| Epilepsy, a brain dysfunctional syndrome with many pathogenies, is characterized by recurrent and temporary disorder of brain funtions. There are about 4,550,000~6,300,000 patients with epilepsy in China, with about 300,000 new epilepsy diagnoses every year. Within this population, about 800,000~1,000,000 patients are sufferring from medically intractable epilepsy, which remains a knotty problem for modern clinical medicine.With the development of stereotactic radioneurosurgery, Gamma Knife Surgery (GKS) turns to be a promising alternative technology for intracranial lesions without invasive operation. Moreover, the application of magnetoencephalogram (MEG), a noninvasive epileptogenic focus-locating device, promotes the use of GKS for the treatment of drug resistant epilepsy. Clinical information from stereotactic radiosurgery indicated that, GKS could decrease the frequence of epileptic seizures without causing subsequent funcitonal impairment.N-methyl-D-aspartate receptor (NMDAR), a subtype of ionotropic receptor for glutamate (N-methyl D-aspartate is a name of its selective specific agonist), could induce slow excitatory effect. Activation of NMDA receptors results in the opening of an ion channel that is highly permeable for Ca2+. The influx of Ca2+ mediates variety of physiological processes, such as synaptic plasticity and synaptic transmission. Althouth the pathogenic mechanisms of epilepsy are complicated, the relationship of NMDAR and epilepsy is affirmative. It was reported that, NMDAR played an important role in the development and mainteinance of epilepsy.In the etiologic studies of epilepsy, several ion channels are identified to be involved in some cryptogenic epilpsy. Especially, the abnormalities in structure and/or functions of voltage-dependent sodium channels play an important role in the development of epilepsy.Part 1 Effects of Gamma knife irradiation on the expression of NMDA receptor subunits in rat forebrainObjective: To examine the effects of GKS on the expression NMDAR subunits in rat forebrain.Methods: Using stereotactic technique, we performed Gamma Knife irradiation on the left forebrain of 18 male Wistar rats with a maximum dose of 30 and 60 Gy respectively for two experimental groups (9 animals for each group). These animals were further raised for 24 h, 30 or 60 d before they were killed. Then immunohistochemstry and in situ hybridization were used to detect the relative levels of NMDAR subunits (NR1, NR2A, and NR2B) in the target region.Results: Compared with control group, the expression (mRNA and protein) of NR1 and NR2A but not of NR2B of the 30 Gy group rats increased significantly in the irradiated cortex at 60 d after operation. For the 60 Gy group, the expression (mRNA and protein) of NR1 and NR2A but not of NR2B increased significantly in the irradiated cortex at both 30 and 60 d after GKS. Nevertheless, no significant differences of these three subunits were detected in the caudate putamen at all time points.Conclusion: Gamma Knife irradiation induced up–regulation of NMDAR subunits, NR1 and NR2A, which might represent a possible mechanism underlying the therapeutic effects of Gamma Knife irradiation on many neurological diseases, including drug resistance epilepsy.Part 2 Effects of Gamma Knife Surgery on cytoplasmic calcium level of rat cortex neuronsObjective: To investigate the effects of GKS on cytoplastic calcium level of rat cortex neurons.Methods: 24 Wistar rats were randomly divided into control group (6 animals) and 2 experiment groups (9 animals for each experiment group). The maximal doses for the two experimental groups are 30 Gy and 60 Gy respectively. After left frontal lobes of experimental group rats were treated with GKS, the animals were decapitated at 24 h, 30 d, and 60 d after GKS respectively. Cortex neurons of the target regions were acutely isolated and loaded with fluorescent dye fluo-3. Then the fluorescence intensity of cytoplasmic calcium was measured with laser scanning confocal microscopy (LSCM).Results: There was no significant difference of cytoplasmic calcium fluorescence intensity between the two experimental groups and control group at 24 h after GKS. At 30 d, the cytoplasmic calcium fluorescence intensity of 60 Gy group, not of the 30 Gy group, increased significantly compared with control group. At 60 d after GKS, the cytoplasmic calcium fluorescence intensitys of both 30 Gy and 60 Gy groups are significantly higher than that of control group.Conclusion: GKS could induce relative calcium overload in target cortex neurons in a moderately longer period through up-regulating NMDAR subunits.Part 3 Effects of Gamma Knife irradiation on NMDA induced current of rat cortical neurons in vitro.Objective: To investigate the effects of GKS on NMDA induced current of primarily cultured rat cortical neurons in vitro.Methods: Primarily cultured neurons were randomly divided into 4 experimental groups and 4 corresponding control groups. Using stereotactic technique and 18 mm collimating device, we performed gamma knife irradiation on primarily cultured cortical neurons with a maximum dose of 30Gy. After irradiation, the neurons of experimental and control groups were cultured for additonal 1, 3, 7 and 10 days respectively before NMDA receptor subunits expression and NMDA induced current were detected. For the examination of NMDA induced current, 6-8 neurons of each group were recorded for each time point.Results: Western blot examinations showed, compared with control group, the expression of NR1 and NR2A, but not NR2B, increased significantly at 3, 7, 10 d after gamma knife irradiation, P<0.05. Whole-cell current recording showed, the NMDA induced current peak densities (pA/pF) of primarily cultured cortical neurons of experimental groups recorded at 3, 7 and 10 d after gamma knife irradiation were significantly increased compared with control groups (P<0.05).Conclusion: Gamma knife irradiation could increase NMDA induced current in primarily cultured rat cortical neurons, which might represent a possible mechanism underlying the therapeutic effects of gamma knife irradiation on epilepsy.Part 4 Effects of Gamma Knife irradiation on voltage-dependent sodium current of rat cortical neurons in vitro.Objective: To investigate the effects of GKS on voltage-dependent sodium current of primarily cultured rat cortical neurons in vitro. Methods: Primarily cultured neurons were randomly divided into 4 experimental groups and 4 corresponding control groups. Using stereotactic technique and 18 mm collimating device, we performed gamma knife irradiation on primarily cultured cortical neurons with a maximum dose of 30Gy. After irradiation, the neurons of experimental and control groups were cultured for additonal 1, 3, 7 and 10 days respectively before voltage-dependent sodium current were detected. For each group, 6-8 neurons were examined at one time point.Results: Voltage-dependent sodium current recording shows, the voltage-dependent sodium current peak densities (pA/pF) of primarily cultured cortical neurons of experimental groups recorded at 1, 3, 7 and 10 d after gamma knife irradiation were 89.94±4.65, 84.13±4.56, 80.27±5.18 and 80.80±5.41, respectively. Compared with control groups (current peak densities at 1, 3, 7 and 10 d were 100.93±4.63,97.89±4.77,100.76±5.90 and 102.84±4.08, respectively), were significantly reduced by GKS in a time-dependent manner (P<0.05). The I-V curves of voltage-dependent sodium current of experimental groups were also upwardly shifted significantly, while the decents of I-V curves shifted to the right.Conclusion: Gamma knife irradiation reduced the excitability of primarily cultured rat cortical neurons through inhibiting the ability of voltage-dependent sodium channels, which might represent a possible mechanism underlying the therapeutic effects of gamma knife irradiation on epilepsy.
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