Effects of Diet and Mating on Sickle Cell Disease Murine
Authors: Tran Huy , Valverde Guevara Yessenia , Jha Ritu , Duong Thu , Ercan-Fang Deniz , Skubitz Lindsey , Nguyen Julia , Gupta Kalpna 
Published: October 2019

Abstract
Patients experiencing chronic and/or recurrent pain show a correlative decline in health and lifespan. In turn, declining health and sensory effects may contribute to persistent pain and poor response to analgesia. We therefore hypothesized that dietary alterations to improve health and stress reduction by mating may reduce chronic pain in male sickle mice.
Methods. We used male HbSS-BERK (sickle) mice, which show hyperalgesia as compared to and HbAA-BERK (control) mice (Kohli et al., Blood 2010). Breeders and pups up to 12 wks of age were fed Sickle Mouse Diet (59M3, TestDiet) and 18% Protein Rodent Diet (2018, Harlan), thereafter, called ‘sickle’ and ‘rodent’ diet. Sickle diet contained 26.4% Protein, 11.1% fat and 27.5% and 26% kcal/g of each, respectively. Rodent diet contained 18.6% protein and 6.2% fat, and 24% and 18% kcal/g of each, respectively. Vitamins were about 2-fold higher in sickle as compared to rodent diet. Sickle mice showing hyperalgesia were recruited and treated as follows: [A] Rodent diet without mating (RD/M-); [B] Sickle diet with mating (SD/M+); [C] Rodent diet with mating (RD/M+); [D] Sickle diet without mating (SD/M-) and [E] mice on SD and mating for 4 weeks, were deprived of SD and mating and fed RD. Control mice which do not show hyperalgesia were fed RD without mating. Sensory testing was performed at baseline (BL) at recruitment and weekly, to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the treatments, release of cytokines from skin biopsies was analyzed by cytokine arrays as described by us earlier (Vincent et al., Blood 2013), and spinal cords were analyzed for nociceptive signaling.
Results. We did not observe difference in the body weight of mice between different groups at any time. White blood cell counts and spleen weight were significantly increased in group E following the withdrawal of SD and mating as compared to group C on SD/M+ (p<0.05), but the liver and kidney did not show any difference amongst groups. Groups B and C involving mating with SD/M+ or RD/M+, respectively, showed maximum decrease in mechanical, and thermal hyperalgesia following 3 weeks of treatment as compared to baseline (p<0.05 and 0.0001 for mechanical, p<0.001 and 0.0001 for thermal, respectively for B and C), suggesting that mating decreases sensitivity to noxious stimuli including touch and temperature. In group E (mice were in mating and with SD for 4 wks), withdrawal of sickle diet and mating for 4 weeks, led to a significant increase in thermal hyperalgesia as compared to the start of the withdrawal (p<0.05 for mechanical and heat). Group D (SD/M-) showed a significant decline thermal hyperalgesia one week after starting the sickle diet as compared to BL (p<0.0001 for cold and <0.005 for heat). Thus sickle diet in the absence of mating reduced hyperalgesia, but the reduction with sickle diet only (SD/M-) was significantly less than the reduction caused by mating (RD/M+) or by sickle diet with mating (SD/M+), suggesting that mating is more effective than diet in ameliorating pain. None of the groups demonstrated a decrease in grip force, suggesting that deep tissue pain was not influenced or that the strength of mice was not influenced and the influence is perhaps restricted to the periphery (skin). Cytokine profile of the skin showed a significant decrease in IL6, MIP-1a, RANTES, MCP-1 and GM-CSF in group B, (SD/M+) as compared to group A, (RD/M-), indicating that the diet and mating decrease inflammation in the periphery, thus reducing the activation of peripheral nerve fibers, resulting in reduced hyperalgesia. Consistent with reduced inflammation, spinal cords of group B (SD/M+) showed a significant decrease in nociceptive signaling of phosphorylation of Stat3 pathway as compared to group A (RD/M-). Since, Stat3 is associated with transcription of inflammatory cytokines, it likely reduces the inflammatory response in the spinal cord and inflammatory pain. These data suggest that high protein/high fat diet and mating reduce inflammation and hyperalgesia in sickle mice. Stress appears to be a critical factor in the perception of pain, because mating attenuated hyperalgesia. Thus, improving general health and happiness and stress reduction may reduce pain in SCD.

Disclosures
No relevant conflicts of interest to declare.