Disruption of normal forelimb regeneration in adult Notophthalmus viridescens by sublethal concentrations of trypan blue

Abstract

The effects of the vital dye trypan blue (TpB) on the regeneration of amputated newt forelimbs were examined. Administration of the dye (10 mug/g body weight) via IP injection during the early wound healing and dedifferentiation phases of regeneration inhibited the normal regenerative response. The accumulation phases of regeneration are similarly halted but only by greater concentrations of TpB (50 mug/g body weight) while redifferentiation and morphogenesis are only affected by still greater concentrations of the dye (100 mug/g body weight). In addition to abolishing the regenerative response, low levels of TpB were also capable of inducing skeletal abnormalities in the regenerates as might be expected from previous reports on the teratogenicity of the dye. The in vitro action of newt hyaluronidase (as well as purified testicular hyaluronidase) on hyaluronate was diminished by TpB, with virtually complete inhibition observed at initial reaction mixture concentrations of 100 mug/ml. The results of this study suggest that TpB acts to disrupt the normal regenerative response by preventing dedifferentiation and remodeling, perhaps by inhibition of various necessary lytic enzyme functions or by interference with normal intercellular communications.