Nguyen Nhi V, Lin Joshua S, Parikh Miti J, Cutri Raffaello M, Shibata Seiji B
Caruso Department of Otolaryngology-Head and Neck Surgery, University of Southern California, Los Angeles, CA 90033, USA.
Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Mol Ther Methods Clin Dev. 2025 Mar 3;33(2):101440. doi: 10.1016/j.omtm.2025.101440. eCollection 2025 Jun 12.
The spiral ganglion neurons (SGNs) are the primary afferent neurons in the cochlea; damage to the SGNs leads to irreversible hearing impairment. Mouse models that allow selective SGN degeneration while sparing other cell types in the cochlea are lacking. Here, we investigated a genetic ablation method of the SGN using a Cre-responsive adeno-associated virus (AAV) vector expressing diphtheria toxin subunit-A (DTA). We microinjected AAV2-retro-FLEX-DTA-mCherry driven by the EF1a or hSYN promoter in neonatal parvalbumin-Cre (PV) and wild-type strains via the posterior semicircular canal. Apoptotic markers were observed in the degenerating SGNs as early as 3 days. After 1 week, we assessed the SGN cell density, revealing an average degeneration of 60% for AAV-DTA driven by the EF1a promoter and 61% for that driven by the hSYN promoter. By 1 month, injected ears demonstrated a nearly complete loss of SGN, while hair cell morphology was intact. The auditory brain stem response result showed significantly elevated threshold shifts at 1 month, while the distortion-product otoacoustic emissions function remained intact. Furthermore, we show that our method did not effectively ablate SGN in adult PV mice. We generated a neonatal mouse model with primary SGN degeneration in PV mice, mimicking auditory neuropathy phenotype using an AAV Cre-dependent expression of DTA.
螺旋神经节神经元（SGNs）是耳蜗中的初级传入神经元；SGNs受损会导致不可逆的听力损伤。目前缺乏能使耳蜗中的SGNs选择性退化而其他细胞类型不受影响的小鼠模型。在此，我们研究了一种使用表达白喉毒素A亚基（DTA）的Cre反应性腺相关病毒（AAV）载体对SGNs进行基因消融的方法。我们通过后半规管将由EF1a或hSYN启动子驱动的AAV2-retro-FLEX-DTA-mCherry显微注射到新生小白蛋白-Cre（PV）和野生型品系小鼠体内。早在3天时就在退化的SGNs中观察到凋亡标记物。1周后，我们评估了SGN细胞密度，结果显示由EF1a启动子驱动的AAV-DTA导致的平均退化率为60%，由hSYN启动子驱动的为61%。到1个月时，注射侧耳朵的SGN几乎完全丧失，而毛细胞形态保持完整。听觉脑干反应结果显示1个月时阈值偏移显著升高，而畸变产物耳声发射功能保持完整。此外，我们发现我们的方法在成年PV小鼠中不能有效消融SGN。我们利用DTA的AAV Cre依赖性表达，在PV小鼠中建立了一个具有原发性SGN退化的新生小鼠模型，模拟听觉神经病表型。
