Age-related hearing loss was accelerated by apoptosis of spiral ganglion and stria vascularis cells in ApoE KO mice with hyperglycemia and hyperlipidemia

Abstract

Age-related hearing loss (ARHL) is associated with diabetes and/or dyslipidemia in humans. However, the detailed mechanism for the development of ARHL by diabetes and/or dyslipidemia has not been elucidated. In this study, we investigated the etiology of ARHL in apolipoprotein E (ApoE)-deficient mice with diabetes and dyslipidemia. The atherosclerotic CD-STZ (mice fed with a control diet and received an STZ injection), WD-con (mice fed with a western diet), and WD-STZ (mice fed with a western diet and received an STZ injection) mice showed a 2.4-, 4.9-, and 6.8-fold larger area, respectively, occupied by lesions throughout the aorta compared with the CD-con mice. A significantly larger area under the curve (AUC) was observed in the STZ-treated groups than in the non-treated groups based on the oral glucose tolerance test (OGTT). At 20 weeks of age, HbA_1c levels were significantly higher in the CD-STZ and WD-STZ mice than in the CD-con and WD-con mice. In all the groups, the auditory brainstem response (ABR) thresholds of the 16-week-old mice were significantly higher compared with those of the 8-week-old mice. In particular, in the WD-STZ mice, the ABR thresholds of the left and right ears reached the maximum decibel peak equivalent sound pressure levels (130 dBpeSPL), which is a sign of deafness. The apoptotic spiral ganglion neurons (SGNs) of the WD-STZ mice were significantly increased compared with those of the other three groups, indicating that SGN apoptosis resulted in hearing loss in STZ-induced diabetic ApoE KO mice fed with a WD. A significant loss of the stria vascularis cells was observed in the WD-STZ group compared with the CD-con mice. In the organ of Corti, few apoptotic hair cells were found in all the groups; however, no significant difference was observed. Therefore, we consider that the reduced hearing ability in the STZ-treated and WD-fed groups was attributed to the damage to the SGN and stria vascularis in the cochlea. Thus, our results indicated that ototoxicity by diabetes and/or dyslipidemia accelerated ARHL in ApoE KO mice, thereby suggesting the importance of appropriate treatment of patients with diabetes and/or dyslipidemia to prevent ARHL.

Keywords: age-related hearing loss; apoptosis; atherosclerosis; hyperglycemia; hyperlipidemia; spiral ganglion neuron; stria vascularis.

Figure 1

Experimental protocol and survival rate. (A) Experimental protocol, including procedures and timeline, for each group. A western diet (WD) was provided at 8 weeks of age, and streptozotocin (STZ) was injected at 12 weeks of age to induce hyperlipidemia and hyperglycemia. (B) Decreased survival rate in the WD-STZ mice due to severe atherosclerosis and diabetes. The WD-STZ mice exhibit a gradual decrease in survival rate starting from 17 weeks of age.

Figure 2

Lipid profiles and atherosclerotic plaques. (A) The plasma total cholesterol level in each group of mice at 16 and 24 weeks of age. (B) Low-density lipoprotein (LDL) cholesterol in each group of mice at 16 and 24 weeks of age. (C) Representative en face images of the atherosclerotic plaques in the whole aorta using Sudan IV staining (red). (D) Percentage of lesion areas relative to the whole aortic area in CD-con, CD-STZ, WD-con, and WD-STZ ApoE KO male mice at 24 weeks of age. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

Fasting blood glucose and hemoglobin A_1c (HbA_1c) levels. (A) Increased fasting blood glucose levels in STZ-treated mice at 16 weeks of age. Blood glucose levels were checked in all groups after 6 hours of fasting. (B) Hemoglobin A1c (HbA_1c) levels in all groups of mice at 16, 20, and 24 weeks of age. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

Oral glucose tolerance test (OGTT). The area under the curve (AUC) after the OGTT was calculated in mice at 16 (A), 20 (B), and 24 (C) weeks of age. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 5

Pancreatic islet size and glucagon/insulin ratio. (A,B) Decreased pancreatic islet size in STZ-treated mice confirmed on hematoxylin and eosin staining. (C,D) Increased α-cell and decreased β-cell intensities in pancreatic islets of STZ-treated mice. The glucagon (green)/insulin (red) ratio was measured and calculated. *** p < 0.001, and **** p < 0.0001. Scale bars correspond to 50 μm.

Figure 6

Hearing ability according to age and experimental conditions. (A,B) Increased auditory brainstem response (ABR) thresholds with age. ABR thresholds were measured in CD-con, CD-STZ, WD-con, and WD-STZ mice at 8, 16, 20, and 24 weeks of age with click sounds to compare age differences between each group in the right (A) and left (B) ears. (C,D) Increased ABR thresholds due to Western diet (WD) feeding and streptozotocin (STZ) treatment. ABR thresholds were measured in CD-con, CD-STZ, WD-con, and WD-STZ mice at 8, 16, 20, and 24 weeks of age with click sounds to compare differences between groups at the same age in the right (C) and left (D) ears. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 7

Histological analysis of the cochlear spiral ganglion neurons (SGNs) and stria vascularis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, green) and propidium iodide (PI, red) staining were used to detect apoptosis in the SGNs (A) and stria vascularis (B) at 24 weeks of age. The TUNEL⁺/PI⁺ ratio of SGNs was significantly different between all groups (A), while that of stria vascularis was significantly different between the CD-con and WD-STZ groups (B). * p < 0.05, *** p < 0.001, and **** p < 0.0001. Scale bars correspond to 50 μm.

Figure 8

Histological analysis of the organ of Corti. No significant difference was noted in the TUNEL⁺/PI⁺ ratio in hair cells. The average count of apoptosis cells was taken from at least eight cross-sectional images. Scale bars correspond to 50 μm (upper panel) and 1 μm (lower panel).