For approximately 200,000 US veterans, the 1991 Persian Gulf War marked the beginning of their experience with Gulf War Illness (GWI). GWI encompasses a cluster of chronic symptoms including memory and cognitive problems, fatigue, and fibromyalgia.
GWI has long been associated with a combination of several possible contributory factors: the stress of deployment, altered immune function, and exposure to acetylcholinesterase inhibitors (AChEI), but the exact cause or causes have remained elusive. The AChEI pyridostigmine bromide (PB) was administered to soldiers as a prophylactic against the risk of nerve agent weapons, but many veterans were also exposed to AChEI based pesticides, further complicating the etiology of this illness.
To elucidate the relationship between these factors, Dr. Victoria Macht, her advisor Prof. Lawrence Reagan, and colleagues at the University of South Carolina School of Medicine studied rats exposed to pyridostigmine bromide and repeated restraint stress. The rats were then given either an immune challenge or an acute immobilization stress challenge during in vivo microdialysis. It is the first study to use an in vivo method (microdialysis) to show that PB changes the response of the central cholinergic system to both stress and immune challenges, and does so in a brain region specific manner.
By measuring acetylcholine levels via microdialysis and subsequent HPLC-ECD, they found that cholinergic responses were attenuated in the PFC and hippocampus after immobilization stress. Lipopolysaccharide (LPS) was administered as an immune challenge, after which cholinergic responses were attenuated in the hippocampus but not the PFC. These results indicate that PB and stress interact to shift the cholinergic response to future psychological and immunological stressors, providing a potential mechanism for the persistent and exacerbated cognitive symptoms evidenced in soldiers with GWI.
Mike Churchill: What story do the different responses to the immune challenge and the immobilization challenge tell?
Victoria Macht: By using two different types of challenges, we were able to test both the diversity and consistency of effects of PB and stress on the cholinergic system. LPS is a novel challenge which specifically elicits a response from the innate immune system. The immobilization challenge is more of a psychological stressor, and as it shares some similar qualities with the prior restraint stress, this allowed us to test if rats with PB and restraint stress had impaired neurochemical adaptations to recurrent stressors.
MC: How might these results relate to changes in fear memory and cognitive function?
VM: ACh is an important regulator for a variety of factors in fear memory including coordination of local circuits to help with sensory and cortical processing of stimuli as well as the consolidation process. Interestingly, regional differences in the cholinergic response of the PFC and hippocampus to immobilization stress suggested that PB impairs cortical processing of novel stressful stimuli and impairs the neurochemical adaptation to recurrent stressful stimuli. In our fear conditioning studies, we similarly found impairments in the way PB and stress interacted to impair context and cue related retrieval. This suggested to us that impairments in the function of the cholinergic systems impacts a variety of psychological stressful stimuli, indicating that this is a global deficit in cognitive function rather than a specific deficit to only one type of stressor.
MC: How do the microdialysis results relate to the tests for inflammation you ran?
VM: ACh is really fascinating because while it is not only central in learning and memory, it is also an important negative regulator for the inflammatory response via α7 nicotinic ACh receptors. We found that PB blunted the central cholinergic response to an innate immune challenge, which could suggest an exacerbated chronic inflammatory response in the brain. Interestingly, these microdialysis results for acetylcholine parallel some of our findings with peripheral inflammatory markers. Peripheral levels of c-reactive protein were elevated after the LPS challenge in rats which had received PB, suggesting a dysregulated inflammatory response. While we need to confirm these results with cytokine levels in the brain, our results suggest that impaired cholinergic feedback to inflammatory stimuli could underlie some of the changes in the sensitivity of the immune system which are evident in clinical populations with GWI.
MC: Does PB have to cross the BBB to cause these effects?
VM: It does not. There has been a big debate on this topic. One suggestion was that stress caused a leaky barrier, allowing PB to get through. However, tests on this have been inconsistent on this. What our studies demonstrate is that PB changes the function of the central cholinergic system regardless of whether it is able to get through the BBB.
MC: What will be the next steps for this project?
VM: Prof. Reagan will continue the project: measuring cytokine responses in the brain to see if they match peripheral cytokine responses. There is also an opportunity to see if aging exacerbates the decline of the cholinergic responses and cognitive deficits in our model of GWI. The goal would be to see if animal models of GWI can predict further changes in veterans as they age, and plan treatment accordingly. We have a unique opportunity with this population for the preclinical research on treatments to get ahead of the patient population as they age.
MC: How did you like using the Eicom HTEC HPLC-ECDs in Prof. Jim Fadel’s lab?
VM: It is amazing! I can’t imagine having done these projects without it, and I miss using it. We used the system daily for two years to measure acetylcholine without any real problems. It made my dissertation a much more pleasant experience!
MC: Had you used HPLCs before using the HTEC?
VM: We used a different system before but it was not reliable, so when it was working people felt they had to immediately run all of their samples before it went down again, and watch it all of the time when it was running.
MC: How many samples do you think you ran over the course of this project?
VM: That makes my head spin! We looked at both ACh and glutamate, in two brain regions, each rat underwent microdialysis 2 separate days, there were approximately 8 animals per group, and 4 groups. So at least 3500 samples – plus the pilot study! Plus there were other studies going on during this time which were also using the HTEC.
MC: Where is your career taking you next?
VM: I am now doing a postdoc at UNC Chapel Hill, working with Prof. Fulton Crews, studying the long term effects of binge drinking in adolescents. Interestingly, while this is a different clinical population, changes in the cholinergic system and innate immune system are also common features here.
The article appears in the April issue of Brain, Behavior, and Immunity:
Pyridostigmine bromide and stress interact to impact immune function, cholinergic neurochemistry and behavior in a rat model of Gulf War Illness
V.A. Macht, J.L. Woodruff, E.S. Maissy, C.A. Grillo, M.A. Wilson, J.R. Fadel, L.P. Reagan