| BackgroundEpilepsy is a condition or a group of conditions involving transient disturbancesin cerebral function caused by abnormal neuronal discharges and characterized byrecurrent seizures. Once the diagnosis is confirmed, long-term treatment withantiepileptic drugs (AEDs) is recommended. Drug therapy has remained the mainapproach so far for treating patients with epilepsy. Approximately 75% of treatedpatients are seizure-free using appropriate AEDs; the remaining 25% of patients,whose paroxysms are not effectively controlled, are said to have drug-resistantepilepsy or intractable epilepsy. Temporal lobe epilepsy (TLE), one of the mostcommon types of intractable epilepsy in adult patients, is characterized with theprogressive development of spontaneous recurrent epileptic seizure originating fromtemporal lobe foci. It is crucial to develop methods for successfully treatingintractable epilepsy. Therefore, exploration of the mechanism leading to intractableepilepsy is imperative.Hippocampal mossy fibers, axons of dentate granule cells, converge in thedentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. From pathological viewpoints, TLE is characterized byseveral histological aberrations in the hippocampus. In the hippocampal tissues fromtemporal lobe epileptic children and experimental animal models, frequently observedis abnormal morphology of the axons of granule cells in the dentate gyrus, i.e.,hippocampal mossy fibers sprouting (MFS).The mechanisms of drug resistance in some patients are far from being clearlyunderstood. Inadequate intraparenchymal drug concentration may be one of apossible mechanism of resistance to AEDs. In the 1970's, the concept of multidrugresistance (MDR) was proposed to describe the phenomenon that once cells becomeresistant to a drug, they often show resistance to a large variety of other drugs as well.A series of studies found that overexpression of the MDR gene contributes to themultidrug resistance phenomenon. The human MDR gene consists of MDR1 andMDR3. Transgenic experiments have shown that MDR1 is most frequentlyimplicated in drug resistance. Some studies showed that combined phenytoin (PHT)and phenobarbital (PB) resistance is correlated with MDR1 gene overexpression inthe cerebral cortex of amygdala-kindled Wistar rats. Therefore, the MDR1 gene maybe responsible for the poor outcome of treatment for intractable epilepsy. However, itremains unknown whether MDR1 gene expression could be found in the brains ofpatients with intractable epilepsy.For a long time, astrocytes have been thought to provide mainly a structural,trophic, and metabolic support to neurons. With the development of researchtechniques, a growing body of evidence has emerged on the existence in the brain of aclose bidirectional communication between neurons and astrocytes. Astrocytes are notinertia cell and can regulated actively neuronal activity. The astrocytes may play akey role in preventing the propagation of an epileptic activty and maintaininghomeostasis in the extracellular microenvironment. Objective1. To observe the mossy fiber sprouting in the resected hippocampal dentategyrus and CA3 area of patients with temporal lobe epilepsy.2. To detect the expression of MDR-1, neuron-specific endase (NSE), and glialfibrillary acidic protein (GFAP) in the temporal lobe and hippocampal dentate gyrus..Methods12 patients with temporal lobe epilepsy intractable epilepsy were selected inFirst clinical Hospital of PLA General Hospital and Neurosurgery Department ofZhujiang Hospital from Apr 2006 to Jun 2006 Patients were included in this study ifthey fulfilled the following: (1) according with clinical diagnostic criteria ofintractable epilepsy. (2) having integrated Preoperative evaluation data. (3) patientand his family member request or agree with operation. (4)Preoperativeinvestigation data of patients were consistent with surgical treatment indication.Operative ways included anterior temporal resection and selectiveamygdalo-hippocampectomy. In control group, the brain tissues were obtained from 4dead patients underwent autopsy. Furthermore, they have been precluded the epilepsy history.By means of routine HE and Nissl's staining, the morphological changes inhippocampus and temporal lobe were detected. Hippocampal mossy fibers sproutingwas observed by Timm's staining under microscopy and electron microscope. Bymeans of anti- multidrug resistance protein 1(MDR1) and GFAP or NSE doubleimmunofluorescence histochemical staining method combining with confocallaser-scanning microscopic technique, the distribution of MDR1 or NSE positive cellsand reactive astrocytes were observed in the temporal lobes and hippocampus.SPSS12.0 statistical package was used for Data evaluation. The measurement data wereindicated by mean±standard differentiation. Compared with control group, mossy fiberssprouting in CA3 area and inner molecular layer of hippocampus or GFAP positive astrocytes in temporal lobe cortex and hippocampal dentate gyrus in experimental group were analyzedby means of independent samples test.Results1. HE and Nissl's staining: No abnormality was shown in hippocampus andtemporal lobe in control group. In experiment group, some pathological changes,neuronal degeneration and reactive proliferation of astrocyte, could be found indentate gyrus, CA3 area of hippocampus and cortex of temporal lobe.2. Timm stain: It was detected that the mossy fibers were stained withbrownish-yellow or brownish-black silver granules under microscopy. In controlgroup, Timm stain positive materials (mossy fibers) mainly distributed in CA3 andhilus of hippocampus dentate gyrus. Most of them were intensive deep color blackparticle. Few of Timm stain particle could be found in inner molecular layer of thedentate gyrus. In experiment group, it was found hippocampal mossy fibers sproutinginto the inner molecular layer of dentate gyrus. The distribution of mossy fibersdisplayed black lamellar Timm particles or successive dense particle zones. Robustsprouting of mossy fibers was also seen in CA3 area of hippocampus. Compared withcontrol group, the scores of CA3 area and inner molecular layer of the dentate gyrusin experiment group were higher according to score standard of mossy fibers. Therewere significant difference in the sprouted mossy fiber scores of CA3 area (t=6.683,P=0.000) and inner molecular layer (t=13.154, P=0.000) of the dentate gyrus betweenexperiment group and control group.Under electronmicroscope, many silver labeled synaptic ends of mossy fiber'swere observed in the hilus of dentate gyrus and few silver labeled ends were observedin the granule cell and inner molecular layer of the dentate gyrus in control group.In epilepsy group, many silver labeled synaptic ends could be found in innermolecular layer of the dentate gyrus and most of them commonly form asymmetric synapse with dendrite.3. Immunohistochemistry stain: In control group, many GFAP positiveastrocytes and neuron-specific endase (NSE) positive neuron could be found in thecortex of temporal lobe and dentate gyrus of hippocampus. No MDR1 positive cellscould be observed in above-mentioned areas. The GFAP positive astrocytes displayedsmall fine reddish cell process and mainly distributed in the subcortex of temporallobe.In experiment group, GFAP positive astrocytes within the cortex of temporallobe and dentate gyrus of hippocampus increased markedly. Most of them were deepred stained. There was a significant difference in number of GFAP positive astrocytesboth the cortex of temporal lobe (t=7.934, P=0.000) and dentate gyrus ofhippocampus(t=7.731, P=0.000) between the experiment group and the control group.The augmented GFAP reactivities took on hypertrophic cell bodies, thicker and longerprocesses.Many MDR1 positive cells could be detected at the cortex of temporal lobe anddentate gyrus of hippocampus. These cells' appearance was quite similar to GFAPpositive astrocytes.Anti-GFAP and -MDR1 double immunofluorescence histochemical stainingshowed that GFAP and MDR1 could co-express in the same astrocyte in the cortex oftemporal lobe and dentate gyrus of hippocampus. Collocalization of MDR1 andGFAP were present at most of reactive astrocytes. The number of GFAP positiveastrocyte was more than MDR1 positive cell in this astrocyte. Anti-MDR1 and -NSEdouble staining showed that there is no MDR1 positive neuron in the cortex oftemporal lobe and dentate gyrus of hippocampus.Conclusions:The present results indicated that: 1. There were common pathological changes including neuronal degenerationand reactive proliferation of astrocyte in the brain tissue of patients with temporallobe epilepsy. It may be resulted from recurrent epileptic seizure.2. Temporal lobe epilepsy could considerably cause mossy fiber sprouting.Mossy fiber sprouting in hippocampus might play an important role in thepathogenesis of intractable epilepsy.3. There was a homeochronous overexpression of MDR-1 and GFAP gene in thereactive astrocytes within brain tissues of patients with intractable epilepsy. MDR1had not been found in NSE postive neuron. It was suggested that multidrug resistancegene of astrocytes might play an important role in the pathogenesis of intractableepilepsy. |
