The Impact of Omega‐3 Fatty Acids on Cardiac Autonomic Nerve Fiber Innervation in High Fat‐Fed Mice

Abstract

Peripheral neuropathy and cardiac autonomic neuropathy (CAN) are common and serious complications of diabetes, characterized by loss of peripheral nerves that innervate the skin and loss of parasympathetic and sympathetic nerves that innervate the heart, respectively. CAN is associated with increased risk of myocardial infarction and mortality, yet often goes undetected in patients. Both prediabetic and diabetic patients develop peripheral neuropathy and CAN, suggesting that the level of hyperglycemia at which peripheral neuropathy and CAN develop precedes the onset of diabetes. Although evidence from human and rodent studies indicate that omega-3-fatty acids prevent and/or reverse the development of peripheral neuropathy, the effects of omega-3-fatty acids on CAN has not been investigated. PURPOSE Assess the effects of omega-3-fatty acids on cardiac autonomic nerve fiber innervation in high fat diet-induced prediabetic mice. We hypothesize that omega-3 fatty acids will attenuate a reduction in cardiac autonomic nerve fiber innervation in high fat-fed mice. METHODS Male C57BL/6 mice were randomized into four diet groups (n = 12-14/group) for 14 weeks: 10% low fat fish oil (LFFO), 54% high fat-fish oil (HFFO), 10% low fat-lard (LFL), or 54% high fat-lard (HFL). The von Frey behavioral test for hindpaw mechanical sensitivity was used to characterize peripheral neuropathy at baseline then biweekly for 14 weeks. At study completion, fasting glucose and insulin values were used to calculate the homeostatic model assessment of insulin resistance (HOMA-IR). Mice were anesthetized, transcardially perfused with 1% PBS, and heart tissue was harvested and frozen in O.C.T. Heart was sectioned in three planes (anterior coronal, posterior coronal, and sagittal) and mounted on slides. Fluorescence immunohistochemistry was performed on heart cryosections using anti-choline acetyl-transferase and anti-tyrosine hydroxylase followed by Alexa Fluor secondary antibodies to visualize parasympathetic and sympathetic nerve fibers. For each mouse, nerve fibers were quantified on three slides (1 from each plane) and at least 3 areas with the highest nerve fiber density for each slide were examined. Nerve fiber density was determined using Nikon Elements and was calculated as the total length of all counted nerve fibers divided by the total area examined (µm/mm2). RESULTS Body weight, fasting insulin and glucose, and HOMA-IR were higher in HFL compared to all other groups. Fasting glucose was also higher in LFL compared to LFFO. Hindpaw withdrawal threshold was lower in HFL compared to HFFO, indicative of hindpaw hypersensitivity in HFL. CONCLUSIONS Omega-3 fatty acids prevented excess body weight, insulin resistance, elevated glucose, and hindpaw hypersensitivity in mice fed a high fat diet. Based on this data, we expect a diet rich in omega-3 fatty acids will attenuate cardiac autonomic nerve fiber denervation. Taken together, these findings would suggest that omega-3 fatty acids might hold therapeutic potential in the prevention of metabolic derangements as well as peripheral and CAN in individuals who consume a high fat, high calorie diet.