Preliminary Studies Of The Effect Of Isoflurane On Rat Cochlea And Isolated Guinea Pig Cochlear Outer Hair Cells

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.