The cochlea of the spontaneously diabetic mouse. I. Electron microscopic observation of KK mice

We examined the cochleae of the spontaneously diabetic KK mice by using transmission electron microscopy. At the age of 3 months, the mice started to show evidence for glycosuria and hyperglycemia, and tissue sections showed beginning cochlear pathology. The pathological changes present were found to be limited to the stria vascularis: protrusions of marginal cells, swellings of intermediate cells and widening of intercellular spaces were the main findings seen. These changes progressed with age, but were not observed in age-matched non-diabetic 57BL/6 mice. The possible mechanism of diabetes causing cochlear pathology is discussed.     

Ultrastructural and physiological defects in the cochlea of the Mpv17 mouse strain. A comparison between young and old adult animals

Ultrastructural investigations were performed in young (approximately 2 months) and old (7 months) Mpv17-negative and wild-type mice. The onset, the severity and the pattern of the degeneration significantly differed between both mice strains. In the wild-type mouse strain the degenerative changes of the cochlear structures were similar to the aging pattern described for other species. In contrast, the Mpv17 mutants showed degenerative changes of the cochlear structures already at the age of 2 months. The degenerative changes were patchy arranged throughout the entire length of the cochlea and involved the organ of Corti as well as the stria vascularis epithelia with alterations of the basement membrane of the capillaries. The severe sensorineural hearing loss and degenerative changes of the cochlear structures indicate that cochlear structures, especially the outer hair cells and the intermediate cells of the stria vascularis, are vulnerable to the missing Mpv17 gene product.     

Spiral Ligament Pathology: A Major Aspect of Age-Related Cochlear Degeneration in C57BL/6 Mice

Data from systematic, light microscopic examination of cochlear histopathology in an age-graded series of C57BL/6 mice (1.5-15 months) were compared with threshold elevations (measured by auditory brain stem response) to elucidate the functionally important structural changes underlying age-related hearing loss in this inbred strain. In addition to quantifying the degree and extent of hair cell and neuronal loss, all structures of the cochlear duct were qualitatively evaluated and any degenerative changes were quantified. Hair cell and neuronal loss patterns suggested two degenerative processes. In the basal half of the cochlea, inner and outer hair cell loss proceeded from base to apex with increasing age, and loss of cochlear neurons was consistent with degeneration occurring secondary to inner hair cell loss. In the apical half of the cochlea with advancing age, there was selective loss of outer hair cells which increased from the middle to the extreme apex. A similar gradient of ganglion cell loss was noted, characterized by widespread somatic aggregation and demyelination. In addition to these changes in hair cells and their innervation, there was widespread degeneration of fibrocytes in the spiral ligament, especially among the type IV cell class. The cell loss in the ligament preceded the loss of hair cells and/or neurons in both space and time suggesting that fibrocyte pathology may be a primary cause of the hearing loss and ultimate sensory cell degeneration in this mouse strain.     

Age-related changes in the murine cochlear lateral wall

Cochleas from C57BL/6 mice were investigated electrophysiologically and histochemically to evaluate the pathology of presbycusis. The average auditory brainstem response thresholds from 6-week-old mice were significantly lower than those of 6-month-old mice and those of 1-year-old mice. Histologic observation revealed changes in the cochlea after age 6 months. Conventional hematoxylin and eosin (H&E) staining showed disorganization of the organ of Corti, a decrease in the number of spiral ganglion cells, and atrophy of the stria vascularis. Although H&E staining and type II collagen immunolabeling did not show obvious changes in the spiral ligament (SL), the density of connexin 26 staining was reduced in this region. Sodium-potassium-adenosinetriphosphatase immunolabeling was increased in the SL, whereas its average density was not significantly altered in the stria vascularis. These results suggest that the SL could be among the regions responsible for cochlear malfunction with aging.     

Bone marrow cells as an origin of immune-mediated hearing loss

The MRL/lpr mouse, which is homozygous for the recessive lpr genes and has a mutation in the Fas gene encoding a cell-surface receptor for apoptosis, exhibits severe lymphadenopathy and develops systemic lupus erythematosus (SLE)-like disease. It has recently been reported that this mouse also manifests sensorineural hearing loss (SHL) with cochlear pathology at 20 weeks of age. We examined the effects of reconstituting severe combined immunodeficient (SCID) mice with MRL/lpr bone marrow on the development of SHL. These mice normally develop neither SHL nor cochlear pathology. Immune-mediated SHL and cochlear pathology did, indeed, occur following transfer of MRL/lpr bone marrow into SCID mice. These findings suggest that the development of SHL and cochlear pathology observed in MRL/lpr mice and in SCID mice receiving MRL/lpr bone marrow are the result of bone marrow defects rather than the result of a problem intrinsic to the cochlea.     

p-ERK5在快速衰老小鼠耳蜗血管纹中的增龄性变化

目的探讨快速衰老小鼠耳蜗血管纹中磷酸化的细胞外信号调节激酶5（phosphorylated extracellular signal regulated kinase 5, p ERK5）表达的增龄性变化。方法选用3、5、7月龄的快速衰老小鼠亚系８（senescence accelerated mouse, ＳＡＭＰ８）各６只,分别进行８ｋＨｚ短纯音听性脑干反应（ＡＢＲ）检测,并用免疫组化染色方法分别检测各月龄组小鼠耳蜗血管纹细胞中p ERK5的表达,分析其增龄性变化。结果3、５、７月龄小鼠耳蜗血管纹细胞中p ERK5平均光密度值分别为0.3838±0.0020、0.3646±0.0010、0.3423±0.0036; p ERK5在不同月龄快速衰老小鼠耳蜗组织中的表达光密度值随着月龄增加而显著降低（P＜０.０５）。结论随着年龄相关性功能减退,快速衰老小鼠耳蜗血管纹中p ERK5的表达水平逐渐降低,推测p ERK5可能与维持正常的耳蜗功能及听觉有关。     

Bone marrow cells as an origin of immune-mediated hearing loss

The MRL/lpr mouse, which is homozygous for the recessive lpr genes and has a mutation in the Fas gene encoding a cell-surface receptor for apoptosis, exhibits severe lymphadenopathy and develops systemic lupus erythematosus (SLE)-like disease. It has recently been reported that this mouse also manifests sensorineural hearing loss (SHL) with cochlear pathology at 20 weeks of age. We examined the effects of reconstituting severe combined immunodeficient (SCID) mice with MRL/lpr bone marrow on the development of SHL. These mice normally develop neither SHL nor cochlear pathology. Immune-mediated SHL and cochlear pathology did, indeed, occur following transfer of MRL/lpr bone marrow into SCID mice. These findings suggest that the development of SHL and cochlear pathology observed in MRL/lpr mice and in SCID mice receiving MRL/lpr bone marrow are the result of bone marrow defects rather than the result of a problem intrinsic to the cochlea.     

Ultrastructural pathology in the stria vascularis of the MRL-Fasl(lpr) mouse.

The MRL-Fas(lpr) mouse, a model of multisystemic, organ nonspecific autoimmune disease, has been proposed as a model of immune-mediated inner ear disease. A preliminary study employing light microscopy indicated that it develops cochlear pathology that appeared most striking in the stria vascularis, where cells underwent edema and degeneration. However, other structures, including the inner and outer hair cells and the supporting cells, also appeared to display pathology. The current study analyzed cochlear ultrastructure using transmission electron microscopy to better delineate the cochlear lesions found in these animals. MRL-Fas(lpr) animals were allowed to develop systemic disease (20 weeks old) and then had auditory brainstem response (ABR) thresholds determined. Animals were then killed and their cochleas prepared for electron microscopy. Age-matched MRL-+/+ and BALB/c mice served as controls. Results indicated that MRL-Fas(lpr) mice demonstrated elevated ABR thresholds. In contrast to a preliminary report, the cochlear pathology was observed exclusively in the stria vascularis, where cells demonstrated hydropic degeneration. Strial capillary structure was normal as were the rest of the cellular cochlear constituents. No inflammatory infiltrate was noted. These studies confirm that the MRL-Fas(lpr) mouse develops cochlear abnormalities focused in the stria vascularis. Whether the mechanism of the cellular degeneration involves autoimmune, genetic, or uremic processes has yet to be determined.     

不同龄CBA/J 小鼠耳蜗毛细胞的自然衰退

目的 为了观察CBA/J小鼠耳蜗毛细胞的自然退化现象，方法 本文采用了耳蜗铺片技术配合耳蜗毛细胞图直观方法对出生后不同月龄小鼠内外毛细胞作了形态学定量分析。结果 从出生6个月开始，随着月龄增加，CBA/J小鼠的内外毛细胞缺失从底回逐渐向顶回发展，以外毛细胞为最明显。结论 本实验证实了CBA/J小鼠耳蜗毛细胞存在自然退化现象，并有一定的规律。为临床研究耳蜗遗传性疾病提出 可靠的对照依据。     

The cochlea of the spontaneously diabetic mouse

Summary We used transmission electron microscopy to examine the cochleae of non-obese diabetic mice as animal models for human type I or non-insulin-dependent diabetes mellitus. Pathological changes were observed in the organ of Corti of the basal turn and in the stria vascularis of each turn. Major findings in the stria vascularis were protrusion or condensation of marginal cells, swelling of intermediate cells, and widening of the intercellular spaces. Principal findings in the organ of Corti involved degenerative changes of the outer and inner hair cells and replacement of hair cells by supporting cells. No prominent pathological changes were observed in the capillaries. The possible mechanism of diabetic involvement in cochlear pathology is discussed.     

The cochlea of the spontaneously diabetic mouse. II. Electron microscopic observations of non-obese diabetic mice

We used transmission electron microscopy to examine the cochlea of non-obese diabetic mice as animal models for human type I or non-insulin-dependent diabetes mellitus. Pathological changes were observed in the organ of Corti of the basal turn and in the stria vascularis of each turn. Major findings in the stria vascularis were protrusion or condensation of marginal cells, swelling of intermediate cells, and widening of the intercellular spaces. Principal findings in the organ of Corti involved degenerative changes of the outer and inner hair cells and replacement of hair cells by supporting cells. No prominent pathological changes were observed in the capillaries. The possible mechanism of diabetic involvement in cochlear pathology is discussed.     

Comparison of the quantity of cochlear melanin in young and old C57BL/6 mice

OBJECTIVE: To elucidate the functional relationship between cochlear melanin and aging. DESIGN: Melanin has been described in the cochlear labyrinth and has been suggested to protect the cochlea from various types of trauma. The quantity of melanin has been shown to change with aging in several organs; however, to our knowledge, aging changes in the cochlea have not been documented. Therefore, we chemically quantified cochlear eumelanin and pheomelanin contents and compared these in young and old C57BL/6 mice using high-performance liquid chromatography. Because melanin deposits in the cochlea present most extensively in the stria vascularis, we morphologically examined the stria using transmission electron microscopy. SUBJECTS: Cochleae from an inbred strain of C57BL/6 male and female mice; 6 at the age of 10 weeks and 5 at the age of 100 weeks were studied. RESULTS: The quantities of cochlear eumelanin and pheomelanin were 421 and 480 ng per cochlea in young mice, and 2060 and 765 ng per cochlea in old mice, respectively. Under transmission electron microscopy, the number of pigmented granules seemed to be greater in older mice compared with younger mice, especially in marginal cells. CONCLUSION: To our knowledge, our findings are the first quantitative evidence to show an age-related overexpression of cochlear melanin and an alteration in the proportion of eumelanin and pheomelanin with aging, suggesting a possible otoprotective function of eumelanin against age-related cochlear deterioration.     

Auditory peripheral influences on calcium binding protein immunoreactivity in the cochlear nucleus during aging in the C57BL/6J mouse

The C57BL/6J (C57) mouse was selected as a suitable model for early presbyacusis to determine if there were correlations between peripheral pathology (spiral ganglion loss, inner and outer hair cell loss) and calcium binding immunoreactivity in the cochlear nucleus during aging. The quantitative stereological method, the optical fractionator, was used for determining the total number of neurons and calcium binding immunopositive neurons (calbindin, parvalbumin and calretinin) during aging in the posteroventral- and dorsal cochlear nucleus (PVCN and DCN) in C57 mice. Comparing 30-month-old to 1-month-old C57 mice, a percent increase in parvalbumin and calbindin immunoreactivity was evident in both the PVCN and DCN. Correlations were made between peripheral pathology (spiral ganglion and inner and outer hair cell loss) and calcium binding protein expression. Significant correlations between cochlear pathology and the percentage of parvalbumin and calretinin immunoreactive neurons were demonstrated in the DCN. Moreover, significant correlations were found between cochlear pathology and parvalbumin and calbindin in the PVCN. In summary, the findings imply that degenerative changes in the auditory periphery can modulate neuronal homeostasis by increasing calcium binding proteins in the PVCN and DCN during aging. Taken together, these findings suggest a role for calcium binding proteins in protecting against age-induced calcium toxicity.     

Reduction in Sharpness of Frequency Tuning but not Endocochlear Potential in Aging and Noise-Exposed BALB/cJ Mice

Schuknecht proposed categories for human age-related hearing loss (ARHL) based upon whether the primary degeneration involves the organ of Corti (sensory ARHL), spiral ganglion cells (neural), stria vascularis (strial), or a combination of these (mixed). Genetically standardized mouse ARHL models can help validate Schuknecht's framework and clarify the underlying cellular processes. Much recent work has focused on the mouse Ahl locus, which promotes both ARHL and noise-induced hearing loss. On the C57BL/6 inbred background, Ahl has been associated with degeneration of organ of Corti, afferent neurons, and stria vascularis/spiral ligament, suggesting that it promotes mixed (sensory/neural/strial) ARHL. Some cochlear degeneration in C57BL/6 mice could be caused by genes other than Ahl, however. The question of what constitutes Ahl-related pathology can be addressed by comparing C57BL/6 mice with other strains that carry the same allele, including BALB/c substrains. We examined the effects of aging and broadband noise exposure in inbred BALB/cJ mice (1.5–13.0 mos) using measures of frequency tuning (compound action potential tuning curves) (CAPTCs), strial function (endocochlear potential recording, EP), and light microscopy. Aging and noise led to generally similar physiological and anatomical changes. Reductions in sensitivity and sharpness of frequency tuning were not consistently linked to hair cell loss, reduction in the EP, or changes in the lateral wall. Instead they appeared best explained by alterations in supporting cells in the basal half of the cochlear and in the spiral limbus in the apex. These results emphasize the importance of cell types other than hair cells in cochlear pathology. They also indicate that Ahl does not necessarily promote a strial form of ARHL.     

Genetic dependence of cochlear cells and structures injured by noise

The acute and permanent effects of a single damaging noise exposure were compared in CBA/J, C57BL/6 (B6), and closely related strains of mice. Two hours of broadband noise (4-45 kHz) at 110 dB SPL led to temporary reduction in the endocochlear potential (EP) of CBA/J and CBA/CaJ (CBA) mice and acute cellular changes in cochlear stria vascularis and spiral ligament. For the same exposure, B6 mice showed no EP reduction and little of the pathology seen in CBA. Eight weeks after exposure, all mice showed a normal EP, but only CBA mice showed injury and cell loss in cochlear lateral wall, despite the fact that B6 sustained larger permanent threshold shifts. Examination of noise injury in B6 congenics carrying alternate alleles of genes encoding otocadherin (Cdh23), agouti protein, and tyrosinase (albinism) indicated that none of these loci can account for the strain differences observed. Examination of CBA x B6 F1 mice and N2 backcross mice to B6 further indicated that susceptibility to noise-related EP reduction and associated cell pathology are inherited in an autosomal dominant manner, and are established by one or a few large effect quantitative trait loci. Findings support a common genetic basis for an entire constellation of noise-related cochlear pathologies in cochlear lateral wall and spiral limbus. Even within species, cellular targets of acute and permanent cochlear noise injury may vary with genetic makeup.     

Cochlear pathology of the circling mouse: a new mouse model of DFNB6

CONCLUSION: The circling mouse (cir/cir) has phenotypes which follow the pattern of neuroepithelial defects of deafness from 10 days after birth. The cir mouse is defective in Tmie gene, the function of which should be further elucidated. OBJECTIVES: We previously reported a recessive mutation of deafness called circling mice (cir/cir). The present study focused on investigating phenotypes and histological findings of the cochlea in circling mice with respect to age. MATERIALS AND METHODS: In order to analyze cochlear pathology over time, five different age groups of circling mice were examined (10, 18, 21, 35, and 90 days old). The organs of Corti and spiral ganglion neurons in basal and middle turns were evaluated. RESULTS: The pathology of the organ of Corti followed the pattern of neuroepithelial defects. Hair cells in organs of Corti had degenerated in circling mice at 10 days old, in a time-dependent manner. Scanning electron microscopy (SEM) showed that stereociliary bundles were irregular in size and had shortened at 10 days, and that this degeneration was complete at 21 days. The number of spiral ganglion neurons significantly reduced with age. RT-PCR analysis indicated that the transmembrane inner ear gene (Tmie) was absent in various organs in circling mice.     

Inner ear pathology in the Palmerston North autoimmune strain mouse.

The Palmerston North autoimmune strain mouse is a model for spontaneous systemic lupus erythematosus. Inner ear structure and function were examined during the onset and progression of systemic autoimmune disease to identify potentially correlated auditory system pathology. The onset of systemic disease occurred at 4 to 5 months of age and was characterized by elevated serum immune complexes, cryoglobulins, and antinuclear antibodies. Coincident with the onset of autoimmune disease was degeneration of the apical turn stria vascularis and outer hair cells. These cochlear changes progressed basalward. At 10 months of age, auditory brainstem response thresholds were elevated and the stria vascularis area was measurably smaller throughout the cochlea. Immunohistochemical staining showed immunoglobulin G deposits within the organ of Corti, the vas spirale of the basilar membrane, the scala tympani, and marrow cavities of the bony otic capsule. These results suggest that cochlear pathology may be immune mediated in this mouse, which would make the strain suitable for the study of the mechanisms relating inner ear abnormalities and autoimmune disease.     

不同月龄BALB/c小鼠耳蜗miR-96表达与ABR阈值及耳蜗形态的关系

目的　研究不同月龄BALB/c小鼠ABR阈值、耳蜗形态学改变及miR-96的表达,明确miR-96对老年性聋小鼠听力的调节作用。方法　通过听性脑干反应测试、荧光染色、扫描电镜,观察3、6、12、18月龄BALB/c小鼠8 kHz听反应阈值及耳蜗形态学改变。实时定量PCR定量检测miR-96在各月龄BALB/c小鼠耳蜗内的表达。SPSS13.0统计软件进行 统计分析。结果　3、6、12月龄组小鼠8 kHz听反应阈值分别为（18.5±8.3）、（45.8±7.8）、（85.6±15.6）dB SPL,18月龄组120 dB SPL刺激声基本测不出听觉反应。荧光染色及扫描电镜发现,自6月龄起毛细胞出现显著缺失,静纤毛出现不同程度缺失、倒伏、融合、变短和转位等改变,并随月龄增大病变逐渐加重。miR-96在3、6、12、18月龄BALB/c小鼠耳蜗中的相对表达量（2-△CT）分别为0.0225±0.0073、0.0162±0.0048、0.0116±0.0048和0.0050±0.0014,与3月龄小鼠相比,差异有统计学意义（P <0.05）。结论　BALB/c小鼠听力损失、耳蜗毛细胞缺失及纤毛损害随月龄增长而逐渐加重,小鼠耳蜗中miR-96表达随月龄增加而减少,提示miR-96可能在老年性聋的发病机制中起重要用。     

Comparison of the auditory sensitivity of neurons in the cochlear nucleus and inferior colliculus of young and aging C57BL/6J and CBA/J mice

Thresholds of neurons to sounds were compared as a function of central auditory structure [ventral cochlear nucleus (VCN), dorsal cochlear nucleus (DCN), and inferior colliculus (IC)] in young and middle-aged C57BL/6J mice (multiple- and single-unit recordings) and in young and old CBA/J mice (single-unit recordings). Middle-aged C57 mice show progressive loss of sensitivity to high frequencies and noise due to cochlear pathology; CBA mice show little loss of sensitivity through most of their lifespan. Multiple-unit threshold curves (MTCs) for tones indicated that neurons in the C57 VCN suffered a greater degree of age-related loss of sensitivity than neurons in the IC (from an earlier study). Furthermore, whereas the low frequency portions of MTCs in IC neurons in high frequency tonotopic regions typically become 'sensitized' in middle-aged C57 mice (i.e., lower thresholds than young mice), such was not the case for VCN neurons. In contrast to VCN neurons, MTCs of the population of DCN neurons studied were statistically indistinguishable from those of the IC. Measurements of single-unit response areas in C57 mice corroborated the MTCs. In CBA mice, little effect of age was found in comparing single-unit response areas of young and old mice. The findings indicate that sensorineural impairment in middle-aged C57 mice is accompanied by threshold changes that are more severe in the VCN than in the IC or DCN. Because the VCN and DCN are believed to play different roles in hearing, the functions they support should, likewise, be affected to different extents by age-related hearing loss.     

Hereditary deafness occurring in cd/1 mice.

Different strains of mice provide a valuable research tool for studying both hereditary and acquired forms of deafness. The cd/1 strain has been found to demonstrate hereditary cochlear pathology. The characteristics of hearing loss in cd/1 mice have not previously been reported. In this investigation auditory thresholds were obtained by measuring evoked brain stem responses in subjects of three different ages: 3 weeks, 10 weeks and 6 months. The results were compared with thresholds obtained from CBA/Ca mice (which have normal hearing) and C57BL/6 mice (which are known to have a genetically determined pre-senile progressive cochlear hearing loss). A significant hearing loss was observed which progressed from high to low frequencies, and with age. Extensive degeneration was observed throughout the organ of Corti. cd/1 mice may provide a useful model for studying genetically determined deafness.