Role Of VEGF In NG2 Cells Proliferation In Rats After Cerebral Ischemia

Background and objective:In recent years,many studies have demonstrated that vascular epithelial growth factor(VEGF) could not only protect the nerve system, but also facilitate the surviving of the injured neurons and regenerate the adult mammalian neurons. VEGF could alleviate the brain injure and could be used in the treatment of ischemia cerebral vascular disease.During the development of the nervous system,VEGF is predominantly expressed in the neuroectodermal ventricular perimeter,where VEGF expression gradually decreases from the inner to the outer. In the nervous system,VEGF is widely distributed in neuroblastoma cells, glial cells and vascular endothelial cells,but expression of VEGF is the highest in neuroblastoma cells. With the development of neutral nervous system,expression of VEGF gradually decreases. In the nervous system of normal adult human and animals,levels of VEGF are very low, however the expression of VEGF up-regulate significantly after cerebral ischemia. The high expression of VEGF induced by cerebral ischemia occurred in neurons and glial cells of ventricular perimeter. Recent studies show that VEGF can not only directly extend neuroprotection on neurons but also lengthen cell survival until the formation of new blood vessels. This protection mechanism is that KDR protects cortex neurons against lack of oxygen and glucose. It is confirmed that VEGF can facilitate proliferation of neuron progenitors in vitro and in vivo model through KDR.In the embryonic stages, VEGF play a important role in the growth of nerve tissue because it can induce angiogenesis and neuronal generation (neumgenesis). When the brain suffers from ischemia and hypoxia, VEGF can extend neuroprotection on brain by inducing proliferation and migration of neural stem cells. The number of neural stem cells increases by 20% to 30% in the present of VEGF. VEGF infusion into the lateral ventricle of rat can causes a dramatic increase in Brdu-positive cells in the rat hippocampus, which shows that VEGF can contribute to the proliferation of neural stem cells.Oligodendrocyte precursor cells (oligodendrocyte progenitor cells OPCs) have been widely found in the gray and white matter of adult mammalian in recent years. As a specific cell markers of OPCs,NG2 is a sulfate proteoglycans (chondroitinsulp peoteoglycan, hate CSPG) expressed on the membrane of OPCs. In the system of cell culture, OPCs have proved to have the potency of cell differentiation, such as differentiating into oligodendrocyte cells, astrocytes, even neurons. In the rat, the amount of the NG2 positive cells increased accompanied by the development of embryo during the embryonic period which can reach the peak at the time of birth. Although the myelinization is almost completed in the first month, a large amount of NG2 positive cells still exist. Adult covers all glial cells in the central nervous system in the proportion of 5%-8%, and the proportion in gray and white matter is slightly different, 8%-9% in white matter., 2% to 3% in gray matter, in the regions of cortex and hippocampal the ratio between NG2-positive cells and oligodendrocytes can reach 1:1. Despite the difference of amount of positive cells, in gray and white matter NG2 positive cells also showed different shapes, such as round or oval shape in the gray area which sent out many protuberance; streak or spindle shape in the white matter which is squeezed into a narrow region by myelinization axon. When the cerebral ischemia happened, the morphological changes of NG2 positive cells surrounding damaged regions took place which included the volume enlargement, protuberance hypertrophy and immunoreactions enhancement.NG2 positive cells is one of cell types which response quickly to the central nervous system damage, but its function in normal central nervous system is not yet clear. Despite of acupuncture and spinal cord injury, ischemia and demyelinating induced by poisoning experiment can induce NG2 positive cell proliferation. The role of the cell in the mature brain and the meanings of its activation under damage and repair situation are not yet clear. The current study have showed that the rapid proliferation of NG2-positive cells is the main characteristic of central nervous system responded to injury, but the signal for the proliferation is still unknown. This response only happened in the injury area, so no matter what the mechanism promoting re-enter the cell cycle of NG2-positive cells must locate in the damage area only. One possibility was that neighboring neurons or oligodendrocytes maintained slow proliferation of NG2-positive cells by Notch signaling pathway.Because VEGF can promote endothelial cells and vascular smooth muscle cell proliferation, it is used on the treatment of ischemic vascular disease. Recent researches have showed that VEGF was a very important neurotrophic factors and protective factors, VEGF receptors distributed on the surface of neurons and astrocytes, and VEGF could be synthesized by nerve cell itself. VEGF is involved in the regulation of signaling pathway. If VEGF is involved in promoting the process of NG2 cell proliferation after the brain ischemia is not yet reported.Objective:To observe the reactive changes of oligodendrocyte progenitor cells(OPCs) in rats of cerebral ischemia resulted from carotid artery stenosis,and discuss explore the effects of vascular epithelial growth factor (VEGF) in on these cells.Method:78 healthy SD rats aged 5~6 months, weighing 280 to 300 g, in half respectively male and female were randomly divided into three groups: the sham operation non-ischemia control group (control group), ischemia group and ischemia + bevacizumab groups. Every group was randomly divided into 4 time units (cerebral ischemia 6,12,24,72 h repectively),10 rats in each. The change of NG2 and VEGF is detected by double immunofluorescent staining. 2 rats were selected randomly for DSA examination in each group.Results:1. After cerebral ischemia resulted from carotid artery stenosis, VEGF positive cells in the cerebral cortex began to proliferate,with the brain ischemic going on, reached the peak in 24h and then declined in 72h.The number of VEGF positive cells was up to(23.33±3.93) in 6h after ischemia, which was significantly higher than the sham operation group (3.17±0.75) (p <0.01); That number got to the peak in 24h after ischemia (57±3.58), significantly increased compared with 6h (p <0.05). The number of 72h's (46.67±2.16) decreased than 24h's (57±3.58) (p <0.05).2. After cerebral ischemia resulted from Carotid artery stenosis, NG2 positive cells in the cerebral cortex began to proliferate. The number of NG2 positive cells was up to(6.50±1.05) in 6h after ischemia, which was significantly higher than the sham operation group(2.83±0.31)(p<0.01); with the brain ischemic going on, NG2 cells increased further more to reach (17.67±2.73) (p <0.05) in 72h.3. The NG2 positive cells in cortex showed red fluorescence under fluorescent- microscope while VEGF positive cells showed green fluorescence, the cells double immunofluorescent labeling overlapped which fully overlapped showed yellow-green fluorescence.4. After cerebral ischemia, the number of NG2 and VEGF double? positive cells increased significantly, up to (5.50±1.05) in 6h after the ischemia, which was significantly higher than the sham operation group (2.83±0.31) (p <0.01); with the ischemia going on, NG2 and VEGF double positive cells increased further more,got to the peak in 72h after the ischemia (17.83±2.99), and the double-positive cells also increased significantly compared with that of 6h group (p <0.05), Overall,the number of NG2 and VEGF double positive cells was higher than the sham operation group in every different time point (p <0.01).5. The number of NG2 and VEGF double? positive cells in Ischemia + bevacizumab group was up to (4.33±1.21) in 6h, which was lower than the ischemic group (5.50±1.05) (p <0.05) and higher than the sham group (2.83±0.31) (p <0.01), after that,the number of the double-positive cells was lower than the ischemic group (p <0.05 )and higher than the sham group (p <0.01) in every different time points.Conclusion:1. The improved bilateral carotid artery stenosis models were capable of simulating the pathophysiology of cerebral ischemia. The model is stable and simple.2. Cerebral ischemia could result in NG2 cells proliferating and VEGF express increase.The mechanism of NG2 cells proliferating induced by cerebral ischemia may be related to the expression of VEGF.