| Epilepsy is a serious neurological disorder in human being that ischaracterized by neuronal death in brains and spontaneous recurrent seizures,butthe neurochemical basis or mechanism remains obscure.Previous studiesindicated that patients with temporal lobe epilepsy (TLE) were subjected tocerebral cortical neuronal loss,recurrent seizures and cognitive impairments,andmostly insensitive to current antiepileptic drugs or medications.Growingevidence has shown that N-methyl-D-aspartate (NMDA) receptors,whichmediate neuron survival,synaptic plasticity and excitatory transmission in thecentral nervous system (CNS),may be involved in the pathogenesis andprogression of epilepsy,and NMDA receptor antagonists exhibit anti-epilepticbenefit effects.However,the serious side-effect of NMDA receptor antagonistsdue to direct and widespread functional blocking of NMDA receptors has limitedtheir clinical application,it sounds necessary to elucidate NMDA receptor-related neurochemical alternative for the treatment of chronic epilepsy.The D-serine molecule is a D-amino acid that has been identified to play animportant role in the regulation of NMDA signaling in CNS neurons.D-serinecan bind to the glycine site of NMDA receptors and acts as an endogenousco-agonist for the NMDA receptor activation.Moreover,release of endogenousD-serine contributed to neuronal damage in the cerebral cortex and hippocampusby excitotoxicity in the neurological diseases or various insults.The altered brainexpression of serine racemase in patients with schizophrenia also impliedinvolvement of D-serine signaling in the disease progression.We hypothesize thatD-serine signaling may also be involved in the pathogenesis and recurrent seizuredevelopment of chronic epilepsy possibly by inducing NMDA receptorover-activation,excitotoxicity and neuronal damage.In our previous study,time-course of massive neuronal death in brains was successfully demonstrated inthe mouse pilocarpine model.It is also well known that systemic injection ofcholinergic agent pilocarpine can induce seizure attack,neuronal loss andspontaneous recurrent seizures that resemble human TLE.Therefore,the mousepilocarpine model was prepared in present study,cellular localization andtime-course of D-serine production were examined in brains byimmunohistochemistry for D-serine,Fluoro-Jade C staining that can specificallyvisualize degenerating neurons,double labeling for NeuN (neuronal marker),glutamic acid decarboxylase-67 (GAD-67,GABAergic neuronal marker) or glialfibrillary acid protein (GFAP,astrocyte marker),in order to elucidate theneurochemical evidence that D-serine signaling might be involved in neuronaldeath and early pathogenesis in the chronic epilepsy.Main results are as follow:1.D-serine-immunoreactivity was identified in neuronal cells of brains in themouse pilocarpine model,and densely observed in the cerebral cortex, hippocampus,amygdala and thalamus at 12 or 24h after moderate seizures.TheD-serine-immunoreactivity was predominately located on cell membranes or incytoplasm of neurons,and D-serine-positive neurons mostly had oval-shapedsomata and were medium-size(24±6μm).These D-serine-positive neurons weremainly distributed in the cerebral cortex and hippocampus,and double labelingfor D-serine/NeuN was analyzed on the sections of cerebral cortex andhippocampus.It showed that a population of neurons displayed D-serineimmunoreactivity in brains of pilocarpine-treated mice.Cell count data indicatedthat about 96% of D-serine-positive neurons showed NeuN-immunoreactivity inthe hippocampus at 3days after moderate seizures.2.By using double immunofluorescent staining and confocal microscopy,D-serine-positive neurons were not found in brains of control or normal mice,while they were numerously observed in the cerebral cortex and hippocampus ofthe mouse pilocarpine model.However,D-serine-immunoreactivity was detectedin the astrocytes of both control and pilocarpine model,which was basicallyconsistent with previous reports.The D-serine-immunoreactivity appearedpunctuate in astrocytic somas or processes.These D-serine-positive astrocyteswere widely distributed in the cerebral cortex and hippocampus,and increasedobviously in their numbers or density in the pilocarpine model.3.Distribution of D-serine-positive neurons was mapped in brains of mousepilocarpine model.It revealed that D-serine-positive neurons were observed in thecerebral motor cortex,somatosensory cortex,orbital cortex,piriform cortex,cingulate cortex,insular cortex,entorhinal cortex,CA1,CA2,CA3 and hilusregions of hippocampus.In addition,they were seen in the dorsal thalamus and afew observed in lateral hypothalamus,but hardly detected in the brain stemregions.Furthermore,time-course changes of the numbers of D-serine positive neurons were examined in the above brains regions from lh to 14days aftermoderate seizures.While they were not detected in brains at 1h,theD-serine-positive neurons appeared in a few brain regions at 4h,increased andreached peak levels at 12-24h in most brain regions,and gradually went down at3-14days.Time-course patterns of D-serine-positive neurons were statisticallysummarized in the cerebral cortex and hippocampus of the pilocarpine modelover the period from 4h to 14days.4.Neurons showing both D-serine and GAD-67-immunoreactivites wereobserved in the cerebral cortex and hippocampus.The D-serine/GAD-67double-labeled neurons were predominately distributed in the pyramidal layer ofcerebral cortex and the polymorph layer of hippocampus.A large portion ofD-serine-positive neurons (98%) showed GAD-67 immunoreactivity,andD-serine/GAD-67 double-labeled neurons constituted 34% of totalGAD-67-positive ones,indicating that most D-serine-positive neurons wereGABAergic in brains of the mouse pilocarpine model.5.Overlapping distribution of D-serine-positive neurons and FJC-positivecells was seen in the double-labeled sections of cerebral cortex and hippocampus.The D-serine/FJC double-labeled neurons were numerously distributed in thecortical pyramidal layer and hippocampal polymorph layer.Cell count datashowed that most of D-serine-positive neurons (93%) showed FJC positivestaining,indicating that D-serine-positive neurons mostly underwent degenerationin the mouse pilocarpine model.6.The pNMDAR1 immunohistochemistry was applied in brain sections ofpilocarpine-treated mice and controls to detect phosphorylation of NMDAreceptor,which may reflect activation state of NMDA receptors in neurons.Largenumbers of pNMDAR1-positive neurons were observed in the cerebral cortex and hippocampus in the pilocarpine model.Immunoreactivity was localized oncell membrane and cytoplasm of neurons.The pNMDARl-positive neuronsshowed certain similarities to the D-serine-positive neurons in their distributionand dynamic changes of their numbers.While they were much fewer or rarelydetected in controls,these pNMDAR1-positive neurons appeared rapidly after SE,and increased to peak levels at 12-24h in cerebral cortex and hippocampus.This study has provided new evidence that up-regulation of D-serineproduction might induce GABAergic neuronal degeneration through excitotoxicmechanism in the pilocarpine model and may be involved in early pathogenesisand recurrent seizure of chronic epilepsy.
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