Evaluation Of The Expression Of Connexin26and Connexin30in Cochlea’s Pathology And Etiology Of Diabetic Deafness In Rats

Part Ⅰ：Reproduction of two types diabetic models in ratsBackground: Along with the development of our national economy,improvement of living quality, life style change and aging in society, theprevalence of diabetes has been climbing up gradually, endangering health ofthe human beings. As a genetic disease with relative or absolute insufficiencyof insulin, diabetes causes elevation of blood glucose level and abnormalmetabolism of protein and lipid. One of diabetic complications is impairmentof auditory function. More and more medical professionals and patients startto pay much attention to diabetic deafness because the disease affects qualityof life greatly. To further study how diabetes damages auditory function, astandard rat model of diabetic deafness has to be established by generallystandard of diabetic rats model. It is basis for the animal models were copiedand test glucose level, body weight, urine glucose to study how experiment.Methods: One-hundred and twenty Wistar rats were randomly dividedinto four groups: T1DM experimental group (n=40),control group (n=20)；T2DM experimental group (n=40),control group(n=20). The rats in theexperimental group were fed in two different ways to induce Type1or type2diabetes. When the models were established, tail blood was drawn atscheduled timepoints to test glucose level with a whole blood glucose meter,With regard to the generally standards of daibetes, and to test body weight andurine glucose. the random glucose level in type1, diabetic rats should behigher than16.65mmol/L in successful models. Whereas, the random fastingglucose level in type2diabetic rats should be higher than7.8mmol/L withevidence of insulin resistance in successful models. The established diabeticrats were evaluated once a month to check their conditions (including overall looking, activity, hairs, eating, urination and the reaction of ala auris auricle ofpinna), body weight, blood glucose level and glucoseuria.Results:1General state of health of experimental rats: In the diabeticgroups, rats developed polydypsia, polyuria, losing weight, yellowish hairswithout normal shining, lesargic appearance, slow body movement, decreasedsensitivity of ala auris auricle of pinna. Otoscopy revealed clean ear canalwithout secretion and clear membrane.2Changes in blood glucose of experimental rats:Type1diabetes: Therewas no significant change the random glucose level between control andexperimental group at baseline(P＞0.05). At1,2,3,4,5month afterestablishing the model, the difference between control and experimentalgroup was significant highter(P＜0.01).Type2diabetes: After feeding special forage8weeks, the glucose levelthe difference between experimental and control group was significanthighter(P＜0.05); After injecting STZ at1to5month it was significanthigher in the experimental group with control(P＜0.01).3Changes in urine glucose of experimental ratsIt was negative for urine glucose in the control groups but persistentlypositive(++or+++) in the diabetic groups, which is consistent with thestandard of diabetic animal model.4Changes in weight of experimental ratsType1diabetic：There was no significant change the body weightbetween control and experimental group at baseline(P＞0.05). At1st,2nd,3rd,4th and5th month, the average body weight of rats in diabetic group wassignificantly lower tha that in n the control group (P＜0.01).Type2diabetic：After feeding special forage for4weeks, the bodyweight the difference between experimental and control group was significanthighter(P＜0.01). So was it for8weeks. The average body weight of rats indiabetic group was little lower than that after injecting STZ at1month(P＜0.05). It was no lower with control group(P＞0.05). After2month, theaverage body weight of rats in diabetic group was significantly lower than that in the control group (P<0.01).Conclusions: Reproduced2types successful diabetic rat models is tofurther study how pathogenesis of diabetic hearing loss is essential forproviding basis for molecular mechanism of the disease in successful diabeticrat models of observing clinic and all of the body complaincation. PartⅡ： Observations on dynamic changes of auditorydysfunction in dibeatic rats and the expression ofCx26and Cx30in the cochleaBackground: There is no cure for diabetes at this moment; the only thingwe can do is to control its progression. For this purpose, the major sttrategy tointerfere with diabetes is to prevent its complications. As one of the majorcomplications, diabetic deafness is characteristized by bilateral and symmetricsensorineural hearing loss; it can be acoustic cochlear or retrocochlear. Theclinical manifestations are often decreasing of hearing at high frequencies withvertigo and insufficiency of vestibular function. Currently, the studies onmechanisms in diabetic deafness are involving neuropathy, microvasculopathy,changes of hemodynamics in blood, immune reaction and genetic alterations.The exact process in the etiology of diabetes-associated deafness is stillunclear, which brings tremendous difficulties to interference and treatment ofdiabetic deafness. For this reason, it is our long-term goal to better understandthe mechanism of diabetic deafness. At this point, most publications about theinner ear changes in diabetic deafness mainly point to the loss of hair cells,which deserves further investigations and discussions. The auditory functionimpairment in diabetes is likely to be caused by the pathogenesis of cochlearfunction and central auditory nervous systems. Here, we’ll study thismechanism at both global and micro levels. Connexins are important factors inthe pathways of signal transduction of cells, among which Cx26and Cx30 have been extensively studied. Gap junction forms the structural basis ofcell-to-cell communications; it’s also the pathway of electrolytes, secondmessengers and metabolites. The interaction between connexins in differentareas is crucial for the permission of gap junction formation. The structure ofconnexins is the basis of their functional expression. Gap junction in the innerear may also involve regulation of sensory epithelial cell function in additionto transporting nutrition, maintaining the stasis of the inner ear. Potassium isan essential electrolyte for auditory initiation in the inner ear endolymph fluid.Cx26and Cx30are pathways of potassium transport; they are essential forauditory initiation with participation of potassium.Normal mammaliancochlear cells express Cx26and Cx30to maintain their function. In the ratmodel of diabetic deafness, there has been no previous report about theexpression of Cx26and Cx30in cochlear cells or the relationship between theexpression and auditory deficiency. We investigated dynamic changes ofauditory function in the diabetic rat models (type1and2) with measurementof auditory brainstem response. Western blot was used in testing theexpression of Cx26and Cx30in the cochlear area of diabetic rats. UsingWestern blot method, the expression of Cx26and Cx30in the cochlear area ofthe diabetic rats were quantitatively studied. The results provided furtherinformation for getting diabetic hearing loss is essential for providing basis formolecular mechanism of the disease.Methods: All rats of control and2types of diabetes at1,2,3,4and5month underwent ABR examination with American ICS-CHARTR auditorybrainstem monitor. The threshold of ABR higher than40dBnHL or prolongedauditory brainstem latency interval and interpeak latency were defined asdiabetic deafness. The animals were put into ABR examination. In thesuccessful diabetic models, at2,3,4and5months, the rats were sacrificed,cochleas were harvested immediately for extraction of proteins. Western blotwas used to test the expression of Cx26and Cx30. To confirm the positivecorrelation between Connexins’ expression and auditory function in diabeticrats. Results:1In type1diabetic rats, The changes in ABR were mainlyin wave II latency, wave V latency and I-V, III-V interpeak latency. There wasno significant change between control and experimental group at baseline and1month after establishing the model (P>0.05). At2,3,4and5months, therewere signicicant differences of II, V wave latency and I-V, III-V interpeaklatency between control and experimental groups (P<0.01and P<0.05,respectively).2The major change of ABR in type2daibetic rats was III and V wavelatencies; I-III and I-V interpeak latencies; it had no change at baseline and at1month in the experimental group compared with control group (P>0.05). At2,3,4and5months, the difference of III and V wave latencies, I-III, I-Vinterpeak latency was significant between control and experimental group(P<0.01).3Image J software was used for analysis of scan results. In type2diabetic rats, the protein expression of Cx26and Cx30in the cochlea wasdifferent from the control group of the same offsprings. Compared with thenormal control group, the protein’s expression of Cx26and Cx30in cochleawas gradually decreased by time. The protein of Cx26decreased moresignificantly than Cx30.Conclusions:1In both rat diabetic models, auditory function decreased gradually at2month. Auditory function decreased gradually with different level of hearingimpairment. ARB could be used as an early diagnostic method.2Cx26and Cx30were essential connextins for auditory initiation basedon our experiment; In the diabetic rats, the protein’s expression of Cx26andCx30in cochlea was gradually decreased compared with control at the secondmonth, it worsened by time. The auditory dysfunction in diabetic deafness ispossibly related to the decreased expression of Cx26and Cx30. Part III：Preliminary study on the loss of Cx26and Cx30expression in hair cell damage in the inner ear ofrats with diabetic hearing lossBackground: Along with the increasing numbers of diabetic patients,there have been more reports about auditory dysfunction caused by diabetes.Auditory dysfunction in diabetes starts gradually and can be confused withaging-induced deafness. When the disease is diagnosed, it is often irreversible,which can significantly affect the quality of patients’ life. Therefore, it is inurgent need to further study the pathogenesis of diabetic deafness. Meanwhile,prevention and treatment of diabetes must be specifically addressed.In the rat model of diabetic deafness especially the type2model, after2months, the rats developed degeneration at basal membrane, stria vascularisand spiral ligament and auditory dysfunction. The auditory dysfunctiondeteriorated over time. Connexins play an important role in maintaining thenormal mechanic-electronic conversion to fulfill auditory transduction. Indiabetic deaf model, the cochlear connexins had significant change at2month.The expression of connexins decreased by time, it positively correlated withauditory dysfunction. To confirm the positive correlation between Connexins’expression and auditory function in diabetic rats, Immunochemistry was usedin testing the expression of Cx26and Cx30in the cochlear area of diabetic rats.Using immunochemistry method, the expression of Cx26and Cx30in thecochlear area of the diabetic rats were semi-quantitatively studied. The resultsprovided further information for etiology, diagnosis and treatment of diabeticdeafness. cochlear pavement slides were also prepared to observe therelationship between hair cell loss and auditory dysfunction. Getting furtherinsight into expression of Cx26and Cx30in the cochlear pathogenesis ofdiabetic hearing loss is essential for providing basis for molecular mechanismof the disease.Methods: The animals were put into ABR examination and thensacrificed by decapitation to prepare cochlea samples, to be fixed, decalcified, paraffin imbedded and sliced for immunohistochemical study. Intact paraffinslides were selected for HE and immunochemical staining. A laser confocalmicroscope was used to observe the structure of cochlea and the expression ofCx26and Cx30. namely the loss of hair cells. Cochlear pavement slides werealso prepared to observe the relationship between hair cell loss and auditorydysfunction.Results:1In2types diabetic rat model, Compared with the control group,the amount of lumens in the stria vascularis were less in the test group. In thediabetic groups the tubel were less than control groups. The amount of spiralganglion cells and the cells of spiral ligament were. less in test group（P<0.05）.2In2types diabetic rat model, the florescent expression of Cx26and CX30in cochlear basal membrane, stria vascularis and spiral ligament was notsignificantly different between control and experimental groups (P>0.05) at1month. At2,3,4and5month, in the experimental group. the florescentexpression of Cx26and CX30in cochlear basal membrane, stria vascularisand spiral ligament was significantly decreased than in the controlgroup(P<0.05).3In2types diabetic rat model, at the first month, there was no damageon the inner and external hair cells as observed on the cochlear slides in bothexperimental and control groups. This result is consistent with the proteinexpression of Cx26and Cx30in the cochlea and the ABR auditory testingresults.4At the second month, the inner and external hair cells on cochlearslides in the2types of diabetic rats were disoriented but not disappeared. Atthe third month, the hair cells were more disoriented without prominent loss.At the fourth and fifth month, disorientation of hair cells were not only seen,but disapperance of some hair cells was noted as well. These changes are allconsistent with the protein’s expression of Cx26and Cx30in the cochlea andthe ABR auditory testing results. Conclusions:1Cx26and Cx30are connexins which are essential auditory initiation.It Is possibly related to hearing loss with erpression of Cx26and Cx30.2In the2types of diabetic rats with hearing loss, their florescentexpression in cochlear basal membrane, stria vascularis and spiral ligamentwas significantly decreased in the diabetic rats at2,3,4and5month.Theseresults suggest changes in Cx26and Cx30expression are possibly related withauditory dysfunction in diabetic rats.3The damage to the inner and external cochlear hair cells were observed,which was deteriorated overtime. The severity of damage was consistent withthe protein’s expression and the ABR auditory testing results. whichcontributes to the inner ear damage, especially to the hair cell in the cochlea.