| Background and objectivesHypoxic-ischemic encephalopathy is one of the most common diseases in neonatal nervous system. It may cause irritability, higher muscle tension, lethargy, convulsion and other symptoms. In severe cases, it can lead to coma, even direct death. Even though survived, but not treated promptly, the neonate may have serious sequelae (such as growth retardation, learning and memory decline, epilepsy, etc). The pathogenesis of hypoxic-ischemic encephalopathy includes dysbolism of energy, over-activation of excitatory amino acid, calcium overloading, apoptosis and so on. Over-activation of excitatory amino acid plays a critical role in the development of hypoxic-ischemic encephalopathy.NMDA (N-Methyl-D-aspartate) is the main receptor combined with excitatory amino acid. In physiological conditions, the excitatory amino acids and its specific receptor of amino acid are combined with each other, which can cause moderate Ca2+ influx and formation of LTP. The postsynaptic LTP are closely related to the learning and the memory. But when the receptor is overactivated, massive influx of Ca2+ would activate and enlarge the function of Ca2+ as the second messenger, which finally causes necrosis and apoptosis. The NMDA receptor is consisted of different subunit protein. NR1 and NR2B are the main subunit protein in hippocampus area. The former regulates the function of calcium ion aisle. The latter is closely related to the learning and the memory.Cell apoptosis induced by hypoxic-ischemic brain damage could lasts for a long time. The lost neurons in the last stage are mainly caused by apoptosis. Mitochondrial plays an important role in regulating apoptosis. Cytochrome C is released into the cytosol, which is the main pathway to cause apoptosis in cell.As a neurotrophic drug, Ganglioside has been applied to the clinical treatment for hypoxic-ischemic encephalopathy. It can improve the cognitive function impaired by hypoxic-ischemic encephalopathy, but there is very little research about it. To observe the expression of NR1, NR2B and cytochrome C in hippocampal CA1 area and investigate the mechanism of ganglioside improving cognitive function, we established the animal models of HIBD.Material and methods1 Prepared the rat models of HIBD and divided into group7-day old Sprague-Dawley rats were randomly divided into three groups:group A (model group); group B (ganglioside intervention group); group C (control group). There were 20 rats in every group. Group A and Group B were heads and extremities to keep a dorsal position after they were marked weight and anesthetized by diethyl ether, then degermed their skin, choosed the median incision of cervicum. Freed and ligatured the left common carotid artery, sew up the cut and taken them to the nest for 1 hour rest. Then, put them in an oxygen deficiency Box (inhaling 8%O2and 92%N2), made the hypoxic-ischemic rat model. Group C, only free the left common carotid artery, neither ligation nor hypoxia. All groups were breeded in the same way.2 Drug application and type collectionThe rats in Ganglioside intervention group were treated with intraperitoneal injection of ganglioside (30mg/kg) immediately after models were prepared, and 1 time a day for 7 days; the rats in model group were treated similarly with intraperitoneal injection of isotonic Na chloride; the control group received no treat. 10 rats were randomly selected from every group and them were killed at the eighth day, dissect the 30 rats and free the brain quickly after infusing heart, fixed the barins, took the center parts to be anhydrated and paraffin imbed, then made histological section from them.3 ObservationHistological sections were immunohistochemisty stained to observe the expression of NMDAR NR1, NR2B and Cytochrome C, and TUNEL stained to observe expression of cell apoptosis. The remaining rats were tested at the 28th days by maze test, observed the message of their study and memory ability.4 Statistical analysisSystematic analysis of the results with SPSS 13.0 software, measurement data were expressed as mean±standard deviation (x±s) and group comparison with one-way ANOVA, statistical significance were signed by (P<0.05)Results1 Learning and Memory ComparisonOn the first and second day, rats in group A need significantly more times to study than those in group B and group C (P<0.05). and the right reation rate were signigicantly lower than those two groups; however, rats in group B need significantly more times to study than those in group C, and the right reaction rate were significantly lower than those of group C.2 Expression of NRI, NR2B and CytochromeCCell membrane of NR1 positive neuron was stained brown, the colour of group A was deeper than others, some colorized nucles can be found. The number of positive neuron in group C was fewer than others. The average gray scale value of NR1 in three groups were (153.23±6.31,115.82±10.43,130.35±4.29, P<0.01), there were no significant difference.In group B and C, the membrane was stained brown.The membrane in Group A was stained lighter, and the number of neurons was few. The average gray scale values of NR2B were (147.86±14.85,142.76±7.11,113.27±9.50). Group B were significantly higher than that in group A (P<0.01). Compared group B with group C, there was no significant difference (P=0.148).In group C, the number of CytochromeC positive cell was rare. A large number of cells in group A were stained brown. Compared with Group A. group B was significantly decreased. The average gray scale value of three groups were significantly different (113.00±7.27,152.55±7.14,127.55±5.83,P<0.01).3 The condition of cell apoptosisThe results indicate that there is less apoptotic cells in group C, a large number of apoptotic cells in group A, the apoptosis cell in group B was less than the group A,but more than group C. The percentage of apoptotic cells in three groups were significantly different (5.12±1.32,59.23±2.73,30.15±3.26, P<0.01).Conclusions1. Ganglioside can improve the learning and memory in rats with the hypoxic-ischemic. Its mechanism may be achieved by increasing the expression of NR2B.2. Ganglioside can reduce apoptosis by hypoxia-ischemia. Its mechanism maybe achieved by reducing the expression of NR1 and CytochromeC. |
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