The Role Of Apoptosis And Bax, Bcl-2, Caspase-3 In Cochlear Injury Of Spontaneous Diabetic Mice And The Protective Effect Of Edaravone

PartⅠ.Morphological and Apoptotic Changes of Cochlear Tissues in Spontaneous Diabetes MiceObjective:To investigate changes in pathological morphology and apoptosis of the early diabetic cochlea,through studying the mouse models of spontaneous diabetes.Materials and Methods:Non-obese diabetic(NOD)female mice were assigned into the 4-week-diabetic group(group D4),12-week-diabetic group(group D12) and normal control group(group C).Toluidine blue staining of the cochlear semi-thin sections was used to observe the morphological changes in cochlear tissues,the scanning electron microscope was employed to observe hair cell loss and the TUNEL assay of cochlear semi-thin sections and fluorescent staining of cochlear hair cell nuclei was applied to observe apoptosis of cochlear cells.Results:1.Toluidine blue staining of cochlear semi-thin sections:the morphology of the cochlear Cortis organ,spiral ganglion,stria vascularis and spiral ligament were normal in group C;in group D4,the Cortis organ shrank,inner and outer hair cell nuclei were occasionally lost but the structure of these cells remained intact,spiral ganglion cell nuclei swelled and decreased in number,nerve fibers were arranged in disorder, stria vascularis revealed edematoid changes and micro-thrombus developed;in group D12,the Cortis organ collapsed,inner and outer hair cells and support cells were lost to some extent,the number of spiral ganglion cells further decreased,nerve fiber bundles loosened,stria vascularis shrank and microaneurysms developed.2.Scanning electron microscopy of the cochlea:the morphology of inner and outer hair cell stereocilia of the cochlea in group C was normal; in group D4,hair cell stereocilia in the lower apical turn lodged and fractured and inner hair cell stereocilia revealed sparing loss,and inner and outer hair cell stereocilia in the upper apical turn displayed no obvious sparing loss;the inner and outer hair cell stereocilia in group D12 revealed obvious sparing loss.3.TUNEL detection of cochlear semi-thin sections:A few apoptotic cells could be observed in the stria vascularis and spiral ganglion in group C and no apoptotic cells could be observed in the spiral ligament and Cortis organ.A few apoptotic cells could be observed in the cochlear Cortis organ,spiral ganglion,stria vascularis,spiral ligament and support cells in NOD mice of group D4.An abundance of apoptotic cells could be observed in structures above the cochlea in NOD mice of group D12, obviously more in group D12 than those in group D4(P＜0.05).At the same time the lower apical turn apoptotic cells in the two diabetic groups were obviously increased than the upper apical turn apoptotic cells (P＜0.05).4.AO-PI fluorescent staining:No obvious apoptotic cells were observed in the upper and lower apical turn of group C.Orange yellow fluorescent apoptotic cells could be observed in the second and third line outer hair cells of the upper apical turn in group D4 and green fluorescent normal cells were observed in the first line outer and inner hair cells;the apoptotic rate was 22.33±1.72%.An abundance of apoptotic cells were observed in lower apical turn inner and outer hair cells and sparing normal hair cells were observed;the apoptotic rate was 26.53±1.96%. More apoptotic cells were observed in the upper and lower apical turn inner and outer hair cells group D12;the apoptotic rates of the upper and lower apical turn were 58.32±1.67%and 84.32±1.77%,respectively. The apoptotic number of cochlear hair cells in group D4 and D12 was significantly higher than that in group C(P＜0.05)and it was more obvious in the lower apical turn than that in the upper apical turn (P＜0.05);the number of apoptotic cells in group D12 was obviously higher than that in group D4 and sparing cell loss was observed(P＜0.05).Conclusion:1.NOD diabetic mice have evident changes in pathological morphology of structures like the Cortis organ,spiral ganglion,stria vascularis and spiral ligament at an early stage.2.The cochlaer tissue injury in animals with spontaneous diabetes gradually worsens from the upper apical turn to the lower apical turn, heavier in inner hair cells than in outer hair cells and becoming heavier with the extension of the diabetic course.3.Apoptosis is a mode of great importance for diabetic cochlear cell injury or death. PartⅡ.The Role of Bax,Bcl-2 and Caspase-3 in Regulating Cochlear Injury of Spontaneous Diabetic MiceObjective:To explore the molecular mechanism of Bax,Bcl-2 and Caspase-3 in cochlear injury of spontaneous diabetes through understanding the spatial distribution characteristics and temporal law of the expression of Bax, Bcl-2 and Caspase-3 proteins in cochlear injury of mice with spontaneous diabetes,providing theoretical evidence for medication and prevention of diabetic cochlear injury.Materials and Methods:Non-obese diabetic(NOD)female mice were allocated into the 4-week-diabetic group(group D4),12-week-diabetic group(group D12) and normal control group(group NC).The immunohistochemical assay and Western blot assay were used to detect the expression of Bcl-2,Bax and Caspase-3 in diabetic cochlear tissues.Results:1.Immunohistochemical detection of Bax,Bcl-2and Caspase-3:The expression of Bax and Caspase-3 in the upper and lower apical turn of Cortis organ,spiral ganglion,stria vascularis and spiral ligament of cochleas in group D4 were positive in cytoplasms,and the expression of Bcl-2 was positive in both nuclei and cytoplasms.Bax and Caspase-3 of the above sites expressed in nuclei and cytoplasms of group D12 positively,especially obviously in the spiral ganglion and spiral ligament, and Bcl-2 expressed only in cytoplasms positively.Bax and Caspase-3 in upper and lower apical turn in cochleas of group NC were negatively expressed and Bcl-2 was positively expressed in cytoplasms.2.Western blot detection of Bax,Bcl-2 and Caspase-3 protein expressions:color bands of Bax,Bcl-2 and Caspase-3 could be observed orderly in molecular weight fractions of-24 kDa,-28 kDa and 35 kDa.In group NC,the optical density(OD)ratios of Bax/β-actin,Bcl-2/β-actin and Caspase-3/β-actin were respectively 30.27%,111.67%and 6.79%;in group D4,the OD ratios of Bax/β-actin,Bcl-2/β-actin and Caspase-3/β-actin were respectively 110.54%,158.37%and 64.42%;in group D12,the OD ratios of Bax/β-actin,Bcl-2/β-actin and Caspase-3/β-actin were respectively 209.87%,50.89%and 80.18%. Compared with group NC,group D4 had significantly higher contents of Bax,Caspase-3 and Bcl-2 in cochlear tissues(P＜0.05),and D12 group had obviously higher contents of Bax and Caspase-3 in cochlear tissues (P＜0.05)but obviously the lower content of Bcl-2(P＜0.05).Compared with group D12,D4 group had evidently lower contents of Bax and Caspase-3 in cochlear tissues(P＜0.05),but evidently the higher content of Bcl-2(P＜0.05).The ratios of Bcl-2/Bax in group NC,group D4 and group D12 were 3.63,1.43 and 0.24,respectively. Conclusion:1.Increases in apoptosis-relating genes of Bax,Bcl-2 and Caspase-3 proteins in cochlear tissues of spontaneous diabetes suggest that apoptosis is involved in cochlea injury and the mechanism is possibly related with the mitochondrial pathway of apoptosis.2.With the extension of the diabetic course,the anti-apoptotic factor Bcl-2 is down-regulated and expressions of the pro-apoptotic factors,Bax and Caspase-3,are up-regulated,indicating that apoptosis worsens with the extension of the diabetic course. PartⅢ.The Protective Effect of Edaravone on the Cochlea of Spontaneous Diabetic MiceObjective:To discuss the protective effect of edaravone auditory vesicle infusion on the early diabetic cochlea and its molecular mechanism, providing theoretical evidence for applying edaravone to local inner ear medication of diabetic cochlear injury.Materials and Methods:Non-obese diabetic(NOD)female mice were divided into the medication treatment group(group MT),diabetic model group(group DM)and normal control group(group NC).The auditory vesicle infusions of edaravone were performed at 4 w of diabetes,once every other day and 4 times in total,and mice were executed at 7 d.Mice in group DM were female sham operation NOD mice of the same batch at 4 w since the occurrence of diabetes.Methylene blue-alkaline fuchsin staining of cochlear semi-thin sections was used to observe the morphological changes in cochlear tissues.TUNEL staining and cochlear hair cell nuclei fluorescent staining were applied to observe apoptosis. Immunohistochemical staining of Bcl-2,Bax and Caspase-3 was performed on paraffin sections near the central axis to observe the effect of edaravone auditory vesicle infusion on expressions of Bcl-2,Bax and Caspase-3 proteins.Results:1.Methylene blue-alkaline fuchsin staining of cochlear semi-thin sections:in group MT,the morphological structure of the Cortis organ in the upper and lower apical turn was intact,inner and outer hair cells revealed no obvious loss,spiral ganglion cells were arranged closely and homogeneous in morphology,nerve fibers were arranged regularly in a fascicular form and the stria vascularis and spiral ligament were normal in morphology,clearly structured and uniform in color development.2.Apoptotic activities of cochlear tissues:compared with group DM, group MT had obviously less sparing apoptotic cells in the cochlear Cortis organ,spiral ganglion,stria vascularis and spiral ligament(P＜0.05)and the comparison between group MT and group NC had no statistical significance(P＞0.05).3.Propidium iodide/acridine orange(PI/AO)fluorescent staining of cochlear hair cell nuclei:in group MT,cochlear hair cell loss was unobvious,nuclei were round,homogeneously constructed,orderly arranged and uniformly stained with green fluorescence,and there were no obvious apoptotic cells.Rates of hair cell loss and apoptosis were respectively 0.05%and 1.31%;compared with group DM,group MT had obviously lower apoptotic cells,and the difference between them had statistical significance(P＜0.05);compared with group C,group MT had no significant increase in apoptosis,and the difference between them had no statistical significance(P＞0.05).4.Immunohistochemical detection of Bax,Bcl-2 and Caspase-3 in cochlear paraffin sections:in group MT,Bax was weakly positively expressed,Caspase-3 was negatively expressed and Bcl-2 was strongly positively expressed in the cochlear Cortis organ,spiral ganglion,stria vascularis and spiral ligament of NOD mice;compared with group NC, group MT revealed no obvious increase in Bax and Caspase-3 expressions and the difference between them had no statistical significance(P＞0.05),but the expression of Bcl-2 was significantly increased(P＜0.05);compared with group DM,group MT had obviously lower expressions of Bax and Caspase-3(P＜0.05)but obviously the higher expression of Bcl-2,and the difference of the Bcl-2 expression between the two groups had statistical significance(P＜0.05).Conclusion:1.Edaravone auditory vesicle infusion can up-regulate the expression of Bcl-2 and down-regulate the expressions of Bax and Caspase-3 in cochlear tissues.2.Edaravone can alleviate apoptosis in cochlear micro-vessels, spiral ganglion and the Cortis organ in NOD mice.