Light and electron microscopic demonstration by the PATS reaction of peripheral nerve regeneration

Abstract

Although periodic acid-Schiff(PAS) type reactions have been applied to nervous tissues for many years, interest has centered upon staining glycolipids, principally myelin constituents such as the class of sphingolipids. The staining of these compounds such as sphingomyelin has generally been attributed to the presence of amino and hydroxyl groups on adjacent carbon atoms of carbohydrate of the sphingosine moiety. But unsaturated lipids also give the reaction and sphingolipids stain even if carbohydrate moieties are absent. This reaction has been used for staining myelin sheaths but lipid solvents must be avoided in processing the specimens. Toluidine blue staining of semi-thin sections of epoxy- embedded nerve specimens has also been widely used to study regenerating fibers after nerve transection or avulsion. A recent study was made in our laboratories of conduits (sleeves) tailored from biodegradable polyester (VicrylR) mesh to guide the reconnection of regenerating fibers from the proximal stump of a rat sciatic nerve, across an 11 mm gap, with fibers in the distal stump of the interrupted nerve. Complete reconnection of the stumps was observed as early as one month after creating the avulsive nerve injury.Comparison of transverse sections of the repaired sciatic with sections of control nerve with the toluidine blue stain, however, showed little evidence of axonal regeneration after one month (Figs. 1,3). A variation of the PAS reaction (depositing silver) for light and electron microscopy developed in our laboratories (PATS reaction, 5) was than applied to the study of the semi-thin sections of the epoxy-embedded control and repaired sciatic nerves of the same rat one month postsurgery. Correlative light and scanning electron microscopy by SEI and BEI modes could then be performed since the PATS reaction produced very satisfactory staining of the semi-thin sections (Figs. 3-5). Myelin was not stained by the PATS reaction in these specimens since the nerves had been processed with lipid solvents for epoxy embedment. Schwann cells, however, were very prominent in control but not in the repaired nerve. The inner layers of endoneurium and all pericapillaries associated with nerve fibers were intensely stained due to their reticulin content in both control and repaired nerve (Figs. 2,4). This was not unexpected because the PATS reaction employs a silver methenamine reagent. Thus, with the PATS reaction axons could be identified in sections of repaired nerve (Fig. 4) that could not be discerned with toluidine blue staining (Fig. 3). In sections of repaired nerve stained with either toluidine blue or the PATS reaction few axons or axis cylinders were observed but more were seen with the PATS stain (Figs. 3,4). In control nerve sections stained with either procedure many were seen (Figs. 1,2).