| Objective: Using a model of Pulsinelli's four-vessel occlusion global cerebral ischemia and initiating nasophar- yngeal cooling at different onsets to detect the concentration of extracellular Glutamate ([Glu]e)in hippocampal CA1 and the expressions of Bcl-2 and Bax, and to investigate the therapeutic window of mild hypothermia for global ischemia-reperfusion.Method: Twenty-four male Wister rats (weight 250±20g) were randomly divided into four groups (n=6) according to the onset time of hypothermia (33°C, 1h). Normothermia group (group A): normothermic control group; Preischemia hypothermia group (group B): temperature was reduced to 33℃before the onset of 20 min ischemia; Intraischemia hypothermia group (group C): hypothermia was initiated at the onset of ischemia; Postischemia hypothermia group (group D): hypothermia was initiated at the onset of reperfusion.Anaesthesia was induced by an intraperitoneal injection of 10% chloral hydrate (0.3ml/100g body weight). 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 (3mm to the left and 3.6mm posterior to the bregma and 2mm below the cortical surface). The right hippocampal temperature was measured by placing a small thermocouple in the CA1 region. Rectal temperature was continuously monitored and controlled at 37.0±0.5°C using a heated water blanket and an infrared lamp.Selective brain hypothermia was initiated by a nasopharyngeal cooling method. In brief, silicon cannulas were inserted into both nasal cavities to a depth of 5mm. Cold physiologic saline (5°C) was infused at a rate of 100 mL·min-1·kg-1 until the hippocampal temperature had decreased to 33°C.The microdialysis probes were 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.After the above, all rats were deeply anesthsized and fixed with 4% PFA which was perfused via ascending aorta. Then, the rats'brain were carefully removed and stored under 4°C. What's more, specimans of the brain were treated for immuno- histochemical test to observe the expression of Bax and Bcl-2 in hippocampal CA1. Dialysates were stored under -70°C and determined the concentration of Glutamate by high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE-LIF).Result:1 The concentration of extracellular Glutamate ([Glu]e)in hippocampal CA1 during ischemia.During the first 10 min, there was no difference of the [Glu]e between group A and groupD (P>0.05); [Glu]e in group B and group C decreased compared with group D (P<0.05); [Glu]e in group B decreased compared with group C (P=0.037).During the second 10 min, there was no difference of the [Glu]e between group A and group D (P>0.05); [Glu]e in group B and group C decreased compared with group D (P<0.05); there was no difference of the [Glu]e between group B and group C (P>0.05).2 The concentration of extracellular Glutamate ([Glu]e)in hippocampal CA1 during reperfusion.During the first and the second 10 min, [Glu]e in group B,C,D decreased compared with group A (P<0.05); [Glu]e in group B and group C decreased compared with group D(P<0.05).During the third 10 min, [Glu]e in group B,C,D decreased compared with group A (P<0.05); there were no differences of the [Glu]e among these three groups.3 The time of [Glu]e in hippocampal CA1 recovering to the level of baselineThe time of [Glu]e in hippocampal CA1 field recovering to the level of baseline was 40 min,20min,20min,30min in group A,B,C,D respectively.4 The expression of Bcl-2 and Bax in hippocampal CA1 The expression of Bcl-2 in group B,C,D was increased compared with group A (P<0.05).The expression of Bax in group B,C,D was decreased compared with group A (P<0.05).Conclusion:1 Nasopharyngeal cooling initiated at different onsets has neuroprotective effect on ischemia-reperfusion injury.2 Observing the change of extracellular glutamate, the earlier nasopharyngeal cooling initiates, the better neuroprotective effect is. |
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