EFFECT OF PYRIDOSTIGMINE BROMIDE AND STRESS ON NEURONAL APOPTOSIS AND MUSCARINIC RECEPTOR DENSITY IN C57Bl MICE

by Mauck, Brena S.

Mauck, Brena S. Ph.D., Biomedical Sciences Ph.D. Program, Wright State University, 2003. Effect of Pyridostigmine Bromide and Stress on Neuronal Apoptosis and Muscarinic Receptor Density in C57Bl Mice. Gulf War Syndrome is a name given to a group of neurological and neuro-muscular symptoms experienced by soldiers of the 1991 Persian Gulf War. These soldiers were prescribed pyridostigmine bromide (PB) as a prophylactic against nerve gases. PB inhibits cholinesterases, enzymes necessary for the termination of cholinergic transmission. One hypothesis is that extended exposure to PB, coupled with stress, allowed the drug to cross the blood brain barrier resulting in neuronal damage. The purpose of this study was to determine if a 7 day exposure to PB and stress was sufficient to alter muscarinic receptor density and to induce apoptosis in male C57Bl mice. Physostigmine, a cholinesterase inhibitor that crosses the blood brain barrier, was used as a positive control. Subcutaneously implanted osmotic mini-pumps delivered a constant dose of drug for 7 days. The stress paradigm involved random shaking of the mice for 2 minutes out of every 30 minutes. This was sufficient to raise blood corticosterone levels. Significant changes were seen in cholinergic receptor densities, primarily in cholinergic nuclei and their projection fields. Stress alone and physostigmine alone produced changes in the diagonal band nuclei and in the projection fields, the amygdala, the limbic cortex, and the hippocampus at the end of a 7 day exposure. PB produced changes in diagonal band nuclei at day 7 but changes in the amygdala and the limbic cortex were not seen until day 37. This delayed effect is important in that soldiers did not experience symptoms until months after their exposure. The combination of stress and drug did not increase the magnitude of density changes as expected. In fact, stress and physostigmine interacted in an antagonistic manner with the combination producing a change in receptor density of smaller magnitude than that produced by either component alone. This neuroprotective effect of stress is supported by studies found in the literature. The regions showing altered receptor densities in this study are known to be involved in memory and learning, areas where veterans experience problems. This study does not provide any evidence that shaker stress altered the leakiness of the blood brain barrier to PB as evidenced by the lack of significant differences between PB treated and PB + stress treated animals. The receptor density changes seen at day 7 due to physostigmine exposure do suggest that if PB is able to cross the blood brain barrier it might be capable of producing cellular changes in areas of the brain responsible for learning and memory. The changes seen at day 37 due to PB exposure suggest that the drug is capable of producing delayed effects, another characteristic of the symptoms experienced by the soldiers. Initial apoptosis studies showed high levels of apoptotic cells, which while consistent with levels found in the literature seemed unlikely to be real. Such high levels of neuronal apoptosis would be expected to lead to modified animal behavior or higher mortality. Modifications of the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) protocol designed to eliminate non-specific binding of the avidin and biotin conjugates eliminated any positive staining in the brains of treated animals. A compound known to produce low levels of apoptosis was used to confirm that the modified assay was capable of detecting low levels of apoptosis. This study shows that exposure to PB or stress leads to altered muscarinic receptor density but neither exposure leads to detectable levels of neuronal apoptosis.