内耳注射药物建立新生鼠耳聋动物模型

A Hearing Loss Model in Mouse Neonate by Direct Drug Delivery to the Cochlea

目的通过纳升级微量注射泵内耳注射给药的实验方法,建立新生鼠耳聋动物模型。方法 42只出生第二天新生鼠,分别以注射速度分组:单纯穿刺组(对照组,6只,仅穿刺耳蜗底回侧壁,不注射液体)、蒸馏水1组(H2O 20组,4只,20nl/min)、蒸馏水2组(H2O 60组,3只,60nl/min)及药物浓度分组(新霉素各组给药速度为60nl/min):新霉素1组(Neo1组,4只,1μmol/ml)、新霉素2组(Neo10组,4只,10μmol/ml)、新霉素3组Neo 50组,5只,50μmol/ml)、新霉素4组(Neo 100组,8只,100μmol/ml)及新霉素5组(Neo 50 1组,8只,50μmol/ml)。各组均采用耳蜗底回微创打孔技术,利用纳升级微量注射泵按上述各组用药、剂量及给药速度进行内耳药物注射。术后1天(Neo 50 1组)及3天(其余各组)采用基底膜免疫荧光染色观察内耳毛细胞及支持细胞的形态改变。结果术后新生鼠存活好,伤口恢复优良。耳蜗毛细胞机械性损伤与注射速度呈正相关,术后3天,H2O 20组和H2O 60组注射区域内、外毛细胞略减少,差异无统计学意义(P>0.05)。毛细胞损伤与新霉素浓度呈正相关,与对照组比较,术后3天Neo1组的外毛细胞数量及Neo10、50、100组内、外毛细胞数量均明显减少(P<0.05)。与H2O 60组比较,术后3天Neo50及Neo100组内、外毛细胞数量均明显减少(P<0.05)。新霉素50μmol/ml注射后1天(Neo 50 1组)与3天(Neo 50组)内、外毛细胞数量差异无统计学意义(P>0.05)。新霉素50μmol/ml注射后3天免疫荧光染色示支持细胞形态良好。结论利用纳升级微量注射泵给药方式注射50μmol/ml新霉素200纳升至内耳,可建立新生鼠耳聋动物模型,具有毛细胞死亡率高,致聋效果显著的优点;而支持细胞保留较好,且动物存活率高。

Objective This study aimed to establish a hearing loss model specific to hair cells loss in neonatal mouse using a nanoliter micro--injection system. Methods We injected a single dose of aminoglycosides antibiotics （200 nl） into the scalar media of 42 neonatal mice cochlea at postnatal day 2. Mice were divided into 8 groups,they were surgery control group（n=6）, water injection group 1 （injection at speed of 20 nl/min, n=4） and water injection group 2（at speed of 60 nl/mim,n=3）, and neomycin group 1 （injection speed at 1 umol/ml,n=4）, neomycin group 2 （ 10 umol/ml, n = 4）, neomycin group 3 （ 50 umol/ml, n = 5）, neomycin group 4 （ 100 umol/ml, n = 8） and neomycin group 5 （50 umol/ml, n= 8, sacrificed day 1 after surgery）. The histology change of the inner ear hair cells and the supporting cells was examined at 1 day or 3 days after surgery. Results After the injection, all 42 mice survived and wound healed. The mechanical damage to hair cells was proportional to the injection speed and volume. A small number of hair cells were lost, but not statistically significant at the injection site in water injection group 1 and group 2 which the injection speed was 20 and 60 nl/mim,respectively. Neomycin injection resulted in a dose--dependent damage to hair cells. Compared to surgery control, 1 umol/ml neomycin induced a significant loss of outer hair cells, 10 umol/ml, 50 umol/ml and 100 umol/ml showed significant loss of both inner and outer hair cells 3 days after injection （P〈0.05）. Compared to the water injection group 2（60 nl/min）, 50 umol/ml and 100 umol/ml neomycin led to a significant loss of inner and outer hair cells （P〈0.05）. Neomycin injection at a concentration of 50 umol/ml induced a quick hair cell loss 1 day after injection, and no significant differences were observed 3 days after. Moreover, 50 umol/ml neomycin resulted in a complete loss of hair ceils in the basal and middle turn, but all the supporting cells remained intact. Conclusion A hearing loss model in neonatal mouse specific to hair cell loss was established by a direct application of 200 nl aminoglycosides at 50 umol/ml into the cochlea using a micro--injection system. Direct application of drugs to the cochlea was an efficient approach to murine neonates. The rate of hair cell loss was high, while the supporting cells were well preserved with a direct delivery of aminoglycosides,and the mice survived.