Characterization of Pain, Vasculature and Innervation in Sickle Cell Disease.
Authors: Gupta Kalpna , Chen Chunsheng , Ericson Marna E. , Hebbel Robert P. , Li Yunfang 
Published: April 2020

Abstract
Pain in sickle cell disease (SCD) is characterized by chronic vasculopathy. Characterization of pain and vasculature may be critical to improve the analgesic ability of opioids in treating pain in SCD. Therefore, we examined the association between vasculature, innervation and pain in a transgenic mouse model of SCD (BERK) and control mice (HbABERK). Pain behavior was analyzed using paw withdrawal latencies (PWL) using Hargreave’s device for thermal hyperalgesia. A radiant heat stimulus was applied to the plantar surface of each hind paw from underneath the glass floor with a projector lamp bulb (CXL/CXR, 8 V, 50 W). PWL to the nearest 0.1 s was automatically recorded upon paw withdrawal. Paws were alternated randomly to preclude “order” effects. We observed that 5 mo old sickle mice showed significantly lower thresholds to noxious heat than controls (p=0.002 for females; and p=0.04 for males; n=5–7 and 15 observations/mouse). Females showed a significantly shorter latency than their respective males (control, p=0.03 and sickle, p=0.01), i.e., they were more sensitive to thermal stimuli. No significant difference was observed between sickle and control nor male and female mice (p &gt; 0.05, n = 5–7) for mechanical allodynia using von Frey filaments, suggesting that sickle mice show increased sensitivity to thermal hyperalgesia. BERK mice were subcutaneously injected with 0.7 mg morphine/Kg mice/day (equivalent to 50 mg/70 kg /day/ human adult) with an added increment of 0.14 mg/day/Kg for each week for three different time periods; Group I, treated for 6 weeks, Group II treated for 6 weeks followed by withdrawal for 6 weeks and Group III treated for 12 weeks. Morphine treatment for 6 weeks as well as 12 weeks resulted in a ∼50–75% increase in PWL vs PBS, suggesting that chronic morphine treatment decreased hyperalgesia in sickle mice. The anti-hyperalgesic effect of morphine was antagonized by simultaneous co-administration of naloxone (2 mcg/Kg/day), suggesting an opioid receptor mediated effect. Naloxone treatment alone did not show any significant effect on PWL. In Group II, withdrawal of morphine for 6 weeks following 6 wks of treatment showed about 35% increase in PWL vs PBS (p&lt;0.005), suggesting that the anti-hyperalgesic effect of morphine continues after its withdrawal. The continued anti-hyperalgesic effect of morphine following withdrawal could be secondary to its vasoregulatory effect. Indeed, confocal microscopy of skin sections revealed disorganized and stringy blood vessels, nerves and lymphatics in sickle mice vs. control. Deep blood vessels and nerve plexus, as well as the skin, were appreciably thicker in controls vs sickle. The diameter of lymphatics and densities of both blood vessels and nerves were significantly lower in sickle vs HbA controls (p= 0.0001 and 0.002, for sub-epidermal and dermal blood vessels, respectively; p=0.04 for lymphatic diameter; p=0.0001 for sub-epidermal, dermal and deep dermal nerve fibers). Functionally, we observed about 30% decrease in subcutaneous O2 measured by Laser Doppler (in real-time) in BERK vs HbA control (p&lt;0.05). Thus, complementary alteration in vasculature and innervation may underlie the chronic pain in SCD. Given that morphine promotes angiogenesis and vasodilation, its prolonged anti-hyperalgesic activity could be due to its vasoregulatory function. Together, these data suggest that intermittent therapy with morphine in SCD may provide analgesia similar to continued therapy and may even be devoid of side-effects.