PROPOSAL ABSTRACT We propose to test the accuracy and feasibility of a new, time-resolved measure of cell death after organophosphate (OP) poisoning in a murine model of OP-induced status epilepticus. This novel assay was developed as part of the grant, ?Mechanisms of cell death during epileptogenesis?, a 7- year R37 (Javits Award) project. We hypothesize that single-timepoint assays of cell death after OP poisoning are not accurate because they cannot determine 1) how many cells have already undergone necrosis and are no longer visible, 2) how many cells have irreversibly committed to apoptotic death but are not yet positive for biomarkers of that pathway, and 3) how many cells have already undergone apoptotic death and have been phagocytosed by microglia so that they are no longer visible. We propose to measure cell death by the transgenic fluorescent protein quenching assay before and after OP poisoning to determine both the extent and the timing of neuronal death. These studies will form critical preliminary data for subsequent rigorous studies measuring the causative role of seizures in neuronal death and the neuroprotective value of various anticonvulsant and other treatment strategies after OP poisoning.
Much of the concern regarding the rigor of neuroscience arose from overestimations of the amelioration of neurotoxic effects by experimental therapies. Here we propose to test whether our assessments of neurotoxicity are an important source of these inaccuracies. We have developed a new, time-resolved assay for neuronal cell death that makes use of the recent advances in the selective expression of fluorescent proteins by neurons. We propose that this continuously assessible measure of neuronal death will resolve many of the inaccuracies arising from standard, single-time-point assays that require sacrifice of the experimental subject.
