The Therapeutic Effect And Mechanism Of Hyperbaric Oxygen On Hypoxic-ischemic Brain Damage In Neonatal Rats

Objective1.To evaluate the therapeutic effect of hyperbaric oxygen(HBO) on hypoxic-ischemic brain damage(HIBD) in neonatal rats.2.To investigate the mechanism of HBO on nestin expression in hypoxic-ischemic brain of neonatal rats. 3.To probe into the mechanism of how dose HBO work in hypoxic-ischemic brain damage(HIBD).So to find out the more effective method to treat HIE.Methods120 SD rats at age 7d were randomly assigned into three groups,they were sham operation group(sham group),hypoxic-ischemic group(control group) and HBO treatment group(trial group).Each group contained 40 rats.HIBD was induced through right common carotid artery legation,and then breathing 8%O₂+92%N₂ for two hours.In this procedure,neurofunction were detected,and rats have 1 to 3 scores been included in this experiment.Sham group,of which the right common carotid of the rats was isolated but not legated;Control group in which the rats was doing nothing after HIBD;And the trial group,hyperbaric oxygen treatment was administered by placing the pups in a chamber after HIBD(0.2Mpa for 20min per day for 5 days).30 rats were taken from each group and sacrificed on the 7th,14th and 21st days after the operation respectively,each time point contained 10 rats.The pathological changes in the brain were observed by optical microscope;the expressions of nestin were examined with immunohistochemical staining.The spatial cognitive capability of other thirty rats which were taken from each group respectively was evaluated by using the Morris water maze from postnatal days 37 to 41.Lastly.at day-42,rats were decapitated and the neuron density in the CA1 subfield of the rat's hippocampus was measured with Nissl staining.Results1.HE staining:In sham group,the structure of hippocampus is normal,the pyramidal cell is lined up in order,and one or two nucleolus were observed conspicuously in round karyoplast.In control group,pyramidal cells were cytolysis and glial cells were proliferated at 7d points,brain atrophy was presented at 14d and 21d points.In different time points of control group,the pyramidal cell layer of cellular demarcation,coagulation necrosis,cytoplasmic eosinophilia and nuclear pyknosis were both seen in trial group and control group,there was significant difference compared with control group.The pathological manifestation of the brain damage in the trial group was milder than that of the control group.While the pyramidal cell layer in trial group was thinner than that of the sham group.2.Expression of nestin in different region:The number of nestin-positive cells both in hippocampal CA1 region and in sensorimotor cortex region of trial group on the 7th day after the operation was the highest,significant higher than those of the sham operation group and the control group.On 14th day after the operation,the expression of nestin becomes fewer and fewer.The nestin-positive cells also can be seen on 21st day after the operation.The mean±standard deviation denotes the number of nestin-positive cells,univariate test was used to analyze the difference.Comparison of shaps of nestin positive cells in hippocampal region:Different shapes of nestin positive cells were found distinctly between the sham group and the other two groups.In sham group,a few nestin positive cells were observed in hippocampal CA1 region,body of nestin positive cells was stained slightly.In control group and trial group,however,nestin positive cells enlarged with thick and long.Comparison of numbers of nestin positive cells in hippocampal region:the number of nestin-positive cells on hippocampal CA1 in the all time points have significantly differences after ischemia-hypoxia at different group(F=342.677,P＜0.001).In all three groups,the number of nestin-positive cells on hippocampal CA1 peak appeared at 7th day after ischemia-hypoxia,and then dropped along with the time going(F=185.654,P＜0.001).The interaction between group and time points is significantly(F=72.167,P＜0.001).Comparison between these 3 groups showed that according to the number of nestin-positive cells on each time point,3 groups arranged from high to low as the trail group＞the control group＞the sham group(P＜0.001). And the comparison within these 3 groups showed that the number of nestin-positive cells in each groups raised along the time going.Comparison of shaps of nestin positive cells in sensorimotor cortex region:In sham group,a small quantity and diffused nestin positive cells were observed in SMC. In control group and trial group,however,nestin positive cells are narrowly distributed.Most of them are located at the pyramidal cell layer and the granular cell layer.Comparison of numbers of nestin positive cells in sensorimotor cortex region: the number of nestin-positive cells in sensorimotor cortex in the all time points have significantly differences after ischemia-hypoxia at different group(F=25.99,P＜0.001).In all three groups,the number of nestin-positive cells in sensorimotor cortex peak appeared at 7th day after ischemia-hypoxia,and then dropped along with the time going(F=10.832,P＜0.001).The interaction between group and time points is not significantly(F=0.509,P=0.729).3.Learning and memory disability:In Morris water maze,mean escape latency in control group were longer(57.05s vs.22.22s,P＜0.001),probe time and probe length were shorter(31.15s vs.51.52s and 553cm vs.988cm respectively.P＜0.001). HBO therapy resulted in protection against both HIBD-induced brain tissue loss and spatial learning and memory disability(mean escape latency:38.68s vs.57.05s,probe time:36.38s vs.31.15s,probe length:688cm vs.553cm.P＜0.001).4.The brain weight of the left side(one side suffered hypoxic-ischemic damage) significantly lighter than the right side.Degree of brain atrophy in the control group was(20.2±11.4)%,significantly higher than in the trial group(8.5±8.3)%(P=0.001). And the degree of brain atrophy was not increased significantly in the trial group in comparison with the sham group(3.5±5.7)%(P=0.560).5.Compared with the sham group,the neuron density in the CA1 subfield of the rat's hippocampus was decreased in the control group(P＜0.001);while the neuron density in the CA1 subfield of the rat's hippocampus was elevated in the trial group in comparison with the control group(P=1.0).Conclusions1.The treatment with HBO can mitigate the HIBD-induced histomorphologic damage of the hippocampal CA1 subregion and improve the ability of learning and memory of neonatal rats with HIBD.2.HBO could enhance the expression of nestin in hippocampal CA1 region and sensorimotor cortex region of neonatal rats with HIBD.3.The activation of neural stem cells(NSC) may play an important role in restoration of brain damage due to hypoxia-ischemia.