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 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.     

KCNQ1在小鼠耳蜗血管纹的表达及在听觉中的意义

目的探讨KCNQ1在耳蜗侧壁血管纹的表达及其在听觉中的作用。方法以不同基因型小鼠KC-NQ1-/-(突变纯合子)、KCNQ1 /-(杂合子)和KCNQ1 / (野生型)以及C57BL/6J小鼠为实验对象,采用免疫组织化学和ABR检测技术,检测KCNQ1在小鼠耳蜗血管纹的表达及其听力。结果KCNQ1蛋白阳性颗粒集中在小鼠耳蜗血管纹边缘细胞顶膜。KCNQ1 / 小鼠的听力正常,短声ABR的阈值为36.67±7.13dBSPL;KCNQ1 /-小鼠听力低于同窝KCNQ1 / 野生型鼠,短声ABR的阈值为38.25±9.35dB SPL;KCNQ1-/-小鼠呈现全聋,ABR在100dB SPL时仍无反应。结论KCNQ1是位于耳蜗侧壁血管纹边缘细胞的重要通道蛋白,在维系耳蜗听觉功能中有重要作用。KCNQ1通道蛋白的缺失或功能受限可以不同程度地影响耳蜗的听觉功能。     

小鼠耳蜗Na-K-2Cl联合转运子-1的定位及其缺失后的耳蜗组织学改变

目的探讨耳蜗钾循环途径中Na-K-2Cl联合转运子-1(Na-K-2Clcotransporter-1,NKCC1)在小鼠耳蜗的分布及NKCC1基因敲除后耳蜗组织学的改变。方法选用10只C57BL/6J小鼠((NCKK1 / )和5只NKCC1基因敲除小鼠(NKCC1-/-),应用听性脑干反应(auditorybrainstemresponse,ABR)分别检测NCKK1 / 小鼠和NKCC1-/-小鼠的听功能,采用免疫组织化学及甲苯胺蓝染色的方法观察NKCC1在NCKK1 / 小鼠耳蜗的定位及NKCC1-/-小鼠耳蜗组织学的变化。结果NCKK1 / 小鼠ABR平均阈值为31±5.36dBSPL,而NKCC1-/-小鼠听力完全丧失。NKCC1在NCKK1 / 型小鼠耳蜗主要分布在血管纹上皮(边缘细胞)和螺旋韧带下部纤维细胞,在纹上区和螺旋缘处的纤维细胞中也有适度表达;NKCC1-/-小鼠耳蜗前庭膜塌陷,中阶完全消失,内毛细胞、外毛细胞、支持细胞减少,Corti隧道消失。结论NKCC1在耳蜗的定位与耳蜗钾循环密切相关,NKCC1缺失会导致耳蜗正常结构的破坏,继而影响耳蜗生理功能。     

C57BL/6J 小鼠耳蜗血管纹Na-K-2Cl联合转运子-1的年龄相关性变化

目的观察不同周龄C57BL/6J(C57)小鼠听力及血管纹Na-K-2Cl联合转运子-1(Na-K-2Cl co-transporter-1,NKCC1)表达的情况。方法应用听性脑干反应(auditory brainstemresponse,ABR)分别检测4、8、16、32、48、64周龄组C57小鼠的听力;采用免疫组织化学染色法观察其血管纹NKCC1表达的变化。结果C57小鼠随年龄增大出现听力下降,自16周龄时ABR阈值出现显著性增高(P<0.05);血管纹NKCC1表达也出现年龄相关性减少,其灰度值自16周龄时显著增高(P<0.01)。结论C57小鼠血管纹NKCC1蛋白表达随年龄增长而减少,可能与年龄相关性听力损失具有一定相关性。     

PDCD5和caspase3在不同年龄段C57BL/6J小鼠耳蜗中的表达

目的 检测程序化细胞死亡分子5（programmed cell death 5,PDCD5）和半胱氨酸天冬氨酸蛋白酶3（caspase 3）在不同年龄段C57BL/6J小鼠耳蜗中的表达,初步探讨其在年龄相关性听力下降发生、发展中的作用。方法 选择3、6、9及12月龄段C57BL/6J小鼠各15只,即按月龄分为四组。检测各组小鼠双侧短声( click)及短纯音(6、8 kHz)听性脑干反应(ABR)阈值。采用免疫组化和蛋白质印迹杂交(Westem blotting)检测各月龄段小鼠耳蜗PDCD5和Caspase3蛋白的表达,实时荧光定量PCR( real-time PCR)检测各月龄段小鼠耳蜗PDCD5和caspase 3基因mRNA的表达。结果 随着年龄的增长,C57BL/6J小鼠各频率ABR阈值逐渐提高,耳蜗PDCD5和Caspase3蛋白的表达亦逐渐增强。3月龄和6月龄小鼠耳蜗毛细胞和血管纹细胞仅出现少量PDCD5和Caspase3蛋白表达,9月龄时表达有明显增加,至12月龄时表达最强,各月龄组间比较,差异具有统计学意义(P值均＜0.05)。Real-time PCR检测显示PDCD5和caspase3基因mRNA随着年龄的增长表达逐渐增强,与其蛋白变化趋势相一致。结论 C57 BL/6J小鼠耳蜗PDCD5和caspase3随着年龄的增长表达增强,与耳蜗的老化密切相关,可能是老年性聋发病机制中的一个重要因素。     

晚期糖基化终产物受体在小鼠耳蜗中表达的初步探讨

目的通过研究晚期糖基化终产物受体(RAGE)在小鼠耳蜗中的表达,初步探讨RAGE与老年性聋关系。方法将C57BL/6J小鼠分为2月龄、10月龄组,分别行ABR阈值检测,组织学切片观察耳蜗形态结构,免疫组化和realtime PCR检测RAGE在耳蜗中的定位和定量表达,半定量RT-PCR检测其配体S100B在耳蜗中的表达情况。结果 :(1)10月龄小鼠较2月龄ABR阈值明显提高;(2)RAGE主要在两组小鼠耳蜗的螺旋神经节细胞,内外毛细胞和血管纹中表达,10月龄C57BL/6J小鼠耳蜗中RAGE平均光密度值比2月龄小鼠大,两组间差异有统计学意义;(3)2月龄组耳蜗中RAGE相对表达量为1±0.15,10月龄组小鼠耳蜗中RAGE相对表达量为2.0816±0.117,两组间差异有统计学意义;(4)配体S100B在10月龄小鼠耳蜗中表达增高。结论 RAGE在C57BL/6J小鼠耳蜗中有表达且随年龄增大表达增强,可能与配体S100B相互作用导致听力下降,提示RAGE可能与老年性聋的发病机制有关。     

XIAP在年龄相关性听力减退C57BL/6J小鼠初级听皮质的表达

目的探讨幼年和10月龄C57BL／6J小鼠听力及初级听皮质（AI）中凋亡抑制蛋白XIAP的年龄相关变化。方法检测两组C57BL/6J小鼠听性脑干反应（ABR）;免疫组织化学法染色检测两组C57BL/6J初级听皮质神经元凋亡抑制蛋白XIAP的表达情况。结果与幼年C57BL／6J小鼠相比,10月龄C57BL/6J的ABR反应阈值更高,凋亡抑制蛋白XIAP的表达显著减少。结论随年龄增长C57BL/6J小鼠的听力减退,同时C57BL／6J小鼠大脑初级听皮质凋亡抑制蛋白表达减少,XIAP的表达水平可能与C57BL/6J小鼠听力减退有关。     

老化型C 57B L/6J小鼠耳蜗外侧壁血管纹Claudin-5、N F-κB、P21的表达

目的 探讨紧密连接蛋白(Claudin-5)、核转录因子(NF-κB)、细胞周期抑制蛋白(P21)在老化型C57BL/6J小鼠耳蜗外侧壁血管纹内的表达及意义.方法 将C57BL/6J小鼠分为青年组(3月龄)和老年组(24月龄),每组各20只,检测两组小鼠听性脑干反应(ABR)的反应阈,分别行组织学切片观察耳蜗外侧壁血管...     

卡那霉素对三种小鼠耳毒性比较及对血管纹Na-K-2Cl联合转运子1表达的影响

目的建立小鼠氨基糖甙类抗生素（aminoglycoside antibiotics，AmAn）耳毒性模型，探讨在不同种小鼠中的AmAn耳毒易感性及其对耳蜗血管纹Na—K-2Cl联合转运子-1（Na-K-2Cl cotransporter-1，NKCC1）表达的影响。方法将72只C57BE／6J、CBA／CaJ、NKCC1＋／-小鼠各自随机分为A、B、C、D4组。A组：卡那霉素组；B组：卡那霉素＋2，3-二羟基苯甲酸组；C组：2，3-二羟基苯甲酸组；D组：生理盐水组。各组连续用药14d。各组动物在用药前、用药后第14天及用药后第35天行脑干诱发电位（auditory brainstem response，ABR）检测听功能；耳蜗琥珀酸脱氢酶组织化学染色观察耳蜗形态学变化；免疫组化法观察血管纹NKCC1表达的变化。结果①A组小鼠ABR闽值明显提高（P〈0．01）并伴随外毛细胞的减少；②B组小鼠ABR阈值变化明显小于A组（P〈0．01），外毛细胞的损害也明显减轻；③A组小鼠耳蜗血管纹NKCCl表达减弱，与D组比较有明显差异（P〈0．01），而B组小鼠血管纹NKCC1表达较A组增强（P〈0．01）；④3种小鼠中CBA／CaJ小鼠对AmAn最敏感，C57BE／6J和NKCC1＋／-小鼠对AmAn的易感性无明显差异。结论应用卡那霉素可建立小鼠AmAn耳毒性模型；卡那霉素可抑制血管纹NKCC1的表达；2，3-二羟基苯甲酸拮抗AmAn耳毒性的途径之-可能是通过减轻AmAn对血管纹NKCC1的抑制作用；具有年龄相关性听力损失特性的小鼠对AmAn耳毒作用并不易感。     

C57BL/6J小鼠听力及耳蜗毛细胞活性的年龄相关性研究

目的 建立年龄相关性听力损失(age-related hearing loss,AHL)的小鼠动物模型,探讨C57BL/6J小鼠发生AHL与毛细胞活性变化的关系,并初步对C57BL/6J小鼠AHL模型进行AHL的病理分类.方法 按3、8、9、10、17、18月龄段分6组培育C57BL/6J小鼠,各组分别进行听性脑干反应(ABR)测试,对耳蜗毛细胞行琥珀酸脱氢酶染色并作基底膜硬铺片,观察各年龄段小鼠内外毛细胞线粒体琥珀酸脱氢酶的活性.结果 C57BL/6J小鼠随年龄增大,ABR阈值明显增高,在3月龄到9月龄期间ABR平均反应阈值增大比较缓慢,差异无统计学意义;在10月龄时,出现明显的听力下降,平均阈值比3月龄时约高18～23 dB,差异有统计学意义(click:t=5.78,P＜0.01;6 kHz:t =3.93,P＜0.01;8 kHz:t=3.01,P＜0.05).10月龄后小鼠听力继续下降,21月龄时平均阈值比3月龄时增高约60～68 dB,差异有显著统计学意义(click:t=31.23,P＜0.01;6 kHz:t=30.44,P＜0.01;8 kHz:t=33.83,P＜0.01).琥珀酸脱氰酶染色显示,随年龄增大,毛细胞线粒体活性丧失逐渐加重:先是底回外毛细胞活性下降,接着发生活性消失,并逐渐向顶回发展,最后累及内毛细胞.结论 C57BL/6J小鼠具有典型的年龄相关性听力损失特点,其听力下降的原因早期可能主要足外毛细胞及内毛细胞活性的丧失,晚期可能是由于基底膜结构混乱,导致电生理屏障消失,致耳蜗内电位(EP)不能维持而引起.C57BL/6J小鼠可作为感音型老年性听力损失动物模型.     

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.     

半拷贝基因NKCC1杂合小鼠与年龄相关性听力下降的关系

目的培育杂合子NKCC1＋／一和野生型NKCC1＋／＋小鼠，观察携带不同拷贝基因的NKCC1小鼠的年龄相关性听力表现，研究NKCC1基因及其离子通道在年龄相关性听力下降（agerelated hearing loss，AHL）中的作用，并初步探讨其可能发生的机制。方法运用NKCC1＋／一杂合子小鼠和NKCC1＋／＋野生型小鼠为平台，于小鼠生长过程中的各个年龄段分别检测小鼠的听性脑干反应（auditory brainstem response，ABR）和耳蜗内电位（endocochlear potential，EP）；同时采用扫描电镜进行耳蜗形态学观察和免疫印迹杂交检测不同鼠龄小鼠耳蜗的NKCC1通道蛋白的含量。结果NKCC1＋／一杂合子小鼠与NKCC1＋／＋野生型小鼠相比较，其ABR检测短声阈值在每一年龄组的各测试频率均升高，各年龄组相比较差异有统计学意义（P值均〈0．05）。NKCC1＋／一杂合子半拷贝基因鼠的听力随年龄增长而逐渐下降，与低龄鼠相比，老龄鼠的ABR阈值显著性升高（P值均〈0．05）。NKCC1＋／-小鼠的EP也随年龄增长而呈下降趋势，老龄小鼠的EP值与其低龄鼠相比明显降低（P〈0．05）。老龄小鼠的耳蜗NKCC1蛋白含量低于低龄鼠。与老龄NKCC1＋／＋鼠相比较，老龄NKCC1＋／-鼠的耳蜗底回外毛细胞出现散在的点状缺失。结论当NKCC1基因呈现半拷贝复制时小鼠可呈现AHL，NKCC1基因可能在AHL的发病中起一定的作用。AHL的发病可能与NKCC1通道蛋白的遗传特性及功能有关，还可能与NKCC1＋／-鼠出现的耳蜗底回外毛细胞缺失有关。     

Age-Related Hearing Loss in C57BL/6J Mice has both Frequency-Specific and Non-Frequency-Specific Components that Produce a Hyperacusis-Like Exaggeration of the Acoustic Startle Reflex

Auditory brainstem-evoked response (ABR) thresholds were obtained in a longitudinal study of C57BL/6J mice between 10 and 53 weeks old, with repeated testing every 2 weeks. On alternate weeks, acoustic startle reflex (ASR) amplitudes were measured, elicited by tone pips with stimulus frequencies of 3, 6, 12, and 24 kHz, and intensities from subthreshold up to 110 dB sound pressure level. The increase in ABR thresholds for 3 and 6 kHz test stimuli followed a linear time course with increasing age from 10 to 53 weeks, with a slope of about 0.7 dB/week, and for 48 kHz a second linear time course, but beginning at 10 weeks with a slope of about 2.3 dB/week. ABR thresholds for 12, 24, and 32 kHz increased after one linear segment with a 0.7 dB slope, then after a variable delay related to the test frequency, shifted to a second segment having slopes of 3–5 dB/week. Hearing loss initially reduced the ASR for all eliciting stimuli, but at about 6 months of age, the response elicited by intense 3 and 6 kHz stimuli began to increase to reach values about three times above normal, and previously subthreshold stimuli came to elicit vigorous responses seen at first only for the intense stimuli. This hyperacusis-like effect appeared in all mice but was especially pronounced in mice with more serious hearing loss. These ABR data, together with a review of histopathological data in the C57BL/6 literature, suggest that the non-frequency-specific slow time course of hearing loss results from pathology in the lateral wall of the cochlea, whereas the stimulus-specific hearing loss with a rapid time course results from hair cell loss. Delayed exaggeration of the ASR with hearing loss reveals a deficit in centrifugal inhibitory control over the afferent reflex pathways after central neural reorganization, suggesting that this mouse may provide a useful model of age-related tinnitus and associated hyperacusis.     

Degeneration of stria vascularis in age-related hearing loss; a corrosion cast study in a mouse model

Conclusion With age, in a mouse model, degenerative changes to the capillaries of the stria vascularis are observed. These range from a narrowing of vessel lumen to complete degeneration of strial vessels. Other vascular beds in the cochlea are relatively unchanged with age. Strial capillaries at the cochlear base are significantly more affected than those in mid-apical turns.

Objectives Previous work suggests that age-related hearing loss is associated with degenerative changes to cochlear vasculature; the term strial presbyacusis is often cited. This study reports on vascular changes observed in a murine model of presbyacusis, analyzed using corrosion cast techniques.

Methods A novel corrosion cast technique was developed to compare cochlear vasculature in control mice (non-presbycusic, CD1) and old (>?6 months) C57BL/6 animals. ABR measures indicated a significant age-related threshold elevation in the C57BL/6 mice. Cochlear vascular casts were imaged using scanning electron microscopy, and vessel degeneration was quantified by measuring capillary diameters.

Results Corrosion casts of cochlear vasculature in 6–12 month old C57BL/6 mice reveal significant degeneration of stria vascularis. Other capillary beds (spiral ligament and the spiral limbus) appear unchanged. Comparison of strial capillary diameters reveals significantly more damage in basal/lower-turn regions compared with the cochlear mid-turn.