【作者】 薛丽；

【导师】 周红；

【作者基本信息】 第三军医大学 ， 药理学， 2006， 硕士

【摘要】 现实生活中,长时连续作业情况较为常见,例如长途司机或飞行员连续驾驶、军事作业等。这些超昼夜周期的工作以及强制性无睡眠或少睡眠的工作对操作人员的行为能力和认知功能造成了严重影响,大大降低了工作效率。因此,如何提高长时连续作业人员的工作效率早已是当今职业医学亟待解决的重要课题。我室以传统的小动物跑台装置为基础,并结合自行设置48小时连续作业模式,以此建立了长时连续作业模型。该模型成功地模拟了长时连续作业过程中的多种影响因素,表明长时连续作业可明显地损害大鼠的觉醒能力和认知功能。在此基础上,我们研究了冰片对觉醒能力损害的干预作用,结果表明冰片可在一定程度上逆转长时连续作业对大鼠觉醒能力和认知功能的损害作用,但冰片作用的神经机制并没有得到阐明。本实验首先观察了冰片对大鼠前额叶皮层兴奋性的影响,并阐明了其神经机制。进而研究了长时连续作业外侧下丘脑区orexin-A、c-Fos表达及冰片的干预作用,从而更进一步认识了冰片作用的神经机制。结果如下:一、冰片对大鼠前额叶皮层电活动兴奋性的影响及其机制（一）冰片对大鼠前额叶皮层（PFC）的电兴奋作用通过PFC神经元单位放电频率改变判断皮层兴奋性高低。研究显示,大鼠PFC神经元自发性放电具有单个不均型、束簇状及混合型三种放电模式,放电活动以混合型为主。单位放电频率（5min平均值）为7.45±1.67Hz（n=48）,放电时程为3～3.5ms。经留置胃管分别注入中、高剂量冰片（1.2g/5ml/kg和2.0/5ml/kg）后,单位放电频率逐渐增高,25～35min时最为明显（n=12,P＜0.05;n=12,P＜0.01）,高剂量冰片增高程度高于中剂量冰片（n=12,P＜0.01）,而低剂量冰片（0.4g/5ml/kg）则无明显改变（n=12,P＞0.05）。结果表明,冰片对大鼠PFC神经元单位放电具有兴奋效应,且存在剂量-效应依赖关系。提示冰片对长时连续作业大鼠受损觉醒能力的改善作用可能与其提高PFC神经元兴奋性有关。（二）冰片对大鼠前额叶皮层电兴奋作用的神经机制1.组胺H₁受体拮抗剂吡拉明（pyrilamine）对冰片兴奋前额叶皮层神经元电活动的抑制效应侧脑室给予pyrilamine 60μg后,与给药前相比,给药后神经元单位放电频率逐渐减慢（n=12,P＜0.05）。随后实验中,pyrilamine 60μg icv微注入预处理并经胃管注入高剂量冰片后,给药前后神经元单位放电频率无显著变化（n=12,P＞0.05）。结果表明,高剂量冰片对PFC神经元放电频率的增加作用被pyrilamine阻断,提示组胺H₁受体途径参与了冰片的皮层兴奋效应。2.去甲肾上腺素α₁受体拮抗剂哌唑嗪（prazosin）对冰片兴奋大鼠前额叶皮层神经元电活动的轻度抑制效应Prazosin 63μg icv微注入后,PFC神经元单位放电频率前后无明显变化（n=12,P＞0.05）。Prazosin 63μg icv微注入预处理并经胃管注入高剂量冰片后,给药后放电频率仍可明显增加（n=12,P＜0.05）,但增加程度要低于高剂量冰片单独兴奋效应,二者有显著差异（n=12,P＜0.05）。结果表明,高剂量冰片对PFC神经元放电频率的增加作用可被prazosin部分阻断,提示去甲肾上腺素α₁受体途径一定程度上参与了冰片的皮层兴奋效应。3.5-羟色胺（5-HT₂）受体拮抗剂利坦色林（ritanserin）对冰片兴奋前额叶皮层神经元电活动的轻度抑制效应Ritanserin 40μg icv微注入后,与给药前相比,给药后神经元单位放电频率减慢（n=12,P＜0.05）。随后实验中,侧脑室给予40μg ritanserin微注入预处理并经胃管注入高剂量冰片后,给药后神经元放电频率仍可明显增加（n=12,P＜0.05）,但增加程度要低于高剂量冰片单独兴奋效应,二者有显著差异（n=12,P＜0.05）。结果表明,高剂量冰片对PFC神经元放电频率的增加作用可被ritanserin部分阻断,提示5-羟色胺受体途径（5-HT₂）一定程度上参与了冰片的皮层兴奋效应。二、冰片对长时连续作业大鼠前额叶皮层神经递质水平的影响（一）长时连续作业大鼠前额叶皮层神经递质水平的变化本实验中,利用高效液相色谱-电化学方法测定了长时连续作业后前额叶皮层去甲肾上腺素（NA）、多巴胺（DA）及5-羟色胺（5-HT）神经递质含量的变化。结果显示,跑台石蜡组去甲肾上腺素前额叶皮层脑区的含量均较低（P＜0.05）,而多巴胺及5-羟色胺则无明显下降（P＞0.05）。结果提示,长时连续作业后,动物的觉醒能力和认知功能的下降可能与前额叶皮层去甲肾上腺素水平的下降有关。（二）冰片对长时连续作业大鼠前额叶皮层神经递质水平的影响大鼠连续48h作业结束后,经自制胃管灌服冰片石蜡油或石蜡油溶液,2小时后测定前额叶皮层上述神经递质含量。较跑台石蜡油组比,跑台冰片组NA及5-HT含量明显升高（n=5,P＜0.05）,而DA则无明显变化（n=5,P＞0.05）。结果表明,冰片对长时连续作业大鼠受损觉醒能力的改善作用可能与前额叶皮层NA及5-HT水平增加有关。三、冰片对长时连续作业大鼠外侧下丘脑区orexin-A及c-Fos表达的影响（一）长时连续作业大鼠外侧下丘脑区c-Fos及orexin-A表达的变化采用免疫组化染色的方法对跑台石蜡及未跑台石蜡组大鼠进行了研究。结果显示:跑台组大鼠外侧下丘脑区c-Fos和orexin-A表达均减少（n=4,P＜0.05）。结果提示长时连续作业后,大鼠觉醒能力和认知功能的下降可能与外侧下丘脑区c-Fos和orexin-A表达减少有关。（二）冰片对长时连续作业大鼠外侧下丘脑区c-Fos及orexin-A表达的影响研究了长时连续作业大鼠冰片干预后外侧下丘脑区c-Fos及orexin-A的表达。结果显示跑台冰片组大鼠外侧下丘脑区c-Fos及orexin-A表达均高于跑台石蜡组（n=4,P＜0.05）。未跑台冰片组大鼠c-Fos表达明显高于未跑台石蜡油组（n=4,P＜0.01）,而orexin-A则与未跑台石蜡油组无明显差异（n=4,P＞0.05）。结果提示,冰片对长时连续作业大鼠受损觉醒能力的改善作用可能与冰片引起的外侧下丘脑区c-Fos及orexin-A表达增高有关。综上所述,本研究结果表明,第一,冰片对大鼠前额叶皮层具有电兴奋作用,兴奋效应与皮层下组胺系统直接相关,而去甲肾上腺素及5-羟色胺系统则部分相关。第二,冰片可增加长时连续作业大鼠前额叶皮层去甲肾上腺素及5-羟色胺水平,这可能参与了冰片的干预作用。第三,冰片可使长时连续作业大鼠外侧下丘脑区c-Fos及orexin-A表达增高,这可能与冰片对受损觉醒能力的改善作用直接相关。更多还原

【Abstract】 Long-term continous operations are becoming more popular in the modern society, including the long-distance drives,the continual military operations and so on.It is well known that these ultra dilirnal periods work as well as compulsory work without enough sleep have serious influences on operator’s capacity and cognitive function,which usually reduces the working efficiency.How to improve the long-term continuous operation personnel’s working efficiency is an important issue which shoule be urgently solved in the modern medical research field.The research has already been carried out in our laboratory. Rat model was established by using a treadmill protocol.Rats were allowed to do continous exercise for 48 h in the conventional treadmill equipment used for small animal.It is suggested that rats’ awake activity and cognitive function could be apparently impaired by stimulating many kinds of complicated environment in long-term continuos operations.Our research indicated that Borneol could reverse the deleterious effects of long-term continuous operations on awake activity,but the neuronal mechanisms of Borneol are not illustrated.In the present study,effects of Borneol on single-unit activity in the prefrontal cortex and its neuronal mechanisms are firstly studied.Subsequently,to investigate its neuronal mechanisms,we examined the expression of orexin-A and c-Fos in the lateral hypothalamic area of rat after long-term contious operations and Borneol applied.1.Effects of Borneol on single-unit activity in prefrontal cortex of rats and its mechanisms（1） Excitory effects of Borneol on PFC of ratsThe excitability of cortex was judged by the frequency of neuron discharge in the prefrontal cortex.Three types of firing patterns were exhibited in neuron:single and irregular discharges,cluster discharges and complex discharges,with a mean frequency of 7.45±1.67 Hz（n=48） and duration of 3～3.5 ms.The mean frequency of unit discharges was gradually increased after application of the middle and high dosages of Borneol（1.2g/5ml/k and 2.0g/5ml/kg） by the placed gastric tube,which reached to a peak level at 20 to 25 minutes（n=12,P＜0.05;n=12,P＜0.01 ）.The degree of increase induced by the high dosage of Borneol was more sigficicant than that of middle（n=12,P＜0.01）.While no obvious difference was observed after application of Borneol at low dose.These data show the excitatory effects of Borneol on single-unit activity with a dose-dependent manner.The improvement of Borneol on the impaired awake activity of rats is probably related to the increased single-unit activity in the prefrontal cortex.（2） Neuronal mechanisms of excitatory effects of Borneol on prefrontal cortex①H₁ histamine antagonist pyrilamine prevented the excitatory effects of Borneol on unit discharges of prefrontal cortex neuronsThe mean frequency of unit discharge of prefrontal cortex was gradually decreased after i.c.v,application of 60μg H₁ histamine antagonist pyrilamine（n=12,P＜0.05 ）.And in the presence of pretreatment 60μg pyrilamine,the high dosage of Borneol failed to exert an excitatory action on unit discharges of cortical neurons（n=12,P＞0.05 ）.These results indicate that the excitatory effects of Borneol on the neurons of prefrontal cortex may be mediated by activation of histaminergie neurotransmission.②α1-adrenoreceptor antagonist prazosin had a few contributions on the effects of Borneol on unit discharges of prefrontal neuronsThe mean frequency of unit discharge of prefrontal cortex showed no evident difference after i.c.v,application ofα1-adrenoreceptor antagonist prazosin at the dose of 63μg（n=12,P＞0.05 ）.And in the presence of pretreatment 63μg prazosin,application of the high dosage of Borneol was administered by the placed gastric tube and increase the mean frequency（n=12,P＜0.05 ）,but it is still lower than that only application of the high dosage of Borneol significantly（n=12,P＜0.05）.Therefore,these results suggest that Borneol induced-discharge activities may be partly blocked by prazosin at the dose of 63μg, and these excitatory effects appear to be partly associated with noradrenaline neurotransmission.③5-serotonin selective receptor antagonist（5-HT₂） ritanserin showed little influence on the effects of Borneol on unit discharges of prefrontal cortex neuronsThe mean frequency of unit discharge of prefrontal cortex was gradually decreased after i.c.v,application of 5-HT₂ antagonist ritanserin at the dose of 40μg（n=12,P＜0.05）. And in the presence of pretreatment 40μg ritanserin,application of the high dosage of Borneol was administered and increased the mean frequency（n=12,P＜0.05）,but it is still lower than that only application of the high dosage of Borneol significantly（n=12,P＜0.05）.Therefore,these results suggest that Borneol induced-discharge activities may be partly blocked by ritanserin at the dose of 40μg,and these excitatory effects appear to be partly associated with serotonin neurotransmission.2.Effects of Borneol on levels of nerurotransmitters in prefrontal cortex（PFC） after long-term continous operations（1） Effects of long-term continous operations on nerurotransmittors in PFCIn the present study,the levels of NA,DA and 5-HT in prefrontal cortex of rats were determined after long-term continous operations by high performance liquid chromatography combined with electrochemical detection（HPLC-ECD）.Our results indicated that the levels of NA decreased significantly as compared to control group （P＜0.05）,but the level of DA and 5-HT did not change（P＞0.05）.Therefore,these data imply that the depressed awake activity and cognitive function induced by long-term continous operations appear to be associated with noradrenergic modulation.（2） Effects of Borneol on levels of neurotransmitters in prefrontal cortex（PFC） after long-term continous operationsBorneol was applied to the operating rats after long-term continous operations by gastric tube,and the control animals were treated with vehicle.The levels of these neurotramsmitters in PFC were determined by HPLC-ECD.To the operational group,the levels of noradrenaline and 5-serotonin in PFC increased significantly after application of borneol（n=5,P＜0.05）,but dopamine not.The results indicate that the improvements of borneol are associated with the increase in noradrenaline and 5-serotonin levels in PFC.3.Effects of Borneol on expression of orexin-A and c-Fos in LHA in rats after long-term continuous operations（1） Using a rat model of long-term continuous oPerations to investigate the effects of long-term continuous operations on orexin-A and c-Fos in LHA.We found the number of orexin-A immunoreactive neurons and c-Fos in LHA significantly decreased as compared with the control group.The results indicate that the depressesd awake activity and cognitive function appear to be associated with the decreased expression of orexin-A and c-Fos.（2） Borneol was administered by the placed gastric tube.We observed the effects of Borneol on the expression of orexin-A and c-Fos in the long-term continuous rat model..We found the number of orexin-A immunoreactive neurons and c-Fos in LHA significantly increased compared with the control group.The results indicate that the improvement of borneol are associated with the increased expression of orexin-A and c-Fos.In summary,the improvement of borneol on impaired awake activity and cognitive function is probably associated with its excitatory effects on PFC,as well as the increased level of noradrenaline and 5-serotonin and increased expression of orexin-A and c-Fos in LHA.更多还原