| Background Cerebrovascular disease (CVD) has been considerd to be a kind of vital disease for its high incidence rate and high disability rate. Amongst them is ischemic cerebrovascular disease seen frequently. Recently, many studies have showed that brain ischemia/reperfusion (I/R) has relationship with many factors, such as free radicals, the excitotoxicity, calcium over-loading, the dysfunction of mitochondria, inflammation and apoptosis. A good body of studies suggests the inflammatory response during reperfusion speed the secondary damages and play a key role in I/R.Astrocytes are the first cell type in the central nervous system to encounter insult after brain ischemia. Then ischemia stimulates hypertrophic and proliferative changes in astrocytes and induces it produce inflammatory factors involved in the initiation of immunologic cascade. The expression of these cytokines which interact in the central nervous system can either damage or protect the brain tissue. Therefore, it is important to study astrocytes and cytokines in order to find a new way to reduce inflammation insults after brain ischemia.Objective (1) To investigate the progression temporal and spatial changes of astrocytes in the focal middle cerebral artery occlusion in rats; (2) To investigate the expression and mechanisms of NF-κB p65,TNF-α, ICAM-1 in astrocytes after focal cerebral ischemia/reperfusion; (3) To further appraise the effect of different dosage of pyrrolidine dithiocarbamate (PDTC) on the expression of inflammatory factors in astrocytes during the process of focal cerebral ischemia/reperfusion in rats, and accumulate experience for clinical treatment.Methods Animal model was made by ligating external carotid artery and inserting a piece of nylon thread into the internal carotid artery among male Wistar rats. The rats were randomly divided into six groups: normal control group (NC group), sham operation control group (SC group), ischemia/reperfusion group (I/R group), ischemia/reperfusion treated with PDTC 50mg.kg-1 group (I/RP1 group), ischemia/reperfusion treated with PDTC 100mg.kg-1 group (I/RP2 group), normal saline group (NS group). Each group which has 6 rats contains six time points, such as 2h, 6h, 12h, 24h, 48h and 72h, except for NC group which has only 3 rats.We use the HE staining method to observe the pathology variety, immunohistochemistry method to detect the expression of GFAP,NF-κB p65,TNF-α,ICAM-1 protein, in situ hybridization to detect the expression of NF-κB p65 mRNA,TNF-αmRNA,ICAM-1 mRNA and double immunofluorescent labeling technique to measure the expression of NF-κB p65,TNF-α,ICAM-1 in astrocytes.Results (1) The ischemic area was peaked after 24 hours'focal cerebral ischemia/reperfusion and can be reduced by PDTC. (2) The result of GFAP by immunohistochemistry method: NC group and SC group found only a little amount of weakly positive GFAP-labeled cells. In I/R group: GFAP-labeled cells started to increase at 6h (p<0.05), reaching a peak value at 72h (p<0.01). Comparing I/RP1 group and I/RP2 group with I/R group, the differences were significant (p<0.05). On the contrary, there was no significant difference between NS group and I/R group (p>0.05). (3) The result of NF-κB p65 by immunohistochemistry method: During 2-12h of reperfusion, comparing NC group with other 5 groups, the differences were not significant (p>0.05). In I/R group: the expression of NF-κB p65 protein in stained cells shaped like astrocytes started to increase at 24h (p<0.05), reaching a peak value at 72h (p<0.01). Comparing I/RP1 group and I/RP2 group with I/R group, the differences were significant (p<0.05). However, no significant changes occured between NS group and I/R group (p>0.05). (4) The result of TNF-αby immunohistochemistry method: During 2-24h of reperfusion, comparing NC group with other 5 groups, the differences were not significant (p>0.05). In I/R group: the expression of TNF-αprotein in stained cells shaped like astrocytes started to increase at 48h (p<0.05), reached a peak value at 72h (p<0.05). Comparing I/RP1 group and I/RP2 group with I/R group, the differences were significant (p<0.05),but no significant differences were observed between NS group and I/R group(p>0.05). (5) The result of ICAM-1 by immunohistochemistry method: There were little changes between NC group and other 5 groups after 2-48h of reperfusion(p>0.05). The expression of ICAM-1 protein in stained cells shaped like astrocytes in I/R group started to increase at 72h (p<0.05). When Compared I/RP1 group and I/RP2 group with I/R group, the differences were significant (p<0.05),but have little difference between NS group and I/R group (p>0.05). (6) The result of NF-κB p65 mRNA by in situ hybridization: During 2-6h of reperfusion, there were no significant differences between NC group and other 5 groups (p>0.05). In I/R group: the expression of NF-κB p65 mRNA in stained cells shaped like astrocytes started to increase at 12h (p<0.05), peaked at 24h (p<0.05) and decreased after 48h.Significant differences were observed in I/RP1 group,I/RP2 group and I/R group (both p<0.05), but have little changes between NS group and I/R group (p>0.05). (7) The result of TNF-αmRNA by in situ hybridization: During 2-12h of reperfusion, compared NC group with other 5 groups, there were no significant differences (p>0.05). In I/R group: the expression of TNF-αmRNA in stained cells shaped like astrocytes started to increase at 24h (p<0.05), reached a peak value at 72h (p<0.05). Comparing I/RP1 group and I/RP2 group with I/R group, the differences were significant (p<0.05). But when compared NS group with I/R group, there were little difference (p>0.05). (8) The result of ICAM-1 mRNA by in situ hybridization: During 2-24h of reperfusion, there were no significant changes between NC group and other 5 groups (p>0.05). In I/R group: the expression of TNF-αmRNA in stained cells shaped like astrocytes started to increase at 48h (p<0.05), reaching a peak value at 72h (p<0.05). Comparing I/RP1 group and I/RP2 group with I/R group, the differences were significant (p<0.05). But have no significant difference between NS group and I/R group, (p>0.05). (9) The result of GFAP and NF-κB p65 double immunofluorescent labeling: NF-κB p65 positive cells were co-staind with GFAP, a astrocyte marker, in the perifocal area, periventricular zone and the neighbourhood of the above-mentioned areas after 24 hours' focal cerebral ischemia/reperfusion, gradually increased at 72h of reperfusion. (10) The result of GFAP and TNF-αdouble immunofluorescent labeling: TNF-αpositive cells were co-staind with GFAP, a astrocyte marker, in the perifocal area and periventricular zone at 48h after focal cerebral ischemia/reperfusion, also gradually increased at 72h of reperfusion. (11) The result of GFAP and ICAM-1 double immunofluorescent labeling: ICAM-1 positive cells were co-staind with GFAP, a astrocyte marker, in the perifocal area and periventricular zone at 72h after focal cerebral ischemia/reperfusion.Conclusion (1) The early astrocytes change the shape and the quantity of reactive astrocyes increase in the ischemic core after focal cerebral ischemia/reperfusion in Wistar rats; (2) Astrocytes start to express NF-κB p65,TNF-α,ICAM-1 in turns in the perifocal region during early period of focal cerebral ischemia/reperfusion; (3) NF-κB p65 becomes activated in astrocytes and triggers induction of TNF-α,ICAM-1 expression after focal cerebral ischemia/reperfusion; (4)The expression of astroglial ICAM-1 may be regulated by TNF-αdependent pathway after focal cerebral ischemia/reperfusion; (5) The expression of NF-κB p65,TNF-α,ICAM-1 in astrocytes are harmful to brain tissue after focal cerebral ischemia/reperfusion; (6) PDTC has an inhibitive effect on the expression of NF-κB p65,TNF-α,ICAM-1 in astrocytes during the process of focal cerebral ischemia/reperfusion; (7) PDTC used in the process of focal cerebral ischemia/reperfusion can ameliorate the motor dysfunction, reduce the infarct size and inflammation. |
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