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目的:建立耳蜗器官体外培养模型,详细观察缺氧对体外培养耳蜗螺旋神经节细胞(SGC)及神经纤维的影响。方法:分离生后3 d Wistar大鼠耳蜗基底膜,平铺在含有DMEM培养液的培养基内(每盘6条基底膜),2盘为1组,随机分为5组。4组作为实验组,按不同时间段(6 h、12 h、24 h、48 h)进行缺氧(37℃,90%N2,5%CO2,5%O2)培养。1组作为对照组放置在37℃、5%CO2培养箱。用抗神经丝蛋白作为一抗,对耳蜗SGC及神经纤维进行免疫荧光染色。激光共聚焦显微镜下观察缺氧时耳蜗SGC及神经纤维形态变化并计数单位面积(24 mm×36 mm)SGC数即细胞密度。结果:缺氧早期(6 h)神经纤维局灶性水肿,SGC变化不明显。12 h神经纤维局灶性断裂及崩解,SGC减少,细胞密度与对照组相比差异有显著性(P<0.01)。随缺氧时间延长,神经纤维崩解和SGC缺失逐渐加重,48 h神经纤维完全崩解破坏,SGC严重缺失,细胞密度与对照组比较差异有显著性(P<0.01)。结论:缺氧造成体外培养耳蜗SGC及神经纤维损伤,神经纤维对缺氧更敏感。
OBJECTIVE: To establish an in vitro culture model of cochlear organs and observe the effects of hypoxia on cultured spiral ganglion cells (SGC) and nerve fibers in vitro. Methods: The basilar membrane of Wistar rats was isolated 3 days after birth and plated in the culture medium containing DMEM medium (6 basal membranes per dish). Two plates were divided into five groups. Four groups were used as experimental group and were cultured in hypoxia (37 ℃, 90% N2, 5% CO2, 5% O2) for different time periods (6 h, 12 h, 24 h, 48 h). Group 1 as control group placed in 37 ℃, 5% CO2 incubator. Anti-neurofilament protein was used as a primary antibody to immunostaining for cochlear SGC and nerve fibers. Under confocal laser scanning microscope, morphological changes of SGC and nerve fibers were observed under hypoxia, and the number of SGCs per unit area (24 mm × 36 mm) was counted. Results: Focal edema of nerve fibers in the early stage of hypoxia (6 h) showed no obvious change of SGC. At 12 h, there was a significant difference in the focal rupture and disintegration of nerve fiber, SGC reduction and cell density compared with the control group (P <0.01). With the prolongation of hypoxia time, the disintegration of nerve fibers and the loss of SGC gradually increased. The nerve fiber completely disintegrated and destroyed at 48 hours, and the SGC was severely absent. The cell density was significantly different from that of the control group (P <0.01). Conclusion: Hypoxia can induce SGC and nerve fiber damage in cultured cochlear in vitro. Nerve fiber is more sensitive to hypoxia.