Chin K W, Lopez I, Lee S C, Honrubia V
Department of Surgery, University of California at Los Angeles, School of Medicine, USA.
Laryngoscope. 1999 Jul;109(7 Pt 1):1037-44. doi: 10.1097/00005537-199907000-00005.
Determine the expression of glutamate by immunohistochemistry in normal and recovering vestibular hair cells in the chinchilla crista ampullaris after gentamicin ototoxicity.
In five groups of three animals each, ototoxicity was produced by placing gentamicin (50 microg)-impregnated Gelfoam pellets within the perilymphatic space of the superior semicircular canal. Animals were sacrificed at 1, 2, 4, 8, and 16 weeks after treatment. A group of normal (n=3) animals was also processed.
For the detection of glutamate the inner ears of these animals were dissected, and the horizontal cristae ampullaris embedded in plastic. Two-micron-thick tissue sections were obtained and incubated with monoclonal antibodies against glutamate. The immunoreaction was detected using the avidinbiotinylated-complex technique and diaminobenzidine was the chromogen.
Normal sensory epithelia demonstrated type I and type II hair cells with moderate glutamate-like immunoreactivity. Supporting cells demonstrated no glutamate-like immunoreactivity. Afferent nerve fibers and calyxes surrounding type I hair cells demonstrated strong glutamate-like immunoreactivity. At 1 and 2 weeks after treatment the few type II hair cells surviving ototoxic treatment (15%-18%) contained moderate glutamate-like immunoreactivity, supporting cells showed no immunoreactivity, and nerve terminals and fibers displayed strong immunoreactivity. At 4 and 8 weeks after treatment, recovered hair cells (80%) had greater glutamate-like immunoreactivity when compared with normal hair cells, supporting cells displayed no glutamate-like immunoreactivity, and afferent fibers contained strong glutamate-like immunoreactivity. At 16 weeks, glutamate-like immunoreactivity in hair cells returned to normal level.
Glutamate may be used as an indicator of hair cell differentiation and as an index of the molecular recovery of hair cells after ototoxicity.
通过免疫组织化学法测定庆大霉素耳毒性后正常及恢复中的豚鼠壶腹嵴前庭毛细胞中谷氨酸的表达。
将五组动物，每组三只，通过在椭圆囊上半规管的外淋巴间隙放置含庆大霉素（50微克）的明胶海绵颗粒来诱导耳毒性。在治疗后1、2、4、8和16周处死动物。还对一组正常动物（n = 3）进行了处理。
为检测谷氨酸，解剖这些动物的内耳，将水平壶腹嵴包埋于塑料中。获取2微米厚的组织切片，并用抗谷氨酸单克隆抗体孵育。使用抗生物素蛋白 - 生物素化复合物技术检测免疫反应，显色剂为二氨基联苯胺。
正常感觉上皮显示I型和II型毛细胞具有中等程度的谷氨酸样免疫反应性。支持细胞未显示谷氨酸样免疫反应性。I型毛细胞周围的传入神经纤维和杯状神经末梢显示强烈的谷氨酸样免疫反应性。治疗后1周和2周，少数经耳毒性治疗后存活的II型毛细胞（15% - 18%）含有中等程度的谷氨酸样免疫反应性，支持细胞无免疫反应性，神经末梢和纤维显示强烈免疫反应性。治疗后4周和8周，恢复的毛细胞（80%）与正常毛细胞相比具有更强的谷氨酸样免疫反应性，支持细胞未显示谷氨酸样免疫反应性，传入纤维含有强烈的谷氨酸样免疫反应性。在16周时，毛细胞中的谷氨酸样免疫反应性恢复到正常水平。
谷氨酸可作为毛细胞分化的指标以及耳毒性后毛细胞分子恢复的指标。
