Subcutaneous Exposure To Carbamate Acetylcholinesterase Inhibitors Does Not Induce Apoptosis In Mouse Brain

Pyridostigmine bromide (PB), a cholinesterase inhibitor used as a prophylactic against nerve agents, has been reported to produce neuronal apoptosis in rats. The goal of this study was to determine if PB produced similar levels of apoptosis in a C57BL/6J mouse model. Since the ability of PB to cross the blood-brain barrier is controversial, we used physostigmine (PHY), a cholinesterase inhibitor that readily crosses the blood-brain barrier, as a positive control. Following subcutaneous exposure to either PB or PHY, we examined coronal sections of the brain for evidence of apoptosis at time points ranging from 8 hours to one week. Results obtained using the standard terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay revealed high levels of non-specific staining in the dentate gyrus, striatum, and cerebral cortex. When the assay was modified by the addition of a lipid extraction step, reductions in incubation times, and modifications to a wash buffer to eliminate nonspecific binding, no apoptosis was detected in any brain area following treatment with PB, PHY, or saline. To verify that the modified protocol was capable of detecting low levels of apoptosis, we used 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) treatment to identify TUNEL positive cells in the substantia nigra of mice with this assay.