Mechanisms of pain in sickle cell disease

Abstract

Objectives:

The hallmark of sickle cell disease (SCD) is acute and chronic pain, and the pain dominates the clinical characteristics of SCD patients. Although pharmacological treatments of SCD targeting the disease mechanisms have been improved, many SCD patients suffer from pain. To overcome the pain of the disease, there have been renewed requirements to understand the novel molecular mechanisms of the pain in SCD.

Methods:

We concisely summarized the molecular mechanisms of SCD-related acute and chronic pain, focusing on potential drug targets to treat pain.

Results:

Acute pain of SCD is caused by vaso-occulusive crisis (VOC), impaired oxygen supply or infarction-reperfusion tissue injuries. In VOC, inflammatory cytokines include tryptase activate nociceptors and transient receptor potential vanilloid type 1. In tissue injury, the secondary inflammatory response is triggered and causes further tissue injuries. Tissue injury generates cytokines and pain mediators including bradykinin, and they activate nociceptive afferent nerves and trigger pain. The main causes of chronic pain are from extended hyperalgesia after a VOC and central sensitization. Neuropathic pain could be due to central or peripheral nerve injury, and protein kinase C might be associated with the pain. In central sensitization, neuroplasticity in the brain and the activation of glial cells may be related with the pain.

Discussion:

In this review, we summarized the molecular mechanisms of SCD-related acute and chronic pain. The novel treatments targeting the disease mechanisms would interrupt complications of SCD and reduce the pain of the SCD patients.

Introduction

Sickle cell disease (SCD) is an inherited group of autosomal recessive disorder in which the sickle mutation (Glu6Val) of β-globin gene is co-inherited with a mutation at the other β-globin allele resulting in sickle haemoglobin (HbS).^1–4 SCD is prevalent in large areas covering sub-Saharan Africa, the Mediterranean, the Middle East and India with the number of patients of the disease being estimated to increase.³,^5–7 Even with the best medical care, it is estimated that life expectancy for SCD patients is reduced by approximately three decades.^6,8 The patients possess sickle shaped red blood cells (RBCs), which cause haemolytic anaemia, acute/chronic pain, organ damages or comorbid conditions.^9–11 A unique complication of this disease is vaso-occlusive crisis (VOC), which causes episodic or recurrent acute pain, and the patients also suffer from chronic pain.^12,13 Although the precise cause of chronic pain is yet to be discovered, both acute and chronic pain are the hallmark of the disease and dominate the clinical characteristics of SCD patients.^9,12 The main aim of this review is to focus on the molecular mechanisms of SCD-related acute and chronic pain, and to concisely summarize SCD-related pain from a scientific point of view. This review can help clinicians to get a better insight into understanding of the mechanisms of pain in SCD.

Clinical manifestations of SCD

The clinical manifestations of SCD are protean.^1,10 The major features include haemolytic anaemia and VOC which relates to acute/chronic pain and tissue ischaemia or infarction leading to a variety of complications from nearly all the organs.¹ For example, cardiothoracic, nervous, reticuloendothelial, musculoskeletal, urogenital or gastrointestinal organs are commonly affected.⁶ Among them, acute chest syndrome, defined as a new radiodensity on chest radiograph with fever and/or respiratory symptoms such as cough and/or wheezing, is a leading cause of death and requires immediate medical attention.^1,14 The patients also suffer from infections caused due to splenic infarction.^8,15 Pain in SCD has unique features and typically occurs in the chest, the abdomen, the back, long bones and joints.^10,16 Although the pathophysiology of pain is complicated and not fully understood, ischaemia and VOC in the microcirculation could be the triggering factors.¹⁶ Some patients suffer from allodynia where severe pain is elicited by innocuous, low threshold stimuli, such as cold, wind or low humidity.^9,17–20

Molecular mechanisms of pain in SCD

Acute pain

Acute pain is caused by VOC, impaired oxygen supply or infarction-reperfusion tissue injury.^16,21 The morphological change in sickle cells is caused by deoxygenation and the deformed cells damage and adhere to endothelial cells of blood vessel walls with the increase in the affinity along with circulating leukocytes and platelets, followed by blocking blood flow all over the body.^22–24 This interaction between sickle cells and leukocytes with vascular endothelial cells is mediated by adhesion proteins such as E-selectin and P-selectin.^3,25–27 In addition, the adherence of sickle cells to vascular endothelium may also result in intimal hyperplasia leading to tissue infarction and injury.^10,28

In the setting of VOC, the absence of vascular flow causes RBC and endothelial cell lysis, and the sites are turned to be ischaemic surrounded by lower oxygen concentration and lower pH.²³ Immune cell infiltration follows at VOC site, and the endothelial cells were activated by producing inflammatory mediators which allow monocytes to extravasate.^22,29 Consequently, released inflammatory cytokines including tryptase from mast cells near the site together with lower pH and hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor activate nociceptors such as the transient receptor potential vanilloid type 1 (TRPV1).^11,13,23,30 Peripheral nerve endings have various receptors which respond to adenosine triphosphate (ATP) and H⁺ including TRPV1.³¹ TRPV1 is one of the key players of pain sensation and is highly expressed in perivascular primary sensory neurons including C and Aδ fibres.^13,23,32 These receptors are activated by the neurotransmitter glutamate which triggers electrical pulses on Aδ and C fibres to the dorsal horn of the spinal cord which is the entry of the central nervous system.³³ At this site, calcitonin gene-related peptide (CGRP), substance P (SP) and norepinephrine are released retrogressively towards the periphery and repeat the pain.^13,34,35 CGRP and SP in the periphery produce neurogenic inflammation.³⁵ Neurogenic inflammation is observed in sickle mice and immunoreactivity of SP and CGRP is increased in the skin of sickle mice.^36,37

In the setting of tissue injury, the secondary inflammatory response is triggered. This response may release norepinephrine, cause further tissue ischaemia and generate more cytokines and pain mediators such as interleukin-1 (IL-1), bradykinin, H⁺, K⁺, CGRP, SP, leukotrienes and others.³⁸ Bradykinin, H⁺ and K⁺ trigger pain by activating nociceptive afferent nerves.³⁹ IL-1 synthesizes prostaglandins E₂ and I_2.^9,38 Prostaglandins, bradykinin and leukotrienes sensitize peripheral nerve endings and promote the transmission of painful stimuli on Aδ and C fibres to the dorsal horn of the spinal cord.^9,40 SP released by activated nociceptors, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the N-methyl-d-aspartate (NMDA) receptor at the dorsal horn, also facilitate the transmission of the stimuli when activated.^9,38,41,42 The interaction between these two sodium channels determines the severity and duration of pain.⁴² Francis et al.⁴³ reported that plasma IL-1 was elevated in six out of 34 adult SCD patients and Graido-Gonzahez et al.⁴⁴ found that plasma prostaglandin E₂ (PGE₂) was elevated during VOC in SCD patients. Plasma leukotriene B₄ and urine leukotriene E₄ levels were also elevated in SCD patients.^45,46

Chronic pain

Chronic pain is often defined as pain that lasts more than 3 months and is experienced by a large number of SCD patients.^11,13 Although the precise aetiology of chronic pain in SCD still remains unclear, the causes of chronic pain may be from extended hyperalgesia after a VOC, leg ulcers, bone infarction, avascular necrosis, compression fractures, arthropathies, chronic osteomyelitis and central sensitization.^10,12,13,42

Central sensitization

Central sensitization induces pain hypersensitivity by changing the sensory response from normal inputs and is found in a variety of diseases with chronic pain situations, such as fibromyalgia, osteoarthritis, irritable bowel syndrome, chronic pancreatitis, SCD and so on.^47–51 Central sensitization occurs when nociceptive signals from the peripheral nerves change the spinal cord and the brain, resulting in continuous enhancement of pain sensation.^52,53 In the brain, the continuous severe pain impulses stimulate a large number of neurons which trigger pain impulses on many other neurons, resulting in the severe pain perception.⁴² The neuroplasticity is then constructed in the brain and it may change the perceptual processing and continuity of pain.^42,54

The activation of glial cells, such as oligodendrocytes, astrocytes and microglia, is also related with pain.^55,56 The glial cells are believed to facilitate the transmission of painful stimuli, and the activated microglia releases proinflammatory cytokines and glutamate which accentuate the pain experience.^42,56 Campbell et al.⁵⁷ described that SCD patients with high central sensitization assessed by quantitative sensory testing experienced heavier clinical pain, more VOC length, catastrophizing and negative mood compared to the patients with low central sensitization.

Transgenic HbSS-BERK (Berkeley) and HbSS-Townes mice expressing human HbS are the mouse models of SCD and exhibit the characteristics of pain in SCD patients.^58–60 Both mouse models express human α-globin and sickle β-globin without mouse α-and β-globins and exhibit hyperalgesia compared to control mice.^36,58,59,61 According to VOC, Cain et al.⁶² incited vaso-occlusion with hypoxia/reoxygenation and reported that BERK mice have cutaneous and deep tissue hyperalgesia which was increased following hypoxia/reoxygenation.⁶³ In addition, Sabaa et al.⁶⁴ reported that pharmacological inhibition of endothelin receptors prevented hypoxia-induced acute VOC in a SCD mouse model. In sensitization, Cataldo et al.⁶⁵ reported enhanced excitability, increased rate of spontaneous activity, greater responsiveness after mechanical stimulus and lower mechanical thresholds in spinal dorsal horn nociceptive neurons of BERK mice. They also reported that these phenomenon were accompanied by increased phosphorylation of mitogen-activated protein kinases, including c-Jun N-terminal kinase, p38 and p44/p42 extracellular signalling-regulated kinase (ERK) in the spinal cords of BERK mice.^65,66 ERK activation increases action potential amplitude of spinal cord dorsal horn neurons leading to hyperexcitability.⁶⁷ Valverde et al.⁶⁸ found elevated level of SP, elevated reactive oxygen species and activated microglial and astrocytic cells with increased glial fibrillary acidic protein in the dorsal horn of the spinal cord of BERK mice. It is assumed that these hyperexcitability might be a continuum of peripheral nociceptor sensitization.¹² This is because increased excitability of primary afferents was detected in skin-nerve preparations of BERK mice after mechanical stimuli by activation of TRPV1 channels.^12,69 Uhelski et al.⁷⁰ reported that C-fibre nociceptors in BERK mice were sensitized, which were supported by increased spontaneous activity and increased responses to mechanical, heat and cold stimuli on C-fibres. PGE₂-glycerol (PGE₂-G) produces mechanical and thermal hyperalgesia, and a P2Y₆ receptor, a receptor for PGE₂-G, antagonist was reported to block hyperalgesia in BERK mice.^71–73

Neuropathic pain

Although pain in SCD is mainly nociceptive, it may also have a neuropathic component.⁷⁴ Neuropathic pain in SCD could be due to central/peripheral nerve injury or nerve dysfunction and is presented as hyperalgesia, allodynia or sensitivity to cold or heat.^13,42,75 Protein kinase C (PKC) has roles in cell differentiation, cell proliferation or long-term potentiation of neuronal activity.^41,76 Mao et al.⁷⁷ reported that PKCγ immunoreactivity was increased after peripheral nerve injury and inflammation in rats, which suggested the association between neuropathic pain and PKC. PKCδ activation was reported to be elevated in the spinal cord dorsal horn in Townes mice compared with control mice, especially in GABAergic inhibitory neurons.^60,78

Regarding hyperalgesia and hypersensitivity, the detailed molecular mechanisms remain to be determined. However, Kohli et al.³⁶ reported that paw withdrawal threshold to mechanical stimuli and withdrawal latency to thermal (cold and heat) stimuli were lower in BERK mice compared with control mice. Furthermore, phosphorylation of signal transducer and activator of transcription 3 (STAT3), p38 mitogen-activated protein kinase and mitogen-activated protein kinase/ERK, which are activators of neuropathic and inflammatory pain, were increased in protein level together with increase in COX-2, IL-6 and Toll-like receptor 4 in the spinal cord of hBERK1 mice which are homozygous for knockout of murine α globin, hemizygous for knockout of murine β globin by carrying a single copy of the linked transgenes for human α and β-globins.^36,58 Cold hypersensitivity was also reported in Townes mice.⁷⁹ Zappia et al.⁸⁰ reported that cold hypersensitivity exacerbated with advanced age in BERK mice and they found elevated mRNA level of endothelin 1 (ET-1) and tachykinin receptor 1 in dorsal root ganglia of BERK mice which might contribute to hypersensitivity.

Current treatment of pain in SCD

Acute pain treatment

A VOC is accompanied by acute pain, and the acute sickle cell painful episode is the most common cause of hospitalization for SCD patients.⁸¹ Pain management should be according to pain severity of SCD patients.^8,55 Pharmacologic management of pain in SCD includes non-opioids, opioids and adjuvants.^9,55,82 Non-opioids used are acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs).⁹ Weaker opioids such as tramadol or routine opioids are mostly prescribed for pain in SCD, especially for severe pain, and parenteral opioids should be included for a VOC with severe pain.^8,9,10,82,83 A retrospective study showed no difference in average length of stay in hospitals between intermittent injection (INT) of opioid and patient-controlled analgesia (PCA) for adult SCD patients with VOC; however, treatment failure was statistically higher in the INT group.⁸⁴ INT might be chosen for patients who are unable to manage PCA including paediatric cases or who may require rapid dose titration.⁸⁵ Although 53% of emergency department physicians believed that more than 20% of SCD patients addicted to opioids, Zempsky estimated the prevalence of opioid addiction among SCD patients at 0.5–8% in the United States.^86,87 Furthermore, the prevalence of opioid abuse among patients who used prescription-type psychotherapeutic drugs nonmedically in the past month was 2.8% in the United States, which might underscore the misconception of health care providers that opioid addiction profoundly prevails among SCD patients.⁸⁸ Opioid tolerance could be developed by opioid receptor phosphorylation and uncoupling, upregulation of the cyclic adenosine monophosphate pathway, agonist-selective μ-opioid receptor internalization or the activation of NMDA receptors.^77,89,90 Low-dose ketamine, a noncompetitive antagonist of NMDA receptors, may be appropriate in SCD patients with VOC not responsive to standard opioid analgesics.⁹¹ The adjuvants include antihistamines, antidepressants, benzodiazepines, anticonvulsants and so on.⁹ The adjuvants are used with opioid analgesics and would enhance their analgesic potential and obviate or reduce opioid side effects.⁹ Hydroxyurea, a ribonucleotide reductase inhibitor, which binds to iron molecules and scavenge the free radicals, is used in the overall management of SCD, especially for the symptomatic SCD patients.^92,93 It inhibits HbS polymerization by inducing haemoglobin F (HbF) and reduces the frequencies of VOC, acute painful episode and acute chest syndrome and lowers mortality.^4,8,94–96 L-glutamine was reported to lower the median number of pain crises over 48 weeks among children and adults with sickle cell anaemia and was approved by the US Food and Drug Administration in 2017.⁹⁷ Nitric oxide (NO) is the endothelium-derived relaxing factor and is synthesized from l-arginine.⁹⁸ Plasma l-arginine levels were significantly low in SCD children with VOC and NO bioactivity is reduced in peripheral conduit vessels; however, inhaled NO for sickle cell crisis did not improve time to crisis resolution in a randomized placebo-controlled study.^98–101 A SCD patient with chronic myeloid leukaemia, who was treated with imatinib, a mast cell and Bcr-Abl tyrosine kinase inhibitor, was reported to experience a reduction of VOC.¹⁰² Adjuvants, such as antidepressants, antihistamines, benzodiazepines and anticonvulsants, are used along with analgesics to either ameliorate the side effects or potentiate the effects of analgesics.¹⁰

Chronic pain treatment

Chronic pain in SCD is different from chronic pain in other settings such as pain in cancer.¹³ Chronic pain treatment should be individualized according to the aetiology, intensity and duration of pain, and its purpose is to maximize functioning and quality of life for a longer period.¹⁰ The pharmacological treatment is usually with long-acting or controlled-release opioids.^13,22 Anticonvulsants could be used for neuropathic pain.¹⁰ SCD adult patients (29%) in a single-centre questionnaire survey answered that cannabis reduced their chronic pain, which might suggest the efficacy of cannabinoids for SCD pain relief.¹⁰³ Stimulating cannabinoid receptors was reported to relieve pain in BERK mice, and vaporized cannabis for chronic pain is under investigation in a double-blind, placebo-controlled clinical trial.^11,37

Non-pharmacological management of pain

In addition to the pharmacological treatments, certain non-pharmacological methods have also been used to manage pain in SCD. Hydration and blood transfusion are the major non-pharmacological modalities used to minimize pain. Hydration helps to reduce the sickling process by increasing blood plasma volume. Usually, between 1 and 1.5 times daily estimated fluid intake is recommended, and hydration is recommended regardless of the patient’s hydration status.^82,104 Blood transfusion helps to reduce pain by reducing the percentage of abnormal RBCs. Transfusion is carried out using simple transfusion or exchange transfusion.⁸² Rodgers-Melnick et al.¹⁰⁵ revealed that standard care and 20 minutes of electronic music improvisation with a board-certified music therapist improved visual analogue scale of pain intensity of SCD patients compared to control using a three-group mixed methods intervention designed study. A retrospective review showed 13 SCD patients who received inpatient acupuncture treatment for acute VOC revealed a reduction in reported pain score immediately after the acupuncture treatment.¹⁰⁶ These and other modalities, such as transcutaneous electrical nerve stimulation (TENS), heat, cold and vibration, relaxation, distraction, massage, music, guided imagery, self-hypnosis, self-motivation, acupuncture and biofeedback have been used in controlling pain in SCD; however, there are no controlled clinical studies to support their use.^{10,105,107–110}

Possible targets of pain treatment of SCD and future possibility

Many pharmacological treatments targeting one or more of the mechanisms that contribute to the pain relief or the disease process of SCD are being explored in clinical trials.^3,111 The targeting mechanisms of SCD include cell adhesion, inflammation, HbF induction, cell sickling, coagulation or platelet function. PGE₂-G and P2Y₆ receptors could be novel pharmacological targets for reducing pain in SCD patients. R-flurbiprofen, which reduces production of PGE₂-G, decreased the mechanical and thermal hyperalgesia, and a P2Y₆ receptor antagonist blocked the hyperalgesia in BERK mice.⁷³ In addition, crizanlizumab and voxelotor are newly approved drugs for SCD.^4,111 Crizanlizumab is a P-selectin-targeted humanized monoclonal antibody, and a phase 2 double-blind randomized clinical trial for SCD patients resulted in a significantly lower rate of sickle cell-related pain crises than placebo and is currently in phase 3 clinical trial.¹¹² Voxelotor is an oral small molecule that inhibits HbS polymerization, and a phase 3 double-blind randomized clinical trial for SCD patients revealed that the drug was associated with a trend towards reduced vaso-occlusive events.¹¹³ It also increased haemoglobin levels and reduced markers of haemolysis of the SCD patients.¹¹³

Conclusion

In this review, we summarized the molecular mechanisms of SCD-related acute and chronic pain. The newly discovered disease mechanisms provides the directions for future efforts to improve pain treatment in SCD.¹² It is crucial to keep exploring how these mechanisms interact with each other and the relative contribution of each to trigger pain in SCD.¹² Although observational studies still show severe morbidity and early mortality of the patients, future treatments targeting the disease mechanisms are believed to hold greater promise to interrupt complications and reduce the burden of SCD patients.