编码Na-K-2Cl的基因缺陷小鼠出现听觉和平衡功能障碍及内耳形态异常

Deficiency in Na-K-2Cl co-transporter impaired hearing and balance in mice

目的:培育突变型纯合子NKCC1-/-、杂合子NKCC1+/-及野生型NKCC1+/+小鼠,将这3种基因型小鼠作为实验平台,研究NKCC1离子通道(Na-K-2Cl,co-transporter)在耳蜗听觉平衡功能中的作用,并观察不同基因型小鼠的内耳形态特征。方法:利用同窝生各个基因型小鼠NKCC1-/-(突变纯合子)、NKCC1+/-(杂合子)和NKCC1+/+(野生型)为实验对象,采用听觉脑干反应(ABR)和耳蜗内电位(EP)检测技术,对各基因型小鼠的听觉功能和EP进行检测;同时利用光学显微镜观察不同基因型小鼠的内耳形态变化。结果:NKCC1+/+野生型小鼠听力正常,ABR检测的click短音阈值为[(23.13±3.78)dB,SPL];EP为(98±16)mV。NKCC1+/-杂合子小鼠听力低于同窝生的NKCC1+/+野生型小鼠,ABR检测其Click短音阈值为[(38.49±12.29)dB,SPL],EP为(78±7)mV。NKCC1+/+和NKCC1+/-小鼠ABR的阈值均值在各个频率的差异显著(P<0.01);两者EP的均值差异也显著(P<0.05)。NKCC1-/-突变型纯合子小鼠呈现全聋,ABR各个频率在100dB均无反应,其EP接近消失(4mV±6mV)。NKCC1-/-突变型纯合子小鼠表现出听觉功能丧失和旋转为主的平衡失调。光镜下NKCC1+/+野生型小鼠和NKCC1+/-杂合子小鼠的耳蜗解剖结构无明显异常;NKCC1-/-突变纯合子小鼠的耳蜗出现前庭膜塌陷、内淋巴腔缩小而致中阶完全消失,并有血管纹萎缩、Corti器缺失、螺旋神经节萎缩;且球囊和椭圆囊膜迷路失去正常结构并出现塌陷等病理改变。结论:NKCC1离子通道在内耳K+循环和维系内耳正常听觉平衡功能中起重要作用。NKCC1通道的缺失或功能受限,均可导致耳蜗内淋巴K+负荷水平,进而影响耳蜗的听觉平衡功能。

AIM： We generated transgenic mice of NKCC1^-/- （homozygous mutant）, NKCC1^＋/- （heterozygous） and NKCC1^＋/＋（wild -type） that have a targeted disruption in the NKCC1 gene to investigate the role of Na -K - 2Cl （NKCC1） channel in auditory function of the inner ear. METHODS： Hearing threshold and endocochlear potential （EP） were measured in the NKCC1^-/- , NKCC1^＋/- and NKCC1^＋/＋ mice by auditory brainstem response （ABR） and EP recordings, respectively. The inner ears of the mice were removed and examined morphologically with the light microscope. RESULTS： The auditory function of NKCC1^＋/＋ mice was normal, the mean value for ABR thresholds in response to click sound was [ （23. 13±3.78）dB, SPL], EP was （98 ±16） mV. The mean value for ABR thresholds to click sound was elevated in NKCC1 ^＋/-mice [（38.49± 12.29） dB, SPL], relative to that significantly increased in NKCC1^＋/＋ mice （P〈0.01）. EP in NKCC1^＋/- mice was about （78±7） mV, significantly decreased than that in NKCC1^＋/＋ mice （P 〈0.05 ）. NKCC1^-/- mice were completely deaf, the ABR waveform was not observed for even 100 dB SPL sound stimuli used and EP was nearly disappeared （EP, 4 mV±6 mV）. NKCC1^-/- mice were deaf and demonstrated difficulties in maintaining their balance. NKCC1^-/- mice exhibited a marked atrophy of the stria vascularis, contraction of the endolymphatic compartments and collapse. CONCLUSION： NKCC1 channel plays a critical role in potassium homeostasis of endolymph in the inner ear. Mice lacking of NKCC1 can lead to changing K^＋ concentration in endolymph and influence auditory function subsequently. NKCCI knockout mice exhibit marked structural abnormalities of the cochlea as well.