Clinical Analysis Of Cognitive Condition Of Patients With Intractable Epilepsy And The Effects Of Low Dose Of Gamma Knife Irradiation On Cognitive Functions Of Epileptic Rats And Its Mechanism

Epilepsy, a brain dysfunctional syndrome caused by abnormal discharge of neurons with many reasons, is characterized by recurrent and temporary disorder of brain functions. Seizure is the core content of epilepsy, but in recent years, the effect of epilepsy on cognitive function has been paid more and more attention. The data showed that about 30~40% of epileptic patients were troubled with various degrees of cognition disorders, which not only seriously affected the patients'life quality, but also brought burden and pressure to their families and society.About 20~30% of epilepsy is medically intractable. With long-term and recurrent seizures, the patients'cognitive function may be damaged easily. The primary treatment method for patients with intractable epilepsy is operation. Although operation may control seizure and lessen the dosage of antiepileptic drugs, which could improve cognitive function of the patient, at the same time, the operation itself may cause harm to cognition in some degree. Therefore, it is very important to pay attention to cognition disorders of patients with intractable epilepsy and to understand their characteristic and influencing factors. Making an objective assessment of cognitive condition before and after operation has significance to evaluate comprehensive efficacy for operation and select reasonable treatment protocols.With the development of stereotactic radiosurgery, gamma knife surgery (GKS) has turned to be a promising alternative technology for intracranial lesions without invasive operation, which has been used in treatment of intractable epilepsy. The data from clinical and basic research suggested that, sublethal dose of gamma knife could produce an antiepileptic effect without causing subsequent functional impairment in target areas, but its mechanism remains unclear, which limits further optimization of treatment. Whether gamma knife surgery could treat epilepsy without damage to cognitive function and even improve cognition, needs further study.At present, cognition disorder of epileptic patients is considered to be related with many factors, but its exact biologic mechanism is still debatable. N-methyl-D-aspartate receptor (NMDAR), a subtype of ionotropic receptors for glutamate, is highly permeable for Ca2+ and could mediate variety of physiological processes, such as synaptic plasticity and synaptic transmission, which is closely associated with learning and memory. The increased intracellular Ca2+ may phosphorylate cAMP response element binding protein (CREB) through several signal transduction pathways such as calmodulin dependent protein kinase (CaMK) and extracellular signal-regulated kinase (ERK). As an important nuclear transcription factor, CREB could regulate the process of learning and memory on gene transcription level. Several study showed that NMDAR, influx of Ca2+ and proteins of signal pathways play important roles in the development and maintenance of epilepsy, but the pathogenic mechanism is unclear. Whether gamma knife surgery could protect cognitive function of epileptic patients by intervening the change of biological molecules, remains to be deeply researched.Our study, on the one hand, analyzed the cognition and influencing factors of patients with intractable epilepsy, and then evaluated the effect of surgery on their cognitive function; and on the other, through establishing epileptic animal model, we performed low dose of gamma knife irradiation on rats and assessed their abilities of learning and memory, and then we observed the changes of NMDAR, intracellular Ca2+ and proteins of CREB signal pathways in cortex and hippocampus, in order to explore the biologic mechanism of cognition disorders and the effects of gamma knife surgery.Part 1 Clinical analysis of cognitive condition and influencing factors of patients with intractable epilepsyObjective: To explore the characteristic of cognition disorders of patients with intractable epilepsy and analyses the effects of various influencing factors on cognitive function.Methods: 62 patients with intractable epilepsy and 60 healthy persons were chosen in the study. The methods of SPM, EPQ and SCL-90 were used respectively to evaluate their intelligence, personality and mentality, and the effects of various influencing factors on cognitive function were analyzed.Results: 1.Compared with control group, the total score and IQ of SPM of the epilepsy group decreased significantly. There were 53.2% of mental deficiency in patients with intractable epilepsy, and the proportion was higher than that of control group(1.7%)(P<0.05). There were only 18 patients with middle intelligence, and the proportion was 29%, which was lower than that of control group(53.3%)(P<0.05). 2. There were no significant differences on scores of EPQ and the proportion of personality type in two groups(P>0.05). 3. Many patients with intractable epilepsy had various degrees of psychological barriers. The total score of SCL-90 and the factor scores, such as somatization, interpersonal sensitivity, depression, anxiety and hostility in patients were higher than those in control group. 4. Various factors, including the onset age, course of disease, seizure types, frequency, duration, number of drug, total score and the factor scores of SCL-90 were correlated to the cognitive functions of epileptic patients, except sex(P<0.05).Conclusion: 1.The cognitive functions of many patients with intractable epilepsy were damaged, and the proportion of patients with mental deficiency was higher significantly than that of healthy people. 2. Many patients with intractable epilepsy had various degrees of psychological barriers, and which had much important effects on the cognitive function. 3. The onset age, course of disease, seizure types, frequency, duration and number of drug were important risky factors of cognitive disorders of epileptic patients.Part 2 Effects of surgery on neuropsychology and cognitive functions of patients with intractable epilepsyObjective: To investigate the effects of surgery on neuropsychology and cognitive functions of patients with intractable epilepsy. Methods: 30 patients with intractable epilepsy were chosen in the study. The methods of SPM, EPQ and SCL-90 were used respectively to evaluate their intelligence, personality and mentality of pre-operation and post-operation, and the comprehensive efficacy of surgery was assessed.Results: 30 patients were followed up from 10 to 24 months. 21 patients had no seizure after the operation, and 5 patients had only a few seizures. The total effective rate of surgery was 86.67%. There were no significant differences on scores of EPQ between pre-operation and post-operation (P>0.05), while the total score of SCL-90 and the factor scores of somatization, interpersonal sensitivity, depression and anxiety decreased obviously after operation. The total score and IQ in SPM of 10 cases (33.33%) improved significantly after operation, 13 cases (43.33%) declined, and 7 cases (23.33%) remained unchanged. Further analysis showed that, the decline rate of intelligence of patients with preoperative IQ≥70 (60%) was significantly higher than that with IQ<70 (26.67%) after operation, and left side operation (50%) was higher than right side operation (35.71%) (P<0.05). Of 26 patients with EngleⅠ-Ⅱ, 10 (38.46%) improved in the postoperative intelligence, and 11 (42.31%) suffered from a decrease . Of 4 patients with EngleⅢ-Ⅳ, 0 improved in the postoperative intelligence, and 2 (50%) decreased. Temporal lobe and non-temporal lobe operation could both lead to cognitive disorders, and there were no significant differences in two groups (P>0.05).Conclusion: 1. At present, surgery is still an effective means to treat intractable epilepsy, which could satisfactorily control epileptic seizure. 2. The mental status of epileptic patients could get improved in some degree after operation, and the cognitive function of some patients could become better, but still many patients suffered damage. 4. The patients who were performed left side operation, higher preoperative IQ and not well controlled, could occur easily cognitive disorders after operation. Temporal lobe and non-temporal lobe operation could both lead to cognitive disorders.Part 3 Effects of low dose of gamma knife irradiation on seizure and cognitive function of epileptic ratsObjective: To investigate the effects of low dose of gamma knife irradiation on seizure and cognitive function of epileptic rats.Methods: Primarily 60 rats were randomly divided into 4 groups:①control group;②control + GS group;③PTZ group;④PTZ+ GS group. The rats were injected intraperitoneally with PTZ (35mg/kg) to establish the epileptic models. Gamma knife irradiation was performed on bilateral frontal cortex of rats with a maximum dose of 30Gy. After irradiation, the changes of the seizure, behavior and EEG were observed and recorded. On the 12th week after irradiation, the step-down test and Morris water maze were used to evaluate the ability of learning and memory of four groups.Results: The rats could be kindled completely after 12~16 injections with PTZ, and the successful kindling rate was 80%. After low dose of gamma knife irradiation, the degree of seizures gradually lightened in the rats of PTZ+GS group with time passing by. Compared with PTZ group, on the 8th~12th week, the degree of seizures significantly reduced, the latency of seizure waves prolonged, and the frequency of seizure waves decreased in rats of PTZ+GS group (P<0.05). The step-down test showed that the response time and the number of errors increased, and the latent time decreased significantly in rats of PTZ group (P<0.05). Compared with control group, the Morris water maze test showed that the escape latency and the swimming distance increased significantly (P<0.05), and the number of platform crossings and the percentage of swimming time in platform quadrant decreased obviously in rats of PTZ group (P<0.05). It showed that the abilities of learning and memory of epilepsy rats were obviously impaired. While compared with PTZ group, the results of step-down test and Morris water maze test improved significantly in rats of PTZ+GS group. There were no significant differences in step-down test and Morris water maze test between control group and control + GS group (P>0.05).Conclusion: 1. PTZ-kindled epileptic model could be easily established, which might lead to damage to the ability of learning and memory of rats, and was an ideal model for investigate cognitive disorders of epilepsy. 2. Low dose of gamma knife irradiation could reduce seizure and improve cognitive function of epileptic rats.Part 4 Effects of low dose of gamma knife irradiation on the expression of NMDA receptor subunits in cortex and hippocampus of epileptic rats Objective: To examine the effects of low dose of gamma knife irradiation on the expression of NMDA receptor subunits in cortex and hippocampus of epileptic rats.Methods: Primarily 60 rats were randomly divided into 4 groups:①control group;②control + GS group;③PTZ group;④PTZ+ GS group. The rats were injected intraperitoneally with PTZ (35mg/kg) to establish the epileptic models. Gamma knife irradiation was performed on bilateral frontal cortex of rats with a maximum dose of 30Gy. On the 12th week after irradiation, the step-down test and Morris water maze were used to evaluate the ability of learning and memory of four groups. And then the rats were killed and immunohistochemstry and western blotting were used to detect the relative levels of NMDAR subunits (NR1, NR2A, and NR2B) in cortex and hippocampus.Results: Compared with control group, the protein expression of NR1, NR2A and NR2B of the PTZ group increased significantly in the cortex and hippocampus, and the positive neurons and their average absorbance value also increased significantly (P<0.05). Compared with PTZ group, the protein expression of NR1, NR2A and NR2B of the PTZ+GS group decreased significantly in the cortex and hippocampus (P<0.05). There were no significant differences between control group and control+GS group (P>0.05).Conclusion: 1. The mechanism of PTZ induced epilepsy might be associated with increased expression of NR1, NR2A and NR2B in the cortex and hippocampus, which also might be one of reasons to cause cognitive disorders after epileptic seizures. 2. Low dose of gamma knife irradiation could decrease expression of NR1, NR2A and NR2B in the cortex and hippocampus of epileptic rats, which might represent a possible mechanism underlying the therapeutic effects of Gamma Knife irradiation. Part 5 Effects of low dose of gamma knife irradiation on cytoplasmic calcium level of neurons in cortex and hippocampus of epileptic ratsObjective: To investigate the effects of low dose of gamma knife irradiation on cytoplasmic calcium level of neurons in cortex and hippocampus of epileptic rats.Methods: Primarily 60 rats were randomly divided into 4 groups:①control group;②control + GS group;③PTZ group;④PTZ+ GS group. The rats were injected intraperitoneally with PTZ (35mg/kg) to establish the epileptic models. Gamma knife irradiation was performed on bilateral frontal cortex of rats with a maximum dose of 30Gy. On the 12th week after irradiation, the step-down test and Morris water maze were used to evaluate the ability of learning and memory of four groups. After that, the rats were killed and neurons of cortex and hippocampus were acutely isolated and loaded with fluorescent dye fluo-3. And then the fluorescence intensity of cytoplasmic calcium was measured with laser scanning confocal microscopy (LSCM).Results: The cytoplasmic calcium fluorescence intensity of neurons in cortex and hippocampus in control group was in the basic level, and there was no significant difference between the control group and gamma knife control group (P>0.05). The cytoplasmic calcium fluorescence intensity of PTZ group increased significantly compared with control group (P<0.05), and that of PTZ+GS group were significantly lower than PTZ group (P<0.05). There was no significant difference between control+GS group and control group (P>0.05).Conclusion: The increased cytoplasmic calcium fluorescence intensity of neurons in cortex and hippocampus might be associated with the mechanism of PTZ induced epilepsy and cognitive disorders after seizures. Low dose of gamma knife irradiation might control seizure and improve cognitive function through regulating calcium level of neurons.Part 6 Effects of low dose of gamma knife irradiation on CREB signal pathways in cortex and hippocampus of epileptic ratsObjective: To observe the effects of low dose of gamma knife irradiation on CREB signal pathways in cortex and hippocampus of epileptic ratsMethods: Primarily 60 rats were randomly divided into 4 groups:①control group;②control+GS group;③PTZ group;④PTZ+GS group. The rats were injected intraperitoneally with PTZ (35mg/kg) to establish the epileptic models. Gamma knife irradiation was performed on bilateral frontal cortex of rats with a maximum dose of 30Gy. On the 12th week after irradiation, the step-down test and Morris water maze were used to evaluate the ability of learning and memory of four groups. And then the rats were killed and immunohistochemstry and western blotting were used to detect the expression of CREB,α-CaMKⅡand ERK1/2 in cortex and hippocampus.Results: Compared with control group, the protein expression of CREB andα-CaMKⅡof the PTZ group decreased, and ERK1/2 increased significantly in the cortex and hippocampus(P<0.05). Compared with PTZ group, the protein expression of CREB andα-CaMKⅡof the PTZ+GS group increased, and ERK1/2 declined significantly in the cortex and hippocampus (P<0.05). There were no significant differences of expression of CREB,α-CaMKⅡand ERK1/2 between control group and control+GS group (P>0.05).Conclusion: 1. The mechanism of PTZ induced epilepsy might be associated with increased expression of CREB andα-CaMKⅡ, and decreased expression of ERK1/2 in the cortex and hippocampus, which also might be the important cause of cognitive disorders after epileptic seizures. 2. Low dose of gamma knife irradiation could regulate the expression of CREB,α-CaMKⅡand ERK1/2 in the cortex and hippocampus of epileptic rats, which might represent a possible mechanism underlying the therapeutic effects of Gamma Knife irradiation.