Am J Physiol Regul Integr Comp Physiol 315: R104–R112, 2018.

First published March 28, 2018; doi:10.1152/ajpregu.00021.2018.

REVIEW 2017 APS Conference: Physiological and Pathophysiological

Consequences of Sickle Cell Disease

Targeting pain at its source in sickle cell disease

Kanika Gupta,* Om Jahagirdar,* and Kalpna Gupta
Vascular Biology Center, Division of Hematology, Oncology and Transplantation, Department of Medicine, University of
Minnesota, Minneapolis, Minnesota
Submitted 25 January 2018; accepted in final form 20 March 2018

Gupta K, Jahagirdar O, Gupta K. Targeting pain at its source in sickle cell

disease. Am J Physiol Regul Integr Comp Physiol 315: R104 –R112, 2018. First
published March 28, 2018; doi:10.1152/ajpregu.00021.2018.—Sickle cell disease
(SCD) is a genetic disorder associated with hemolytic anemia, end-organ damage,
reduced survival, and pain. One of the unique features of SCD is recurrent and
unpredictable episodes of acute pain due to vasoocclusive crisis requiring hospi-
talization. Additionally, patients with SCD often develop chronic persistent pain.
Currently, sickle cell pain is treated with opioids, an approach limited by adverse
effects. Because pain can start at infancy and continue throughout life, preventing
the genesis of pain may be relatively better than treating the pain once it has been
evoked. Therefore, we provide insights into the cellular and molecular mechanisms
of sickle cell pain that contribute to the activation of the somatosensory system in
the peripheral and central nervous systems. These mechanisms include mast cell
activation and neurogenic inflammation, peripheral nociceptor sensitization, mal-
adaptation of spinal signals, central sensitization, and modulation of neural circuits
in the brain. In this review, we describe potential preventive/therapeutic targets and
their targeting with novel pharmacologic and/or integrative approaches to amelio-
rate sickle cell pain.
analgesia; mast cell; neurogenic inflammation; opioid; pain; sickle cell disease;
substance P; vasoocclusive crises

INTRODUCTION
Sickle cell disease (SCD) affects millions of people world-
wide (65). Although the molecular basis of this genetic disease
is a point mutation in the ␤-chain of hemoglobin, its conse-
quences are manyfold (7, 86). One of the major comorbidities
of SCD is pain. In SCD, pain can start in infancy and continue
throughout life, often leading to hospitalization and poor qual-
ity of life (1, 77). Two types of pain have been characterized in
SCD: acute pain and chronic pain. Acute pain is associated
with vasoocclusion. Under low oxygen tension, sickle hemo-
globin polymerizes into rigid fibers and makes red blood cells
(RBCs) less deformable. Such stiff RBCs occlude microcircu-
lation and cause vasoocclusive crisis (VOC) characterized by
episodic, recurrent, and unpredictable acute pain (7, 8, 68).
During VOC, there is impaired oxygenation of the distal tissue
and ischemic-reperfusion injury. This injury leads to inflam-
mation, oxidative stress, and endothelial dysfunction, resulting
in acute pain (1, 8). In addition to the acute pain of VOC,
* Kanika Gupta and Om Jahagirdar contributed equally to this work.
Address for reprint requests and other correspondence: Kalpna Gupta,
Vascular Biology Center, Medicine - Hematology, Oncology and Transplan-
tation, Univ. of Minnesota, Mayo Mail Code 480, 420 Delaware St., SE,
Minneapolis, MN 55455 (e-mail: gupta014@umn.edu).
R104 0363-6119/18 Copyright © 2018 the American Physiological Society
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patients with SCD may also experience chronic pain. Chronic
pain may be the result of maladaptive changes in pain path-
ways. Current understanding of pain in SCD suggests that pain
occurs by nociceptive, inflammatory, and neuropathic mecha-
nisms (77). Emerging observations suggest several pain path-
ways that could be targeted to ameliorate or reduce pain.
In this review, we describe the mechanisms of acute and
chronic pain in SCD, focusing on how sickle pathobiology
evokes pain by activating the peripheral and central nervous
systems (Fig. 1). We provide brief insight into the unique
humanized transgenic mouse models of SCD, which have been
critical in defining the mechanisms of sickle cell pain. We also
elaborate on the targeting of these mechanisms with specific
pharmacologic and/or integrative approaches with the potential
to treat sickle cell pain more effectively.
HUMANIZED MOUSE MODELS OF SCD
Transgenic mouse models of SCD expressing human sickle
hemoglobin have played a key role in understanding the
pathobiology of pain in SCD, reviewed by Tran et al. (77).
There are two broad categories of such mice. The first type of
mice expresses human ␤-sickle hemoglobin alongside mouse
␣- and ␤-globin; these include the NY1DD and S⫹SAntilles
mice with a mild-to-moderate SCD phenotype (29, 30). The
second type of mice expresses exclusively human ␣- and
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 TARGETING SICKLE CELL PAIN
Fig. 1. Pain pathways from the periphery to the brain and from the brain and
central nervous system to the periphery. We propose that sickle pathobiology
replete with vasoocclusion, ischemia-reperfusion injury, inflammation, and
oxidative stress activates mast cells, which in turn leads to peripheral nocice-
ptor sensitization. Signals from the periphery are transmitted to the dorsal horn
of the spinal cord via primary afferents, which are modulated and further
transmitted to the brain. However, sickle cell disease pathobiology may
influence each of these components of pain independently. Moreover, sus-
tained hyperexcitability of second-order neurons in the spinal cord may lead to
an antidromic release of action potentials and neurotransmitters from the
central nervous system to the periphery, leading to a feedback loop of
peripheral and central sensitization. In addition to receiving signals from the
spinal cord, the brain may modulate pain by releasing neurotransmitters
through the descending inhibitory pathway into the dorsal horn, which may
have an inhibitory or facilitatory effect on pain. DRG, dorsal root ganglion.
␤-globin chains and no mouse ␣- and ␤-globin chains. These
include the HbSS-BERK and HbSS-Townes sickle mice,
which exclusively express ⬎99% human sickle hemoglobin
(HbS), and control HbAA-BERK or HbAA-Townes mice,
which express normal human hemoglobin (HbA; 64, 69).
HbSS-BERK and HbSS-Townes mice constitutively show me-
chanical, thermal, and deep tissue hyperalgesia, characteristic
features of chronic pain in patients with SCD (1, 17, 37, 38, 45,
47, 56). Using hypoxia-reoxygenation treatments, we have
been able to evoke acute pain in these sickle mice to simulate
pain due to VOC (17, 81). BERK sickle mice exhibit a
significantly greater degree of hyperalgesia for all behavioral
measures compared with sex- and age-matched Townes sickle
mice (39). These models have been highly instructive in
examining the pathobiology of sickle cell pain and show
translational potential. The genetically distinct “knock-in”
strategy of human ␣- and ␤-transgene insertion in Townes
mice compared with BERK mice may provide relative advan-
tage for further genetic manipulations to examine specific
mechanisms of pain (39).
PATHOBIOLOGY OF CHRONIC PAIN IN SCD
Chronic pain is defined as pain that continues for ⬎3 mo
(14). A significant majority of patients with SCD develop
chronic pain with increasing age (54, 73). Development of new
treatments is hampered by the long-term requirement of opi-
oids, lack of objective pain measures, and relatively poor
understanding of symptoms and mechanisms. A collaborative
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R105
effort between the Analgesic, Anesthetic, and Addiction Clin-
ical Trail Translations, Innovations, Opportunities, and Net-
works (ACTTION) and the American Pain Society has led to
an evidence-based classification system to facilitate the diag-
nosis and management of chronic sickle cell pain (24), the
ACTTION-American Pain Society Pain Taxonomy (AAPT).
These criteria may improve the diagnosis of chronic pain, but
new treatments are dependent on a better understanding of the
sickle pathobiology that perpetuates pain.
SICKLE PATHOBIOLOGY UNDERLYING THE ACTIVATION
OF SOMATOSENSORY MECHANISMS LEADING TO PAIN
The unique sickle pathobiology of vasoocclusion, inflamma-
tion, and oxidative stress along with ischemia-reperfusion
injury may create a noxious environment in the periphery,
spinal cord, and/or brain, contributing to a complex pathology
underlying pain in SCD (Fig. 1). Two key processes that may
drive chronic as well as acute pain are neurogenic inflamma-
tion and mast cell activation.
Neurogenic Inflammation in SCD
Inflammation caused by nerve stimulation is called “neuro-
genic inflammation” (36). It leads to the release of cytokines
and neuropeptides including substance P (SP) and calcitonin
gene-related peptide (CGRP) from nerve fibers, which stimu-
late vascular dilatation and increased venular permeability.
Sickle BERK mice show neurogenic inflammation as evi-
denced by an increase in vascular leakage compared with
control BERK mice (84). This process may underlie enhanced
peripheral nociceptor sensitization observed by us in these
mice (79) described in CENTRAL AND PERIPHERAL SENSITIZATION OF
CHRONIC PAIN PATHWAYS IN SCD. Significantly higher circulating
SP has been described in patients with SCD at steady state
compared with healthy controls (55). One of the sources of SP
appears to be mast cells as described below.
Mast Cell Activation in SCD
Mast cells are tissue-resident leukocytes, unlike neutrophils,
which are found in circulation (36). Mast cells display tremen-
dous heterogeneity in their activation, which is specific to the
surrounding microenvironment (36). An important feature of
mast cell activation is their ability to release a variety of
substances by degranulation and/or de novo synthesis, includ-
ing proteases, adhesion molecules, neuropeptides, and cyto-
kines, which act in a paracrine as well as autocrine manner.
Notably, mast cells also release extracellular traps, vesicles,
podia, and nanotubes, which physically interact directly with
other cellular structures. Since mast cells are located in the
vicinity of vasculature and nerve fibers, their activation has
direct influence on circulatory and neural physiology. We
found degranulating mast cells in the skin in close proximity to
nerve fibers, particularly in the nerve plexus of BERK sickle
mice (36, 84). We found that mast cells release tryptase, which
activates protease-activated receptor 2 (PAR2) and transient
receptor potential cation channel V1 (TRPV1) channels on
nerve endings, leading to the release of neuropeptides SP and
CGRP. These neuropeptides act on the vasculature leading to
increased permeability and neurogenic inflammation and a
vicious cycle of mast cell activation and pain. Treatment of
sickle mice with imatinib, a c-kit inhibitor, or cromolyn so-
R106 TARGETING SICKLE CELL PAIN
dium, a mast cell stabilizer, led to reduced inflammation,
neuroinflammation, and hyperalgesia. Deletion of mast cells
from sickle mice ameliorated chronic pain; this finding sug-
gests that mast cell activation contributes to pain. As described
below in MECHANISM-BASED STRATEGIES TO TARGET SICKLE CELL
PAIN, imatinib treatment completely ameliorated VOC in pa-
tients with SCD (61, 75). It is likely that mast cell activation in
SCD underlies the stimulation of selectins on the endothelial
surface and activation of neutrophils, as described below,
which orchestrate sickle RBC adhesion and vasoocclusion, as
well as contribute to coagulopathy by activating platelets.
PATHOBIOLOGY OF ACUTE PAIN IN SCD
A characteristic and unique feature of SCD is unpredictable
and recurrent VOC due to occlusion of venules accompanied
by ischemia-reperfusion injury, inflammation, hemolysis, and
oxidative stress (8, 68, 73). Acute pain, considered worse than
the pain of childbirth, requires hospitalization and is often
challenging to treat (8). Under low oxygen tension, sickle
erythrocyte HbS polymerizes into rigid fibers resulting in the
typical sickle shape of erythrocytes (16). Rigid sickle RBCs
aggregate and adhere to activated endothelium, along with
circulating leukocytes and platelets (90, 92). Leukocytes, spe-
cifically neutrophils, are activated in sickle mice and patients
with SCD (50). These interactions of sickle RBC and leuko-
cytes with vascular endothelium are facilitated by adhesion
molecules including E- and P-selectin (52, 66). Vasoocclusion
is prevented in sickle mice lacking E- and P-selectin (78).
Blockade of P-selectin with heparin improved microvascular
blood flow in patients with SCD (46, 52). Crizanlizumab, an
antibody to P-selectin, significantly reduced pain crises in
patients with SCD in a phase II trial, described in detail in
MECHANISM-BASED STRATEGIES TO TARGET SICKLE CELL PAIN (6).
However, it remains to be determined how vasoocclusion leads
to acute pain.
Upregulation of selectins on the endothelial surface is crit-
ical for adhesion of sickle RBCs and leukocytes. Mast cell
activation induces P-selectin-dependent leukocyte rolling and
adhesion in postcapillary venules in vivo (32, 76). Activation
of mast cells leads to endothelial leukocyte adhesion molecule
(ELAM) expression on vasculature in the skin (44). In the
Klein et al. (44) study, neonatal human foreskins, incubated
with morphine, a known activator of mast cell degranulation,
led to increased ELAM expression. We observed extremely
large aggregates of activated mast cells releasing extracellular
traps in close association with the intact vasculature in the skin
of sickle mice (36). Thus, activation of mast cells in SCD may
play a critical role in VOC and acute pain in addition to chronic
pain.
CENTRAL AND PERIPHERAL SENSITIZATION OF CHRONIC
PAIN PATHWAYS IN SCD
In chronic pain, constitutively hyperexcitable nociceptors on
peripheral afferents and/or second-order neurons in the spinal
cord continue to transmit action potentials on a sustained basis
in the absence of noxious insult. Hypersensitivity to noxious
and innocuous stimuli— called “hyperalgesia” and “allodynia,”
respectively— has been observed in sickle mice and patients
(12, 17, 18, 38, 42, 45, 47, 84). Electrophysiological recordings
in the dorsal horn neurons of sickle BERK mice compared with
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control BERK mice demonstrate a higher rate of spontaneous
discharge, enlarged receptive fields, reduced mechanical thresh-
old, and prolonged after-discharges after mechanical stimulation
(20). These electrophysiological changes are accompanied by
increased phosphorylation of the MAPK family members p42/44
MAPK, ERK, JNK, and p38 MAPK in the spinal cord, which is
consistent with chronic pain pathways (20).
In addition to central sensitization, we observed sensitization
of cutaneous nociceptors in the periphery in sickle BERK mice
(79). Electrophysiological recordings of the tibial nerve in vivo
in live anesthetized mice demonstrated spontaneous activity in
the A␦ and C fiber nociceptors. The C fibers showed increased
response to mechanical and thermal stimuli in sickle BERK
compared with control BERK mice. Complementary to these
observations, TRPV1 channels on the primary afferent termi-
nals and somata have been shown to be activated and contrib-
ute to mechanical hypersensitivity in sickle BERK mice (38).
The molecular signatures of these electrophysiological out-
comes are emerging. We found that oxidative stress, SP, glial
activation, glial fibrillary acidic protein, and inflammation are
increased significantly in the dorsal horn of sickle BERK mice
compared with control BERK mice (84, 85). The spontaneous
nociceptor activity in the periphery and spinal cord and
hypersensitivity to mechanical and/or thermal stimuli are
suggestive of central sensitization in sickle mice, which may
underlie chronic pain and be recalcitrant to analgesic ther-
apy. It is likely that this hyperexcitability of dorsal horn
neurons is a continuum of peripherally evoked sensory
activity and/or may be due to the glial activation by sickle
pathobiology involving mast cells and ischemia-reperfusion
injury in the spinal cord (Fig. 1).
NEUROMODULATORY PATHWAYS
Signals in the dorsal horn of the spinal cord are also
modulated by neurotransmitters including dopamine, endoge-
nous opioids, ␥-aminobutyric acid, glycine, and others released
from the nerve fiber projections from the brain stem. These
top-down mechanisms originating in the brain may be influ-
enced by perception and lead to affective modulation, which
may have an inhibitory or facilitatory effect on pain transmis-
sion. Neuroimaging studies in sickle cell subjects with chronic
pain have shown abnormal activity in two key neuronal net-
works of the brain, namely, the default mode network (DMN)
and executive control network (ECN; 19, 25). The DMN is a
network of highly correlated areas of the brain that is active
when the brain is engaged in nonpurposeful actions, such as
daydreaming, not conscious and purposeful action. The key
zones of the DMN are the ventral medial prefrontal cortex, the
inferior parietal lobule, the posterior cingulate cortex, the
precuneus, areas of the medial frontal gyri, the hippocampal
formation, and the posterior lateral temporal cortex (15). In
patients with SCD, the neuronal circuits that enhance pain are
sensitized, and the circuits that suppress pain are desensitized.
In SCD subjects with increased pain, pronociceptive connec-
tions between the anterior cingulate cortex and areas of the
DMN such as the precuneus and the inferior parietal lobule are
increased (25). In contrast, SCD subjects with infrequent pain
show increased connectivity between the opioid-rich antinoci-
ceptive perigenual and subgenual cingulate and the DMN.
There is also increased connectivity between the perigenual
 TARGETING SICKLE CELL PAIN
anterior cingulate area and the salience network (SLN; 25).
The SLN is the main area for pain processing and includes the
bilateral primary and secondary somatosensory cortexes, the
anterior insula, the primary motor cortex, and the sensory
motor area (9). The ECN is the part of the brain that is active
when the brain is engaged in conscious, purposeful action. The
ECN is involved in external processing such as nociception and
decision making. The ECN comprises the dorsolateral prefron-
tal cortex and the posterior parietal cortex (53). In patients with
SCD, this part of the brain is hyperactive and may contribute to
exaggerated pain perception by the patient. Sociopsychological
factors leading to cognitive impairment have been described to
influence pain and opioid requirement in patients with SCD
(31). In addition, cerebral infarction in SCD subjects also
results in a significant decline of neuropsychological functions,
IQ, and cognitive score (5, 10, 13, 22, 26, 71, 87). However,
molecular events underlying these neural processes in the brain
remain unknown. It is likely that local ischemia-reperfusion
injury, mast cell activation, and other inflammatory processes
may directly influence the neural circuits and/or may be a result
of somatosensory inputs from the periphery.
MECHANISM-BASED STRATEGIES TO TARGET SICKLE
CELL PAIN
Opioids have been the mainstay of symptomatic manage-
ment of sickle cell pain. Use of opioids is fraught with side
effects including hyperalgesia, dependence, and tolerance, re-
R107
viewed by Gupta et al. (37). Furthermore, opioids adversely
influence RBC rheology by altering their membrane structure,
increase mast cell degranulation-induced inflammation, and in-
fluence organ pathology via their coactivation of receptor tyrosine
kinases leading to mitogenic signaling (35, 37). Importantly,
opioids do not treat the underlying mechanisms evoking pain, and
considerable concerns regarding opioid use have led to opioid
phobia, often leading to undertreatment of sickle cell pain (8).
Recent understanding of the mechanism-based targets of pain
described above has the potential to lead to improved analgesic
therapies in SCD, described below (Table 1).
Targeting VOC in SCD
During the initial phases of VOC, P-selectin initiates the
adhesion of sickle erythrocytes and leukocytes to the endothe-
lium and mediates the adhesion of activated platelets to neu-
trophils, resulting in vasoocclusion and inflammation (68). The
anti-P-selectin antibody, crizanlizumab, inhibits P-selectin-me-
diated cell-cell adhesion and has been shown to reduce the
frequency of painful VOC by 45% and triple the median time of
first VOC in a phase II clinical trial (6). Crizanlizumab may be the
first drug to be approved by the US Food and Drug Administra-
tion (FDA) to prevent VOC and subsequent pain at its source.
Inhibiting Inflammation
Elastase released from activated leukocytes mediates inflam-
mation in the peripheral tissues and the dorsal root ganglion

Table 1. Mechanism-based therapies to target sickle cell pain

Intervention Mechanism Known Outcomes and Current Clinical Trials

Crizanlizumab Anti-P-selectin antibody Reduced the frequency of painful VOC by 45% and tripled the median time of first VOC in
patients with SCD; current clinical trial (ClinicalTrials.gov identifier NCT03264989:
“Pharmacokinetics and Pharmacodynamics Study of Crizanlizumab in Adult SCD Patients
with VOC”)
Sivelestat Leukocyte elastase inhibitor Decreased neuronal injury to the DRG and reduced neuropathic pain in BERK sickle mice
Imatinib cKIT/mast cell inhibitor Reduced neurogenic inflammation and prevented hypoxia-reoxygenation-induced hyperalgesia
in sickle mice; decreased frequency of VOC in patients with SCD
Cromolyn Mast cell stabilizer Increased the analgesic effect of a suboptimal dose of morphine in BERK sickle mice
Trifluoperazine CaMKII␣ inhibitor; reduces In a phase I clinical trial, ameliorated neurogenic pain and caused 50% reduction in chronic
calpain-1 activity pain in patients with SCD without severe sedation or supplemental opioid analgesics
Simvastatin Decreases calpain-1 activation? In patients with SCD, reduced frequency of pain, oral analgesic use, and markers of
inflammation, acting synergistically with hydroxyurea; 4 completed clinical trials are cited
on the ClinicalTrials.gov website
CP55,940 Cannabinoid receptor 1 and 2 Ameliorated chronic and hypoxia-reoxygenation-induced hyperalgesia in sickle mice; mitigated
agonist mast cell activation, inflammation, and neurogenic inflammation in sickle mice; current
clinical trial (ClinicalTrials.gov identifier NCT01771731: “Vaporized Cannabis for Chronic
Pain Associated with SCD”)
Rapamycin mTOR inhibitor: increases fetal Ameliorated the nociception phenotype in sickle mice; in 1 renal transplant recipient, increased
everolimus hemoglobin levels fetal hemoglobin levels from 4.8 to 15% and was well tolerated
Omega-3 fatty Antioxidant Reduced frequency of VOC and transfusion requirements in a randomized study of 140
acids patients in Sudan; current clinical trial (ClinicalTrials.gov identifier NCT02947100:
“Omega–3 Fatty Acids in Sickle Cell Disease”)
Curcumin and Anti-inflammatory and antioxidant Attenuated glial activation, neuroinflammation, and oxidative stress in spinal cords of BERK
CoQ10 sickle mice, resulting in attenuation of hyperalgesia; reduced inflammation, oxidative stress,
and VOC-associated pain in patients with SCD
AT-200 Nociceptin opioid receptor agonist Ameliorated chronic and hypoxia-reoxygenation-induced mechanical, thermal, and deep tissue/
and mast cell inhibitor musculoskeletal hyperalgesia in BERK sickle mice without causing tolerance
Acupuncture Inhibits inflammation peripherally In awake BERK sickle mice, reduced inflammatory cytokines, substance P, and neurogenic
and in the spinal cord inflammation in the periphery and signaling pathways of nociception in the spinal cord and
potentiated the effect of a suboptimal dose of morphine; acupuncture significantly reduced
VOC-associated pain in patients with SCD
Hypnosis Modulates vascular physiology Decreased pain intensity and increased peripheral blood flow during anticipation and
experience of pain in patients with SCD
CoQ10, coenzyme Q10; DRG, dorsal root ganglion; mTOR, mammalian target of rapamycin; SCD, sickle cell disease; VOC, vasoocclusive crisis.

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R108 TARGETING SICKLE CELL PAIN
(DRG), contributing to pain. Vicuña et al. showed that upon
peripheral nerve injury in rodents, elastase is released from
infiltrating T cells in the DRG, contributing to neuropathic pain
[82a; see Weyer and Stucky (88)]. SerpinA3N, an endogenous
inhibitor of serine proteases, and the drug sivelestat, a leuko-
cyte elastase inhibitor, alleviated pain in this mouse model of
neuropathic pain as measured by the paw withdrawal threshold
and responses to von Frey filaments (P ⬍ 0.05). We found that
leukocyte elastase activity is significantly increased in the
DRG and lungs of sickle mice (2). Sivelestat treatment de-
creased neuropathic pain in BERK sickle mice (2). The safety
of sivelestat is already established in humans, and it has been
approved for treatment of acute lung injury in Japan (3). Trials
of this drug in the United States were previously halted because
of concerns that it increased long-term mortality rate in me-
chanically ventilated patients with acute lung injury (93) Sive-
lestat and other similar drugs in development require preclin-
ical evaluation for sickle-specific pain and associated adverse
effects.
Inhibiting Mast Cell Activation
Activation of mast cell plays a critical role in neurogenic
inflammation and nociceptor activation via the release of SP in
the skin and DRG of sickle mice. We found that imatinib, a
mast cell inhibitor, significantly reduces neurogenic hyperal-
gesia and prevents hypoxia-reoxygenation-induced hyperalge-
sia in BERK sickle mice (P ⬍ 0.05; 84). In separate case
reports of two patients with chronic myeloid leukemia and
SCD, imatinib significantly reduced painful episodes, hospital-
izations, and daily pain (21, 74). Imatinib is FDA approved for
several indications. At this time, we are not aware of any
clinical trials of imatinib in SCD, but the rationale for such a
trial would be compelling. We also found that pretreatment
with the mast cell stabilizer cromolyn sodium potentiated the
analgesic effect of otherwise ineffective low doses of morphine
in BERK sickle mice (84). Therefore, it is likely that morphine
activates mast cells, thereby contributing to pain, but concom-
itantly acts as an analgesic via its activity in the nervous
system. Mast cell targeting thus offers an opioid-reducing
strategy in the management of sickle cell pain.
Calpain-1 contributes to IgE-mediated mast cell activation
and plays an important role in neurotransmission and neuro-
genic pain (89). We have demonstrated that genetic ablation of
calpain-1 in Townes sickle mice ameliorates tonic hyperalgesia
in sickle mice, but not that evoked by hypoxia-reoxygenation
(62). Trifluoperazine, an FDA-approved antipsychotic agent,
acts as a potent CaMKII␣ inhibitor and reduces calpain-1
activity (49). It ameliorated neurogenic pain and caused 50%
reduction in chronic pain in patients with SCD without severe
sedation or supplemental opioid analgesics in a phase I clinical
trial (57). Statins also decrease calpain-1 activation (91). In a
single-center pilot study in adolescents and adults with SCD,
simvastatin treatment for up to 3 mo led to an 85% reduction
in the rate of sickle cell-related pain and oral analgesic use,
improved soluble biomarkers of inflammation, and acted syn-
ergistically with hydroxyurea (39). Despite the marked decline
in the rate of occurrence of pain, the intensity of pain did not
change with simvastatin (39). To explain this disparate effect
of simvastatin on frequency and intensity of pain, the authors
of this study hypothesized that the visual analog scale, used as
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the sole measure of intensity of pain in this study, may not have
fully captured the multidimensional nature of intensity of pain
in SCD (39).
Stimulating Cannabinoid Receptors
Cannabinoid receptor type 1 (CB1R) and cannabinoid re-
ceptor type 2 (CB2R) activation on mast cells has been shown
to inhibit degranulation and inflammation, respectively (72).
CB1R and CB2R are expressed in both the central nervous
system and non-central nervous system tissues, including in-
flammatory cells (59, 82). Activation of CB2R peripherally
generates an antinociceptive response in inflammatory and
neuropathic pain (41). Targeting CB1R has psychotropic ef-
fects, but targeting CB2R does not have psychotropic effects
(27). CB2R agonists and/or knockout mice provide compelling
evidence that CB2R activation mitigates neuropathic and in-
flammatory pain and is protective against ischemia-reperfusion
injury by decreasing the endothelial expression of adhesion
molecules and secretion of chemokines (58, 60, 67) and by
attenuating leukocyte adhesion to the endothelium, transendo-
thelial migration, and interrelated oxidative nitrosative damage
(63), all of which are consistent with the pathobiology of SCD.
We showed that cannabinoids mitigate mast cell activation,
inflammation, and neurogenic inflammation in BERK sickle
mice via both CB1R and CB2R, but activation of CB1R is
required to ameliorate hyperalgesia (83). In 86 human patients
with HbSS, HbSC, and HbS-␤ thalassemia, a structured self-
administered anonymous questionnaire found that that 31
(36%) had used cannabis in the previous 12 mo to relieve
symptoms associated with SCD (40). The main route in all but
two patients was by smoking. The most common reasons for
use were to reduce pain (in 52%) and to induce relaxation or
relieve anxiety and depression (in 39%). We are currently
performing a double-blind, placebo-controlled, FDA-approved
clinical trial to test the effect of vaporized cannabis on chronic
pain in SCD (ClinicalTrials.gov identifier NCT01771731).
Elevating Fetal Hemoglobin
Chronic pain has been found to be inversely associated with
circulating fetal hemoglobin (HbF) in patients with SCD (25).
Notably, higher HbF in patients with SCD has been shown to
be associated with reduced frequency of painful crises (51).
HbF-increasing strategies have been examined in preclinical
studies. The mammalian target of rapamycin (mTOR) plays a
central role in regulating many fundamental cell processes,
from protein synthesis to autophagy, and deregulated mTOR
signaling is implicated in a variety of pathological conditions
(70). The mTOR inhibitor, rapamycin, increases HbF levels
and ameliorates the nociception phenotype in sickle cell mice
(43). Gaudre et al. reported a case of a kidney transplant
recipient with SCD who was treated with everolimus, an
mTOR inhibitor (33). At 10 mo after initiating therapy, the
patient’s HbF level increased dramatically from 4.8 to 15%,
and there was excellent tolerance to treatment. Reinduction of
HbF by depleting DNA methyltransferase 1 with a combina-
tion of decitabine and tetrahydrouridine led to a significant
increase in HbF and improved RBC quality as well as hemo-
lysis and inflammation in patients with SCD in a phase I
clinical trial (NCT01685515). Together, these preclinical and
 TARGETING SICKLE CELL PAIN
clinical observations suggest the potential of HbF-enhancing
strategies in reducing the activation of pain pathways.
Anti-inflammatory and Antioxidant Strategies
Omega-3 fatty acids have antiaggregatory, antiadhesive,
anti-inflammatory, and vasodilatory properties. Omega–3 fatty
acid was shown to reduce frequency of VOC and transfusion
requirements in a randomized study of 140 patients in Sudan
(23). In the United States, a phase I/II clinical trial to determine
the safety of a new formulation of the omega–3 fatty acids
docosahexaenoic acid and eicosapentaenoic acid and to assess
whether it decreases inflammation and inflammatory pain in
children and young adults with SCD has been undertaken
(NCT02947100).
Central nociceptive mechanisms contribute to chronic pain
and hyperalgesia in SCD, releasing reactive oxidative species,
inflammatory cytokines, neurotrophic factors, and prostaglan-
dins that excite nociceptive neurons by activated microglia
contributing to the persistence of chronic pain in SCD (34). We
found that glial activation, neuroinflammation, and oxidative
stress in spinal cords of sickle mice are ameliorated with
curcumin and/or coenzyme Q10 (CoQ10), resulting in attenua-
tion of hyperalgesia in BERK sickle mice (80). Curcumin and
CoQ10 are likely to be relatively nontoxic adjuvants. In a
preliminary study in a subpopulation of patients with SCD,
CoQ10 treatment reduced inflammation and oxidative stress as
measured by circulating C-reactive protein and thiobarbituric
acid reactive substances, respectively, and reduced pain during
VOC (75).
Nociceptin Opioid Receptor Agonists
Once pain has been evoked, analgesic strategies are re-
quired. Opioids exert most of their antinociceptive effect via
the ␮-opioid receptor (MOP/R). In addition to MOP/R, the
opioid receptor family includes the nociceptin opioid receptor
(NOP/R), which is involved in nociceptive signaling via its
endogenous peptide ligand nociceptin/orphanin FQ. NOP/Rs
are found in the DRG, spinal cord, and supraspinal regions of
the brain involved in nociception. We have shown that AT-
200, a high-affinity NOP/R agonist with low efficacy at the
MOP/R, ameliorated chronic pain and hypoxia-reoxygenation-
induced mechanical, thermal, and deep tissue/musculoskeletal
hyperalgesia in HbSS-BERK sickle mice (81). Daily treatment
for 7 days with AT-200 did not cause tolerance and showed a
sustained antinociceptive effect, which improved over time and
led to reduced plasma serum amyloid protein, neuropeptides,
inflammatory cytokines, and mast cell activation in the periph-
ery. These data suggest that AT-200 ameliorates pain in sickle
mice via NOP/R by reducing inflammation and mast cell
activation without causing tolerance. Thus, NOP/R agonists are
promising drugs for treating pain in SCD because of their
effect on sickle pathobiology as well as nociceptive mecha-
nisms.
Electroacupuncture
We found that electroacupuncture in awake BERK sickle
mice influenced the sickle microenvironment as well as noci-
ception centrally. We observed varied antinociceptive response
among identical electroacupuncture-treated mice. To analyze
individualized responses, we categorized all electroacupunc-
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ture-treated mice into three groups on the basis of their increase
in pain threshold after electroacupuncture treatment: high re-
sponders (⬎200%), moderate responders (~100 –200%), and
nonresponders (ⱕ100%). Electroacupuncture led to reduction
in inflammatory cytokines, SP, and neurogenic inflammation in
the periphery and signaling pathways of nociception in the
spinal cord and potentiated the effect of a suboptimal dose of
morphine in moderate responders, resulting in analgesia equiv-
alent to that in high responders (85). In a retrospective review
and descriptive analysis of 24 patients with SCD with pain
treated at the National Institutes of Health, 9 patients under-
went only inpatient acupuncture for VOC, 11 patients received
only outpatient acupuncture treatment for chronic pain, and 4
patients received both inpatient and outpatient treatments (48).
For the patients who received inpatient acupuncture treatment
for VOC, there was a significant reduction of reported pain
score immediately after acupuncture treatment with an average
pain reduction of 2.1 points on the 0 –10 numeric pain scale
(P ⬍ 0.0001). Five of the 15 outpatients had only 1 session per
course of acupuncture and thus did not contribute to outpatient
data. Of the 10 outpatients who accepted ⬎1 session per course
of acupuncture, 2 patients accounted for 79.2% of the 231
sessions. The investigators therefore analyzed the results from
these two patients separately from the other eight patients. Six
out of the remaining eight outpatients described their pain as
improved. The two high-usage patients, who had pain mostly
related to chronic ulcers, reported improved pain compared
with the prior session in 36.1% of sessions, stable pain in
28.5%, and worse pain in 11.5%. Data were not available for
23% of the sessions (48). This analysis raises the possibility of
acupuncture as an adjuvant for pain management in SCD (48).
Perception-Based Therapies
Nonpharmacological techniques such as guided imagery,
mindfulness, relaxation, hypnosis, and cognitive behavioral
therapy are effective in alleviating chronic pain in SCD (4, 11,
28). Activation of the descending neuromodulatory pathway by
nonpharmacologic interventions (e.g., acupuncture, medita-
tion, and yoga) has been shown to reduce peripheral and
central sensitization. In a pilot study of patients with SCD and
healthy controls, a single 30-min hypnosis session decreased
pain intensity by a moderate amount in patients with SCD (11).
Pain threshold and tolerance increased after hypnosis in the
control group, but not in patients with SCD. Patients with SCD
exhibited lower baseline peripheral blood flow and a greater
increase in blood flow after hypnosis than controls, suggesting
that hypnosis may increase peripheral vasodilation during both
the anticipation and experience of pain in patients with SCD.
FUTURE PERSPECTIVES
Emerging pain therapies are in development that target SCD
pathobiology, peripheral and central sensory mechanisms, and
descending neuromodulatory pathways. Notably, many thera-
peutic strategies have the potential to reduce opioid require-
ment and improve opioid analgesia. Targeting pain at its source
is perhaps the most effective method of preventing the pain of
SCD compared with treating pain once it has been evoked.
Targeting mast cells with imatinib, an FDA-approved drug, to
ameliorate the evoking of somatosensory mechanisms by
sickle pathobiology appears to be a highly promising pharma-
R110 TARGETING SICKLE CELL PAIN
cotherapeutic strategy to prevent/treat sickle cell pain. In-
creased attention is required to apply perception-based affec-
tive modulation to minimize the use of analgesics. A major gap
still exists in diagnostic criteria for quantifying and phenotyp-
ing pain on the basis of circulating biomarkers, physiological
measures, and neuroimaging to guide the use of specific
therapies and/or combination therapies. Finally, well-designed
clinical trials are required to test these pharmacological and
integrative approaches.
ACKNOWLEDGMENTS
We thank Michael Franklin for editorial suggestions and Barb Benson for
assistance with manuscript preparation. We apologize for our inability to
include many studies due to space limitations, but we deeply appreciate their
contribution to the study of pain/SCD.
GRANTS
This effort was supported by National Heart, Lung, and Blood Institute
Grant U01-HL-117664 to Kalpna Gupta.
DISCLAIMERS
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of Health.
DISCLOSURES
Kalpna Gupta is a consultant for TauTona Group.
AUTHOR CONTRIBUTIONS
K.G. and K.G. prepared figures; K.G., O.J., and K.G. drafted manuscript;
K.G., O.J., and K.G. edited and revised manuscript; K.G., O.J., and K.G.
approved final version of manuscript; Kalpna G. conceived and designed
research.
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