Biochemical and behavioral effects of decreasing dietary linoleic acid and increasing eicosapentaenoic acid and docosahexaenoic acid in a rat chronic monoarthrits model (2025)

Abstract

Clinical studies have demonstrated that decreasing linoleic acid (LA) while increasing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in diets evokes an analgesic effect in headache sufferers. We utilized a rat chronic monoarthritis model to determine if these analgesic effects can be reproduced in rats and to and further probe potential analgesic mechanisms. We fed 8 rats a control diet (with fatty acid levels similar to standard US diets) and 8 rats a low LA diet with added EPA and DHA (H3L6 diet) and after 10 weeks, performed a unilateral intraarticular injection of Complete’s Freund Adjuvant (CFA). We evaluated thermal and mechanical sensitivity as well as hind paw weight bearing prior to and at 4 and 20 days post CFA injection. At 28 days post CFA injection rats were euthanized and tissue collected. H3L6 diet fed rats had higher concentrations of EPA and DHA, as well as higher concentrations of oxidized lipids derived from these fatty acids, in hind paw and plasma, compared to control fed rats. LA and oxidized LA metabolites were lower in the plasma and hind paw of H3L6 compared to control fed rats. Diet did not affect thermal or mechanical sensitivity, nor did it affect hind paw weight bearing. In conclusion, the H3L6 diet evoked biochemical changes in rats but did not impact pain related behavioral measures in this chronic monoarthritis model.

Keywords: Polyunsaturated fatty acid, Diet, Pain, Arthritis, Linoleic Acid, Omega-3

1. Introduction

Consumption of polyunsaturated fatty acids (PUFAs) increased approximately 3-fold during the 20^th century, largely due to the increased consumption of vegetable oils [1], The increase in PUFA consumption has been accompanied by a decrease in saturated fat consumption [2] mimicking public health initiatives advising Americans to replace saturated fats with PUFAs [3], PUFAs can be converted, via enzymatic or non-enzymatic processes, to oxidized lipids (oxylipins), and oxylipins have been reported to impact a number of processes such as inflammation, nociception, cell death and neurodegeneration. The most consumed PUFA in US diets is Linoleic Acid (LA, 18:2ω6), an omega 6 (n-6) PUFA that can serve as a substrate for Arachidonic Acid (AA, 20:4ω6). Oxylipins derived from LA (e.g. 9-hydroxyoctadecenoate, 9-HODE) and AA (e.g. Prostaglandin E2, PGE2) have been reported to impact nociception which led us to hypothesize that decreasing dietary n-6 PUFA may impact pain. In support of this hypothesis, a small clinical trial in 67 participants with chronic daily headaches demonstrated that decreasing dietary LA while increasing the dietary omega 3 (n-3) PUFAs, Eicosapentaenoic acid (EPA, 20:5ω3) and Docosahexaenoic acid (DHA, 22:6ω3) was associated with decreases in headache frequency and severity [4].

This preliminary finding led to the initiation of several other controlled trials testing whether dietary alterations can alter nociceptive mediators and decrease pain in other chronic pain conditions (NCT01251887 and NCT03272399, NCT02531711). The largest of these trials (n=182) confirmed the analgesic effect of decreasing dietary LA and increasing dietary EPA and DHA in migraine sufferers [5], The mechanisms underlying this diet induced analgesia are unclear and ability to sample human pain circuit tissues, to interrogate these mechanisms, is limited. Clinically, circulating concentrations of oxylipins derived from LA have been positively associated with pain while oxylipins derived from DHA have been shown to be inversely associated with pain. We have reported on the impact of oxylipins on pain in rats and found oxylipins are present throughout pain circuit tissue making them well situated to affect pain [6], Additionally, oxylipins derived from LA have been reported to evoke pain [7–11] while oxylipins derived from DHA have been reported to be anti-nociceptive [12] [13,14] in preclinical models.

Here we aimed to: (1) determine whether these analgesic/anti-nociceptive effects of decreasing dietary LA while increasing dietary EPA and DHA observed in humans can be replicated in a rat model of chronic monoarthritis; and (2) to probe the mechanism(s) underlying the diet-induced pain reduction. The long-term goal of this research is to better understand mechanisms linking diet to pain in order to help refine analgesic diet interventions and suggest novel targets for drug development. We hypothesized that diet would evoke changes in oxylipins in pain circuit tissue leading to a decrease in pro-nociceptive oxylipins and an increase in antinociceptive oxylipins. Since we were studying arthritic pain in hind paw, and LA is particularly concentrated in skin, we emphasized measuring many LA derived oxylipins in inflamed hind paw. We found that tissue concentrations of oxylipins were affected by diet, however, we were unable to reproduce the analgesic/antinociceptive effect of decreasing dietary LA and increasing EPA and DHA.

2. Methods

2.1. Diet

Diets were manufactured by Dyets Inc (Bethlehem, PA). Diet ingredients are listed in Supplementary Table S1. The High LA Control Diet (H6CTL diet) was designed to mimic average US intakes of PUFA and contained approximately 23% of fatty acids as LA (Table 1) which amounted to approximately 8% of energy (%en). Oxidized corn oil was purchased from Cayman Chemical (Ann Arbor, MI). The experimental High n-3 PUFA + Low LA Diet (H3L6 diet) lowered LA about 7-fold (compared to the H6CTL diet) and contained added EPA and DHA (Table 1).

Table 1 -.

Diet fatty acid composition as measured by Gas Chromatography Flame Ionization Detection

Fatty Acid (Carbon Atoms : Number of Double Bonds)	H6CTL Diet	H3L6 Diet
8:0 μ g/mg	2.1	2.9
10:0 μg/mg	2.4	4.0
12:0 μg/mg	22.0	36.8
14:0 μg/mg	8.4	15.9
16:0 μg/mg	10.8	11.0
18:0 μg/mg	9.1	12.7
16:1ω7 μg/mg	0.1	0.6
18:1ω9 μg/mg	39.6	38.3
18:1ω7 μg/mg	0.5	0.5
18:2ω6 (LA) μg/mg	28.4	3.7
20:4ω6 (AA) μg/mg	0.0	0.1
18:3ω3 (ALA) μg/mg	1.9	1.9
20:5ω3 (EPA) μg/mg	0.0	0.8
22:5ω3 μg/mg	0.0	0.1
22:6ω3 (DHA) μg/mg	0.0	0.8

Total Sat	53.8	81.4
Total Mono	40.5	39.7
Total ω6	28.4	3.9
Total ω3	1.9	3.6
n-3 HU FA μg/mL	0.0	1.7
Sat %	43.2	63.3
Mono %	32.5	30.9
ω6 %	22.8	3.0
ω3 %	1.5	2.8

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2.2. Animals

Experiments were conducted in accordance with the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and other Communication Disorders Animal Care and Use Committee and the International Association for the Study of Pain guidelines for the care and use of experimental animals. Male, Long Evans rats (Charles River) arrived at our facility at 6 weeks of age. Upon arrival rats were pair housed and each cage was allocated to either H6CTL or H3L6 diets (n=8 rats per group). Rats were maintained on an inverted, 12-hour light/dark cycle and were given ad libitum access to food and water. After 8 weeks of feeding, rats were subjected to a sensory testing battery. After 10 weeks of feeding, rats were injected intra-articularly at the ankle, with 50 μL of Complete Freund’s Adjuvant (Sigma-Aldrich, MO) using a 29G insulin syringe (Becton Dickinson, NJ).

The sensory testing battery was repeated 4 days and 20 days post injection. At 28 days post injection, relative hind paw weight bearing was evaluated, hind paw edema was measured with a caliper and rats were euthanized and brain, hind paw (ipsilateral and contralateral) and plasma were collected.

2.3. Sensory Testing Batteiy

Rats were regularly handled by the investigators who performed the sensory testing. For 1 week prior to baseline sensory testing, rats were habituated to the behavior testing suite by familiarizing them with the testing suite and allowing them to explore the testing boxes as well as apparatuses (i.e. allowing rats to see the testing lights and Von Frey filaments while in the testing boxes). All testing was performed during the rats dark cycle and the behavior suite was illuminated with red lights.

To examine thermal hypersensitivity, rats were placed in 10 × 20 cm Perspex testing boxes (2 sides opaque, arranged so animals unable to see each other). Testing boxes were placed on the glass surface heated to 30 °C of the Hargreaves testing device (IITC Life Science, model 400). On testing days, animals were placed in testing boxes for 20 min and latency to withdraw from a radiant light stimulus, directed at the ventrocaudal aspect of the hind paw was measured. Withdraw was assessed by 2 investigators (AFD and BCW) following methods previously reported by our group [15], At least 2 withdrawal latencies per paw were recorded.

To examine mechanical hypersensitivity, testing boxes were placed on a raised wire mesh surface. Animals were placed in testing boxes for 20 min then probed with a 1 g (#4.08) Von Frey Filament (North Coast Medical, CA) by pressing the filament to the ventrocaudal aspect of the paw until the filament bent. A withdraw response was recorded if the rat actively removed its paw from the filament. Rats were probed 5 times (minimum of 30s between each stimulus) on each paw then the process was repeated with a 6 g (#4.74) and 15 g (#5.18) filament. Withdrawal frequency to each filament was recorded.

Static weight bearing (i.e. relative amount of weight placed on each paw) was measured using an incapacitance meter (IITC Life Science, model 600). Following the methods of Pitcher et al. [16] we placed the rats on an inclined plane and gently held the animal’s tail. Once both feet were flat on their respective scale a 5s measurement was taken. Data are expressed as an average of 3 measurements of the relative amount of weight on the ipsilateral paw vs the contralateral paw.

2.4. Tissue Collection

Twenty-eight days post CFA injection, following the static weight bearing assessments described above, animals were anesthetized with sevoflurane and decapitated with a guillotine. Trunk blood was collected into a 2 mL microcentrifuge tube and vortexed for 10 min at 2910 G (microONE, Tomy, Japan) to isolate plasma. Plasma was collected into a new 2mL microcentrifuge tube and placed on dry ice. The ventral surface of the hind paw was collected following the method previously published by our group [6], weighed and placed in a 2 mL microcentrifuge tube and placed on dry ice. Brain was collected by removing the skull bones with a rongeur and carefully scooping out the brain, including the brainstem. Brain was bisected sagitally at the midline and placed in 5 mL tubes and frozen on dry ice. Tissue remained frozen at −80°C until analysis.

2.5. Fatty Acid Measurements

To determine if diet affected tissue concentrations of oxylipin precursors, ipsilateral brain and contralateral hind paw were homogenized by mixing tissue with methanol then pulverizing tissue with a glass dounce homogenizer. Fatty acids were then extracted following the methods of Folch et al. 1957 [17] and measured using Gas Chromatography Flame Ionization Detection following as previously described [4].

2.6. Oxylipin Analysis

Hind paw was homogenized using a Percellys with temperature maintained below 8°C by a Cryolys (Bertin Corp. MD, USA). Tissue was placed in a 7 mL tube and CkMix50 lysing matrix were placed on top of tissue. Samples were shaken at 8000 RPM for 10 seconds (6 cycles with 2 min between shaking). After shaking, internal standard was added then the sample was extracted twice with methanol containing butylated hydroxy toluene and ethylenediaminetetraacetic acid (both added at 0.02% by volume), transferred to a microcentrifuge tube and stored at −80°C overnight. Protein pellets were precipitated by centrifuge (17000g for lOmin at 4°C). Supernatants were collected then purified by solid phase extraction. Oxylipins were then identified and quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) as previously reported [18,19].

Plasma samples (200 μL) were aliquoted to new microcentrifuge tubes, combined with internal standard and proteins were precipitated by incubating with antioxidant containing methanol (described above) for 1 hour at −80°C followed by centrifugation as previously reported [19][18,19]

2.7. Statistics

Group differences in fatty acids and oxylipin concentrations were analyzed by 2-tailed t-test and only p-values that remained significant after Benjamini-Hoochberg correction, with a false discovery rate of 5% are reported as significant [20], Metaboanalyst was used for principal component analyses (PC As) [21], For plasma oxylipin and hind paw concentrations, PC A was performed on cube root transformed, pareto scaled oxylipin concentration data. Pathway figures were generated using Cytoscape (Version 3.8.2, San Diego, CA). Sensory testing results were analyzed by 2-way ANOVA (Graph Pad Prism, La Jolla, CA) followed by Sidak’s multiple comparison test. Data are presented as mean ± standard deviation unless otherwise indicated.

3. Results

3.1. Effect of diet on body weight

There were no differences in final body weight of rats fed the H3L6 diet compared to the H6CTL diet (612 ± 100 g vs. 515 ± 219 g, respectively, p=0.28).

3.2. Effect of diet on hind paw and brain fatty acid concentrations

The H3L6 diet decreased n-6 PUFA concentrations and increased n-3 PUFA concentrations in hind paw (Figure 1). Notably, alpha-linolenic acid (ALA, 18:3ω3) and docosapentaenoic acid n-6 (DPAn-6, 22:5ω6) were not significant altered after correcting for FDR (Supplemental Table S2). In hind paw, concentration of lauric acid (12:0) was increased after feeding the H3L6 diet but concentration of no other non-PUFA was altered by diet.

Biochemical and behavioral effects of decreasing dietary linoleic acid and increasing eicosapentaenoic acid and docosahexaenoic acid in a rat chronic monoarthrits model (1)

In brain, only PUFA concentrations were affected by diet (Supplemental Table S3), however, overall changes were less pronounced than in hind paw. Notably, after correcting for the FDR, the H3L6 diet did not increase brain concentration of DHA, though AA and EPA concentrations were decreased and increased, respectively.

3.3. Effect of diet on plasma oxylipin concentrations

We were able to quantify 14 oxylipins in the plasma total lipid pool of rats fed the H6CTL and H3L6 diets (Supplemental Table S4). The H3L6 diet produced major changes in plasma oxylipins (Figure 2 and Supplemental Table S4). Of the oxylipins measured only 12-hydroxyeicosatetraenoic acid (12-HETE) and 14-hydroxydocosahexaenoic acid (14-HDHA), both of which are synthesized by Arachidonate 12-Lipoxygenase, were not significantly altered by diet. PC A indicated that plasma oxylipin concentrations were significantly different in rats fed the H3L6 diet (Figure 2A). PCA indicated that 9-HODE, 9,10-DiHOME and 12,13-DiHOME were important features that were decreased by the H3L6 diet (Figure 2B and C). Additionally, 4-hydroxydocosahexaenoic acid (4-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) were important features that were increased in plasma by the H3L6 diet (Figure 2B and C). Interestingly, 19,20-EDP was only above the limit of quantitation in plasma of rats fed the H3L6 diet.

3.4. Effect of diet on oxylipin concentrations in inflamed hind paw

We quantified 24 oxylipins in hind paw of rats fed the H6CTL and H3L6 diets (Table 2). PCA indicated that hind paw oxylipin concentrations were significantly different between rats fed the different diets (Figure 3A) and after correcting for Benjamini-Hochberg FDR we found that hind paw concentrations of 15 oxylipins were significantly altered in rats fed the H3F6 diet (Table 2). Specifically, oxylipins synthesized through the cytochrome p450 epoxygenase (CYP) pathway, such as FA derived epoxyoctadecenoates (EpOMEs), dihydroxy octadecenoates (diHOMEs) and DHA derived EDPs were significant variables in the PCA model and most significantly different between diet groups (Figure 3B and C and Table 2). The significance of the effect of the H3L6 diet on CYP derived oxylipins is illustrated in Figure 3C, which presents the diet evoked changes in oxylipin concentration presented as part of the molecular pathways through which oxylipins are synthesized. Additionally, monohydroxy DHA derivatives 4-, 14-, and 17-hydroxy docosahexaenoic acid (4-, 14, and 17-HDHA, respectively) were significantly more concentrated in hind paw of rats fed the H3F6 diet while monohydroxy FA derivatives 9- and 13-hydroxyoctadecenoate (9- and 13-HODE, respectively) were less concentrated (Figure 3C and Table 2). Notably, Prostaglandin E2 (PGE2) and Thromboxane B2 (TXB2) were not significantly different in hind paw of rats fed the H3F6 diet compared with H6CTF fed rats.

Table 2 -.

Hind paw oxylipin concentrations

	H6CTL Diet	H3L6 Diet	p-value	Significant after FDR? (Y/N)
	Mean Concentration ± Standard Deviation (ng/g)	Mean Concentration ± Standard Deviation (ng/g)	p-value	Significant after FDR? (Y/N)	Limit of quantitation (ng/ml)
9-HODE	517.2 ± 194.2	220.8 ± 99.0	0.002	Y	7.1
13-HODE	757.0 ± 294.6	291.4 ± 106.7	0.001	Y	35.7
9,10-EpOME	101.3 ± 54.3	25.1 ± 10.7	0.002	Y	7.1
12,13-EpOME	109.5 ± 55.5	26.1 ± 10.0	0.001	Y	7.1
9,10-DiHOME	341.0 ± 124.4	57.1 ± 15.9	2E-05	Y	1.4
12,13-DiHOME	215.4 ± 94.5	44.4 ± 13.8	2E-04	Y	7.1
9,12,13-TriHOME	322.7 ± 110.7	341.2 ± 219.5	0.834	N	35.7
9,10,13-TriHOME	48.6 ± 18.0	49.5 ± 25.6	0.941	N	35.7
9,10,11-TriHOME	13.7 ± 3.4	11.0 ± 4.5	0.192	N	0.4
PGE2	130.9 ± 57.3	81.7 ± 26.7	0.045	N	0.4
TXB2	23.0 ± 8.9	16.4 ± 10.5	0.199	N	0.7
5-HETE	36.7 ± 11.8	30.6 ± 9.8	0.278	N	1.4
12-HETE	575.4 ± 152.0	343.3 ± 131.7	0.006	Y	1.4
15-HETE	304.4 ± 88.1	205.1 ± 55.4	0.017	Y	3.6
4-HDHA	3.4 ± 1.6	10.2 ± 3.3	1E-04	Y	1.4
14-HDHA	40.2 ± 21.1	94.0 ± 53.5	0.019	Y	1.4
17-HDHA	77.0 ± 42.6	197.0 ± 128.5	0.025	Y	3.6
PDX	0.2 ± 0.1	0.7 ± 0.5	0.021	Y	0.4
16,17-EDP	1.5 ± 0.6	3.4 ± 0.9	3E-04	Y	0.7
19,20-EDP	2.6 ± 0.8	6.5 ± 1.9	1E-04	Y	1.4
9H-12E-LA	52.2 ± 19.0	29.9 ± 14.7	0.020	Y	7.1
11H-12E-LA	260.7 ± 90.9	154.7 ± 102.7	0.046	N	7.1
11H-9E-LA	137.7 ± 39.4	94.1 ± 49.5	0.071	N	1.4
13H-9E-LA	125.4 ± 45.8	84.5 ± 35.4	0.065	N	1.4

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3.5. Effect of diet on Complete Freund’s Adjuvant (CFA) induced hind paw swelling and hypersensitivity

At the end of the study, CFA injected paws were approximately 40% thicker compared to contralateral paws. However, we did not observe group differences in hind paw thickness at either paw (Figure 4A).

CFA evoked increases in thermal and mechanical hypersensitivity. In the Hargreaves test (a measure of thermal hypersensitivity) we observed a significant effect of time. Compared to baseline, relative latency to withdrawal from the noxious heat stimulus decreased by approximately 60% at 4 days post CFA (Figure 4B) indicating CFA injection evoked a thermal nociceptive hypersensitivity. The CFA injected paw remained hypersensitive to heat stimuli even 18 days post injection (Figure 4B). There was no effect of diet on thermal nociceptive withdrawal latencies.

We assessed mechanical hypersensitivity by assessing withdraw frequency to 3 different sized Von Frey filaments. For the 2 smallest filaments we tested we observed a significant effect of time (Figure 4C and D). We observed an increased withdraw frequency to both of these filaments at 4 days post CFA (approximately 10- and 2-fold increases for the #4.08 and 4.74 filaments, respectively) indicating CFA evoked mechanical hypersensitivity. Mechanical hypersensitivity was not observed at 18 days post CFA injection and we did not observe an effect of diet on sensitivity to these 2 filaments. For, the largest filament we tested (#5.18), we did not observe an effect of time but did observe a main effect of diet on withdraw frequency (Figure 4E). However, the between diet-group post-hoc tests were not statistically significant at any timepoint. It should be noted that overall, there was low withdraw frequency from the filaments chosen here (highest mean withdraw frequency = 0.4).

We also assessed the relative amount of weight that rats voluntarily placed on each hind paw. We observed an effect of time, with rats reducing the amount of voluntary weight bearing on the injected paw by approximately 50% at 4 days post CFA (Figure 4F). Relative weight bearing on the injected paw remained decreased for the duration of the study (28 days post CFA). No effect of diet was observed on relative voluntary weight bearing (Figure 4F).

4. Discussion

In this report we assessed the effect of lowering dietary LA and increasing dietary DHA and EPA on pain related hypersensitivity in a rat model of rodent monoarthritis. We found that the H3L6 diet evoked changes in hind paw PUFA concentrations, as well as changes in plasma and hind paw oxylipin concentrations. Similar to previous reports in humans [4,22–24], feeding rats the H3L6 diet decreased tissue concentrations of LA and LA derived oxylipins while increasing tissue concentrations of DHA and DHA derived oxylipins. Despite decreasing hind paw concentrations of pronociceptive, LA-derived oxylipins such as 9-HODE, the H3L6 diet did not impact thermal or mechanical hyperalgesia and did not affect the voluntary hind paw weight following a unilateral, intraarticular injection of CFA. In light of recent reports indicating the analgesic effect of the H3L6 diet in humans [5], and the small sample size of the current study, the present findings should be interpreted with some caution. That said, if these results are reproduced with a larger sample size of rats, it would suggest that the mechanisms by which dietary PUFA impact chronic pain may not be conserved between humans and rodents or that the rodent monoarthritis model used in this report evokes pain through mechanisms that differ from human headache pain. Indeed it is conceivable that there are mechanistic differences between headache and arthritis pain, as interventions that are beneficial for arthritis (such as exercise) are not necessarily beneficial for headache and may even evoke migraine [16], Additionally, a recent report confirmed a role of PUFA in pain and nociception in inflamed (and not inflamed) mouse hind paw [25], indicating at least some of the potential mechanism of the H3L6 diet is conserved across species and modalities.

It was recently reported that feeding mice a high n-6 PUFA diet (H6 diet) evoked thermal and mechanical hypersensitivity which was accompanied by decreased intraepidermal nerve fiber density (a marker of neuropathy) [25]. Additionally, these authors found that feeding mice a H6 diet prolonged hypersensitivity evoked by an intraplantar CFA injection [25]. The effect the H6 diet appeared to be mediated through increased tissue concentrations of LA leading to increased non-membrane-bound forms of LA. In our study, we did observe increased tissue concentrations of LA, however, we did not observe increased hypersensitivity in our rats.Besides the use of rats in our study, there are other notable differences from the recent report. In particular, our H6CTL diet provided LA at 8 %en, which is in line with average US consumption, whereas the mouse study provided LA at 11 %en [25] an amount that is being consumed by a smaller number of US adults (<15% of US adults) [26]. Furthermore, when evaluating the effect of LA on CFA evoked hypersensitivity, we opted for an intraarticular CFA injection at a dosage higher than the report of Boyd et al; therefore, our methodology is likely relevant to monoarthrits while Boyd et al. may have examined inflammation more generally [27]. These combined findings may indicate that the effect of dietary n-6 PUFA on pain may be modality specific. Alternatively, effects of dietary n-6 PUFA on pain may differ between rats and mice. Therefore, future work investigating this should take care when selecting a pain model/condition and rodent species in which to study these effects.

The H3L6 diet evoked intriguing changes in hind paw unesterified oxylipin concentrations. While almost all lipoxygenase derived DHA derivatives were increased by the diet, changes in n-6 PUFA derived oxylipins were more nuanced. The H3L6 diet did decrease hind paw concentrations LA derived cytochrome p450 products, but only one of the LA derived epidermal lipoxygenase products that we measured (9-hydroxy-12,13-epoxy-octadecenoate, 9H-12E-LA) was significantly decreased by diet. In addition, eicosanoids derived from the cyclooxygenase pathway were unchanged by the H3L6 diet. Notably, PGE2, a pain inducing prostanoid [15], was not significantly changed by diet which may have contributed to the lack of diet induced analgesia observed here. That said, PGE2 was also not altered by the H3L6 diet in plasma of human migraine sufferers, despite decreases in headache pain in these subjects[5]. Unlike our findings in hind paw, the H3L6 diet evoked robust changes in plasma oxylipin concentrations indicating a longer duration of dietary feeding may be necessary to maximize changes in tissue oxylipin concentrations. It should be stressed, however, that the plasma oxylipin concentrations presented here were measured from the total lipid pool which we were unable to assess in hind paw. Additionally, the plasma oxylipin changes observed here, in rodents, were much stronger than what we recently observed in humans [26] which indicates the potential for more intensive dietary interventions (i.e. longer dietary intervention or greater LA lowering targets) targeting oxylipins in humans.

We also investigated the impact of the H3L6 diet on tissue PUFA concentrations. As expected we observed striking changes in brain and hind paw PUFA concentrations with n-3 PUFA increasing and n-6 PUFA decreasing in H3L6 fed rats, compared to control diet fed rats. One difference between hind paw and brain was that brain DHA concentrations were not significantly different between H3L6 and control diet fed rats. However, EPA concentrations increased 10-fold in brains of rats consuming the H3L6 diet compared to control fed rats (the largest fold-change among brain fatty acids). This is notable because EPA concentrations are tightly controlled in brain [28] and in light of findings that EPA supplementation may be beneficial in the treatment of neurological disorders [29]. However, our results do not support a role of EPA, in brain, for pain and nociception.

This work has several limitations and strengths that should be discussed. Firstly, our work was limited to using a rodent chronic pain model to evoke pain, and rodent behavioral sensory testing to assess pain. While this is a widely used approach, these techniques have been criticized for potential lack of relevance to human pain or chronic pain conditions [30,31]. Additionally, our feeding duration and diet composition was modeled after human headache studies [4], and therefore, may not have maximally altered oxylipins in the tissues we studied here. Finally, our small sample size may not have been sufficient to detect subtle differences in behavior, although it was sufficient to detect differences in PUFA concentrations. One strength of our study was that we utilized LC-MS/MS to measure oxylipins in plasma and hind paw. While the limitations associated with mass spectrometry apply (i.e. lack of resolution of closely related isomers), LC-MS/MS provides superior accuracy, sensitivity and specificity to other methods measuring oxylipins. Furthermore, we added oxidized corn oil to our H6CTL diet instead of the purified corn or soybean oils usually used in rodent diets. Compared to purified corn oil, oxidized corn oil may be more reflective of the type of LA source in typical US diets (which is often heated for cooking). Finally, we utilized a conservative false discovery rate in our statistical analysis which increase the odds of finding false negatives. Though this is certainly likely, only 2 oxylipins were significantly different (p<0.05) that did not meet the false discovery rate threshold. These oxylipins were PGE2 and 11H-12E-LA, both of which were decreased in the H3L6 diet group which supports our conclusion that altering pronociceptive oxylipin concentrations in hind paw did not affect pain in our rat monoarthritis model.

5. Conclusions

In conclusion, compared to the H6 diet (modelled to mimic US fatty acid consumption), the H3L6 diet altered tissue PUFA and oxylipin concentrations in a manner predicted to decrease physical pain. Despite these biochemical changes, the H3L6 diet did not mitigate the hypersensitivity or allodynia evoked by a rat chronic monoarthritis model.

Supplementary Material

NIHMS1849650-supplement-1.docx^{(76.7KB, docx)}

Highlights.

We fed rats a diet that was higher in omega 3 polyunsaturated fatty acids and low in linoleic acid, compared to the control diet designed to mimic US fatty acid intakes.
Compared to rats fed the control diet, the H3L6 diet-fed rats had lower concentrations of linoleic acid, and oxidized lipids derived from linoleic acid, as well as higher concentrations of omega 3 polyunsaturated fatty acids, and omega 3 derived oxidized lipids.
The H3L6 diet did not impact pain related behavior in a rat chronic monoarthritis model.

6. Acknowledgements

Support for this work included funding from Department of Defense in the Center for Neuroscience and Regenerative Medicine, Brain and Behavior Research Foundation NARSAD Independent Investigator Award and the intramural programs of the National Institute on Aging, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health Clinical Center and the National Center for Complimentary and Integrative Health.

Footnotes

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