| Objective:Leukoaraiosis(LA)is demyelinating lesions of the deep brain white matter,which is closely related to ischemic cerebrovascular disease and the most important clinical imaging signs of cerebral microvascular disease.Chronic hypoperfusion is one of the main reasons of cerebral white matter damage.Hypoperfusion can lead to cerebral ischemia and hypoxia,and hyperbaric oxygen therapy is an effective means to correct ischemia and hypoxia.To observe the neuroprotective effects of hyperbaric oxygen on leukoaraiosis in rats and to explore its mechanism,we established leukoaraiosis in model rats to provide basis for hyperbaric oxygen in the treatment of chronic hypoperfusion of cerebral white matter disease in clinical application.Methods:1.Animal models:Sixty male Wistar rats were randomly divided into 3 groups:sham operated group,operation group and postoperative HBO treatment group,20 rats in each group.The rat model of chronic hypoperfusion white matter injury was made by bilateral common carotid artery ligation.After 14 days of ligation,the models were success.2.Treated by HBO:Fourteen days after surgery,chronic hypoperfusion white matter damages had formed.2.0 ATA hyperbaric oxygen was administered once a day for 14 days.The oxygen washing time was 10 min,the pressure time 15 min,and the oxygen was pressurized for 60 min,the final decompression time 15 min,Cabin temperature(23.3±2)?.3.Detection index:3.1 Behavioral tests:14 days after the formation of white matter damage,The navigation test was tested and the mean escape latency(MEL)was measured to evaluate the learning and spatial memory abilities.3.2 Staining and immunohistochemistry:On the 28th day after operation(fourteen days after HBO treatment),rats were killed,and the hippocampus was isolated from the brain tissue of the rats.The morphological structures of hippocampus and the apoptosis of neurons were observed by optical microscope after hematoxylin-eosin staining(HE)and tunel staining.Oligodendrocyte apoptosis is an important indicator of leukoaraiosis.The myelin basic protein(MBP),oligodendrocyte marker(CC1)and Oligodendrocyte precursor cell marker(NG2)were observed after immunofluorescence staining.3.3 Quantitative detection of cytokines and gene proteins:The protein and mRNA levels of TNF-?,IL-2 and IL-6 were analyzed by Western Blot and RT-PCR.The expression of caspase-3,Akt and Bcl-2 protein in downstream of TNF-a was determined by Western Blot.The signal pathway of hyperbaric oxygen was determined.3.4 Statistical analysis:Data were means±SD(standard deviation),unless otherwise indicated.Analysis of Variance(ANOVA)with repeated measures designs was used to compare the mean latency and mean step size of the rats in each group and SNK for comparison between groups.According to a = 0.05 test level,p<0.05 was considered to be statistically significant difference.Results:1.Behavioral aspects:The water maze test was performed 14 days after bilateral common carotid artery ligation.The MEL of the operation group was significantly longer than that of the normal control group 14 and 21 days after surgery(p<0.05).Compared with the surgery group,MEL in the HBO treatment group was significantly shorter 21 days and 28 days after surgery(p<0.05).There was no significant difference in MEL between the HBO treatment group and the normal control group(p>0.05).2.Staining and immunohistochemistry:2.1 Hippocampus HE staining and TUNEL staining:On the 28 day after operation,compared with sham operation group,the hippocampus tissues of operation group showed karyopyknosis,enhanced eosinophilic cytoplasm and widened perivascular space.Compared with the operation group,the tissue structure of the hippocampus was more complete,and the number of TUNEL positive cells significantly decreased,which meaned that the apoptotic neurons significantly decreased.The number and degree of nuclear pyknosis and anachromasis were similar in HBO treatment group and sham group.2.2 Immunofluorescence staining of MBP,NG2,CC1 and test of the inflammatory factors:MBP and CC1 significantly reduced and NG2 significantly increased in the corpus callosum area of the surgery group compared with that of the sham operation group.Western Blot results showed that IL-2,IL-6,TNF-alpha and other inflammatory factors were elevated after the surgery(p<0.05).The MBP and CC1 were significantly higher than that of the operation group but NG2 was significantly reduced in HBO group.IL-2,IL-6 did not change significantly after HBO treatment(p>0.05).TNF-alpha was significantly reduced after hyperbaric oxygen treatment,compared with the operation group(p<0.05).RT-PCR results showed that TNF-alpha increased significantly at the gene level after chronic cerebral ischemia,and decreased after HBO treatment.2.3.The results of Western Blot showed that the expression of Caspase-3 in operation group was higher than that of sham operation group,and caspase-3 expression in HBO group was lower than that in operation group(p<0.05).The expression of Bcl-2 and p-Akt in the operation group was not significantly higher than that in the sham operation group.After HBO treatment,the expression of Bcl-2 and p-Akt increased significantly(p<0.05).Conclusions:1.Ligation of bilateral common carotid artery to produce low perfusion model could lead to decreased learning and memory ability in rats and cause damage to the hippocampus,increased apoptosis of cells,inflammatory factors,pro-apoptotic factors and decreased myelin basic protein and oligodendrocyte.2.HBO can reduce the damage of hippocampus,inhibit the apoptosis of nerve cells,and protect the nerve cells and glial cells.3.HBO attenuated the reduction of myelin basic protein(MBP)due to chronic cerebral ischemia.It improved the progressive reduction of oligodendrocyte and promoted the proliferation of oligodendrocyte precursor cells.4.HBO may inhibit the apoptosis of oligodendrocyte through down-regulation of TNF-alpha that is involved in inflammatory reaction,inhibiting Caspase-3 apoptosis pathway and upregulation AKt pathway to reduce apoptosis of oligodendrocytes. |
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