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Comprehensive Review

Sex differences in neuroimmune and glial mechanisms of pain

Gregus, Ann M.a,*; Levine, Ian S.a; Eddinger, Kelly A.b; Yaksh, Tony L.b,c; Buczynski, Matthew W.a

Author Information
doi: 10.1097/j.pain.0000000000002215

Abstract

1. Introduction

1.1. Therapeutic challenges

The International Association for the Study of Pain (IASP) Task Force recently proposed a new definition of pain as an aversive sensory and emotional experience typically caused by, or resembling that caused by, actual or potential tissue injury.188 Importantly, acute pain serves a critical Darwinian protective function: to initiate an escape response from noxious stimuli that in the future should be avoided for personal safety. However, chronic intractable pain is maladaptive and constitutes a widespread public health issue, significantly impairing quality of life and costing nearly $600 billion per year in the United States alone.1 Current efforts to develop novel pain therapeutics are guided by the following observations: (1) pain may arise from multiple mechanisms, and this complexity reflects the difficulty in achieving significant relief; (2) chronic pain states may reflect an important sex covariate in the development of the pain phenotype; and (3) there is a growing appreciation that secondary to tissue and nerve injury, elements of the immune system are recruited in a sex-dependent manner to influence the chronic pain phenotype. In the following sections, we will discuss aspects of these 3 points.

1.2. Categorization of pain phenotypes

Mechanistically, pain states evolving into a chronic pain phenotype may be classified heuristically into 4 categories: (1) nociceptive pain resulting from activation of high threshold sensory neurons (nociceptors); (2) inflammatory pain resulting from persistent inflammation in soft tissue (viscera, fascia, and muscle), joints (arthritis), or other specific tissues (eg, dental, meningeal, and bone); (3) neuropathic pain resulting from direct (trauma, compression, and ischemia) or indirect (chemotoxins, radiation, or autoimmune attacks, as with paraneoplastic syndromes) injury to the peripheral afferent nerve or ganglia; or (4) dysfunctional/centralized pain occurring in the absence of a noxious stimulus, detectable inflammation, or structural damage to the primary afferent.228 It should be noted that in accord with the IASP guidelines outlined above, these 4 categories are associated with the generation of an aversive state accompanied by changes not only in physiology (eg, blood pressure and hormone release42) but also in reward and cognition (eg, formation of a negative association leading to avoidance and development of a positive appetitive response to drugs that diminish the negative affect169). Chronic pain syndromes with neuropathic etiology are often challenging to manage because they tend to be refractory to treatment with anti-inflammatory drugs, and many patients report inadequate or variable relief from commonly used first-line therapies such as anticonvulsants and antidepressants.239 Although it is informative to consider types of pain individually from a mechanistic standpoint, many chronic pain conditions represent multiple phenotypes expressed simultaneously. For instance, effective management of cancer pain may require several functionally distinct medications to target various underlying processes. Furthermore, there is increasing support for the assertion that acute pain states secondary to tissue injury may evolve into a chronic condition with peripheral and central neuropathic components.183

1.3. Sex as a covariate in the evolution of chronic pain

Evidence is accumulating in support of quantitative and qualitative sex differences in pain sensitivity and analgesia. Pain syndromes with high prevalence in humans—such as arthritis, temporomandibular disorder, migraine, and fibromyalgia—disproportionately affect females, occurring with significantly greater incidence than in males.158,194 Such disparities largely have been attributed to genetic and hormonal differences between males and females in preclinical and clinical studies, as explored in depth by several elegant reviews.20,53,62,73,74,158,177 Historically, most preclinical studies of pain hypersensitivity have focused on assessment of evoked behaviors in young adult male rats or mice. This approach largely derived from perceived challenges posed by evaluating effects of estrus cycle phases (which change every 4-5 days) on nociception and analgesic responsiveness.157 Surprisingly, evidence suggests that variability associated with different stages of the estrus cycle is no greater than that occurring intrinsically in males.15 There is a high failure rate of analgesic investigational new drugs in clinical trials, particularly for pain conditions with greater incidence in women.17 Thus, elucidation of the molecular underpinnings of chronic pain states in females and the mechanisms underlying sex-dependent differences in pain signaling is critical for successful development of novel therapeutics.159 Recent efforts are emphasizing inclusion of females and of spontaneous painful disease models such as osteoarthritis in companion animals to identify novel druggable targets for pain symptoms,111,158 although the literature still remains biased toward males.159 Recognizing this issue, the NIH specifically mandated that studies must use both males and females unless there are organ-specific reasons to exclude one sex or the other. Several preclinical studies suggest that interactions between the immune and nervous systems contribute to sex differences in many chronic pain syndromes and may serve as a source of novel drug targets that are specific to either males, females, or both.10,40,54,133,178,195,199,206,242 In the present review, we provide a comprehensive synthesis of reported sex differences in neuroimmune mechanisms of pain hypersensitivity in rodent models, suggesting potential high-value targets to pursue for sex-specific treatments of chronic pain in men and women.

2. Assessment of nociception in rodent models

In humans, pain is difficult to assess reliably using objective clinical measures because of its highly subjective and individualized nature.45 Thus, diagnosis of pain syndromes and subsequent evaluation of therapeutic efficacy relies heavily on patients' descriptions of their pain levels, features, and location. As nonverbal organisms (infants and rodents) lack this capacity, one endpoint that can be isolated and examined easily in behavioral models is nociception, or the neural process of encoding noxious stimuli constituting the sensory, nonaffective component of pain (Fig. 1). For excellent reviews of pain circuitry in development and adulthood, see Treede, Fitzgerald et al., and Basbaum and Fields.14,77,223 Injury-induced or disease-induced pain hypersensitivity results from peripheral or central sensitization, or the increased responsiveness of nociceptive neurons in the peripheral and central nervous systems (CNS) to normal or subthreshold primary afferent input,122 a process mediated by several mechanisms described herein. Pain hypersensitivity presents both in humans and in animals as allodynia, wherein stimuli that do not normally produce pain are perceived as painful, or hyperalgesia, a state of enhanced sensitivity to noxious stimuli that is often coupled with spontaneous (nonevoked) pain.196 If present at the site of injury, hyperalgesia is considered as primary, whereas that which occurs in the surrounding area is termed secondary. The development of secondary hyperalgesia is attributed to central sensitization.

Figure 1.
Figure 1.:
Nociceptive sensory primary afferent pathways. Nociceptors specialized for detection of high-intensity mechanical, thermal, and/or chemical stimuli originate in the sensory ganglia (dorsal root, trigeminal, and nodose) of the peripheral nervous system generally possess small-diameter to medium-diameter, thinly myelinated Aδ fibers or small-diameter unmyelinated C fibers and terminate predominantly in spinal superficial laminae I, II, and V of the dorsal horn.180,238 Nociceptors can be described using the following categories: red, Neurofilament H (NFH)+ Aβ large, high threshold mechanoreceptor (HTMR)/heat; orange, peptidergic Aδ small/medium, HTMR/heat; green, nonpeptidergic C small, HTMR/itch/chemical; blue, peptidergic or nonpeptidergic C small polymodal or mechanoheat/cold; purple, peptidergic Aδ small/medium HTMR or C small polymodal. In recent years, several subclassifications of nociceptors have been proposed, and the reader is directed to several excellent references for more detailed information on evolving designations of primary afferent sensory neuron subtypes.48,63,120,124,125,175,220,225

Classically, nociception in animals is measured as nocifensive (reflexive withdrawal) behaviors in response to evoked stimuli. However, the low probability of clinical success for candidate analgesic molecules based on evoked endpoints alone has sparked efforts to improve the face validity of preclinical paradigms of chronic pain.235 Several groups have pursued various methods of also capturing affective and motivational components of the pain state in rodents and in larger animals,27,79,96,131,146,162,171 although these approaches are still undergoing refinement. In contrast to a spinally organized nociceptive reflex, the perception and expression of pain unpleasantness depend on higher order functions in cognitive and limbic regions of the brain (Fig. 2). Because the affective component of a pain state manifests as spontaneous as well as time-dependent, species-dependent, and paradigm-dependent behaviors, there is no singular assay in existence that encapsulates the human experience of pain in an animal. Nonetheless, some testing approaches can capture specific aspects of emotional and motivational responses to noxious stimuli. When used concurrently with evoked measures, these methods may provide a stronger assessment of candidate analgesic drug efficacy in preclinical pain models. For example, rodents in pain display coping behaviors such as licking the site of injury or emitting ultrasonic vocalizations.96 Context-dependent approach or avoidance responses after injury include conditioned place aversion to a location linked with an aversive experience (ie, a pain state) and conditioned place preference for a site in which the pain state is alleviated.170 Similarly, operant paradigms for self-administration of analgesic drugs display motivated and goal-directed behaviors to obtain relief from pain,90,93 although interpretation of the results is complicated if the drug itself is intrinsically rewarding. Because depression often is comorbid with chronic pain, incorporation of assays of depression-like behaviors comprised of both evoked measures, such as the forced swim test, and spontaneous assessments of anhedonia, including motivation for naturally reinforcing substances such as sucrose.221

Figure 2.
Figure 2.:
Supraspinal nociceptive circuits. Nociceptive information transmitted through the spinal cord dorsal horn is communicated to the brain along several ascending pathways (merged together in black). Laterally projecting systems to the somatosensory (SSC) and insular cortices (IC) correspond to the classical somatosensory pathway, with a highly preserved body image mapped at several synaptic links. This tract mediates the sensory/discriminative (red) component of the pain phenotype. By contrast, medially projecting systems underlying the affective (green) and motivational (purple) aspects of pain have relatively crude somatosensory mapping and project to limbic structures appreciated for their roles in emotional responses such as the parabrachial nucleus (PBN), amygdala (AMYG), anterior cingulate cortex (ACC), nucleus accumbens (NAcc), and ventral tegmental area (VTA).5;33;48;150 Cognitive (blue) interpretation of nociceptive information is mediated by the ACC, prefrontal cortex (PFC), and NAcc. Together, these structures contribute to pain processing by integrating information about its sensory, cognitive, and affective/motivational components. The activity of ascending pathways is in turn regulated by descending facilitatory and inhibitory systems, which send projections down to the spinal cord mainly from the ACC or periaqueductal gray (PAG) by way of serotonergic neurons of the nucleus raphe magnus in the rostroventral medulla (MED) or by noradrenergic neurons in the locus coeruleus to modulate excitability of dorsal horn neurons.176 ACC, anterior cingulate cortex.

3. Influence of sex on evoked and spontaneous nocifensive behaviors

Several rodent models of chronic pain states exhibit sex differences that parallel findings in many human disorders, with greater sensitivity to nociceptive stimuli in females (Table 1). For example, tactile allodynia is more pronounced and/or of longer duration in female rodents in nerve injury paradigms of chronic constriction injury (CCI)226,227 (but see also216), partial sciatic nerve ligation (pSNL),51 spinal nerve ligation (SNL),32,210,212 intra-articular lysophosphatidic acid–induced neuropathy,174 and endoneurial injection of functionally active myelin basic protein fragment (84-104).40 Hyperalgesic priming, an age-dependent model of the acute to chronic pain transition characterized by the prolongation of hyperalgesia by repeated nociceptive insults, also exhibits sexual dimorphism. This paradigm elicits increased allodynia and facial grimacing in female compared with male rodents in a dural calcitonin gene–related peptide model of migraine.10 Female mice also experience earlier onset of pain-related functional deficits with systemic lipopolysaccharide (LPS),106 intra-articular (IA) Complete Freund's Adjuvant (CFA)-induced arthritis,46 muscle hyperalgesia,86 and femoral bone cancer, correlating with faster progression of disease.64,121 Both mechanical and cold allodynia are more pronounced in female vs male mice in models of multiple sclerosis (experimental autoimmune encephalitis)185 and of complex regional pain ayndrome (CRPS).213

Table 1 - Rodent models exhibit sex-dependent differences in neuroimmune-mediated pain hypersensitivity.
Sex Model Pain/Nociception Rodent Ref
Females Nerve injury (SNI) Tactile allodynia and withdrawal frequency C57BL/6 mice 24
Nerve injury (SNL) Tactile and cold allodynia C57BL/6, SWR/J mice; Wistar rats 32,210,212
Nerve injury (CCI) Tactile allodynia CD-1 mice 226,227
Nerve injury (L5 radiculopathy) Tactile allodynia Sprague–Dawley, Long–Evans rats 116
Nerve injury (SNT) Tactile allodynia Sprague–Dawley rats 57
Nerve injury (pSNL) Tactile allodynia Sprague–Dawley rats 51
Systemic bacteremia (IP LPS) Grip force SWR/J mice 106
Spinal TRL4 activation (IT LPS) Grip force SWR/J mice 106
Serum-transfer arthritis (K/BxN) Grip force C57BL/6 mice 22
Complete Freund's adjuvant (CFA) Tactile allodynia Lewis rats 46
Fatigue-enhanced muscle insult Muscle hyperalgesia C57BL/6 mice 86
Joint neuropathy (IA LPA) Tactile allodynia Wistar rats 174
Femoral bone cancer Limb use Balb/c mice 64
Complex regional pain syndrome (CRPS) Tactile allodynia C57BL/6 mice 213
Migraine (IC CGRP) Tactile allodynia, grimace CD-1 mice; Sprague–Dawley rats 10
Multiple sclerosis (EAE) Tactile and cold allodynia C57BL/6 mice 185
Autoimmune demyelination Tactile allodynia C57BL/6 mice 40
Males Complete Freund's adjuvant (CFA) Tactile allodynia Sprague–Dawley rats 26
Nerve injury (SNI) Tactile allodynia CD-1, C57BL/6 mice 97,205,206
Context-dependent pain hypersensitivity Tactile allodynia CD-1 mice 145,207
Osteoarthritis (DMM) Tactile allodynia CD-1, C57BL/6 mice 139
Spinal TLR4 activation (IT LPS) Tactile allodynia CD-1, C57BL/6 mice 205,206,243
Serum-transfer arthritis (K/BxN) Tactile allodynia C57BL/6J mice 242
CCI, chronic constriction injury; IC CGRP, intracisternal calcitonin gene-related peptide; CFA, Complete Freund's Adjuvant; DMM, destabilization of the medial meniscus; EAE, experimental autoimmune encephalitis; IA LPA, intra-articular lysophosphatidic acid; LPS, lipopolysaccharide; pSNL, partial sciatic nerve ligation; SNT, spinal nerve transection; SNI, spared nerve injury; SNL, spinal nerve ligation.

By contrast, some studies report greater expression of pain hypersensitivity in males. For example, the development of allodynia after intrathecal (IT) LPS or the initiation of arthritis (K/B × N serum transfer-induced or intraplantar IPLT CFA-induced) is more pronounced in male mice,26,205,206,242,243 yet also see Ref. 22. Similarly, males also exhibit increased allodynia vs females in the spared nerve injury (SNI) model,97,205,206 yet also see Ref. 24). The discrepancies in magnitude of allodynia reported by these studies may be explained in part by different strains of rodents or paradigms used. Evidence of sex differences also is emerging both in mice and in humans for the expression of cued pain-related fear memory mediated by limbic, mesolimbic, and cortical circuits.11,153 For example, context-dependent pain hypersensitivity is increased in males relative to females when tested by a male experimenter or when examined in an environment previously associated with an aversive tonic pain experience.145,207

In other injury paradigms, male and female rodents develop equivalent severity of evoked or spontaneous pain-like behaviors. Studies using collagen antibody–induced arthritis,67 IA CFA-induced arthritis,66 chemotherapy-induced peripheral neuropathy,69 adult reincision after neonatal paw incision,165 and intraplantar (IPLT) formalin243 models all report allodynia of similar magnitude in males and females. Interestingly, despite sex differences observed in allodynia during arthritis or after IT LPS, both male and female mice exhibit deficits in grip strength—a widely used rheumatology measure sensitive to analgesics and a frequently reported deficit known to correlate with pain in rheumatoid arthritis.106,164 Arthritis-induced declinations of functional measures such as the locomotor activity and home cage wheel running also are observed in mice of both sexes.75,104 Both males and females exhibit postsurgical or arthritis injury–induced grimace behaviors as well as depressed nesting and burrowing.101,102,209 Likewise, sucrose consumption and social exploration after systemic delivery of low dose LPS are transiently reduced181 or unchanged246 in both sexes. The effects of morphine on conditioned place preference and conditioned place aversion during peripheral inflammation also are not significantly different between males and females.8,92 Despite similar levels of pain-like behaviors in both sexes, it is important to note that the mechanisms underlying these behaviors sometimes differ in males vs females.26,50,89,130,149,178,195 Thus, caution should be exercised when drawing conclusions about positive or negative effects of treatments for pain when males and females are not stratified.158

3.1. Influence of gonadal hormones

Sex differences in nociceptive thresholds and opioid analgesia depend largely on organizational effects of the gonadal hormone status—that is, hormone action during critical periods of gestation. Specifically, neonatal exposure to testosterone seems necessary for the phenotype of decreased nociceptive sensitivity and increased morphine analgesia observed in adult males relative to females.23,43,114,117 Nonetheless, the presence of testosterone also can exert pronociceptive actions.205 The acute, or activational, effects of estrogens on pain and analgesia are decidedly more complex. Activational effects can vary according to the type, level, stability, and route of administration of estrogens, whether they are administered alone or in combination with progestins, as well as the nociceptive paradigm used and the chronicity of pain state. Particular caution should be exercised in the interpretation of studies in which supraphysiological doses of these hormones are administered.52,53

For example, systemic administration of estradiol decreases nociceptive behaviors in the second phase (10-60 minutes postinjection) of IPLT formalin-induced acute pain in gonadectomized male or female rats81,115,140 and in nerve-injured intact mice.227 By contrast, some chronic pain states that emerge days to weeks after injury or inflammation may be exacerbated by estrogens25,46 or are unaffected by hormones.12 Mu opioid receptor–mediated analgesia in cycling females also depends on the phase of the estrus cycle, as morphine potency is greatest in metestrus, diestrus, and proestrus phases but is lowest during estrus.107,219 The intricate effects of estrogens are perhaps best illustrated by the observations that estradiol suppresses the induction yet facilitates the expression of hyperalgesic priming.68,70,103 By contrast, progesterone seems mainly to serve a protective function, in that it mediates pregnancy-related analgesia192 and attenuates hyperalgesia precipitated by IPLT CFA-induced or carrageenan-induced monoarthritis,189,214 excitotoxic spinal cord injury,84 and peripheral diabetic neuropathy.126 For in-depth discussions of these processes, the reader is directed to several extensive reviews.20,47,52,105,147,158,234

3.2. Influence of stress pathways

Similarly, stress also exerts paradoxical analgesic and hyperalgesic effects that are sexually dimorphic and likely are mediated by estradiol and testosterone99 as well as stress hormones of the hypothalamic pituitary adrenal axis such as corticotrophin releasing factor, adrenocorticotropic hormone, and glucocorticoids.80,132 Interestingly, antisense knockdown of spinal β2 adrenergic receptors attenuates chemotherapy-induced peripheral neuropathy in female but not male rats, whereas the inverse is observed after knockdown of spinal glucocorticoid receptors.69 After early life stress, female rats exhibit increased central amygdala corticotrophin releasing factor–mediated visceral pain hypersensitivity184 and augmented expression of hippocampal tumor necrosis factor alpha (TNFα) and IL-6 concomitant with greater SNL-induced allodynia.32 These findings indicate sex-specific dependence on stress mediators of the sympathetic nervous system and the hypothalamic pituitary adrenal axis in addition to gonadal hormones.

3.3. Influence of genetics

In a rodent model of lumbar L5 radiculopathy, female Sprague–Dawley and Long–Evans but not Holtzman rats developed more severe mechanical allodynia than their male counterparts.116 These findings are corroborated by the observation that L5 spinal nerve transection (SNT) produced greater allodynia in female vs male Sprague–Dawley rats, but no significant sex difference in Holtzman rats.57 Swim stress–induced analgesia is greater in female Wistar and spontaneously hypertensive but not Lewis rats.230 By contrast, stress-induced analgesia is enhanced in male C57BL/6 and Swiss Webster mice compared with isogenic females.160 Similarly, morphine antinociception also is greater in several strains of male rats and mice, as reviewed in depth.161 Although allodynia is expressed in both sexes of CD-1 mice, it is evident in male but not female C57BL/6 mice in the destabilization of the medial meniscus (DMM) model of knee osteoarthritis.139 Of note, substrain differences of C57BL/6J vs C57BL/6N mice in nociceptive behaviors are found after IPLT formalin, but not with IPLT CFA or CCI models.28 Quantitative trait locus mapping in a cross of these strains uncovered a difference between B6J vs B6N, with thermal nociception being more pronounced in males. These observations indicate that rodent strain also should be considered when drawing conclusions about sex differences in pain hypersensitivity.

4. Sex-dependent neuroimmune mechanisms of pain hypersensitivity

Tissue damage or infection initiates an immune response that can ultimately lead to a chronic pain state. Acute inflammation serves a dual purpose in that a wide variety of mediators are secreted to prevent the organism from incurring further injury and to recruit peripheral immune cells for containing and repairing the damage. Historically, these factors have been characterized as either maladaptive “proinflammatory” or beneficial “proresolving,” and are released sequentially to promote active healing. Our current understanding of pathogen-associated or damage-associated molecular pattern–induced inflammatory responses may best be described as an organized progression of interactions between immune cells. Accordingly, the secretion of factors by each cell type influences the timing and destination of chemotaxis by another cell type.19,29,82 Under typical circumstances, pain remains acute as the injury is repaired and inflammation is resolved, allowing the organism to resume homeostasis.

It is widely recognized that infiltrating as well as resident immune cells likely contribute to the transition from acute to chronic pain in instances where either the damage cannot be repaired or dysregulated inflammatory signaling continues even after the injury is resolved (see Fig. 3 for definitions of immune cell types).13 For example, infiltrating neutrophils, macrophages, and T-lymphocytes as well as activated Schwann cells and satellite cells secrete factors to communicate with resident astrocytes, microglia, and oligodendrocytes in the CNS to release mediators that sensitize nociceptors. These processes in turn trigger adjacent glia and neurons to drive maintenance of hyperalgesia and allodynia.31,98,148,155,198,200,231,247 Among the molecules contributing to central sensitization are neurotransmitters (glutamate and ATP), peptide signals (cytokines, chemokines, and neuropeptides), and bioactive lipids generated from cyclooxygenases (COX-1/2), 12/15-lipoxygenases (12/15-LOX), and endocannabinoid system enzymes.87,88,108,138,236,237,241

Figure 3.
Figure 3.:
Immune and glial cells linked to sex differences in pain hypersensitivity. Neuroimmune cells are generally derived from 4 different progenitor stem cell types (blue circles): N, neuroepithelial112; D, mesodermal72; M, myeloid7; L, lymphoid.6 Neuroepithelial cells can differentiate into astrocytes,203,204 oligodendrocytes,190 or Schwann cells.163 Mesodermal cells can be programmed into endothelial cells.144 Myeloid-derived cells include microglia,128 macrophages,123 neutrophils,172 or mast cells113 and are referred to as splenocytes127 (circled in purple). Lymphoid cells differentiate into B-cells4 and T-cells,65 and collectively these cells are referred to as lymphocytes (circled in green).

Emerging evidence supports a profound role for sex-specific immune responses that may underlie disparities in incidence of pain and other neurological disorders.18,60,62,141,186,191,208 Because of specific challenges unique to each sex, the male and female immune systems have different requirements, with the female immune system specifically requiring the flexibility to allow for pregnancy without attacking the fetus or sperm required for procreation.187 Consequently, females have larger populations of most immune cells, higher levels of immunoglobulins, and exhibit stronger responses to infection.110,168 Conversely in males, the Y chromosome contains multiple genes involved in epigenetic regulation of the immune system and susceptibility to autoimmune diseases.37 Although some neuroimmune interactions underlie nociceptive processing in both males and females, some pain states exhibit clear sex-specific mechanisms that likely affect their responsiveness to current analgesics and adjuvant therapeutics.9

4.1. Male-specific nociceptive mechanisms

Chronification of pain states in males is believed to be facilitated largely by the innate immune system through neutrophil recruitment to the injury site197 and to the spinal vasculature,156 along with CNS infiltration of monocytes and activation of microglial-neuronal crosstalk by several mechanisms179 (Table 2). Although significant spinal microgliosis is evident in both sexes of rodents after injury,3,38,206,242 allodynia in males is believed to be mediated by several mechanisms including, but not limited to: stimulation of purinergic P2X4 receptors142,224 likely on CX3CR1- (Fractalkine receptor)-positive microglia,224,248 phosphorylation of P38 mitogen-activated protein kinase,100,137,165,216 and release of cytokines such as brain-derived neurotrophic factor (BDNF) either from spinal microglia206 or dorsal root ganglion (DRG) nociceptors,166 acting on tropomysin receptor kinase B receptors in spinal dorsal horn neurons. In a model of pain chronification, hyperalgesic priming with IT BDNF or IPLT interleukin 6 (IL-6) is mediated by activation of spinal dopamine D5 receptors (DRD5) in male but not female spinal neurons.149 Furthermore, the NOD-like receptor 3 (NLRP3) inflammasome drives IL-1β release likely from non-neuronal cells, leading to subsequent activation of neuronal transient receptor potential ankyrin 1 (TRPA1) in males but not females in a postoperative pain model of paw incision.50 Likewise, in CCI or SNT paradigms of neuropathic pain, the cytokine TNFα mediates allodynia by spinal TNF receptor 1 (TNFR1) alone in male mice despite similar expression of allodynia in both sexes.56,211 TNFα and IL-1β are unchanged supraspinally in anterior cingulate cortex (ACC) after common peroneal nerve injury, indicating potential local release and site-specific involvement of these mediators. However, these studies were performed in a mixed-sex cohort of mice.135

Table 2 - Sex-dependent cellular and molecular neuroimmune mechanisms driving pain hypersensitivity.
Sex Model Cell Type(s) Mediator Rodent Ref
Females Nerve injury (CCI) Schwann cells MHC-II activation of helper Th-cells 129/FVB mice 91
Microglia, astrocytes Phospho-P38 MAPK in the spinal cord CD-1 mice 226,227
Nerve injury (SNI) T-cells CD4+ and CD8+ T-cell infiltration into lumbar spinal dorsal horn CD-1 mice 206
Nerve injury (SNL) Mast cells Increased mast cell infiltration into lumbar spinal dorsal horn SWR/J mice 212
Nerve injury (pSNL) T-cells Not specified; T-cell infiltration into DRG Sprague–Dawley rats 49,133
Nerve injury (L5 radiculopathy) Not specified Spinal NRG1, ErbB4, and TAC1; progesterone Sprague–Dawley rats 118,119
Femoral bone cancer Microglia; not specified TLR4, CD11b, CD14; not specified Balb/c mice; Sprague–Dawley rats 64,121
Migraine (IC nitroglycerin) Mast cells, endothelial cells P2X3 Sprague–Dawley rats 71
Migraine (IC CGRP with priming) Not specified RAMP1, CLR, RCP ICR mice; Sprague Dawley rats 10
Fatigue-enhanced muscle insult Lymphocytes Lymphocyte migration to muscle C57BL/6 mice 86
Autoimmune demyelination (IA MBP) T-cells T-cell migration into DRG and spinal cord; PLC in females C57BL/6 mice 40
Males Nerve injury (SNT) Microglia CD40+ spinal microglia interaction with infiltrating CD4+ T-cells Balb/c mice 34,36
Nerve injury (SNI) T-cells CD2+ cell migration into ipsilateral spinal dorsal horn; Th1-mediated Sprague Dawley rats 49
CD4+/CD8+ cell infiltration into ipsilateral spinal cord C57BL/6 mice 44
Neurons TRPV1 C57BL/6 mice 44
Microglia, macrophages TLR4, P2X4, BDNF, Phospho-P38 MAPK CD-1, C57BL/6 mice; Sprague Dawley rats 142,205,206
Nerve injury (SNL) Microglia, neurons P2X4, Phospho-P38 MAPK Sprague Dawley, Wistar rats 100,224
Oral cancer Neutrophils Ly6G+ neutrophil migration to tumor C57BL6 mice 197
IPLT carrageenan Neutrophils; not specified S100A8+ and S100A9+ neutrophil migration to spinal vasculature; gonadal hormones Sprague Dawley, Fischer 344 FBNF1 rats 156,214
Spinal TLR4 activation (IT dsHMGB1) Microglia, macrophages Spinal TNF, IL-1b, CCL2, CxCl1, CxCl2, gfap, CD11b, females recover faster C57BL/6 mice 2,3
Spinal TLR4 activation (IT LPS) Not specified Spinal TLR4 CD-1, C3H/HEK, C3H/HeN, C57BL/6J, C57BL/10ScNJ, C57BL/10ScSnj mice 205,243
Serum-transfer arthritis (K/BxN) Glia Spinal TLR4 and TNFa, spinal TRPV1 C57BL/6 mice 22,242
Chemotherapy-induced peripheral neuropathy Not specified Spinal TLR4, females recover faster C57BL/6 mice 240
BDNF, brain-derived neurotrophic factor; CCI, chronic constriction injury; IC CGRP, intracisternal calcitonin gene-related peptide; DRG, dorsal root ganglion; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; IA MBP, intra-articular myelin basic protein; pSNL, partial sciatic nerve ligation; SNT, spinal nerve transection; SNI, spared nerve injury; SNL, spinal nerve ligation.

Thus, it is important to consider that male-specific involvement of macrophages and neuroimmune mediators in allodynia is likely dependent on the paradigm used, the activation of specific circuits (spinal vs supraspinal), hormone status, or be influenced by other factors such as age94,95,129,134 and strain.152,202 Injury-induced activation of toll-like receptor 4 (TLR4) is a prominent example of this controversy. IT delivery of LPS or endogenous ligands (eg, high mobility group box 1, HMGB1) and models of peripheral neuropathy or rheumatoid arthritis elicit spinal TLR4-dependent allodynia that in some, but not all, cases is more pronounced in males than in females.2,195,205,206,240,242,243 The observed reduction in responsivity of females to spinal TLR4 activation seems to be dependent on estrogen, as ovariectomy in conjunction with testosterone replacement restores expression of TLR4-mediated allodynia in CD-1 female mice to levels comparable with that observed in intact males.205 Estrogen also attenuates LPS-induced inflammatory signaling and prevents expression of the proinflammatory phenotype of microglia during development.229,232,233 Interestingly, the sex difference observed in spinal TLR4-mediated allodynia is absent when LPS is administered either at supraspinal (intracerebroventricular) or peripheral (IPLT) sites in uninjured CD-1 mice,205 or intramuscularly in a model of noninflammatory acidic saline-induced muscle hyperalgesia.83 In addition, systemic delivery of LPS produces pain hypersensitivity in both male and female Sprague–Dawley rats as neonates and as adults,21 correlating with decreased expression of Oprm1 encoding Mu opioid receptor in the periaqueductal gray (PAG)246 and IL-1β mRNA in the spinal cord, ventrolateral PAG, and hippocampus.182 These observations are consistent with the finding that intra-PAG LPS in rats significantly decreases morphine antinociception in both sexes.61 Similarly, in both sexes of C57BL/6 mice, IPLT formalin–induced delayed tactile hypersensitivity is prevented by global deletion of TLR4,243 and spinal blockade of HMGB1 reverses collagen antibody-induced arthritis–induced mechanical allodynia.2

Furthermore, in some paradigms, crosstalk between macrophages and sensory neurons contributes to allodynia in both sexes (Table 3). For example, IPLT angiotensin II activates its receptors (AT2R) in peripheral Iba1(+) leukocytes, leading to TRPA1 transactivation in nociceptors concurrent with pain hypersensitivity in males and females.199 IT delivery of BDNF elicits allodynia in both sexes of CD-1 mice,143 whereas IL-6 contributes to enhanced hyperalgesia in males and females after muscle injury83 and peripheral inflammation.83,217 In a model of hyperalgesic priming for migraine, intracisternal (IC) IL-6-induced dural inflammation is BDNF-dependent in both male and female Sprague–Dawley rats.30 K/B × N arthritis elicits time-dependent increases in spinal and circulating TNFα in males and females, and IPLT delivery of TNFα produces spinal transient receptor potential vanilloid 1 (TRPV1)-dependent allodynia in both sexes.22,66

Table 3 - Sex-independent cellular and molecular neuroimmune mechanisms driving pain hypersensitivity.
Model Cell Type(s) Mediator Rodent Ref
Nerve injury (SNT) Spinal glia Spinal soluble TNF and IL-1b Holtzman rats 211
Not specified IL-6; allodynia F = M; autotomy behavior F > M C57BL/6 x 129/BV/SV mice 245
Microglia CXCR1+ microglia C57BL/6 mice 179
C40+ spinal microglia interaction with infiltrating CD4+ T-cells Balb/c mice 34,36
T-cells Cd4+ T-cell infiltration into ipsilateral spinal cord Balb/c mice 35
Nerve injury (CCI) Microglia Phospho-P38 MAPK in males CD-1 mice; Sprague–Dawley rats 216
CXCR1+ microglia CD-1 mice; Sprague–Dawley rats 216,248
P2X4+ microglia Sprague–Dawley rats 142
TNFR1-induced NMDA1 activation in the spinal cord, cortex C57BL/6 mice 56
Macrophages CD68, Cd11b+ macrophages; tactile allodynia F = M C57BL/6 mice 44
T-cells TNFR2 essential for recovery C57BL/6 mice 76
Nerve injury (SNI) Microglia P2X7 Sprague–Dawley rats 54
Nerve injury (pSNL) Not specified P2X7 C57BL/6 mice 41
Postoperative pain DRG neurons PRLR in females C57BL/6.129 mice; Sprague–Dawley rats 178
Microglia Phospho-P38 MAPK in males Sprague–II–Dawley rats 165
Macrophage, mast cells, neutrophils, NLRP3-dependent IL-1b release in DRG and peri-incisional skin in males; NLRP3-independent in females C57BL/6j, C57BL/6N/129 mice 50
Mast cells Mrgprb2 C57BL/6 mice 85
Complex regional pain syndrome (CRPS) Microglia (males), T-cells (females), B-cells (both) Spinal IL-6, NK1 in males; delayed adaptive immune response in females C57BL/6J mice 89
IPLT Zymosan Not specified TLR2 CD-1 mice 205
IPLT CFA Neurons High-affinity tonic GABAA current in PAG Sprague–Dawley rats 222
Not specified Spinal TLR4, serum, TNFa, IL-1b, and IL-6 in males CD-1 mice; Sprague–Dawley rats 26,205
IPLT formalin Glia, neurons (males); T-cells (females) DRD1, DRD3 in females; DRD4 in males; BDNF, TLR4 in both sexes CD-1, C57BL/6J, C57BL/6/129.P2 mice 130,149,193,202,243
Muscle hyperalgesia Macrophages P2X4+ resident macrophages, TLR4, IL-6 C57BL/6 mice 83
IPLT TNFa Not specified Peripheral TRPA1, central TRPV1; mixed males and females C57BL/6J.129.SvJ, CD-1 mice 66
IPLT angiogensin II Macrophages, neurons AT2R in skin macrophages transactivates TRPA1 in neurons C57BL/6J, FVB/NJ mice 199
Hyperalgesic priming Neurons Spinal DRD5 in males; DRG PRLR in females C57BL/6J.129, C57BL/6J mice 149,178
IT BDNF Microglia Spinal KCC2 downregulation CD-1 mice 143
IT NRG-1 Astrocytes, neurons Spinal ErbB Sprague–Dawley rats 118
IT LPS Not specified Spinal TLR4 SWR/J mice 106
ICV LPS Not specified TLR4 CD-1 mice 205
Intra-PAG LPS Microglia TLR4 Sprague–Dawley rats 61
IPLT LPS Not specified TLR4 CD-1 mice 205
Systemic bacteremia (IP LPS, neonatal) Microglia, not specified Spinal TLR4, spinal COX2; downregulation of Oprm1 in PAG, PFC Sprague–Dawley rats 21,95,246
Migraine (intracisternal IL-6) Not specified BDNF Sprague–Dawley rats 30
IA carrageenan Immune cells (in joint) P2X2 and P2X3 in both sexes; P2X7 in females Wistar rats 217,218
IA CFA Not specified TRPA1, TRPV1, and TNFa CD-1, C57BL/6J mice 66
Collagen antibody-induced arthritis (CAIA) Glia, neurons (spinal); macrophages Macrophage HMGB1/TLR4 in males; nociceptor TLR4 in females Balb/c mice 2,67,195
BDNF, brain-derived neurotrophic factor; CCI, chronic constriction injury; CFA, Complete Freund's Adjuvant; DRG, dorsal root ganglion; ICV, intracerebroventricular; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; PAG, periaqueductal gray; pSNL, partial sciatic nerve ligation; SNT, spinal nerve transection; SNI, spared nerve injury; SNL, spinal nerve ligation.

4.2. Female-specific nociceptive mechanisms

Sustained allodynia in females is believed to derive in part from the adaptive immune system by activation and infiltration of cluster of differentiation 4 (CD4)+ T-lymphocytes to either central206 or peripheral sites after nerve injury91,133 (Table 2). Interestingly, intrasciatic (IS) injection of myelin basic protein (84-104) elicits T-cell migration to the DRG and spinal cord concurrent with tactile allodynia in female but not in male mice, in which T cells remain localized to the sciatic nerve.40 Voluntary wheel running attenuates experimental autoimmune encephalitis-induced allodynia, correlating with reduced release of inflammatory cytokines from myelin-reactive T cells and attenuated DRG neuron excitability in female but not in male mice.154 However, a female-specific role of the adaptive immune system remains to be clarified and may be paradigm-dependent or strain-dependent. Several investigators have demonstrated that infiltrating CD4+ T-cells also contribute to tactile hypersensitivity after SNT or SNI in male Balb/c or C57BL/6 mice and Sprague–Dawley rats, respectively.34–36,44,49 CD4+ T-cells mediate reduced formalin-mediated nociceptive sensitivity and increased morphine analgesia in male compared with female CD-1 mice.193 In addition, T-regulatory cells (Tregs) are essential for recovery from CCI-induced tactile allodynia by TNFα Receptor 2 (TNFR2) in both sexes.76

Alternatively, other immune cells may be involved in female-specific mechanisms of neuropathic pain because the number of mast cells is increased in lumbar spinal dura mater during intradermal (ID) capsaicin–induced or IPLT carrageenan–induced inflammation244 as well as on the side of the thalamus receiving nociceptive input following SNL,212 concurrent with allodynia in female but not in male rodents. Mast cells also mediate ID nitroglycerin–induced hyperalgesia, which is more pronounced in female rats.71 However, paw incision–induced or CFA-induced activation of the mast cell receptor mas-related G protein–coupled receptor b2 (Mrgprb2) elicits inflammation and pain hypersensitivity that is not different between male and female mice,85 so a female-specific involvement of mast cells may depend on the pain model used.

Microglia are believed to drive allodynia predominantly in males, yet several reports suggest that females can switch to a microglia-dependent pathway in some models of pain hypersensitivity when adaptive immune mechanisms are suppressed.89,205,206 For example, microglial P2X7 is activated in females during IA carrageenan–induced or collagen–induced arthritis (CIA)173,218 yet not after IPLT CFA monoarthritis or partial SNL-induced or SNI-induced nerve injury.41,54 Female-specific progesterone-dependent upregulation of neuregulin-1 (NRG-1) in astrocytes has been observed in a model of experimental L5 lumbar radiculopathy, whereas exogenous spinal delivery of NRG-1 produces allodynia in both sexes.118,119 Mice heterozygous for NRG-1 express sex-specific reductions in serum cytokines in conjunction with increased hotplate latency, including IL-6, IL-8, and IL-10 in females and IL-1β in males.58 However, IL-6 may also exert a female-specific protective effect, as female IL-6 deficient mice experience increased autotomy behavior after nerve injury.245 Other mechanisms of nociception in females include inflammation-induced activation of CNS DRD3130 or DRD1 receptors,149 gamma aminobutyric acid receptor subtype A (GABAA) in the PAG222 or spinal cord,78 and prolactin receptors (PRLR) in sensory neurons.39,178

5. Future directions

Chronic pain affects up to 33% of the population and surpasses cancer, diabetes, and heart disease in terms of societal burden.59 Management of persistent pain is largely an exercise of trial and error, and the scarcity of viable treatment options places undue burden on the patient.183 There is a considerable body of evidence demonstrating that an interaction between the nervous and immune systems underlies many pain syndromes at the molecular and cellular level. Because immune cells play a major role in the development of mood disorders,151 it is likely that they also may contribute to the averse emotional experience of pain. Most clinically relevant pain states have a tonic component that is not captured by standard-evoked paradigms, so continued incorporation of spontaneous and functional measures of pain behaviors in preclinical studies will be critical for a deeper understanding of sex-related differences in chronic pain states and the future development of analgesics.109,169,171,215 Taken together, studies suggest that neuroimmune signaling events altered by injury, disease, or aberrant central nociceptive processing may serve as a rich resource of novel druggable targets and of predictive biomarkers suitable for patient stratification in trials55 examining efficacy of potential pain therapeutics.

Given the challenges of developing safe and effective drugs reaching the CNS,201 one potential therapeutic avenue is to neutralize release of cytokines and chemokines released from circulating leukocytes and/or to intercept these cells before they cross the blood–brain barrier. Accordingly, the development of monoclonal antibody–based interventions targeting immune system mediators for the treatment of cancer and autoimmune diseases has grown exponentially over the past decade.136 As our understanding of the role of specific immune cells in the development and maintenance of central sensitization continues to evolve, some of the newer-generation FDA-approved biologics may be repurposed for treating chronic pain either of peripheral origin by systemic delivery, or by routes of administration that bypass the blood–brain barrier (eg, intranasal or intrathecal). Alternatively, humanized single-domain antibodies could be harnessed as therapeutics, diagnostic agents, or as delivery devices of small molecules targeting specific leukocytes because of their stability, small size, and low production cost.16 Nonetheless, small molecules still lead the field in percent of FDA approvals despite challenges encountered with safety and tolerability profiles.167 Ultimately, it is imperative for more preclinical and clinical pain studies to draw direct comparisons between males and females. Including sex as a biological variable will allow experts both to better predict which therapeutic strategies may be effective in each sex and to achieve true progress in the discovery of novel nonopioid analgesics.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Supplemental video content

A video abstract associated with this article can be found at http://links.lww.com/PAIN/B286.

Acknowledgments

This work was supported by NIH AR075241 (A.M.G.); Cayman Biomedical Research Institute (CABRI) Undergraduate Research Award (I.S.L.); NIH NS099338 and DA015353 (T.L.Y.); NIH DA035865 (M.W.B.).

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              Keywords:

              Dimorphism; Hyperalgesia; Leukocyte; Glia; Nociceptor; Cytokine

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