| Objective: Currently, there are more and more cases in the treatment of epilepsy using Gamma Knife, although therapeutic efficacies were clinically gained in some degree ,the arguments were existed,especially the basic radiobiological research of Gamma Knife in the treatment of epilepsy, was comparatively unsubstantial. In our experiment, motor cortexes of healthy cats'frontal lobe as a target, were given different doses of Gamma Knife irradiation. observation of The changes of morphology, the quantity of cell apoptosis and the changes of bcl-2 protein expression of the motor cortex in the frontal lobe were observed in different time after irradiation. The relationship between the time and dose-effect, and the molecular mechanism of apoptosis inducing by Gamma ray were explored. This research provides theoretical and experimental basis for choosing the suitable therapeutic dose for clinical treatment for epilepsy in functional area, tumors etc by using Gamma Knife. Furthermore, radiation-induced brain injury may be reduced.Methods: 96 healthy domestic cats, weigh 2500~3500 grams, were randomly divided into control group, Gamma Knife 15Gy irradiation group and 50 Gy group(with 32 cats in each group), regardless of sex. There were five observation time points including 1 day, 2 days, 3 days, 7 days, and 14 days. 6 healthy cats were randomly selected at each time point in each group, the control group was not irradiated with Gamma Knife, and the other two groups were given different doses of Gamma irradiation. The selected cats were sacrificed at the above mentioned different time points to make paraffin slice. Then we used immun-ohistochemical method to determine the bcl-2 protein and Tdt-mediated dUTP nick end labeling (TUNEL) method to detect the dynamic changes of apoptosis. Additionally, we selected two animals on the 14th day in each group for MRI detection and specimens sampling to observe changes in the ultrastructure using transmission electron microscopy (TEM). All these data were expressed as mean±standard deviation( x±s), and analyzed with SAS statistical software. P<0.05 is considered significantly different.Results:1 General state of animal: all the trial animals completely sobered up and began to restore diet gradually and didn't show symptoms such as hemiplegia at 6~8 hours after irradiation with Gamma Knife. The animals in 50Gy irradiation group were listless, poor appetite and less active within 7 days and recovered completely 7 days later.2 Changes of brain imaging with the MRI scan: the MRI scan for animals neither in 15Gy nor 50 Gy irradiation groups 14 days after irradiation showed obvious necrotic foci. 3 Changes of brain cells under the optical microscope: there's no degenerative change in neurones under optical microscope within 14 days after radiosurgery with Gamma Knife in 15Gy irradiation animals, while neuronal degeneration and nerve cells devouring were found in 50Gy group.4 Changes of TEM cell morphology observation: The neuronal cells karyon in 15Gy irradiation group was morphologically normal, but the endoplasmic reticulum expanded within 14d under electron microscope. The following phenomena were observed in 50Gy group under electron microscope: vacuoles in neuronal cells karyon, karyon pyknosis, endoplasmic reticulum and Golgi complex expansion.5 Changes of brain cell apoptosis: (1) The apoptosis after irradiation in Gamma-Knife irradiation group showed an increasing trend. Compared with the control group, the positive expressions percentage of apoptotic cells in 15Gy irradiation and 50Gy irradiation group at various time points increased, and statistical analysis showed significant difference(P<0.05). (2) The dynamic expression of apoptotic cells in each Gamma Knife irradiation group was certainly regular, that is, the positive expression of apoptotic cells begin to increase from 1 day after irradiation, reached a peak at 2 days, then decreased again gradually, which is still higher than that in the control group at 14 days. (3) At the same time point, the positive expression percentage of apoptotic cells was correlated with irradiation dose, that is, the higher the dose, the more obvious the decrease of apoptotic cells. The positive expression percentage of apoptotic cells in 50Gy group were higher than that in 15Gy group, but q test showed significant difference between the two irradiation groups only at 2 days after irradiation (P<0.05).6. Changes of the bcl-2 protein expression in brain cell (1) The bcl-2 protein after irradiation in Gamma Knife irradiation group showed an decreasing trend. Compared with the control group, the bcl-2 protein expression in the 15Gy and 50Gy irradiation group at various time points were lower , and statistical analysis showed significant difference (P<0.05). (2) The dynamic expression of bcl-2 protein in each Gamma Knife irradiation group had certain regularity, that is, bcl-2 protein expression decressed from 1 day after irradiation, attained the minimum value at 2 days, then rised again gradually, which was still lower than that of the control group at 14 days. (3) At the same time point, the positive expression percentage of bcl-2 protein was correlated with irradiation dose, that is, the higher the dose, the more obvious the decrease of bcl-2 protein. The positive expressions percentage of bcl-2 protein in 50Gy group was lower than that in 15 Gy group, but q test showed no significant(P>0.05).Conclusion:1 The peripheral dose of 15Gy with isodose curve of 50% were safe dose for Gamma Knife treatment. The peripheral dose of 50Gy with isodose curve of 50% can cause neuronal degeneration after irradiated for 14 days, which long-term aleration need to be observed more deeply.2 The research suggested the incidence of radiation- induced brain injury was lower in 15Gy irradiation group after irradiated with gamma knife, which provided theoretical and experimental basis for the clinical application of gamma knife at low-dose (peripheral dose of 10~15Gy, isodose of 40~60%) in the treatment of epilepsy.3 The changes of cell apoptosis quantity of cat's normal prefrontal motor cortex were correlated positively to irradiation dose within 14days after gamma knife irradiation. The weaken of the bcl-2 protein expression in the area may be one of the molecular mechanisms of cell apoptosis caused by gamma knife irradiation.
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