| Background:Nowadays in China, apoplexy has surpassed tumor and cardiovascular disease to become the first cause of death, of which cerebral infarction presents a major part by about 70% to 80%. While due to an fast aging population, the number of cerebral ischemia patients grows larger and larger year by year with a tendency of younger age. It causes a very high rate of lethality and disability, epileptic seizure is one of the common clinical manifestations of cerebral ischemia, which often occurs as an early state or initial symptom of some cerebrovascular diseases such as cerebral vascular sclerosis, moyamoya disease and cerebral arteriovenous malformation owning to cerebrovascular occlusion or intracerebral steal phenomenon. Furthermore, epileptic seizure is apt to result in brain anoxia exacerbating such condition and casting a severe impact on its prognosis. Particularly in newborns, epileptic seizure is the major symptom and clinical manifestation in vascular cerebral infarction, which will protract the course of disease. At a growing rate, ischemic epileptic seizure cast not only severe impact on the living quality and social function of the patients, but also heavy cost of manpower and financial resources to the society and nation. Therefore, it will probably produce a huge social and economical benefit to , study the mechanism of epileptic seizure after cerebral ischemia and seek for news pertinent treatment.Epilepsy is a sudden abnormal discharge of neurons in the brain, resulting in a short of a chronic disease of its dysfunction. Since the beginning of an abnormal discharge and delivery, epileptic seizure of complex and varied clinical manifestations, can be characterized by paroxysmal movement, sensory and autonomic nerve, consciousness and mental disorders[1-4]. According to the pathogenesis, epilepsy can be classified into two categories, that is, essential epilepsy and secondary epilepsy. There is no concrete pathogenesis in essential epilepsy, except for a close connection of genetic facto. While in secondary epilepsy, there are various pathogeneses with acquired characteristics. Ischemic brain injury is one important factor in secondary epilepsy. However, its potential mechanism is not clear, so far, the study has found oxidative stress reaction and inflammatory reaction after cerebral ischemic injury, excitatory neurotransmitter and inhibitory neurotransmitter imbalance and the damage to the function of ion channels play an important regulating role in the occurrence of ischemic epilepsy[5-9]. Gamma-aminobutyric acid (GABA) is a natural amino acid, which is a nonprotein amino acid and an important conveying material of inhibitory nerve in mammalian central nervous system, accounting for about 30-40% of the central nervous synapse [10,11]. play an important roles in the human cerebral cortex, hippocampus, thalamus, basal ganglia and cerebellum, exercising a regulatory role to a variety of functions of the body. In the nervous system, GAT-1 and GAT-3, the two subtypes of GABA transporter are the main factors of regulating extracellular GABA levels[12-14]. Studies have found that GAT-1 and GAT-3 in parietal cortex and hippocampus all have regulating effect on the GABA transshipment, and GABA in the brain’s internal transference is closely related to the occurrence of epilepsy[11-16].The epileptic seizure process often associated with production of oxidatiye stress and proinflammatory cytokines (PICs) rise in cerebrospinal fluid, neurons and neurogliocyte, is consistent with the evident increase of Proinflammatory factor IL-1β, IL-6 and TNF-a in the parietal cortex/hippocampus/amygdala organization in MCAO+NEF, while further studies have also found in these specific areas of the brain, the increase of IL-1β,IL-6, and TNF-a changes GAT-1/GAT-3 expression[17,18].Nefiracetam (NEF), a pyrrolidone derivative, commonly used to improve the cognitive function in patients with ischemic brain injury, is a nootropic medicine and a nerve exciter to enhance the development and repair of nerve cells in the hippocampal area. Nefiracetam promote the recovery of the nervous system mainly through the following mechanism:(1) By activating the signal channel to increase memory, N-Methyl-D-aspartic Acid receptor can improve the cognitive function[20]; (2) Moriguchi and some other scientists have discovered in the hippocampal area of rats’study that nefiracetam can activate the signal channel of metabotropic glutamate receptor 5 so as to enhance long term memory, thus improving the learning ability[21]. (3) Han and some other scientists have discovered that nefiracetam can help the phosphorylation of cAMP response element-binding protein, to decrease rats’ depressive symptom and improve cognitive competence[22]. Many studies have found nefiracetam can reduce epileptic symptom induced by the amygdala excitement or chemical drugs (e.g.veratridine, glutaminate)[9, 23-25].Some experts have also found that a small dose of nefiracetam is enough to restrain the epileptic seizures of electric shock in rats, but not good enough to the therapy of the epileptic seizures of chemical induction, probably due to the different starting courses of epileptic seizures.So nefiracetam could be an important medicine used to prevent seizures or stroke and improve cognitive function. However, there is few study reports on therapeutic effect in the treatment of epileptic seizures caused by mechanical interdiction of blood vessels. Therefore, this research based on rats models with postischemic seizures, proposes a discussion about the protective effect and its pertinent mechanism of nefiracetam in treating brain tissue injury and concurrent epilepsy.Objective:Through the establishment of a rat model of post-ischemic nonconvulsive seizure, this paper analyses the organic protection of nefiracetam on the parietal cortex/hippocampus/amygdala, as well as the mechanism of GAB A in the treatment of nefiracetam to brain tissue damage, so as to clarify the^theoretical support and its clinical application of nefiracetam in improving concurrent epilepsy mechanism.Methods:Part I The Protective Mechanism of Nefiracetam on Injured Brain of Rats with Postischemic Seizures.1. Establishment of rat models with postischemic seizures:55 male Sprague-Dawley rats (250-300g) were randomly divided into three groups, respectively the sham control group (n=15) and the MCAO group (n=20) with saline injection and the MCAO group with NEF injection (n=20). NEF (Sigma Co.) was dissolved in a saline solution at a final concentration of 15 mg/ml and given by intraperitoneal injection (i.p.,30mg/kg, twice). In experiments, suture-occluded method is adopted to producing rat models with postischemic seizures. After anesthesia by intraperitoneal injection of pentobarbital (dose of 45mg/kg), four electrodes were implanted on the rat skull symmetrically through burr holes over bilateral frontal and parietal regions of the cortex (1 mm anterior and 4 mm posterior to bregma,3.5mm lateral to midline. A reference electrode was placed posterior to lambda over the transverse sinus. The electrodes were soldered to a multi-pin connector. A computerized EEG recording was obtained via a Grass polygraph amplifier and digitizing system. The NCSs were detected via stainless steel electrodes implanted on the rat skull. The off-line EEG traces were analyzed using the NCS criteria defined previously1261. The frequency of NCS was considered as the number of NCS events detected in each animal.After 3 days of recovery from the above-mentioned procedures, each rat was fixed in supine position, incised from the middle of neck to separate the left common carotid artery (CCA), external carotid artery (ECA), its branches and internal carotid crotch. Temporary arteriole clip was fixed to interdict ICA, its affiliate lingual artery and maxillary artery. Then both CCA and ECA were ligated at near the end of heart. An incision was made at the crotch of CCA beside the heart to insert one 3-0 single nylon suture with paraffin at the leading end temporary clip over the pterygopalatine artery,PPA,to avoid mistaken plug, through the ICA into skull at a depth of 20mm to the near end of arteria cerebri anterior, so as to completely interdict blood supply at the original part of middle cerebral artery.During the surgery,laser-doppler flowmeter was used to monitor cerebral blood flow in cortex, as a flow reduction over 70% and NCS found inlhour was defined as molding success. As to the sham control group, no blood vessel occlusion was needed as the incision was sutured after exposure. Note that the first dose was injected as the first NCS occurrence (15mg/ml as per 30mg/kg, intraperitoneal injection) and the second one was injected 12 hours after the surgery. The control group rats and the sham control group rats received the same volume of i.p. injection of saline. EEG recordings were performed and observed during 30 minutes before operation and 24 hours after induction of MCAO as described previously. The rats’ brains were removed 24 hours after the end of the MCAO, to obtain the brain tissues of parietal cortex/hippocampus/amygdala and keep them in a -80 ℃ refrigerator. Casualties at operation were replaced if any.2. Record each frequency of seizure in rats according to the EEG3. Injured tissue detection: (1) Observe the general morphous, neuron damage and inflammatory cell infiltration in the parietal cortex/hippocampus/amygdale as the targeting brain tissue after formaldehyde fixation, embedding, paraffin and HE staining.(2) Use ELISA method to measure the content of TNF-a, IL-1β and IL-6 in the parietal cortex brain/hippocampus/amygdala and test the inflammatory degree of it(3) Use colorimetry to detect tissue, the content of MDA, GSH and GSSG to identify the degree of oxidation reaction.(4) Use TUNEL staining method to detect necrotic and apoptosis cell in the parietal cortex/hippocampus/amygdala.Part II Mechanism of GABA in Nefiracetam Treatmen against Brain Tissue Damage1.Extract protein by grinding the parietal cortex/hippocampus/amygdala tissue. Use Western Blot (Western Blot) to detect GAT-1/GAT-3 proteins in the parietal cortex/hippocampus/amygdala.2. Use ELISA method to determine GABA levels in the parietal cortex/ hippocampus/amygdala of brain.Results:1. Protective Effect of nefiracetam on Brain of Rats with Postischemic Seizures.(1) After ischemia handling the control group of rats suffered a significantly higher frequency of epilepsy than that of the sham control group, which shows that the ischemia handling successfully induce seizure in rats indicating its pathogenesis in epileptic seizure; while after nefiracetam treatment, the cerebral ischemic rats’ epilepsy seizure frequency decreased to an obviously lower level than that of the control group, statistically indicating that nefiracetam can obviously reduce the frequency of NCS in rats.(2) The tissue morphology results show that the sham control group maintained a normal tissue state in parietal cortex/hippocampus/amygdale. however, the ischemia rats in the control group after processing reflected a obvious tissue damage to the parietal cortex/hippocampus/amygdala of brain, with obvious phenomena of inflammatory cell infiltration, dissolution damage and necrosis/apoptosis to the histocyte, and incomplete tissue structure. However, after nefiracetam treatment, significant decreases were observed intissue injury of cerebral ischemia in parietal cortex/hippocampus/amygdale of brain,inflammatory cell infiltration, dissolution damage and necrosis/apoptosis to the histocyte and incomplete tissue structure.(3) In the control group of rats, TNF-a, IL-1β and IL-6 levels in the parietal cortex/hippocampus/amygdala of the brain remained normal; but obtained a significantly higher level repesctively in that of the control group of rats after ischemia process, showing an inflammatory reaction caused by ischemia to the parietal cortex/hippocampus/amygdale of brain. However, after nefiracetam treatment, significantly decrease of TNF-a, IL-1β and IL-6 levels were observed in the parietal cortex/hippocampus/amygdale of cerebral ischemic rats in contrast to the control group, statistically indicating that nefiracetam can cause a significant reduction in the inflammatory reaction of rats’ parietal cortex /hippocampus/amygdale of brain.(4) In the sham control group, the levels of MDA, GSH and GSSG in the parietal cortex/hippocampus/amygdale of brain were normal, but in the ischemia rats both MDA and GSSG rose significantly while GSH dropped evidently in the control group after processing showing a stronger oxidative stress in the parietal cortex/hippocampus/amygdale of brain after ischemia processing and that the nefiracetam treatment can significantly lower MDA and GSSG level, and increase GSH. Therefore, it is likely that nefiracetam can effectively restrain the oxidative stress caused by ischemia to the parietal cortex/hippocampus/amygdale of brain.(5) The TUNEL staining results show less apoptosis in the sham control group, but a remarkable increase of apoptosis after ischemia processing in the sham control group in contrast to the control group in the parietal cortex/hippocampus/significantly amygdale of brain while nefiracetam treatment in the experimental reduced the apoptosis neurons obviously compared with the control group.2. The Function of GABA Mechanism in Nefiracetam Treatment against Brain Tissue Damage to Ischemic Epilepsy Rat.(1) After ischemia processing, expression level of GAT-1 and GAT-3 in the parietal cortex/hippocampus/amygdala of rat brain in the control group increased significantly in contrast to the sham control group, but after nefiracetam treatment in the rat models with postischemic seizures they remained a relatively lower level in contrast to the control group, indicating a significant statistical difference.(2) After ischemia handling, GABA level in the parietal cortex/hippocampus/amygdale of brain in the control group rats descended significantly the in contrast to the sham control group, but evidently elevated after nefiracetam treatment in the rat models with postischemic seizures in contrast to the control group, indicating a significant statistical difference.Conclusion:This research adopts the rat models with postischemic seizures with post-operation nefiracetam treatment to detect and analyze brain tissue damage, expression of inflammatory factors, oxidative stress, the GAT-1/GAT-3/GABA levels in the parietal cortex/hippocampus/amygdale of rats’ brains, and concludes as the following that:1. Nefiracetam can significantly reduce the frequency of epileptic seizure in ischemic rats;2. Nefiracetam can significantly reduce the brain tissue damage in parietal cortex/hippocampus/amygdala to ischemic rats caused by epilepsy.3. Nefiracetam can significantly reduce tissue inflammatory reaction in parietal cortex/hippocampus/amygdala to ischemic rats caused by epilepsy.4. Nefiracetam can significantly reduce oxidative stress in parietal cortex/hippocampus/amygdale to ischemic rats caused by epilepsy.5.Nefiracetam can significantly reduce necrotic and apoptosis cell in parietal cortex/hippocampus/amygdala to ischemic rats caused by epilepsy.6.Nefiracetam can reduce the brain tissue damage in parietal cortex/hippocampus/amygdala to ischemic rats caused by epilepsy through lowering the levels of GAT-1/GAT-3 and elevating the levels of GAB A molecular expression.By creating rat models with postischemic seizures through mechanical interdiction of middle cerebral artery, the research has found that Nefiracetam can reduce the oxidative stress reaction (changing the level of MDA, GSSG and GSH) and inflammatory reaction (increasing the synthesis secretion of TNF-a, IL-10 and IL-6) in brain tissues as well as epileptic seizure frequency in ischemic rats, and furthermore, Nefiracetam can also lower the levels of GAT-1/GAT-3 and increase GABA concentration in the brain tissues in the parietal cortex/hippocampus/amygdale to reduce the frequency of epileptic seizures to rats in MCAO+NEF, which may illustrate a new approach of working mechanism of Nefiracetam in reducing brain tissue damage in parietal cortex/hippocampus/amygdala to ischemic rats caused by epilepsy and providing theoretical support for the application of Nefiracetam to clinical treatment to ischemic seizure. |
PDF Full Text Request