Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.

Polyclonal antibodies raised against mouse 2.5S NGF (mNGF) and against synthetic peptides made from hydrophilic portions of mNGF have been used to compare the immunological properties of mNGF, human recombinant brain-derived neurotrophic factor (hrBDNF), and human recombinant neurotrophin-3 (hrNT-3). Affinity-isolated antibodies raised against intact mNGF reacted with all three neurotrophins when tested by ELISA and totally or partially blocked the bioactivities of the proteins in survival assays of embryonic chicken sensory and sympathetic neurons. On Western blots, mNGF antibodies reacted with all three neurotrophins but less well with hrBDNF and hrNT-3 than with mNGF. Antibodies to hydrophilic peptides within NGF (amino acids 23-35, 59-67, 69-79, and 91-100) showed partial reactivity with some but not all of the neurotrophins when tested by ELISA and on Western blots. The peptide antibodies were also selectively effective in reducing the survival-promoting activity of the neurotrophins on sensory neurons. Results show that mNGF, hrBDNF, and hrNT-3 are immunologically related proteins and that mNGF antibodies react also with other members of the neurotrophin family.

Antibodies to channel proteins and specific peptide sequences have been previously used to localize voltage-activated sodium channels in the rat brain. Here we describe the first localization of sodium channels in an insect nervous system using a site-directed antibody. The mesothoracic ganglion of the cockroach was stained with an antibody to the highly conserved SP19 sequence. Antibody labelling was visualized by light microscopy using the avidin/biotin method on wax sections, and transmission electron microscopy of immunogold-labelled thin sections. Central ganglia of insects contain clearly separated regions of cell bodies, synaptic neuropil, axon tracts, and nerves. Antibody staining by light microscopy was limited to neurons, and was intense in axons throughout the ganglion and nerves. Staining was also strong in the cytoplasm, but not the nuclei, of many neuronal cell bodies. Neuropil regions were relatively lightly labelled. These findings can be correlated with the known electrophysiology of the ganglion. Electron microscopy detected sodium channels in areas surrounding axons, probably including axon membranes and enveloping glial cell membranes. Axonal mitochondria were also heavily labelled, suggesting a sodium channel transport function for these organelles.