【作者】 杨静；

【导师】 刘莉洁；

【作者基本信息】 东南大学 ， 生物化学与分子生物， 2019， 硕士

【摘要】 研究背景:听力损失(Hearing Loss,HL)和痴呆已成为全球范围内的严重健康问题。HL被提出可能是痴呆发生发展的促进因素。本课题组前期通过建立噪声性听力损失(noise induced hearing loss,NIHL)小鼠模型,观察到NIHL动物出现了海马相关认知功能减退、以及海马神经新生减少、海马神经元树突复杂度降低,不仅为HL与痴呆之间的因果关联提供了动物实验证据,还提出NIHL的认知损害效应可能与海马神经可塑性的降低有关。本课题将继续就NIHL影响海马神经可塑性的机制展开研究。组织微环境(microenvironment)是机体内外环境变化影响神经可塑性的必经媒介,小胶质细胞(microglia)是中枢神经系统(the central nervous system,CNS)微环境的重要决定者。小胶质细胞是CNS中的固有免疫调节细胞,也被认为是CNS的免疫“双刃剑”,其功能失衡已被证明是多种脑退行性疾病的关键病理基础。组织慢性损伤、慢性炎性反应(包括小胶质细胞持久激活所引起的慢性炎性)是引起中枢小胶质细胞功能失衡的主要因素。近年已有研究报道,获得性HL能引起耳蜗核小胶质细胞的持久激活。鉴于中枢小胶质细胞功能转化的生物规律、以及听觉中枢与海马之间丰富的解剖学与功能学联系,我们提出了这样的科学假说:NIHL发生后,包括耳蜗核在内的听觉中枢发生了小胶质细胞的持久激活,进而引起了海马小胶质细胞功能失衡;海马小胶质细胞功能失衡参与介导了NIHL动物的海马神经可塑性降低。根据这一科学假说,本课题将围绕小胶质细胞和神经新生对NIHL影响海马神经可塑性的机制进行初步探索。方法:本实验继续采用6-8周龄健康雄性CBA小鼠作为实验动物,随机分为对照组(Control组)和噪声性听力损失组(NIHL组)。NIHL组动物给予123 d B SPL宽带白噪声暴露2h。各组动物再分为多个亚组,分别于噪声后6小时(6HPN)、1天(1DPN)、2天(2DPN)、4天(4DPN)、1个月(1MPN)、3个月(3MPN)、6个月(6MPN)和12个月(12MPN)进行样本取材。在1MPN时通过听觉脑干诱发电位(auditory brainstem response,ABR)的记录检测动物听觉阈值。各组动物于实验终点进行心脏灌流、取脑,制作脑冠状面冰冻切片。对脑冰冻切片进行免疫荧光标记(Immunofluorescence,IF),以内源性新生神经元标志物双皮质素(Doublecortin,DCX)、细胞增殖标志物核蛋白Ki67为观察靶点,分析NIHL对海马神经新生的影响;利用小胶质细胞特异性标志物钙离子接头蛋白分子1(ionized calcium binding adapter molecule 1,Iba1)及活化标志物清道夫受体CD68(cluster of differentiation 68,CD68)的免疫荧光标记,分析噪声暴露或NIHL对听觉中枢及海马小胶质细胞数量、形态及功能的影响;通过对目标脑区白细胞介素-1β(interleukin-1β,IL-1β)的免疫荧光标记,分析NIHL对海马组织炎性因子水平的影响。结果:(1)1MPN时的ABR结果显示,经历噪声暴露的动物听力阈值显著高于对照动物,表明已成功建立永久性中重度NIHL模型。(2)在1MPN、3MPN、6MPN时,NIHL动物在各时间点均有不同程度新生神经元数量和增殖细胞数量的减少。这些结果与本课题组前期数据一致,再次表明NIHL动物海马神经新生显著减少,且表现出神经新生的加速衰退。(3)在1MPN至12MPN期间,NIHL动物听觉中枢多个脑区出现了小胶质细胞数量增加、胞体增大、CD68细胞占有率增高(从1MPN持续至少达6MPN),提示NIHL后听觉中枢出现了小胶质细胞的持久广泛激活;同期海马齿状回(denate gyrus,DG)及角状突起区域3(cornu ammonis area 3,CA3)小胶质细胞则表现为细胞数量减少、胞体减小、但CD68细胞占有率增高,提示NIHL动物出现了海马小胶质细胞功能失衡。(4)在1MPN、3MPN时,NIHL动物海马中炎性因子IL-1β的表达水平增高。(5)在6HPN、1DPN及2DPN时,经历噪声暴露的动物听觉中枢及海马中的小胶质细胞有不同程度的胞体面积增加,然而4DPN时则与对照动物无显著差异。提示本实验室采用的噪声暴露对目标脑区小胶质细胞的功能仅产生了一过性影响。(6)对1MPN、3MPN动物海马DG区小胶质细胞功能表型相关指标(胞体面积、CD68平均光密度)与海马神经新生相关指标(DCX~+细胞数、Ki67~+细胞数)进行Pearson相关性分析的结果显示,海马DG区Ki67~+增殖细胞的减少与海马小胶质细胞胞体面积显著相关,海马DCX~+新生神经元、Ki67~+增殖细胞的减少与CD68平均光密度显著相关,提示海马小胶质细胞功能失衡可能参与介导了NIHL动物海马神经可塑性的降低。本研究结果为深入揭示HL与痴呆之间的关联机制、以及相关疾病防治靶点的挖掘提供了新的研究线索和较为系统的实验依据。更多还原

【Abstract】 Background:Hearing loss（HL）and dementia have become serious public health problems worldwide.A number of large-scale epidemiological surveys suggest that HL be a promoting factor in the development of dementia.Using of a mouse model,our previous study showed that long after the establishment of noise-induced hearing loss（NIHL）,animals exhibited impaired hippocampus-related cognitive function,along with a decline in hippocampal neurogenesis and dendritic complexity.The result not only provided direct evidence for the causal association between HL and dementia,but also pinpointyed the declined hippocampal neuroplasticity as the mechanim for this connection specifically for NIHL.The present study will continue to investigate how NIHL affects hippocampal neuroplasticity.Neuroplaticity is under a close regulation of microenvironment.Microglia plays a critical role in the microenvironment formaton in the central nervous system（CNS）,where they serve as the innate immune cells.However,the dysfunction of those cells is the also pathological reason in a variety of neurodegenerative diseases.Therefore,they are called immunal“double sword”.Sustained microglia activation is often seen as a major phenotype of microglia dysfunction,which is often evident in chronic tissue injury and inflammation in CNS.In recent years,acquired HL has been reported to induce sustained activation of microglia in the cochlear nucleus.Based upon the biological laws of microglia and the abundant anatomical and functional connections between the auditory pathway and hippocampus,we hypothesize that NIHL results in a sustained microglia activation that is initiated and spread across the nuclie in the auditory pathway and;further extended to hippocampus,where it consequently cause the reduction of neuroplasticity.The present study therefore was intended to explore if the functional change of microglia in hippocampus is the connected with the declined neuroplasticity in NIHL model.Method:In this study,healthy male CBA mice of 6-8 weeks old were used as experimental animals and were randomly divided into control and NIHL groups.Animals in the NIHL group were exposed to 123 d B SPL broadband noise for 2 h,and were divided into multiple subgroups according to the time points at which the animals were sacrifieced for brain sample taken at 6 hours（6HPN）,1 day（1DPN）,2 days（2DPN）,4 days（4DPN）,1 month（1MPN）,3months（3MPN）,6 months（6MPN）,and 12 months（12MPN）post the noise.The control group was also divided into time-matched subgroups.On 1MPN,auditory brainstem response（ABR）was tested to determine NIHL.At the ending time,every animal was given heart infusion under deep anesthesia.The brain was taken and frozed for coronal sectioning.Immunofluorescence labeling（IF）was performed against the endogenous neonatal neuron marker Doublecortin（DCX）and the cell proliferation marker nucleoprotein Ki67 to analyze the effects of NIHL on hippocampal neurogenesis.IF of ionized calcium binding adapter molecule 1（Iba1）and the microglia activation marker cluster of differentiation 68（CD68）were used to analyze the effects of noise exposure or NIHL on the morphology and function of microglia in both auditory pathway and hippocampus.IF against interleukin-1β（IL-1β）in targeted brain regions was also performed as a preliminary exploration of the possible mechanism of NIHL affecting the function of microglia in hippocampus.Results:（1）The ABR threshold,as verified at 1MPN,was significantly higher in the NIHL group than in the control animals,suggesting a successful establishment of NIHL of moderately-to-severe degree.（2）At 1MPN,3MPN and 6MPN,mice with NIHL showed a varied decline in the number of proliferating cells and neonatal neurons.In consistent with our previous data,this result again confirmed that the hippocampal neurogenesis is significantly declined in subjects with NIHL as the concequence of an accelerated decline in neuroneogenesis.（3）Mice with NIHL showed a varied but increase of Iba1⁺cell density,the area of cell soma and the rate of CD68-positive cells across multiple auditory nuclei at each time point observed from 1MPN to 12MPN（consistent at least up to 6MPN）,suggesting a sustained and wide-sprea activation of microglia by NIHL in the auditory pathway.Mice with NIHL showed a varied but decrease of Iba1⁺cell density,the area of cell soma but increase of the rate of CD68-positive cells across DG and CA3 at each time point observed from 1MPN to12MPN,suggesting dysfunction of microglia by NIHL in hippocampus.（4）At 1MPN and 3MPN,mice with NIHL showe up-regulated IL-1βin hippocampus.（5）At 6HPN,1DPN and 2DPN,the area of microglia soma was found to be increased in the auditory nuclei and hippocampus of the mice exposed to the noise.However,the difference was not seen between the NIHL subgroup of 4DPN and the time-match control subgroup,suggesting that the noise exposure caused a transient and acute effect on the microglia function in the auditory pathway and hippocampus,which could be differentiated from the long-term effect of NIHL.（6）Pearson correlation performed at 1MPN and 3MPN showed that in mice with NIHL,the area of microglia soma was significantly correlated with the decrease of Ki67⁺cells（for proliferation）,microglia activation ratio(CD68⁺/Iba1^+d)was significantly correlated with the decrease of both DCX⁺cells（for neonatal neurons）and Ki67⁺cells,suggesting that microglia dysfunction play a role in mediating the reduction of neuroplasticity in the hippocampus of those mice.These results reveal a new approach to investigate the nature of the relationship between HL and dementia.The potential involvement of microglia activation may also serve as a new therapeutic target to reduce the promoting effect of HL on the development of dementia.更多还原