Dietary Supplementation with Docosahexanoic Acid (DHA) Improves RBC Flexibility and Reduces Cold Hypersensitivity in Mice with Sickle Cell Disease.
Authors: Wandersee Nancy J. , Hanson Madelyn S. , Maciaszek Jamie L. , Larson Michael C. , Giger Katie M. , Zheng Suilan , Retherford Dawn M. , Mickelson Barbara , Stucky Cheryl L. , Low Philip S. , Lykotrafitis George , Hogg Neil , Hillery Cheryl A. 
Published: October 2019

Abstract
Abstract 2116
Humans and mice with sickle cell disease (SCD) have RBC stiffness, multiorgan and vascular pathology, and complex pain syndromes. Omega-3 fatty acids, such as docosahexanoic acid (DHA), are essential fatty acids that have anti-inflammatory and anti-thrombotic activities. As dietary supplements, omega-3 fatty acids are beneficial in many cardiovascular diseases. Several studies demonstrate that dietary supplementation with omega-3 fatty acids results in increased incorporation of these fatty acids into the RBC membrane, which can influence RBC deformability. In this study, SCD mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control matched in total fat with a similar distribution of saturated, monounsaturated, and polyunsaturated fatty acids (CTRL diet). After 8 weeks of feeding, we examined the RBCs for: 1) deformability, as measured by ektacytometry; 2) stiffness, as measured by atomic force microscopy; 3) osmotic fragility, using a flow cytometric method; and 4) percent irreversibly sickled RBCs on peripheral blood smears. Consistent with other studies, RBCs from SCD mice fed Control diet exhibit low deformability by ektacytometry as compared to RBCs from wild-type mice (0.075 Max EI, SCD mice Control Diet, versus 0.285 Max EI, wild-type mice). Correspondingly, RBC stiffness, as measured by atomic force microscopy, is increased in SCD mice fed Control diet as compared to wild-type mice (1911 Pa, SCD mice Control Diet, versus 831 Pa, wild-type mice). In contrast, RBCs from SCD mice fed DHA diet had improved deformability (0.135 Max EI) compared to RBCs from SCD mice fed Control diet (p<0.02). Furthermore, RBCs from DHA diet-fed SCD mice had markedly decreased stiffness (798 Pa) to nearly normal levels compared to RBCs from Control diet-fed SCD mice (p<0.0001). In addition, RBCs from SCD mice fed DHA diet had decreased osmotic fragility as compared to RBCs from SCD mice fed Control diet (p<0.001). Examination of peripheral blood smears also revealed less irreversibly sickled RBCs in SCD mice fed DHA diet as compared to Control Diet (2.5% versus 4.1%, p<0.04). These results suggest that DHA supplementation has modified RBC membrane properties of SCD mice. Individuals with SCD as well as murine models of SCD display increased sensitivity to pain. Stiffened RBCs are thought to contribute to pain in SCD by stimulating vasoocclusive events. Importantly, a small-scale human SCD trial indicated that dietary supplementation with omega-3 fatty acids reduced pain episodes by almost 50% (Tomer, et al, 2001). Therefore, we measured sensitivity of SCD mice to mechanical and cold stimuli both prior to dietary feeding and after 8 weeks of feeding on Control or DHA diet. Mechanical hypersensitivity was assessed using von Frey filaments, with the paw withdrawal threshold assessed using the up-down method of Dixon (1980). Mechanical hypersensitivity thresholds are reduced in SCD mice (Hillery, et al, 2011), and were not altered by either the Control or DHA diet. Cold hypersensitivity was assessed using a 2-temperature choice assay and measuring the amount of time spent on the colder plate (23°C) versus the warmer plate (30°C). Similar to our previous findings, SCD mice exhibit hypersensitivity to cold. There was no change in pre- versus post-diet cold hypersensitivity in SCD mice fed Control diet. In contrast, post-diet cold hypersensitivity was improved compared to pre-diet cold hypersensitivity in SCD mice fed DHA diet (p<0.007). In summary, our findings indicate that DHA supplementation improves RBC flexibility, decreases osmotic fragility and irreversibly sickled cells, and reduces cold hypersensitivity in a murine model of SCD. The reduction in cold hypersensitivity may reflect the increased flexibility of the RBCs, making them less likely to cause vasoocclusive events that can activate inflammatory pain mechanisms. Additionally, resolvins D1 and D2, anti-inflammatory lipid mediators derived from DHA, inhibit the activity of the transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) channels, which contribute importantly to inflammatory pain sensitivity to mechanical and thermal stimuli.

Disclosures:
Mickelson: Harlan Laboratories, Inc.: Employment. Stucky:Purdue Pharma: Research Funding. Hillery:Purdue Pharma: Research Funding; Bayer Pharmaceuticals: Consultancy.