ENHANCED IMMUNOHISTOCHEMICAL DETECTION OF AUTONOMIC NERVE-FIBERS, CYTOKINES AND INDUCIBLE NITRIC-OXIDE SYNTHASE BY LIGHT AND FLUORESCENT MICROSCOPY IN RAT SPLEEN

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat sple en. This includes norepinephrine-synthesizing enzymes (dopamine-beta e ta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (N PY), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-ga mma), c-fos protein, inducible nitric oxide synthase (iNOS), and the m acrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-alpha, iNOS, IFN-gamma and c-fos, animals were injected IV with saline or 100 mu g of lipopolysaccharide (LPS) and w ere sacrificed at various time intervals post injection. Rats were per fused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-mu m floating sections were cut on a freezing microtome. Immunodet ection was performed with various detection systems and substrate/chro mogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-gamma, iNOS, and ED1, and the improvement was c oncentration-dependent Using NPY immunostaining to index the signal-to -noise ratio for various substrates and detection systems, we found th at an alkaline phosphatase detection system with NBT/BCIP as a substra te was the best procedure for light microscopy, whereas the CY3-labele d secondary antibody technique proved optimal for fluorescent microsco py. Surgical transection of the splenic nerve eliminated all nerve fib er staining for DBH, TH, and NPY. TNF-alpha, IFN-gamma, c-fos, and iNO S proteins were observed in the spleen in a time-dependent manner afte r LPS stimulation. Fluorescent double labeling, visualized with fluore scent confocal scanning laser microscopy, revealed many NPY fibers dis tributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effect s of an immune challenge on splenocytes in situ and verifies that sple nic immune cells are innervated by the sympathetic nervous system.