Sex differences in pain along the neuraxis

Abstract

Despite the overwhelming female-predominance in chronic pain disorders, preclinical pain studies have historically excluded females as research subjects. Male-biased explanations of pathological pain mechanisms may not fully translate to pain processes in females, necessitating the exploration of pain processing and modulation in both sexes at the preclinical and clinical levels. This review highlights historical trends in the study of sex differences within the pain field and examines the current literature regarding new techniques for the mechanistic analysis of pain modulation in males and females. A large body of evidence suggests that sex differences exist at the molecular, cellular, and systems levels of pain processing, likely influenced by a combination of genetic, hormonal, and neuroimmune factors that may differ at distinct levels of the neuraxis.

1. Introduction

Chronic pain is a pervasive health care issue that impacts over 20% of the global population each year (1–3). Sex differences in pain have become an increasingly important topic of research in recent years. Females greatly outnumber males as chronic pain patients (4), yet female animals have historically been underrepresented as preclinical research subjects (5). This is usually attributed to concerns regarding potential confounding effects of the female estrous cycle on experimental results. However, a recent-meta analysis showed that female mice tested throughout the hormonal cycle do not show any greater variability than males, weakening the argument against their inclusion in biomedical research protocols (6). Subsequently, calls for the National Institutes of Health (NIH) to require consideration of sex as a biological variable in preclinical experiments resulted in the requirement of sex and gender inclusion plans in preclinical research (7). Though some changes have been applied to preclinical practices (8), other reports have indicated that the prevalence of male-only rodent studies remains largely unchanged in behavioral research disciplines (9). Thus, a more widespread inclusion of female subjects in biomedical research, particularly in differentially impacted areas such as chronic pain, is warranted.

Together, the interactions of sensory, cognitive, and emotional-affective dimensions form the highly complex and intense experience of pain. This intricate interplay presents a challenge to identifying effective treatment strategies, as therapeutic options are limited and often associated with variable efficacy and side effects (10,11). Difficulties in finding effective solutions arise in part from a lack of full understanding of the mechanisms and targets involved in chronic pain development. As sex differences in response to pain interventions have been widely described (12), it is reasonable to infer that sex-specific pain mechanisms contribute to the differential effectiveness of treatments in males and females. Attempts to identify the biological basis of sexually dimorphic pain behaviors and therapeutic efficacies have been investigated across pain subdisciplines. Sex differences in preclinical pain mechanisms have been thoroughly reviewed by others (13–19), with many groups emphasizing the concept of complex neuroimmune interactions that contribute to differences in synaptic modulation and neuroplasticity (20–24). The aim of this review is to highlight historical evidence for preclinical sex differences in pain processing at all levels of the neuraxis and to provide an update regarding up-and-coming techniques that are being implemented in the exploration of sexually dimorphic pain mechanisms across the field. All preclinical references in this review were collected from Pubmed. Sex-specific pain behavior and implicated cellular and molecular factors are shown in Tables 1 and 2.

Table 1.

Sex-specific pain-related behaviors

Pain model	Behavior	Sex difference	Ref.
CCI	Mechanical allodynia, slow recovery	F-predominant	64,65
CFA arthritis	Thermal hypersensitivity	F-predominant	66
CIBP	Early pain emergence	F-predominant	71
CIPN	Cold allodynia, reduced voluntary wheel running	F-predominant	62,63
CPIP	Mechanical allodynia	F-predominant	70
EAE	Mechanical and cold allodynia	F-predominant	69
HIV-1- associated neuropathic pain	Mechanical and cold allodynia	F-predominant	61
K/C arthritis	Anxiety-like behavior	F-predominant	73
MIA osteoarthritis	Mechanical allodynia, susceptibility to persistent pain	F-predominant	67,68
MMT	Mechanical allodynia, hindlimb weight bearing deficit	M-predominant	72
Sciatic nerve constriction	Anxiodepressive-like behavior	M-predominant	76
SNI	Cognitive deficits	M-predominant	78
SNL	Affective responses, anxiety-like behavior	F-predominant	73
SNT	Cognitive deficits	M-predominant	77
Varicella zoster pain	Affective responses	F-predominant	74

CCI, chronic constriction injury; CFA, Complete Freund’s Adjuvant; CIPB, cancer-induced bone pain; CIPN, chemotherapy-induced peripheral neuropathy; CPIP, chronic post-ischemia pain; EAE, experimental autoimmune encephalomyelitis; HIV, human immunodeficiency virus; K/C, kaolin/carrageenan; MIA, monoiodoacetate; MMT, medial meniscus transection; SNI, spared nerve injury; SNL, spinal nerve ligation; SNT, spinal nerve transection. M- and F-specific indicate male- and female-predominant sex differences.

Table 2.

Cellular and molecular factors implicated in sex-specific pain processing

Region		Factor	Pain model	Outcome measure	Sex difference	Ref.
Periphery		α1-adrenoceptor	CCI	Behavior	F-predominant	118
		α2-adrenoceptor	CCI	Behavior	M-predominant	118
		β-endorphin	MMT	ELISA	F-predominant	72
		BDNF	IL-6 hyperalgesic priming	Behavior	M-predominant	113
		CCL5	CIPN	Behavior	M-predominant	165
		COX-2	CFA arthritis	Behavior	M-predominant	163
		Dopamine D3 receptor	KO	Behavior	F-predominant	119
		GIRK	KO	Behavior	M-predominant	107,108
		Glucocorticoid receptor	CIPN	Behavior	M-predominant	116
		Glutathione peroxidase	CCI	Behavior	F-predominant	109
		HCAR2	CCCI	Behavior	F-predominant	117
		HDAC2	SSRI-induced hypersensitivity	Behavior	F-predominant	122
		HMGB1	Collagen antibody-induced arthritis	Behavior	M-predominant	167
		IFN-ɣ	Tibia fracture	Behavior	F-predominant	158
		IL-1β	Tibia fracture, CCI	Behavior	F-predominant	158,159
		IL-6	CIPN	Behavior	M-predominant	165
		IL-10	Tibia fracture, CCI	Behavior	F-predominant	158,159
		MBP(84–104)	MBP(84–104)-induced hypersensitivity	RNA-Seq	F-predominant	201
		Melanocortin-1 receptor	κ-opioid analgesia (acute)	Behavior	F-predominant	93,94
		mGluR2	SSRI-induced hypersensitivity	Behavior	F-predominant	122
		Myelin-reactive T cells	EAE	Behavior, Ca2+ imaging	F-predominant	185
		Nav1.8 channel	LPA-induced joint neuropathy	Electrophysiology	F-predominant	186
		NMDA receptor	Swim stress-induced analgesia, κ-opioid analgesia (acute)	Behavior	M-predominant	88–91
		NMDA receptor	κ-opioid analgesia (chronic)	Behavior	F-predominant	92
		PKCε	Streptozocin-induced diabetic peripheral neuropathy	Behavior	M-predominant	110
		PKCδ	Streptozocin-induced diabetic peripheral neuropathy	Behavior	F-predominant	110
		Prolactin receptor	IL-6 hyperalgesic priming, κ-opioid analgesia (acute)	Behavior	F-predominant	114,115
		PTGDS	-	RNA-Seq	F-predominant	200
		Ryanodine receptor	Ryanodine-induced priming	Behavior, Ca2+ imaging	F-predominant	111,112
		Serotonin 5HT1B receptor	Sumatriptan-induced hypersensitivity	Behavior	M-predominant	121
		Serotonin 5HT1D receptor	Sumatriptan-induced hypersensitivity	Behavior	F-predominant	121
		Serotonin 5HT2A receptor	5HT-induced inflammation	Behavior	F-predominant	120
		TLR4	Collagen antibody-induced arthritis	Behavior	M-predominant	167
		TLR9	CIPN	Behavior	M-predominant	166
		VEGF	Tibia fracture	Behavior	F-predominant	158
Spinal Cord		α5-GABAA receptor	SNL, SNI	Behavior	F-predominant	130
		Angiotensin IV	Sciatic nerve ligation	Behavior	M-predominant	125
		Bhlha9	KO	Behavior, Electrophysiology	M-predominant	187
		Caspase6	Formalin test, CCI	Behavior	M-predominant	174
		Cav2.3 channel	Capsaicin-induced hyperalgesia	Behavior	F-predominant	123
		Dopamine D1 receptor	SNI	Behavior	F-predominant	129
		Dopamine D5 receptor	SNI	Behavior	M-predominant	129
		Dynorphin/κ-opioid receptor	κ-opioid analgesia (acute)	Behavior	F-predominant	96,97
		GFAP	EAE	IHC	M-predominant	177
		HMGB1	HMGB1-induced hypersensitivity	Behavior	M-predominant	179
		Iba-1	SNI	IHC	M-predominant	178
		IFN-β	K/BxN arthritis	qPCR	M-predominant	176
		κ-opioid receptor	-	Electron microscopy	F-predominant	95
		LVV-hemorphin 7	Sciatic nerve ligation	Behavior	M-predominant	125
		Melanocortin-1 receptor	κ-opioid analgesia (acute)	Behavior	F-predominant	99
		NMDA receptor	κ-opioid analgesia (acute)	Behavior	M-predominant	99
		Oxytocin	Sciatic nerve ligation	Behavior	M-predominant	125
		P2X4	SNI, CCI, IL-6 hyperalgesic priming	Behavior	M-predominant	170–173
		p38	SNI, CCI, IL-6 hyperalgesic priming	Behavior	M-predominant	170–173
		PI3K	Plantar incision	Behavior	M-predominant	127
		PKCι/λ	Acidic saline-induced allodynia	Behavior	M-predominant	126
		PKMζ	Acidic saline-induced allodynia	Behavior	M-predominant	126
		PPARα	SNI	Behavior	M-predominant	170
		PPARγ	SNI	Behavior	F-predominant	170
		RCP	Noxious meningeal stimulation	Western blot	F-predominant	128
		Substance P	Formalin test	Behavior	F-predominant	124
		TLR4	CFA arthritis, SNI, LPS-induced allodynia	Behavior	M-predominant	168,169
		TNF	K/BxN arthritis	qPCR	M-predominant	176
Brain	Amygdala (BLA)	PKCζ	Plantar incision	Behavior	M-predominant	134
	Amygdala (BLA)	PKMζ	Plantar incision	Behavior	M-predominant	134
	Amygdala (CeA)	CRF	ELS-induced hypersensitivity	Behavior	F-predominant	133
	Amygdala (CeA)	Glucocorticoid receptor	ELS-induced hypersensitivity	Behavior	F-predominant	133
	BNST	Dopamine D1 receptor	Formalin test	Behavior	F-predominant	137
	Cortex	TNFR1	CCI	Behavior	M-predominant	181
	Cortex	TSP2	-	Electrophysiology	M-predominant	189
	Dura mater	CGRP	CGRP-induced hypersensitivity	Behavior	F-predominant	135
	mPFC (PL)	PVINs	SNI	Electrophysiology	M-predominant	188
	mPFC (PL)	SOM	SNI	Electrophysiology	F-predominant	188
	PAG	TLR4	κ-opioid analgesia (acute)	Behavior	F-predominant	180
	vlPAG	GABAA δ receptor	CFA-induced inflammation	Behavior, Electrophysiology	F-predominant	138
	n.s.	NMDA receptor	κ-opioid analgesia (acute)	Behavior	M-predominant	99
	n.s.	Melanocortin-1 receptor	κ-opioid analgesia (acute)	Behavior	F-predominant	99

BDNF, brain-derived neurotrophic factor; bhlha9, basic helix-loop-helix family member A9; BLA, basolateral nucleus of the amygdala; BNST, bed nucleus of the stria terminalis; Cav2.3, R-type voltage-gated calcium channel; CCI, chronic constriction injury; CCL5, chemokine (C-C motif) ligand 5; CeA, central nucleus of the amygdala; CFA, Complete Freund’s Adjuvant; CGRP, calcitonin gene-related peptide; CIPN, chemotherapy-induced peripheral neuropathy; COX-2, cyclooxygenase-2; CRF, corticotropin releasing factor; EAE, experimental autoimmune encephalomyelitis; ELISA, enzyme-linked immunosorbent assay; ELS, early life stress; GFAP, glial fibrillary acidic protein; GIRK, G protein-coupled inwardly-rectifying potassium channel; HDAC2, histone deacetylase 2; HMGB1, high motility group box 1; Iba-1, ionized calcium-binding adapter molecule 1; IFN, interferon; IL, interleukin; KO, knockout; MBP(84–104), 84–104 myelin basic protein fragment; LPA, lysophosphatidic acid; LPS, lipopolysaccharide; mGluR2, metabotropic glutamate receptor 2; MMT, medial meniscus transection; mPFC, medial prefrontal cortex; Nav1.8, voltage-gated sodium channel; NMDA, N-methyl-D-aspartate; P2X4, P2X purinoreceptor 4; p38, p38 mitogen-activated protein kinase; PAG, periaqueductal gray; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PKM, protein kinase M; PL, prelimbic area; PPAR, peroxisome proliferator-activated receptor; PTGDS, prostaglandin-H2 D-isomerase; PVINs, parvalbumin-positive inhibitory neurons; qPCR, quantitative real-time polymerase chain reaction; RCP, receptor component protein (of CGRP); SNI, spared nerve injury; SNL, spinal nerve ligation; SOM, somatostatin-expressing interneurons; SSRI, selective serotonin reuptake inhibitor; TLR, toll-like receptor; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TSP2, thrombospondin 2; VEGF, vascular endothelial growth factor; vlPAG, ventrolateral periaqueductal gray. M- and F-specific indicate male- and female-predominant sex differences. “-“ denotes no pain model was used. “n.s.” denotes no subregion of the brain was examined following intracerebroventricular drug injection.

2. Sex differences in clinical pain conditions

At the systems level, sexual dimorphisms in pain perception have been established in both clinical and preclinical studies. Many reports have found that female patients show increased sensitivity to noxious stimuli. Females have consistently demonstrated lower pain thresholds, decreased pain tolerance, and higher pain perception ratings than male counterparts (4,14,15,25,26). Such findings have been attributed to factors ranging from biological [increased female predominance in the most prevalent chronic pain syndromes (27) and the existence of female-specific pain syndromes (28)] to psychosocial [increased willingness by females to report pain or seek health care services (4,29)] to statistical [female underrepresentation as experimental subjects across biomedical disciplines (30–32)]. Sex differences among pain sensitivities may also have an anatomical or physiological basis, as one study reported a larger volume of pain-related brain structures such as amygdala nuclei in healthy males compared to females (33). Furthermore, neuroimaging studies of pain-evoked brain activity revealed a male-specific deactivation of ventromedial prefrontal cortical control regions but a female-specific recruitment of anterior cingulate cortical emotion-processing areas (34). Sex differences in clinical pain conditions have been well reviewed (28,35–39), and many epidemiological reports have highlighted an increased risk among females for chronic pain conditions such as migraine, arthritis, and low back pain (40–43).

Though there is little doubt that such differences exist, the nature of the underlying mechanisms remains unclear. The most common explanation for sex differences in pain responses lies in the influence of gonadal hormones (37) as they can impact the peripheral and central nervous systems both directly via receptor-based mechanisms and indirectly through neurotransmitter interactions in nociceptive pathways (44–48). Further evidence for hormonal influences is provided by experimental pain studies that found no significant sex differences in young children but more sensitive pain responses in girls versus boys after the onset of puberty (49). Despite this straightforward hypothesis, hormonal effects may not fully encompass the complexity of pain responses between males and females. Genetic mediators of pain may also play a contributory role, as genome-wide association studies of pain have reported sex-specific associations with two chromosomal regions in diabetic neuropathic pain (50) and with single-nucleotide polymorphisms (SNPs) in temporomandibular disorder (51,52).

As the experience of pain is comprised of important cognitive and affective dimensions, social factors likely also influence pain perception differently between males and females (53–57). In contrast to the reports of female-specific increased pain sensitivity, one study found the relationship between chronic low back pain and pain-related anxiety to be stronger in male than female patients (58). Another group reported a positive association between pain intensity and negative affect following total knee arthroplasty among males but not females (59), and male patients referred to a pain rehabilitation clinic reported more mood disturbances than their female counterparts (60).

Taken together, clinical reports suggest that a more comprehensive explanation of the sex differences in pain processing considers a combination of biological and psychological elements. Therefore, preclinical pain studies serve to explore potential biological bases for differential pain responses in humans, as they offer critical insight into underlying mechanisms that may be influenced by not only hormonal but also genetic and molecular factors.

3. Sex differences in preclinical pain-related behavior

Quantitative sex differences at the preclinical level seem to reflect the trend in human studies, with female animals showing greater sensitivity to acute and chronic pain (summarized in Table 1). Female mice had increased mechanical allodynia and increased cold sensitivity relative to males in a model of HIV-1-associated neuropathic pain (61), and females demonstrated increased cold allodynia (62) and shorter distance traveled in voluntary wheel running (63) in a model of chemotherapy-induced peripheral neuropathy (CIPN). In both the graded (64) and regular (65) chronic constriction injury (CCI) models of neuropathic pain, female rodents showed increased allodynia and slower recovery than males. Female rats exhibited increased nociceptive sensitivity following injection of pro-inflammatory Complete Freund’s adjuvant (CFA) into the hindpaw (66), increased mechanical hyperalgesia in the monoiodoacetate (MIA)-induced osteoarthritis model (67), and increased susceptibility to persistent pain development in an MIA-induced model of temporomandibular joint (TMJ) pain (68). Female mice demonstrated increased mechanical and cold hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (69), earlier and more robust mechanical allodynia in a model of complex regional pain syndrome (CRPS) (70), and earlier pain emergence in a model of cancer-induced bone pain (71) when compared to male counterparts. Surprisingly, male rats exhibited more robust mechanical allodynia and larger hindlimb weight bearing deficits compared to female rats in the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (72). Though the literature suggests a female predominance in pain hypersensitivity that may be model-dependent, sex differences in pain-related affective behaviors are more complex and may not follow a similar trend.

Important sex differences have been reported for not only sensory but also affective and cognitive pain behaviors. Female rats exhibited increased emotional-affective responses and anxiety-like behaviors compared to males in the spinal nerve ligation (SNL) model of neuropathic pain, and increased anxiety-like behaviors in the kaolin/carrageenan (K/C) arthritis pain model (73). Female rats also showed longer affective responses in an orofacial model for varicella zoster-associated pain (74). Functional brain activation patterns following noxious visceral stimulation revealed greater activation of the ventromedial prefrontal cortex and limbic areas in female rats but broad cortical activation in male rats, supporting the concept of male-predominant cortical inhibition on limbic structures and female-predominant engagement of affective mechanisms during pain responses (75). Interestingly, one study reported that only male mice developed anxio-depressive-like behavior following sciatic nerve constriction (76), while another found that spinal nerve transection (SNT)-induced neuropathic pain caused similar depressive- and anxiety-like behavior in both sexes but male-specific cognitive deficits (77). Males have also been reported to have increased cognitive deficits compared to females in an attentional set-shifting task following spared nerve injury (SNI) (78).

Taken together, the literature suggests that preclinical data align well with clinical reports of increased pain sensitivity in female subjects, though sex differences in emotional-affective and cognitive responses to pain may not be as clear.

3.1. Opioid-mediated analgesic responses

Periphery.

From the study of baseline sex differences in pain arose the investigation of sex differences in analgesic responses, particularly to opioid drugs. These studies initially focused on analgesic efficacies of predominately κ-opioid receptor agonists, where female patients showed greater analgesic responses than males (79). This finding was later questioned in non-human primates (80), and further preclinical studies found striking evidence for the opposite phenomenon, with opioids producing stronger analgesic effects in male rather than female animals (81–85), though others have reported no sex differences in analgesic effects of morphine for formalin-induced pain (86) or following κ-opioid receptor blockade with nor-binaltorphimine (nor-BNI) in a model of restraint stress to induce latent sensitization (87). While the mechanisms underlying this apparent difference between preclinical and clinical studies remain unidentified, several preclinical studies suggested that sex differences in analgesia could be explained by male-selective N-methyl-D-aspartate (NMDA) receptor-related mediation of κ-opioid analgesia (88–90). One study demonstrated this sex difference only via non-competitive NMDA receptor antagonism (91), and another found that sexually dimorphic NMDA-opioid interactions may differ in acute versus persistent pain (92). Further investigations of κ-opioid analgesic mechanisms revealed involvement of the melanocortin-1 receptor gene for only female animals and humans (93,94).

Spinal Cord.

Sex differences in spinal κ-opioid receptor density and axon terminal distribution have been found (95), and intrathecal morphine administration was shown to recruit a female-specific spinal dynorphin/κ-opioid receptor pathway for antinociception (96), possibly through the formation of a μ-/κ-opioid receptor heterodimer (97). Selective activation of the κ-opioid receptor in the spinal cord produced antinociception in female rats that was estrogen-dependent (98), and NMDA receptor and melanocortin-1 receptor antagonists selectively reversed hyperalgesia in males and female mice, respectively, after both spinal and supraspinal administration (99).

Brain.

Sex differences in the opioid system pain response have been demonstrated in distinct brain regions. Injection of a κ-opioid receptor agonist into the rostral ventromedial medulla (RVM) produced an antinociceptive effect only in female rats, and female rats were found to be more sensitive to a μ-opioid receptor agonist injected into the ventrolateral periaqueductal gray (PAG) (100), though another study found that males experienced higher analgesic responses than females after a microinjection in the same region (101). Interestingly, intracerebroventricular injections of a μ-opioid receptor agonist produced greater antihyperalgesic effects in female rats (102), a finding that is more consistent with clinical studies rather than preclinical data on sex differences in peripheral antinociception. For example, a clinical study found that men experiencing pain showed higher endogenous opioid release and μ-opioid system activation in brain regions that modulate sensory and affective aspects of pain, such as the amygdala, while women in pain showed suppression of μ-opioid mechanisms in the nucleus accumbens, an area that tonically releases endogenous opioids at baseline (103). This could indicate that the central nervous system is the major site of opioid analgesia and therefore may more closely mimic the human condition than peripheral targeting.

It is important to note that the observed sex differences in nociceptive thresholds and opioid analgesia are likely influenced by gonadal hormones. The decreased nociceptive sensitivity and increased morphine analgesia in males may be established by exposure to testosterone during development (104–106), though the role of estrogen on analgesic responses is not as clear and seems to depend on the stage of pain, pain model, and the presence or absence of concurrent progesterone administration. Overall, sex differences in analgesic responses are impacted by an assortment of hormonal and mechanistic factors that may be region-dependent.

3.2. Neuronal signaling

Pain research has traditionally focused on the role of neuronal signaling as a mechanism of pain development and persistence; therefore, a potential explanation for the observed sex differences in pain behavior may implicate differences in underlying neural mechanisms. Some initial studies focused on the role of G protein-coupled inwardly rectifying potassium channels (GIRKs), which are involved in the regulation of synaptic transmission. In GIRK2-null mutant mice, the antinociceptive effects of several drugs were reduced to a greater extent in males rather than females, suggesting that females may recruit additional signal transduction mechanisms (107). The GIRK2-null mutation also reduced pain withdrawal thresholds in male but not female mice (108).

Periphery.

Peripheral nerve fibers are vulnerable to oxidative stress reactions, and neuropathic female but not male rats showed improved antioxidant capacity following exercise (109). Mechanistic studies on the contribution of second messenger signaling to peripheral neuropathy found that inhibitors of protein kinase Cε (PKCε) and protein kinase Cδ (PKCδ) selectively attenuated hyperalgesia in males and females, respectively (110). In a hyperalgesic priming model that activates second messengers downstream of PKCε, peripheral nociceptor plasticity was influenced by a reciprocal interaction between ryanodine (111) and inositol 1,4,5-triphosphate (IP₃) (112) receptors in a female-specific regulatory mechanism. Another hyperalgesic priming study showed that brain derived neurotrophic factor (BDNF) signaling through its cognate receptor TrkB is sexually dimorphic in dorsal root ganglion (DRG) nociceptors (113). Prolactin receptor (PRLR) expression on sensory neurons was shown to be necessary for hyperalgesic priming in female, but not male, mice (114), and long and short prolactin receptor isoforms (PRLR-L and PRLR-S, respectively) were more highly expressed in female than male rat DRGs, where overexpression of PRLR-L reversed opioid-induced hyperalgesia and decreased PRLR-L elicited allodynia selectively in females (115). Peripheral nociceptors may also be influenced by sex differences in their glucocorticoid receptors, as reduced expression of these greatly attenuated hyperalgesia in male but not female rats in a CIPN model (116). The role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) has similarly been investigated due to its upregulation in the sciatic nerve and DRG in neuropathic mice. One study found that the antiallodynic properties of dimethylfumarate (DMF), an agonist of HCAR2, were more pronounced in female compared to male mice in a CCI pain model (117). Sex differences in neurotransmission may also contribute differently to nociceptive behaviors in males and females. Peripheral nerve damage differentially alters descending noradrenergic modulation in male and female rats (118). Absence of the dopamine D3 receptor induced more analgesic behavior in female knockout (KO) mice than their male counterparts (119), and peripheral serotonin evoked stronger and longer lasting pain behaviors in female rats, though effects were dependent on fluctuations in the estrous cycle (120) and may present a caveat to the meta-analysis of trait variability mentioned previously (6). The serotonin receptor (5-HT_1B/D) agonist sumatriptan, traditionally used for migraine relief, induced sex differences in mechanical hyperalgesia after injection into the hindpaw (121). Administration of a selective serotonin reuptake inhibitor (SSRI) in the formalin pain model produced analgesic responses only in female mice, possibly through the reduction of histone deacetylase 2 (HDAC2) expression in the DRG and upregulation of metabotropic glutamate receptor 2 subtype (mGluR2) in the spinal dorsal horn (122).

Spinal Cord.

Sex differences in pain-related neuronal signaling in the spinal cord have also been reported. Voltage gated calcium channels play a critical role in central sensitization, and inhibition of the Cav2.3 channel in the spinal cord produced antinociceptive effects in female but not male mice (123). Spinal release of substance P from primary afferents, measured by internalization of neurokinin 1 receptors in spinal dorsal horn neurons, was greater in female rats than male rats in the formalin pain model (124). Other studies illustrated sex differences in the anti-allodynic effects of peptides such as angiotensin IV, LVV-hemorphin 7, and oxytocin, or in the pro-allodynic effects of molecules such as atypical PKC isoforms and phosphatidylinositol 3-kinase (PI3K), at the spinal level (125–127). Of particular clinical relevance is calcitonin gene-related peptide (CGRP), which plays a critical role in migraine pathophysiology. Expression of CGRP receptor component protein (RCP) in the spinal trigeminal nucleus caudalis and upper spinal cervical cord was significantly higher in female rats, offering a possible explanation for the increased incidence of migraine in female patients, though knock-down of RCP had similar anti-allodynic effects in females and males in a preclinical migraine model induced by noxious stimulation of the meninges with inflammatory mediators (128). Neurotransmitters may also play sexually dimorphic roles at the spinal level. Dopaminergic modulation of spinal cord plasticity in neuropathic pain (spared nerve injury) may rely on different receptors in male (D5) and female (D1) mice (129). Spinal α₅-GABA_A receptors have been shown to play sexually dimorphic roles in neuropathic rats (spinal nerve ligation) and mice (spared nerve injury), with upregulation in DRG and spinal cord in females but methylation-induced downregulation in males, and greater pronociceptive effects in females than males (130). These effects were also found to be influenced by hormonal variability, as ovariectomy downregulated spinal α₅-GABA_A receptors but treatment with 17β-estradiol (E2) enhanced them.

Brain.

Rodent studies have revealed sex differences in functional connectivity patterns of the insular cortex in a visceral pain model (131) and sex-dependent neuronal reshaping in the ventral posterolateral (VPL) nucleus of the thalamus in an orofacial pain model (132). Within the amygdala, a limbic region important in pain modulation and affective pain responses, female but not male rats showed upregulation of the stress modulators corticotropin-releasing factor (CRF) and glucocorticoid receptor (GR), along with a significant increase in visceral hypersensitivity, in an early life stress model (133). Another study highlighted a male-specific role for an atypical PKC isoform in the amygdala during pain development and maintenance (134). Similar to spinal cord studies, CGRP-related studies in the brain showed female-specific effects. A migraine model of dural CGRP administration produced hypersensitivity responses only in female rats(135), whereas systemic progesterone administration increased CGRP release in the trigeminal ganglia of both sexes (136). This suggests that CGRP-based mechanisms may contribute to the female bias of migraine disorders, potentially through sex-dependent hormonal influences. Sexual dimorphisms in dopaminergic transmission have also been implicated in the brain, as delivery of a dopamine D1 receptor antagonist into the bed nucleus of the stria terminalis (BNST) caused hyperalgesia only in female rats in the formalin pain model (137).

Sex differences in behavior related to descending pain modulatory mechanisms have also been documented. PAG projections to the rostral ventromedial medulla (RVM) comprise a key pathway of the brainstem that modulates pain signaling through projections to the dorsal horn of the spinal cord. In CFA-treated female but not male rats, morphine-induced antinociception was reversed by a positive allosteric modulator (PAM) of the GABA_A δ subunit (4-chloro-N-[2-(2-thienyl)imidazo[1,2-α]pyridine-3-yl]benzamide; DS2) in the ventrolateral PAG (vlPAG), suggesting that this area may represent a site of potentially differential regulation between the sexes in a persistent inflammatory state (138). This aligns well with clinical reports that suggest inhibitory functions of the descending pain modulatory system are higher in females, possibly through BDNF modulation (139).

Together, these studies provide potential mechanistic bases for reported sex differences within clinical studies, where sex-specific patterns in brain activity were found following noxious visceral stimuli (140) and at rest (141) in chronic pain patients. There is good evidence to suggest that neural mediation of pain is influenced by sex differences at all levels of the neuraxis with regard to many different neurochemicals and signaling molecules.

3.3. Neuroimmune Signaling

A rapidly growing domain of pain research focuses on the role of non-neuronal factors in pain development and maintenance. Both preclinical and clinical studies revealed that pain-related peripheral and central sensitization is not due solely to neuronal communication but instead influenced by intricate crosstalk between immune cells, glia, and neurons (142–146). Despite these advances, an important knowledge gap in pain neuroimmunology lies in the role of sex-specific neuroimmune mechanisms that contribute to chronic pain. As immune system contributions to pain disorders likely differ between males and females (20,21,147–152), hormonal interactions with neuroimmune regulatory mechanisms present one likely contributor to sex differences in pain-related behavior. The anti-inflammatory actions of sex hormones, particularly estrogens, have been well-reviewed (24,46,153,154), with much evidence citing their beneficial effects against neurodegeneration and pain-related processing. Intravenous administration of E2 has been shown to reduce mechanical allodynia and thermal hyperalgesia in rats following spinal cord injury (SCI), likely through the inhibition of astrocyte and microglia activation (155). Similarly, a single systemic dose of E2 following electrolytic injury to the spinothalamic tract (STT) attenuated mechanical hypersensitivity and suppressed microglial activation in the VPL (156). Similarly, subcutaneous progesterone administration prevented mechanical and thermal allodynia in male SCI rats (157). Animals receiving progesterone showed significantly lower levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNFα in the spinal cord dorsal horn, supporting progesterone’s capability to suppress reactive gliosis following CNS injury (157). Such hormonal contributions to pain-related neuroimmune mechanisms render sex differences in this field a critical area of ongoing investigation with important therapeutic implications.

Periphery.

Following peripheral nerve injury, cytokine profiles demonstrate sex differences in immune responses. Female mice showed increased tissue levels of the proinflammatory cytokines IL-1β and IFN-ɣ, the anti-inflammatory cytokine IL-10, and the pro-angiogenic factor VEGF compared to males following tibial fracture (158). Up-regulation of proinflammatory IL-1β and anti-inflammatory IL-10 have also been demonstrated in sciatic nerve lysates of CCI female but not male mice (159). Marked sex differences in efficacies for inflammation-related therapeutic strategies have also been reported. A tetracycline derivative (tigecycline), which possesses anti-inflammatory properties, produced antinociceptive effects in males but pronociceptive effects in females in the formalin pain test in a mouse model of binge drinking (160), and another derivative (minocycline) reduced hindpaw nociceptive sensitization for males only in a CRPS model (161). In the carrageenan inflammatory pain model, the neuroprotective anti-inflammatory agent cerebrolysin reduced mechanical allodynia in female but not male rats (162), and in the CFA inflammatory pain model, COX-2 inhibition of macrophages produced stronger and longer lasting antinociceptive effects in males but not females (163). Resolvin D5, which downregulates the production of IL-6 and chemokine ligand 5 (CCL5) (164), selectively reduced mechanical allodynia in male mice in a CIPN model (165). Several mechanistic studies have illustrated sex differences in peripheral neuroimmune responses. In the CIPN model, toll-like receptor 9 (TLR9)-deficient males did not show upregulated pronociceptive inflammatory mediators in DRGs or increased mechanical allodynia after pain induction, an effect that was not observed in females (166). A recent study found that inhibition of the proinflammatory high motility group box 1 (HMGB1) protein in the periphery reversed arthritis-mediated hypersensitivity only in male mice, and preventing myeloid cell activation protected male but not female mice from developing HMGB1-induced hypersensitivity (167). Together, the peripheral data support earlier reports from the spinal cord (reviewed below) that suggest male rodents rely on microglia-related pain processing mechanisms to a much greater extent than females, as illustrated by male-specific responses to inhibitors of microglia or of microglia-specific molecular targets.

Spinal Cord.

The overwhelming majority of pain-related neuroimmune studies have analyzed mechanisms at the spinal level. One early study that centered around the role of toll-like receptor 4 (TLR4) in pain found that its activation in the spinal cord caused robust mechanical allodynia in female but not male rodents. Pain behaviors were also shown to be TLR4-dependent in males but TLR4-independent in females for both inflammatory and neuropathic pain models (168), though a later study reported that TLR4 signaling is necessary for tactile allodynia in both males and females (169). This led to the exploration of whether cell types that expressed TLR4 (glia) were differentially activated with regard to sex in pain conditions. While males and females showed similar activation of spinal microglia in the SNI model of neuropathic pain, the response to injury in females was found to be preferentially mediated by cytokine release from T cells despite their ability to utilize the microglia system (170). CCI induced similar microgliosis between the sexes, though pronociceptive p38 signaling in microglia was enhanced only in male mice, and inhibition of this signaling reduced mechanical allodynia in male but not female mice (65,171). Spinal inhibition of p38 signaling or of the microglia-activating ionotropic purinergic receptor P2XR also disrupted hyperalgesic priming in a male-selective fashion (172), and P2XR blockade attenuated CCI-induced pain hypersensitivity in male rats only (173). Despite evidence for male-dominated microglial signaling in the spinal cord, inhibitors of astrocytic signaling produced sex-independent effects in formalin and CCI pain models (174). Sex differences in the timeline of spinal glial signaling have also been reported, as females showed a delayed and longer-lasting activation of microglia and astrocytes following CCI induction (65); however, hormonal treatment with E2 significantly attenuated this difference and induced complete functional recovery in the injured limb (175). Sex-specific patterns of spinal cord inflammation and resultant tactile hypersensitivity have also been implicated in arthritis (176) and EAE (177) pain models, and sexually dimorphic effects of therapeutic strategies have been attributed to patterns of spinal glial activation in neuropathic pain (178). Administration of HMGB1 evoked higher cytokine and chemokine responses in microglia derived from males compared to females, and intrathecal administration of both HMGB1 and a microglial inhibitor only prevented pain behavior in male mice (179). Thus, microglia may play a more critical role in spinal pain mechanisms for males while T cells may act as a more predominant player for females, though the underlying mechanisms are complex and a clear understanding of sex-specific neuroimmune modulation at the spinal level has yet to emerge.

Brain.

The role of pain-related neuroimmune signaling within the brain is less clear, and sex differences at this level are largely unknown. One study showed that, in response to morphine administration, there were no sex differences with regard to microglia density in the PAG, though microglia surprisingly exhibited a more activated phenotype at baseline in females. While morphine binding to TLR4 induces a neuroinflammatory cascade that can reduce its analgesic efficacy, blockade of this binding in the PAG of female rats reversed the sex differences that have previously been observed in morphine responsiveness (180). Inhibition of the proinflammatory tumor necrosis factor receptor 1 (TNFR1) was therapeutic for CCI-induced neuropathic pain in male but not female mice, as measured by NMDA receptor changes in the cerebral cortex and spinal cord (181). This provides support for claims that microglia-related pain processing may be a more relevant mechanism in males. Hippocampal microglia were primed by exposure to stress in male but not female rats (182), illustrating that sex differences in neuroinflammatory mechanisms may potentiate behavioral responses in many disease conditions. As male rats have been found to have increased number and complexity of astrocytes compared to females in important pain modulatory regions such as the amygdala (183), a baseline finding that has not been illustrated in the spinal cord (174), sex differences in brain-related pain processing may not follow spinal nociceptive mechanisms. Despite the rapid growth of novel findings in this pain research domain, mechanisms of male and female neuroimmune modulation have yet to be clearly characterized, and there is a great need to further investigate these sex-specific pathways at all levels of the neuraxis.

4. Sex differences in pain-related neurophysiology

An important knowledge gap exists regarding the functional mechanisms implicated in the differences in observed pain behaviors between male and female rodents. This is partly due to the limited preclinical evidence available regarding potential peripheral or central elements involved in the sexual dimorphism of the pain system.

Periphery.

A handful of studies investigated the periphery, as it was assumed to serve as the main site responsible for sex differences. However, emerging evidence suggests that different regions along the pain neuraxis make a critical contribution as well. In-vitro recordings of cultured DRG neurons innervating the gastrocnemius muscle showed a more hyperpolarized resting membrane potential in females than males, but no differences in other cellular properties were detected. In in-vivo single fiber electrophysiology, this translated into higher mechanical nociceptive thresholds of muscle afferents in females, but no differences in other neuronal characteristics such as conductance velocity and responses to sustained and suprathreshold stimulation were found between sexes (184). The results were interpreted to suggest that central mechanisms are needed to explain the lower pain thresholds found in females and they would need to be large enough to overcome the opposite sex differences in peripheral pain mechanisms. In the EAE model, effects of the daily voluntary wheel exercise on Ca²⁺ responses of isolated small and large lumbar DRG neurons were investigated in male and female mice. The running EAE female mice showed similar Ca²⁺ responses as a CFA-injected control group, while the non-running EAE females had increased Ca²⁺ release that was mediated by proinflammatory cytokines, suggesting that exercise had beneficial effects in the pathology that were attributed to the reduction of inflammatory responses in females (185). Surprisingly, in the running EAE male group, increased Ca²⁺ responses of the large, but not small, diameter neurons were detected when compared to the non-running and CFA control groups, supporting the idea that exercise aggravated the neuronal damage and corroborating the sex-specific effects of exercise (185). In different studies, the potentiation of the Ca²⁺ responses mediated by the ryanodine receptors and IP₃ partnership were specific to the small female DRG cultured isolectine B4-positive (IB4+, nociceptors that develop hyperalgesic priming) neurons incubated with estradiol. No effects were observed in the male DRG neurons, suggesting that females are more sensitive to hyperalgesic priming, a critical process of the transition to chronic pain (111,112). In a lysophosphatidic acid (LPA)-induced (joint neuropathy) rodent pain model, female rats showed a more prominent demyelination of small and large diameter joint afferent fibers 7 and 21 days post-induction compared to males. Single-unit electrophysiological recordings from joint afferents did not show sex differences in the baseline firing activity from the joint neuropathy animals, but stronger inhibitory effects of a selective voltage gated sodium channel (Nav1.8) antagonist (A-803467) were found in females than males (186). Electron microscopy analysis showed joint afferent damage caused by medial meniscus transection (MMT) surgery (model of post-traumatic osteoarthritis, PTOA) in male, but not in female, rats compared to their respective control groups, although no saphenous nerve demyelination was observed in either sex in the pain condition (72). Additionally, in-vivo single-unit recordings determined that PTOA-induced peripheral sensitization of joint afferent fibers occurred in MMT male, but not female, rats and suggested that MMT surgery may not represent a good PTOA model in female rodents. Interestingly, the same study showed that local application of amitriptyline (acting as a sodium current blocker in the periphery) had inhibitory effects on joint afferents in both male and female PTOA rats, but systemic application had greater effects on pain behaviors in males, pointing to central mechanisms of sex differences; for example, the engagement of the descending pain modulatory system by systemic amitriptyline (acting as serotonin and noradrenaline reuptake inhibitor in the brain and spinal cord) may be impaired in females (72).

Spinal Cord.

To the best of our knowledge, only one study has investigated sex-differences at the spinal level in a pain condition. Recordings from lamina II neurons in spinal cord slices showed that the application of a GABA_A receptor PAM (NS11394) decreased the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) in female, but not male, KO mice lacking the transcription factor bhlha9 (Fingerin), which is critically involved in the digit growth and highly expressed in C-LTMR’s (C-low-threshold mechanoreceptors). The electrophysiological data match the results of behavioral tests (von Frey test) that showed only male KO mice lacked antinociceptive effects of NS11394 in the formalin test, supporting the idea that the bhlha9 deficit caused an impairment of the GABAergic system only in males. Importantly, no significant sex differences in neuronal (amplitude, frequency and decay) and synaptic (sEPSCs) properties were observed before drug administration (187).

Brain.

Whole-cell patch-clamp recordings of parvalbumin-positive inhibitory neurons (PVINs) in the prelimbic (PL) region of the medial prefrontal cortex (mPFC) in brain slices showed that neurons from female sham control mice had greater neuronal excitability (number of action potentials elicited by increasing depolarizing current), input resistance, and sEPSC frequency compared to male control mice (188). Sex-differences were specific to the layer 5 (L5) of the PL cortex. In the SNI model (7 days after induction) neuronal excitability and input resistance of L5 PVINs increased in males but not in females. In contrast, no significant sex-differences were found in somatostatin (SOM) neurons of the PL cortex in sham control mice, and no significant changes were observed between SNI and sham control males. Interestingly, reduced input resistance and frequency of sEPSCs in layer 2/3 SOM neurons were observed in SNI females (188), suggesting that the sex differences could be subtype- and region-specific and even time-dependent, as recordings were made only 7 days after SNI surgery (an early stage for significant neuronal changes to occur). Whole-patch clamp recordings determined that application of TSP2, a particular isoform of astrocyte-secreted factors, increased the frequency of miniature EPSCs (mEPSCs) in male, but not female, cultured cortical neurons, providing evidence for sex-specific astrocyte influence on synaptic formation (189). In the amygdala, an aromatase (enzyme converting testosterone to E2) inhibitor (letrozole, 10⁻⁶ M) impaired long-term potentiation (LTP) induced in the basal (BA) nucleus by stimulation of the lateral (LA) nucleus in brain slices from female but not male mice, suggesting a sex-specific modulation of the neuron-derived E2 on synaptic plasticity in the basolateral amygdala (BLA) (190). In-vivo electrophysiology showed greater firing activity of LA and BA neurons in anesthetized female than male rats (191). Morphological analysis performed using Golgi-Cox staining revealed a higher number of LA and BA neuronal spines in female mice, suggesting greater excitatory glutamatergic input onto these neurons in females consistent with the enhanced neuronal activity observed in the in-vivo recordings. Moreover, LA-BA neurons of female rats showed higher mEPSC frequency in the in-vitro brain slice preparation and greater responses to the iontophoretic application of glutamate in in-vivo electrophysiology than males, confirming sex differences in the glutamatergic drive of LA-BA neurons (191). However, the same study also reported increased inhibitory responses (spontaneous and miniature inhibitory post-synaptic currents, sIPSCs and mIPSCs) of LA and BA neurons and greater sensitivity of BA neurons to the iontophoretically applied GABA in females compared to males. Thus, both excitatory and inhibitory transmission in the amygdala appears to be greater in females than males.

A few studies have investigated potential sex differences in neurophysiological mechanisms of the descending pain modulatory system. Patch-clamp recordings from the vlPAG neurons demonstrated that induction of the CFA inflammatory pain model had differential effects on the GABA_A signaling in male and female rats (138). Increased mIPSC frequency but not amplitude, suggesting enhanced GABA release at the presynaptic terminals, and decreased high-affinity tonic GABA_A currents were reported in females. Activation of the GABA_A receptors containing δ subunits (GABA_A δ receptors) by the co-application of a selective PAM, DS2, with exogenous GABA increased tonic GABA_A currents in CFA-treated female but not male rats, supporting the behavioral evidence that suggests GABA_A δ receptors in the vlPAG are activated by the endogenous ligand and counteract the morphine-mediated antinociceptive effects in the inflammatory model (138). Therefore, vlPAG GABA_A signaling may represent a potentially valuable therapeutic target for pain management. Under normal physiological conditions, the RVM pronociceptive (ON) and antinociceptive (OFF) cells exhibited similar responses to peripherally applied heat noxious stimuli in both sexes, suggesting no basal functional differences in the pain modulatory circuitry. Moreover, the ON and OFF neurons were similarly recruited 3–6 days after CFA induction and by acute systemic morphine injection in male and female rats, providing evidence that RVM nociceptive cells are equally sensitized in acute inflammatory pain and opioid-mediated responses (192–194).

Based on the evidence reviewed here, important functional differences between the sexes emerged at the central level, corroborating the idea that neuronal or non-neuronal spinal or supraspinal elements play a critical role in the dimorphic pain-related mechanisms that may occur during sex determination or during growth and development.

5. Sex differences in the pain-related transcriptome

The urgent need to understand sex-specific mechanisms in pain modulation has been driving research on sex differences at the transcriptomic level. Sexually dimorphic gene expression changes can shape sex-dependent physiology and behavior in animals, and molecular mechanisms that drive pain-related processing can have a profound influence on disease prevalence, susceptibility, and pathophysiology.

Periphery.

In a clinical study, DRGs from patients undergoing spinal tumor resection were separated into groups based on the presence or absence of associated dermatomal radicular or neuropathic pain. Extensive differences in transcriptional signatures were found between male and female pain cohorts, and many differentially expressed genes (DEGs) in the male pain cohort were macrophage-related (195). This supports evidence for sexually dimorphic mechanisms in neuropathic pain pathology. Further evidence for sex-specific gene expression in the peripheral nervous system was found when analyzing the human tibial nerve transcriptome, as macrophage-associated genes were preferentially expressed in the male cohort and indicated that sex-differential expression patterns of inflammatory markers may differ even under normal conditions (196). Preclinical studies also demonstrated unique gene expression profiles between males and females under chronic pain conditions. Following CCI of the infraorbital nerve in rats, expression changes of 84 preselected, pain-related genes were measured in the trigeminal ganglia. Of 32 genes that were significantly regulated for both males and females, four of these showed significant differences in magnitude of regulation between the sexes (197). Another study found that CCI of the sciatic nerve caused significant transcriptomic changes in the DRG, with 1513 DEGs identified between the sexes. 146 genes were specifically upregulated in females and belonged to functional pathways related to glucocorticoid and corticosteroid responses, while 859 genes were specifically upregulated in males and were associated with ion channel activity, gated channel activity, and metal ion membrane transporter activity (198). Other sex-specific gene expression profiles have been characterized in sensory neuronal populations at baseline in mice, where female-predominant DEGs were related to inflammatory, synaptic transmission and extracellular matrix reorganization processes which may exacerbate neuroinflammatory severity, while male-predominant DEGs were linked to oxidative phosphorylation and protein metabolism that may serve protective functions for male sensory neurons in chronic pain development (199). Striking sex differences in pain-related gene expression changes have been demonstrated with regard to prostaglandin signaling (200), after myelin basic protein fragment exposure (201), and after knockout of bhlha9 which has been implicated as a pain modulator in mice and humans (187). Though further exploration is warranted, the literature suggests that sex differences in nociceptive behavior or the differential occurrence of pain disorders in male and female patients may be influenced by sex-dependent DEGs in the peripheral nervous system.

Spinal Cord.

Interestingly, transcriptomes of spinal tissue from CCI rats that underwent repetitive spinal cord stimulation showed upregulation of genes involved in immune-related processes and downregulation of genes associated with synaptic signaling for both male and female rats (202). Other gene expression studies on spinal cord tissue have not found evidence for transcriptional differences between males and females (203,204). The lack of sex differences in DEGs at the spinal level may indicate that in both males and females, pain mechanisms may become independent from synaptic regulation with the central sensitization underlying chronic pain being maintained by a predominately neuroimmune response.

Brain.

Though sex differences in pain-related transcriptomic changes in the brain have yet to be explored, some evidence suggests that, at baseline, male-derived microglia exhibit a more proinflammatory transcriptome while female-derived microglia exhibit a stronger neuroprotective transcriptome (205,206). This is surprisingly different from the previously mentioned peripheral finding that proinflammatory DEGs were female-predominant while neuroprotective DEGs were male-predominant (199), supporting the concept of sex-differential pain mechanisms that vary between the different levels of the neuraxis. Future avenues of exploration in this field include sequencing of cell type-specific populations and single-cell transcriptome sequencing at peripheral, spinal, and supraspinal levels in male and female subjects, particularly at different stages of pain development.

6. Conclusions

The existing literature regarding sex differences in pain conditions provides compelling evidence that pain modulation is influenced by sex-specific processes. Studies performed at the molecular, cellular, and systems levels have demonstrated that pain processing in males and females may be strongly and differentially impacted with regard to genetic expression, hormonal influences, and neuroimmune interactions (as shown in Table 2). Additionally, sexually dimorphic pain mechanisms may be dependent on the level of the neuraxis (see Fig. 1 for a simplified depiction of the pain system). While there is abundant evidence that female subjects may be more sensitive to pain in both the preclinical and clinical setting, some studies suggest that this finding may not extend to emotional-affective or cognitive pain components. Therefore, it is difficult to draw firm conclusions surrounding which sex is more strongly affected by or more resilient to pain, as the underlying mechanisms driving these dimensional differences are largely unknown.

Figure 1. Sex-predominant cellular and molecular factors within the pain system.

First order nociceptive neurons are located in the dorsal root ganglia (DRG) and carry nociceptive information from the periphery into the CNS. These neurons synapse in the spinal cord dorsal horn (DH), and axons from second order neurons decussate in the anterior white commissure to ascend in the contralateral spinal cord. Pain-related information is conveyed through at least three main pathways: the spinothalamic tract (STT, shown in yellow), the spinoparabrachioamygdala tract (SPbA, shown in green), and the postsynaptic dorsal column (PSDC) pathway (not shown). Sensory information is conveyed through the STT to the ventral posterolateral and -medial (VPL and VPM) nuclei of the thalamus (TH) before synapsing in the somatosensory cortex (SS-Ctx) and posterior aspect of the insular cortex (INS-Ctx), allowing for the localization and intensity detection of pain. In the SPbA pathway, also known as the limbic pathway, nociceptive information relays in the parabrachial nucleus (PB) in the brainstem before reaching the amygdala (AMY). AMY integrates multimodal sensory and nociceptive information to alter central autonomic functions and emotional-affective states through associative processing and provides value-based information to prefrontal cortical regions (PF-Ctx) and anterior INS-Ctx. Various brain regions engage the descending pain modulatory system, including efferents from AMY and PF-Ctx that synapse onto the periaqueductal gray and rostral ventromedial medulla (PAG-RVM) system (shown in orange). These brainstem regions then modulate spinal nociceptive processing through descending monoaminergic projections. Cellular and molecular factors that have been reported to have sexually dimorphic effects are illustrated by pink (representing female-predominant effects) and blue (representing male-predominant effects) boxes. BNST, bed nucleus of stria terminalis.

A considerable body of work suggests that the coordination of the nervous and immune systems is a predominant contributor to pain modulation, and future directions in pain research will likely focus on this area. Though robust sex differences have already been implicated, the concept of sex-specific neuroimmune signaling has yet to be fully characterized. Many molecules (such as HMGB1) and even entire cell types (such as microglia) have been identified as key players in a male-specific neuroimmune response to pain in the periphery and spinal cord. However, the roles of such factors in the brain have been overwhelmingly understudied, and there is some evidence to suggest that pain-related neuroimmune signaling may be region dependent, as seen by differences in sex-specific DEGs between the brain and the periphery. This is an excellent illustration of the critical need to explore the role of pain modulatory factors in both males and females at all levels of the neuraxis.

Important knowledge gaps to address include potential sexual dimorphisms in the role of neuroimmune signaling as it contributes to the transition from acute protective pain to chronic pathological pain, and the impacts of these mechanisms within currently understudied levels of the neuraxis, such as brain regions that are critically involved in pain and pain modulation. The current literature provides ample support for the need to study female subjects in future pain research for a complete understanding of pain. Ultimately, the investigation of sex differences in pain will lay the groundwork for the development of novel, sex-specific therapeutic strategies for chronic pain relief.