Animal Study About The Hypothermia Treats Rats After Delayed Ischemic Neurological Deficits

Objective: On the bases of stable and reliable animal model, to study the affectof applying the hypothermia treatment to the rats after the delayedischemic nerurological deficits (DIND) which were induced bysubarachnoid hemorrhage. The index we used to contrast were thediscrepancy of the cortex blood flow, the pathology changes of thebasilar artery, the apoptosis of brain cell and expression different ofits related genes, and the change of cerebral metabolism.Material and Methods: 1,100 rats are divided into 3 groups, normal, hypothermia and controlrespectively. Introduce delayed vasospasm animal model by twostages blood injection. The contrastive study is beginning at the 5thday after the second inject. The hypothermia group was maintain31-32℃for 8 hours and the other two groups were maintain atnormal temperature.2,Using the LDF to inspect the different cortex blood flow among threegroups, in company of the pathology change of the basilar artery,which were checked by electronic microscope, to confirm thepresence of vasospasm.3,Discussing the effect of hypothermia to the DIND by the differentiaof cortex blood flow and the pathology changes of the basilar artery. 4,According to the HE staining and TUNEL staining to check the cellapoptosis of the district of CA1; contrast the different expression ofthe related protein Bcl-2 and Bax in CA1 by immunohistochemicalstaining; By the way of fluorescent PCR to study the differentia ofapoptosis related genes (Bcl-XL, ICE) in cortex, hippocampus andbrain stem.5,Study the changes of Glucose, Lactate, Pyruvic acid and the ratio ofL/P by microdialysis.6,Use one-way ANOVA to analysis the difference between groups, anduse LSD to study difference between every two groups.Result:1,The mortality of our rats is 10%, and the distribution of blood clotwas seen in the basal cisterns, interpeduncular cisterns,on the base ofthe frontal lobe. Methylthioninium Chloride with blood was mainlyobserved in the subarachnoid space on the base of the brain,bloodwas seen in the ventricle and diastematia cistern as well. However, incontrol group none of the rats developed blood and no blooddistribution was seen. The model of SAH was induced successfullyaccording to the distribution of blood, the obviously reduce of cortexblood flow and the pathological changes of the basilar artery.2,There was only slight decrease of cortex blood flow in normal group,obvious decrease were seen in both hypothermic and controlgroups(p＜0.05). The statistic study also confirmed the discrepancy inevery hour.3,Light microscopic examination: Different degrees of cerebralvasospasm were observed in hypothermic and control groups,especially severe in control group (p＜0.05). The basal artery revealedluminal narrowing, reduction in diameter, increased wall thickness, corrugation of the internal elastic lamina (IEL), and cellularproliferation were observed in the spastic BA as well. However, thehypothermic group is better then the control group. In control group,the mean diameter of BA was reduced from control levels by 50.59%;the increases in wall thickness of BA were 159.27%; the meanluminal perimeter reduced by 62.50%. As far as the wall thickness ofBA, obvious difference was seen between the hypothermic and thecontrol group. Transmission electron microscopic examination:Apoptotic-like changes were noted in endothelial cells in both thehypothermic and the control groups, consisted of blebbing of the cellmembrane, condensation of the cytoplasm, condensation ofperipheral nuclear chromatin, and cytoplasmic vacuolization. Themorphological changes in endothelial were most severe and widespread in control group with some endothelial cells and smoothmuscle cells showed evidence of necrotic changes. Detachment ofendothelial cells was widespread resulting in large areas ofdenudation from the IEL. The same pathological changes were alsoseen in hypothermic group, but the degree were obviously alleviated.4,The HE staining of CA1 showed that more necrotic and metamorphicneuron can be seen in control group, there nucleolus were seenconcentration, heavy coloration, dissolution and disappearance.Although these changes also can be seen in hypothermic group, thedegree was obviously mitigated than control group. The moreapoptosis cells were seen in control group by TUNEL staining. Therewas obvious difference between hypothermic and control groups(p＜0.05). Immunohistochemical staining showed that the sequence ofprotein bcl-2 among three groups was hypothermia, normal, control.There was statistic difference between hypothermic and control groups (p＜0.05). However, more protein Bax was seen in controlgroup, and also has obvious different with hypothermic group. PCRresult: As to ICE, there is obviously difference between three groups(p＜0.05). Most obvious expression was seen in control group, andleast was seen in normal groups. As far as Bcl-XL was concerted,obviously differences between three groups were seen in cortical andbrain stem. Most obvious expression was seen in hypothennia group,and least was seen in normal groups. According LSD tests, obviousdifferences were seen between each two groups too (p＜0.05).However in the zone of hippocampus, obvious difference was onlyseen between hypothermia group and the other two groups (p＜0.05).5,Result of microdialysis: As to lactate, pyruvic acid and L/P, there isno difference between normal and hypothermia groups. However,obvious differences were seen between control group and other twogroups (p＜0.05). The difference of glucose was only seen betweennormal group and the other two (p＜0.05).Conclusion:1,According to our modification, we think our model can be used as astable and reliable rat model of study the DIND.2,Hypothermic treatment will not decrease the cortex blood flow inDIND rat. This can guaranty the exertion of the brain protectionmechanism of hypothermia treatment, for example decrease themetabolism and protect the ischemic cell.3,According to our study, hypothermia treatment can alleviate or slowdown the pathological changes of BA during the DIND, which maybe can good for maintain the blood supply and protect the neuronsfrom ischemia.4,Depend on the result of TUNEL, Immunohistochemical staining and PCR, we consider that the hypothermia treatment can mitigate orslow down the course of cell apoptosis in DIND rat.5,Hypothermia treatment could protect the cerebral injury fromvasospasm by slow down the oxygen consumption, maintain themetabolism and alleviate pyruvic acid accumulation.