Anxiety, neuroinflammation, cholinergic and GABAergic abnormalities are early markers of Gulf War illness in a mouse model of the disease

Abstract

Gulf War Illness (GWI) is a chronic disease that affects the 1991 Gulf War (GW) veterans for which treatment is lacking. It has been hypothesized that drugs used to protect military personnel from chemical attacks and insects during the war: pyridostigmine bromide (PB),N, N-diethyl-m-toluamide (DEET), and permethrin (PER) together with stress may have contributed collectively and synergistically to generate GWI. There is a need to find markers of pathology to be used in pre-clinical trials. For this purpose we employed a previously validated mouse model of GWI evoked by daily exposure to PB (1.3 mg/kg), DEET (40 mg/kg), PER (0.13 mg/kg), and 5 min of restraint stress for 28 days to analyze behavior, brain pathology and neurochemical outcomes three months later. GWI-model mice were characterized by increased anxiety, decreased hippocampal levels of N-acetyl aspartate, GABA, the GABA-producing enzyme GAD-67 and microglial activation. We also observed that GWI model was sexually dimorphic on some measures: males had increased while females had decreased protein levels of the acetylcholine-synthesizing enzyme, choline acetyltransferase, in the septum and hippocampus and decreased levels of the receptor for brain-derived neurotrophic factor, TrkB140, in the hippocampus. Increased hippocampal levels of nerve growth factor were detected in males only. Together the data show behavioral and neuropathological abnormalities detected at 3 months post-exposure and that some of them are sexually dimorphic. Future preclinical studies for GWI may take advantage of this short latency model and should include both males and females as their response to treatment may differ.

Keywords: Anxiety; Cholinesterase inhibitors; Gulf war illness; Magnetic resonance spectroscopy.

Figure 1

Effect of exposure to GW-related chemicals and stress on anxiety, learning and memory. A. Anxiety. Exposure causes increased anxiety revealed in the Plus Maze test as both GW-exposed female and male mice did not enter as often and did not spent as much time as control mice in open arms. Two-way ANOVA for the number of entries into the open arms found a main effect of exposure (F= 4.31; p<0.05) with no effect of sex and no interactions. For time spent in open arms there was a main effect of exposure (F=4.9; p = 0.03) and no effect of sex and no interaction between factors. B. Learning and memory in the water maze. No significant differences in the escape latency and comparable swim speed between exposed and control female and male mice were observed during the 6 days of training trials. The probe trial performed twenty-four hours after the last training trial to test mice’s memory also showed no significant differences between groups for any of the analyzed parameters.

Figure 2

Sexually dimorphic cholinergic abnormalities in GW exposed mice. The effect of exposure to GW-related chemicals and stress on the levels of the ACh-synthesizing enzyme, ChAT, and GFP in the septum and hippocampus of CHGFP mice. Two-way ANOVA using the two brain regions, hippocampus and septum, as a repeated measure, revealed significant interaction terms between exposure and sex (p = 0.003 for ChAT; p = 0.016 for GFP assayed by western blot; and p = 0.025 for GFP determined by ELISA). This interaction is illustrated by the consistently diverging blue (male) and pink (female) lines for all of the measures.

Figure 3

Microglial activation in the hippocampus of GW-exposed mice. A. Representative microphotographs showing that a larger number of activated microglia, prominent by their distinctive cell morphology and increased levels of Iba1 expression, were detected in the CA1-3 regions of the hippocampus of exposed CHGFP mice compared to controls in both female and male mice. Original pictures were taken at 100x and 400x, as indicated. The inset graphs are the results from the quantitation of the number of activated microglia in the CA1-3 region showing that in this region of the hippocampus the number of activated microglia is significantly larger in exposed female and males than in their respective control mice. B. Western blot analysis of protein extracts from the whole hippocampus with Iba1 antibodies. The level of Iba1 increased in exposed males and females to 135.5% and 107.8% of controls, respectively. Two-way ANOVA showed a significant effect of exposure for Iba1 (F = 4.63; p = 0.038), and no effect of sex.

Figure 4

Abnormalities in NGF, BDNF and TrkB in GW exposed mice. Increased hippocampal NGF levels were detected in male, but not female, mice 3 months after exposure to GW related chemicals and stress. No changes in BDNF levels after exposure were detected in the hippocampus of either male or female mice. Sexual dimorphism was evident in the effect of exposure on TrkB receptor forms. Data are presented as mean ± SEM. *p<0.05; **p<0.01.

Figure 5

A. Decreases in NAA and GABA, measured using HRMAS from punches of the hippocampus, in GWI model male mice compared to control not exposed mice. There is an 18% decrease in NAA and a 20% decrease in GABA following exposure to the chemicals and stress. * p < 0.05 for a one-way ANOVA comparing the two groups with a Student–Newman–Keuls post hoc test for multiple comparisons. B. Comparative % decrease to GABA was detected for GAD67, but not GAD65. A two-way ANOVA for GAD67 showed a main effect of exposure (F = 4.6; p<0.04) with no effect of sex and no interaction.