缺氧对乳鼠耳蜗螺旋神经节神经元外向电流的影响及其环磷酸腺苷、环磷酸鸟苷含量变化的研究

Effect of hypoxia on outward current and change in content of cAMP and cGMP in cochlear spiral ganglion neurons

摘要:

目的  观察缺氧时间对体外培养的耳蜗螺旋神经节神经元（SGNs）外向电流的影响及其细胞内第二信使环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）含量变化。方法  体外原代培养新生1～3 d SD大鼠SGNs并进行免疫荧光鉴定。用膜片钳全细胞记录模式观察缺氧外液灌流SGNs时，外向电流的变化趋势和特点。用酶联免疫吸附实验检测缺氧5、15和20 min时SGNs内cAMP和cGMP的浓度变化。结果  ①酶解分离可获得生存状态良好的神经元，经免疫荧光鉴定为SGNs。②当电压钳制在-60 mV时，急性缺氧增强SGNs外向电流，主要增强0～+60 mV电压区间的激活电流幅度。缺氧5 min时，+60 mV激活电流幅度从（971.2±50.3）pA增强到（2361.0±207.4）pA，差异有统计学意义（P <0.01）。随后出现下降趋势，11～15 min时，增强的电流幅度与对照组基础电流比较，差异无统计学意义（P >0.05）。③分别检测缺氧5、15和20 min后SGNs中cAMP和cGMP的含量变化。缺氧5 min组与正常对照组比较，cAMP浓度升高，差异有统计学意义（P <0.01）。缺氧15 min组与正常对照组比较，cAMP浓度变化差异无统计学意义；缺氧20 min组与正常组对照组比较，cAMP浓度水平下降（P <0.01）。cGMP浓度随着缺氧时间的延长，总体呈下降趋势，但在20 min后有部分回升（缺氧15 min vs 缺氧20 min，P <0.01）。结论  在SGNs急性缺氧损伤过程中，外向电流增强，增强幅度随缺氧时间变化先升后降，推测在短期缺氧时细胞通过降低兴奋性，影响听觉信息的传导，且该过程与胞内第二信使cAMP和cGMP浓度的改变相关。

Abstract:

Objective To observe the effect of hypoxia time on outward current in spiral ganglion neurons (SGNs) cultured in vitro and changes in the intracellular second messenger systems. Methods SGNs of 1-3 d neonatal Sprague Dawley rats were cultivated in vitro and identified by immunofluorescence. Whole-cell patch clamp technique was used to record the variation trends and characteristics of outward current in SGNs perfused with hypoxic external solution. The changes in concentrations of cAMP and cGMP in SGNs were detected by enzyme-linked immunosorbent assay at 5, 15 and 20 min of hypoxia. Results Good neurons could be isolated by enzyme which were identified by immunofluorescence to be SGNs. Acute hypoxia mainly enhanced outward current at 0-(+60) mV with the holding potential of -60 mV. At 5 min of hypoxia, the amplitude of outward current was increased from (971.2 ± 50.3) pA to (2361.0 ± 207.4) pA at +60 mV (P < 0.01); then tended to decline. From 11 to 15 min of hypoxia, the increasing amplitude of outward current had no significant difference from the base current in the control group (P > 0.05). Compared with the control group, the concentration of cAMP in SGNs significantly increased in the 5-min hypoxia group (P < 0.01), but was not statistically different in the 15-min hypoxia group (P > 0.05); however, the concentration of cAMP decreased in the 20-min hypoxia group (P < 0.01). The concentration of cGMP was totally in a downward trend with prolonged hypoxia time, but partially went up after 20 min (15-min hypoxia group vs 20-min hypoxia group, P < 0.01). Conclusions In the process of acute hypoxia injury in SGNs, outward current increases and the enhancing amplitude increases at first and then decreases along with hypoxia time. So we deduced the conduction of auditory information is influenced by reduced excitability in short-term hypoxia of SGNs, which is related to the change in the concentrations of intracellular second messengers cAMP and cGMP.