【作者】 王小成；

【导师】 张作明； 冯立宁；

【作者基本信息】 第四军医大学 ， 航空、航天与航海医学， 2009， 博士

【摘要】 正常情况下，人体各感觉系统接受到的感觉信息是相互匹配的。不同感觉通道间信息的整合和匹配是维持机体正常生理和心理活动的前提。当不同通道信息间发生冲突时将会出现不同程度的生理和心理反应，可能导致工作能力降低，有时甚至会导致机体失能。视觉系统和前庭系统是人体两个非常重要的感觉系统，在维持机体正常的生理和心理活动中发挥着重要的作用。据统计80％-90％的外界信息是通过视觉系统传入的，而前庭系统在人体平衡、各种运动状态的感知和空间定向等方面发挥着重要的作用。视觉系统与前庭系统是与航空航天活动最密切相关的两个感觉系统。视觉系统信息和前庭系统信息的冲突或不匹配是导致运动病(motionsickness，MS)和空间定向障碍(sp atial disorientation，SD)发病的重要原因。运动病和空间定向障碍长期以来是困扰全世界航空航天活动的两大难题，由于具体的发病机制至今仍未阐明，使得预防和治疗措施缺乏针对性而难以奏效。因此，关于视觉系统信息与前庭系统信息互相作用和整合机制的研究是各国航空航天医学界研究的重点和热点问题。但由于缺乏相应的视觉缺陷研究模型，使得视觉信息与前庭系统信息的整合机制以及视觉信息在MS和SD发病机制中作用的研究受到极大限制。视觉缺陷模式动物是一类视觉功能先天存在缺陷的模式动物，在国内外被广范地应用于视觉相关的研究中。我们实验室发现和培育了1种视觉功能缺陷的模式动物—视网膜快速变性(retinal degeneration fast，rdf)小鼠，遗传特性稳定，并已建立了近交系。研究证实rdf小鼠出生后早期视锥细胞和视杆细胞发生凋亡，出生后3周时外层视网膜已消失，因此成年rdf小鼠明视觉和暗视觉丧失。由于其视觉功能的先天性缺陷，为我们从视觉系统入手开展视觉信息在SD和MS发病机制中的作用，以及视觉系统信息与前庭系统信息在中枢神经系统的整合机制的相关研究提供了独特的优势。本课题拟利用rdf小鼠先天性视觉功能缺陷的特性，观察旋转运动刺激对正常野生型昆明小鼠和rdf小鼠中枢神经系统Fos蛋白及降钙素基因相关肽和已酰胆碱表达的影响。初步阐明视觉系统信息与前庭系统信息在小鼠中枢神经系统整合的机制，为预防和治疗SD和MS提供理论依据。材料与方法1．采用标准化视觉电生理检查方案和HE染色对F 19(Filial 19)代成年rdf小鼠和正常野生型成年昆明小鼠的视觉电生理学特征和视网膜组织结构特点进行观察。2．分别给予rdf小鼠和正常野生型昆明小鼠旋转运动刺激(旋转半径0．6m，角速度180°／S，顺时针／逆时针交替刺激，每周期3 min，持续时间共30 min)，通过观察它们条件性厌食症(conditioned taste avetsion，CTA)的程度，即旋转运动刺激前后对糖精水饮用量的变化，研究两种小鼠运动病敏感性的差异。3．运用免疫组织化学染色的方法观察旋转运动刺激(刺激模式同上)30 min后，两种小鼠中枢神经系统前庭相关的区域(前庭核、舌下神经前置核、下橄榄体β亚核和K帽、小脑绒球和旁绒球)Fos表达的差异。通过观察视觉信息对以上区域Fos蛋白表达的影响，明确视觉系统信息与前庭系统信息在小鼠神经中枢整合的关键区域。4．运用免疫组织化学染色的方法观察多次旋转运动前庭刺激(刺激模式同上，每次刺激30 min，间隔24 h，共3次)后，降钙素基因相关肽(calcitonin gene-related peptide，CGRP)和胆碱乙酰转移酶(choline acetyltransferase，ChAT)在视觉系统信息与前庭系统信息整合区域的表达差异，明确CGRP和乙酰胆碱(acetylcholine，Ach)是否参与了视觉系统信息与前庭系统信息在小鼠中枢神经系统的整合。结果1．野生型昆明小鼠ERG五项检查——视杆细胞反应视网膜电图(scotopic electroretinogram，Scot ERG)、振荡电位(oscillatory potentials，Ops)、最大混合反应视网膜电图(maximal combined responseelectroretinogram，Max ERG)、明适应视网膜电图(photopic electroretinogram，Phot ERG)、闪烁光视网膜电图(flicker electroretinogram，Flicker ERG)均可引出正常波形。在同样条件下rdf小鼠均未引出诱发电位波形。视网膜组织HE染色结果显示，正常野生型昆明小鼠视网膜各层均存在，层次清楚；而rdf小鼠视网膜外核层、光感受细胞层和外网状层消失，内核层也变薄。2．旋转运动刺激30 min后，两种小鼠24 h糖精水的饮用量与刺激前相比均显著减少[rdf小鼠：10．94±0．92 g(刺激前)vs 6．87±0．79 g(刺激后第1个24 h)，n=7，P＜0．01；昆明小鼠：11．16±0．96 g(刺激前)vs 9．37±0．86 g(刺激后第1个24 h)，n=7，P＜0．01]。但rdf小鼠比正常野生型昆明小鼠减少的程度更大[刺激后第1个24 h饮用量／刺激前饮用量：63．07±8．35(％，rdf小鼠)vs84．49±10．16(％，昆明小鼠)，n=7，P＜0．05]。3．旋转运动刺激30 min后，rdf小鼠舌下神经前置核(prepositushypoglossal nucleus，PrH)和下橄榄体内侧核K帽(Kooy cap ofinferior olivemedial nucleus，IOK)Fos阳性神经元数目显著少于正常野生型昆明小鼠[PrH：35．33±2．19(昆明小鼠)vs 20．67±1．45(rdf小鼠)，n=6，P＜0．01；IOK：12．17±1．19(昆明小鼠)vs 6．67±0．42(rdf小鼠)，n=6，P＜0．01]。神经中枢与前庭相关的其他区域，两种小鼠Fos蛋白表达也存在差异，但差异无显著性。4．3次间隔24 h的30 min旋转运动刺激后，CGRP和ChAT在rdf小鼠和正常野生型昆明小鼠前庭相关区域的表达模式均与旋转运动刺激后Fos蛋白的表达模式相似。rdf小鼠PrH和IOK的CGRP阳性神经元[PrH：87．33±1．96(昆明小鼠)vs 62．67±1．65(rdf小鼠)，n=6，P＜0．01；IOK：26．50±1．23(昆明小鼠)vs 17．00±0．89(rdf小鼠)，n=6，P＜0．01]和ChAT阳性神经元[PrH：78．33±1．78(昆明小鼠)vs 56．67±1．73(rdf小鼠)，n=6，P＜0．01；IOK：17．17±1．05(昆明小鼠)vs 10．33±0．92(rdf小鼠)，n=6，P＜0．01]均明显少于正常野生型昆明小鼠。除前庭内侧核rdf小鼠ChAT阳性神经元稍多于正常野生型昆明小鼠外[62．67±3．84(昆明小鼠)vs 66．67±2．54(rdf小鼠)，n=6，P=0．03]，神经中枢与前庭相关的其他区域，两种小鼠CGRP和ChAT表达的差异无显著性。结论野生型昆明小鼠ERG五项检查——Scot ERG、Ops、Max ERG、PhotERG Flicker ERG均可引出正常波形，证实其视网膜功能正常。在同样条件下rdf小鼠五项检查均未引出诱发电位波形，为熄灭型表现，说明其视杆体细胞与视锥体细胞功能均丧失。视网膜组织学观察表明，正常野生型昆明小鼠视网膜各层均存在，层次清楚。而rdf小鼠视网膜外核层、光感受器细胞层和外网状层消失，内核层也变薄，说明其视锥细胞和视杆细胞均完全凋亡。从视觉电生理特征和视网膜组织结构两方面证明，我们实验室发现的rdf小鼠是一种先天性视觉功能缺陷小鼠，其明视觉和暗视觉功能完全丧失。旋转运动刺激后，rdf小鼠糖精水饮用量的减少程度大于正常野生型昆明小鼠，rdf小鼠比野生型昆明小鼠发生了更加严重的厌食症。由于实验中，给予两种小鼠的旋转运动刺激的刺激模式、刺激时间和旋转角速度均完全相同，因此，rdf小鼠的运动病敏感性高于正常野生型昆明小鼠。这说明视觉系统信息在运动病的发病过程中发挥了一定的作用，视觉信息通过某种途径与前庭信息发生了关系，影响了中枢神经系统对运动刺激的反应性。30 min旋转运动刺激后，rdf小鼠舌下神经前置核和下橄榄体内侧核K帽Fos阳性神经元数目显著少于正常野生型昆明小鼠。说明视觉系统信息影响了这两个与前庭系统相关区域的神经元的激活，以上两个区域可能是视觉系统信息与前庭系统信息在小鼠中枢神经系统整合的关键部位。3次间隔24 h的30 min旋转运动刺激后，rdf小鼠舌下神经前置核和下橄榄体内侧核K帽的CGRP阳性神经元和ChAT阳性神经元数目均显著少于正常野生型昆明小鼠。结果表明旋转运动刺激后CGRP和ChAT在rdf小鼠和正常野生型昆明小鼠前庭相关区域的表达模式均与Fos蛋白的表达模式相似。这说明舌下神经前置核和下橄榄体内侧核K帽的神经元可能通过生成、释放CGRP和Ach参与了视觉系统信息与前庭系统信息在小鼠中枢神经系统的整合，CGRP和Ach可能做为神经递质或神经调质参与了视觉系统信息与前庭系统信息在小鼠神经中枢的整合。为今后探索运动病和空间定向障碍的发病机制及预防和对抗措施提供了重要的依据。更多还原

【Abstract】 Normally,sensory information accepted by different sensory systems iscompatible and complementary.Sensory conflict would induce varying degreesof physiology or psychological reactions,even incapacitation.Visual system andvestibular system are two very important sensory systems in maintaining normalphysical and mental activities.80%-90% of enviromental information isaccepted through the visual system,and the vestibular system plays animportment role in the body balance,sensing sports state and spatial orientation.Therefore,the match of visual system information and vestibular systeminformation is essential to maintain normal psychological and physiologicalactivities.Motion sickness and spatial disorientation has been two problems impedingaviation and aerospace activities.Due to their unclear pathogenesis,it is difficultto prevent and treat them effectivly.The conflict of visual system informationand vestibular system information is one of the main reasons which inducemotion sickness and spatial disorientation.It become hot issue to explore integration and impact mechanism of visual information and vestibularinformation in aerospace medicine.However,for lacking animal model withvisual defect,the role of visual system information in mechanisms of spatialdisorientation and motion sickness is rarely reported.A kind of mouse with dysopia named rdf (retinal degeneration fast)mousehas been detected and nurtured in our laboratory.In this study,we may initiallyclarify the integration mechanism of visual information and vestibularinformation in the central nervous system using animal model with visual defect—rdf mouse under motion sickness,and provide basic theory for the preventionand treatment of spatial disorientation and motion sickness.Materials and Methods1.Electrophysiological characteristics of rdf mice and normal wild-typeKunming mice were investigated using international standardization schedule ofelectroretinogram test,and organizational structures were observed by HEstaining.2.Rdf mice and normal wild-type Kunming mice were subjected to rotarystimulation (turning radius:0.6 m;angular volicity:180°/s;3 min per cycle;clockwise rotation alternated with the anticlockwise rotation)for 30 minutes toinduce motion sickness.The conditions taste anorexia (CTA)to saccharinsolution was used to observe the differences in the sensitivity of motion sicknessof the two kinds of mice.3.The critical regions of sensory integration of visual information andvestibular information in the central nervous system were determined throughobserving the differences in the expressions of Fos in the vestibular-relatedregions (vestibular nucleus,prepositus hypoglossal nucleus,Kooy cap of inferior olive medial nucleus,βsubnucleus of olive inferior,flocculus andparaflocculus)of rdf mice and normal wild-type Kunming mice after rotatorystimulation (turning radius:0.6 m;angular volicity:180°/s;3 min per cycle;clockwise rotation alternated with the anticlockwise rotation)30 min.4.The differences in the expressions of calcitonin gene-related peptide(CGRP)and choline acetyl transferase (ChAT)in the vestibular-related regionsof rdf mice and normal wild-type Kunming mice after rotatory stimulation(turning radius:0.6 m;angular volicity:180°/s;3 min per cycle;clockwiserotation alternated with the anticlockwise rotation;30 min every time;interval:24 h)3 times were used to determine whether the calcitonin gene-related peptideand diacetyl-choline play the roles in the sensory integration of visualinformation and vestibular information in the central nervous system of mice.Results1.The results of electroretinogram tests indicted that ScotERG,Ops,MaxERG,PhotERG and Flicker ERG induced normal waves in the normal wild-typeKunming mice,But under the same conditions,the five tests failed to inducenormal waves in the rdf mice.The HE staining of retina displayed that all layersexisted clearly in normal wild-type Kunming mice.The photoreceptor layer,outer nuclear layer and outer plexiform layer of retina were invisible,and theinner nuclear layer became thining in the rdf mice.2.The intake of saccharin solution was significantly reduced in rdf mice[10.94±0.92 g (before stimulation)vs 6.87±0.79 g (first 24 h after stimulation),n=7,P＜0.01] and normal wild-type Kunming mice [11.16±0.96 g (beforestimulation)vs 9.37±0.86 g (first 24 h after stimulation),n=7,P＜0.01] afterrotary stimulation.The intake volumes first 24 h after stimualation of rdf mice and normal wild-type Kunming mice were 62.07% and 84.49% of intake beforestimulation respectively,the decrease of rdf mice was more greater than that ofnormal wild-type kunming mice [the intake volume in first 24 h afterstimulation/the intake volume before stimulation:63.07±8.35 (%,rdf mice)vs84.49±10.16(%,Kunming mice),n=7,P＜0.05]3.The numbers of Fos-positive neurons in prepositus hypoglossal nucleusand Kooy cap of inferior olive medial nucleus were significantly less in rdf micethan those in normal wild-type Kunming mice after rotary stimulation[PrH:35.33±2.19 (Kunming mice)vs 20.67±1.45 (rdf mice),n=6,P＜0.01;IOK:12.17±1.19 (Kunming mice)vs 6.67±0.42 (rdf mice),n=6,P＜0.01].In othervestibular-related regions,the differences in the numbers of Fos-positiveneurons between the two experimental groups were not significant.4.After rotatory stimulus 3 times at interval 24 h,the expressions of CGRP[PrH:87.33±1.96 (Kunming mice)vs 62.67±1.65 (rdfmice),n=6,P＜0.01;IOK:26.50±1.23 (Kunming mice)vs 17.00±0.89 (rdfmice),n=6,P＜0.01] and ChAT[PrH:78.33±1.78 (Kunming mice)vs 56.67±1.73 (rdfmice),n=6,P＜0.01;IOK:17.17±1.05 (Kunming mice)vs 10.33±0.92 (rdf mice),n=6,P＜0.01] inprepositus hypoglossal nucleus and Kooy cap of inferior olive were significantlyless in rdf mice than those in normal wild-type Kunming mice.There were somedifferences in expression of CGRP and ChAT in the other vestibular-regions inthe two kinds of mice after rotatory stimulus,but the differences were notsignificant except the differences of ChAT in the medial vestibularnucleus[62.67±3.84 (Kunming mice)vs 66.67±2.54 (rdfmice),n=6,P=0.03].ConclusionThe five electroretinogram tests of Scot ERG、Ops、Max ERG、Phot ERG and Flicker ERG failured to induce normal waves in the rdf mice,and the HEstaining of retina displayed that photoreceptor layer,outer nuclear layer andouter plexiform layer were not visible and the inner nuclear layer became thin inthe rdf mice.All those indicated that the rdf mouse is a kind of mouse withdysopia.After rotatory 30 min,the reduction of intake volune of saccharinsolution was greater in rdf mice compared to that of normal wild-type Kunmingmice.That indicated that anorexia in the rdf mice was more seriously than thatin the normal wild-type Kunming mice.It also manifested that the rdf mice weremore sensitivie to rotatory stimulation than normal wild-type Kunming mice.Sowe came to a conclusion that visual information plays a role in the mechanismof motion sickness.Visual information and vestibular information impact eachother through certain channels.After rotatory stimulation 30 min,the numbers of Fos-positive neurons inprepositus hypoglossal nucleus and Kooy cap of inferior olive were significantlyless in rdf mice than those in normal wild-type Kunming mice.The expressionof Fos protein reflects activation of neurons.The results indicated that visualinformation may affect the activation of neurons by rotatory stimulation in thosetwo regions.Therefore,the prepositus hypoglossal nucleus and Kooy cap ofinferior olive medial nucleus are key regions where the visual information andvestibular information integrate in the central nervous system in mice.Theexpression patterns of CGRP and ChAT were similar to that of Fos in thevestibular-related regions in the normal wild-type Kunming mice and rdf miceafter rotatory stimulus 3 times at interval 24 h.So we speculated that CGRP andAch generated and released by neurons in the hypoglossal nucleus and Kooy capof inferior olive may paly a role in the sensory integration of visual systeminformation and vestibular system information in the central nervous system in mice with motion sickness.更多还原