Effect Of Hyperbaric Oxygen On The Brain Edema And Expression Of Aquaporin-4 Surrounding Brain Tissue In Rats Following Intracerebral Hemorrhage

Objective: Intracerebral hemorrhage (ICH) is a frequently occurring and common disease of the central nervous system , with higher fatality rate in the acute phase in China, there is no effective treatments. Hyperbaric oxygen (HBO) therapy is a therapy which can treat various diseases , refers to the body breathing in high pressure environment (above standard atmospheric pressure) in which is full of equal pressure pure oxygen . The effect of this treatment in the ischemic stroke has assured , with regard to intracerebral hemorrhage, at the present stage lacks of evidence-based medicine evidence.The brain edema after intracerebral hemorrhage is one of the fatal reasons for the patients .At present some clinical studies show that HBO can effective relief the secondary brain edema after intracerebral hemorrhage in patients, which could help patients neural function recovery, but the specific mechanism is not clear.Aquaporins (AQPs) is a kind of membrane protein, widely distributed in different tissues and organs of the body, which mediate water transportation through different types of membranes . From the mammal organizations have already identified 13 aquaporins, including aquaporin-4 (AQP-4), which is the hightest aquaporin of the brain tissue in content and distribution .AQP-4 is similar to other aquaporins in the structure, its main function is to transport water from internal and external cells. The present researches demonstrate that in normal brain tissues AQP-4 is in the lower expression state, cells oedema and vasogenic edema can affect its expression, getting through its expression of level can reflect that hyperbaric oxygen is whether related or not in reducing the cerebral edema.The experiments using SD (Sprague– Dawley) rats ,with two different methods , establish the intracerebral hemorrhage models, adopt hyperbaric oxygen and hyperbaric oxygen preconditioning methods to intervene. Evaluate the neurological scores, measure the brain edema and the expression of aquaproin-4 surroungding brain tissue. Aim to compare the effect of hyperbaric oxygen for two kinds of intracerebral hemorrhage models and evaluate the tolerance of hyperbaric oxygen preconditioning on the intracerebral hemorrhage in rats,determine the relationship between HBO decreasing cerebral edema and expression level of AQP-4.Methods:1 Healthy and male, weighing 350-380g, Sprague–Dawley 156 rats were randomly divided into six groups: Sham-operated group (SHG, 6 rats), autologous blood induced intracerebral hemorrhage model control group (A-ICH, 30 rats), collagenase-induced intracerebral hemorrhage model control group (C-ICH, 30 rats), autologous blood induced intracerebral hemorrhage model with hyperbaric oxygen experimental group (A-ICH+HBO, 30 rats), collagenase-induced intracerebral hemorrhage model with hyperbaric oxygen experimental group(C-ICH+HBO,30rats), hyperbaric oxygen preconditioning experimental group (HBO-PC,30 rats) .All the groups except the sham-operated group were divided into 24h, 48h, 72h, 5d, 7d five time points(6 rats in each time point).2 Intracerebral hemorrhage experimental animal models:Adopting autologous blood induced and collagenase-induced two methods to establish right-side intracerebral hemorrhage models.3 Hyperbaric oxygen therapy: The experimental groups rats, once a day,were exposed in the single infant oxygen-cabin. At the beginning of experiment , pressurized 15min to get the pressure to 0.10 MPa and sustain 60min in the cabin , controlled the oxygen concentration more than 90% and temperature in 24℃,decompressed 15min at the end of the test. The control groups were exposed in the normal atmosphere.4 Neurologic deficits scores test was carried out by an examiner blinded to experimental groups after 6h with hyperbaric oxygen therapy and the control groups at the five time points. The deficits were scored on a modified scoring system based on that developed by Longa follows: 0, no deficits; 1, difficulty in fully extending the contralateral forelimb; 2,unable to extend the contralateral forelimb; 3,mild circling to the contralateral side; 4, severe circling; and 5, falling to the contralateral side.5 Using the standard wet–dry method cut a coronal brain slice 4mm from the frontal pole to measure the brain water content.6 Specific experiment contents:Experiment1:using immunohistochemistry methods to determine AQP-4, observed the effect of hyperbaric oxygen on the expression of the AQP-4.Experiment2:compared the strengths and weaknesses with hyperbaric oxygen therapy in two different intracerebral hemorrhage models.Experiment3:established intracerebral hemorrhage model after hyperbaric oxygen preconditioning for 5 days,evaluated the tolerance of intracerebral hemorrhage by hyperbaric oxygen preconditioning.Results:1 The success rate of autologous blood induced ICH model was 50% , which was lower than the domestic and overseas reported 71% and 79%. The success rate of collagenase-induced ICH model was 55%, which was higher than autologous blood induced ICH model.2 Neurologic observation: The A-ICH+HBO group compared with the A-ICHgroup, differences of neurologic scores were not statistically significant (P > 0.05). In the experimental groups, the differences between A-ICH +HBO group and C-ICH+HBO group were statistically significant (P<0.05). The HBO-PC group compared with the A-ICH group ,the neurologic scores were not statistically significant (P > 0.05).3 Brain water content: In general the water content of the experimental groups were obviously reduced, compared with the control groups and were significant difference (P < 0.05) in cerebral edema peak. In the experimental groups, differences of the brain water content between A-ICH +HBO group and C-ICH+HBO group were statistically significant (P< 0.05). The HBO-PC group compared with the A-ICH group ,the neurologic scores were statistically significant (P< 0.05).4 The expression of the AQP-4: Aquaporin-4 could express in all groups after intracerebral hemorrhage,with low expression in the sham operated group . In the control groups, the AQP-4 began to increase at 24h following intracerebral hemorrhage , reached the peak at 48h , which gradually reduced after 72h but still higher than the sham operated groups .In general the hyperbaric oxygen groups were obviously reduced, compared with the control groups and were significant difference(P<0.05) in cerebral edema peak(48h). In the experimental groups ,the expression of the AQP-4 in A-ICH+HBO group were lower than the C-ICH+HBO group ,were statistically significant (P<0.05) .The HBO-PC group compared with the A-ICH group ,the expression of the AQP-4 were statistically significant at 24h, 48h, 7d three time points (P< 0.05).Conclusion:1 Hyperbaric oxygen therapy could effectively reduce neurologic deficits following intracerebral hemorrhage , impelled neural function recovery.2 Edema could be decreased in cerebral edema peak at 48h time point by hyperbaric oxygen therapy after intracerebral hemorrhage.3 Hyperbaric oxygen therapy could affected the expression of AQP-4 and edema for the two ICH models, down-regulation of AQP-4 expression were related to the decrease of water content in the brain .4 Compared with collagenase induced ICH models ,autologous blood induced ICH models were more sensitive and suitable in study edema after hyperbaric oxygen therapy.5 Hyperbaric oxygen preconditioning could reduce the cerebral edema, down-regulation of AQP-4 expression, enhance the tolerance for the intracerebral hemorrhage.