| Objective :To explore the neuro-protection and neuro-protective mechanism of cyclophosphamide by studing its effect on expressions of glial fibrillary acidic protein(GFAP), tumor necrosis factor-alpha(TNF-α)and neuroglobin(Ngb) in hippocampi and the survive of neuro after local cerebral ischemia-reperfusion injury in rats.Methods:Seventy healthy male Sprague-Dawley rats which avoirdupois from 180g to 220g provided by experimental animal department of South China University were randomly devided into 4 groups:normal control group(N group) , sham operation group(S group) , model control group (model group)and cyclophosphamide treated group(T group).Each groups had 18 rats,each of the later groups(S group,model group and T group) were randomly divided into 3 subroups by three time spots.Each of subgroup had 6 rats.The model of middle cerebral artery occlusion (MCAO) by improved suture embolus was established according to Longa's report. Treatment group was given intraperitoneal injection of cyclophosphamide one hour later after ischemia;while both I/R group and sham operation group received same doses of saline.The formation of reperfusion following cerebral ischemia two hours later;each group was observed according appointed time spots—24 hours, 72 hours and 5 days.The scores of behavior obstacle in rats were rated by Longa's report at different time after reperfusion, and then executed them to obtain the brains.Normal group were executed and obtained brains.All the brains were formed by paraffin section.HE staining and immunohistochemistry staining were used to observe pathological changes and the expression of GFAP,TNF-αand Ngb in cerebral cortex and hippocampi of ischemic side.All the data were proceessed through two-way ANOVA analysis of SPSS 13.0 for windows.The significant testing standard was P<0.05 or P<0.01.Results1.The scores of neurologic impairmentIn N group and S group were 0;the scores of T group were less than model group,it had significant difference between them(P<0.05).2.HE stainingThe neurons of cortex are morphologically normal quantity and arrange in neat rows in N group or S group. In model group,there is sparse nerve cells, widened cells gaps, and a large number of cells degeneration and necrosis,that manifest as inclusions shrinkage, nuclear pyknosis deeply stained, nucleolus disappeared; while the number of survival nerve cells is significantly increased and the degree of injury is reduced in T group.3.Nissl stainingThe cortical membrane is blue-purple and light blue nucleus was full in N group and S group; we can see a lot of purple and blue Nissl body, the cells arranged in neat rows; in model group, Nissl body dissolution disappeared or is little,while Nissl body increase significantly in T group.4.GFAP expression of hippocampusWe can see a small amount of GFAP-positive cells in hippocampus after I/R 24 hours in model group;the expression of GFAP-positive cells increases after 72 hours; while five days later, it reaches the peak. After cyclophosphamide treated, the expression of hippocampus GFAP decrease that shows a statistical significance (P <0.05) comparing T group with model group among the same time point (24 hours, 72 hours, 5 days)respectively. There can not see the expression of GFAP-positive cells in hippocampus in S group and N group .5.TNF-αexpression of hippocampusWe can see the expression of TNF-αamong each time point both model group and T group, 24 hours after I/R, the highest growth over time, gradually reduce with the time passed;there can not see any expression of TNF-αboth N group and S group.Comparing T group with model group,it shows a statistical significance (respectively P <0.01 and P <0.05) between 24 hour point and 72 hour point, and 5 day point between the two groups shows no statistical significance(P>0.05).6.Ngb expression in brain tissueThere can be seen the expression of Ngb both N group and S group in the brain tissue of rats, no statistical significance between the two groups; brain ischemia-reperfusion later,it significantly increase, and comparing with N group or S group, there are statistics significance (P <0.05); while the expression of Ngb after cyclophosphamide treated further increased with N group or S group, there are statistically significant (P <0.01); compared with the model group, there are statistical significance (P <0.05).Conclusion1. in cerebral ischemia-reperfusion injury in rat model, cyclophosphamide can inhibit neuronal degeneration and necrosis, inhibit activation of astrocytes, so that reduction of GFAP expression, inhibit the expression of TNF-αincreased the expression of Ngb reduce cerebral ischemia-reperfusion injury, to play a role in cerebral protection.2.cyclophosphamide neuroprotective mechanism may be related to cerebral ischemia-reperfusion injury inhibit astrocyte activation, inhibited TNF-αexpression increased Ngb on, thereby reducing cerebral ischemia-reperfusion injury in the inflammatory cascade reaction.
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