Decherchi P, Cochard P, Gauthier P
Département de Physiologie et de Neurophysiologie, URA CNRS 1832, Faculté des Sciences et des Techniques de Saint-Jérôme, Marseille, France.
J Neurosci Methods. 1997 Feb;71(2):205-13. doi: 10.1016/s0165-0270(96)00146-x.
A membrane permeant nucleic acid stain, ethidium homodimer was used in combination with calcein-AM to document the viability of Schwann cells (SCs) in whole nerves after cold storage assays. Segments of peripheral nerves were, (i) kept intact in buffer (viability controls), (ii) thawed after a cryopreservation, according to a protocol which has been previously shown to maintain the integrity of most nerve components [Ruwe and Trumble, J. Reconstr. Microsurg., 1990, 6: 239-244; Gauthier et al., In 3rd International Symposium on Axonal Regrowth in the Mammalian Spinal Cord and Peripheral Nerve, Deauville, France, 1995, p. 24, abstract], (iii) killed by chemical injury, or (iv) by successive freezing-thawing. Teased preparations of nerve fibers were prepared from the various types of nerve segments and incubated with calcein-AM and ethidium homodimer, which stain, respectively, living and dead cells. In control or cryopreserved nerves, staining with calcein-AM resulted in bright green fluorescence in the cytoplasm of SCs, with no red fluorescence of ethidium homodimer. In contrast, in killed nerve preparations, intense ethidium red fluorescence was observed in SC nuclei, with negligible green calcein cytoplasmic fluorescence. Thus, the combination of calcein-AM/ethidium homodimer appeared as an effective tool for assessing the viability of SCs and determine the quality of cold stored nerve preparations used in graft repair procedures. In addition, the generated fluorescence enabled clear visualization of myelinated fibers by confocal imaging.
一种膜渗透性核酸染料——溴化乙锭同二聚体，与钙黄绿素-AM联合使用，以记录冷储存试验后全神经中雪旺细胞（SCs）的活力。外周神经段：（i）在缓冲液中保持完整（活力对照）；（ii）根据先前已证明能维持大多数神经成分完整性的方案进行冷冻保存后解冻[鲁韦和特朗布尔，《重建显微外科杂志》，1990年，6: 239 - 244；高蒂尔等人，第三届哺乳动物脊髓和外周神经轴突再生国际研讨会，法国多维尔，1995年，第24页，摘要]；（iii）通过化学损伤致死；或（iv）通过连续冻融致死。从各种类型的神经段制备神经纤维的 teased 制剂，并与分别对活细胞和死细胞进行染色的钙黄绿素-AM和溴化乙锭同二聚体一起孵育。在对照或冷冻保存的神经中，用钙黄绿素-AM染色导致雪旺细胞胞质中出现亮绿色荧光，而溴化乙锭同二聚体无红色荧光。相反，在致死的神经制剂中，在雪旺细胞核中观察到强烈的溴化乙锭红色荧光，而钙黄绿素胞质绿色荧光可忽略不计。因此，钙黄绿素-AM/溴化乙锭同二聚体的组合似乎是评估雪旺细胞活力和确定用于移植修复程序的冷储存神经制剂质量的有效工具。此外，产生的荧光通过共聚焦成像实现了有髓纤维的清晰可视化。
