Immunological Identification of Dying Neurons
Neuronal death is a prominent event during normal development ofanimals and in may human neurological disorders. Immunochemicalmarkers for dying neurons would be valuable reagents for they wouldallow a simpler, more direct way to identify dying neurons than iscurrently available. In the future they might also be developedas a diagnostic tool to detect neuronal death from samples ofbodily fluids.The specific aims of this project are to obtain detailed data aboutthe cytological features and time course of neuronal death afterneuronal axotomy in the rat. Using that information monoclonalantibodies will be developed that specifically identify dyingcentral and peripheral neurons.Spinal cord and ganglion will be examined histologically between1 and 50 days after unilateral neonatal axotomy in rats. Thatprocedure results in a massive depletion of motor neurons andganglionic neurons. Comparisons will be made between theexperimental and sham-operated sides of each rat to identify thecytological features of those neurons stained with dyes that havealso ceased to incorporate tritiated leucine into protein asdetermined autoradiographically. Markedly diminished proteinsynthesis in neuronal somata is an early characteristic of neuronaldeath. The time course of neuronal death will be assessed directlyfrom those tissue sections by counting dying neurons and,independently in a separate series of rats, by counting surviving,retrogradely labeled neurons.Mice will be immunized with either spinal cord or ganglionic tissueobtained from rats at a time when neuronal death is maximal afterneonatal axotomy. Lymphocytes from the immunized mice will befused with myeloma cells to make hybridomas which will be grown intissue culture medium selective for the hybrid cells. Supernatantswhich contain primary antibody will be assayed on tissue sectionsin conjunction with a second antibody that will contain a marker.Hybridomas that produce antibodies which give a positive reactionfor dying neurons relative to controls will be isolated bylimiting-dilution to obtain monoclonal antibodies.