Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer

Abstract

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.