Protective Effects Of Xenon Combined With Hypothermia Intervention On Inhibition Of Autophagy In HIBD Neonatal Rats

ObjectiveWe established a model of hypoxic-ischemic brain damage in 7-day-old neonatal rats that was intervened by xenon combined with hypothermia to decteced Beclin-1 and LC3 realted with autopathy to explore the neuroprotective mechanism.Metheods7-day-old SD rats(n=100)were divided into sham group(n=20),hypoxic ischemic(HI)group(n=20),xenon(Xe)group(n=20),hypothermia(HT)group(n=20)and xenon combined with hypothermia(Xe+HT)group(n=20)randomly.The rats in the sham group did not have any treatment but ligatured of the left common carotid artery.The rats in the HI group were ligatured of the left common carotid artery and hypoxia for 1.5h.The rats in the Xe group were treated as HI group,rats breathed with 50% xenon mixed with 30%oxygen and 20% nitrogen for 3 hours.The rats in the HT group were treated as HI group and maintained the anal temperature at 32℃ for 3 hours.The rats in the Xe+HT group were treated as HI group,rats breathed with 50% xenon mixed with 30% oxygen and20% nitrogen and maintained the anal temperature at 32℃ for 3 hours.Monitored index of this groups of neonatal rats after operation:(1)Neurobehavioral function was measured by Longa score and Negative geotaxis test after 72 hours and Open field test and Trapeze test after 28 days;(2)Histopathological morphology changes in the cerebral tissues were detected using hematoxylin and eosin stain,transmission electron microscope and TTC stain;(3)Detected the beclin-1 and LC3 protein expressions in neurons of left cerebral tissues by immunohistochemistry and western blot;(4)Detected the beclin-1 and LC3 m RNA expressions in neurons of left cerebral tissues by Real-Time PCR.Results(1)Neurobehavioral: Rats was monitored after 72 h.The rats in the control group with flexible limb and normal motor function,the rats in the HI,HT,Xe and Xe+HT groups all showed of right limb weakness,poor right forepaw extension and even dumping.The Longa score results,the HI group(2.3±1.2),HT group(2.4±0.8),Xe group(2.2±0.8)and Xe+HT(2.4±0.9)were higher than the sham group(0.2±0.4).The Negative geotaxis test results,the HI group(10.6±2.8),HT group(9.2±2.8),Xe group(9.5±2.4)and Xe+HT(8.9±3.2)were higher than the sham group(5.2±1.3)(P<0.05).Rats was monitored after28 days.The Open field test results,the HI group(7.8±3.5),HT group(10.3±3.2)and Xe group(11.5±3.9)were lower than the sham group(21.3±6.5).The Xe+HT group(15.8±5.3)was higher than the HI group.The Trapeze test results,the HI group(1.5±0.5)was lower than the sham group(3.0±0.8)(P<0.05).(2)Histopathological morphology changes: In the HI group left cerebral tissue appeared swelling,gray and demarcation,blood stasis and necrosis;HE staining showed that neuron swelling,arrangement disorder,some karyopyknosis and fragmentation;characteristic auto-phagosomes with double membranes structure were discovered under the electron microscopy.In the Xe group and HT group left cerebral tissue showed a lower degree about swelling,gray and demarcation,blood stasis,necrosis.In the Xe+HT group,the left cerebral tissue showed a approximately normal histopathological morphology.The area of cerebral infarction with TTC staining(%),the HI group(55.62±17.94%)was higher than the sham group(1.13±1.81%);the Xe group(33.72±7.45%)and Xe+HT group(13.77±6.56%)were lower than the HI group;and the Xe+HT group was lower than the HT group and Xe group(P <0.05).(3)Immunohistochemistry results: The positive area ratio of Beclin-1 and LC3(%),the HI group(25.78±2.34%,30.50±1.78%),HT group(15.41±0.54%,22.04±1.54%),Xe group(20.40±1.63%,20.50±1.70%)and Xe+HT group(10.49±1.02%,14.55± 1.03%)all were higher than the sham group(5.70±1.10%,5.51±0.90%);the HT group,Xe group and Xe+HT group were lower than the HI group;and the Xe+HT group was significantly lower than the Xe group and HT group(P<0.05).(4)Western blot results: The proteins expression of Beclin-1 and LC3-Ⅱ in the left brain tissues,the HI group(1.29±0.11,1.05±0.06)was higher than the sham group(0.42±0.04,0.29±0.02);the Xe group(1.04±0.13,0.68±0.05),HT group(1.11±0.10,0.83±0.07)and Xe+HT group(0.59±0.07,0.41±0.03)were lower than the HI group;and the Xe+HT group was significantly lower than the Xe group and HT group(P<0.05).(5)The Real-Time PCR results: The m RNA expression of Beclin-1 and LC3 in the left brain tissues,the HI group(3.77±0.31,3.23±0.28),HT group(2.37±0.11,2.62± 0.19),Xe group(1.88±0.15,2.56±0.53)and Xe+HT group(1.29±0.06,1.43±0.20)were higher than the sham group(1.01±0.18,1.00±0.68);the HT group,Xe group and Xe+HT group were lower than the HI group;and the Xe+HT group was significantly lower than the Xe group and HT group(P<0.05).Conclusions(1)In the neonatal rats,neuron autophagy was involved in the pathophysiology of Hypoxie-ischemic brain damage.(2)Xenon combined with Hypothermia intervention can significantly down-regulated the proteins and m RNA expression of Beclin-1 and LC3,and performanced neuroprotective effects by alleviated the neuron autophagy.