The Effect Of Hypoxia On The Growth Of Rat Intracranial Glioma

Part1. The Establishment of C6Glioma ModelPurpose:To establish a stable and reliable glioma animal model.Methods:40Wistar male rats were randomly divided into the experimental group (20) and control group (20).10μl C6cell suspension containing1×106cells was stereotaxically injected into the right caudate nucleus of the experimental group rat brain. In control group, lOμl DMEM medium was stereotaxically injected into the right caudate nucleus of the rat brain. After C6inoculation, we observed the growth of glioma. MRI scan were conducted after implantation at7d,21d. Para formaldehyde perfusion for2group rats having received implantation was carried out at21d. The tumor-containing samples were prepared histologically by hematoxylin and eosin stains. The expression of glial fibrillary acidic protein (Glial fibrillary acidic protein, GFAP) in the tumors was determined by immuohistochemistry.Results:1. After establishing Wistar-C6intracranial glioma model of rats through the stereotactic caudate injection, ponderal growth rate of experimental group was significant lower than that of control group. There was a decrease of weight in experimental group on15-17d. The experimental rats spirits are drooping and bilateral leg or left limbs are paralyzed. 2. After para formaldehyde perfusion, there was tumor mass in the right caudate nucleus of the experimental rats brain, which the boundarise were still clear. There was visible neovascularization within the tumor.3. HE staining confirmed tumor specimen in all tested tissues. There was patchy necrosis in the tumor mass. The tumor cells showed infiltrative growth, pathological mitosis, nuclear condensation and formation of apoptotic bodies. There was the strongly positive expression of GFAP in the tumors4. MRI scan prompted that the experimental group rats have intracranial tumor on day7after implantation. On day21, tumor volume and surrounding brain tissue edema were increased.Conclusions:The successful rate of C6brain tumor model established by the intracerebral inoculation of C6cells is very high in rats. The tumor established in the experiment is similar to human glioma in histopathological features and it can be used as a good model to study the glioma.Part2. Hypoxia on VEGF expression, proliferation and apoptosis of tumor cells in rat intracranial glioma modelsPurpose:To study effect of hypoxia on expression of VEGF, proliferation and apoptosis of tumor cells in rat intracranial glioma models.Methods:50Wistar male rats were randomly divided into five groups(ten rats in each group).A group(control):animals were injected with10μl of DMEM nutrient solution; B group(Hypoxia control):animals were injected with10μl of DMEM nutrient solution and hypoxia treatment; C group (Glioma induced+Hypoxia):animals were injected with cell suspension of C6glioma cells and hypoxia treatment; D group (Glioma induced):animals were injected with cell suspension of C6glioma cells and no hypoxia; E group(Hypoxia preconditioning):animals were injected with cell suspension of C6glioma cells after completion of a3-week repetitive hypoxic preconditioning. The oxygen concentration was8%, lh/d. At14d,35d after implantation, the brains of the group A, B,C,D rats were obtained for VEGF and Ki-67by immunohistochemistry. At14d after implantation, the brains of the group E rats were obtained for VEGF and Ki-67by immunohistochemistry. Used TUNEL staining to detect the apoptosis of tumor in rat.Results:1. There was no difference of intratumoral expression of VEGF between C group and D group. The intratumoral expression of VEGF in E group was lower than that of C group, but there was no statistical difference. There was rare expression in normal tissues.2. The intratumoral expression of Ki-67in D group was significantly higher than that of A group(P<0.05). There was no difference of intratumoral expression of Ki-67between C group and D group. The intratumoral expression of Ki-67in E group was lower than that of C group, but there was no statistical difference.3. There was no difference of intratumoral expression of TUNEL in C group,D group and E group.4. The balance between proliferation and apoptosis of the cells of glioma was deranged. But the proliferation/apoptosis index (P/AI) was no statistical difference between C group and D group.Conclusions:1. The effect of hypoxia on expression of VEGF, proliferation and apoptosis of tumor cells was ineffective in rat intracranial glioma models.2. Repetitive hypoxic was not harmful to normal rats.3. To some degree, the hypoxia tolerance could be improved by repetitive hypoxic preconditioning. But the effect was not obvious.