| Objective:This study was to build cerebral ischemic reperfusion (I/R) injury model in SD rats; measure the expression variations of the relationship between, lipoprotein-associated phospholipase A2(Lp-PLA2) and tumor necrosis factor-α (TNF-α) in brain tissues and blood after I/R injury by immunohistochemistry (IHC), reverse transcription PCR (RT-PCR), and enzyme-linked immunosorbent assay (ELISA); with morp-hological observations, and primarily discussing the mechanisms of TNF-α and Lp-PLA2in cerebral I/R injury.Methods:1. Sixty SD male rats (250g-330g) were randomly divided into six groups (ten for each group):control group (CG), sham-operated group (SG), I/R6h, I/R1d, I/R3d and I/R7d groups. The middle cerebral artery occlusion (MCAO) model in rats was built by applying Longa thread cork. Grading and screening were conducted according to relevant standards.2. The expressive levels of TNF-α and Lp-PLA2in rat blood were detected by ELISA.3. The pathomorphological changes of cerebral tissues were observed through HE staining.4. The protein expressions of TNF-α and Lp-PLA2were determined by IHC.5. The mRNA expres-sions of TNF-a and Lp-PLA2in cerebral tissues were examined by RT-PCR.Results:1. Histological and Morphology Analyses 1. Histological and Morphology AnalysesThe cerebral tissues of rats were observed in light microscope. It was found that the morpha and structure of CG and SG were normal; The rats in the I/R6h group showed slight hyperplasia in glial cells, while some rats had slight dropsy in connective tissues and neurons, and some had destruction in cellular structure; In addition to medium hyperplasia in glial cells, the I/R1d group showed ischemic necrosis, cellular swelling and deformation, as well as a few inflammatory cells, and apoptosis in neurons; The I/R3d group displayed a large number of necrotic neurons, obvious dropsy in cerebral tissues, as well as extensive ischemic liquefactive necrosis; The I/R7d group displayed encephalomalacia, sheet-like necrosis and apoptosis in neurons, as well as severe hyperplasia in glial cells.2. ELISA ResultsCompared with CG and SG, the I/R6h group had higher expressive level of serous TNF-a and the differences were significant (P<0.01); The I/R1d group culminated in TNF-a level, and its differences from CG, SG and the I/R6h group were all significant (P<0.01); The difference between the I/R7d group and CG, or SG, was insignificant (P<0.05) The difference of Lp-PLA2between the I/R6h group and CG, or SG, was significant (P<0.01). The I/R Id group had lower Lp-PLA2than the I/R6h group, but its differences with CG and with SG were both significant (P<0.05). The difference between the I/R3d group and the I/R1d group was insignificant (P>0.05). The differences between the I/R7d group and CG, SG, and the I/R3d group were all significant (P<0.05).3. Immunohistochemical ResultsTNF-a had obvious positive expressions in I/R6h group, I/R1d group and I/R3d group, where the first group had the largest positive staining area percentage, and the three groups were significantly different (P<0.01) from CG or SG. Values did not significantly differ between I/R7d group and CG (P>0.05).The difference of Lp-PLA2positive staining area percentage between the I/R6h group and CG, or SG, was significant (P<0.01). The difference between the I/R1d group and CG was insignificant (P>0.05); For the I/R3d and the I/R7d groups, their differences with CG or with SG were significant (P<0.01).4. RT-PCR ResultsCG and SG had TNF-α mRNA expressions; The I/R6h, the I/R1d and the I/R3d groups had higher levels of TNF-α mRNA expressions than CG and SG, and the differences were all significant (P<0.01); The difference between the I/R7d group and CG was insignificant (P>0.05).The I/R6h group had higher levels of Lp-PLA2mRNA expression than CG and SG, and the differences were significant (P<0.01); The difference between the I/R1d group and CG was insignificant (P>0.05); For the I/R3d and the I/R7d groups, their differences with CG or with SG were significant (P<0.01).Conclusions:1. Lp-PLA2involved ischemia-reperfusion injury, expression a "high-low-high" effect in ischemia reperfusion injury.2. TNF-a expression was one of important role for the inflammatory reaction of I/R injury, and there are inconsistent relationship between Lp-PLA2. |
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