Activation of the Central Serotonergic System Reduces Hyperalgesia in Sickle Mice

Pain in sickle cell disease (SCD) often requires chronic opioid use and remains a major challenge to treat, necessitating a need for alternate strategies to manage pain. We examined whether altering the activity of descending anti-nociceptive pathway could produce analgesia in sickle mice. Strategies tested included dietary modification, mating and the use of a serotonergic agent. We used homozygous HbSS-BERK (sickle) mice, fed customized Sickle Mouse Diet (SD; 59M3, TestDiet) and Rodent Diet (RD; 2018, Harlan). SD contains 26.4% protein, 11.1% fat and 27.5% and 26% kcal/g from each, respectively. RD contains 18.6% protein and 6.2% fat, and 24% and 18% kcal/g from each, respectively. SD also contains higher minerals, vitamins and omega-3 fatty acids compared to RD. Male and female sickle mice on RD show tonic hyperalgesia as compared to "control" mice expressing normal human hemoglobin A (Kohli et al., Blood 2010). Mice were treated as follows: [A] SD with mating for 6 - 8 months (called "SD/M+" group), [B] mice on SD and mating for 6-8 months were deprived of SD and switched to RD as well as withdrawn from mating for 3 weeks (called "Withdrawal" group), and [C] mice on RD without mating (called "RD" group). Mechanical, thermal and musculoskeletal/deep tissue hyperalgesia were examined before and after these treatments. Since serotonin (5-hydroxytryptamine: 5-HT) is an important mediator of the descending anti-nociceptive system originating in large part from the rostral ventromedial medulla (RVM) of the brainstem and providing a source of serotonergic input that modulates nociceptive transmission in the spinal cord dorsal horn, we examined 5-HT-immunoreactivity in the spinal cord dorsal horn and RVM. Finally, mice on "RD", which show tonic hyperalgesia, were treated with an intraperitoneal injection of 3 or 10 mg/kg body weight of duloxetine, a serotonin-norepinephrine reuptake inhibitor (SNRI). Tolerance to duloxetine was examined by assessing hyperalgesia in mice receiving 3 mg/kg/day of duloxetine for 10 days. In parallel, HbAA-BERK ("control") mice expressing normal human hemoglobin A were included with each experiment.

Mice fed sickle diet and allowed to mate ("SD/M+") showed significantly reduced mechanical and thermal hyperalgesia as compared to those on "RD" (p<0.0001). Uponswitching the SD to RD and withdrawal from mating for 3 weeks ("Withdrawal" group), we observed a significant increase in mechanical and thermal hyperalgesia (p<0.0001), suggesting that sickle (high calorie) diet and mating attenuate hyperalgesia in sickle mice. These behavioral changes were accompanied by a significant decrease in the level of 5-HT upon metabolomic analysis in spinal cords after withdrawal of sickle diet and mating privileges (Withdrawal group) as compared to the pre-withdrawal state ("SD/M+") (p=0.0136). Validation with HPLC confirmed a decrease in 5-HT in spinal cords (p=0.0288) and a significant decrease in 5-HT-immunoreactivity in lamina I of the spinal cord dorsal horn (p=0.0003) and RVM (p=0.0190) in Withdrawal group compared to "SD/M+" group of sickle mice, suggesting that SD and mating reduce hyperalgesia by activating the descending anti-nociceptive pathway. Withdrawal of these interventions led to a reversal of the analgesic effect and decrease in 5-HT. Therefore, we next tested if a pharmacological strategy to increase 5-HT with an SNRI would ameliorate hyperalgesia. This strategy resulted in a significant decrease in heat and cold hyperalgesia in mice on RD. The effect was noted as early as 30 min after treatment with 3 or 10 mg/kg duloxetine (p<0.05 Vs baseline) in both sexes of sickle mice. Male mice showed a significant decrease in deep tissue hyperalgesia at the dose of 3 mg/kg (p<0.001 Vs baseline), but female mice did not. Duloxetine treatment did not lead to a significant change in mechanical hyperalgesia in either sex. Importantly, no analgesic tolerance occurred in response to chronic treatment with duloxetine (3 mg/Kg/day) over a period of 10 days.

This study has for the first time demonstrated that hyperalgesia in sickle mice involves the descending serotonergic anti-nociceptive system. Our data show that hyperalgesia can be attenuated with dietary, behavioral (mating) and pharmacologic (duloxetine) approaches in sickle mice. Clinical trials are required to test the potential efficacy of dietary modification and SNRIs as adjuncts to opioid analgesics in patients with SCD.