儿童局灶性皮质发育不良Ⅱ型病例脑组织中阳离子-氯离子共转运体(NKCC1/KCC2)的表达

Expression of cation chloride cotransporter(NKCC1/KCC2)in brain tissue of children with focal cortical dysplasia typeⅡ

目的探讨儿童局灶性皮质发育不良(FCD)Ⅱ型病例的脑病灶中形态异常神经元(dysmorphic neuron)和气球细胞(balloon cell)的阳离子-氯离子共转运体(NKCC1/KCC2)的表达情况,提示可能存在的癫痫发生的形态学基础。方法收集2017年2月至2019年12月在北京市海淀医院(8例)及首都医科大学宣武医院(12例)癫痫手术后石蜡包埋的脑组织,选择患者年龄在14岁以下的诊断为FCDⅡa型10例、FCDⅡb型10例。采用免疫组织化学、免疫组织化学双染的方法,检测1型Na-K-2Cl共转运体(NKCC1)、2型K-Cl协同转运体(KCC2)在FCDⅡa型、FCDⅡb型中的表达情况。对NKCC1在FCDⅡa型的形态异常神经元和正常神经元免疫组织化学染色测定平均吸光度(IA)值。结果(1)正常大脑皮质神经元细胞质表达NKCC1,细胞膜表达KCC2;(2)在FCDⅡa型的形态异常神经元中,NKCC1表达量增加,与正常神经元表达比较IA值差异有统计学意义(t′=7.618,P<0.01)。KCC2在形态异常神经元异常表达为细胞质阳性。(3)在FCDⅡb型中NKCC1/KCC2在形态异常神经元的表达与FCDⅡa型相同。气球细胞中NKCC1异常表达为阴性或少许稀疏细胞膜着色,KCC2异常表达为阴性。结论形态异常神经元及气球细胞中NKCC1/KCC2的表达模式与成熟神经元表达模式完全不同,其蛋白表达变化可能影响到神经元跨膜氯离子流,从而影响了抑制性神经递质γ-氨基丁酸的作用,导致神经元兴奋性增加,这个过程可能与临床癫痫症状发生相关。

Objective To investigate the expression of cation chloride cotransporter(NKCC1/KCC2)in the neurons from cerebral lesions of children with focal cortical dysplasia(FCD)typeⅡ,to provide a morphological basis for revealing the possible mechanism of epilepsy.Methods Eight cases of FCD typeⅡdiagnosed at Beijing Haidian Hospital,Beijing,China and 12 cases diagnosed at Xuanwu Hospital,Capital Medical University,Beijing,China from February 2017 to December 2019 were included.The expression of NKCC1 and KCC2 in FCD typeⅡa and FCD typeⅡb was detected using immunohistochemistry and double immunohistochemical stains.The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD typeⅡa(10 cases).Results The patients were all younger than 14 years of age.Ten cases were classified as FCD type IIa,and 10 cases as FCD typeⅡb.NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes.In dysmorphic neurons of FCD typeⅡa,expression of NKCC1 increased,which was statistically higher than that of normal neurons(P<0.01).Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm.In the FCDⅡb type,the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD typeⅡa.The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane,while the aberrant expression of KCC2 was absent.Conclusions The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons.The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons,modify the effect of inhibitory neurotransmittersγ-aminobutyric acid and increase neuronal excitability.These effects may be related to the occurrence of clinical epileptic symptoms.