Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War Illness

Abstract

Gulf War Illness (GWI) is a multi-symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N-diethyl-meta-toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti-inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7-14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain-wide neuroinflammation assessed by qPCR of tumor necrosis factor-α, IL6, chemokine (C-C motif) ligand 2, IL-1β, leukemia inhibitory factor, and oncostatin M. Pre-treatment with high physiological levels of CORT greatly augmented (up to 300-fold) the neuroinflammatory responses to DFP. Anti-inflammatory pre-treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI. Gulf War (GW) veterans were exposed to stressors, prophylactic medicines and, potentially, nerve agents in theater. Subsequent development of GW Illness, a persistent multi-symptom disorder with features characteristic of sickness behavior, may be caused by priming of the CNS resulting in exaggerated neuroinflammatory responses to pathogens/insults. Nerve agent, diisopropyl fluorophosphate (DFP), produced a neuroinflammatory response that was exacerbated by pre-treatment with levels of corticosterone simulating heightened stressor conditions. While prophylactic treatments reduced DFP-induced neuroinflammation, this effect was negated when those treatments were combined with corticosterone.

Keywords: CORT; DFP; GWI; microglia; minocycline; neuroinflammation.

Figure 1

Dosing paradigm. Time‐line for administration of diisopropyl fluorophosphate (DFP) (4 mg/kg, i.p.) with and without prior administration of corticosterone (CORT) (200 mg/L in drinking water) and/or pyridostigmine bromide (PB) (2 mg/kg/day, s.c.) and N,N‐diethyl‐meta‐toluamide (DEET) (30 mg/kg/day, s.c.) (a), or minocycline (MINO) (100 mg/kg/day, s.c.) (b).

Figure 2

Diisopropyl fluorophosphate (DFP) causes neuroinflammation in multiple brain regions. Mice were administered DFP (4 mg/kg, i.p.) with saline (0.9%) as control and were killed at 6 h post dosing. Total RNA was prepared from frontal cortex (FC), hippocampus (HIP), striatum (STR), hypothalamus (HYPO), olfactory bulbs (OB), and cerebellum (CB) and for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean ± SEM, n = 5. Group differences between saline and DFP exposed mice were measured by Student's t‐test, and statistical significant was measured at p < 0.05 is denoted by * as compared to saline controls.

Figure 3

Diisopropyl fluorophosphate (DFP) causes a time‐dependent neuroinflammation in frontal cortex (FC) and hippocampus (HIP). Mice were administered DFP (4 mg/kg, i.p.) with saline (0.9%) as control and were killed at 2–12 h post dosing. Total RNA was prepared from FC or HIP for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean ± SEM, n = 5. Differences between mice in saline and DFP groups and time course data were analyzed using a two‐way anova, with Fisher's LSD Method of post hoc analysis, p < 0.05 is denoted by *as compared to saline control, ^#as compared to the 6 h time point within the appropriate brain region, and ^§as compared to the 2 h time point within the appropriate brain region. Symbols used to mark differences observed in the FC alone are in red, differences in HIP alone are in blue, and differences in both groups are marked in green.

Figure 4

Pre‐treatment with corticosterone (CORT) enhances diisopropyl fluorophosphate (DFP)‐induced neuroinflammation in multiple brain regions. Mice were administered CORT (200 mg/L) in the drinking water for 1 week prior to administration of DFP (4 mg/kg, i.p.) with saline (0.9%) as control and were killed at 6 h post dosing. Total RNA was prepared from frontal cortex (FC), hippocampus (HIP), striatum (STR), hypothalamus (HYPO), olfactory bulbs (OB), and cerebellum (CB) for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean ± SEM, n = 5. Group differences between mice exposed to DFP and mice exposed to CORT+DFP treatments was measured using a Student's t‐tests, and statistical significant was measured at p < 0.05 is denoted by * as compared to DFP alone.

Figure 5

Pre‐treatment with corticosterone (CORT) enhances diisopropyl fluorophosphate (DFP)‐induced neuroinflammation in a time‐dependent manner in frontal cortex (FC) and hippocampus (HIP). Mice were administered CORT (200 mg/L) in the drinking water for 1 week prior to administration of DFP (4 mg/kg, i.p.) with saline (0.9%) as control and were killed at 2, 6, and 12 h post dosing. Total RNA was prepared from FC or HIP for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean ± SEM, n = 5. Differences between mice in DFP and CORT +DFP groups and time course data were analyzed using a two‐way anova, with Fisher's LSD Method of post hoc analysis, p < 0.05 is denoted by *as compared to DFP, ^#as compared to the 6 h time point and ^§as compared to the 2 h time point. Symbols used to mark differences observed in the DFP alone are in red, differences in CORT + DFP alone are in blue, and differences in both groups are marked in green.

Figure 6

Pyridostigmine bromide (PB)/N,N‐diethyl‐meta‐toluamide (DEET) pre‐treatment can reduce diisopropyl fluorophosphate (DFP)‐induced neuroinflammation. Mice were administered PB (4 mg/kg/day, s.c.) and DEET (30 mg/kg/day, s.c) prior to administration of DFP (4 mg/kg, i.p). See Fig. 1 for dosing timeline. Saline (0.9%) was used as control and mice were killed at 6 h post dosing. Total RNA was prepared from frontal cortex (FC), hippocampus (HIP) and striatum (STR) for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean ± SEM, n = 5. The treatment effect of PB/DEET on mice exposed to DFP was analyzed using a two‐way anova, with Fisher's LSD Method of post hoc analysis, p < 0.05 is denoted by *as compared to control, ^#as compared to DFP.

Figure 7

Corticosterone (CORT) exposure negates the amelioration of diisopropyl fluorophosphate (DFP)‐induced neuroinflammation by pyridostigmine bromide (PB)/N,N‐diethyl‐meta‐toluamide (DEET). Mice were administered PB (4 mg/kg/day, s.c.) and DEET (30 mg/kg/day, s.c), with and without co‐administration of CORT (200 mg/L) in drinking water prior to administration of DFP (4 mg/kg, i.p). See Fig. 1 for dosing timeline. Saline (0.9%) was used as control and mice were killed at 6 h post dosing. Total RNA was prepared from frontal cortex (FC), hippocampus (HIP), and striatum (STR) for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean ± SEM, n = 5. Statistical significance was measured by one‐way anova with Fisher's LSD Method of post hoc analysis, p < 0.05 is denoted by *as compared to DFP, ^#as compared to CORT+DFP.

Figure 8

Diisopropyl fluorophosphate (DFP) with and without prior pyridostigmine bromide (PB)/N,N‐diethyl‐meta‐toluamide (DEET)+corticosterone (CORT) did not cause remarkable neurodegeneration or gliosis. Mice were administered PB (4 mg/kg/day, s.c.) and DEET (30 mg/kg/day, s.c), with and without co‐administration of CORT (200 mg/L) in drinking water prior to administration of DFP (4 mg/kg, i.p). See Fig. 1 for dosing timeline. Saline (0.9%) was used as control and mice were killed at 24‐h post dosing. Neurodegeneration as indexed by silver disintegration staining (a) or Fluoro‐Jade B staining (b) did not reveal evidence of neuronal damage in the hippocampus (HIP). Astroglial staining with glial fibrillary acidic protein (GFAP) (c) did not show evidence of astrogliosis; microglial staining with ionized calcium‐binding adapter molecule 1 (Iba1) (d) did not reveal microglial activation in the HIP. Scale bars = 100 μm (Silver) and 200 μm (Fluoro‐Jade B, GFAP, and Iba1).

Figure 9

Diisopropyl fluorophosphate (DFP) with and without prior pyridostigmine bromide (PB)/N,N‐diethyl‐meta‐toluamide (DEET)+corticosterone (CORT) did not increases glial fibrillary acidic protein (GFAP) in multiple brain regions. Mice were administered PB (4 mg/kg/day, s.c.) and DEET (30 mg/kg/day, s.c), with and without co‐administration of CORT (200 mg/L) in drinking water prior to administration of DFP (4 mg/kg, i.p). See Fig. 1 for dosing timeline. Saline (0.9%) was used as control and mice were killed at 72‐h post dosing. Immunoassay of GFAP levels in frontal cortex (FC), hippocampus (HIP) or striatum (STR), as a quantitative index of astrogliosis did not reveal any significant effects as a function of any of the treatments. All data points represent mean + SEM, n = 5. Statistical significance was measured by one‐way anova with Fisher's LSD Method of post hoc analysis, p < 0.05 is denoted by *as compared to control.

Figure 10

Pre‐treatment with minocycline (MINO) partially suppresses neuroinflammation induced by corticosterone (CORT)+diisopropyl fluorophosphate (DFP) in frontal cortex (FC) and hippocampus (HIP). Mice were pre‐treated with CORT (200 mg/L in drinking water) with and without co‐administration of MINO (100 mg/kg/day, s.c.) prior to administration of DFP (4 mg/kg, i.p). See Fig. 1 for dosing timeline. Saline (0.9%) was used as control and mice were killed at 6‐h post DFP dosing. Total RNA was prepared from FC and HIP for q‐PCR analysis of tumor necrosis factor‐alpha (TNF‐α), IL6, chemokine (C‐C motif) ligand 2 (CCL2), IL‐1β, leukemia inhibitory factor (LIF), and oncostatin M (OSM). All data points represent mean + SEM, n = 5. Statistical significance was measured by one‐way anova with Fisher's LSD Method of post hoc analysis, p < 0.05 is denoted by *as compared to control and ^#as compared to CORT+DFP. Previously, we have shown that MINO alone does not affect cytokine or chemokine expression using the regimen employed in this study (Sriram et al. 2006; O'Callaghan et al. 2008).