Brain-Specific Increase in Leukotriene Signaling Accompanies Chronic Neuroinflammation and Cognitive Impairment in a Model of Gulf War Illness

Abstract

Persistent cognitive impairment is a primary central nervous system-related symptom in veterans afflicted with chronic Gulf War Illness (GWI). Previous studies in a rat model have revealed that cognitive dysfunction in chronic GWI is associated with neuroinflammation, typified by astrocyte hypertrophy, activated microglia, and enhanced proinflammatory cytokine levels. Studies in a mouse model of GWI have also shown upregulation of several phospholipids that serve as reservoirs of arachidonic acid, a precursor of leukotrienes (LTs). However, it is unknown whether altered LT signaling is a component of chronic neuroinflammatory conditions in GWI. Therefore, this study investigated changes in LT signaling in the brain of rats displaying significant cognitive impairments six months after exposure to GWI-related chemicals and moderate stress. The concentration of cysteinyl LTs (CysLTs), LTB4, and 5-Lipoxygenase (5-LOX), the synthesizing enzyme of LTs, were evaluated. CysLT and LTB4 concentrations were elevated in the hippocampus and the cerebral cortex, along with enhanced 5-LOX expression in neurons and microglia. Such changes were also associated with increased proinflammatory cytokine levels in the hippocampus and the cerebral cortex. Enhanced CysLT and LTB4 levels in the brain could also be gleaned from their concentrations in brain-derived extracellular vesicles in the circulating blood. The circulating blood in GWI rats displayed elevated proinflammatory cytokines with no alterations in CysLT and LTB4 concentrations. The results provide new evidence that a brain-specific increase in LT signaling is another adverse alteration that potentially contributes to the maintenance of chronic neuroinflammation in GWI. Therefore, drugs capable of modulating LT signaling may reduce neuroinflammation and improve cognitive function in GWI. Additional findings demonstrate that altered LT levels in the brain could be tracked efficiently by analyzing brain-derived EVs in the circulating blood.

Keywords: 5-lipoxygenase; blood-brain barrier; cysteinyl leukotrienes; cytokines; gulf war illness (GWI); gulf war-related chemicals; leukotriene signaling; neuroinflammation.

Figure 1

Animals with chronic Gulf War Illness (GWI) displayed recognition memory and temporal pattern processing impairments. Cartoon (A) illustrates different trials (T1-T3) involved in a novel object recognition test (NORT). The bar charts (B, C) compare percentages of time spent with the familiar object (FO) and the novel object (NO) in naïve (B) and GWI rats (C). (D) compares the novel object discrimination index between naïve and GWI rats. The bar charts in (D–G) compare the total object exploration time (TOET, E), the distance moved (F), and the velocity of movement (G) between the two groups in T2. The cartoon H depicts the sequence of trials (T1-T5) in the temporal pattern processing task (TPPT). The bar charts (I, J) compare the percentages of time spent with the recently explored object (REO) vis-à-vis the previously explored object (PEO) in naïve (I) and GWI rats (J). (K) compares the PEO discrimination index between naïve and GWI rats. The bar charts in (L–N) compare TOETs between the two groups in T2 (L), T3 (M), and T4 (N). *, p<0.05; **, p<0.01; ****, p<0.0001; NS, not significant.

Figure 2

Neurons in the hippocampus and cerebral cortex displayed increased 5-lipoxygenase (5-LOX) expression in rats with chronic Gulf War Illness (GWI). Figures (A–L) illustrate 5-LOX expression (red) in NeuN+ neurons (green) from a naïve control rat in the granule cell layer (GCL, A–C), the hippocampal CA1 (D–F) and CA3 (G–I) subfields, and the cerebral cortex (J–L). Figures (M–X) demonstrate 5-LOX expression (red) in NeuN+ neurons (green) from a rat with chronic GWI in the GCL (M–O), the hippocampal CA1 (P–R), and CA3 (S–U) subfields, and the cerebral cortex (V–X). The bar charts (Y-AB) compare percentages of NeuN+ neurons expressing 5-LOX between naïve and GWI rats in the GCL (Y), the hippocampal CA1 (Z), and CA3 (AA) subfields, and the cerebral cortex (AB). ***, p<0.001; ****, p<0.0001; Scale Bar = 25µm.

Figure 3

Microglia in the hippocampus and cerebral cortex displayed increased 5-lipoxygenase (5-LOX) expression in rats with chronic Gulf War Illness (GWI). Figures (A–H) illustrate 5-LOX expression (red) in IBA-1+ microglia (green) from a naïve control rat in the dentate hilus (DH, A, B), the hippocampal CA1 (C, D) and CA3 (E, F) subfields, and the cerebral cortex (G, H). Figures (I–P) demonstrate 5-LOX expression (red) in IBA-1+ microglia (green) from a rat with chronic GWI in the GCL (I, J), the hippocampal CA1 (K, L) and CA3 (M, N) subfields, and the cerebral cortex (O, P). Arrows indicate 5-LOX expressing microglia. The bar charts (Q–T) compare percentages of IBA-1+ microglia expressing 5-LOX between naïve and GWI rats in the DH (Q), the hippocampal CA1 (R) and CA3 (S) subfields, and the cerebral cortex (T). **, p<0.01; ****, p<0.0001; Scale Bar = 25µm.

Figure 4

The soma of astrocytes (the area where multiple processes emanate) rarely displayed 5-LOX expression in both naïve control and GWI animals. Figures (A–F) illustrate representative examples of dual immunofluorescence staining for GFAP (red) and 5-LOX (green) in the cerebral cortex of a naive rat (A–C) and a GWI rat (D–F). Arrows indicate a lack of 5-LOX expression in astrocytes. Scale Bar = 12.5 µm.

Figure 5

Animals with chronic Gulf War Illness (GWI) displayed an increased concentration of leukotrienes (LTs) in the brain and brain-derived extracellular vesicles (EVs) in the circulating blood but not in the serum. The bar charts (A–C) compare 5-LOX (A), LTB4 (B), and cysteinyl LT (CysLT; C) concentrations in the hippocampus, (D, E) compare LTB4 (D) and CysLTs (E) concentrations in the cerebral cortex, and (F, G) compare LTB4 and CysLT concentrations in the serum between naïve and GWI rats. The bar charts in (H–K) compare LTB4 and CysLT concentrations in neuron-derived extracellular vesicles (NDEVs H, I) and astrocyte derived EVs (ADEVs; J, K) between naïve and GWI rats. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; NS, not significant.

Figure 6

Animals with chronic Gulf War Illness (GWI) displayed an increased concentration of proinflammatory cytokines in the hippocampus, cerebral cortex, and circulating blood. The bar charts A–H compare tumor necrosis factor-alpha (TNF-α; A, E), interleukin-1 beta (IL-1β; B, F), macrophage inflammatory protein-1 alpha (MIP-1α; C, G), and interleukin 6 (IL-6; D, H) concentrations in the hippocampus (A–D) and the cerebral cortex (E–H) between naïve and GWI rats. The bar charts (I–P) compare TNF-α (I), IL-1β (J), MIP-1α (K), monocyte chemoattractant protein-1 (MCP-1; L), transforming growth factor-beta (TGF-β; M), fibroblast growth factor-beta (FGF-β; N), interleukin-1 alpha (IL-1α; O), and vascular endothelial growth factor (VEGF; P) concentrations in the serum between naïve and GWI rats. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.