| The incidence of cerebral ischemia disease is very high and it is still the main reason that causes death of old people. So it is very important to carry out studies on the mechanisms and therapies of this disease. The best way for the treatment of stroke is to restore reperfusion. But when the duration of cerebral ischemia is too long, the ischemia/reperfusion injury becomes worse after the restoration of reperfusion. Recently, besides studying the protective treatment for cerebral ischemia, great attention and efforts have been paid to the exploration of the cerebral endogenous protective mechanisms. In 1990, Kitagawa et al first observed the phenomenon of ischemic preconditioning (IPC), which indicated that one or more brief episodes of sublethal cerebral ischemia and reperfusion may induce significant protection against a subsequent lethal ischemic insult. However, so far the neuroprotective mechanism of IPC remains largely unknown and needs to be further studied.It has been reported that IPC may reduce the ischemia/reperfusion injury of hippocampal neurons in the CA1 region of rats by regulating the expression of the apoptosis-related genes Bcl-2 and Bax. However, till now, no report has described the detailed study of the mitochondrial apoptotic pathway concerned in the neuroprotective action of IPC. Our present experiments for the first time carried out a general and systematic study on the upstream and downstream expression of apoptosis- related genes in the mitochondrial pathway after ischemia/reperfusion and the effect of IPC on the expression of these genes.Uncoupling protein 2 (UCP2) exists in many kinds of cells and it is the only kind of UCP that is expressed in brain (including the hippocampus). UCP mediates the transfer of the fatty acid anion from the inside to the outside of the inner mitochondrial membrane, then the fatty acid anion reacts with protons, thereby leading to dissipation of the electrochemical proton gradient, and indirectly leading to reduced ROS production by the respiratory chain. It has been reported that UCP2 may protect neurons from acute brain trauma and epilepsy, but whether it participates in the neuroprotection of IPC remains unclear. The present experiments for the first time investigated the relationship between IPC and UCP2 expression and demonstrated the role of UCP2 in the protection of IPC against ischemia/reperfusion injury of hippocampal neurons.Ghrelin is a new brain-gut peptide, synthesized in stomach and some neural structures including hippocampus, hypothalamus and pituitary. Ghrelin regulates the release of growth hormone, energy metabolism and functioning of the gastrointestinal tract. It was demonstrated that intracerebroventricular administration of a growth hormone secretagogue (GHS)--hexarelin-- reduced the injury of cerebral cortex, hippocampus, and thalamus after brain hypoxia-ischemia in neonatal rats. However, whether ghrelin itself protects neurons against ischemia/reperfusion injury and whether IPC regulates the release of ghrelin are still unknown. In the present experiments, we studied for the first time the effect of IPC on the release of ghrelin and the role and mechanism of ghrelin in the protection of hippocampal neurons against ischemia/reperfusion injury.Objective1. To investigate if IPC protects hippocampal neurons against ischemia/ reperfusion injury through inhibition of the mitochondrial apoptotic pathway.2. To study the role of UCP2 in the neuroprotective process induced by IPC against ischemia/reperfusion injury of hippocampal neurons.3. To explore the role of ghrelin in the neuroprotection of IPC against ischemia/reperfusion injury of hippocampal neurons and the relationship between ghrelin and UCP2. Through the three sets of experiments, we aimed to provide new aspects for further study of the mitochondrial mechenism of the neuroprotective role of IPC. Methods1. Global forebrain ischemia modelMale adult Wistar rats were used. Forebrain ischemia/reperfusion was induced by the modified four-vessel occlusion model (4-VO) as described by Pulsinelli et al. Briefly, the rats were anesthetized with pentobarbital (40 mg/kg, i.p.). Vertebral arteries of both sides were electrocauterized and both common carotid arteries were exposed and threads were placed around each of them. 24 h later, the rats were re-anesthetized. The common carotid arteries were occluded with aneurysm clips for 8 min, followed by 3 d of reperfusion. During the ischemia, the body temperature of the rats was maintained above 37.0℃.2. GroupsRats were divided into the following several groups: (1) Control group (CON group): Rats were prepared by performing sham operations, exposing the foramen alares and carotid arteries, which were not occluded. (2) Ischemia/ reperfusion group (I/R group): Rats underwent an 8-min lethal ischemia followed by 72 h of reperfusion without ischemic preconditioning. (3) Ischemic preconditioning group (PC group): Rats were preconditioned with an 3-min ischemia and 72 h of reperfusion. (4) Ischemic preconditioning + ischemia/reperfusion group (IPC+I/R group): 24 h after vertebral electrocauterization, the animals underwent a 3-min sublethal ischemia and then 72 h later underwent an 8-min lethal ischemia and 72 h of reperfusion. (5) Ischemic preconditioning+SOD group (PC+S group): In a PC group, 5 min before the 3-min sublethal ischemia, superoxide dismutase (SOD) at 10000 U/kg was injected i.p. (6) Ischemic preconditioning+saline group (PC+N group): In a PC group, 5 min before the 3-min sublethal ischemia, the same volume of saline as SOD solution was injected i.p. (7) Ischemia/ reperfusion+ghrelin group (I/R+G group): Rats in I/R group received a daily i.p. injection of ghrelin after the ischemic insult at a dose of 0.4 mg/kg for 3 d. (8) Ischemia/ reperfusion+saline group (I/R+N group): Rats in the I/R group received an i.p. injection of saline after the ischemic insult of the same volume and frequency as ghrelin treatment. (9) Control+saline group (CON+N group): Rats were prepared by performing sham operations of exposing foramen alares and carotid arteries, which were not occluded. Rats received an i.p. injection of saline of the same volume and frequency as ghrelin treatment.3. Parameters and techniques for study(1) With the technique of HE staining, TUNEL staining and anti-NeuN immunohistochemical study, the pathological histology and apoptosis of neurons in the hippocampal CA1 region after IPC or ghrelin injection were examined.(2) RT-PCR was applied to detect the expression of UCP2 m RNA of hippocampal tissue after IPC or ghrelin injection.(3) Immunohistochemical staining was used for assay of the expression of Bcl-2, Bax, Cyt c and UCP2 of neurons in the hippocampal CA1 region after IPC and examined the expression of Bcl-2 after ghrelin injection.(4) Western blot was used for detection and measurement of the expression of actived caspase-9 of hippocampal tissue after IPC.(5) The ghrelin level in the serum of rats after IPC was assayed with ELISA.4. Statistical analysisThe data were expressed as mean±standard error (x±SEM). Comparisons of 3 or 4 groups were made by one-way or two-way ANOVA analysis of variance (ANOVA) followed by t test. Differences were considered to be statistically significant when p≤0.05.Results1. Effect of IPC on the pathological histology and apoptosis of neurons in hippocampal CAl region after ischemia/reperfusionHE staining results showed that in the I/R group, cell loss in the hippocampal CAl region was severe and most cells showed shrunken cytoplasm and degeneration of the nucleus. In the IPC+I/R group, most of the cells had normal shapes and a clear outline, a few cells had some characteristics of degeneration such as deep staining of nucleus and disappearance of part of cytoplasm. The number of surviving neurons in the CON group, I/R group and IPC+I/R group was 250.5+10.5, 30.2±3.8 and 221.0±8.0/mm respectively. The number of surviving neurons in the IPC+I/R group was significantly less than that in the CON group (p<0.001), but significantly greater than that in the I/R group (p<0.001).Anti-NeuN immunohistochemical staining demonstrated that the neurons of the hippocampal CA1 region in the I/R group showed shrunken cytoplasm and aggregation of chromatin, whereas in the CON group, IPC+I/R group and PC group, the neurons in the same region had normal shapes and clear outline. The number of surviving neurons in these three groups was 231.3±5.2, 194.0±10.0 and 237.2±12.3/mm respectively, and there were no significant differences among these groups. In the I/R group, the cell loss in the hippocampal CA1 region was severe, the number of surviving neurons was 41.1±7.7/mm, much less than that in the other three groups (p<0.001).TUNEL staining showed that in the I/R group, most of the cells in the hippocampal CA1 region showed a shrunken nucleus and chromatin condensation in the perinuclear regions. In the IPC+I/R group, only occasional apoptotic neurons were observed. The number of apoptotic neurons in the CON group, I/R group and IPC+I/R group was 6.8±0.6, 137.4±4.5 and 33.3±6.0/mm respectively. The number of apoptotic neurons in the IPC + I/R group was greater than that in the CON group (p <0.001), but less than that in the I/R group (p<0.001).2. Effect of IPC on the expression of apoptotic genes of the mitochondrial pathway in the neurons of the hippocampal CA1 regionData of immunohistochemical staining showed that compared with the I/R group, the frequency of expression of Bcl-2 in the neurons of the hippocampal CA1 region in the IPC + I/R group was greater (p<0.05), whereas the frequency of expression of Bax and Cyt c was more lower (p<0.05).Western blot examination showed that compared with the I/R group, the expression of activated caspase-9 in the hippocampal tissue was decreased in the IPC + I/R group (p<0.001).3. Effect of IPC on the expression of UCP2 in the neurons of the hippocampal CA1 regionRT-PCR analysis revealed that there was a high expression of UCP2 mRNA in the hippocampal neurons in the PC group, much more than that in the CON group, I/R group and IPC+I/R group (p<0.001). The levels of UCP2 mRNA in the IPC+I/R group and I/R group were similar, but were both significantly higher than that in the CON group (p<0.05). Immunohistochemical staining of UCP2 showed that the expression of UCP2 protein in the neurons of the hippocampal CA1 area was low in the CON group, I/R group and IPC+I/R group. However, in the PC group, the expression was greater than that in the other three groups (p<0.05). In the PC+S group, only weak expression of UCP2 was detected, which was significantly lower than that in the PC group (p<0.001) and in the PC+N group (p<0.01).4. Effect of IPC on the level of ghrelin in serumELISA assay results suggested that the ghrelin levels in serum in IPC+I/R group was increased at 24 h after the lethal ischemia, about double that in the CON group and I/R group (p<0.001). This high level continued to 72 h (p<0.001) after lethal ischemia. In the I/R group, the ghrelin level in serum was 'constant, and not significantly different compared with the CON group.5. The protective role of ghrelin on the ischemia/reperfusion injury of the neruons in the hippocampal regionHE staining showed that the number of surviving neurons in the hippocampal CA1 area after injection of ghrelin in the CON+N group, I/R+N group and I/R+G group was 251.3±8.7, 28.5±7.3 and 179.5±11.5/mm respectively. The number of surviving neurons in the I/R+G group was greater than that in the I/R+N group (p<0.001) and less than that in the CON +N group (p<0.001).The number of TUNEL-positive staining neurons of hippocampal CA1 region in the CON+N group, I/R+N group and I/R+G group was 7.5±0.9, 135.0±5.7 and 28.7±1.9/mm respectively. The number of apoptotic neurons in the I/R+G group was greater than that in the CON+N group (p<0.05) and less than that in the I/R+N group (p<0.001).6. The effect of ghrelin on the expression of UCP2 and Bcl-2Immunohistochemical staining showed that the expression of Bcl-2 in the neurons of hippocampal CA1 region in the above three groups was not obvious.RT-PCR examination results showed that there was a significantly higher expression of UCP2 mRNA in the hippocampal tissue in the I/R+G group, much more than that in the CON+N group, I/R+N group and IPC+I/R group (p<0.001). In the IPC+I/R group and I/R+N group, the expression of UCP2 mRNA was similar, but still... |
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