| Objective: To study the effects of Nrf2 and HO-1 in delayed encephalophathy after acute carbon monoxide poisoning in rats by detecting their behavior changes,morphological changes and the expressions of the two proteins in brain hippocampus,in order to provide a certain experimental foundation and direction for the further research about the pathogenesis of DEACMP.Methods:1.One hundred and sixty-eight rats were randomly divided into three groups: with forty-eight rats in blank control group(BC group),sixty rats in air control group(AC group)as well as in DEACMP group(CO group).The animals were valuated at 1st,3rd,7th,14 th,21th,28 th day after acute carbon monoxide poisoning(there were ten rats in every subgroup of AC and CO groups,eight rats in every subgroup of BC group).2.The DEACMP model was established by Improved intraperitoneal injections in CO group,the AC group injected an equal amount of air in the same way,and the BC group without any processing.3.Using Morris water maze test to measure the ability of learning and memory of rats before and after the infected,and testing the concentration of HbCO in rats at the same time to judge whether the DEACMP model were successfully established.4.HE method was used to observe the pathological changes of rat hippocampal CA1 area.Detecting the pyramidal cell apoptosis of hippocampal CA1 area by TUNEL method.The expressions of Nrf2 and HO-1 were tested by immunohistochemical method and Western Blot method.Results:1.The establishment of DEACMP model:(1)Symptoms of acute carbon monoxide poisoning: The rats of CO group appeared restless,deep and short of breath,the skin and mucous membranes were cherry red,then became weakness,transient paralysis,bradykinesia,some rats appeared body stiffness tic and even coma or death at last.The survival rats of CO group ate less than the other groups and became not sensitive.The other groups rats were without such abnormal performance.(2)The changes of HbCO concentration in arterial blood: The HbCO concentration in rats maintained more than 50% above 16 hours throughout the whole model building process.(3)The results of Morris water maze test:The average escape latency was no statistical differences among the three groups rats either before the infected or in the first seven days after infected(P>0.05),but the average escape latency of CO group rats was significantly prolong than the other two groups,the time in platform quadrant and the number of crossing the platform reduced obviously in 14 th to 28 th day(P<0.01).2.HE staining: The celluar forms and structures of the pyramidal cells in hippocampal CA1 area are mainly normal in BC and AC groups before or after infected.However,there were different levels of pathological changes in hippocampal CA1 area of CO group over time:cell edema,nuclear pyknosis,nuclear fragmentation,pyramidal cell reducing and cells arrangement sparse and disorders.3.TUNEL test: The apoptosis phenomenon in hippocampal CA1 area of BC group and AC group were not obvious.on the contrary,the apoptosis cells in hippocampal CA1 area started to increase from first day gradually and achieved its peak(20.20±1.78)at the seven day and then began to decrease slowly in CO group,but the apoptosis phenomenon was existed till 28 th day,and that the apoptosis index(AI)of the CO group was markedly higher than the other two groups at each time point(P<0.01).4.The expressions of Nrf2 and HO-1:(1)Immunohistochemical detection: Both the expressions of Nrf2 and HO-1 were not obvious in hippocampal CA1 area of BC and AC groups,and the expression differences among rats at each time point had no statistical significance(P>0.05).However,both the expressions of the two proteins began to increase at first day after infected in CO group,achieved their peaks(8.20±1.08,9.04±1.13)at third day,then reduced gradually but maintained at a relatively high level at 28 th day,and the expressions of Nrf2 and HO-1 were obviously higher than BC group and AC group at each time point(P<0.01).(2)Western Bloting:The expression thends of Nrf2 and HO-1 were similar to immunohistochemical detection in the three groups,moreover,it was the detection object(the complete brain hippocampal)that made the results more convincing.Conclusions:1.The DEACMP model established by the improved intraperitoneal injections is mostly similar to the pathological process in persons,and it is an ideal method because of its easy operation,relative security,common requires of materials and the simply controlled dosage of CO.2.Detecting the behavioral changes(the ability of cognitive,learning and memory)and the HbCO concentration to preliminarily judge whether the rats occurred DEACMP.3.The genesis and development of DEACMP in rats are more likely to be related to the delayed pyramidal cell apoptosis in hippocampus.4.Both the Nrf2 and HO-1 proteins played a dual role on DEACMP:The Nrf2 rapidly increased in first three days to against apoptosis by promoting the expression of HO-1.However,not only the excessive expression of Nrf2 but also the accumulations of the downstream factor HO-1 and its metabolites in 7th to 28 th day probablely promoted the delayed cell apoptosis in hippocampus.That may be the significant pathogenesis of DEACMP. |
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