Radioprotective Effects Of VPA On The Normal Brain Tissue Of Rat C6Glioma Models

Object Cerebral glioma, which is also called neurogliocytoma, is the most common primary tumor in central nervous system (CNS) with the character of refractory, local spread and tumor recurrence. At present, radiotherapy has beome one of the main adjunctive treatments in human glioma after surgery. However, the dose gradient of radiation is smaller between curing or controlling tumor and causing damage of normal tissue. To increase irradiation effect we can choose conformal radiotherapy to decrease the radiation dose normal tissues received, and increase the treatment dose. On the other hand, we can enhance lethality of radiation via improving radiosensitivity. It is a major challenge that reduces the damage of normal tissues if possible during the radiotherapy on tumors. Histone deacerylase inhibitors (HDACIs) are new antitumor agents, and a number of studies has shown that HDACIs could alleviate normal tissue injury as the radioprotector. Valproic acid (VPA) is widely used in glioma as antiepileptic drug with the feature of relaxative and harmfulless. We choose VPA as object of our study to observe radioprotective effects on the normal brain tissue of the rat C6glioma models following irradiation, which may provide experimental evidences for VPA to the therapy of glioma.Methods C6glioma cells were cultured commonly in vitro, and then we implanted C6glioma cells into Wistar rats’brains. We verified whether the C6rat glioma models were established successfully via MRI one week later. Rats (with tumor) were randomized into four groups (n=12). That is control group, VPA group, radiotherapy group, combined group. Control group received sham irradiation plus physiological saline. VPA group received sham irradiation plus VPA150mg/kg. Radiotherapy group received X-ray irradiation of3Gy as a single dose plus physiological saline. Combined group received X-ray irradiation of3Gy as a single dose plus VPA150mg/kg.24hours post-irradiation, six rats of each group were sacrificed by the heart perfusion with4%paraformaldehyde and brains were harvested. Cell apoptosis of the normal brain tissue on the contralateral cortex of the tumor was determined by Immunohistochemistry using an antibody for protein Caspase-3.We record the survival time of the rest24rats. Rats (without tumor) were randomized into four groups (n=6), which received the same and corresponding treatment. Then we record the animal bodyweight changes of2weeks start from the injection of VPA every day.6months after radiotherapy, the remaining rats were sacrificed for transmission electron microscope analyses to observe the vascular changes, neuron nucleus changes and synaptic changes.Results It was proved that the C6rat glioma models were established successfully via MRI. Immunohistochemistry results demonstrated that Caspase-3was significantly increased in radiotherapy groups compared to the combined group on the contralateral cortex of the tumor. The expression of Caspase-3is pale brown in cell nucleus. Survival was prolonged in VPA group, radiotherapy group and combined group compared to the control group (17d), and the median survival was19d,23d,28d separately. There were significant differences in survival time between radiotherapy group and combined group(p<0.05),while the combined group produced long term survivors (>50d). There was a trend toward lower body weight of radiotherapy group after irradiation compared to the rest three groups. There were significant differences in body weight change between radiotherapy group(without tumor) and combined group (without tumor)(p<0.05). Combined group (without tumor) weight change was characterized by a plateau and a steady increase during the irradiation compared radiotherapy group (without tumor). Both of combined group (without tumor) and radiotherapy group (without tumor) had a tendency of an increase with body weight after irradiation.TEM (transmission electron microscope) analysis demonstrated that vascular abnormalities of radiotherapy group after irradiation showed a severe structural disorder, loss and thickening of microvascular wall, hyaline degeneration and fibrosis generally changed. It appeared that focal swelling of the capillary endothelial cells in the part containing white matter could be detected with TEM in combined group. Mild swelling of the capillary endothelial cells in the irregular lumen could be observed. However control group and VPA group were normal vessel walls. Radiotherapy group neuron nuclear membrance surface was conveoconcave and irregular, even some was broken. However, combined group neuron nuclear membrance surface was a little conveoconcave and no broken. The structure of synappses was clearer and the number was larger in combined group than in the radiotherapy group.Conclusions VPA could reduce the damage of rat normal tissues from radiation, and the function that VPA relived the apoptosis of nerve cell arising from radiation played an important role. The treatment of combined group could prolong the survival of C6glioma rat. The mechanism was considered to be the radioprotictive of VPA on normal brain tissue, while we couldn’t exclude the radio sensitivity of VPA on tumor cells.