PB (99.4%) was purchased from Fisher Scientific (Hanover Park, IL), and permethrin (PER) (98.3% mixture of 27.2% cis and 71.1% trans isomers) was purchased from Sigma Aldrich (St. Louis, MO). As there is no information currently available on the exact cis/trans ratio of PER that was used in the 1990–1991 Gulf War, we used this commercially available ratio since it was similar to that recommended by the World Health Organization (25% cis and 75% trans; WHO, 2009 ). We used 0.7 mg/kg of PB and 200 mg/kg of PER doses that have been used in previous mouse studies showing adverse behavioral or pathological outcomes (Gillette and Bloomquist, 2003 (link); Abdullah et al., 2011 (link); Ojo et al., 2013 (link); Zakirova et al., 2015 (link)).
We acknowledge the limitations of this animal model of GW agent exposure, specifically, the route of administration of GW agents, PB and PER, via intraperitoneal administration. Given that, clinical literature on GWI reports that PB was taken orally by GWVs, and that PER exposure likely occurred through inhalation and/or through skin exposure. However, we would like to reiterate that this work was an extension of our previously published model. A daily consumption of 120 mg of PB for an average weight of 75 kg per individual would approximate 1.6 mg/kg. This higher dose range has been shown to inhibit AChE and activate pathways involved in long-term memory retention (Friedman et al., 1996 (link); Vythilingam et al., 2005 (link)). However, for our current studies the dose of PB was systematically scaled down from 2 to 0.7 mg/kg in the C57BL6/J mice as higher dose(s) of PB caused insurmountable death in these mice (Dr. Ait-Ghezala, pers. comm.), this is due to the fact that the C57BL6/J mouse strain is known to exhibit cholinergic deficits (Schwab et al., 1990a (link),b (link)). PER was provided to enlisted personnel as 0.5% spray, and its usage far exceeded that recommended on the PER label (Binns et al., 2008 ). Given the paucity of information on doses and routes (i.e., inhaled, skin absorption) of PER delivery, there is no accurate way of estimating the exact dose of PER exposure to GW veterans. Thus, we used 200 mg/kg of PER to mimic a high-level exposure that is similar to doses administered to mice in previous studies showing adverse behavioral or pathological outcomes (Pittman et al., 2003 (link); Dodd and Klein, 2009 (link)). While we agree that investigation into the effects of different doses of GW agents in different preclinical models is critical in order to fully capture the heterogeneity of exposure and to recapitulate the clinical presentation seen in veterans with GWI, the doses for PB and PER used in this study are approximately less than one fifth and less than half of the reported LD50 dose for mice, respectively (Williamson et al., 1989 (link); Chaney et al., 2002 (link))and are therefore relevant to modeling GWI disease pathophysiology.
We acknowledge the limitations of this animal model of GW agent exposure, specifically, the route of administration of GW agents, PB and PER, via intraperitoneal administration. Given that, clinical literature on GWI reports that PB was taken orally by GWVs, and that PER exposure likely occurred through inhalation and/or through skin exposure. However, we would like to reiterate that this work was an extension of our previously published model. A daily consumption of 120 mg of PB for an average weight of 75 kg per individual would approximate 1.6 mg/kg. This higher dose range has been shown to inhibit AChE and activate pathways involved in long-term memory retention (Friedman et al., 1996 (link); Vythilingam et al., 2005 (link)). However, for our current studies the dose of PB was systematically scaled down from 2 to 0.7 mg/kg in the C57BL6/J mice as higher dose(s) of PB caused insurmountable death in these mice (Dr. Ait-Ghezala, pers. comm.), this is due to the fact that the C57BL6/J mouse strain is known to exhibit cholinergic deficits (Schwab et al., 1990a (link),b (link)). PER was provided to enlisted personnel as 0.5% spray, and its usage far exceeded that recommended on the PER label (Binns et al., 2008 ). Given the paucity of information on doses and routes (i.e., inhaled, skin absorption) of PER delivery, there is no accurate way of estimating the exact dose of PER exposure to GW veterans. Thus, we used 200 mg/kg of PER to mimic a high-level exposure that is similar to doses administered to mice in previous studies showing adverse behavioral or pathological outcomes (Pittman et al., 2003 (link); Dodd and Klein, 2009 (link)). While we agree that investigation into the effects of different doses of GW agents in different preclinical models is critical in order to fully capture the heterogeneity of exposure and to recapitulate the clinical presentation seen in veterans with GWI, the doses for PB and PER used in this study are approximately less than one fifth and less than half of the reported LD50 dose for mice, respectively (Williamson et al., 1989 (link); Chaney et al., 2002 (link))and are therefore relevant to modeling GWI disease pathophysiology.
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