【作者】 张林；

【导师】 黄矛； 张晓冬； 郑杰民；

【作者基本信息】 第二军医大学 ， 药理学， 2010， 硕士

【摘要】 晕动病(motion sickness)是因机体暴露于被动运动(passive motion)环境中,受不适宜的运动环境刺激,引起定向(orientation)和平衡(body balance)功能失调,伴随严重的前庭和植物神经反应。发生晕动病时,可出现头晕、头痛、出冷汗、面色苍白、恶心、呕吐等综合征,严重者可丧失自理能力或作业能力,或因反复呕吐而发生全身脱水和电解质紊乱等。晕动病的发病机制尚不完全清楚。晕动病的发生与机体在被动运动环境中的前庭(vestibular)刺激、视觉(visual perception)刺激和本体感觉(proprioceptive sense)刺激的整合有关。二十世纪50年代以来,其病因及发病机制分别有十余种学说或假说。目前大多数学者接受“感觉冲突假说”或“神经错配假说”。在乘车、乘船、乘飞机旅行,或驾驶坦克、空投跳伞等情况下,乘员晕动病的发生率可达70%甚至更高,严重阻碍乘员的作业能力。人们由于不能乘用某种交通工具必然影响其工作和日常生活,其中女性乘员将占相当比例。女性的晕动病发病率高于男性,早已是许多调查研究报告的结论和日常生活中的常见现象。既然晕动病易感性(susceptibility)与性别有关,其性别差异(gender difference)原因何在?病理生理机制如何?与性激素及其受体有何关联?对此尚无相关研究。开展对此课题的研究,首次以实验研究解析晕动病易感性性别差异的生物学基础,在晕动病病因学的基础研究方面进行新的探索。既有认识卵巢功能与前庭功能之间的功能生理学联系提供新认识,还可能为临床女性晕动病预防策略和措施、或改进自我保健方法,启发新的应用价值,具有重要的实际意义。目的：结合小鼠旋转-热板晕动病模型,大鼠“异食癖”晕动病模型探讨自身雌激素水平对晕动病易感性的影响,观察雌激素受体调节剂他莫昔芬在不同晕动病模型上对小鼠和大鼠晕动病指标的影响,为“雌激素易化晕动病易感性假说”提供实验室依据。方法和结果：实验一：在小鼠热板模型上雌激素对晕动病易感性的影响1小鼠热板反应晕动病模型的优化晕动病研究之所以难以取得突破性进展,缺乏模拟人体实际的简便实验性晕动病动物模型和客观评价指标是其瓶颈之一。以本实验室完成的国家自然科学基金项目,用于研究小鼠晕动病的旋转-热板模型上,从不同角度证实小鼠旋转后热板潜伏期变化,可以作为研究雌激素水平与晕动病易感性关系的指标之一。(1)适宜旋转刺激条件的选择本实验采用自制可调变速旋转诱发仪,比较不同旋转时间和不同旋转速度刺激方式下诱发的小鼠晕动病反应,并在热板上测定小鼠旋转前后的舔后足潜伏期作为评价晕动病的指标。结果显示,在以70rpm旋转40min或者105rpm旋转20min的刺激条件下,其中变速周期为旋转(R)15s+暂停(S)5s,热板温度为55±0.5℃,小鼠热板反应潜伏期随旋转刺激强度比基础潜伏期显著延长,适宜旋转刺激强度控制在热板反应潜伏期40s左右(过短晕动病不够明显,过长则易烫伤小鼠)。(2)小鼠旋转时精氨酸加压素水平与晕动病的联系晕动病发生时会伴随着一系列神经内分泌激素水平的改变,其中精氨酸加压素(arginine vasopressin, AVP)是其最早、最显著的神经内分泌变化。同时,小鼠在旋转刺激后一般状态会发生改变,可通过小鼠旋转后排便(fecal granules)、排便(urination)、竖毛(piloerection)、震颤(tremor) 4项评分,对小鼠晕动病程度进行综合评分(motion sickness score, MSS)。本实验选择晕动病综合评分和AVP水平变化来直接证实该模型的可行性。结果表明,在70rpm×40min或者105rpm×20min刺激条件下,旋转组小鼠旋转后热板潜伏期显著延长,且晕动病评分以及血浆AVP含量都显著高于对照组,证明在该模型上,给予适当的旋转刺激确实可以诱发小鼠晕动病,即小鼠热板反应潜伏期可以作为评价晕动病的指标之一。(3)小鼠旋转-热板模型的药理学验证首先,东莨菪碱是经过多年临床实践证实的经典防晕动病药物；临床观察早就发现丧失前庭功能或前庭器官发育不全者不易发生晕动病。对小鼠给予有效剂量的东莨菪碱,或进行前庭毁损,再在该模型上诱发晕动病,最后研究结果显示,旋转后东莨菪碱的组别和前庭毁损的组别其垂体和血浆中AVP含量降低,热板潜伏期以及晕动病评分也明显低于对照组,说明东莨菪碱在该模型上具有防晕效果,以及前庭毁损后在该模型上可完全阻滞晕动病反应。其次,异食癖是被较多采用的啮齿目动物的晕动病症状。本实验将旋转-热板模型和小鼠异食癖模型结合,用前者诱发小鼠晕动症状,测定小鼠在旋转前后食用高岭土的量,结果显示旋转刺激下小鼠高岭土食用量明显增多,模拟异食癖模型成功,同时也反证了旋转-热板模型的可行性。2在小鼠旋转-热板模型上雌激素易感性的初步研究本实验设计几种处理方式,主要针对内源性雌激素水平对小鼠晕动病易感性的影响展开研究。采用手术摘除小鼠卵巢,给予雌激素受体拮抗剂他莫昔芬来人为改变小鼠内源性的雌激素水平,并通过测定单次给药后血浆和脑垂体中AVP含量的变化,再与东莨菪碱和生理盐水对照组进行比较来定性和定量雌激素对小鼠晕动病易感性的影响。实验结果显示,给予他莫昔芬和卵巢切除后,对晕动病的影响不同,血浆和垂体AVP水平相反,这与他莫昔芬是选择性的雌激素受体调节剂,具有拮抗和激动双重作用以及AVP在体内的分布区域有关,证实小鼠自身雌激素水平的变化与晕动病易感性之间确实存在一定关联。实验二：大鼠动情周期雌激素水平变化在“异食癖”模型上的研究1大鼠“异食癖”模型上性别差异的研究比较不同性别大鼠以及手术去势的大鼠在旋转前后摄入高岭土的量,探讨大鼠晕动病的性别差异与雌激素水平的关系。采用大鼠“异食癖”模型,将SD大鼠分为4组,正常雌雄组以及去势雌雄组,用放免法测定血浆精氨酸加压素(AVP)水平。结果显示,旋转诱发后,正常雌性大鼠高岭土食用量的增加程度,血浆中AVP水平升高幅度也明显高于正常雄性大鼠。而去势雌性大鼠血高岭土摄取量,以及AVP变化幅度也没有正常雌性组大,结果提示大鼠晕动病敏感性存在性别差异,且与雌激素水平有关。2动情周期中雌激素水平的变化对大鼠晕动病的影响研究大鼠的雌激素水平随着动情周期呈现一定规律的波动,结合大鼠的异食癖晕动病模型,通过测定旋转前后血浆AVP及E2和P的含量,可以发现动情周期的波动与大鼠食用高岭土的量存在某种关联。故采用手术去势和化学去势的方式对大鼠内源性雌激素水平进行干扰影响动情周期,观察在雌激素水平变化的情况下大鼠异食癖的情况,探讨雌激素水平与晕动病易感性之间的联系。实验结果显示阻断雌激素受体或降低雌激素水平,减弱大鼠晕动病易感性,而大鼠自身的动情周期波动与晕动病易感性之间的关系没有统计学意义,但是有趋势显示在雌激素水平最高的动情期时晕动病反应稍严重。结论：分别在小鼠热板潜伏期、综合评分及大鼠“异食癖”晕动病模型上,雌性小鼠及大鼠旋转后血浆AVP水平随晕动病反应程度加重而升高；雌性大鼠对晕动刺激比雄性大鼠敏感,且晕动病反应程度随动情周期雌激素水平波动而起伏；外科去势和化学去势雌性大鼠的晕动病反应减弱,其血浆E2和AVP水平随之降低。结果表明晕动病易感性的性别差异与雌激素水平之间确实存在关联性。更多还原

【Abstract】 Motion sickness (MS) is a result of the body exposed to passive movement environment, causing orientation and body balance dysfunction, with severe vestibular and autonomic reaction. There may be dizziness, headache, cold sweats, pale, nausea, vomiting syndrome that can be too serious to loss of self-care ability or operational capacity, or systemic dehydration and disturbance of electrolyte balance.The pathogenesis of motion sickness is not yet fully understood, motion sickness is considered with the integration of vestibular stimulation, visual perception stimulation and proprioception stimulation. Since 1950’s, there were more than ten of theories or hypotheses on the pathogenesis of motion sickness. Most scholars accepted the "Sensory Conflict Hypothesis" or "Neural Mismatch Hypothesis".Many research findings have reported that women are more susceptible to motion sickness than men. Why is the motion sickness susceptibility gender-related? What is the pathophysiological mechanism? What is associated with the sex hormone and its receptor? There is no systematic study on these questions. According to the present subject classification, motion sickness and female hormones, especially estrogen, is two difficult issues to link.Carrying out research on this subject, can provide new understanding on etiology of motion sickness, and has important practical significance.Objective:Combinating of mouse hot-plate motion sickness model and rat "pica" motion sickness model, we explore the effects of their own estrogen level and tamoxifen on the susceptibility of motion sickness, and provide experimental basis in laboratory on "Hypothesis on susceptibility of motion sickness mediated by estrogen".Methods and Results:Part I:Effects of estrogen on susceptibility of motion sickness in the mouse rotation-hot-plate model1 Optimize the rotation-hot-plate motion sickness model in mice(1) Select the most optimal rotation stimulationWe compared motion sickness responses in mice induced by different rotation time and rotation speed, and measured the latency of paw lick in the hot plate before and after rotating for evaluating motion sickness. The results showed that 70rpm×40min or 105rpm×20min, including variable period of rotation (R) 15s+suspended (S) 5s, hot plate temperature of 55±0.5℃, mouse hot-plate response latency about 40s, too short for motion sickness was not obvious, too long, easy to burn pad of mice) was significantly longer than basic latency.(2) The relation between arginine vasopressin (AVP) and motion sickness in the mouse rotation-hot-plate motion sickness modelIn this study, we used AVP, and motion sickness score to directly confirm the feasibility of the model. The results showed that 70rpm×40min or 105rpm×20min stimulus conditions, the mice after rotating latency was significantly prolonged, and plasma AVP concentration, motion sickness score were significantly higher than control group. The results proved that the appropriate rotation stimulation can indeed induce motion sickness in mouse, so mouse hot-plate response latency as an evaluation of motion sickness can be one of the indicators.(3) Pharmacological validate the mouse rotation-hot-plate motion sickness modelFirst of all, mouse were given an effective dose of scopolamine or dealed with by chemical labyrinthectomy, and then the model-induced motion sickness, the final study results showed that mouse given scopolamine or with labyrinth damaged whose plasma AVP concentration decreased, hot plate latency as well as the motion sickness score was significantly lower than the control group. So scopolamine and chemical labyrithectomy in the model has the anti-motion sickness effects. Secondly, we combined hot-plate model with pica model in mice. Motion sickness were induced in rotation-hot-plate model, and measured by the consumption of kaolin in "pica" model. Rotation stimulated mouse showed significantly increased kaolin consumption, indicating the success of pica model, but also proving the feasibility of hot-plate model in reverse.2 Effects of estrogen on MS susceptibility in the mouse rotation-hot-plate modelWe designed several experimental approaches, aiming at the study of the effects of the levels of endogenous estrogen on mouse susceptibility of motion sickness. Mouse with surgical removal of ovaries, or given estrogen receptor antagonist tamoxifen (single dose) to artificially change the mouse endogenous estrogen levels, and by measuring plasma and pituitary AVP contents changes, and then were compared with the scopolamine and saline control group to evaluate the effects of estrogen on mouse susceptibility to motion sickness qualitatively and quantitatively.PartⅡ:the study of rats estrous cycle and estrogen levels in the "pica" model1 gender differences in rat "pica" modelSD rats were divided into four groups, the normal male and female groups, and castrated male and ovariectomized female groups. Plasma estrogen (E2) and progesterone (P) levels were determinated by ELISA, arginine vasopressin (AVP) levels were determinate by radioimmunoassay. The results showed that rotation-induced, normal female rats increased kaolin intake, meanwhile, plasma E2 and P levels were higher than normal male rats as well as plasma AVP. The ovariectomized female rats’ plasma E2 and P content, as well as AVP variations lower than the normal female group, the results suggest that the motion sickness susceptibility related with gender differences and the level of estrogen in rats.2 Influences of estrous cycle and estrogen levels on rats MSWe used surgical and chemical castration ways to change endogenous estrogen levels in the estrous cycle disturbances, observed changes in estrogen levels in the case of pica in rats to explore the relation between the estrogen and motion sickness susceptibility. The results showed that the ovariectomy or estrogen receptor antagonist tamxifen may lead to lower susceptibility to motion sickness, but there was no statistically significant between the rat estrous cycle fluctuations and their motion sickness susceptibility, but there is a trend that motion sickness is serious during estrus at the highest level of estrogen.Conclusion:The mice rotation-hot-plat model using hot-plate response latency as an indicator of motion sickness is a simple, rapid and feasible experimental model of motion sickness. In combination with the model and the rat "pica" motion sickness model, we know that estrogen receptor modulators tamoxifen, endogenous estrogen secreted from ovarian have link with the susceptibility to motion sickness in rats.更多还原