The sudanophilic lipid of normal and regenerating limb tissues of the adult newt, Diemictylus viridescens

Abstract

The most intense and widely distributed sudanophilic responses of cryostat-sectioned newt limb tissues were obtained with a simultaneous fix and stain procedure of 1:l 10% formal-calcium and sudan black B. Droplets and globules of lipid mixtures and rodlets (mitochondria) were typical responses distributed within the epidermis, subcutaneous glands, dermis and other connective tissues, striated muscle (also with positive fibrils), tunics of blood vessels, and blood cells. A prominent droplet response was located subjacent to the adepidermal basement membrane. The myelin of brachial nerve stained intensely.In regenerating limbs, the wound epithelium response was comparable to that of epidermis. Post-amputational lipophanerosis of injured muscle and brachial nerves was observed. The retrograde degeneration of nerve myelin was extensive, and continued into the early differentiative phase of regeneration. Lipid-engorged macrophages were prominent among the injured tissues, distal to these, and within the wound epithelium.The regeneration blastema revealed a large quantity of sudanophilic lipid.Prominent droplet and rodlet responses were typical of the myelinating regenerating nerves. The response of regenerating muscle equaled that of the mature stump fibers.The cells of the regenerating chondroskeleton contained sudanophilic lipid.Organic solvents such as acetone, ether, chloroform and ch1oroform:methanol reduced or prevented the sudanophilic responses. Sudan red 7B revealed less lipid than did sudan black B. A fixation effect was demonstrated with post-chromated formalcalcium, and chromic-formalin fixed sections. In the latter preparations, swollen-bodies, identified as mitochondria, stained intensely.The lipid content of the normal and regenerating tissues of the adult newt has received only casual attention (Salpeter and Singer, '60; Schmidt, '62a; '63a, b). Hess ('59) has reported briefly on the distribution of unsaturated lipids in the regenerating tail of Xenopus laevis tadpoles. In view of these few observations, and the suggestion (Schmidt and Weidman, '64) that lipids may play a significant role in the events of regeneration, we undertook the investigation of the presence and distribution of lipids in the normal and regenerating tissues of the adult newt limb.This communication includes studies that have appeared in preliminary reports (Schmidt, '64a, b), as well as additional investigations that have been conducted since. MATERIALS AND METHODSThe course of regeneration was followed in the amputated enucleated forelimbs of J. MORPH., 118: 57-78. the adult newt, Dieinictylus viridescens, obtained from the vicinity of Petersham, Massachusetts. The humerus was extirpated from both forelimbs as previously described in detail (Schmidt, '62b). Following an interim of 21 days, both forelimbs were amputated through the enucleated brachium just proximal to the elbow.The animals were maintained in individual fingerbowls containing aerated distilled water. For the first post-operative week, one or two drops o...