Effects Of Minocycline On Rats Following Cerebral Injury By Ischemia-reperfusion

Objective:To observe the effects of minocycline on rats following focal ischemia-reperfusion injury by assaying the the neurological function deficit,cell apoptosis in the hippocampal CA1region,and expression of Caspase-3and PARP-1protein, explore the protective effects of minocycline on neuronal injuries induced by focal ischemia-reperfusion.Methods:108health male Sprague-Dawley(SD) rats were randomly assigned into3groups, sham-operation group (n=36)(Sham),cerebral I/R group (n=36)(I/R), minocycline-treatment group (n=36)(MT). According to the treatment time Minocycline-treatment group was divided into24h group (n=18) and72h group (n=18), so did the cerebral I/R group and the minocycline-treatment group.Except of Sham operation group, other groups used the suture method to establish with ischemia-reperfusion model. Minocycline (45mg/kg) was immediately injected intraperitoneally after cerebral ischemia-reperfusion, and then intraperitoneal injection (22.5mg/kg)after each12h, till put to death.The equal volume of normal saline was injected intraperitoneally in the sham-operation and cerebral I/R group.The neurological behavioral deficits were immediately evaluated after waking, using Water-maze test and the Step-down test to observe the rat’s change about the function of learning and memory. The volume of cerebral infarction was measured by TTC staining. Morphologic changes were observed by HE staining. TUNEL staining was used for detecting cell apoptosis. Immunohistochemical staining and Western blot assay were applied to detect the change about the expression of Caspase-3and PARP-1protein in the hippocampus. Results (1) Sham operation group:0; cerebral I/R group:2.25±0.44. The rats induced by middle cerebral artery occlusion showed obvious symptoms of neurological function through neurological behavioral deficits before injecting drug.(2) Through the Water-maze test after1day and3day treatment, the escape latency in sham operation group are61.5±10.6and65.5±11.2; the escape latency in cerebral I/R group are161.7±7.0and140.8±7.8; the escape latency in MT group are120.7±9.2and97.3±6.6. The escape latency in MT group is shorter than that in cerebral I/R group.(P<0.05).(3) Through the Step-down test after1day and3day treatment, the latency in sham operation group are241.3±22.5and218.0±25.8, the number of mistakes are3.3±1.8and3.7±1.4; the latency in cerebral I/R group are81.5±13.4and86.8±14.1, the number of mistakes are9.0±1.8and8.0±1.8; the latency in MT group are166.0±17.8and125.2±13.8, the number of mistakes are6.0±1.4and5.2±2.1. The latency in MT group is longer than that in cerebral I/R group, and the number of mistakes in MT group is less than that in cerebral I/R group.(P<0.05).(4) Through TTC staining after1day and3day treatment, the volume of cerebral infarction in sham operation group (mm3) are0. The volume of cerebral infarction in cerebral I/R group (mm3) are0.29±0.02and0.25±0.01; the volume of cerebral infarction in MT group (mm3) are0.15±0.01and0.13±0.01. The volume of cerebral infarction in MT group is smaller than that in cerebral I/R group through TTC staining (5)The number of the neuron in MT group is more than that in cerebral I/R group through HE staining.(6) Through TUNEL staining after1day and3day treatment, the apoptosis index(%) in sham operation group are1.73±0.62and2.37±0.51; the apoptosis index(%) in cerebral I/R group are55.43±0.62and46.10±1.99; the apoptosis index(%) in MT group are22.67±2.43and15.63±1.30. The number of cell apoptosis in minocycline treatment group is less than that in cerebral I/R group(P<0.05).(7)The expression of Caspase-3and PARP-1protein in minocycline treatment group is less than that in cerebral I/R group through immunohistochemical staining in the hippocampal CAl region.(8) Through Western blot in the hippocampal region after1day and3day treatment, the expression of the Caspase-3protein in sham operation group are0.38±0.07and0.23±0.04, in cerebral I/R group are0.71±0.04and0.76±0.04, in MT group are0.47±0.02and0.38±0.02; the expression of the PARP-1protein in sham operation group are0.49±0.07and0.45±0.04, in cerebral I/R group are1.49±0.03and1.56±0.06, in MT group are0.81±0.38and0.94±0.10. The expression of the Caspase-3and PARP-1protein in MT group is less than that in cerebral I/R group(P<0.05).Conclusion:Minocycline could protect the neuronal injuries induced by focal ischemia-reperfusion effectively, the mechanism underlying the neuroprotection maybe by inhibiting the Caspase-3and PARP-1protein expression.