| Objective:Delayed encephalopathy is a rare complication of carbon monoxide(CO) intoxication.But the mechanism of the delayed encephalopathy was still unknown to us all.In this paper,we observed the effect of carbon monoxide intoxication on memory impairment and expression of Bcl-2,Bax,Caspase-3,Cyt-C,and NF in the cortex and hippocampus of the rat.Methods:Wistar male rats(provided by the Laboratory Animal Center of Shandong University),weighing 200-280 g,were kept with free access to a commercial animal feed and tap water.The animal room was maintained at approximately 22℃and 50% humidity with a 12-h light-dark cycle.The animals(n=144) were kept in the Laboratory Animal Center of Shandong University for 7 days for acclimatization.CO poisoning was performed by peritoneal injection(4 times with a 4 hrs interval).The first dosage was 100 ml/Kg and the dosage of the last three times dropped to 50 ml/ Kg.After the test of the step down type passive avoidance,the rats(n=12 in each group) were chosen randomly and sacrificed on day 1,3,7,14,28,respectively.All animals' care was in accordance with institutional guidelines. The tissues of cortex and hippocampus were excised,and the total proteins were isolated from flash frozen tissues using Trizol reagent.The levels of NF-L,NF-M, NF-H,andβ-actin proteins were quantified using western blotting.Results:1.The establishment of the animal modelWistar male rats were treated with carbon monoxide poisoning by intraperitoneal injection(4 times with a 4 hrs interval).The first dosage was 100 ml/Kg and the dosage of the last three times dropped to 50 ml/Kg.After the test of the step down type passive avoidance,the rats(n=12 in each group) were chosen randomly and sacrificed on day 1,3,7,14,28,respectively.All animals' care was in accordance with institutional guidelines.2.Memory dysfunction occurred after carbon monoxide poisoning,as evaluated by shortened escape latency and increased numbers of error times in step-down passive avoidance test.The step-down latency was considerably shortened in the first 3 days after CO poisoning.In the following 18 days,the latency maintained at the lower level,the step-down test error times significantly increased in the first 3 days,compared with the control value.The error times were all significantly more than that of the control value in the following days,except for a slight decrease at the day 14.3.Time-dependent changes of LPO and antioxidative statusesThe level of MDA in the serum and nerve tissues significantly was increased. Compared to the level of day 0,the serum MDA levels were increased by 162%, 134%,125%and 120%(P＜0.01) on day 1,3,7,14 and 21,respectively,while MDA levels in cerebral cortex were increased by 176%,140%,125%(P＜0.01) on day 1,3 and 7 after CO poisoning.The hippocampus MDA levels altered in a similar manner, which were increased by 150%,136%,127%and 121%on day 1,3,7 and 14 after CO poisoning(P＜0.01).The activities of anti-ROS,GR and GSH-Px gradually were decreased as shown in the Figs.The anti-ROS activity in the serum was decreased by 90%comparing with day 0 at the end of 21 days of CO poisoning(P＜0.01).Significant drop of the anti-ROS activity was observed at the end of the day 7,14 and 21 in the cerebral cortex,while those exhibited at the end of the day 14 and 21 in the hippocampus.Compared to the level of day 0,the anti-ROS activity in the cerebral cortex and hippocampus was declined by 19%and 22%(P＜0.01),respectively,on day 21.The activities of GR and GSH-Px were progressively decreased in the serum and cortex.Compared with the level of day 0,the activities of GR and GSH-Px in serum dropped significantly after 3 days of CO poisoning(P＜0.05).In the cerebral cortex, GR and GSH-Px activities declined by 14%and 22%at the end of 3 days of CO treatment,respectively.The similar decreases were also observed in the hippocampus at the end of 14 and 21 days of CO poisoning(P＜0.05).The GSH level changed in a different way,which was increased firstly on day 1 but then decreased in the following days.Compared to the level of day 0,the levels of GSH were increased by 138%,139%,and 129%in serum,cerebral cortex and hippocampus,respectively,on day 1,while that decreased by 103%,69%,63%,and 65%in cerebral cortex at the end of the day 3,7,14 and 21,and decreased by 97%, 75%,71%,and 71%in hippocampus at the end of the day 3,7,14 and 21.4.TUNEL assayNumber of TUNEL positive cells counted in the cerebral cortex and hippocampus of the rats after carbon monoxide poisoning.TUNEL staining expressed as the ratio of positive staining area to negative staining.Values are mean±SD.The percentage of the TUNEL-reactive cells in cerebral cortex increased significantly on day 14 and 28. In hippocampal CA1,the percentage of the YUNEL-reactive cells was noticed markedly on day 28.5.Immunohistochemistry of Bcl-2,Bax andCaspase-3The immunostaining Index(ISI) shows the significantly decreased expression of Bcl-2.The immunoreactive staining pattern exhibited a coalesced granular signal which was exclusively perinuclear and appeared as beaded lobule encircling the nucleus.Compared to the level of Day 0,the expression of Bcl-2 decreased significantly on day14 and 28in cerebral cortex and hippocampus of the rats after carbon monoxide poisoning.ISI shows the gradually increased expression of Bax.Compared to the level of day 0, the cytoplasmic staining patterns can be notable in the immunoreactive cells of the cerebral cortex and hippocampus of the rats on day 7 and 14 after carbon monoxide poisoning.On day 28,the expression of Bax had a little decline but still higher than that of day 0.Staining pattern characteristic of Caspase-3 included an intense cytoplasmic expression.Compared to the level of day 0,the cytoplasmic staining patterns can be notable in the immunoreactive cells of the cerebral cortex and hippocampus of the rats on day 14 and 28 after carbon monoxide poisoning.6.Apoptosis-related proteins expression in the cortex and hippocampus Western blot analysis of Bcl-2 expression was determined in cerebral cortex and hippocampus.In comparison to the level of day 0,the levels of Bcl-2 in the cortex were elevated by 28%,37%,44%,75%,and 77%(p＜0.01) in rats on day 1,3,7, 14and 28 after carbon monoxide poisoning.In hippocampus,the levels of Bcl-2 declined by 22%,50%,59%,67%,and 70%(p＜0.01).The expression of Bax had a notable change in the cerebral cortex and hippocampus after carbon monoxide poisoning.The graph indicated that the expression of Bax gradually increased,especially on day 14 and 28.The levels of Bax in the cortex were elevated by 30%,49%,55%,61%,and 62%.In hippocampus,the levels of Bax increased by 32%,49%,47%,54%,and 52%.It indicated that the expression of Caspase-3 increased markedly in the cerebral cortex and hippocampus after carbon monoxide poisoning,especially on day 14 and 28.Its levels in the cortex were elevated by 13%,64%,65%,90%,and 89%.In hippocampus,the levels increased by 6%,25%,27%,56%,and 58%. 7.The alterations of cytoskeleton protein in the cortex and hippocampusCyt-C:Compared with the level of day0,the level of Cyt-C firstly decreased by16%,43%on dayl and3,and then increased by42%,46%and133%on day 7,14 and28,and in the hippocampus,the level of Cyt-C also firstly decreased by 39%, 47%,20%,and then increased by 4%and20%on day 14 and28.NF-L:Compared to the level of day0,the level of NF-L decreased by23%,42%,36%on dayl,3 and 7;And then increased by 66%,13%on dayl4 and28 in the supematant fraction of the cortex.Meanwhile,in the pellet fraction,it decreased by48%,40%and33%,and then increased by 11%和15%(P＜0.01).In the hippocampus,the level of NF-L firstly decreased by12%,and then increased by4%,20%,65%和124%in the supernatant fraction;In the pellet fraction,it firstly decreased by22%,16%,15%,and then elevated by 28%和148%.NF-M:Compared to the level of day0,the level of NF-LM decreased by1%,6%,and then increased by 39%,30%in the supematant fraction of the cortex.But there was a notable decline on day28.Meanwhile,in the pellet fraction,it decreased by 12%,4%, 21%,6%,148%。In the hippocampus,there were similar alternations as compared to that of the cortex.NF-H:In the cortex,the level of NF-H firstly increased by 14%,and then decreased by 1%,31%,22%,and 6%in the supernatant fraction of the cortex.Differently,in the pellet fraction,the concention of NF-H declined by 24%,34%,22%,and then increased by12%和26%.The level of NF-H varied in a similar manner in the hippocampus. 8.Agarose gel electrophoresis of DNANo typical 180-200bp banding pattern characteristic of apoptotic cell death was observed in DNA extracted from the cortex and hippocampus of the rats after carbon monoxide poisoning.Conclusions:1.Memory dysfunction occurs after carbon monoxide poisoning which suggestes that delayed neuron damage happened after carbon monoxide poisoning.2.Lipid peroxidation is involved in the delayed neuron damage induced by carbon monoxide poisoning.3.Apoptosis is likely involved in the mechanisms of delayed neuron damage induced by carbon monoxide poisoning,which may be the basis of delayed encephalopathy after carbon monoxide intoxication.4.Alternations of neurofilament proteins expression are related to the delayed neuron damage. |
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