Quantitative evaluation of axonal regeneration by immunochemical assay for neurofilament protein

In experiments on nerve regeneration requiring assessment of the rate and extent of axonal outgrowth, the availability of a simple and accurate method of quantification would be extremely useful. We approached this issue by modifying the conventional ELISA procedure so as to provide a sensitive, specific, and quantitative biochemical assay of the phosphorylated neurofilament content of homogenates or sections of nerve tissue. The technique involves four sequential steps: (i) adhesion of fixed or fresh homogenates or tissue sections to wells of microtiter plates, (ii) binding of a monoclonal antibody against phosphorylated neurofilament to the tissue, (iii) secondary binding to the anti-phosphorylated neurofilament of a phosphatase-labeled second antibody (antimouse IgG), and (iv) enzymatic assay of alkaline phosphatase activity using a fluorescent substrate (4-methylumbelliferyl phosphate). The technique is sufficiently sensitive to measure the phosphorylated neurofilament content of a 1:100,000 (w/v) homogenate of brain, spinal cord, or peripheral nerve and of single 10-microns paraffin sections of Bouin-fixed rat spinal cord. To validate the applicability of the procedure to the study of nerve regeneration, the sciatic nerve of adult rats was either crushed (to permit regeneration) or transected and ligated (to preclude regeneration). The animals were autopsied 1 to 16 weeks later, when four segments 3-mm in length taken from regions proximal and distal to the lesion site were assayed for phosphorylated filament content. The temporal course of its disappearance during degeneration and its reappearance during regeneration coincided with the known histologic changes in crushed and transected nerves. These findings demonstrate the validity of using the immunochemical assay for PNF in studies of nerve regeneration in the peripheral nervous system and the potential applicability of this procedure to studies on regeneration in the central nervous system.