| Background and objectivesHypoxic-ischemic encephalopathy (HIE) is one of the common diseases during the neonatal period, which usually causes the neonatal deaths and developmental disorders of the nervous system. According to WHO datum in 2005, there were 400 million neonatal deaths each year in the world, and 23% of asphyxia-related. Even in Europe, America and other developed countries, the mortality and severe disability rates of moderate and severe HIE were also high to 53%～61%, which was worse in developing countries. At present, it is lack of safe and effective neuroprotection for this disease. In clinical, the main way is the supportive therapy of stable internal environment.Mouse nerve growth factor (mNGF) is extracted from mouse submandibular gland nerve, which is a relative safe and less-toxic drug. It does not affect any organ by using high dose and long time, and it is the first one found and studied thoroughly in neurotrophin, which has trophic and protective nerve, promotes the neurite outgrowth, and so on. It is of great importance to grow, differentiate, repair, regenerate and express in functional properties for central and peripheral nerve.Through establishing the hypoxic-ischemic brain damage (HIBD) model of neonatal rats, we observed the learning and memory ability of rats treated by mNGF, and the effects of expression vascular endothelial growth factor (VEGF) in cerebral cortex and neurofilament protein (NFP) in dentate gyrus. To investigate the mNGF protective mechanism for HIE from the regeneration of nerve and blood vessels provides an experimental basis for clinical.Materials and methods1 Animals and groups 126 seven-day-old Sprague-Dawley (SD) rats were randomly divided into three groups which subdivided to seven time points (3h,12h, Id,3d,7d,14d,53d):the sham group, the model group and the treatment group, and 6 rats in each point.2 Model preparation and drug application Model preparation was to ligate the left common carotid artery and give 2 hours of hypoxia. Nothing was given The sham group used, and the model group were injected intraperitoneally with normal saline solution, the treatment group with MNGF.3 Specimens The rats were killed by heart perfusion at each time point after surgery(except 53 days), and got the brains after fixation. Then gave para-formaldehyde fixation, conventional dehydrated, transparent, embedded in paraffin, and consecutive coronal sections for HE staining and immunohistochemistry.4 Results determined Using the light microscope in 400 times, VEGF expression showed brown in the cytoplasm belongs to positive cell, and it is also NFP expression in the cytoplasm, processes, axons. With Qwin550CW image processing and analysis system to test the mean gray value of the positive material.5 Y-maze test To test the learning ability of rats (53 days after surgery) by Y-maze, it reached the learning standard if nine correct responses to ten consecutive tests. The number as the norm of learning ability was recorded. To repeat ten times after 24 hours for observing memory retention.6 Statistical analysis To analyze all the data by SPSS 17.0 statistical software, the form of x±s stood for results. ANOVA and LSD-t were used. The difference is statistically significant when P<0.05.Results1 Results of pathology In the light microscope, brain tissue of the sham group at different time points showed clear hierarchy, regular arrangement and normal cell morphology. On the contrary, the model group showed disordered arrangement, dissolved cell bodies, lightly stained cytoplasm, nuclear condensation and forming cavities. And cells regular arrangement, patchy nerve cell degeneration and necrosis could be seen in the treatment group.2 Results of immunohistochemistry The expression of VEGF in the sham group was maintained at a low level as compared to the model group and the treatment group. At different time points after HIBD, the expression of VEGF in the treatment group was higher than that in the model group. The average integrated optical density (IOD) of VEGF-positive area in the treatment group was higher than that in the model group from 3h to 3d (P<0.05), and the time to peak was 3 hours there were no significant differences at 7d or 14d time point. The average IOD of NFP-positive area in the model group and the treatment group were significantly lower than that in the sham group from 3h to 1d (P<0.01), but it was significantly higher in the treatment group than that in the model group at 3d,7d and 14d time points(P<0.01).3 Results of Y-maze test The model group needed the highest number to reach the required standards, and the lowest in the sham group, but converse in the memory ratio after 24 hours. Two differences in three groups had statistically significance (P<0.05).Conclusions1 mNGF can improve learning and memory in rats.2 mNGF may induce endothelial cell proliferation, promote angiogenesis and improve blood and oxygen supply to brain tissue by increasing the expression of VEGF.3 mNGF may promote axonal sprouting and regeneration by increasing the expression of NFP, and contribute to the repair and regeneration of nerve cells in brain injury. |
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