Comparison of cochlear morphology and apoptosis in mouse models of presbycusis

Objectives

Morphological studies on presbycusis, or age-related hearing loss, have been performed in several different strains of mice that demonstrate hearing loss with auditory pathology. The C57BL/6 (C57) mouse is a known model of early onset presbycusis, while the CBA mouse is characterized by relatively late onset hearing loss. We performed this study to further understand how early onset hearing loss is related with the aging process of the cochlea.

Methods

We compared C57 cochlear pathology and its accompanying apoptotic processes to those in CBA mice. Hearing thresholds and outer hair cell functions have been evaluated by auditory brainstem response (ABR) recordings and distortion product otoacoustic emission (DPOAE).

Results

ABR recordings and DPOAE studies demonstrated high frequency hearing loss in C57 mice at P3mo of age. Cochlear morphologic studies of P1mo C57 and CBA mice did not show differences in the organ of Corti, spiral ganglion, or stria vascularis. However, from P3mo and onwards, a predominant early outer hair cell degeneration at the basal turn of the cochlea in C57 mice without definitive degeneration of spiral ganglion cells and stria vascularis/spiral ligament, compared with CBA mice, was observed. Additionally, apoptotic processes in the C57 mice also demonstrated an earlier progression.

Conclusion

These data suggest that the C57 mouse could be an excellent animal model for early onset ''sensory'' presbycusis in their young age until P6mo. Further studies to investigate the intrinsic or extrinsic etiologic factors that lead to the early degeneration of organ of Corti, especially in the high frequency region, in C57 mice may provide a possible pathological mechanism of early onset hearing loss.     

Low-Frequency Tone Pips Elicit Exaggerated Startle Reflexes in C57BL/6J Mice with Hearing Loss

The strength of the acoustic startle reflex (ASR) as a function of age was studied in adult C57BL/6J and CBA/CaJ mice, because altered ASR levels are a potential behavioral consequence of the neural reorganization that accompanies the early-onset hearing loss of the C57BL, in contrast to the normal-hearing CBA. For C57BL mice at 14–36 weeks of age, compared with 7-week-old mice, high-frequency thresholds measured with the auditory brainstem response (ABR) were less sensitive by about 25–30 dB while the hearing loss at low frequencies was 10–15 dB, but by 60 weeks losses of 45–50 dB were present across the entire spectrum. Their ASR amplitudes for 16 kHz tone pips were highest at 7 weeks and then declined with age, but, for 4 kHz tones the ASR increased in strength at 18 weeks and beyond to levels above that of the younger mice. This hyperreactivity persisted even in 60-week-old mice. The ASR for 16 kHz stimuli was positively correlated with hearing sensitivity, but the ASR for 4 kHz stimuli was positively correlated with hearing loss for mice that were 18–36 weeks of age. Furthermore, ASR amplitudes for 4 kHz stimuli were positively correlated with the 16 kHz ASR in young C57BL mice but negatively correlated in older mice. There were no similar ASR or ABR changes in adult CBA mice through 19 weeks of age. Correlations between ASR and ABR scores were always weakly positive, and correlations between 4 kHz and 16 kHz ASR amplitudes were always strongly positive. The ASR data in older C57BL mice with hearing loss are consistent with reports describing their increased neural representation of low-frequency sounds and reinforce the value of this strain for studying the functional consequences that accompany age-related cochlear degeneration.     

Age-related loss of auditory sensitivity in two mouse genotypes.

Frequency-specific auditory brainstem responses (ABR) (frequency range 2-31.5 kHz) were used to describe age-dependent alterations in frequency sensitivity in CBA/ca (to 30 months) and C57BL/6J (to 16 months) mouse genotypes. The two strains displayed an age-related loss similar to that of humans, with a primary decline in high-frequency sensitivity. CBA mice showed a slow, yet gradual decrease in sensitivity to high- followed by low-frequency until 18 months of age and a rapid deterioration at all frequencies thereafter. C57 mice demonstrated precocious aging in auditory sensitivity in an unsteady rather than gradual manner. By testing the same individuals on repeated occasions it was shown that, for C57 mice, the degenerative process was more precipitous with three phases, and that the individual variability was larger than that of CBA mice. It is suggested that the two mouse genotypes can be used to express the different aspects of human presbycusis in individual variability and modes of gradual or staggered progression.     

Prolonged exposure to an augmented acoustic environment ameliorates age-related auditory changes in C57BL/6J and DBA/2J mice.

The effects of exposure to an augmented acoustic environment (AAE) on auditory function were evaluated in mouse strains that exhibit high-frequency hearing loss beginning during young adulthood (the C57BL/6J strain [C57]) or around the time of weaning/ adolescence (the DBA/2J strain [DBA]). Beginning at age 25 days, mice were exposed 12 h every night to a 70 dB SPL broad-band noise AAE. The AAE was maintained until age 14 months in C57 mice and 9 months in DBA mice. Control mice were age-matched and maintained under normal vivarium acoustic conditions. The auditory brainstem response (ABR), acoustic startle response amplitude, and prepulse inhibition (PPI) were used to assess the auditory system. Exposure to the AAE resulted in improved auditory performance in both strains (better PPI, lower ABR thresholds, bigger startle amplitudes).     

Inner ear morphology in CBA/Ca and C57BL/6J mice in relationship to noise,age and phenotype

Summary CBA/Ca mice showing moderate hearing losses with onset late in life and C57BL/6J mice with progressive hearing losses starting when animals were young adults were exposed to a 2–7 kHz, 120 dB SPL noise band for 5 min in order to investigate morphological consequences to noise as a function of age and genotype. Permanent threshold shifts were determined by auditory brainstem responses 1 month after noise exposure at 1, 3 and 6 months of age. CBA mice had a decreasing susceptibility to noise with increasing age, while C57 mice remained equally susceptible throughout all ages tested. The threshold shifts were then analyzed in relation to morphological changes of the organ of Corti as visualized by light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). CBA mice exposed at 1 month and sacrificed at 2 months had seemingly normal cochlear morphologies under LM. In these animals SEM findings demonstrated mild stereocilia damage to noise trauma, but not when mice were exposed at 3 and 6 months. There was no visible morphological aging of hair cells found during the period tested. In contrast; the C57 mice had early hair cell changes including bent and fused stereocilia, bulging of the cuticular plates, hair cell loss and swelling of afferent dendrites. These changes became more pronounced throughout the test periods with the variability of damage in this strain more evident over time. This pattern was also aggravated in all C57 age groups tested after noise exposure. Quantification with cytocochleograms demonstrated a statistically different reaction pattern to noise trauma between the two different genotypes of mice.     

不同品系正常成年小鼠听性脑干反应特点

目的 研究正常成年CBA、C57BL、昆明系和129系四种品系小鼠的听性脑干反应(auditory brainstem response,ABR),分析其ABR反应阈和潜伏期,得到正常值范围,标准化测试流程,为小鼠的听觉相关研究提供参考.方法 对6周龄正常CBA、C57BL、昆明系和129系四种品系小鼠各20只(20耳),完全麻醉后,进行短声(click)和短纯音(tone burst,tb) ABR测试,观察四种品系小鼠ABR各波的引出率,记录引出率最高的波反应阈值及各波潜伏期.结果 ①在80 dB SPL强度下,CBA、C57BL、昆明系和129系四种品系小鼠ABR各波引出率均为100％,CBA小鼠ABR波形分化最好;在30 dB SPL强度下,除129小鼠外,其余3种小鼠波Ⅱ引出率最高,129小鼠在阈值附近50 dB SPL强度下也是波Ⅱ引出率最高;129小鼠ABR反应阈明显高于其它三种小鼠,差异均有统计学意义(P均＜0.05);②四种品系小鼠的短纯音听觉敏感频率为8、12、16 kHz,其中对12 kHz的短纯音最敏感;③麻醉情况下,ABR各波潜伏期随着测试时间延长而延长,尤其波Ⅲ以后各波潜伏期延长更明显.80 dB SPL强度下CBA、C57BL、昆明系、129系四种小鼠ABR波形在波工前均可分化出SP(summating potential,SP)波,引出率分别为100％、90％、80％、50％,相同刺激条件下,SP波出现率高的小鼠其ABR反应阈相对较低.结论 麻醉状态下,CBA、C57BL、昆明系和129系四种品系小鼠短声及短纯音ABR波Ⅱ引出率最高,故以波Ⅱ刚刚出现的刺激声强度确定为反应阈值;C57BL、昆明系和129系三种品系小鼠的ABR结果均没有CBA小鼠稳定,四种品系小鼠中CBA小鼠更适合用来建立听觉功能相关研究的动物模型;SP波可间接反映毛细胞功能.     

The effect of noise exposure on the aging ear.

The effect of noise exposure on auditory sensitivity and inner ear morphology was compared in aged and young mature mice. Hearing thresholds were obtained by auditory evoked brain stem responses (ABR) before and after noise exposure, and hair cell loss was quantified. The study was done in two parts: first to assess the effect of noise exposure on subjects with presbycusis, and second to assess its effect on aged subjects without measurable presbycusis. In the first experiment C57BL/6 mice, with an age-related hearing loss, were used as a model for presbycusis. C57BL/6 mice exhibiting presbycusis were more susceptible to noise injury than age-matched CBA/Ca mice. In the second experiment CBA/Ca mice were used. These mice retain normal hearing even with advancing age. The aged CBA/Ca mice had the same susceptibility to noise injury as young CBA/Ca mice.     

Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses

The common occurrence of hearing loss in both humans and mice, and the anatomical and functional similarities of their inner ears, attest to the potential of mice being used as models to study inherited hearing loss. A large-scale, auditory screening project is being undertaken at The Jackson Laboratory (TJL) to identify mice with inherited hearing disorders. To assess hearing sensitivity, at least five mice from each inbred strain had auditory brainstem response (ABR) thresholds determined. Thus far, we have screened 80 inbred strains of mice; 60 of them exhibited homogeneous ABR threshold values not significantly different from those of the control strain CBA/CaJ. This large database establishes a reliable reference for normal hearing mouse strains. The following 16 inbred strains exhibited significantly elevated ABR thresholds before the age of 3 months: 129/J, 129/ReJ, 129/SvJ, A/J, ALR/LtJ, ALS/LtJ, BUB/BnJ, C57BLKS/J, C57BR/cdJ, C57L/J, DBA/2J, I/LnJ, MA/MyJ, NOD/LtJ, NOR/LtJ, and SKH2/J. These hearing impaired strains may serve as models for some forms of human non-syndromic hearing loss and aid in the identification of the underlying genes.     

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.     

Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice

Inbred strain variants of the Cdh23 gene have been shown to influence the onset and progression of age-related hearing loss (AHL) in mice. In linkage backcrosses, the recessive Cdh23 allele (ahl) of the C57BL/6J strain, when homozygous, confers increased susceptibility to AHL, while the dominant allele (Ahl+) of the CBA/CaJ strain confers resistance. To determine the isolated effects of these alleles on different strain backgrounds, we produced the reciprocal congenic strains B6.CBACa-Cdh23^Ahl+ and CBACa.B6-Cdh23^ahl and tested 15-30 mice from each for hearing loss progression. ABR thresholds for 8 kHz, 16 kHz, and 32 kHz pure-tone stimuli were measured at 3, 6, 9, 12, 15 and 18 months of age and compared with age-matched mice of the C57BL/6J and CBA/CaJ parental strains. Mice of the C57BL/6N strain, which is the source of embryonic stem cells for the large International Knockout Mouse Consortium, were also tested for comparisons with C57BL/6J mice. Mice of the C57BL/6J and C57BL/6N strains exhibited identical hearing loss profiles: their 32 kHz ABR thresholds were significantly higher than those of CBA/CaJ and congenic strain mice by 6 months of age, and their 16 kHz thresholds were significantly higher by 12 months. Thresholds of the CBA/CaJ, the B6.CBACa-Cdh23^Ahl+, and the CBACa.B6-Cdh23^ahl strain mice differed little from one another and only slightly increased throughout the 18-month test period. Hearing loss, which corresponded well with cochlear hair cell loss, was most profound in the C57BL/6J and C57BL/6NJ strains. These results indicate that the CBA/CaJ-derived Cdh23^Ahl+ allele dramatically lessens hearing loss and hair cell death in an otherwise C57BL/6J genetic background, but that the C57BL/6J-derived Cdh23^ahl allele has little effect on hearing loss in an otherwise CBA/CaJ background. We conclude that although Cdh23^ahl homozygosity is necessary, it is not by itself sufficient to account for the accelerated hearing loss of C57BL/6J mice.     

小鼠自然老化过程中耳蜗的糖代谢障碍

应用定量组织化学技术分析了ＣＢＡ和Ｃ５７ＢＬ小鼠耳蜗中糖元代谢的改变，发现１８个月和３６个月龄ＣＢＡ小鼠的毛细胞和血管纹中，糖元含量比６周龄ＣＢＡ正常小鼠明显增高，说明在自然老化ＣＢＡ小鼠耳蜗中存在糖元储积现象。     

Early bilateral deafening prevents calretinin up-regulation in the dorsal cortex of the inferior colliculus of aged CBA/CaJ mice

This study was conducted to test the hypothesis that age-related calretinin (CR) up-regulation seen in the dorsal cortex of the inferior colliculus (ICdc) of old hearing CBA mice is dependent upon neural activity within the auditory pathway. We tested this hypothesis by bilaterally deafening young CBA/CaJ mice with kanamycin, and then aging them until 24 months. This manipulation mimics the lack of sound-evoked auditory activity experienced by old C57BL/6J mice, who are deaf and do not show CR up-regulation with age. Cell counts revealed that the density of CR+ cells in the ICdc of old hearing CBA mice was statistically different from old deafened CBA mice raised under identical conditions. Old hearing CBAs possessed an average of 27.54 more CR+ cells/100 microm2 than old deafened CBAs. When old deafened CBAs were compared to young hearing CBAs, young hearing C57s, and old deaf C57s, there was no significant difference in mean CR+ cell density in ICdc. Thus, only the old normal hearing CBAs showed an increase in CR+ cells with age, supporting the hypothesis that CR up-regulation depends upon sound-evoked activity. Moreover, these results demonstrate that up-regulation of CR expression was not simply due to a mouse strain difference.     

Age-Related Changes in the Relationship Between Auditory Brainstem Responses and Envelope-Following Responses

Hearing thresholds and wave amplitudes measured using auditory brainstem responses (ABRs) to brief sounds are the predominantly used clinical measures to objectively assess auditory function. However, frequency-following responses (FFRs) to tonal carriers and to the modulation envelope (envelope-following responses or EFRs) to longer and spectro-temporally modulated stimuli are rapidly gaining prominence as a measure of complex sound processing in the brainstem and midbrain. In spite of numerous studies reporting changes in hearing thresholds, ABR wave amplitudes, and the FFRs and EFRs under neurodegenerative conditions, including aging, the relationships between these metrics are not clearly understood. In this study, the relationships between ABR thresholds, ABR wave amplitudes, and EFRs are explored in a rodent model of aging. ABRs to broadband click stimuli and EFRs to sinusoidally amplitude-modulated noise carriers were measured in young (3–6 months) and aged (22–25 months) Fischer-344 rats. ABR thresholds and amplitudes of the different waves as well as phase-locking amplitudes of EFRs were calculated. Age-related differences were observed in all these measures, primarily as increases in ABR thresholds and decreases in ABR wave amplitudes and EFR phase-locking capacity. There were no observed correlations between the ABR thresholds and the ABR wave amplitudes. Significant correlations between the EFR amplitudes and ABR wave amplitudes were observed across a range of modulation frequencies in the young. However, no such significant correlations were found in the aged. The aged click ABR amplitudes were found to be lower than would be predicted using a linear regression model of the young, suggesting altered gain mechanisms in the relationship between ABRs and FFRs with age. These results suggest that ABR thresholds, ABR wave amplitudes, and EFRs measure complementary aspects of overlapping neurophysiological processes and the relationships between these measurements changes asymmetrically with age. Hence, measuring all three metrics provides a more complete assessment of auditory function, especially under pathological conditions like aging.     

Age-related changes in the brainstem auditory evoked potentials of the marmoset

Changes in brain-stem auditory evoked potentials (BAEPs) with age were recorded in common marmosets (Callithrix jacchus) at the age of 1-2, 6-8 and 10-12 years. The auditory function was assessed by thresholds, latencies and amplitudes of BAEPs evoked by use of tone burst stimulations with audible frequencies ranging from 1 to 99 kHz. Prolongation of the latencies of later waves was observed in the animals at the age of 6-8 and 10-12 years at high frequencies, suggesting that aging in marmosets, as reported previously in humans and other animals, may cause earlier hearing loss at high frequency than at low frequency within the hearing range. At 10-12 years of age, the elevations of BAEP thresholds and the declines of BAEP amplitudes in older animals were also observed. As the differences in the parameters are small, it was suggested that only a moderate hearing loss occurred with onset late in life in common marmoset similar to that in CBA/Ca mice. Based on the results obtained in this study, BAEP latencies appear to be more sensitive indicators than BAEP thresholds and amplitudes for the early detection of hearing impairment.     

Effects of 3,3′-Iminodipropionitrile on Hair Cell Numbers in Cristae of CBA/CaJ and C57BL/6J Mice

This study examines absolute hair cell numbers in the cristae of C57BL/6J mice and CBA/CaJ mice from weaning to adulthood as well as the dose required for 3,3′-iminodiproprionitrile (IDPN)-injury of the cristae in C57BL/6J mice and CBA/CaJ mice, the two mouse strains most commonly used by inner ear researchers. In cristae of CBA/CaJ and C57BL/6J mice, no loss of hair cells was observed up to 24 weeks. In both strains, dose-dependent loss of hair cells was observed 7 days after IDPN treatment of 2-month-old mice (IC₅₀?=?16.1 mmol/kg in C57BL/6J mice vs. 25.21 mmol/kg in CBA/CaJ mice). Four-month-old C57BL/6J mice exposed to IDPN developed dose-dependent vestibular dysfunction as indicated by increased activity and circling behavior in open field tests and by failure to swim 7 days after treatment. IDPN-hair cell injury in C57BL/6J mice and CBA/CaJ mice represents a fast and predictable experimental model for the study of vestibular degeneration and a platform for the testing of vestibular therapies.     

Males lose hearing earlier in mouse models of late-onset age-related hearing loss; females lose hearing earlier in mouse models of early-onset hearing loss

Gender-related differences in human hearing have been attributed to genetic, environmental, and/or genetic×environmental interactive factors. These differences tend to increase with age, with males showing greater high frequency threshold elevations. An appropriate animal model could aid in prediction, treatment, and prevention of some of these losses. This paper examines inbred strains of mice that are widely used as models of late- (CBA/J and CBA/CaJ) and early- (C57BL/6J) onset age-related hearing loss. In the former two genotypes, the thresholds to high frequency stimuli of the auditory brainstem response (ABR) are higher in the male than in the female. This gender difference was less pronounced in thresholds to the cochlear nerve envelope response of the CBA/CaJ, although this response was more sensitive to the influence of age than was the ABR. In contrast, the male C57BL/6J had more sensitive thresholds than the female, with both measures showing massive loss of sensitivity with increasing age. The data are discussed in terms of the applicability of these animals as tools for examining factors that degrade cochlear function.     

Influence of genotype and age on acute acoustic trauma and recovery in CBA/Ca and C57BL/6J mice.

Two inbred strains of mice, CBA/Ca (with a moderate auditory impairment starting late in life) and C57BL/6J (with a progressive auditory degeneration starting during young adulthood), were exposed to a 120 dB SPL broadband noise (2-7 kHz) for 5 min at the age of 1, 3, or 6 months. Compound and permanent threshold shifts (CTS and PTS) were determined by auditory brainstem response during the first day (CTS) and 1 month (PTS) after exposure. With increasing age, susceptibility to CTS at middle frequencies (8 and 12.5 kHz) decreased in CBA mice, yet was retained in C57 mice. C57 mice were more severely affected by CTS than age-matched CBA mice. Noise-induced CTS was positively correlated to pre-exposure thresholds in C57 mice and to PTS over all ages and strains. The amount of recovery from CTS to PTS was, however, independent of age and strain. There was only a 2% risk of classifying CBA mice as C57 mice by pre-exposure thresholds at high frequency, while there was about 40% risk by CTS. The results indicate that genetic predisposition can affect susceptibility to auditory degeneration and noise impairment in a systematic manner, allowing the identification of susceptible individuals by pre-exposure audiometric examination, especially at high frequencies.     

Protection by low-dose kanamycin against noise-induced hearing loss in mice: dependence on dosing regimen and genetic background

We recently demonstrated that sub-chronic low-dose kanamycin (KM, 300 mg/kg sc, 2×/day, 10 days) dramatically reduces permanent noise-induced hearing loss (NIHL) and hair cell loss in 1 month old CBA/J mice (Fernandez et al., 2010, J. Assoc. Res. Otolaryngol. 11, 235-244). Protection by KM remained for at least 48 h after the last dose, and appeared to involve a cumulative effect of multiple doses as part of a preconditioning process. The first month of life lies within the early 'sensitive period' for both cochlear noise and ototoxic injury in mice, and CBA/J mice appear exquisitely vulnerable to noise during this period (Ohlemiller et al., 2011; Hearing Res. 272, 13-20). From our initial data, we could not rule out 1) that less rigorous treatment protocols than the intensive one we applied may be equally-or more-protective; 2) that protection by KM is tightly linked to processes unique to the sensitive period for noise or ototoxins; or 3) that protection by KM is exclusive to CBA/J mice. The present experiments address these questions by varying the number and timing of fixed doses (300 mg/kg sc) of KM, as well as the age at treatment in CBA/J mice. We also tested for protection in young C57BL/6J (B6) mice. We find that nearly complete protection against at least 2 h of intense (110 dB SPL) broadband noise can be observed in CBA/J mice at least for ages up to 1 year. Reducing dosing frequency to as little as once every other day (a four-fold decrease in dosing frequency) appeared as protective as twice per day. However, reducing the number of doses to just 1 or 2, followed by noise 24 or 48 h later greatly reduced protection. Notably, hearing thresholds and hair cells in young B6 mice appeared completely unprotected by the same regimen that dramatically protects CBA/J mice. We conclude that protective effects of KM against NIHL in CBA/J mice can be engaged by a wide range of dosing regimens, and are not exclusive to the sensitive period for noise or ototoxins. While we cannot presently judge the generality of protection across genetic backgrounds, it appears not to be universal, since B6 showed no benefit. Classical genetic approaches based on CBA/J × B6 crosses may reveal loci critical to protective cascades engaged by kanamycin and perhaps other preconditioners. Their human analogs may partly determine who is at elevated risk of acquired hearing loss.     

NKCC1和Na-K-ATPase通道蛋白与老年性耳聋的关系及其在老年性耳聋不同阶段中的作用

目的：通过对C57BL/6J小鼠耳蜗中NKCC1和Na-K-ATPase的年龄相关性表达的研究，分析其与老年性耳聋的关系并进一步探讨其在老年性耳聋发生发展不同阶段中的作用。方法通过听性脑干反应（ABR）分别检测C57BL/6J小鼠在4、24和48周年龄段的听力水平。采用实时免疫荧光定量逆转录聚合酶链反应（RT-PCR）法分别检测NKCC1和Na-K-ATPase mRNA在各年龄段小鼠耳蜗中的表达水平。结果随着C57BL/6J鼠龄的增加，其ABR反应阈值逐渐升高（P〈0.05）。RT-PCR显示NKCC1和Na-K-ATPase mRNA在耳蜗的表达水平存在随鼠龄增加逐渐下降的趋势（P＜0.01）。结论 C57BL/6J小鼠的ABR反应阈值随鼠龄增加逐渐增高，具有老年性耳聋的特征；NKCC1和Na-K-ATPase两种通道蛋白随鼠龄的增加逐渐下降，与C57BL/6J小鼠的年龄相关性听力下降密切相关，可能与老年性耳聋后期发展及恶化有关。     

Effects of exposing C57BL/6J mice to high- and low-frequency augmented acoustic environments: auditory brainstem response thresholds, cytocochleograms, anterior cochlear nucleus morphology and the role of gonadal hormones

Gonadectomized and intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to an augmented acoustic environment (AAE): repetitive bursts of a 70dB SPL noise band. The high-frequency AAE (HAAE) was a half-octave band centered at 20kHz; the low-frequency AAE (LAAE) was a 2-8kHz band. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (a trait of B6 mice) were evaluated by obtaining auditory brainstem response (ABR) thresholds at ages 3-, 6-, and 9-months. At 9-months of age, hair cell counts (cytocochleograms) were obtained, and morphometric measures of the anteroventral cochlear nucleus (AVCN) were obtained. LAAE treatment caused elevation in ABR thresholds (8-24kHz), with the highest thresholds occurring in intact females. LAAE treatment caused some loss of outer hair cells in the basal half of the cochlea (in addition to losses normally occurring in B6 mice), with intact females losing more cells than intact males. The loss of AVCN neurons and shrinkage of tissue volume that typically occur in 9-month-old B6 mice was lessened by LAAE treatment in intact (but not gonadectomized) male mice, whereas the degenerative changes were exacerbated in intact (but not gonadectomized) females. These LAAE effects were prominent in, but not restricted to, the tonotopic low-frequency (ventral) AVCN. HAAE treatment resulted in some loss of neurons in the high-frequency (dorsal) AVCN. In general, LAAE treatment plus male gonadal hormones (intact males) had an ameliorative effect whereas HAAE or LAAE treatment plus ovarian hormones (intact females) had a negative effect on age-related changes in the B6 auditory system.