| Epilepsy, with the morbidity only second to stroke in neurologic diseases, is a common chronic disase. There are nearly 50 million people sufferring from epilepsy. EP, caused by repeated highly synchronized dischage of neurons, is an abnormal brain disfunction syndrome affecting cognition, sensation, and motor function. Among the EP patients, 30% patients have various degress of cognitive impairment with decline in learning and memory function, abnormal emotion and behavior, and limited social adaption ability. It had been recognized that deterioration in the cognition and mental state would definitly occur in epilepsy as early as in the 19 th century. In the recent years, how seizures affect cognition becomes the focus in epileptic studies. It has been widely accetped that, epilpsy is more than seizures, but is a syndrom affectting many brain functions. Seizure is transient, however, cognitive impairment caused by seizures is permanently present in interictal period. Further more, cognition impairment caused by epilepsy burdens patients and their families more than epilepsy per se.However, our knowledge about the mechanisms related to cognitive impairment in epilepsy is limited. Potential etiology of seizure, brain damage resulted by repeated seizures, and sideeffects of antiepilepsy drugs are all considered related to cognitive impairment in epilepsy. Cognitive impairment persists and develops along with repeated seizures. Besides that, some of the antiepileptic drugs have untoward sideeffects. In light of the above, new compounds which posses both antiepilepsy function and protective effects on cognition are urgently needed.Oxidative stress is an imbalance between oxidative burden and antioxidative system. Oxidative burden, for example: excessive generation of reactive oxygen species, exceeds endogenous antioxidative system and repair capacity, which is named oxidative stress. There is increasing evidence supporting the important role of oxidative stress in epileptogenesis and in the development of epilepsy. Oxidative stress damages celluar protein, membrane lipid and nucleus DNA, leads to structural and functional changes in mitochondria, destroies mitochondrial oxidative phosphorylation, and results in further increased generation of ROS, which forms an vicious circle between generation of ROS and epileptogenesis. Studies in human and animal both demonstrate that antioxidants can prevent progression of epilepsy. Oxidative stress may be a candidate target for managing epilepsy.Metformin(MET), discovered from Gallega officinalis in Paris, has been used as the most prescribed antidiabetic oral agent since 1950. Recently, metformin stands out not only for its antihyperglycemic properties but also for its effects beyond glycemic control such as improvements in oxidative stress, inflammation, aging, and cancer prevention. Previous stuies demonstrated that metformin can rapidly cross the blood brain barrier and had neuroprotective effects. MET can reduce generation of ROS by suppressing mitochondrial respiration, and has protecitve effects on antioxidative system. MET can activate Nuclear factor erythroid 2-related factor(Nrf2) pathways, upregulate the expression of glutathione and uncoupled proteins 2(UCP2). Besides that, MET can enhance spatial learning in normal C57BL/6 mice and in high-fat diet rats. MET shows neuroprotective effects in various animal models of neurological diseases, including multiple sclerosis, stroke and Alzheimer’s disease. Despite its potential benefits, the effect of metformin on epilepsy has not been investigated.The present study established an animal epilepsy model by pentylenetetrazole-induced kindling in mice and was aimed to evaluate if metformin can suppress the progression of pentylenetetrazole-induced kindling, ameliorate the cognitive deficits and oxidative stress, and protect neurons in the hippocampus from damage. PartⅠ Effects of metformin on seizure and cognition in pentylenetetrazole-induced kindling in miceObjective: To establish a chronic epileptic mice model by repeated pentylenetetrazole injections with subconvulsive dose and observe the effects of metformin on seizures and the performance in the Morris water maze.Methods: Adult male C57BL/6 mice 4-6 weeks old were obtained from Hebei Medical University. Animals were randomly divided into four groups, including the CON group, the PTZ group, the PTZ+MET group and the MET group. The CON group received 0.9% Nacl(10ml/kg) every other day for a total of 14 injections. The PTZ group received saline pretreatment once daily along with PTZ(37mg/kg) every other day. The PTZ+MET group received MET pretreatment in dose of 200mg/kg once daily and alternate day treatment of PTZ for 14 injections. Metformin was given 30 min before PTZ. The MET group received 200mg/kg of metformin once daily to study any effect of metformin on the cognitive functions and seizures. Metformin and PTZ were dissolved in physiological saline freshly prior to the injections. A subconvulsive dose of PTZ(37mg/kg, i.p.) was injected on alternating days for a total of 14 injections. The animals were observed for 30 min after each PTZ administration. Seizure stage was evaluated using the following scale: stage 0, no response; stage 1, mouth and facial jerks; stage 2, convulsive waves axially through the body; stage 3, myoclonic jerks and rearing; stage4, clonic convulsions with the animal falling on its side; and stage 5, generalized tonic clonic seizures(GTCS) or lethal convulsions. The severity of seizures were recorded. Twenty four hours after the last PTZ injection, Morris water maze tests were performed. Learning and memory behavior evaluations were performed in a 120-cm diameter water pool and virtually divided into four quadrants. Each test consisted three parts, learning trials(existed platform), probe trials(non-existed platform), and visible platform trials. The acquisition test was performed for five consecutive days of training with four trials per day. Animals were given 60 s to locate the hidden platform, and any animals that did not find the platform within 60-second period were placed on the platform for 15 s. The acquisition time was recorded at the time the animal got into the water and ending at the time the animal reached the submerged platform. On the sixth day, probe trials without platform were assessed with only one starting point, and the time spent in the target quadrant which the platform had been located was recorded. In order to exclude sensorimotor or motivational factors affecting performance in the Morris water maze, visible trials was conducted on the last day. The platform was located 1.5cm above the water surface. Mice were given four trials per day similarly to the course described above for the hidden platform trial, and the escape latency and swimming speed were recorded.Results:1 Repeated injections of subconvulsive-dose PTZ resulted in increasing convulsive activities leading to generalized clonic-tonic seizure. Administration of metformin effectively delayed the development of PTZinduced kindling in mice, and decreased the mean seizure stage as compared with the PTZ group(P<0.01).2 Morris water maze results showed that, all animals showed a progressive decline in the escape latency with training. Mice in the PTZ group exhibited significantly prolonged escape latency as compared to the control group(P<0.01). However, the poor performance was mitigated by treatment with MET(P<0.01). In the probe trial without the platform, mice in the PTZ group spent significantly less time in the target quadrant in the probe trial than the control group(P<0.01), while treatment with MET significantly improved the performance(P<0.01). The number of crossing the platform in the PTZ group obviously decreased as compared to the control group(P<0.01), while treatment with metformin significantly increased the number of crossing(P<0.01). In addition, metformin per se had no significant effect on the preformance in the Morris water maze.Conclusion: In the PTZ-induced kindling mice, metformin effectively decreased the mean seizure stage, delayed the kindling process, which demonstrated that metformin possesses anticonvulsive property. Additionaly, metformin markedly improved the performance in the Morris water maze.Part Ⅱ Effects of metformin on oxdative stress in the brain in pentyleneterazole-induced kinding in miceObjective: To observe whether metformin has protective effects against oxidative stress and mitochondrial injury in the epileptic mice brain.Methods: Adult male C57BL/6 mice 4-6 weeks old were obtained from Hebei Medical University. Animals were randomly divided into four groups, including the CON group, the PTZ group, the PTZ+MET group and the MET group. The CON group received 0.9% Nacl(10ml/kg) every other day for a total of 14 injections. The PTZ group received saline pretreatment along with PTZ(37mg/kg) every other day. The PTZ+MET group received MET pretreatment in dose of 200mg/kg once daily in addition to alternate day treatment of PTZ for a total of 14 injections. Metformin was given 30 min before PTZ. The MET group received 200mg/kg of metformin once daily to study any effect of metformin on the oxidative stress and mitochondrial. Metformin and PTZ were dissolved in physiological saline freshly prior to the injections. A subconvulsive dose of PTZ(37mg/kg, i.p.) was injected on alternating days for a total of 14 injections. Twenty-four hours after the last injection of PTZ, mice were executed by cervical dislocation, and the brain was harvested. The contonts of malonidialdehyde(MDA) and glutathion(GSH) were detected using commercial assay kits. The levels of ROS and the mitochondrial transmembran potential(△Ψm) in the hippocampus were determinated by flow cytometry. The ultrastructure of mitochondia in hippocampal CA1 subfield was examined by transmission electron microscopy.Results:1 Compared with the control group, the cerebral content of GSH in the PTZ group was significantly lower, however, treatment with MET significantly increased the content of GSH in the epileptic mice brain(P<0.01). The level of cerebral MDA in the PTZ group markedly increased as compared to the CON group(P<0.01), however, treatment with MET significantly decreased the level of MDA in the epileptic mice(P<0.01). There were no significant differences in the levels of cerebral GSH and MDA between the CON group and MET group(P>0.05). PartⅡ Effects of metformin on oxdative stress in the brain in pentylenetetrazole-induced kindling in mice2 Compared with the CON group, in the PTZ group, the level of hippocampal ROS significantly increased(P<0.01), and the level of hippocampal △Ψm significantly decreased(P<0.01). However, treatment with MET markedly decreased the level of hippocampal ROS(P<0.01), and increased the level of hippocampal △Ψm in the epileptic mice(P<0.01). There were no significant differences in the levels of hippocampal ROS and △Ψm between the CON group and MET group(P>0.05).3 In the hippocampal CA1 subfield of the PTZ group,there were obvious morphological abnormalities, including vacuolization and ridge fracture. However, treatment with MET effectivly revesed the changes. In addition, ultrastructure of mitochondrion in the normal control gruop mice and in the mice treated only with metformin were normalConclusions:Oxidative stress was induced in the brain by repeated injections of PTZ, and mitochondrion was the target of free radicals and oxidative damage. Metformin had neuroprotective effetcs by regulating the oxidative state in the mice brain, and improving the structure and functions of mitochondrion. Part Ⅲ The protective effects of metformin on hippocampal neurons in pentylenetetrazole-induced kindling in miceObjective: To evaluate the effects of metformin on hippocampal Nissl staining and, apoptosis rate and expressions of apoptotic related proteins.Methods: Adult male C57BL/6 mice 4-6 weeks old were obtained from Hebei Medical University. Animals were randomly divided into four groups, including the CON group, the PTZ group, the PTZ+MET group and the MET group. The CON group received 0.9% Nacl(10ml/kg) every other day for a total of 14 injections. The PTZ group received saline pretreatment along with PTZ(37mg/kg) every other day. The PTZ+MET group received MET pretreatment in dose of 200mg/kg once daily and alternate day treatment of PTZ for a total of 14 injections. Metformin was given 30 min before PTZ. The MET group received 200mg/kg of metformin once daily to study any effect of metformin on the oxidative stress and mitochondrial. Metformin and PTZ were dissolved in physiological saline freshly prior to the injections.A subconvulsive dose of PTZ(37mg/kg,i.p.)was injected on alternating days for a total of 14 injections.Twenty-four hours after the last injection of PTZ,mice were executed by cervical dislocation,and the brain was harvested.Nissl staining and flow cytometry were respectively used to detect the sturcture and apoptosis of neurons in the hippocampus.The expressions of Bcl-2 and Bax in the hippocampus were detected by western-blot.Results:1 Nissl staining showed that: In the CON group and MET group, the morphology of neurons were normal, with rich nissl bodies in the cytoplasm, and there was no siginificant neuronal loss. While, in the PTZ group, neuronal loss was apparent in the CA1 and CA3 subfields of the hippocampus, accompanying with pyknotic nuclei and decreased nissl bodies. However, in the PTZ+MET group, morphology of the neurons was improved. Cellular counting showed that, the numbers of suviving neurons in the PTZ+MET group and in the MET group were not significatnly different from the CON group(P>0.05), while significant decrease was observed in the PTZ group as compared with the CON group(P<0.05).2 Neuronal apoptotic rate: Compared to the CON group, the apoptotic rate of the hippocampal tissue in the PTZ group was significantly increased(P<0.05). However, treatement with MET significantly decreased neuronal apoptotic rate in the epileptic mice(P<0.05). There was no significant difference between the MET group and the CON group(P>0.05).3 Bcl-2 and Bax expressions: Compared to the CON group, the expression of Bcl-2 in the hippocampal tissue of the PTZ group was significantly decreased(P<0.05), and Bax was significantly increased(P<0.05). However, treatement with MET significantly increased the expression of Bcl-2(P<0.05) and decreased Bax(P<0.05) in the epileptic mice. There was no significant difference between the MET group and the CON group(P>0.05).Conclusions:In the pentylenetetrazole-induced kindling mice model, obvious damage occured in the hippocampal tissue of the epileptic mice. Metformin could effectively protect the morphology of the neurons in the hippocampus, and decreased the nuronal apoptosis. |
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