Peripheral Neurons with More Long-Chain Omega-3s Better Protected from Injury

Peripheral nerves connect the brain and spinal cord to all other parts of the body. If disease or injury damages the peripheral nerves, communication between the brain and the affected area is disrupted and sensory information, such as temperature, may not be appropriately relayed. How an individual detects nerve damage depends on the type of injury or disease and which parts of the body are affected. Some of the dangers from peripheral neuropathy are increased susceptibility to burns and infections, poor control of movements and possible permanent nerve damage. With peripheral neuropathy, one may experience tingling, numbness, pricking sensations or muscle weakness (Figure). More extreme symptoms include burning pain, muscle wasting, paralysis or gland dysfunction. Peripheral nerve disorders can arise from diabetes and other systemic diseases, alcohol abuse, tumors, carpal tunnel syndrome (repetitive strain injuries), thoracic outlet syndrome, traumatic injury, infections and autoimmune conditions. Peripheral neuropathies can often be treated, sometimes cured and usually managed to prevent new damage. As long as the nerve cells have not been destroyed, peripheral nerves can regenerate. A top question for clinicians treating patients with nerve disorders is what can be done to hasten or improve nerve regeneration and ease patient symptoms? Several medications may ease pain, but improvements in muscle strength and function, neuropathic pain and nerve conduction may be more challenging to accomplish. A promising avenue for improved nerve growth and regeneration is the provision of long-chain omega-3 PUFAs (n-3 LC-PUFAs), particularly DHA. Research has shown that animals fed diets restricted or deficient in n-3 LC-PUFAs have lower levels of DHA in several regions of the brain (see article in this issue, “DHA Restriction Leads to Distortions in Visual Pathways in Early Development”), including the hippocampus, cerebral cortex, striatum and retina. In turn, n-3 LC-PUFA restriction or deficiency is associated with impaired visual function, reduced neurotrophin content and lower mitogen-activated protein kinase activity in brain. On the other hand, DHA supplementation is associated with improved visual acuity, neurogenesis, neurite outgrowth, neuroprotection against retinal pigment epithelial cell damage through its derivative neuroprotectin D1 and protection of cholinergic neurons against glutamate cytotoxicity. Further, DHA-derived neuroprotectin D1, along with pigment epithelial-derived growth factor and DHA promoted the regeneration of corneal nerves after experimental surgery. Studies from the laboratory of Adina Michael-Titus have reported that DHA or n-3 LC-PUFAs were associated with improved recovery after spinal cord injury, increased production of nuclear receptors in aged animals, reduced axonal dysfunction and decreased the myelin damage in spinal cord injury. In this report, the authors describe improved outcomes following peripheral nerve damage in fat-1 mice that convert n-6 PUFAs to n-3 PUFAs, which humans cannot do. The fat-1 mouse contains a gene that encodes a fatty acid desaturase, which mammals lack. The result of this gene activity is a reduction in n-6 PUFAs and an increase in n-3 PUFA levels in tissues. The effect of the gene was verified by determining the phospholipid fatty acid composition of the isolated dorsal root ganglion and spinal cord neurons. The study was designed to investigate the effect of mechanical stretch and hypoxia on dorsal root ganglion cells harvested from wild-type and fat-1 mice and cultured in vitro. In other experiments, the investigators assessed in vivo nerve recovery after sciatic nerve injury. Three groups of animals were studied: wild-type mice fed a standard balanced diet; wild-type mice fed a high n-6 PUFA diet low in n-3 PUFAs (~0.1% of total fat); and transgenic fat-1 mice fed the high n-6 PUFA diet, which results in higher tissue levels of n-3 PUFAs and less n-6 PUFAs. To create the injury in vitro, a 20% tensile strain for 1 hour was applied to cultured dorsal root ganglion neurons from adult mice grown on elastic membranes and then left unstrained for 24 hours. Control cells were cultured similarly but without tensile strain. Hypoxic injury was created by culturing cells for 48 hours and then maintaining them in an atmosphere of 2% O2, 5% CO2 for 24 hours. The extent of cell death was assessed by measuring the uptake of ethidium homodimer-1, a fluorochrome that is taken up only by the nuclei of cells with compromised membranes. Neurite growth was assessed in L4 and L5 dorsal root ganglion neurons dissected from wild-type and fat-1 mice. Images of 100 neurons from each of 3 animals were recorded and the length of the longest neurite and number of branches were measured for each neuron. Sciatic nerve damage was induced by a 15-second compression to the isolated left sciatic nerve in anesthetized animals. To assess threshold limb sensitivity, baseline measurements of the hind-paw reflex in response to punctate mechanical stimuli were recorded. Von Frey nylon filaments were applied in ascending order to the mid-plantar surface of the hind-paws. Application of the filament causes the paw to withdraw. The lowest force leading to at least 3 withdrawals in 5 trials was defined as the withdrawal baseline. The von Frey tests were performed 1, 4 and 7 days after sciatic nerve injury. The investigators also assessed motor function recovery by calculating the sciatic functional index before and 7 days after injury. The index was determined from three measurements of inked footprints, which included print length, toe spread and intermediary toe spread. Calculation of the index was performed for the experimental and normal prints by an established formula. Spinal cord phospholipid fatty acid profiles differed substantially from those in the dorsal root ganglia. Spinal cord contained 10.6 mol% DHA compared with 4.1 mol% in the dorsal root ganglia, but arachidonic acid levels were similar (Table). Docosapentaenoic acid (DPA n-3) was twice as high in the spinal cord as in the dorsal root ganglia (2.0 vs 0.9 mol%, respectively). Mice with the fat-1 gene had significantly higher concentrations of DHA in the dorsal root ganglia and spinal cord phospholipids compared with the concentrations in the wild-type mice (5.7 vs 4.1 mol% for dorsal root ganglia and 13.4 vs 10.6 mol% for spinal cord, respectively). Interestingly, concentrations of DPA n-3 were substantially higher in both tissues in the transgenic compared with the wild-type animals. Only dorsal root ganglia had significantly lower concentrations of arachidonic acid in the transgenic compared with the wild-type animals (5.1 vs 5.5 mol%, respectively). Total n-6 PUFAs were lower in both tissues, but not significantly so, in the transgenic mice compared with the wild-type animals. These observations are consistent with previous reports that the fat-1 gene leads to higher concentrations of DHA in tissues. Mechanical injury to the cultured dorsal root ganglion cells from wild-type mice fed either the standard or high-n-6 PUFA diet, led to a 2.5-fold increase in neuronal cell death compared with uninjured controls. In contrast, cells from the fat-1 mice exhibited only a 13% rate of neuronal cell death after injury compared with a 9% loss in the controls. This difference was not statistically significant. The response to hypoxia by these cells was an increase in cell death from 8% to 21% in wild-type animals on the standard diet and from 11% to 25% in wild-type mice fed the n-6 PUFA-enriched diet. The difference between the two groups was not statistically significant. However, the effect of hypoxia on the neurons from the fat-1 mice was nearly completely abolished, with the rate of cell death similar to that observed in the untreated fat-1 controls. When the outgrowth of the dorsal root ganglion neurites from the transgenic and wild-type mice fed the high n-6 PUFA diet was compared, growth of the fat-1 gene neurons was more complex, displayed longer neurites (40 μm vs 20 μm) and more branches (7 vs 3) than the neurons from the wild-type mice. For the in vivo studies, the investigators compared the stimulation withdrawal responses after sciatic nerve injury in the wild-type and fat-1 mice, both fed the high n-6 PUFA diet. Baseline response to von Frey stimulus was similar in both groups. After injury, both groups required a greater force to induce a withdrawal response, indicating a loss of touch sensation. One day after injury, the force required by the wild-type and transgenic mice was 8 vs 5 g in each group, respectively. At post-injury day 4, the difference in force for response between the two groups was significantly different, 5.4 and 2.5 g for the wild-type and transgenic mice, respectively, (P < 0.05). Both groups continued to recover sensation and by the 7th day after injury the response in the fat-1 mice remained significantly better than in the wild-type animals (P < 0.05). Assessment of the loss in motor function by the sciatic functional index in both groups of mice prior to injury at baseline showed no difference between the two groups. Seven days after sciatic nerve injury the functional loss in the wild-type animals was twice as great as observed in the fat-1 mice (25-fold vs 12-fold decrease, respectively). However, the functional loss in the transgenic animals remained significantly higher compared with baseline values. Collectively, these observations suggest that the increased n-3 LC-PUFA content (DHA and DPA) in spinal cord and dorsal root ganglion neurons observed in the fat-1 mice compared with the wild-type mice was associated with significantly greater resistance to peripheral nerve injury. Less is known about the effects of DPA (n-3) compared with DHA, but DPA (n-3) was reported to have neurorestorative effects in the hippocampus of aged rats. Greater protection from neuronal damage was also observed in the intact animals with higher n-3 LC-PUFAs during recovery from sciatic nerve injury. The fat-1 mice recovered sensitivity more quickly and extensively than the control animals. Both groups of animals exhibited functional loss in the injured limb 7 days after injury, but the loss was twice as great in the control animals (25-fold reduced score) as in the fat-1 mice (12-fold reduced score). Use of the fat-1 transgenic mouse, which has higher tissue concentrations of DHA and lower concentrations of arachidonic acid than wild-type animals, permitted the demonstration of a significant association between improved responses to peripheral nerve injury and higher neuronal concentrations of DHA and DPA. For future clinical applications of these observations, it will be necessary to know whether DHA from diet or infusion could alter neuronal concentrations sufficiently for an effective in vivo response. It will be critical to learn whether higher levels of DPA are required with DHA for effective neuronal protection and whether either of these n-3 LC-PUFAs would be effective alone. These and other recent studies on the effectiveness of DHA in promoting nerve regeneration and limiting neuronal damage from injury are promising developments on the path to more effective treatments for patients with impaired nerve function. Gladman SJ, Huang W, Lim SN, Dyall SC, Boddy S, Kang JX, Knight MM, Priestley JV, Michael-Titus AT. Improved outcome after peripheral nerve injury in mice with increased levels of endogenous omega-3 polyunsaturated fatty acids. J Neurosci 2012;32:563-571. PubMed WORTH NOTING Tan ZS, Harris WS, Beiser AS, Au R, Himali JJ, Debette S, Pikula A, Decarli C, Wolf PA, Vasan RS, Robins SJ, Seshadri S. Red blood cell omega-3 fatty acid levels and markers of accelerated brain aging. Neurology 2012;78:658-664. PubMed Luchtman DW, Meng Q, Song C. Ethyl-eicosapentaenoate (E-EPA) attenuates motor impairments and inflammation in the MPTP-probenecid mouse model of Parkinson's disease. Behav Brain Res 2012; 226:386-396. PubMed