| During cerebral ischemia-reperfusion injury, many changes will occur in the brain, which can change the expression of some genes related to neuron apoptosis. bcl-2 and bax are one pair of genes which have most correlation with cell apoptosis. bcl-2 can inhibit the occurrence of cell apoptosis, while bax can accelerate the cells to death. So the ratio of Bcl-2 and Bax in the neural tissue can control the destiny of neurons. HSP27 is a member of heat shock proteins family, which synthesis increased in brain after exposure to injury. Studies showed that HSP27 plays a protective role in brain.Erythropoietin (Epo) is an important stimulator to erythropoiesis. Some studies showed that Epo can protect neurons from apoptosis. But the molecular mechanism of Epo-induced protection has not been known at present, and no remarks were reported on how different the protection with different dosage of exogenous Epo is. This experiment aimed to explore the mechanism of Epo-induced protection by means of detection of Bcl-2, Bax and HSP27 as well as apoptosis in the cortex of rats with focal cerebral ischemia-reperfusion injury, and observe the difference of Epo-induced protection with different dosage. All of this will provide some experimental proofs for the clinical administration of Epo.Materials and methods1. The model of middle cerebral artery (MCA) occlusion by suture embolus was established according to Longa's report. The suture embolus was inserted in left MCA about 17.5± 1.0mm from the origination of internal carotid artery to occlude the blood supply of MCA. When the suture was put out of the endocranial part of internal carotidartery, reperfusion occurred. Sham-operation group received the same operation except that no suture embolus was inserted. The standards of successful model included Homer's sign in left side, paralysis in right limbs and Longa's scores.2. 48 male Sprague-Dawley rats each weighing 300±20g were randomly divided into 6 groups: Group A: Low-dose Epo group; Group B: mid-dose Epo group; Group C: high-dose Epo group; Group D: ischemia-reperfusion group; Group E: sham-operation group and Group F: normal group. Group A, B and C received an intraperitoneal injection of Epo(1000U/kg, 3000U/kg and 5000U/kg respectively) just at very outset of ischemia, while Group D and E received the same doses of saline. After the first four groups experienced ischemia for 2 hours and reperfusion for 24 hours, the brains were removed. Group E was killed 24 hours after operation while Group F was removed without delay. All the brains were prepared for paraffin sections.3. Sections of every rat were processed with HE staining, TUNEL detection, and Bcl-2, Bax, HSP27 immunohistochemistry staining. The average number of positive cells in every section was calculated.4. All the dates were expressed with X ± s and analyzed with one-way analysis of variance among groups and t-test between two groups. The significant testing standard was a = 0.05Results1. Longa's scores in Group E and F were 0, while rats in Group D got scores 2.63±0.52. The scores of rats with Epo administration are 1.63±0.52, 1.50±0.54 and 1.38±0.52, all of which were much lower than Group D (P<0.05). But among the groups with Epo treatment, there were no significant difference.2. The pathological changes: The hemispheres in Group D swelled heavily and seemed pale. HE staining showed that the neurons in ischemic cortex were very little in size and amount, and the space between them became large. There existed a lot of degenerated or necrotic neurons. With Epo administration, the hemispheres swelled gently and the infarct size was decreased. The shapes of neuron were relatively normal.3. Bcl-2 expression was intensive in Group D (55.40±5.08/HP, high power lens),but there was more Bcl-2 positive cell observed in Epo groups (Group A: 68.80±6.34/HP; Group B: 78.70±5.55/HP; Group C: 87.40±11.94/HP). On the contrary, Bax positive cells in Group D (78.20±4.74/HP) is the most intensive in all the groups, while Bax expression decreased significantly in Epo groups (Group A: 47.10±5.29/HP; Group B: 48.60±4.56/HP; Group C: 49.15±4.16/HP). And the ratio of Bcl-2/Bax was also significantly different between Group D (0.71 ±0.08) and Epo groups (Group A: 1.48±0.23; Group B: 1.64±0.26; Group C: 1.79±0.22).4. The expression of HSP27 was just like that of Bax, which was intensive in cortex in Group D(92.10±1.92/HP) and less intensive in Epo groups(Group A: 62.05±1.72/HP; Group B: 55.35±3.30/HP; Group C: 72.95±3.49/HP), and all the difference were significant(P<0.05). Among the groups with Epo treatment, there was significant difference between every two groups.5. No apoptosis neurons were observed in Group E and F. More apoptosis neurons were observed in penumbral region in Group D (64.80±2.84/HP) than that in Epo groups (Group A: 54.18±4.93/HP; Group B: 47.30±2.73/HP;Group C: 51.40±3.54/HP). Among the Epo groups, Group B was less than the other two groups (F<0.05).Conclusion1. The administration of Epo could lessen brain tissue pathological changes and inhibit neurons apoptosis after cerebral ischemia-reperfusion injury in rats, which partially mediated by increasing the expression of Bcl-2 and decreasing the expression of Bax.2. HSP27 has a non-protective effect after cerebral ischemia-reperfusion injury; Epo could decrease the expression of HSP27 in the early phase of brain ischemia-reperfusion injury in rats.3. Different doses of Epo administration can produce a different effect of neuroprotection against brain ischemia-reperfusion injury in rats, a dose of 3000U/kg maybe the one most close to the optimal dose.
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