Oleoylethanolamide treatment reduces neurobehavioral deficits and brain pathology in a mouse model of Gulf War Illness

Abstract

There are nearly 250,000 Gulf War (GW) veterans who suffer from Gulf War Illness (GWI), a multi-symptom condition that remains untreatable. The main objective was to determine if targeting peroxisomal function could be of therapeutic value in GWI. We performed a pilot study that showed accumulation of very long chain fatty acids (VLCFA), which are metabolized in peroxisomes, in plasma from veterans with GWI. We then examined if targeting peroxisomal β-oxidation with oleoylethanolamide (OEA) restores these lipids to the normal levels and mitigates neuroinflammation and neurobehavioral deficits in a well-established mouse model of GWI. In GWI mice, treatment with OEA corresponded with cognitive benefits and reduced fatigue and disinhibition-like behavior in GWI mice. Biochemical and molecular analysis of the brain tissue showed reduced astroglia and microglia staining, decreased levels of chemokines and cytokines, and decreased NFκB phosphorylation. Treatment with OEA reduced accumulation of peroxisome specific VLCFA in the brains of GWI mice. These studies further support the translational value of targeting peroxisomes. We expect that OEA may be a potential therapy for treating neurobehavioral symptoms and the underlying lipid dysfunction and neuroinflammation associated with GWI. Oleoylethanolamide is available as a dietary supplement, making it appealing for human translational studies.

Figure 1

Plasma FFA profiles in veterans with GWI compared to GW control veterans. Mean ± SEM (n = 10 for controls and n = 12 for GWI). (A) When all VLCFA (C 22), were combined as one category, there was an overall increase in GWI compared to control GW veterans. (B) Quantification of lipid peroxidation product TBARS (n = 8 per group for these analyses only) in plasma showed that levels were significantly elevated in GWI compared to control veterans. (C) Profiling of FFA showed that several saturated (no double-bonds) and monounsaturated fatty acids (one double-bond) were decreased in the blood of veterans with GWI compared to controls. Pristanic acid was decreased in GWI compared to controls. The remaining long-chain fatty acids (14–21 carbons) and VLCFA species particularly omega-3 and omega-6 species, were significantly elevated in veterans with GWI compared to controls. *p ≤ 0.05.

Figure 2

The experimental paradigm. The experimental timeline details the timing of GW chemical administration, treatment with OEA, behavioral testing, and tissue collection.

Figure 3

OEA treatment improves cognitive function and reduces fatigue and disinhibition type behavior in GWI mice. Mean ± SEM of control (n = 12), GWI mice (n = 11), control + OEA (n = 12) and GWI + OEA (n = 12). (A) One month after OEA treatment (6-months post-exposure to GW chemicals), acquisition trials were conducted to train mice to escape into the TH. For GWI mice treated with OEA, total distance to the TH was smallest, particularly on days 1 and 2, indicative of learning. As expected, GWI mice on normal chow had the worst performance with the largest total distance to the TH. Control mice treated with OEA had a higher latency to find the TH compared to control mice on the normal diet on day 2 only. (B) For the probe trials, conducted at 2-months post-treatment (7-months post-exposure), OEA treated GWI mice had a similar frequency of visits to the TH as control mice on normal chow and on OEA but a higher frequency of visits than GWI mice. (C) The immobile time was significantly increased in GWI mice compared to controls and was reduced in OEA treated GWI mice. (D) There was no change in the average speed among the different groups. (E) An examination of the time in the open arms of the EPM showed an increase of disinhibition in GWI mice on normal chow compared to both control groups and OEA treated GWI mice. (F) Total time in the closed arms was decreased in GWI mice compared with the control groups and OEA treated GWI mice. *p ≤ 0.05.

Figure 4

Brain lipid profiles show that OEA significantly decreases the VLCFA in the brains of GWI mice. Mean ± SEM expressed as percent control of mice on normal chow (n = 4/5 per group). (A) Levels of VLCFA were increased in the brains of GWI mice compared to control mice. OEA treated GWI mice had similar levels as in control mice. However, control mice treated with OEA had significantly higher levels of VLCFA compared to control mice that received normal diet. (B) Quantification of lipid peroxidation product TBARS showed that levels were significantly elevated in GWI compared to control mice on normal chow but were lower in OEA treated GWI mice compared to all other groups. (C) Compared to controls, GWI mice had higher levels of several major omega-6 and omega-3 FA (FA20:4, FA22:4. FA22:5, FA24:5 and FA24:6). Treatment with OEA decreased the levels of these FA in GWI mice. *p ≤ 0.05.

Figure 5

OEA treatment reduced elevated astroglia activation in GWI mice. Confocal microscopy images showing staining of astroglia with GFAP (n = 4 per group, 4 serial sections for each animal). Scale bar 50 μm. (A) Images show 20x GFAP staining (red) and DAPI (blue) in the cortex and the DG of mice from all 4 treatment groups. (B) Images show 100x magnification of GFAP stained astroglia in the cortex of each study treatment group. (C) Quantification of GFAP staining from 20x images. There are significant increases in GFAP staining for both the cortex and the DG within the hippocampus of GWI compared to control mice. GWI mice treated with OEA had similar levels as control mice on normal chow. Control mice treated with OEA had higher levels than control mice on the normal diet. *p ≤ 0.05.

Figure 6

OEA treatment reduced microglia proliferation in GWI mice. Light microscopy images showing staining of microglia with IBA-1 (n = 4 per group, 4 serial section for each brain). Scale bar 50 μm. (A) Images show 20x Iba1 staining in the cortex and DG of mice from all 4 treatment groups. (B) Images show 100x magnification of Iba1 stained astroglia in the cortex of each study treatment group. (C) Quantification of Iba1 staining from 20x images. Levels of Iba1 staining within the DG was lower in OEA treated GWI mice compared to GWI mice on normal chow. Levels of Iba1 in GWI mice were higher in the DG compared to control mice. There were no differences in the cortices of GWI mice compared to control or OEA treated GWI mice *p ≤ 0.05.

Figure 7

Levels of phosphorylated NFκB and STAT3 were reduced by OEA treatment in GWI mice. Mean ± SEM (shown as arbitrary units, n = 4 per group). (A,B) The ratio of p-P65/P65 was elevated in GWI mice at 11-months post-exposure. At this time-point, which corresponded with 6-months of OEA treatment, GWI mice having OEA treatment had a significantly lower ratio compared to GWI mice on normal chow. Control mice treated with OEA had non-significantly lower ratios compared to control mice on the regular diet. (C,D) Similarly, p-STAT3/Actin levels were significantly elevated in GWI compared to control mice and were also lower in OEA treated GWI mice compared to those on normal chow. Levels of STAT3 were also elevated in control mice treated with OEA compared to control mice on the normal diet. (E) Among the cytokines examined in the brain, IFN-γ, IL-6, and IL-1β were lower in OEA treated GWI mice compared to GWI mice on normal chow. Mice with GWI had elevated levels of these pro-inflammatory cytokines compared to control mice. These cytokines did not differ between control mice treated with OEA compared to control mice on the normal diet. (F) Treatment with OEA decreased the levels of CCR2 and CCL2 in the brain of GWI mice compared to GWI mice on normal chow. *p ≤ 0.05.

Figure 8

Gulf War chemicals dysregulate peroxisomal lipid metabolism in astroglia, contributing to microglia activation and inflammation which corresponds with neurobehavioral deficits. Our working hypothesis is that GW chemicals affect peroxisomal lipid metabolism in the astrocytes since astroglia activation occurs earlier than microglia activation in this GWI mouse model. We also propose that the failure of astroglia to perform its neuroprotective function contributes to the subsequent activation of microglia and neuroinflammation. Targeting peroxisomes with OEA is expected to reduce astroglia activation which corresponds with normalization of peroxisomal lipid metabolism. We also propose that OEA may also reduce NFκB activation in microglia, thereby reducing neuroinflammation. In another scenario, OEA may undergo degradation by FAAH and oxygenation by other enzymes, generating additional bioactive metabolites which may target the cannabinoid receptors and also promote anti-inflammatory responses via synergistic activation of NFκB.