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.

Introduction

Complex peripheral nerve injuries that result in extended neurectomy require reconstructive surgery with nerve grafts (Mackinnon and Hudson, 1992). Appropriate size and rapid availability of the graft material are necessary to optimize and simplify the reconstruction procedure. Nerve allografts are often used to solve this problem (Mackinnon et al., 1987; Bain et al., 1988; Mackinnon and Hudson, 1992; Midha et al., 1993) but transplantations require tissue typing of both donor and recipient, and are often complicated by transportation of the graft material between cities (Mackinnon and Hudson, 1992). A possible solution to the pre-operative requirements and surgical problems could be afforded by the development of a nerve bank (Lewis and McLaurin, 1969; Hare et al., 1993) where abundance of graft material of known antigenicity would be available. Procedures permitting the cold storage of this material require preservation of the viability of Schwann cells (SCs), one of the major components of peripheral nerves required for substantial regeneration (Zalewski and Gulati, 1982; Mackinnon et al., 1984; Gulati, 1988). Although the most common practice used to preserve isolated tissues and cells is cold storage in physiological buffers at 2–6°C, it leads to progressive morphological and functional deteriorations after prolonged time periods (Morrissey et al., 1991a, Morrissey et al., 1991b; Levi et al., 1994). The prospect of virtually infinite time storage of the material, with the only damage being that associated with the freezing and thawing processes, is offered by cryopreservation techniques which have been recently applied on peripheral nerves (Ruwe and Trumble, 1990; Gauthier et al., 1995). So far, the viability and functional capacity of Schwann cells within cold stored nerves was assessed with immunostaining techniques after their dissociation and culture (Morrissey et al., 1991a, Morrissey et al., 1991b; Levi et al., 1994). However, these studies require the use of several elaborate techniques and results can be obtained only several days after the end of the storage period.

The aim of the present study was to devise a simple and rapid method to evaluate cell survival of cold stored nerves. For this, we have adapted on whole peripheral nerve SCs a recently described cytotoxicity assay (`Live/Dead®' Viability/Cytotoxicity Kit, Molecular Probes, Inc.) used on cultured cells (Moore et al., 1990; Kaneshiro, 1993; Sontheimer et al., 1994). This assay utilises fluorescence dyes to discriminate between live and dead cells. Dead cells (membrane damaged) are identified by a red fluorescence generated by the ethidium homodimer bound to nucleic acids, a compound excluded from live cells. On the other hand, viable cells are identified by a green fluorescence generated by the enzymatic hydrolysis of calcein-AM that only occurs in living cells as a result of esterase activity. To compare cell survival in various situations, nerves were placed either in conditions where inside cells were killed (chemically or cryo-mechanically) or in conditions known to preserve cells, i.e. cryopreservation (Ruwe and Trumble, 1990; Gauthier et al., 1995) or control treatment.