Study On Protective Effect Of Combined Administration Of NGF And GM₁ On Hippocampal Neuron In Neonatal Rats With HIBD

Objectives: Hypoxic-ischemic brain damage(HIBD)in the human perinatal period often leads to the death of neonates or long-term neurobehavioral dysfunction.The mechanisms of HIBD is very complified.Apoptosis is very important in the death of cells and it can be prevented and treated.Rat HIBD model was used in this research to study the expression of Bcl-2 and Bax protein,number of apoptosis cells of hippocampal in the hemisphere ipsilateral to the injury, we also test spatial memory abilities and observe their changes after therapy with NGF and GM₁, and combined administration of NGF and GM₁,to vestigate the short-term protection and effective effect of long-term learning ability of administration NGF and GM₁ on hippocampal neuron rat models with HIBD.Methods:SD rats(postnatal day 7)were randomly divided into five groups: sham-operation group,HIBD group,NGF group,GM₁ group and NGF+GM₁ group.The animal model was constructed according to Rice method. NGF (200u/ml,2000u/kg+saline 10ml/kg) and GM₁ (2mg/ml,20mg/kg+saline 10ml/mg) were intraperitoneal injected each day for 7 days.Only saline was applied in HIBD group. All the subjects were sacrificed at 6h, 24h, 72h,7d after HI (Hypoxic-ischemic). HE stain, Immunohistochemical stain and Tunel stain were employed to discover the moophorlogical change: bcl-2, Bax protein expression and apoptosis in hippocampal neuron. The learning and memory ability of subject was tested from postnatal day 42 (P42)through Morris water maze.Results:1 The morphological changes of hippocampal neuron.The neuron in both sides of hippocampal of sham-operation group and right-sided hippocampal of HIBD group arranged orderly and remained normal. The neuron in left-sided hippocamal swollen and disorder arrangement were observed after 6h of HI, and necrosis were observed after 24h, which was most apparent after 7 days in HIBD group.The pathological changes of NGF group, GMi group and NGF + GMi group.2 The apoptosis of hippocampal neuron.Tunel positive cells were observed as brown cytoplasma. They appeared after 6h in HIBD group and lasted for 7 days. Tunnel positive cells in HIBD group were highest than that of other groups(p<0.01), Tunnel positive cells were less observed in NGF group and GMi group (p<0.01),the positive cells in NGF + GMi group decreased when compared with NGF group and GMj group(p<0.01; p<0.05).3. Bcl-2, Bax protein expression in hippocampal region in Neonatal rats after treatment.Bcl-2, Bax positive cells were observed as brown cytoplasma.Bcl-2 protein expression began to display after 6 hours of HI,and lasted for 7 days in HIBD group. The Bcl-2 protein was more apparent in the NGF group and GMi group(p<0.01; p<0.05). In the combined adminstration of NGF and GMi group that was most apparent(p<0.01). Bax protein expression could be found after 6 hours of HI in HIBD group and lasted for 7 days. In NGF group and GMi group(p<0.01), Bax positive cells were apparent in NGF + GMi group when compared with NGF group and GMi group(p<0.01; p<0.05).4. The effect of treatment with drugs on study and memory ability in neonatal rat with HIBD The latency period of the place navigation test in the group of HIBD was longer thanthe group of NGF and GMi (p<0. 01).The period was least in the NGF+GMi group when compared with NGF group and GMi group (p<0. 05;p<0. 01) and there were no significant difference between NGF+GMi group and sham-operation group(p>0. 05). In the spatial probe test ,the time that was observed in the sham-operation group of both staying on the platform and crossing the platform is the most(p<0. 01). It was the least in HIBD group(p<0.01) and other treatment group were more(p<0.01),they were more in NGF+GMi group when compared with NGF group and GMi group (p<0. 01; p<0. 05).Conclusion:1 The animal models were constructed through suture of common carotid artery of rats of postnatal 7 days, and that the animals were placed in hypoxic environment (8%) for 2 hours. The brain damage of neural necrosis, apoptosis, and decrease of long-term learning ability was apparent.2 Apoptosic cells and Bax expression in hippocampal region in rats increased significantly after HIBD, and Bcl-2 expression increased the amount of apoptosic cells and Bax expression decreased after treatment with NGF and GMi.3 The combined administration of NGF and GMi improved more Bcl-2 expression and decrease less Bax expression in hippocampal region.4 The decrease of long-term learning ability in neonatal rats maybe relate to hippocampal cell apoptosis. Application of NGF and GMi could improve the learning ability significantly maybe relate to less apoptosis of neuron.5 The combined application of NGF and GMi are more effective than them single use in the treatment of brain damage.