The Update committee has also evaluated selected studies of the effects of potential Gulf War intoxicants on animals. While this was not within its original charge, and a comprehensive examination of all of the pertinent literature was beyond its expertise, the committee nonetheless felt that this effort has merit, both for defining potential health hazards of Gulf War service and in designing potential future investigations. Accordingly, members of the committee reviewed the animal studies listed in Table 6 of the VA Research Advisory Committee’s report (RAC, 2008), focusing on two questions. First, do data from those animal studies demonstrate that stress can amplify the effects of intoxicants or drugs such as PB, in either the peripheral or the CNS? Second, do these animal studies document long-term adverse effects from short-term exposures to these agents?

With respect to the first question, perhaps the two most salient observations are studies by Friedman et al. (1996) and Abdel-Rahman et al. (2002). Friedman’s group examined the inhibition of brain AChE levels in FVB/N mice without and with stress (induced by forced swimming). They reported that the forced swimming stress paradigm was associated with enhanced inhibition of brain AChE by intraperitoneal pyridostigmine (0.1 mg/kg), concurrently with diminished integrity of the blood-brain barrier (measured by enhanced entry of Evans Blue dye and plasmid DNA). Independently of stress, intraperitoneal pyridostigmine also increased the level of RNA encoding brain c-fos and AChE. In hippocampal slice cultures, pyridostigmine enhanced expression of c-fos (but not synaptophysin) and produced a concomitant increase in evoked CA1 electrical activity. These investigators proposed that by altering the permeation properties of the blood–brain barrier, stress can amplify the CNS effects of peripherally delivered pyridostigmine, a compound with a quarternary amine thought to not normally enter the CNS. This model might explain how doses of pyridostigmine that do not normally produce CNS symptoms—such as nervousness, headaches, drowsiness, attention deficits, subnormal cognition—can trigger such symptom complexes. Aspects of the Friedman study are reinforced by the finding of Abdel-Rahman et al. (2002) that in rats stress imposed by restricting movement enhances neural effects of three simultaneously administered toxins: pyridostigmine (1.3 mg/kg/d), DEET (40 mg/kg/d), and permethrin (0.13 mg/kg/d). When superimposed on a stress paradigm, these compounds reduced the integrity of the blood-brain barrier in several regions (cingulate cortex, dentate gyrus of the hippocampus, lateral dorsal nucleus of the thalamus, and the dorsomedial nucleus of the hypothalamus); in each region, there was evidence of neuronal death and astrogliosis.

The model developed in the Friedman report has obvious relevance to understanding how brief exposures to low doses of intoxicants might have unexpected adverse effects on the CNS in Gulf War veterans. It is of interest, therefore, that Lallement et al. (1998, 2001) who also investigated the impact of stress on brain permeation by pyridostigmine, reached somewhat different conclusions. In the Lallement studies, stress was induced by heat rather than exercise in guinea pigs. Using two methods to significantly increase core temperatures, no evidence of loss of integrity of the blood-brain barrier was found. The authors urged caution in translating the results of the Friedman report to Gulf War phenomena in humans (Lallement et al., 1998). In a follow-up study of pyridostigmine administered subcutaneously to guinea pigs over 6 days, only rare and focal evidence of disruption of the blood-brain barrier was found (Lallement, 2001). In studies of CD-1 and FVB/N mice, Grauer et al. (2000) were not able to demonstrate brain permeation by pyridostigmine (0.4 mg/kg, intramuscular or intraperitoneal), using two

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