【作者】 李元涛；

【导师】 姚尚龙；

【作者基本信息】 华中科技大学 ， 麻醉学， 2007， 博士

【摘要】 对于全身麻醉来说,麻醉药物作用部位是从脊髓到大脑皮层这一段广阔的区域,包括传入神经、传出神经、神经中枢的多种神经元。研究表明全麻药物对外周神经系统中外周伤害性感受器、神经轴传导几乎没有任何作用,但现已证实对神经突触有明显作用,如多数吸入全麻药可对兴奋性突触传递产生抑制,而抑制性突触传递产生增强作用。听觉的传导路径比较复杂,声波传入内耳,刺激耳蜗螺旋器,使耳蜗毛细胞兴奋,并以神经冲动形式经听神经传向大脑皮层听觉中枢(auditory center),产生听觉。听觉是全身麻醉中最后消失的一个感觉,也是清醒时恢复的第一感觉;视觉和本体感觉等很容易被全麻药物所抑制,而听觉在麻醉中并不是突然消失的,随着麻醉程度的加深逐渐被抑制。在全身麻醉中听觉系统除了听皮层、听性脑干受到抑制外,全麻药物对外周感受器耳蜗的作用至今仍不清楚。目前关于全麻后听力损害的报道越来越多,发生机制不清楚。那么全麻药物是否与耳毒性药物(如链霉素、顺铂、水杨酸钠等)一样,可以使耳蜗基因表达发生变化,进而形态学发生病理学改变导致耳损害?基于以上两点原因,本研究通过观察异氟醚和氧化亚氮对大鼠耳蜗总RNA的影响、异氟醚对大鼠耳蜗一氧化氮合酶活性的影响,以及对异氟醚豚鼠耳蜗外毛细胞内钙离子浓度变化的影响,初步了解吸入麻醉药对耳蜗的作用机制。本课题共分三部分:第一部分异氟醚和氧化亚氮对大鼠耳蜗总RNA提取和含量的影响目的通过检测吸入麻醉药异氟醚(Isoflurane)、氧化亚氮(N2O)对大鼠耳蜗总RNA含量的影响,探讨异氟醚、氧化亚氮对大鼠内耳有无损害作用以及耳损害的可能机制。方法30只健康Wistar大鼠随机等分为三组:C组(对照组,n=10)持续吸入50%氧气(O2) 3小时;N组(实验组,n=10)持续吸入50% N2O+50% O2 3小时;I组(实验组,n=10)持续吸入2.5%异氟醚3小时。然后将所有大鼠断头处死,取出双侧听泡,剥离耳蜗,各组标本编号记录后分开液氮低温保存。联用TRIzol和RNeasy法分别抽取三组大鼠耳蜗的总RNA,用分光光度法测总RNA的含量及电泳检测其质量。结果检测C组大鼠耳蜗得到总RNA含量7.69μg;N组大鼠耳蜗得到总RNA含量6.51μg,与C组相比减少15%;I组大鼠耳蜗得到总RNA含量7.32μg,与C组相比几乎没变化。C组、N组和I组的A260/A280值分别为2.07、2.04和2.04,提示RNA纯度高;电泳结果提示总RNA无降解。结论吸入氧化亚氮的大鼠耳蜗总RNA较正常大鼠耳蜗总RNA含量降低,而吸入异氟醚的大鼠耳蜗总RNA含量无明显变化。提示氧化亚氮干扰耳蜗RNA含量,使大鼠耳蜗基因表达发生变化可能是造成耳损害的原因之一,而异氟醚对耳蜗RNA含量没有明显影响。第二部分异氟醚麻醉对大鼠耳蜗一氧化氮合酶活性的影响目的通过观察不同浓度的吸入麻醉药异氟醚(Isoflurane)对大鼠耳蜗Corti器、血管纹、螺旋神经节内各型NOS(nitric oxide synthase)活性的影响,探讨吸入麻醉药对大鼠听觉系统外周感受器的可能作用机制。方法30只健康Wistar大鼠随机等分为三组:C组(对照组,n=10)持续吸入O2 30分钟;I1组(实验组,n=10)持续吸入1.5%异氟醚+O2 30分钟;I2组(实验组,n=10)持续吸入3.0%异氟醚+O2 30分钟。待各组动物实验时间完成后,立即断头处死,迅速取出听泡。充分暴露耳蜗后,在解剖显微镜下刺破蜗窗及前庭窗,蜗尖钻孔并缓慢注入含4%多聚甲醛固定,脱钙后标本制作石蜡切片。HE染色观察各组大鼠耳蜗组织学改变;用免疫组织化学方法检测各组大鼠耳蜗Corti器、血管纹、螺旋神经节内诱生型NOS(iNOS)、内皮型NOS(eNOS)、神经元型NOS(nNOS)的表达,采用Motic image advance图像分析仪,分别测量各组大鼠耳蜗组织不同部位NOS平均灰度值;灰度值越小,表示阳性反应越强。以观察不同浓度异氟醚麻醉对大鼠耳蜗NOS活性的影响。结果HE染色观察各组大鼠耳蜗Corti器毛细胞无损伤,血管纹清晰,螺旋神经节结构正常。C组大鼠耳蜗Corti器、血管纹、螺旋神经节内各型NOS均有强弱不等的阳性表达;与C组比较,I1组大鼠耳蜗Corti器、血管纹、螺旋神经节内各型NOS阳性表达均有减弱、各型NOS平均灰度值均有升高趋势,除血管纹iNOS外其余均有显著性差异(P<0.05);与C组比较,I2组大鼠耳蜗Corti器、血管纹、螺旋神经节内各型NOS阳性表达进一步减弱、各型NOS平均灰度值升高明显(P<0.05),其中以螺旋神经节各型NOS平均灰度值升高显著(P<0.01);与I1组比较,I2组大鼠耳蜗Corti器、血管纹、螺旋神经节内各型NOS阳性表达减弱、各型NOS平均灰度值升高趋势,但除螺旋神经节中eNOS、nNOS外其余均没有显著性差异(P>0.05)。结论吸入异氟醚麻醉大鼠耳蜗NOS活性较正常大鼠耳蜗降低,且耳蜗NOS活性降低程度与吸入异氟醚浓度呈正相关。推测异氟醚可抑止耳蜗Corti器、血管纹、螺旋神经节NOS活性,减少NO(nitric oxide)产生,从而抑止听觉外周感受器功能。第三部分异氟醚对豚鼠耳蜗外毛细胞内钙离子浓度变化的影响目的观察不同浓度异氟醚对氯化钾和咖啡因诱发的豚鼠耳蜗外毛细胞内钙离子移动的影响,探讨其对听觉外周感受器(耳蜗)作用的可能机制。方法采用酶孵育后机械分离法急性分离豚鼠耳蜗外毛细胞,在倒置相差显微镜下观察外毛细胞的形态,辨认其活性。用Fluo-3AM荧光指示剂染色活性良好的豚鼠耳蜗外毛细胞,在激光共聚焦显微镜下动态观察使用异氟醚预处理前及不同浓度异氟醚即1.0最低肺泡有效浓度(minimal alveolar concentration,MAC)、2.0MAC预处理后,用氯化钾和咖啡因诱发的耳蜗外毛细胞内钙荧光强度的变化,记录5min荧光强度峰值;并用激光共聚焦图形分析软件Profile功能测各细胞内的荧光强度进行分析,从而了解异氟醚预处理前、后对氯化钾和咖啡因诱发的耳蜗外毛细胞内钙离子浓度的变化的影响。结果异氟醚可使氯化钾诱发的外毛细胞内钙荧光染色强度的峰值下降,较对照组有明显差异,并且降低程度与异氟醚的浓度呈正相关。但该两种浓度的异氟醚对咖啡因诱发的外毛细胞内钙荧光染色强度升高无明显影响。结论异氟醚浓度依赖性地降低耳蜗外毛细胞内钙离子浓度,可能部分与抑止细胞外钙内流有关,而对肌浆网内钙离子移动无明显影响。更多还原

【Abstract】 With regard to general anesthesia, the nature site of general anesthetic action is extensive district from spinal cord to cerebral cortex, such as afferent nerve, centrifugal nerve, multitude neuron of nerve center. As the research indicated: general anesthesia medicine has no effect on peripheral nociceptor and conduction of neural axis, but has an obvious effect on neurapophysis. It was confirmed that most inhalation anesthesia may inhibit excitatory synaptic transmission, and enhance inhibitory synapse transmission. The pathway of auditory conducting is complex: the sound wave imports auris interna, excites cochlea acoustic organ , stimulates cochlear hair cell, imputs cerebral cortex auditory center through acoustic nerve by nerve impulse, produce sense of hearing. Sense of hearing is the last obsolescent aesthema in general anesthesia, and the first recuperative aesthema in conscious. general anesthesia The sense of sight and proprioceptive were easy to be inhibited by general anesthesia drugs. The sense of hearing was not disappeared abruptly in anaesthesia, but inhibited gradually with the degree of anesthesia. How did the general anesthesia produce a marked effect on peripheric receptor cochlea was still unkown except that the auditory cortex and auditus brain stem in auditory system are held during general anesthesiaMore and more reports about hearing impairment after general anesthesia were reported recently, however the mechanism was not clear . Supposed that It may change gene expression of cochlea, and contribute to pathology change in morphology and damage of ear such as the ototoxicity medicine phytomycin, C-DDP, sodium salicylate .So, We can understand the possible mechanism of inhalation anesthetic effect to Cochlea by observing that Isoflurane and N2O anesthesia on yield of total RNA in Cochleas of the rats, Isoflurane anesthesia on activities of nitric oxide synthase in Rat Cochlea, and Isoflurane on Ca2+ mobilization in isolated outer hair cells in guinea pigThe present research is composed of three sections.The first Section: Isoflurane and N2O anesthesia on Yield of Total RNA in ochleas of the RatsObjective To extract high quality total RNA from cochleas of normal rats or the rats induced anesthesia by N2O and Isoflurane, and determine their RNA yield,in order to explore the reason of hearing impairment by inhalation anesthetic. Methods 30 Wistar Rats were randomly divided into 3 groups as follows:C group (control group,n=10) rats were endlessly inhaled 50% O2 for 3 hours;N group (test group,n=10) rats were endlessly inhaled 50% N2O+50% for 3 hours; I group (test group,n=10) rats were endlessly inhaled 2.5% Isoflurane for 3 hours. Then the cochlear materials from 3 groups of rats were pooled and homogenized ,total RNA was extracted from the homogenized tissues combination TRIzol method with RNeasy method respectively. Finally spectrophotometric analysis was used to determine the yield and purity of totalRNA and gel electrophoresis was used to test whether there existed RNA degeneration. Results Total RNA extracted from C group rats was 7.69μg; Total RNA extracted from N group rats was 6.51μg,it was reduced 15% than C group; Total RNA extracted from I group rats was 7.32μg,it was unchanged than C group. The results of spectrophotometric analysis showed the values of A260/A280 were 2.07、2.04 and 2.04. gel electrophoresis showed no degeneration sign. Conclusion N2O could significantly inhibit the Yield of Total RNA in Cochleas of the Rats and Isoflurane could not,The change of Total RNA during anesthesia induced by N2O suggests it play an important role for N2O -induced hearing impairment, Isoflurane could not damage hearing.The second section:Anesthesia of Isoflurane on activities of nitric oxide synthase in Rat CochleaObjective To investigate the possible mechanism of action of inhalation anesthetic Isoflurane on peripheric receptor of auditory system by observing its effection on the activity of NOS in the cochlea of rat. Methods Thirty healthy Wistar rats were divided into three groups randomly: C group (control group n=10) inhaling oxygen for 30 minututes continuly ; I1 group (experimental group,n=10) inhaling 1.5% Isoflurane and oxygen ; I2 group (experimental group,n=10) inhaling 3% Isoflurane and oxygen.,all for 30 minuts . The histologic changes in the cochlea of the rat were observed by HE staining; The expression of iNOS, eNOS, nNOS in the cochlea of the rats were detected by immunocytochemical staining and microphotography methods; In order to observe the effect of different concentration Isoflurane on NOS in the cochlea of rat, the gray scales of NOS in different part of the cochlea were measured by Motic image advance picture ananlysis respectively. Results There were no lesions of hail cells and stria vascularis in each group by HE staining, There were positive expression of all kinds of NOS in the organ of Corti in cochlea,the stria vascularis and the spiral ganglion ; Comparision with the control group , the expression of all kinds of NOS decreased but the everage of gray scales increased . There were significiant differences except the iNOS of stria vascularis in I1 group(P<0.05). Comparision with the control group , the positive expression of all kinds of NOS decreased either, but the everage of gray scales increased obviously(P<0.05),the everage of gray scales in spiral ganglion increased significantly in I2 group (P<0.01); Comparision with I1 group, the expression of all kinds of NOS decreased,the mean gray scales increased. However, there were no significiant differences except the eNOS、nNOS of the spiral ganglion(P>0.05). Conclusions the activity of NOS decrease in the cochlea of inhalation anesthetic Isoflurane rats comparing with the normol rats, we infer that Isoflurane can depress the activity of NOS in the cochlea and reduce the NO lever, accordingly it can depress the function of peripherial receptor of auditory system.The third section:The effects of Isoflurane on Ca2+ mobilization in isolated outer hair cells in guinea pigObjective To elucidate the effect of Isoflurane on cochleas by measuring intracellular Ca2+ contraction in isolated outer hair cells in guinea pig. Methods outer hair cells in guinea pig were acutely isolated ,then were load with Fluo-3AM,a new Ca2+ indicator.The effects of Isoflurane (1MAC、2MAC)on changes of intracellular Ca2+ fluorescent intensity induced by KCL or caffeine were investigated. Results Isoflurane caused dose–dependent inhibit Ca2+ transsarcolemmal influx by KCL,peak Ca2+ fluorescent intensity decreased significantly.But Isoflurane didn,t alter the amount of Ca2+ release from intracellular stores in response to caffeine. Conclusion These results that Isoflurane dose-dependent decreased intracellular Ca2+ contraction in isolated outer hair cells in guinea pig,at least in part,may be mediated by a decrease of Ca2+ transsarcolemmal influx through calcium channel.But it is not relate to sarcoplasmic reticulum Ca2+ release.更多还原