Association of Non-Healing Wounds, Pain and Neurochemical Alterations In Sickle Cell Disease
Authors: Anuforo Josephine , Nguyen Julia , Luk Kathryn , Wallen-Friedman Silas , Kohli Divyanshoo , Vang Derek , Ericson Marna , Simone Donald , Hebbel Robert P , Gupta Kalpna 
Published: October 2019

Abstract
Abstract 842
The mechanisms underlying non-healing leg ulcers in sickle cell disease (SCD) remain unknown and their treatment is difficult. We hypothesized that leg ulcers in SCD cause injury to nerve fibers leading to neurogenic inflammation, and peripheral and central sensitization resulting in chronic pain and impairment of wound healing. We examined our hypotheses by creating 4 mm punch biopsies on the lower thigh of the left leg (LL) of 12 wk old BERK and hemizygous BERK (hBERK) mice expressing sickle hemoglobin (HbS). Since BERK did not survive wounding, hBERK and control HbA-BERK, expressing normal human HbA and wild type C57/BL6, were used. hBERK appeared to be a suitable model for this study because we found that >5 mo old BERK and hBERK show deep tissue and cutaneous hyperalgesia similar to pain in human SCD (Blood 116:456-65, 2010). Wounds were treated with topical morphine (3 mg/g Eucerin base cream) or with Eucerin base cream twice a day. Behavioral measures of hyperalgesia were assessed by paw withdrawal frequency (PWF) per 10 applications of a 1.0 g von Frey monofilament and paw withdrawal latency (PWL) in response to a radiant heat stimulus, applied to the plantar surface of the hind paw of wounded left leg and unwounded right leg. No hyperalgesia was observed in 12 wk old hBERK (baseline), but wounding led to a sharp increase in PWF and decrease in PWL, in LL for 24h, suggestive of wound-induced pain. Topical application of morphine significantly decreased mechanical and thermal hyperalgesia after 1h and 24h as compared to the base cream treated wound (p<0.05) in LL. Pain measures returned to baseline 2 wks post-wounding in morphine- but base cream-treated hBERK mice exhibited hyperalgesia for 4 wks (last measurement). The right leg exhibited increased mechanical but not thermal hyperalgesia following the wounding of left leg in hBERK. HbA-BERK and C57/BL6 wounds treated with base cream showed an increase in pain which was significantly less than hBERK and reached baseline 2 wks post-wounding, suggesting that wounding results in chronic pain in hBERK mice. Increased pain was accompanied by decreased wound closure in hBERK mice. Morphine-treated mice showed 100% wound closure at 4d Vs 8d in base cream treated hBERK, indicative of morphine-induced augmentation of wound healing. Laser scanning confocal microscopy (LSCM) of whole dorsal root ganglion (DRG) showed that, activating transcription factor 3 (ATF3), a marker of neuronal injury, was highly expressed in left lumbar DRG of base cream Vs morphine treated wounded hBERK, 30d post-wounding (p<0.05), suggestive of ongoing wound-induced neuronal injury which is abrogated by topical morphine. LSCM of 100 micron thick sections of healed wounds showed that substance P (SP), a neuropeptide expressed in nociceptive primary afferents, was significantly increased in base cream treated Vs morphine treated healed wounds 30d post wounding (p<0.01). Increased SP was accompanied by reduced and disorganized protein gene product (PGP 9.5)-ir nerve fibers and vasculature in base cream treated wounds. In contrast, morphine treated wounds showed normally organized dense PGP 9.5-ir nerve fibers and blood vessels replete with nerve and vascular plexus similar to their presentation in normal skin. Together, these data show that wounds stimulate chronic pain accompanied by nerve injury, central sensitization and neurogenic inflammation; and that morphine ameliorates this noxious insult and promotes wound healing. In addition, morphine may directly influence wound healing by promoting angiogenesis. It is possible that chronic pain may contribute to impaired wound healing and that non-healing wounds contribute to chronic pain in SCD. We propose that wound-induced nerve signal conduction (ATF3 in DRG) and neurogenic inflammation/sensitization of primary afferents (SP in skin) may contribute to chronic pain and in turn interfere with wound healing. Our observations suggest that neurochemical and behavioral alterations orchestrated by wounding may contribute to the impairment of healing of leg ulcers in SCD. We speculate that topical application of morphine may ameliorate pain and promote healing of painful leg ulcers in SCD.

Disclosures:
No relevant conflicts of interest to declare.