Effect Of Gamma Knife Radiosurgery On DBP-ERK1/2 Signaling Pathway In Rat With Epilepsy Induced By Pilocarpine

BackgroundThe epilepsy is a common nervous system diseases. It has been reported there are at least 50,000,000 persons involved in epilepsy in the worlds. In china, the occurrence rate of epilepsy was is about 4%o, while 20% of them belongs to intractable epilepsy. This kind of patients need to be treated with neurosurgical treatment. Considering the neurosurgical method, the key point was the accuracy in localizing the epileptogenic foci. With the advances of medical science, MRS, CoEEG, Dee, PET and MEG etc, have been used for accurately localizing epileptogenic foci. In the areas of neurosurgery, stereotactic radiosurgery has been employed for intractable epilepsy due to the charateristics of minimally invasive and accurate lacalizing epileptogenic foci. Gamma Knife radiosurgery has a positioning accuracy, high-energy rays converge on target, the radiation attenuation of the steep gradient of the characteristics of the surrounding tissue injury. Use of different diameter collimator, a number of other central radiation, has become the treatment of brain tumors, vascular malformations and functional diseases such as epilepsy, an important way. People want to find a low-risk, low cost and the obvious effect of the treatment of epilepsy treatment. Gamma Knife in the treatment of temporal lobe epilepsy were some attempts, it has overcome previous trauma craniotomy, bone window limited by the complications of high disadvantage, in the treatment of epilepsy did not show invasive craniotomy effective, The advantages of fewer complications, surgical treatment of epilepsy can also be ineffective as a supplement to treatment, have increasingly been recognized by clinicians. In spite of extensive use of stereotactic radiosurgery for intractable epilepsy, the mechanisms about pathogenisis of epilepsy and radiosurgery have not been clear at present.To date the pathogenesis of human epilepsy research is still mainly rely on animal experiments. Currently there are over one hundred kinds of animal models of epilepsy as much, but in full compliance with human temporal lobe epilepsy model characteristics and pathological changes of less lithium-pilocarpine model of temporal lobe epilepsy is one of them. With in-depth research and test technology, temporal lobe epilepsy in the hippocampus of the pathological changes of physiological processes and pathological aspects, such as molecular mechanism has made considerable progress, but there are still many key issues to be resolved. Hippocampus is the anatomical origin of temporal lobe epilepsy. Hippocampal sclerosis is the most common temporal lobe epilepsy, pathological changes, including nerve cell loss, gliosis, mossy fiber sprouting and synaptic reorganization. Synaptic reorganization of hippocampal neurons and dentate granule cell axons (mossy fiber) sprouting formation of abnormal recurrent neural network is a major part of epilepsy. The formation of temporal lobe epilepsy hippocampal plasticity in neural networks is actually the process of reconstruction. Previous studies have shown that, MFS and synaptic reorganization in the pathogenesis of epilepsy plays an important role.Albumin D-site-binding protein in the pathogenesis of temporal lobe eilepsy have a certain role. Albumin D-site-binding protein is a transcription factor with a zipper to form a dimer with a promoter to promote transcription. Albumin D-site-binding protein to contain basic/leucine zipper (PARb Zip) structure is characterized by a conserved region with PARb Zip transcription factor involved in gene transcription regulation. Overexpression of albumin-D site-binding protein can induce extracellular signal-regulated protein kinase (ERK1/2) activation albumin D-site-binding protein may also be through the extracellular signal-regulated protein kinase pathway is involved in the occurrence of epilepsy. Extracellular signal-regulated protein kinase signaling pathway is mitogen-activated protein kinase family member. Extracellular signal-regulated protein kinase signaling pathway on neural activity is highly sensitive, we can adjust the downstream gene expression and protein synthesis of synaptic remodeling, such as mossy fiber reorganization. Extracellular signal-regulated protein kinase signaling pathway may in refractory epilepsy play a role in the pathogenesis.In this study,lithium-pilocarpine induced epileptic SD rats successfully after the albumin-D site-binding protein as an entry point to the classic means of molecular biological methods to detect low-dose gamma irradiation in rat hippocampus after TLE albumin D-site-binding protein and extracellular signal-regulated protein kinase expression in order to further understand the DBP-ERK1/2 pathway in pilocarpine kindling role of the pathological process,and to explore Gamma knife radiosurgery pilocarpine epileptic rats mechanism for the clinical treatment of epilepsy to find a better role in target.Objective1.Production of SD rats induced by pilocarpine epilepsy model, observation and messenger-related epilepsy indicators,such as pathological changes of the hippocampu to investigate the mechanism of epilepsy. 2.Gamma Knife radiosurgery in the treatment of epilepsy using mouse models to study the mechanism of gamma knife treatment.Method1.SPF level 100 healthy adult SD rats were divided into saline control group (normal saline), non-epileptic Gamma knife radiosurgery group (pilocarpine rat model of epilepsy in rats given low doses of gamma knife is not Radiation), gamma irradiation induced epilepsy model (rat pilocarpine model of epileptic rats treated with low doses of gamma irradiation), each group was killed by the time 3h,7d,15d, 30d,60d, divided into 5 sub-groups, each sub-group of six lines of albumin-D site binding protein, extracellular signal-regulated protein kinase expression detection, and the other non-epileptic Gamma knife radiosurgery for each model group, sub-group 2 for pathological testing.2.Lithium-pilocarpine epilepsy model followed by experimental rats given lithium chloride, atropine sulfate, intraperitoneal injection of pilocarpine induced status epilepticus, continued to 1h after the injection of diazepam to terminate seizures. Day thereafter observed spontaneous seizures. Control group with lithium pilocarpine ntal group the same time point, rats were given normal saline 125mg/kg, injected rats immediately after behavioral change.3.Positioning the rat gamma knife head design aircraft. Animal holder used high-strength non-magnetic plexiglass plate, cloth sheet, hard Xiangjiao Bang, etc., as material, the main piece of plexiglass for the fixed plate, the plate above the head-end devices with fixed animal head, a 3 point positioning fixed From both sides of the ear and the front of the unit with the fixed teeth, can be adjustable. Fixed plate cleverly designed the following four pillars can be firmly fixed in the Leksell frame behind the two vertical columns.4.Gamma Knife treatment. Gamma irradiation induced epilepsy model group at 10% chloral hydrate anesthesia, rats were fixed on the knife holder on the dedicated, magnetic resonance imaging (MRI) of brain coronal scan continuously calculate and determine the target irradiation for the left hippocampus of the XYZ coordinates. The rats were then connected to the bracket 4mm collimator, the adjusted coordinates the implementation of irradiation with gamma knife, peripheral dose of 12Gy,50% isodose curve, the central dose of 24Gy, irradiation time 6min.5.Gamma irradiation of non-epileptic hippocampus of model group, pathological examination. The five were sacrificed at different time points taken hippocampus tissue in rats, coronal frozen sections, cut 8um 25um thick slices and directly attached to polylysine coated glass slides, line Nissl staining. Hippocampal region were observed pathological changes.6.Western blotting assay DBP, p-ERK1/2 protein expression. Each group in the five rats sacrificed at different time points taken hippocampal tissue samples by Pik-day conventional extraction kit to extract total protein, electrophoresis, membrane transfer, hybridization and immunological color. ECL chemiluminescence showed specific bands up to the film cassette exposure, developing, fixing, will be collected with the use of computer imaging system, using Image Tool 5.0 Determination of band gray value, gray value compared with the reference materials, are relatively ratio of protein expression as a target, take the results of repeated experiments were statistically analyzed the mean.7.Experimental data analysis using statistical software SPSS13.0. Measurement information and data to show. Completely random design on the measurement data analysis using One-way AVON A, when homogeneity of variance between groups using LSD multiple comparison method, Test level of significanceα= 0.05, P<0.05 indicated a statistically significant difference.Results 1.Behavioral observation. Observed behavior can be seen rats had gradeⅠ-Ⅳrecurrent spontaneous seizures, and sometimes even see theⅤ-level SRS. Usually lasts about 30s, can be self-terminated. The latter part of this study, the treatment group compared with non-treated group reduced the number of spontaneous seizures.2.Nissl staining. Gamma irradiation of non-epileptic dentate gyrus of rats in model group left the door area, CA1 area, CA3 area shows reduced cytoplasmic Nissl bodies, cell outline vague, disorganized, reduction in the number of neurons, dentate gyrus granule cell layer border irregular, widened, arranged loosely, disorder, the above change is more obvious with time.3.Pilocarpine rat hippocampus after kindling model albumin D-site-binding protein is significantly activated and regularly over time changes in the model group at different time points in hippocampus of rats with albumin D-site-binding protein expression compared with control group with statistical difference (P<0.05), and gamma knife radiation therapy can significantly inhibit this activation (P<0.05). Hippocampus p-ERK1/2 showed the change albumin D-site-binding protein the same rule.Conclusion1. Pilocarpine hippocampal kindling model of DBP-ERK1/2 signaling pathway is activated.2.The gamma knife radiation therapy can inhibit DBP-ERK1/2 activation, gamma knife on the DBP-ERK1/2 signaling pathway may be the treatment of temporal lobe epilepsy one of the mechanisms.