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You Feel What You Eat: ω-6 Polyunsaturated Fatty Acids Drive Painful Neuropathy in Mice

A similar mechanism may be at play in people with diabetic neuropathy, suggesting the translational potential of the new findings.

by Fred Schwaller


20 September 2021


PRF News

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A similar mechanism may be at play in people with diabetic neuropathy, suggesting the translational potential of the new findings.

In The Physiology of Taste, a classic first published in 1825, Jean Anthelme Brillat-Savarin wrote: “Tell me what you eat and I will tell you what you are.”

 

That sentiment has perhaps never been more relevant than it is today, in particular with the popularity of the so-called Western diet. This diet is characterized by an enrichment of ω-6 polyunsaturated fatty acids (PUFAs) and has been linked to a range of conditions including metabolic diseases, atherosclerosis, neurodegeneration, and cancer. New findings now suggest that a diet with high levels of ω-6 PUFAs is also a strong risk factor for chronic pain.

 

Researchers led by Kenneth Hargreaves, University of Texas Health Science Center at San Antonio, US, report that an ω-6 PUFA-rich diet leads to persistent nociceptive hypersensitivity in mice. This hypersensitivity was associated with high levels of spontaneous activity in primary afferent neurons in the skin, and with histological markers of peripheral nerve damage. Remarkably, switching to a low ω-6 PUFA, high ω-3 PUFA diet reversed the hypersensitivity in the animals. The authors also report an association between skin levels of linoleic acid and pain in patients with diabetic neuropathy; linoleic acid is a pro-nociceptive metabolite of ω-6 PUFAs.

 

“This study adds to growing evidence that targeted diet interventions could be a viable adjunct approach for managing chronic pain,” said Christopher Ramsden, National Institute on Aging, National Institutes of Health, Bethesda, US. “The findings match well with human findings showing that targeted dietary alterations can decrease physical pain in patients with daily headaches and chronic migraines,” according to Ramsden, referring to clinical trial findings from him and his colleagues published July 1, 2021, in BMJ and in 2013 in PAIN. (Ramsden et al., 2021Ramsden et al., 2013). Ramsden studies the roles of lipid mediators and lipid-related mechanisms in chronic pain and neurodegenerative conditions, but was not involved with the new work.

 

“This comprehensive and elegant study from [co-first author Jacob] Boyd et al. may serve as a foundation for new clinical trials and ultimately provide new avenues for the clinical treatment of neuropathies,” wrote Aidan McGinnis and Ru-Rong Ji, Duke University Medical Center, Durham, US, in an accompanying News & Views.

 

The study and News & Views appeared in the June 2021 issue of Nature Metabolism.

 

The pain of a Western diet

The central hypothesis of the research, Hargreaves told PRF, was that diet could be a risk factor for pain.

 

“We thought that high ω-6 PUFA diets might cause ω-6 PUFAs to accumulate in the cell membrane in the nociceptive pathways. This would mean that when a cell is activated, like during nerve injury or inflammation, ω-6 PUFAs would be rapidly released from the membrane and turn into pro-nociceptive mediators that sensitize neurons.”

 

Hargreaves, Boyd, co-first author Peter LoCoco and colleagues started by putting wild-type male and female mice on a high ω-6 PUFA diet (H6D) or a low ω-6 PUFA diet (L6D) for 24 weeks. Over that time, the group measured withdrawal thresholds to mechanical, heat and cold stimuli.

 

The behavior analysis found that mice developed severe nociceptive hypersensitivity to noxious thermal and mechanical stimuli, as well as mechanical allodynia in response to non-noxious skin brushing, after eight weeks of the H6D.

 

This nociceptive hypersensitivity profile was reminiscent of peripheral neuropathic pain-like behavior, leading the authors to test whether primary afferent neurons from H6D mice displayed electrophysiological hyperexcitability and histological signs of nerve damage. For these experiments, the group used an ex vivo skin-nerve preparation to measure the activity of tibial nerve primary afferent neurons in response to hindpaw stimulation of the cells' receptive fields at the eight-week timepoint of behavioral nociceptive hypersensitivity.

 

These recordings showed that nociceptive Aδ and C-fibers in H6D mice had high levels of spontaneous activity. Moreover, nociceptors showed higher action potential firing frequencies in response to noxious mechanical and heat stimuli. Meanwhile, after eight weeks of H6D, histological analyses showed a reduction of intra-epidermal nerve fiber (IENF) density in the skin, and elevated expression of the neuronal stress marker ATF3 in dorsal root ganglia (DRG), both of which are markers of peripheral neuropathy.

 

Strangely, the H6D only seemed to cause changes in peripheral DRG neurons, and not in neurons in the spinal dorsal horn. In this case, an H6D did not increase immunolabeling of c-Fos and IBA1 in the spinal cord, which are markers of neuron and microglial activation, respectively.

 

“This was a really interesting finding,” Hargreaves said. “We have a number of hypotheses why this might be the case. It may be something to do with pharmacokinetics in terms of how the blood-brain barrier gives access to PUFAs, or it might be because turnover of PUFAs in the CNS [central nervous system] might be so fast that they don’t accumulate in neurons. Either way, the H6D-induced neuropathy seems to be a peripheral phenomenon.”

 

 

ω-6 PUFA-loaded membranes are a painful accident waiting to happen

ω-6 PUFAs are incorporated into the plasma membrane by enzymes called acyltransferases, and can be released by the enzyme phospholipase A2 (PLA2). This knowledge led the authors to investigate the potential role of PLA2 in the nociceptive hypersensitivity observed in their experiments.

 

Initial experiments found that PLA2 activity was elevated in DRG neurons from H6D mice. This indicated that PLA2 might be working overtime to release pro-nociceptive ω-6 PUFA metabolites like linoleic acid and arachidonic acid.

 

The logical next question was whether inhibiting PLA2 activity could reverse nociceptive hypersensitivity caused by H6D. Earlier work from other researchers (e.g., Usoskin et al., 2015) had determined that PLA2 group 7 (PLA2G7) is the dominant isoform of PLA2 in mouse DRG, so that became the researchers' focus.

 

The authors administered the PLA2G7 inhibitor darapladib directly into hindpaw skin of H6D mice, which reversed the behavioral hypersensitivity to both mechanical and heat stimuli in the animals. Similarly, intrathecal injection of small interfering RNA (siRNA) that reduced Pla2g7 expression in DRGs and the spinal cord also reversed nociceptive hypersensitivity in H6D mice.

 

“These findings show that the sensitization of peripheral nociceptors we saw in H6D mice is almost certainly a consequence of the release of pro-inflammatory metabolites from accumulation of ω-6 PUFAs in DRG neuron plasma membranes,” said LoCoco. “When ω-6 PUFAs are released by the enzyme PLA2, say by high neuronal excitability caused by painful stimuli, fatty acids are rapidly oxidized into metabolites that activate ion channels such as TRPA1 and TRPV1 that drive neuronal excitability.”

 

Boosting dietary ω-3 fatty acid levels relieves pain

Hargreaves was keen to explore non-pharmacological therapeutic options to treat H6D-related persistent pain.

 

“We give dietary recommendations to patients with autoimmune disease, diabetes, and cardiovascular disease. But what about chronic pain? The obvious thing to try was to switch diets and see if this reversed the pain behavior in H6D mice,” Hargreaves said.

 

ω-3 PUFAs are known to have anti-inflammatory and anti-nociceptive effects that directly counter the pro-nociceptive effects of ω-6 PUFAs. Therefore, the authors tested if restoring the ratio of ω-3 to ω-6 PUFAs in the diet could reduce nociceptive hypersensitivity in mice.

 

Switching from the H6D to a diet high in ω-3 PUFAs (H3D) completely rescued nociceptive hypersensitivity in the animals. Meanwhile, switching to a diet low in ω-6 PUFAs(L6D) without increasing ω-3 PUFAs reduced nociceptive hypersensitivity in mice but did not reverse it.

 

Switching to the H3D also reversed histological and electrophysiological hallmarks of peripheral neuropathy. This included restoration of ATF3 levels in DRG neurons and a reduction in both spontaneous activity and hyperresponsivity to mechanical and heat stimuli in peripheral afferent neurons from H3D mice.

 

“It was exciting to see the magnitude of reductions in pain-related behaviors produced by the diets. The findings provide proof of concept that peripheral neuropathic pain could be amenable to dietary intervention and provide some new mechanistic insights,” Ramsden said.

 

Because of the success of the H3D, the authors investigated whether nutritional supplementation (as opposed to daily regulation of dietary PUFAs) with ω-3 PUFAs in H6D mice (without lowering ω-6 PUFA intake) could also reverse nociceptive hypersensitivity. Here, mice received ω-3 supplements daily from eight weeks after the H6D began.

 

Four weeks later, mice that had received daily ω-3 supplements had a partial reversal of pain behaviors. This argued that increasing ω-3 PUFA levels in the diet only partially reverses persistent pain caused by the H6D; a dual increase in ω-3 and decrease in ω-6 PUFAsis required for full reversal.

 

“Nevertheless, it’s encouraging to see that something as simple as taking ω-3 supplements could improve susceptibility to long-term pain. It was remarkable that we could see a near-complete reversal of pain hypersensitivity in mice just with a dietary change, and I think this is very promising for its clinical relevance,” LoCoco said.

 

A similar mechanism in humans?

To begin to show translational relevance of their findings, the authors turned to a cohort of patients with diabetes and painful neuropathy. They looked for possible correlation of pain scores from the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) and Neuropathic Pain Symptom Inventory (NPSI) questionnaire with levels of the pro-nociceptive ω-6 PUFA metabolite linoleic acid in skin biopsies.

 

These findings showed a robust correlation between skin linoleic acid levels and neuropathic pain scores. In addition, 75% of participants with diabetes and elevated linoleic acid levels required neuropathic pain pharmacotherapy, while the remaining participants who did not require pharmacotherapy had low levels of linoleic acid in the skin.

 

“We think that this correlation between skin levels of linoleic acid and neuropathic pain was significant as a potential biomarker for pain hypersensitivity. We’re testing this hypothesis further. We’re not suggesting it’s relevant for all models, but in those pain conditions related to peripheral sensitization that is caused by ω-6 fatty acid build up,” according to Hargreaves.

 

Ultimately, the study adds to growing evidence that targeted dietary interventions could be a realistic way to manage different kinds of chronic pain. 

 

“The biochemical hypotheses that underpin these studies could potentially be relevant for many other types of chronic pain. With additional evidence from human trials, it may ultimately be possible to integrate targeted dietary changes alongside medications to improve the lives of patients with chronic pain,” Ramsden said.

 

Fred Schwaller, PhD, is a freelance science writer based in Germany.

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