D-serine在匹罗卡品致癎小鼠脑GABA能神经元变性中的作用研究
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摘要
癫痫是一种严重的神经系统疾病，其特点是自发的反复发作以及脑内神经元丢失。但其发病的神经化学基础或机制仍不清楚。以往的研究提示，颞叶癫痫的特点是病人脑内神经元丢失，反复发作，常常有认知功能障碍以及大多数颞叶癫痫病人对现有的抗癫痫药物或其他抗癫痫治疗方法不敏感。越来越多的研究表明，NMDA受体在中枢神经系统中对于神经元的存活、突触的可塑性以及兴奋性突触传递中具有重要作用，同时NMDA受体可能参与了癫痫的发生和发展，一些NMDA受体拮抗剂也显示具有抗癫痫效应。然而，由于NMDA受体拮抗剂直接的和广泛的功能性阻断NMDA受体所导致的严重的不良反应限制了NMDA受体拮抗剂在临床的应用。因此阐明NMDA受体相关的神经化学递质的机制对于临床慢性癫痫的药物治疗方案的选择非常必要。
D-serine分子是一种D-氨基酸即右旋-丝氨酸，在中枢神经系统神经元NMDA信号传递的调节中具有重要作用。D-serine与NMDA受体的甘氨酸位点相结合，在NMDA受体激活中充当NMDA受体的内源性配体的角色。而且内源性D-serine的释放在兴奋性毒性导致的大脑皮质和海马的神经元损伤中具有重要作用，因此D-serine参与了一些神经系统疾病的发生。精神分裂症的病人大脑中丝氨酸消旋酶表达的变化也表明D-serine信号与其疾病的发展有关。我们猜想D-serine信号途径可能通过NMDA受体的过度活化、兴奋性毒性和神经元损伤参与慢性癫痫的发病和慢性癫痫的反复癫痫发作。在我们的前期研究中，已经明确了小鼠匹罗卡品模型中大量的神经元死亡的时程变化特点；而且已证实连续腹腔注射拟胆碱剂匹罗卡品可以诱导癫痫发作、神经元丢失和自发的反复癫痫发作，上述特点与人类的颞叶癫痫十分相似。因此，本课题采用小鼠匹罗卡品模型，免疫组织化学染色检测脑内D-serine的细胞内定位和D-serine阳性细胞的时空变化特点，D-serine与特异性标记变性神经元的Fluoro-Jade C染色、神经元的标记物NeuN、GABA能神经元的标记物GAD-67和星形胶质细胞的标记物GFAP进行双标记等方法，以阐明D-serine信号可能参与慢性癫痫的神经元死亡和慢性癫痫发病。主要结果如下：
1．癫痫发作12-24h后在大脑皮质、海马、杏仁核和丘脑观察到大量D-serine阳性细胞。它们分布于细胞的胞浆、胞膜上，阳性细胞呈椭圆形，平均直径为24±6μm。癫痫发作后3天的海马切片观察显示，96％的D-serine阳性细胞为NeuN阳性。
2．免疫荧光双标记和激光共聚焦显微镜显示，对照组没有D-serine阳性神经元。但是，在对照组和匹罗卡品模型组同时检测到了星形胶质细胞阳性的D-serine阳性细胞。D-serine阳性细胞在星形胶质细胞的细胞体和突起都有分布。这些D-serine阳性的星形胶质细胞在大脑皮质和海马上广泛分布，它们的数目和密度在匹罗卡品模型中显著提高。
3．D-serine阳性神经元分布在大脑的运动皮质、躯体感觉皮质、额眶部皮质、梨状皮质、扣带回皮质、岛叶皮质、内嗅皮质和海马的CA1、CA2、CA3和齿状回。此外，丘脑背侧也可以观察到阳性神经元，外侧丘脑下部也能观察到少许阳性神经元，但是脑干几乎没有检测到阳性产物。而且D-serine阳性神经元的数目在癫痫发作1h到14d的范围内是随时间变化的。1h时没有检测到阳性神经元，4h时D-serine阳性神经元开始在脑的一些区域出现，大多数区域在12-24h时逐渐升高并达到高峰，3-14d时逐渐下降。4．D-serine／GAD-67的双标记神经元主要分布在大脑皮质的锥体层和海马的多形细胞层，98％的D-serine阳性细胞为GAD-67阳性，D-seri：ne／GAD-67的双标记神经元占了GAD-67阳性细胞总数的34%，说明小鼠匹罗卡品模型中大多数D-serine阳性细胞为GABA能神经元。
5．D-serine／FJC双标记神经元多数分布在大脑皮质的锥体层和海马的多形细胞层。93％D-serine阳性神经元为FJC阳性，说明小鼠匹罗卡品模型中D-serine阳性神经元大多数发生了变性死亡。
6．pNMDAR1的免疫组织化学染色用来检测NMDA受体的磷酸化，它可以反映神经元中NMDA受体的活化状态。小鼠匹罗卡品模型的大脑皮质和海马切片中观察到了大量的pNMDAR1阳性神经元。pNMDAR1分布于细胞膜和细胞浆上。pNMDAR1阳性神经元与D-serine阳性神经元在分布和数目的动态变化上显示出了一定的相似性。
以上研究结果提示，癫痫发作后脑内D-serine的表达增多，D-serine的上调可能通过NMDA受体介导的兴奋性毒性机制导致GABA能神经元的变性死亡。因此，D-serine作为NMDA受体的内源性配体，可能通过NMDA受体的过度活化参与癫痫的发生和发展。
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.
D-serine分子是一种D-氨基酸即右旋-丝氨酸，在中枢神经系统神经元NMDA信号传递的调节中具有重要作用。D-serine与NMDA受体的甘氨酸位点相结合，在NMDA受体激活中充当NMDA受体的内源性配体的角色。而且内源性D-serine的释放在兴奋性毒性导致的大脑皮质和海马的神经元损伤中具有重要作用，因此D-serine参与了一些神经系统疾病的发生。精神分裂症的病人大脑中丝氨酸消旋酶表达的变化也表明D-serine信号与其疾病的发展有关。我们猜想D-serine信号途径可能通过NMDA受体的过度活化、兴奋性毒性和神经元损伤参与慢性癫痫的发病和慢性癫痫的反复癫痫发作。在我们的前期研究中，已经明确了小鼠匹罗卡品模型中大量的神经元死亡的时程变化特点；而且已证实连续腹腔注射拟胆碱剂匹罗卡品可以诱导癫痫发作、神经元丢失和自发的反复癫痫发作，上述特点与人类的颞叶癫痫十分相似。因此，本课题采用小鼠匹罗卡品模型，免疫组织化学染色检测脑内D-serine的细胞内定位和D-serine阳性细胞的时空变化特点，D-serine与特异性标记变性神经元的Fluoro-Jade C染色、神经元的标记物NeuN、GABA能神经元的标记物GAD-67和星形胶质细胞的标记物GFAP进行双标记等方法，以阐明D-serine信号可能参与慢性癫痫的神经元死亡和慢性癫痫发病。主要结果如下：
1．癫痫发作12-24h后在大脑皮质、海马、杏仁核和丘脑观察到大量D-serine阳性细胞。它们分布于细胞的胞浆、胞膜上，阳性细胞呈椭圆形，平均直径为24±6μm。癫痫发作后3天的海马切片观察显示，96％的D-serine阳性细胞为NeuN阳性。
2．免疫荧光双标记和激光共聚焦显微镜显示，对照组没有D-serine阳性神经元。但是，在对照组和匹罗卡品模型组同时检测到了星形胶质细胞阳性的D-serine阳性细胞。D-serine阳性细胞在星形胶质细胞的细胞体和突起都有分布。这些D-serine阳性的星形胶质细胞在大脑皮质和海马上广泛分布，它们的数目和密度在匹罗卡品模型中显著提高。
3．D-serine阳性神经元分布在大脑的运动皮质、躯体感觉皮质、额眶部皮质、梨状皮质、扣带回皮质、岛叶皮质、内嗅皮质和海马的CA1、CA2、CA3和齿状回。此外，丘脑背侧也可以观察到阳性神经元，外侧丘脑下部也能观察到少许阳性神经元，但是脑干几乎没有检测到阳性产物。而且D-serine阳性神经元的数目在癫痫发作1h到14d的范围内是随时间变化的。1h时没有检测到阳性神经元，4h时D-serine阳性神经元开始在脑的一些区域出现，大多数区域在12-24h时逐渐升高并达到高峰，3-14d时逐渐下降。4．D-serine／GAD-67的双标记神经元主要分布在大脑皮质的锥体层和海马的多形细胞层，98％的D-serine阳性细胞为GAD-67阳性，D-seri：ne／GAD-67的双标记神经元占了GAD-67阳性细胞总数的34%，说明小鼠匹罗卡品模型中大多数D-serine阳性细胞为GABA能神经元。
5．D-serine／FJC双标记神经元多数分布在大脑皮质的锥体层和海马的多形细胞层。93％D-serine阳性神经元为FJC阳性，说明小鼠匹罗卡品模型中D-serine阳性神经元大多数发生了变性死亡。
6．pNMDAR1的免疫组织化学染色用来检测NMDA受体的磷酸化，它可以反映神经元中NMDA受体的活化状态。小鼠匹罗卡品模型的大脑皮质和海马切片中观察到了大量的pNMDAR1阳性神经元。pNMDAR1分布于细胞膜和细胞浆上。pNMDAR1阳性神经元与D-serine阳性神经元在分布和数目的动态变化上显示出了一定的相似性。
以上研究结果提示，癫痫发作后脑内D-serine的表达增多，D-serine的上调可能通过NMDA受体介导的兴奋性毒性机制导致GABA能神经元的变性死亡。因此，D-serine作为NMDA受体的内源性配体，可能通过NMDA受体的过度活化参与癫痫的发生和发展。
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.
引文
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