Cannabinoids as Analgesics for Pain in Sickle Cell Disease.
Authors: Kohli Divyanshoo Rai , Li Yunfang , Khasabov Sergey G , Ericson Marna E , Gupta Pankaj , Simone Donald A , Hebbel Robert P , Gupta Kalpna 
Published: October 2019

Abstract
Abstract 822
Neural mechanisms underlying severe pain in sickle cell disease (SCD) remain unknown. Opioids, the primary medications for pain in SCD, are frequently associated with development of tolerance and side effects. We used transgenic heterozygous BERK mice expressing human sickle hemoglobin (hBERK) and age/sex matched mice of similar genetic background expressing normal human hemoglobin (HbA-BERK) to examine changes in skin morphology and innervation that may relate to behavioral measures of pain. Since cannabinoids have shown promise in treating inflammatory and neuropathic pain, we examined the ability of cannabinoids to treat pain in hBERK (sickle) and control mice. The epidermis and dermis were thinner in hBERK mice (p< 0.01). Confocal microscopy showed that pan-neuronal marker protein gene product 9.5 (PGP) immunoreactive nerve fibers in the epidermis and dermis were disorganized and that the dermis had fewer nerve fibers in hBERK than HbA-BERK mice. Expression of Calcitonin Gene Related Peptide and Substance P, neuropeptides found in nociceptive afferent fibers, was increased in hBERK skin, suggesting neuronal sensitization. We next examined pain sensitivity in hBERK and HbA-BERK mice. Paw Withdrawal Frequency (PWF) per 10 applications of a 1.0 g von Frey monofilament and 50% paw withdrawal threshold evoked by von Frey monofilaments ranging from 0.4 to 8.0 g were used to assess mechanical sensitivity. hBERK mice showed 277.6 ± 83% higher PWF and 35.9 ± 26% lower paw withdrawal threshold than HbA-BERK (p<0.05). PWF increased consistently with age in hBERK (p<0.01; 5 vs. 15 mo) but not in HbA-BERK mice, suggesting increasing mechanical hyperalgesia with age in sickle mice. We observed a 24.4 ± 2% decrease in paw withdrawal latency (PWL) in response to a radiant heat stimulus applied to the plantar surface of the hind paw in hBERK vs. HbA-BERK (p<0.01). Both HbA-BERK and hBERK females exhibited lower PWL than their male counterparts (p<0.01 & p<0.05 in hBERK & HbA-BERK, respectively). Thus, mice with SCD show increased sensitivity to a noxious heat stimulus. When placed on a cold plate at 4°C for 2 min, hBERK mice exhibited a 72 ± 17% increase in PWF and 45.1 ± 11% decrease in PWL compared to HbA-BERK mice (p<0.01 for PWF and PWL). Finally, we examined deep tissue/musculoskeletal hyperalgesia by measuring maximum forepaw grip force. hBERK mice had 26.9 ± 10% lower grip force response compared to age/sex matched HbA-BERK mice (p<0.01). Hence, hBERK mice exhibited increased deep tissue hyperalgesia and sensitivity to mechanical and thermal stimuli. We evaluated the ability of the cannabinoid receptor agonist CP 55,940, to decrease deep tissue hyperalgesia. Intra-peritoneal injection of 0.3 mg/kg CP 55,940 improved grip force responses in hBERK mice in a time dependent manner (p<0.01; baseline vs. 0.5, 1, 1.5 and 3 h post-injection), while no change was seen in age/sex matched HbA-BERK mice, or mice injected with vehicle. Anti-nociception following CP 55,940 was not due to catalepsy. Increase in grip force response following CP 55,940 injection demonstrates that hBERK mice have tonic hyperalgesia that is sensitive to cannabinoids. To simulate the inflammatory pain of SCD, we induced inflammatory pain using Freund's Complete Adjuvant (CFA) and then evaluated the analgesic effect of the cannabinoid receptor agonist CP 55,940 using the von Frey test. Intra-plantar injection of 10 mg CFA resulted in increased PWF in the inflamed paw (p<0.01; baseline vs. 24 h post-injection in hBERK and HbA-BERK mice). Subsequent intra-plantar injection of 10 mg CP 55,940 (24 h after CFA injection) in the inflamed paw resulted in time-dependent reduction in PWF (p<0.01; before vs. 1, 3, 6 h post CP 55,940 injection) in both hBERK and HbA-BERK mice, while no change was seen in mice injected with vehicle, suggesting that cannabinoids attenuate inflammatory pain in SCD. In conclusion, we show that mice with SCD exhibit nociceptive behaviors and hyperalgesia similar to pain characteristics in human SCD. It is likely that vasculopathy and inflammation in SCD lead to alterations in nerve structure and nociceptor sensitization, causing enhanced pain sensitivity. Importantly, we show that low doses of the cannabinoid receptor agonist CP 55,940 attenuate tonic hyperalgesia as well as inflammatory pain in a mouse model of SCD. We speculate that cannabinoids may be effective analgesics for pain in SCD.

Disclosures:
No relevant conflicts of interest to declare.