Neural Rehabilitative Action And Its Mechanisms' Studies Of Enriched Environment On Neonatal Rat With Hypoxic-ischemic Brain Damage

Neural rehabilitative action and its mechanisms' studies of enriched environment on neonatal rat with hypoxic-ischemic brain damageHypoxic-ischemic brain damage(HIBD) is a serious disease in neonatal period, which could lead to death and neurodevelopmental sequelae. There is no efficient therapy so far and what we could do now is just routine care and dealing with the symptoms. Nowadays, multicenter studies from China and overseas have verified that early intervention in convalescence stage for infants with HIBD could improve neural and ethological prognoses and relieve sequelae of nervous system..The mechanisms of the relationship between neural rehabilitation and early intervention are still the mystery of medicine. As the animal model of early intervention, the model of enriched environment(EE) for neonatal rat with HIBD makes us understand the mechanisms of early intervention better. EE includes abundant and variable breeding environment, more spontaneous movements and close communication among individuals etc. EE should have bigger cage with toys, such as ladder, roller, rope, platform, tunnel, box, ball, brick and trapeze and so on. The habits of circadian rhythm and cibation should be considered. The settings should be changed twice or three times per week.The brain of neonatal rat has plenty of neural stem cells(NSC) and precursor cells and would possess more neural plasticity theoretically. But the NSC and precursor cell are more vulnerable to HIBD. It is worth studying if EE could improve the neural prognosis of neonatal with HIBD and promote the proliferation and differentiation of NSC. In addition, the intervention window stage of EE for adult rat with brain damage is one week after surgery. The intervention would be effective after one month. Neonatal rat has more powerful neural plasticity, so the window stage could be longer. The preliminary purpose of the study was to investigate the neural prognosis, the proliferation and differentiation of NSC in dentate gyms(DG), the formation of myelin sheath, and the contents of neurotrophic factor of hippocampus, which subjected HIBD and different beginning time and intensity EE intervention. Part one The impacts of Enriched environment on the neural and ethological prognosis of neonatal rats with HIBDObjective To explore the impacts of Enriched environment, which had different initiation times and intensity, on the neural and ethological prognosis of neonatal rat with HIBD. Methods 7 days old neonatal rats were ligated right carotid artery and placed in hypoxic box, which contained 8% oxygen, for 2 hours to produce HIBD model. Rats were divided into early, intermediate and late intervention groups, which were experienced EE from 7, 14 and 21 days after HIBD for 14 days, in order to explore the impact of different initiation times of EE on the intervention effect. The early and intermediate intervention groups were divided into 1, 6 and 24 hours group, which were experienced EE intervention for 1, 6 or 24 hours separately everyday in order to explore the impact of different intensity of EE on the intervention effect. Trapeze tests were carried out 29 and 36 days after hypoxic-ischemic(HI) surgeries for early and intermediate groups and late group separately. Water maze tests were undertaken from 29 to 32 after HI surgeries for early, intermediate group and 36 to 39 days after HI for late group. Results As 1 hour EE intervention for early and intermediate groups, their scores of trapeze tests were 2.86±1.23 and 2.74±0.97, and had no difference with HI group(2.35±1.02). The scores of water maze test of early and intermediate 1 hour group were 53.28±21.2 and 51.23±21.23 seconds, and no different with HI group(56.66±10.96). The trapeze test's scores of early 6 and 24 hours groups and intermediate 6 and 24 hour groups and late intervention group were 3.67±1.12, 3.50±1.41, 3.50±0.93, 3.56±1.13, 3.50±0.92 separately, and scores of water maze of early 6 hours groups and intermediate 6 and 24 hours groups and late intervention group were 46.49±19.27, 38.20±18.36, 47.96±20.65, 35.93±22.45 seconds separately. These scores were significantly higher than HI groups of same phase. But the scores of trapeze tests and water maze tests among early and intermediate 6 and 24 hours groups had no statistical significance. Conclusion EE intervention with proper intensity could improve the abilities of anterior limb and space retention of rat with HIBD significantly. EE intervention had a long window phase for young rats with HIBD probably. Part two The effects of enriched environment on the neural regeneration in dentate gyrus and cerebullar myelination for neonatal rats with HIBDObjective To explore the impacts of EE on macroscopically pathological changes and neural regeneration in indentate gyms and cerebullar myelination. Methods Animal model preparation and EE intervention were mentioned above. Rats were divided into early, intermediate and late intervention groups, which were experienced EE since 7, 14 and 21 days after HIBD for 14 days. The early and intermediate intervention groups were divided into 6 and 24 hours group, which were experienced EE intervention 6 or 24 hours everyday. And late intervention group experienced 24 hours intervention everyday. At the same time of intervention, rats were received peritoneal injection of 50mg/kg 5-bromodeoxyuridine(BrdU) everyday for 14 days. HI groups and sham groups were injected at corresponding time. Early and intermediate groups were sacrificed at 32 days after HI and late group at 39 days after HI. Pathological and immunohistochemical detections were carried out at DG plane. The cerebral transverse diameter and areas of half brains were measured to investigate the impacts of EE on cerebral pathology. Double fluorescence immunity technology was used to detect nascent NSC in DG through marking BrdU and Nestin positive cells and detected nascent astrocytes through marking BrdU and glial fibrillary acidic protein(GFAP) positive cell, meanwhile detected nascent neurons through marking BrdU and microbule associated protein 2(MAP2). Through these methods to identify the impact of EE on neural regeneration in dentate gyrus. Myelin basic protein (MBP)was marked to detect the effects of HIBD and EE on cerebral myelination. ResuLts There were no statistical significance of cerebral transverse diameters and areas of half brains when compared each EE groups with HI groups. The results of fluorescent histochemical detections showed that the total nascent cell in DG of early, intermediate and late HI groups were 102.23±24.44, 100.67±31.45and 72.45±26.38, and higher than the sham groups of the same period, which were 22.34±5.71, 25.45±6.78 and 27.45±4.57. The results had statistical significance. The nascent neurons in DG of early, intermediate and late HI groups were37.25±20.06, 44.57±13.95and 28.67±14.57 respectively, and higher than sham groups of the same period, which were 6.17±2.71, 10.17±2.64 and 10.50±2.89. The results had statistical significance. The total nascent cells in DG of early 6 and 24 hours groups, intermediate 6 and 24 hours groups and late EE group were 142.56±21.56, 152.57±29.67, 157.05±25.78, 162.79±29.92 and 115.34±25.79 respectively, and higher than the same period HI group with statistical significance. The nascent neurons in DG of early 6 and 24 hours groups, intermediate 6 and 24 hours groups and late EE group were 99.85±27.43, 94.00±26.97, 99.75±20.49, 114.85±24.74and 72.00±24.91, were significantly higher than the same period HI groups. It has been demonstrated that EE could facilitate the neural regeneration in DG, and most nascent cell were neurons. The amounts of nascent NSCs and astrocytes in DG had no difference among each EE groups and HI groups. The results of MBP marked cerebral myelin showed that the ratios of MBP positive cells of early, intermediate and late HI groups were 6.32±1.63% and 6.74±2.19% respectively, and less than the sham groups of same periods significantly, which were 9.09±1.69% and 9.37±2.46% respectively. The ratios of MBP positive cells of each EE groups were higher than the HI groups, especially the 6 and 24 hours of intermediate groups, which were 8.89±2.29 and 8.48±2.67, but had no statistical significance as compared with HI groups.Conclusions EE intervention had no significant effects on macroscopically pathological changes. EE could increase the neural regeneration in dentate gyrus, especially the neurogenesis of neurons. HI could detriment the cerebral myelination and EE may have beneficial effects on myelination after HIBD.Part three The effects of enriched environment on contents of NGF and BDNF in hippocampus of rats with HIBDObjective To explore the effect of EE on contents of nerve growth factor(NGF) and brain derived neurotrophic factor(BDNF) in hippocampus of rats with HIBD.Methods The methodologies were the same as above. Early and intermediate groups were sacrificed at 29 days after HI and late group at 36 days after HI. The hippocampus were removed to measure the contents of NGF—βand BDNF using western blot. Results The relative abundance of NGF—βin hippocampus of early, intermediate and late HI groups were 0.4939±0.1837 and 0.3500±0.1095 respectively, and significantly higher than sham groups of the same periods, which were 0.1433±0.0536 and 0.1003±0.0777. The relative abundance of BDNF in hippocampus of early, intermediate and late HI groups were0.9352±0.1961 and 0.4274±0.1764, and significantly higher than sham groups of the same periods, which were 0.1758±0.1058 and 0.1086±0.0688. The relative abundance of NGF—βof early 6 and 24 hours groups, intermediate 6 and 24 hours groups and late EE group were 1.0419±0.1972, 1.1849±0.3396, 1.2250±0.4331, 1.4587±0.5351 and 0.7799±0.2533 respectively, were significantly higher than the HI groups of the same periods. The relative abundance of BDNF of early 6 and 24 hours groups, intermediate 6 and 24 hours groups and late EE group were 1.9112±0.4088, 1.7472±0.2484, 1.6027±0.6925, 1.8560±0.4891 and 1.0565±0.4415 respectively, were significantly higher than the HI groups of the same periods. Conclusion HI may increase the contents of NGF and BDNF in hippocampus and EE intervention may more significantly increase the contents of NGF and BDNF than HI.Conclusions:1. EE intervention could improve the neural and ethological prognosis of rat with HIBD. No matter began at early, intermediate or late period, the intervention had beneficial effects. EE intervention had a long window stage for young rats probably.2. The effects of 6 or 24 hours intervention were better than 1 hour intervention everyday. But there was no significant difference between 6 and 24 hours intervention groups, it showed that the intensity of 6 hours intervention everyday were enough for such neonatal rats models and had no benefits even prolong it.3. EE could increase the neural regeneration in dentate gyrus, especially the neural regeneration of neurons, no matter for early, intermediate or late EE groups. EE may have beneficial effects on myelination after HIBD.4. Early, intermediate and late EE intervention may increase the contents of NGF and BDNF in hippocampus of rats with HIBD.5. EE intervention could facilitate the neurogenesis of DG and elevate the contents of NGF and BDNF of hippocampus, which could be one of the mechanisms of EE to improve the neural and ethological prognosis of rats with HIBD.