| Cerebrovascular diseases was the disease of nervous system, which was commonly seen and frequently occurred. It tother with malignant tumor and heart disease, constituted the three biggest fatal killers in the world. Among all the diseases, its death rate reached 10 percent, only next to tumor, and it has been one of the three main killers that lead to death.50-70 percent of the survivors had some serious sequelas, such as paralysis, aphasia. It not only tormented the patients themselves, but also brought the society and the patients families heavy burdens. In acute stroke, only a small fraction of patients benefited from intravenous administration of recombinant tissue plasminogen activator, which was the only drug with proven effectiveness in reducing the size of infarct in humans. Even though a large number of different compounds have been proven to reduce the size of brain infarct in animal studies, replication of the experiments with the neuroprotection in humans have regularly failed. The reasons for the unsuccessful clinical trials have been either the toxic side effects, which have overridden the neuroprotective potential of the compounds determined in animals, or a limited time window for human therapy. Many scholars were interest in searching compounds with no or tolerable side effects combined with a protective potential. In the hours following stroke onset, ischemia-induced inflammation caused activation of microglia. The role of microglia in the neuroinflamatory response was complex and may involve tissue repair through release of neurotrophins and removal of necroticdebris. However microglia activation also involved release of toxic substances such as nitric oxide and free radicals that could damage healthy neurons. This microglial response is maintained even weeks following reperfusion. Thus secondary injury caused by neuroinflammation may be a target for neuroprotective intervention. Minocycline was a second-generation, semi-synthetic tetracycline. It was effective against gram-positive and gram -negative infections. Minocycline first introduced in 1967, and it shared a basic chemical structure consisting of a tetracyclic naphthacene carboxamide ring system. It original action mechanisms for antimicrobial activities were based on the characteristics that tetracyclines inhibited protein synthesis by acting ribosome levels. Minocycline that was a highly lipophilic molecule could easily penetrate the blood brain barrier compared to first-generation tetracyclines. So the minocycline played a very important role in curing the nervous system disease. Recently the minocycline has been reported to exert neuroprotective effects over various experimental models through its action on anti apoptosis, and inflamation and antioxidation. In our study, the cerebral-reperfusion injury model was made by middle cerebral artery occlusion to explore the neuroprotective effects and the potential mechanism of the minlcycline on the ischemical reperfusion injury from the following aspects.1 Effect of minocycline on the inflammatory reaction after cerebral ischemia-reperfusion1.1 ObjectivesTo study and research the effect of minocycline on the inflammatory reaction after focal cerebral ischemia-reperfusion injury in rats with immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and radioimmunity.1.2 MethodsMiddle cerebral artery occlusion in rats was established with the suture method. The effect of minocycline on the inflammatory reaction after focal cerebral ischemia-reperfusion injury was researched when the treatment with minocycline (45mg/kg) was started 12 h before ischemia,30 min,4 h,12 h after the onset of ischemia. The effect of low dose minocyclie(10mg/kg) on the inflammatory reaction after cerebral ischemia-reperfusion was also studied. The expression of cyclooxygenase-2 and prostaglandin E2 was detected with immunohistochemistry and radioimmunity. Interleukin-1βconverting enzyme was detected with reverse transcription polymerase chain reaction (RT-PCR).1.3 Results1.3.1 Cyclooxygenase-2 immunohisochemistry positive expressionThere were only a few cyclooxygenase-2 protein positive cells in sham-operation group's cerebral cortex. A significant increase in the number of COX-2 positive cells was observed in the area adjacent to ischemia in control group. The light density values in control group were significantly higher than that of in sham-operation. Compared with the sham-operation group the difference was statistical significance(P=0.000). In those groups that minocycline was administered started 12 h before ischemia and 30 min,4 h after the onset of ischemia, the light density values COX-2 was significant lower than that of in control group. Compared with the control group, the difference was statistical significance (P=0.000,0.007,0.037). The light density values in low dose group was also lower than that of in control group, compared with the control group the difference was statistical significance(P=0.025). The difference was not significant between control group and the group that treatment with minocycline 12 h after the onset of ischemia (P=1.000).1.3.2 The expression of prostaglandin E2 in cerebral cortexThe level of prostaglandin E2 was low in the sham-group's cerebral cortex. The ischemia-reperfusion injury could induce the expression of prostaglandin E2. The level of prostaglandin E2 in control group was higher than that of in sham-group. The difference was statistical significance(P=0.000). All the goups except the group that treated with minocycline 12 h after the ischemia could reduce the accumulation of prostaglandin E2 to a different extent. Compared with the control group, the difference was statistically significant (P=0.003,0.032,0.047,0.048).The group that minocycline was treated 12 h after ischemia could not decrease the level of prostaglandin E2. The difference was not statistically significant, compared with the control group (P=1.000)1.3.3 The expression of the interleukin-1βconverting enzymeThe expression of the interleukin-1βconverting enzyme was low in sham-operation group. The expression of the interleukin-1βconverting enzyme increased significantly in control group. compared with sham-operation, the difference was statistical significance (P=0.012). Significant differences were observed between the control group and those groups that minocycline were treated 12 h before ischemia and 30 min,4 h after the onset of ischemia (P=0.022,0.026,0.030). The expression of the interleukin-1βconverting enzyme in low dose group that treatment with mincycline(10mg/kg) 30 min after ischemia was also lower than that of in control group. Compared with the control group, the difference was statistical significance (P=0.029). The expression of the interleukin-1βconverting enzyme did not decrease in the group that mincycline was treated 12 h after the ischemia. Compared with the control group, the difference was not significant (P=0.918) 1.4 Conclusion(1)The ischemia-reperfusion injury could induce the expression of cyclooxygenase-2, interleukin-1βconverting enzyme and prostaglandin E2.(2)The minocycline(45mg/kg) could inhibit the expression of cyclooxygenase-2 in rats when it was treated 12 h before ischemia and 30min,4h after the onset of ischemia. The low dose minocycline(10mg/kg)could also inhibit the expression of cyclooxygenase-2 when was treated at 30min after the onset of ischemia.(3) The minocycline(45mg/kg) could decrease the expression of interleukin-1βconverting enzyme gene in rats when treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline(10mg/kg) could also inhibit the expression of interleukin-1βconverting enzyme gene when treatment was started at 30 min after the onset of ischemia.(4) The level of prostaglandin E2 could be reduced by minocycline(45mg/kg) when the treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The group that was treated with low dose minoncycline(10mg/kg) could also reduce the level of prostaglandin E2 when treatment was started at 30 min after the onset of ischemia.2 Effect of minocycline on the neuronal apoptosis after cerebral ischemia-reperfusion2.1 ObjectivesTo research the effect of minocycline on the neuronal apoptosis after focal cerebral ischemia-reperfusion injury with immunohistochemistry, Hoechst stain and T2 weighted magnetic resonance imaging.2.2 MethodsFocal cerebral ischemia was produced by introduction of an intraluminal nylon. The expression of caspase-3 was detected with immunohistochemistry. Hoechst stain was used to observe the neuronal apoptosis, T2WI was used to detect the infarction volumes. The effcet of minocycline on the expression of caspase-3, the neuronal apoptosis and the infarction volumes was studied when the treatment was strated 12 h before the ischemia and 30 min,4 h,12 h after the onset of ischemia. The effect of low dose minocycline(10mg/kg) on the the expression of caspase-3, the neuronal apoptosis and the infarction volumes was also studied. The minocyclne was given 30 min after the ischemia. Dates were analysed using SPSS 13.0 software. Results were expressed as mean±standard deviation of the mean. Multiple comparisons were evaluated by analysis one-way ANOVA. Comparisons between two groups were evaluated by Bonferroni when the variances was homogeneity.The comparisons between two groups were value by Welch and Dunnett's when the variances was inhomogeneity. the P<0.05 was considered statistically significant.2.3 Result2.3.1 caspase-3 immunohisochemistry positive expressionThere were only a few caspase-3 protein positive cells in sham-group's cerebral cortex. Lots of caspase-3 protein positive cells were seen in the area adjacent to ischemia in control group. The light density values and the number of caspase-3 in control group were significantly higher than that of in sham group. Compared with the sham-operation group the difference was statistical significance (P=0.000). In those groups that treatment with minocycline was started 12 h before ischemia and 30 min,4 h after the onset of ischemia the light density values and were significant lower than that of control group. Compared with the control group, the differences were statistical significance (P=0.000,0.000,0.003). The light density values in low dose group was also lower than that of control group. Compared with the control group the difference was statistical significance (P=0.020). The difference was not significant between control group and the group that treatment with minocycline 12 h after the onset of ischemia (P= 1.000).2.3.2 Hoechst stainBoth normal nerve cells and the apoptotic cells could emit the blue fluorescence. The normal nuclei were dispersed, homogeneous distribution and the fluorescence was faint. The apoptotic cells had classic condensation and fragmentation of their nuclei and emited bright blue fluorescence. There were only a few of apoptotic cells in sham-operation group's cerebral cortex. Apoptotic cells increased significantly in control group. Compared with the sham group, the difference was statistical significance (P=0.000). In those groups that minocycline was treated 12 h before ischemia and 30 min,4 h after the onset of ischemia, the rates of apoptotic cells was significant lower than that of in control group. Compared with the control group, the differences were statistical significance (P=0.000,0.000,0.000). The rate of the apoptotic cells in low dose group was also lower than that of in control group. Compared with the control group the difference was statistical significance (P=0.000). The difference of the rate of the apoptotic cells was not significant between control group and the group that treatment with minocycline 12 h after the onset of ischemia(P=0.645).2.3.3 T2 weighted magnetic resonance imagingNone of the sham-operated animals showed signal changes on T2 weighted magnetic resonance imaging. There were large abnormal signal areas in control group when cerebral ischemic areas were visualized with with a T2-weighted. All model groups showed abnormal signal areas on T2 weighted magnetic resonance imaging. Differences of the relative infarction volumes between the control group and those groups that minocycline was respectively treated 12 h before ischemia and 30 min,4 h after the onset of ischemia were significant (P=0.010,0.016,0.021). The diffences of the relative infarction volumes between the control group and the group that treatment with minocycline 12h after the onset of ischemia was not significant (P=0.998). And the diffence of the relative infarction volumes between the control group and low dose group was also significant (P=0.029)2.4 Conclusion(1)The number of the positive of caspase-3 and the apoptotic cells increased obviously after the ischemia-reperfusion.(2)The minocycline(45mg/kg) could decrease the expression of caspase-3 in rats when it was treated at 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline (10mg/kg) could also inhibit the expression of caspase-3 when treatment was started at 30 min after the onset of ischemia.(3) The minocycline(45mg/kg) could inhibit the apoptosis caused by the cerebral ischemia-reperfusion injury in rats when treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline(10mg/kg) could also inhibit the apoptosis when treatment was started at 30 min after the onset of ischemia.(4) The minocycline(45mg/kg) could decrease the relative infarction volumes in rats when it was treated at 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline(10mg/kg) could also decrease the relative infarction volumes when it was treated 30 min after the onset of ischemia.3 Effect of minocycline on the brain edema after cerebral ischemia-reperfusion3.1 ObjectivesTo research the effect of minocycline on the brain edema, blood brain barrier and pathological changes of brain tissue, after focal cerebral ischemia with diffusion weighted imaging, enhanced T1-weighted imaging, T2-weighted magnetic resonance imaging and hematoxylineosin staining.3.2 Methods Male Sprague-Dawley rats were randomly divided into sham-operation group, control group, minocycline preconditioning group that treated with minocycline 12 h before ischemia; 30 min group that treated with minocycline 30 minutes after ischemia; 4 h group that the treatment was started at 4 hours after ischemia; 12 h group that the treatment was started at 12 h hours after ischemia and the low dose group that minocycline 10 mg/kg was given 30 minutes after the ischemia. The cytotoxic edema, vasogenic edema, blood brain barrier disruption, and the pathological changes of brain tissue were monitored with diffusion weighted imaging, T2 weighted magnetic resonance imaging, enhanced T1-weighted imaging and hematoxylineosin staining. Date were analysed using SPSS 13.0 software. Results were expressed as mean±standard deviation of the mean. Multiple comparisons were evaluated by analysis one-way ANOVA. Comparisons between two groups were evaluated by Bonferroni when the variances was homogeneity. The comparisons between two groups were value by Welch and Dunnett's when the variances was inhomogeneity. the P<0.05 was considered statistically significant.3.3 Results3.3.1 Diffusion weighted magnetic resonance imagingIn sham group no abnormal signal of diffusion weighted imaging changes were found. In control group there were large hyper-intensity signal areas when were visualized with diffusion weighted imaging. The relative aparent diffusion coefficient decreased obviously. Compared with the sham-operation group the diffence was statistical significance (P=0.000). All model groups showed hyper-intensity signal areas on diffusion weighted imaging. The relative aparent diffusion coefficient in preconditioning group,30 min group, low dose group increased markedly. Compared with the control group, The diffences were statistical significance (P=0.000,0.000,0.036). The relative parent diffusion coefficient in 4 h group and 12 h group also increased. But the differences were not statistically significant, when compared with the control group (P=1.000,1.000).3.3.2 Enhanced T1-weighted magnetic resonance imagingIn sham group no abnormal signal was found when was visualized with enhanced T1-weighted magnetic resonance imaging. Hyper-intensity signal areas were found to different extent in all groups except sham-operation group when taken with enhanced T1-weighted magnetic resonance imaging. The signal intensity reduced gradually from the ischemia core to the area adjacent to ischemia. Hyper-intensity signal areas were observed in control group when were monitored with enhanced T1-weighted imaging. The relative signal intensity obviously increased in control group. The difference was statistically significant when compared with the sham-operation group (P=0.000). The relative signal intensity significantly reduced in preconditioning group,30 min group,4 h group and low dose group when were monitored with enhanced T1-weighted imaging. Compared with control group the differences were statistically significant (P=0.003,0.019,0.043,0.027). The relative signal intensity also reduced slightly in 12 h group. But the difference of the relative signal intensity between the control group and 12 h group was not significant (P=1.000)3.3.3 T2-weighted magnetic resonance imagingIn sham group no abnormal signal was found when was monitored with T2 weighted magnetic resonance imaging. Hyper-intensity signals were found to different extent in all model groups. The relative signal intensity increased in control group. Compared with the sham-operation group the diffence was statistical significance (P=0.000). The relative signal intensity of on T2 weighted magnetic resonance imaging decreased markedly significantly in preconditioning group,30 min group,4 h group and low dose group. Compared with control group, the difference was statistically significant (P=0.003,0.004,0.032,0.048). The relative signal intensity on T2 weighted magnetic resonance imaging also reduced slightly in 12 h group. But the difference of the relative signal intensity between the control group and 12 h group was not significant (P=0.499).3.3.4 hematoxylineosin stainingIn sham group no change of tissue morphology in cerebral cortex was found. The cellular membrane was integrity, chromoplasm was homogeneous. A lot of necrotic neurons, proliferation of colloid cells, Swollen of connetive tissue and neurons, stroma highly edema were found in control group. The change in preconditioning group,30 min group,4 h group and low dose group was mitigated obviously. The change that was observed in 12 h was the same as that of in control group. A great deal of necrotic neurons, proliferative glial cells were observed and the stroma, neurons and glial cells were evident edema in 12 h group.3.4 Conclusion(1) The ischemia-reperfusion injury could cause the cytotoxic edema and vasogenic edema. The damge of the blood brain barrier was evident after the ischemia-reperfusion.(2) The minocycline could decrease the cytotoxic edema after the focal cerebral ischemia reperfusion injury in rats when it was treated by intraperitoneal 12 h before ischemia and 30 min, after the onset of ischemia. The low dose minocycline could also decrease the cytotoxic edema when treatment was started at 30 min after the onset of ischemia.(3) The minocycline could relief vasogenic edema after the focal cerebral ischemia reperfusion injury in rats when treatment was started at 12h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also relief vasogenic edema when treatment was started at 30 min after the onset of ischemia.(4)The minocycline could ameliorate the disruption of blood-brain barrier after the focal cerebral ischemia reperfusion injury when treatment was started at 12 h before ischemia and 30 min,4h after the onset of ischemia. The low dose minocycline could also ameliorate the disruption of blood-brain barrier when it was treated 30 min after the onset of ischemia.(5) The minocycline could ameliorate pathological changes of brain tissue after the focal cerebral ischemia reperfusion injury when treatment was started at 12 h before ischemia and 30 min,4h after the onset of ischemia. The low dose minocycline could also ameliorate pathological changes of brain tissue when it was treated 30 min after the onset of ischemia.Conclusion(1) The ischemia-reperfusion injury could induce the expression of cyclooxygenase-2, interleukin-1βconverting enzyme and prostaglandin E2,.(2)The minocycline could inhibit the expression of cyclooxygenase-2 in rats when it was treated at 12 h before ischemia and 30 min,4 h after ischemia. The low dose minocycline could also inhibit the expression of cyclooxygenase-2.(3) The minocycline could decrease the expression of interleukin-1βconverting enzyme in rats when the treatment was started 12 h before ischemia and 30 min,4 h after ischemia. The low dose minocycline could also inhibit the expression of interleukin-1βconverting enzyme.(4) The level of prostaglandin E2 in rats could be reduced by minocycline when the treatment was started 12 h before ischemia and 30 min,4h after the onset of ischemia. The group that treated with low dose minoncycline could also reduce the level of prostaglandin E2 in rats.(5)The number of the positive of caspase-3 and the apoptotic cells increased obviously after the ischemia-reperfusion.(6) The minocycline could decrease the expression of caspase-3 in rats when it was treated at 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also inhibit the expression of caspase-3.(7) The minocycline could inhibit the apoptosis caused by the cerebral ischemia-reperfusion injury in rats when the treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also inhibit the apoptosis.(8) The minocycline could decrease the relative infarction volumes in rats when treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also decrease the relative infarction volumes.(9) The ischemia-reperfusion injury could cause the cytotoxic edema and vasogenic edema. The damge of the blood brain barrier was evident after the ischemia-reperfusion.(10) The minocycline could decrease the cytotoxic edema in rats after the focal cerebral ischemia reperfusion injury when intraperitoneal injection was started 12 h before ischemia and 30 min after the onset of ischemia. The low dose minocycline could also decrease the cytotoxic edema.(11) The minocycline could relief vasogenic edema after the focal cerebral ischemia reperfusion injury when treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also relief vasogenic edema.(12) The minocycline could ameliorate the disruption of blood-brain barrier after the focal cerebral ischemia reperfusion injury in rats when treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also ameliorate the disruption of blood-brain barrier.(13) The minocycline could ameliorate pathological changes of brain tissue after the focal cerebral ischemia reperfusion injury when treatment was started 12 h before ischemia and 30 min,4 h after the onset of ischemia. The low dose minocycline could also ameliorate pathological changes of brain tissue.
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