Identification of the Neurotransmitter Substance P in Sural Nerve of Rats: An Immunohistochemistry Approach

Abstract

The sural nerve in rats is mainly sensory, providing innervation for the lateral side of the hind paws [1]. The substance P (SP) is a neurotensmitter known to be present in small myelinated and unmyelinated fibers, being closely related to pain tranmission to the central nervous system [2]. Thus, the presence of SP is expected in small fibers of the sural nerves. Nevertheless, morphological demonstration of the presence of SP in sural nerves of rats were not yet performed by immunohistochemestry (IHC). We developed an IHC protocol to identify the SP in sural nerve of male and female Wistar‐Kyoto rats. The animals were anesthetized and had the sural and phrenic nerves (positive control) surgically removed, immersed in Karnovsky solution (4% paraformaldehyde, 2% glutaraldehyde) and cryoprotected in solutions of sucrose with increasing concentrations. Transversal cryosections (18μm) of the nerves were incubated in rabbit polyclonal antibody anti‐substance P (1:10.000, 24h), followed by biotinylated anti‐rabbit IgG (1:500; 24h) and finally Extravidin‐HRP (1:1500, 24h). A reaction with diaminobenzidine intensified with nickel revealed immunoreactivity by light microscopy, confirming the effectiveness and viability of the IHC technique proposed by our protocol for identification of the SP in sural nerve. These results indicate the possibility of investigating neurotransmitter contents in different fiber pupulations in normal nerves and their correlation with peripheral neuropathies such as diabetes and hypertension.Support or Funding InformationFAPESP, CNPq, CAPES and FAEPA