| Objective: Four-vessel occlusion was used to establish a model of globalcerebral ischemia-reperfusion in rats, on this basis nasopharyngeal cavitycooling method was used to reduce the rats’head temperature, andmitochondrial permeability transition pore(MPTP)opener atractyloside wasinjected in the lateral ventricle before global cerebral ischemia. The purpose ofthis study was to observe the opening of MPTP and the expressions of Cyt C,Bax, Bcl-2protein in hippocampal CA1region during reperfusion. The effectsof head mild hypothermia on the opening of MPTP and cell apoptosis in theglobal cerebral ischemia-reperfusion injury were investigated and themechanism of cerebral protection with hypothermia was discussed in rats.Methods:1Experimental groupsSeventy-two healthy male SD rats weighing250~300g were provided bythe Experimental Animal Center of Hebei Medical University and randomlydivided into six groups (n=12): Sham group (group S); Global cerebralischemia-reperfusion injury group (group IR); Mild hypothermia group (groupH); Atractyloside group (group A;) Combination of Atractyloside and mildhypothermia group (group HA); Combination of normal saline and mildhypothermia group (group HN).2Preparation of global cerebral ischemia-reperfusion modelThe global cerebral ischemia-reperfusion model was established by usingmodified Pulsinelli four-vessel occlusion. The rat was anesthetized viaintraperitoneal injection with10％chloral hydrate（400mg/kg）after fasting8hours, but water free, and then fixed on the experimental table with proneposition. The skin was incised on the center of the first and second cervical vertebra after shearing and disinfecting, the double flank holes were exposedand the bilateral vertebral arteries were cauterized to occlusion. Aftertwenty-four hours, the rat was anesthetized again with the same method andfixed on the experimental table with supine position, The spontaneousbreathing was kept after endotracheal intubation. The neck skin of anteriormidline was incised after shearing and disinfecting, the bilateral commoncarotid arteries were separated and a silk was threaded. In the process, the ratwas maintained the anesthesia with inhaling sevoflurane and the caudal veinwas inserted trocar to maintain circulation by pumping lactated Ringer’ssolution.3Experimental treatments and monitoringGlobal cerebral ischemia time was15min and reperfusion time was24h.Different groups were given different treatments: In group S, rats wereexposed to the double flank holes without cauterizing the bilateral vertebralarteries, and just separated the bilateral common carotid arteries, but were notoccluded. In the other five groups, the bilateral vertebral arteries werecauterized and the common carotid arteries were occluded to make the globalcerebral ischemia-reperfusion model. In group H, group HA and group HN,the rats were treated by head hypothermia. Two cotton balls and a sputumsuction tube was placed into the pharynx to avoid aspiration after endotrachealintubation. Bilateral nasal cavities were inserted in two20G silicone tubes(depth of20mm) and cold saline (4-5℃) was continued to pump into at a rateof100mL·min-1·kg-1to apply nasopharyngeal cooling. When the hippocampaltemperature was dropped to (33.0±0.5)℃, bilateral common carotid arteriesclamped for15min and the target hypothermia was maintained for1h, thenrewarmed naturally. In group HA and group A,15ul atractyloside was slowlyinjected into the rats’left ventricle and the same volume of saline was given10min before reperfusion in group HN. The electroencephalogram (electrodepierced into the scalp) and the electrocardiogram (electrode pierced into thesubcutaneous of limbs) was monitored during the experiment. A temperatureprobe was inserted into the right hippocampal CA1region (anterior fontanel 3.6mm, the midline right side3mm and subcortical2mm) to monitor thehippocampal temperature. The rectal temperature was maintained at(37.0±0.5)℃.4Sample preparation and detection4.1Using ultraviolet spectrophotometry to detect the opening ofmitochondrial permeability transition pore (MPTP)Six rats were selected from each group after they were reperfused for24h. After anesthetizing with10％chloral hydrate（400mg/kg）, thehippocampal tissues were rapidly decollated and obtained, the hippocampalmitochondria was extracted according to qproteome mitochondria isolation kit.The whole process were completed under0~4℃. The mitochondria wereused to detect the opening of MPTP.4.2Using immunohistochemical method to determine the expression ofCyt C, Bax, Bcl-2protein in hippocampal CA1regionThe other six rats were taken from each group. The rats were perfusedwith4％paraformaldehyde (PFA) to fix tissues after they were anesthetized.Then the brain tissues were taken out carefully and continued to fix in4％PFAand stored at4℃. These samples were used for immunohistochemicaldetection.Results:1The rats’ weight of six groups were no statistically significant difference(P>0.05).2The comparison of opening of MPTP in hippocampus regionCompared with group S, the decreased degree of MPTP absorbance valuewas increased and the MPTP opening was increased in the other groups（P<0.05）; Compared with group IR, the decreased degree of MPTPabsorbance value was decreased and the MPTP opening was decreased ingroup H、 group HN and group HA（P<0.05）; Compared with group H, thedecreased degree of MPTP absorbance value was increased and the MPTPopening was increased in group HA,(P<0.05).3The comparison of Cyt C protein in rat’s hippocampus CA1region Compared with group S, the expression of Cyt C protein was raised in theother five groups（P<0.05）； Compared with group IR, the expression of CytC protein was decreased in group H, group HA and group HN（P<0.05）；Compared with group H, the expression of Cyt C protein was higher in groupHA (P<0.05); There was no statistically significant difference between groupH and group HN, group IR and group A (P>0.05).4The comparison of Bcl-2、 Bax protein and Bcl-2/Bax ratios inhippocampus CA1region.Compared with group S, the expression of Bcl-2and Bax protein werehigher in the other groups, Bcl-2/Bax ratio was higher in group H and groupHN, Bcl-2/Bax ratio was lower in group IR and groupA (P<0.05); Comparedwith groupIR, the expression of Bcl-2protein、Bcl-2/Bax ratio was higher,Bax protein was lower in group H and group HN (P<0.05); Compared withgroup H and group HN, the expression of Bcl-2protein、Bcl-2/Bax ratio waslower and the expression of Bax protein was higher (P<0.05). There was nostatistically significant difference between group H and group HN，group IRand group A(P>0.05).Conclusions：1Mild head hypothermia can inhibit cell apoptosis and availably relieveglobal ischemia-reperfusion injury.2The protection of mild head hypothermia against cerebralischemia-reperfusion injury is related to inhibit the MPTP opening. |
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