两种听力老化小鼠听力特征及优缺点分析

目的 利用听性脑干反应（ABR）阈值作为检测项目,系统总结C57BL/6J(B6J)小鼠与SAMP8小鼠在不同月龄时听力动态变化规律及其作为老年性听力损失研究模型的特点,并对比不同月龄C57BL/6J小鼠与SAMP8小鼠ABR反应阈值差异,为老年性听力损失研究动物模型选择提供经验。方法 选用67只不同月龄C57BL/6J小鼠以及15只SAMP8小鼠,采用ABR阈值测试方法,以引出可辨认的ABR波I波的最小声强定为阈值,统计ABR反应阈值结果。结果 C57BL/6J小鼠与SAMP8小鼠均表现出听力早衰问题。其中C57BL/6J小鼠在6月龄便有听力下降表现,但以高频听力衰退为主,直至18月龄时,出现双侧对称性全聋。该品系小鼠在听力衰老过程中表现出个体差异较大的特征,部分小鼠表现出双侧听力不对称性损失。SAMP8小鼠在5月龄时仅表现出高频听力损失,在7月龄时表现出全频听力下降,以高频听力损失为著。该品系小鼠个体差异较小,且表现为双侧听力对称性损失。结论 C57BL/6J小鼠和SAMP8小鼠均可作为老年性听力损失研究的动物模型。C57BL/6J小鼠的优点在于：获取方式简单,对麻醉耐受性较好。缺...     

C57BL/6J小鼠在不同剂量氨基糖甙类药物暴露下听力损害的特点

目的探讨C57BL/6J小鼠在不同剂量氨基糖甙类药物毒性作用下听力损害的特点。方法采用不同剂量的庆大霉素对成年C57BL/6J小鼠进行腹腔注射,小鼠随机分成五组,药物剂量浓度分别为300mg/kg,200mg/kg,100mg/kg和50mg/kg。对照组为等量的注射用0.9%生理盐水,药物注射连续应用7天。分别于用药前(p0,对照组)以及用药后第2天(P2)、第4天(P4)、第7天(P7)检测动物的听功能(Click&Tonebrust),并进组间差异的比较分析。结果 (1)在50mg/kg组,用药后第7天的听阈值和对照组听阈分别为48.33±10.33(P7)和41.67±11.20(P0),两者相比具有显著性差异(P<0.05)。在100mg/kg组,用药后第4天的听阈值和对照组相比具有显著性差异(P4:48.89±9.16;P0:40.83±10.07,P<0.05)。在200mg/kg和300mg/kg组,用药后第2天的听阈值和对照组相比有显著上升(200mg/kg,P2:50.56±5.39;P0:40.42±9.66,P<0.05),(300mg/kg,P2:50.50±6.85,P0:40.00±11.00,P<0.05)。(2)各剂量组药物在Click和Toneburst(4kHz,8kHz&t16kHz)频率中,听力损害程度存在区别,损害程度依次为:200mg/kg组>300mg/kg组>100mg/kg组>50mg/kg组。(3)在8kHz&16kHz频率上,①50mg/kg与300mg/kg剂量组在给药后第2、4、7天均无明显听力变化(P>0.05)。②在100mg/kg组:与对照组相比,给药后第4天、第7天的听阈出现明显升高(P<0.05)。③在200mg/kg组:给药后第2、4、7天的听阈值亦出现显著升高(P<0.05)。(4)在给药后第7天,各剂量组的听阈均达到峰值。结论本研究表明:(1)随着给药剂量的增加,C57BL/6J小鼠听力损害出现的时间提前,但听力损害的程度并非同步加重;(2)以200mg/kg浓度给药后,小鼠听力在Click&Toneburst上均可以产生明显变化,同时小鼠的生理状态依然保持良好,因而200mg/kg(庆大霉素)浓度可能是C57BL/6J小鼠药物性致聋的理想剂量。     

C57BL/10J小鼠内耳形态学观察

目的 探讨不同月龄C57BL／10J小鼠内耳形态学变化。方法 取C57BL/10J小鼠2月龄3只（6耳）、8月龄3只（6耳）、12月龄2只（4耳）、27月龄2只（4耳），采用耳蜗铺片和耳蜗图技术，观察耳蜗和前庭毛细胞的变化。结果 2月龄鼠在耳蜗底回和顶回可见有少量的外毛细胞缺失，内毛细胞正常。8月龄鼠外毛细胞缺失主要由耳蜗底回向顶回发展，少量内毛细胞缺失主要在耳蜗底回，对应于20kHz的基底膜区域显示外毛细胞缺失在20％左右。12月龄鼠外毛细胞缺失明显加重，耳蜗底回部分几乎完全缺失，外毛细胞损失区域发展到基底膜中部，并且蜗尖区域外毛细胞缺失达到40％，耳蜗底回内毛细胞缺失达到60％～80％，蜗尖部分内毛细胞基本正常，对应于20kHz的基底膜区域显示外毛细胞缺失近80％，内毛细胞缺失近30％。27月龄鼠耳蜗外毛细胞从基底膜中部至底回完全缺失，顶回缺失60％～80％，内毛细胞缺失逐渐向蜗尖扩展，对应于20kHz的基底膜区域显示外毛细胞完全缺失，内毛细胞缺失超过50％。前庭选取球囊斑，椭圆囊斑和壶腹嵴三个部位进行观察，不同月龄的C57BL／10J鼠前庭毛细胞均正常。结论 本研究首次对C57BL／10J小鼠的内耳形态学进行观察，发现该鼠同C57BL／6J小鼠比较在外毛细胞缺失规律和时间上有明显的差别，明确了内耳毛细胞的变化规律；同时首次观察了前庭毛细胞，为C57BL／10J小鼠作为遗传性和老年性聋研究的动物模型提供了形态学理论依据。     

EFFECTS OF MODERATE NOISE EXPOSURE ON HEARING FUNCTION IN C57BL/6J MICE

Objective To study characteristics of hearing loss after exposure to moderate noise exposure in C57BL/6J mice. Methods Male C57BL/6J mice with normal hearing at age of 5-6 weeks were chosen for this study. The mice were randomly sclccted to be studied immediately after exposure （Group P0）, or 1 day （Group P1）, 3 days （Group P3）, 7 day （Group P7） or 14 days （P14） after exposure. Their before exposure condition served as the normal control. All mice were exposed to a broad-band white noise at 100 dB SPL for 2 hours, ABR thresholds were used to estimate hearing status at each time point. Results ABR threshold elevation was seen at every tested frequency at P0 （P〈0.01）. Elevation at high-frequencies （16 kHz and 32 kHz） was greater than at lower frequencies （4 kHz and 8 kHz, P〈0.05）. From P1 to P14, ABR thresholds continuously improved, and there was no significant difference between P14 and before exposure （P〉0.05）. Conclusion There is a frequency specific re- sponse to 100 dB SPL broad-band white noise in C57BL/6J mice, with the high-frequency being more susceptible. Hearing loss induced by moderate noise exposure appears reversible in C57BL/6J mice.     

Oxidative Stress in Aging in the C57B16/J Mouse Cochlea

Presbycusis is a complex of high frequency hearing loss and disproportionate loss of speech discrimination that is seen concomitantly with physical signs of aging. Among the most extensively characterized strains of mice that show an early hearing loss is the C57B16/J strain, a strain that shows early onset of high frequency hearing loss at age 6 months and complete hearing loss by 1 year of age. The histopathology of this strain consists of loss of hair cells and spiral ganglion neurons in the basal turn, with a progression of loss of hair cells and ganglion neurons towards the apical portion of the cochlea as the animal ages. The process of aging has been extensively studied and although details differ in various organisms the consensus today is that oxidative stress, i.e. free radical-mediated tissue damage, is one of the core mechanisms of aging. Aerobic metabolism results in the creation of hydrogen peroxide and reactive oxygen species. These are normally detoxified by a variety of enzymes and free radical scavengers, including superoxide dismutase (SOD), catalase and glutathione. To determine whether oxidative stress plays a role in the pathophysiology of hearing loss in this mouse model of presbycusis we determined the relative change in mRNA production for selected free radical detoxifying enzymes in the C57B16/J mouse cochlea. Using semi-quantitative RT-PCR with tubulin mRNA as a control, relative levels of antioxidant enzyme mRNAs were determined. There was an overall increase in SOD1 mRNA levels when comparing 1 and 9 month time points, and a transient increase in the expression level of catalase mRNA. B6.CAST +Ahl mice, which carry the C57B16/J genome but receive their Ahl gene from CAST mice, do not show these alteractions in antioxidant enzyme production. Our results suggest that at an age of 9 months, at which point significant hearing loss has developed, the C57B16/J mouse cochlea is exposed to increased levels of free radicals and that the Ahl gene of the C57B16/J mouse mediates this decrease in protective enzymes and therefore increase in levels of oxidative stress.     

Down-regulation of the large conductance Ca2+-activated K+ channel expression in C57BL/6J cochlea

Conclusion: The large conductance Ca²⁺-activated K^+?channels (BK) expression is decreased in the cochleae of age-related hearing loss (AHL) mice. BK channel may be associated with AHL.

Objective: AHL is the most common among elderly persons. BK channels act as sensors for membrane voltage and intracellular Ca^2+?and are essential for hearing. To investigate the distribution of BK channel in the cochleae of C57BL/6J mice, and the relationship between the expression of BK channel and the etiology of AHL.

Methods: BK expression was studied in the cochleae of C57BL/6J mice at various ages (4, 12, 26, 52 weeks). The expressions of BK at the protein and mRNA levels were detected by immunofluorescence technique, western blot and quantitative real time PCR.

Results: In comparison with 4-week-old mice, BK expressions in the cochleae at 12, 26 and 52 weeks of age were significantly and gradually decreased at both the protein and the mRNA levels. The immunofluorescence technique showed the BK channel was located in the hair cells and cells of the spiral ganglion, spiral ligament and stria vascularis and its expression also decreased with aging.     

C57BL/6小鼠内耳形态学和年龄相关性听力损失的研究

目的探讨不同周龄C57BL/6小鼠内耳形态学及其ABR阈值变化。方法取C57BL/6小鼠3周、4周、12周、26周各10只,听性脑干反应(ABR)测试双侧2、4、8、16、20 kHz ABR阈值。采用基底膜铺片MyosinⅥ、Neurofilament免疫组化染色,观察耳蜗毛细胞和神经丝的变化。扫描电镜观察耳蜗毛细胞及其静纤毛随年龄的变化。结果随着年龄增长,C57BL/6小鼠各频率ABR阈值明显提高,顶转和底转内毛细胞缺失逐渐增多,神经丝染色渐淡,毛细胞静纤毛逐渐发生数量减少、增粗融合、倒伏等变化。到26周龄时已达到重度聋,各频率较3周组有显著统计学差异。顶转和底转内毛细胞有连续缺失,外毛细胞完全缺失,内毛细胞只有残存的少量静纤毛,粗细不均,倒伏明显。结论本研究对国产C57BL/6小鼠的内耳形态进行观察,明确了其ABR阈值和内耳毛细胞的变化规律,为用国产C57BL/6小鼠进行老年性聋研究提供了依据。     

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.     

腱糖蛋白-W在不同发育阶段C57BL/6小鼠耳蜗中的表达

目的研究Tnn基因编码的腱糖蛋白-W(tenascin-W,TN-W)在出生后不同发育阶段小鼠耳蜗内的表达,初步探讨tenascin-W在听觉系统中的功能。方法以出生后(postnatal day,P)1、3、14、28天(P1、P3、P14、P28)各6只及出生后7天(P7)9只C57BL/6小鼠作为研究对象,通过免疫荧光标记观察tenascin-W在耳蜗中的表达及定位,运用实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测各发育阶段小鼠耳蜗中Tnn mRNA相对表达水平,Western blot法检测tenascin-W在小鼠耳蜗中的表达。结果免疫荧光标记显示tenascin-W在P1、P3、P7、P14、P28小鼠耳蜗中均有表达。在P1、P3、P7小鼠耳蜗螺旋神经节、Corti器中均有表达,在P14、P28小鼠仅在螺旋神经节高表达,在Corti器中不表达,在各发育阶段小鼠耳蜗血管纹均有表达;共染显示其与Ⅲ型β-tubulin共定位在螺旋神经节神经元胞体的胞浆中。RT-qPCR显示Tnn mRNA在P7小鼠耳蜗相对表达量最高,Western blot法检测到tenascin-W在P7小鼠耳蜗中有表达。结论Tenascin-W在小鼠耳蜗各发育阶段均有表达,在P7表达量最高,且主要定位于螺旋神经节神经元,提示该蛋白可能影响耳蜗螺旋神经元发育及功能,对小鼠正常听觉的维持发挥重要作用。     

年龄相关性听力损失小鼠听皮层神经元凋亡及听功能变化

目的探讨年龄相关性听力损失C57BL/6J小鼠初级听皮层神经元凋亡及听功能变化。方法选取年轻组（2月龄）和老年组（10月龄）C57BL/6J小鼠为研究对象,通过听性脑干反应（ABR）检测并比较年轻组与老年组小鼠听力变化;用HE染色法、Tunel-POD法观察并比较两组小鼠初级听皮层神经元大体形态及凋亡比例的差异。结果老年组小鼠较年轻组小鼠听阈明显提高。老年组C57BL/6J小鼠初级听皮层神经元凋亡比例较年轻组增多。结论初级听皮层神经元的凋亡与C57BL/6J小鼠年龄相关性听力损失密切相关。