperature from 37℃to 33℃It took 31±2.7min in group B and 6.5±0.7min in group C for reducing to 33℃from 37℃of hippocampus, this onset latency in hippocampal temperature reaching 33℃was about 5 times longer than that in the nasopharyngeal cooling group.3. There were no statistical differences of the [Glu]e before ischemia in the three groups.4. The comparison of [Glu]e in hippocampus CA1 field during ischemiaAt 10min and 20min of ischemia, the [Glu]e was significantly lower in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P>0.05). 5. At 10min, 20min, 30min after ischemia, the [Glu]e in hippocampus CA1 field was significantly lower in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P>0.05).6. The time of [Glu]e in hippocampal CA1 field returning to the level of control The time of [Glu]e in hippocampal CA1 field returning to the level of control was 40 min,20min,20min in group A,B and C respectively.7. The expression of Bax in hippocampal CA1 field The expression of Bax level in hippocampal CA1 field was significantly lower in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P>0.05).8. The expression of Bcl-2 level in hippocampal CA1 field The expression of Bcl-2 level in hippocampal CA1 field was significantly higher in both group B and group C compared with group A (P<0.05). There were no statistical differences between group B and group C(P>0.05).Conclusion:1. Nasopharyngeal cooling protects brain from global cerebral ischemia-reperfusion same as whole body cooling does. Both of cooling methods reduce [Glu]e, weaken the expression of Bax and strengthen the expression of Bcl-2 in hippocampus CA1 field.2. The cooling speed of nasopharyngeal cooling method is more quicker than that of the whole body cooling method, and this onset latency in hippocampal temperature reaching 33℃was about 5 times longer than that in the nasopharyngeal cooling group.