| Cerebral ischemia is a leading cause of death and permanent disability for which there is currently no effective treatment. At present, thrombolysis is still the major therapeutic means clinically for acute stoke. But some patients after thrombolysis may aggravate cerebral injury. Thus it has been becoming a new subject to research and produce effective neuroprotectors against cerebral ischemic injury. Nitric oxide (NO) is a small, light molecule and the first gas known to act as a biological messenger. There is a growing body of evidence that NO participates in the mechanisms of cerebral ischemia. However, the role that NO plays in the pathogenesis of ischemic brain damage has not been cleanly defined. In recent years, many studies have showed that the role of NO might induce "dual effects"during focal cerebral ischemic injury, namely neuroprotective and neurotoxic effects. NO is synthesized in the brain from L-arginine by a constitutive NO synthase (NOS). Aminoguanidine (AG), the selective inducible NOS (iNOS) inhibitor, showed neuroprotection by inhibiting the neurotoxic effect of NO, improving neurological recovery, decreasing the cerebral infarct volume, relieving cerebral edema. In present experiment, the middle cerebral artery occlusion (MCAO) model was prepared. By evaluating the expression of three NOS, changes of contents of some important excitatory and inhibitory amino acids, apoptosis, mitochondrion and primary cultures of rat cerebral neurons in vivo, the effect of aminoguanidine on ischemia-induced neuronal damage and the possible mechanisms were further observed. Part 1 Changes of nitric oxide synthase gene expression after focal cerebral ischemia in rat brain Objective: To investigate the changes of three nitric oxide synthase (NOS), endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) gene expression after focal cerebral ischemia in rats. Methods: Adult SD rats, male, were randomly divided into five groups: a normal control group and four ischemic groups, with five rats every group. Rats in the ischemic groups were killed at 2, 6, 12, 24 h after ischemia. We made the focal cerebral ischemic model with thread embolism of left middle cerebral artery occlusion (MCAO). Gene expression of eNOS, nNOS and iNOS were examined by reverse transcription polymerase chain reaction (RT-PCR). Results: 1. Gene expression of eNOS mRNA was detectable in the normal control group (0.44±0.08). eNOS mRNA was increased at 2 h (0.93±0.04)after MCAO compared with the normal control group (P<0.01), and reached the maximum. The expression of eNOS mRNA at 6 h (0.60±0.03)and 12 h(0.58±0.05) after MCAO was tapered, but was still higher than that in the normal group (P<0.05). There were no significant differences between ischemic 24 h group(0.48±0.06) and the normal group (P>0.05). 2. Gene expression of nNOS mRNA (0.35±0.03) was detectable in the normal control group. eNOS mRNA at 6 h after MCAO reached the maximum (0.87±0.07), and was higher than that in the normal control group (P<0.01). The expression at 12 h (0.48±0.03) after MCAO was tapered, but was still higher than that in the normal group(P<0.05). There were no significant differences between ischemic 24 h group (0.37±0.04) and the normal group(P>0.05). 3. Gene expression of iNOS was detectable only in the ischemic group. It reached the maximum at 12 h (0.81±0.08)after ischemia, and was higher than that in the ischmeic 2 h group(0.41±0.04)(P<0.01). The expression at 24 h (0.74±0.07) after MCAO was tapered, but was still higher than that in the ischemic 2 h group(P<0.05). Conclusion: Ischemia leads to early up-regulation of eNOS and nNOS, late up-regulation of iNOS.Part 2 Effect of nitric oxide synthase inhibitor aminoguanidine on amino acid contents of ischemic brain in rats Objective: By evaluating the effect of selective inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine(AG), on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid (GABA) from striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of AG on cerebral ischemic injury and the possible mechanisms. Methods: Rats were divided into sham-operated group, ischemic 2, 6,12 h group and AG treatment group (AG was administrated at 2, 6 and 12h after ischemia respectively). N=6 for each. AG (100 mg ·kg-1, ip) was administrated, 2 times a day, for 3 consecutive days. Same volume of normal saline was administrated in ischmic group. We made the focal cerebral ischemic model with thread embolism of left middle cerebral artery occlusion (MCAO). In sham-operated rats, the external carotid artery was surgically prepared for insertion of the filament, but the filament was not inserted. Rats were killed. The changes of infarcted volume were analyzed by TTC. The contents of aspartate, glutamate, glycine and GABA from striatum, hippocampus and cortex in the rat brain following cerebral ischemia were assayed by high performance liquid chromatogram (HPLC). Results: 1. Effect of aminoguanidine on infarcted volume of rat brain after ischemia: When AG was administrated at 2, 6 and 12h after MCAO, the infarcted volume was much decreased (15.1±3.4, 18.4±5.1, 25.7±3.5) compared with that of ischemic group (23.2±2.9, 28.0±3.9, 37.2±2.9) respectively (%, P<0.05). 2. Effect of aminoguanidine on amino acid contents of rat brain after ischemia: The contents of aspartate, glutamate, glycine and GABA from striatum, hippocampus and cortex in ischemic group were significantly increased compared with sham-operated group (P<0.05, P<0.01). Moreover the contents of glutamate from striatum, hippocampus and cortex weremarkedly decreased when AG was administrated at 2, 6 and 12 h after ischemia respectively (P<0.05 or P<0.01). The contents of aspartate from striatum, hippocampus and cortex were markedly decreased when AG was administrated at 2 and 6 h, and the contents of aspartate from hippocampus and cortex were decreased when AG was administrated at 12 h after ischemia (P<0.05, P<0.01). However, the contents of GABA from hippocampus and cortex were increased in AG group compared with ischemic group when AG was administrated at 2 and 6h, and the contents of GABA from striatum and cortex were increased when AG was administrated at 12 h after ischemia (P<0.05, P<0.01). Similarly, the contents of glycine were also increased from striatum, hippocampus and cortex when AG was administrated at 2 h, the contents of glycine were increased from cortex when AG was administrated at 6 h, and the contents of glycine from hippocampus and cortex when AG was administrated at 12 h after ischemia respectively (P<0.05, P<0.01). Conclusion:It may be concluded that AG have beneficial effect on ischemic cerebral injury. The possible mechanism is that AG could decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA. Part 3 Effect of aminoguanidine on neuronal apopotosis induced by focal cerebral ischemia in rats Objective: By evaluating the effect of aminoguanidine (AG) on neuronal apopotosis induced by focal cerebral ischemia in rats, to investigate the beneficial effect of AG on cerebral ischemic injury and the possible mechanisms. Methods: SD rats weighing 250-290 g were randomly divided into sham operation group (n=6), ischemic 2, 6, 12 h group (n=18) and AG treatment group (n=18, AG was administrated at 2, 6 and 12h after ischemia respectively). In AG group, AG (100 mg/kg, ip) was administrated, 2 times per day, for 3 consecutive days. Saline was administrated in ischemic group. Thefocal cerebral ischemic model was prepared with thread embolism of left middle cerebral artery (MCAO). In sham operation group, the external carotid artery was surgically prepared for insertion of the filament, but the filament was not inserted. Rats were killed at the scheduled time. Pathological changes were observed with light microscope by HE staining. The cell apoptosis were assayed by FCM. The expression of Bcl-2 and Bax proteins were analyzed by FCM and immunohistochemical method. The expression of Caspase-3 was observed by immunohistochemical method and Western blot. Results: 1. In HE staining, cerebral tissue structure in ischemic group showed vascular dilatation, some of neuron cellular swelling, cell nucleus concentrating,cytoplasmic rarefaction, staining weakly, vacuole formation in striatum, hippocampus, cortex. The structure in sham group showed no pathological changes described above. AG could ameliorate these pathological alterations. 2. Apoptosis of brain tissues was determined by FCM. Significant DNA fragmentation was detected after MCAO. The tissue from the ischemic group showed remarkably high apoptotic percentages with (13.9±2.3,11.3±1.2,9.7±0.1)% compared with sham group(5.2±0.5)% (P<0.01). AG could significantly reduce the apoptosis with (9.0±0.7,8.8±0.3,7.9±0.8)% (P<0.05,P<0.01). 3. The expressions of Bcl-2 and Bax studied by immuhistochemical staining showed that positive staining was buffy. They were expressed in endochylema of cellula nervosa and blood vessel endothelium. There were a few positive expression of Bcl-2 and Bax in sham group. In ischemic group, a few positive expression of Bcl-2 and considerable positive expression of Bax were observed.Administration of AG increased Bcl-2 expression, decreased Bax expression. The determination of Bcl-2 and Bax by FCM indicated that the expression of Bax protein(5.6±0.2,5.7±0.8,6.0±0.5)in ischemic group was increased compared with sham group(3.5±0.3)(P<0.01). There were no significant differences in Bcl-2 protein between ischemic group(5.4±0.2,5.5±0.1,5.2±0.5) and sham group(5.2±0.2).The ratio of Bcl-2 and Bax inischemic group (1.0±0.1,1.0±0.2,0.9±0.1) was lower than that in sham group(1.5±0.2) (P<0.01). The response was inhibited by AG. Administration of AG increased the expression of Bcl-2(6.2±0.1,6.9±0.3,6.2±0.3)(P<0.01), decreased the expression of Bax(4.9±0.2,5.0±0.2,4.7±0.3)(P<0.05,P<0.01). The ratio of Bcl-2 and Bax (1.2±0.1 , 1.4±0.1 , 1.3±0.1)was also increased(P<0.01). 4.The results by Western blot showed that the expression of Caspase-3 in ischemic group was remarkably higher than that in sham group. The expression in AG group was lower than that in ischemic group. The expressions of Caspase-3 by immuhistochemical staining showed that positive staining was buffy. They were expressed in cell nucleus. There were a few positive expression of Caspase-3 in sham group. In ischemic group, considerable positive expression of Caspase-3 was observed. Administration of AG decreased Caspase-3 expression. Conclusion:Administration of AG significantly reduced the neuronal cell apopotosis induced by focal cerebral ischemia in rats. Anti-apoptotic effect of AG was considered relating to the increase of the Bcl-2 expression, decrease of the Bax and Caspase-3 expression. Part 4 Effect of aminoguanidine on mitochondria injury after focal cerebral ischemia in rats Objective: To examine the effect of selective inducible nitric oxide synthase inhibitor, aminoguanidine (AG), on mitochondria injury after focal cerebral ischemia in rats and the possible mechanisms. Methods: SD rats weighing 250-290 g were randomly divided into sham operation group (n=6), ischemic 2, 6, 12 h group (n=18) and AG treatment group (n=18, AG was administrated at 2, 6 and 12h after ischemia respectively). In AG group, AG (100 mg/kg, ip) was administrated, 2 times per day, for 3 consecutive days. Saline was administrated in ischemic group. The focal cerebral ischemic model was made with thread embolism of left middlecerebral artery (MCAO). In sham operation group, the external carotid artery was surgically prepared for insertion of the filament, but the filament was not inserted. Rats were killed. Ultrastructure changes of neuronal mitochondria were examined by Electronic microscope. Mitochondria of cerebral tissue were isolated by differential centrifugation. The swelling and the activity of mitochondria, the activities of ATPase, SOD and GSH-Px in mitochondria and the contents of NO and MDA in mitochondria were measured. Results: 1. Electronic microscope showed the neuronal cytoplasm and the mitochondria swelled, the cristae disrupted, dissolved or disappeared in MCAO rats. The above changes were not observed in sham group. Administration of AG could ameliorate these injury induced by cerebral ischemia in rats. 2. In ischemic 2, 6, 12 h group, the OD value at 540nm was decreased, the swelling of mitochondria was markedly increased and the activity of mitochondria was decreased (P<0.01). The value were respectively (0.204±0.024,0.198±0.027,0.186±0.026) and (0.461±0.048,0.460±0.035,0.454±0.037). Compared with ischemic group, the swelling of mitochondria was decreased and the activity of mitochondria was increased after administration of AG (P<0.05). The value were respectively (0.256±0.031,0.252±0.030,0.253±0.036)and (0.544±0.033,0.530±0.034,0.524±0.027). 3. The contents of mitochondria NO were markedly increased in ischemic 2, 6, 12 h group (P<0.01). The value were respectively (3.86±0.49,3.40±0.65,3.32±0.58). Compared with ischemic group, the contents of NO were decreased (2.69±0.91,2.22±0.21,2.17±0.52) after administration of AG (P<0.05). 4. The activities of ATPase (3.03±0.30,2.77±0.38,2.74±0.40), SOD (26.44±2.66,26.37±3.93,25.11±6.35) and GSH-Px (28.70±3.93,27.93±4.64,25.68±5.08) in mitochondria were markedly decreased (P<0.01), the contents of MDA(3.38±0.79,3.43±0.85,3.47±0.52)in mitochondria were markedly increased (P<0.05) in MCAO rats. Compared with ischemic group, the activities of ATPase(3.72±0.46,3.61±0.38,3.47±0.29), SOD(35.38±3.91,36.96±3.44,34.64±4.93) and GSH-Px(38.71±5.55,37.22±5.16,35.87±4.39) in mitochondria were distinctly increased(P<0.01) and the contents of MDA (2.25±0.30,2.37±0.46,2.44±0.41)in mitochondria were distinctly decreased (P<0.05) in AG group. Conclusion: AG showed protective effect by inhibiting the production of NO, beneficially improving the integrity of form and function, ameliorating energy metabolism of mitochondria after focal cerebral ischemia in rats. Part 5 Effect of aminoguanidine on ischemic injury in cerebral neuronal cell cultures Objective: To investigate the effect of selective inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on ischemic injury and NO toxicity in cerebral neuronal cell cultures. Methods: 1. The neuronal cell of fetal rat were cultured by mechanical dispersion method in vitro. The culture plates were randomly divided into 5 groups: normal group, ischemic group, AG 10 μM, AG 20 μM, AG 100μM group, n=6. The model of ischemic neurons was made by treating cells with sodium hydrosulfite in glucose-free medium. 2. The culture plates were randomly divided into 7 groups: normal group, NO donor 1, 10, 50, 100, 500, 1000 μM group, n=6. 3. The culture plates were randomly divided into 5 groups: normal group, NO toxical group, AG 10 μM, AG 20 μM, AG 100μM group, n=6. The model of NO toxicity was established with 100 μM NO donor in medium. Intracellular lactate dehydrogenase(LDH) release, NO content, the cell viability by MTT stain and cellular morphology were used to evaluate the effect of AG. Results: 1. The cell morphology was not altered by NO donor 1, 10μM. There were no significant differences between NO donor 1, 10μM group and normal group in NO content, the cell viability and LDH release. In NO donor 50,100,500,1000 μM group, part of nervous cells were gathered, even to be dead. With the increasing concentration of NO donor, the number and activity...
|
PDF Full Text Request