Autoradiographic study of RNA and protein synthesis in sectioned peripheral nerves

RNA and protein metabolism of injured peripheral nerves were studied as follows: the right and left sciatic nerves of 24 adult rats were cut 4 cm below L4. [ 3H]Uridine (12 rats) and a tritiated amino acid mix (12 rats) were administered in vivo and in situ only to the right proximal stumps. Incubations were made 30 min before removal and at the following times after severing: 0, 3.5, 23.5, and 71.5 h. The nonlabeled, left proximal stumps were used as controls. Light microscope autoradiographies of right and left proximal stumps were carried out. A light microscope provided with a closed TV circuit, and an X-Y plain digitizer coupled to a DEC PDP-11 computer were used to measure axoplasm, myelin, and Schwann cell areas. Silver grains over these areas were counted and grain density in the three compartments were determined. Control experiments affirmed that silver grains corresponded to precursors incorporated in RNA and proteins. This was checked by counting radioactivity in the buffers used for washing the samples and by RNase digestion, inhibition of RNA synthesis by actinomycin D, and inhibition of protein synthesis by cycloheximide. Label was found in the right stump but not in the left stump which means that no diffusion through the body pool occurred. This fact and the method of label administration indicated that the neuronal soma was not the source of labeled macromolecules. The course of label incorporation paralleled the substructural changes reported to occur in the tips of severed axons. The peak of incorporation with both precursors was found 24 h after sciatic transection. For both precursors label incorporation followed a gradient from Schwann cell to axoplasm. A parallel relationship between density in axons and in myelin was observed. A close relationship between newly synthesized RNA and proteins in injured nerves was suggested.