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目的建立大鼠离体耳蜗切片技术,结合红外可视膜片钳技术探讨耳蜗螺旋神经元(spiral ganglion neuron,SGN)的电生理特性。方法 0～2日龄、3～6日龄及7～14日龄的健康 SD 大鼠各10只,快速制作耳蜗切片,应用红外微分干涉相差技术,结合膜片钳记录,观察 SGN 的基本电生理特性,并分析影响耳蜗切片制作质量及膜片钳记录成功的因素。结果 3～6日龄大鼠耳蜗切片的成功率最高,每只耳蜗可制备2～4张切片;保持部分颅骨与耳蜗的连接及耳蜗骨壳的完整是影响切片成功的关键,切片时蜗轴与刀片的位置及切片取材的时间也是影响切片质量和细胞活性的重要因素。红外可视膜片钳技术可准确找到状态良好的 SGN,有助于对封接过程的判断。全细胞记录 SGN静息膜电位平均为(-45.6±5.3)mV(±s,n=52),可记录到 Na~+和 K~+电流。结论耳蜗切片技术能够较完整地保持耳蜗结构以及各种构成细胞的活性及其相互之间的联系,红外可视膜片钳技术具有实时、直视的特点,可操作性强,二者结合能为深入研究 SGN 的电生理特性及听觉传导机制提供良好的手段和平台。
OBJECTIVE: To establish an in vitro rat cochlear slicing technique and to explore the electrophysiological characteristics of spiral ganglion neuron (SGN) by using infrared visual patch clamp technique. Methods 10 healthy SD rats of 0 to 2 days, 3 to 6 days, and 7 to 14 days were selected and 10 rats were used to make cochlear sections. The difference of the technique of infrared differential interferometry and patch clamp recording were used to observe the basic electricity of SGN Physiological characteristics, and analysis of the impact of cochlear slice production quality and patch clamp record of success factors. Results The cochlear sections from 3 to 6 days old had the highest success rate with 2-4 cochlear sections per cochlear. The key to the success of the section was to maintain the connection of part of the skull and cochlea and the cochlear bone and shell. The location of the blade and the time taken for the sectioning are also important factors that influence the quality and cell viability of the sections. Infrared visual patch clamp technology can accurately find a good state of SGN, contribute to the judgment of the sealing process. The mean resting membrane potential of SGN was (-45.6 ± 5.3) mV (± s, n = 52). The currents of Na ~ + and K ~ + were recorded. Conclusion The cochlear slice technique can completely maintain the activity of the cochlear structure and various constituent cells and their connection with each other. The infrared visual patch clamp technology has the characteristics of real-time and direct vision and strong maneuverability. It provides a good means and platform for further study of electrophysiological characteristics and auditory conduction mechanism of SGN.