| Objective:By establishing neonatal rat hypoxic ischemic brain damage model, we observe the effects of selective adenosine A(2A) receptor antagonist SCH58261 on the short-term neurological behavior evaluation, the expression of ICAM-1 and neuron apoptosis, in order to explore whether it has a neuroprotective effect in neonatal rat hypoxic ischemic brain damage and the best drug dose. Methods:1. Grouping and Model building: A total of 40 seven-day old neonatal Sprague-Dawley(SD) rats were randomly divided into 5 groups: Sham group, HIBD group, SCH58261 0.01 mg/kg group, SCH58261 0.2 mg/kg group, SCH58261 2 mg/kg group, each group containing 8 rats. Model of HIBD was established by the Rice method, sham group was merely subjected to the free left common carotid artery without hypoxic ischemic treatment. HIBD was given 0.1ml of DMSO immediately after the hypoxic-ischemic. 3 different doses of SCH58261 group were given intraperitoneal injection respectively with 0.01 mg.kg, 0.2 mg/kg, 2 mg/kg(dissolved in 0.1 ml of DMSO) immediately after hypoxic ischemic, while HIBD group was injected 0.1 ml of DMSO.2. Observation index: Every groups were carried out neurological behavior 24 hours after HBD, then decapitated and slices for observeing the changes of brain tissues with HE staining, the expression of ICAM-1with immunohistochemical method, neuron apoptosis by TUNEL method. Results:1. Short-term neurological behavior evaluation: Compared with Sham group, the achievement of completing the three nerve reflection in the remaining 4 groups were significantly poor, and the difference was statistically significant( P<0.05), it suggested that short-term neurologic was damaged after hypoxic-ischemic. The time of righting reflex, cliff aversion reflex and geotaxis reflex in three administration groups with different doses of SCH58261 shorter than HIBD group(P<0.05). Furthermore, the time of each reflection likely and dose of SCH58261 showed a dose-effect relation. Accompanying dose decrease, time of each reflection was decreased(P<0.05), which 0.01 mg/kg was strongest.2. Pathomorphological change of brain tissue: In Sham group, the brain cell structure was integrity and eumorphism, clear margin and nucleolus markedly. In HIBD group, the brain tissue structure was disordered, the cell number significantly reduced, nerve cell were swelling and degenerative, necrosis of neurons was obvious. In SCH58261 2 mg/kg group, the cell were deeply stained and karyopyknosis, but had no difference compared with HIBD group. In 0.2 mg/kg group, cell were arranged in disorder, visible part cell eumorphism, the number of nerve cell were more than that of 2mg/kg group. In 0.01 mg/kg group, the number of survival neurons was inceresed, cell had regular arrangement, only little nuclear condensation and fragmentation, thus brain damage was lightened compared with 0.2 mg/kg and 2 mg/kg group.3. Immunohistochemical to observe ICAM-1 expression: ICMA-1 positive staining was brown fine particle sediment and was seen in the neurons cytoplasm and nuclei. ICAM-1 was low expressed in sham group, and the expression of ICAM-1 in remaining 4 groups were increased compared with Sham group, and the difference was significant( P<0.05). The expression of ICAM-1 in three dose groups of SCH58261 was lower than HIBD group(P<0.05). Accompanying dose decrease, the expression of ICAM-1 decreased gradually(P<0.05), which 0.01 mg/kg group is the lowest.4. TUNEL technique to detect apoptosis: Few apoptotic cells were seen in sham group, but apoptotic cells increased in remaining 4 groups, apopt osis index(AI) was 5higher than Sham group, and the difference was significant(P<0.05). The AI in three dose groups of SCH58261 was decreased compared with HIBD group(P<0.05). There may be a dose dependent between AI and the dose of SCH58261, accompanying dose decrease, the AI decreased gradually(P<0.05). Which it is the lowest in 0.01 mg/kg group.5. Correlation analysis between AI and ICAM-1: In all model groups(including HIBD group and three administration groups), AI and ICAM-1 had a significantly positive correlation(r1=0.825, P1<0.05; r2=0.937, P2<0.01; r3=0.868, P3<0.01; r4=0.830, P4<0.05), it prompted that the AI increased with the expression of ICAM-1.6. Correlation analysis between short-term neurological behavior and ICAM-1: In all model groups(including HIBD group and three administration groups), there was positive correlations respectively between the time of righting reflex, cliff aversion reflex, geotaxis reflex and ICAM-1(r1=0.888, P1<0.01; r2=0.734, P2<0.05; r3=0.863, P3<0.01; r4=0.902, P4<0.01),(r1=0.766, P1<0.05; r2=0.897, P2<0.01; r3=0.831, P3<0.05; r4=0.769, P4<0.05),(r1=0.878, P1<0.01; r2=0.817, P2<0.05; r3=0.762, P3<0.05; r4=0.804, P4<0.05). it all prompted that the time of short-term sensorimotor reflection increased with the expression of ICAM-1. Conclusions:1. Significant brain damage was caused by hypoxic-ischemic in neonatal rats, mainly performed in: the brain tissue structure was disordered, the cell number significantly reduced, cell morphologic change such as swelling and degenerative and necrosis. This confirmed that the model of HIBD was successfully established in neonatal rats.2. The expression of ICAM-1 in brain tissue in neonatal rats was not only increased significantly after hypoxic-ischemic, but also there was a significantly positive correlation between ICAM-1 and apoptosis index, short-term sensorimotor reflection, it showed that ICAM-1 was involved in the disease process.3. Using selective adenosine A(2A) receptor antagonist SCH58261 in neonatal rats after hypoxic-ischemic has a neuroprotective effect, including alleviation of the degree of degenerative and necrosis in nerve cell, improvement short-term neurological behavior and decrease of apoptosis, which possibly works through reducing the expression of ICAM-1.4. Giving three different doses of SCH58261 after HIBD in neonatal rats all can alleviate the brain damage, which low dose of SCH58261(0.01 mg/kg) is more obvious. |
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