Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer
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.
Section snippets
Surgical procedures
Adult female Sprague–Dawley rats (IFA CREDO, Les Oncins, L'Arbresles) weighing 280–300 g were deeply anaesthetized with sodium pentobarbital (Nembutal®, i.p. 60 mg/kg). Both peroneal (peroneus or fibularis) nerves were removed under sterile conditions using a muscle splitting incision and donor animals were then sacrificed. Nerve segments were about 4 cm in length.
Nerve treatments
The nerves segments obtained from each donor were stored at +4°C and randomly divided into four experimental groups. The control
Incubation times and dye concentrations
The dye concentrations were adjusted to achieve distinct labeling of live SCs with calcein-AM and dead SCs with ethidium homodimer. Using samples of dead cells within nerves treated with Triton X-100, (0.5%, 30 min), both the saturating concentration of homodimer ethidium and concentration of calcein-AM that gave negligible staining were determined. Conversely, using samples of control nerves, the lowest concentration of calcein-AM still yielding maximal fluorescence was determined, in the
Discussion
The present study shows that the Live/Dead® cytotoxicity assay (Viability/Cytotoxicity Kit, Molecular Probes, Inc.) provides an accurate test to evaluate Schwann cell viability within a whole peripheral nerve. This assay uses two fluorophores (calcein-AM/ethidium homodimer) to selectively distinguish between live and dead cells. Calcein-AM is a lipophilic compound that diffuses through the intact cell membrane and stains the cytoplasm (green fluorescence) after the calcein moiety is freed by
Acknowledgements
The authors gratefully acknowledge Drs. G. Novakovitch and P. Ladaique of the Laboratoire de Cryobiologie, Institut Paoli Calmette de Marseille and P. Nannini of the Centre Régional de Transfusion Sanguine de Marseille, for their helpful assistance in nerve cryopreservation. This work was supported by CNRS (URA 1832), IRME (Institut pour la Recherche sur la Moelle Epinière), DRET (Grant 92-053) and by the University Aix-Marseille III.
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