Pain mechanisms in the transgender individual: a review.

Specific Aim: Provide an overview of the literature addressing major areas pertinent to pain in transgender persons and to identify areas of primary relevance for future research. Methods: A team of scholars that have previously published on different areas of related research met periodically though zoom conferencing between April 2021 and February 2023 to discuss relevant literature with the goal of providing an overview on the incidence, phenotype, and mechanisms of pain in transgender patients. Review sections were written after gathering information from systematic literature searches of published or publicly available electronic literature to be compiled for publication as part of a topical series on gender and pain in the Frontiers in Pain Research. Results: While transgender individuals represent a significant and increasingly visible component of the population, many researchers and clinicians are not well informed about the diversity in gender identity, physiology, hormonal status, and gender-affirming medical procedures utilized by transgender and other gender diverse patients. Transgender and cisgender people present with many of the same medical concerns, but research and treatment of these medical needs must reflect an appreciation of how differences in sex, gender, gender-affirming medical procedures, and minoritized status impact pain. Conclusions: While significant advances have occurred in our appreciation of pain, the review indicates the need to support more targeted research on treatment and prevention of pain in transgender individuals. This is particularly relevant both for gender-affirming medical interventions and related medical care. Of particular importance is the need for large long-term follow-up studies to ascertain best practices for such procedures. A multi-disciplinary approach with personalized interventions is of particular importance to move forward.

Gordon Holmes Syndrome Model Mice Exhibit Alterations in Microglia, Age, and Sex-Specific Disruptions in Cognitive and Proprioceptive Function.

Gordon Holmes syndrome (GHS) is a neurological disorder associated with neuroendocrine, cognitive, and motor impairments with corresponding neurodegeneration. Mutations in the E3 ubiquitin ligase RNF216 are strongly linked to GHS. Previous studies show that deletion of Rnf216 in mice led to sex-specific neuroendocrine dysfunction due to disruptions in the hypothalamic-pituitary-gonadal axis. To address RNF216 action in cognitive and motor functions, we tested Rnf216 knock-out (KO) mice in a battery of motor and learning tasks for a duration of 1 year. Although male and female KO mice did not demonstrate prominent motor phenotypes, KO females displayed abnormal limb clasping. KO mice also showed age-dependent strategy and associative learning impairments with sex-dependent alterations of microglia in the hippocampus and cortex. Additionally, KO males but not females had more negative resting membrane potentials in the CA1 hippocampus without any changes in miniature excitatory postsynaptic current (mEPSC) frequencies or amplitudes. Our findings show that constitutive deletion of Rnf216 alters microglia and neuronal excitability, which may provide insights into the etiology of sex-specific impairments in GHS.

Perigestational Opioid Exposure Alters Alcohol-Driven Reward Behaviors in Adolescent Rats.

Every fifteen minutes, a baby is born in the U.S. experiencing neonatal opioid withdrawal syndrome (NOWS). Since 2004, the rate of NOWS has increased 7-fold. Clinical studies have established intrauterine exposure to drugs of abuse as a risk factor for adverse health outcomes in adult life, including the propensity for future illicit drug use. Despite extensive knowledge about common mechanisms of action in the neural circuitry that drives opioid and alcohol reward, there is little data on the risks that those born with NOWS face regarding alcohol use later in life. Here, we investigate the impact of perigestational opioid exposure (POE) on the mesolimbic reward system of male and female Sprague Dawley rats at postnatal and adolescent ages. Our laboratory has developed a clinically relevant model for morphine exposure spanning pre-conception to the first week of life. Using this model, we found that POE increased alcohol consumption in female rats under noncontingent conditions, and inversely, reduced alcohol consumption in both male and female rats during operant conditioning sessions. Operant responding was also reduced for sucrose, suggesting that the impact of POE on reward-seeking behaviors is not limited to drugs of abuse. Expression of µ-opioid receptors was also significantly altered in the nucleus accumbens and medial habenula, regions previously shown to play a significant role in reward/aversion circuitry.

Increased LPS-Induced Fever and Sickness Behavior in Adult Male and Female Rats Perinatally Exposed to Morphine.

As a result of the current opioid crisis, the rate of children born exposed to opioids has skyrocketed. Later in life, these children have an increased risk for hospitalization and infection, raising concerns about potential immunocompromise, as is common with chronic opioid use. Opioids can act directly on immune cells or indirectly via the central nervous system to decrease immune system activity, leading to increased susceptibility, morbidity, and mortality to infection. However, it is currently unknown how perinatal opioid exposure (POE) alters immune function. Using a clinically relevant and translatable model of POE, we have investigated how baseline immune function and the reaction to an immune stimulator, lipopolysaccharide, is influenced by in utero opioid exposure in adult male and female rats. We report here that POE potentiates the febrile and neuroinflammatory response to lipopolysaccharide, likely as a consequence of suppressed immune function at baseline (including reduced antibody production). This suggests that POE increases susceptibility to infection by manipulating immune system development, consistent with the clinical literature. Investigation of the mechanisms whereby POE increases susceptibility to pathogens is critical for the development of potential interventions for immunosuppressed children exposed to opioids in utero.

Perinatal Morphine Exposure Induces Long-Term Changes in the Intestinal Microbiota of Male and Female Rats.

The increased use of opioids by women of reproductive age has resulted in a dramatic rise in number of infants exposed to opioids in utero. Although perinatal opioid exposure (POE) has been associated with an elevated risk of infection and hospitalization later in life, the mechanism(s) by which opioids influence immune development and maturation is not fully elucidated. Alterations in the intestinal microbiota composition, which leads to changes in immune training and maturation, could be at play. Chronic opioid use in adults is associated with a proinflammatory and pathogenic microbiota composition; therefore, we hypothesized here that in utero morphine exposure could negatively affect intestinal microbiota composition, leading to alterations in immune system function. We report that a clinically-relevant model of perinatal opioid exposure, in rats, induces profound intestinal microbiota dysbiosis that is maintained into adulthood. Furthermore, microbial maturity was reduced in morphine-exposed offspring. This suggests that increased risk of infection observed in children exposed to opioids during gestation may be a consequence of microbiota alterations with downstream impact on immune system development. Further investigation of how perinatal morphine induces dysbiosis will be critical to the development of early life interventions designed to ameliorate the increased risk of infection observed in these children.

Perinatal opioid exposure leads to decreased social play in adolescent male and female rats: Potential role of oxytocin signaling in brain regions associated with social reward.

Over the last two decades, the number of infants exposed to opioids in utero has quadrupled in the United States, with some states reporting rates as high as 55 infants per 1000 births. Clinical studies report that children previously exposed to opioids during gestation show significant deficits in social behavior, including an inability to form friendships or other social relationships. To date, the neural mechanisms whereby developmental opioid exposure disrupts social behavior remain unknown. Using a novel paradigm of perinatal opioid administration, we tested the hypothesis that chronic opioid exposure during critical developmental periods would disrupt juvenile play. As oxytocin is a major regulator of sociability, the impact of perinatal morphine exposure on oxytocin peptide expression was also examined. Juvenile play was assessed in vehicle- or morphine-exposed male and female rats at P25, P35, and P45. Classical features of juvenile play were measured, including time spent engaged in social play, time not in contact, number of pins, and number of nape attacks. We report that morphine-exposed males and females spend less time engaged in play behavior than control males and females, with a corresponding increase in time spent alone. Morphine-exposed males and females also initiated fewer pins and nape attacks. Together, these data suggest that male and female rats exposed to morphine during critical developmental periods are less motivated to participate in social play, potentially due to alterations in oxytocin-mediated reward signaling.

Perinatal Opioid Exposure Leads to Decreased Social Play in Adolescent Male and Female Rats: Potential Role of Oxytocin Signaling in Brain Regions Associated with Social Reward.

Over the last two decades, the number of infants exposed to opioids in utero has quadrupled in the United States, with some states reporting rates as high as 55 infants per 1000 births. Clinical studies report that children previously exposed to opioids during gestation show significant deficits in social behavior, including an inability to form friendships or other social relationships. To date, the neural mechanisms whereby developmental opioid exposure disrupts social behavior remain unknown. Using a novel paradigm of perinatal opioid administration, we tested the hypothesis that chronic opioid exposure during critical developmental periods would disrupt juvenile play. As oxytocin is a major regulator of sociability, the impact of perinatal morphine exposure on oxytocin peptide and receptor expression was also examined. Juvenile play was assessed in vehicle- or morphine-exposed male and female rats at P25, P35, and P45. Classical features of juvenile play were measured, including time spent engaged in social play, time not in contact, number of pins, and number of nape attacks. We report that morphine-exposed females spend less time engaged in play behavior than control males and females, with a corresponding increase in time spent alone. Morphine-exposed females also initiated fewer pins and nape attacks. Oxytocin receptor binding was reduced in morphine-exposed females in the nucleus accumbens, a brain region critical for social reward. Together, these data suggest that females exposed to morphine during critical developmental periods are less motivated to participate in social play, potentially due to alterations in oxytocin-mediated reward signaling.

Erratum: The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis.

[This corrects the article DOI: 10.1016/j.isci.2022.104386.].

Age-Induced Changes in µ-Opioid Receptor Signaling in the Midbrain Periaqueductal Gray of Male and Female Rats.

Opioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. The present study investigated the impact of advanced age and biological sex on opioid signaling in the ventrolateral periaqueductal gray (vlPAG) in the presence of chronic inflammatory pain. Assays measuring µ-opioid receptor (MOR) radioligand binding, GTPγS binding, receptor phosphorylation, cAMP inhibition, and regulator of G-protein signaling (RGS) protein expression were performed on vlPAG tissue from adult (2-3 months) and aged (16-18 months) male and female rats. Persistent inflammatory pain was induced by intraplantar injection of complete Freund's adjuvant (CFA). Adult males exhibited the highest MOR binding potential (BP) and highest G-protein activation (activation efficiency ratio) in comparison to aged males and females (adult and aged). No impact of advanced age or sex on MOR phosphorylation state was observed. DAMGO-induced cAMP inhibition was highest in the vlPAG of adult males compared with aged males and females (adult and aged). vlPAG levels of RGS4 and RGS9-2, critical for terminating G-protein signaling, were assessed using RNAscope. Adult rats (both males and females) exhibited lower levels of vlPAG RGS4 and RGS9-2 mRNA expression compared with aged males and females. The observed age-related reductions in vlPAG MOR BP, G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in RGS4 and RGS9-2 vlPAG expression, provide potential mechanisms whereby the potency of opioids is decreased in the aged population.SIGNIFICANCE STATEMENTOpioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. In the present study, we observed age-related reductions in ventrolateral periaqueductal gray (vlPAG) µ-opioid receptor (MOR) binding potential (BP), G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in regulator of G-protein signaling (RGS)4 and RGS9-2 vlPAG expression, providing potential mechanisms whereby the potency of opioids is decreased in the aged population. These coordinated decreases in opioid receptor signaling may explain the previously reported reduced potency of opioids to produce pain relief in females and aged rats.

Altered PVN-to-CA2 hippocampal oxytocin pathway and reduced number of oxytocin-receptor expressing astrocytes in heart failure rats.

Oxytocinergic actions within the hippocampal CA2 are important for neuromodulation, memory processing and social recognition. However, the source of the OTergic innervation, the cellular targets expressing the OT receptors (OTRs) and whether the PVN-to-CA2 OTergic system is altered during heart failure (HF), a condition recently associated with cognitive and mood decline, remains unknown. Using immunohistochemistry along with retrograde monosynaptic tracing, RNAscope and a novel OTR-Cre rat line, we show that the PVN (but not the supraoptic nucleus) is an important source of OTergic innervation to the CA2. These OTergic fibers were found in many instances in close apposition to OTR expressing cells within the CA2. Interestingly, while only a small proportion of neurons were found to express OTRs (~15%), this expression was much more abundant in CA2 astrocytes (~40%), an even higher proportion that was recently reported for astrocytes in the central amygdala. Using an established ischemic rat heart failure (HF) model, we found that HF resulted in robust changes in the PVN-to-CA2 OTergic system, both at the source and target levels. Within the PVN, we found an increased OT immunoreactivity, along with a diminished OTR expression in PVN neurons. Within the CA2 of HF rats, we observed a blunted OTergic innervation, along with a diminished OTR expression, which appeared to be restricted to CA2 astrocytes. Taken together, our studies highlight astrocytes as key cellular targets mediating OTergic PVN inputs to the CA2 hippocampal region. Moreover, they provide the first evidence for an altered PVN-to-CA2 OTergic system in HF rats, which could potentially contribute to previously reported cognitive and mood impairments in this animal model.

Sex differences in the amygdaloid projections to the ventrolateral periaqueductal gray and their activation during inflammatory pain in the rat.

Preclinical and clinical studies have reported sex differences in pain and analgesia. These differences may be linked to anatomical structures of the central nervous system pain modulatory circuitry, and/or hormonal milieu. The midbrain periaqueductal gray (PAG) is a critical brain region for descending inhibition of pain. The PAG projects to the rostral ventromedial medulla (RVM), which projects bilaterally to the spinal cord to inhibit pain. In addition to pain, this descending circuit (or pathway) can be engaged by endogenous opioids (i.e., endorphins) or exogenous opioids (i.e., morphine), and we have previously reported sex differences in the activation of this circuit during pain and analgesia. Forebrain structures, including the amygdala, project to and engage the PAG-RVM circuit during persistent inflammatory pain. However, there are limited studies in females detailing this amygdalar-PAG pathway and its involvement during persistent inflammatory pain. The objective of the present study was to delineate the neural projections from the amygdala to the PAG in male and female rats to determine if they are sexually distinct in their anatomical organization. We also examined the activation of this pathway by inflammatory pain and the co-localization of receptors for estrogen. Injection of the retrograde tracer fluorogold (FG) into the ventrolateral PAG (vlPAG) resulted in dense retrograde labeling in both the central amygdala (CeA) and medial amygdala (MeA). While the number of CeA-vlPAG neurons were comparable between the sexes, there were more MeA-vlPAG neurons in females. Inflammatory pain resulted in greater activation of the amygdala in males; however, females displayed higher Fos expression within CeA-vlPAG projection neurons. Females expressed higher ERα in the MeA and CeA and the same was true of the projection neurons. Together, these data indicate that although the MeA-vlPAG projections are denser in females, inflammatory pain does not significantly activate these projections. In contrast, inflammatory pain resulted in a greater activation of the CeA-vlPAG pathway in females. As females experience a greater number of chronic pain syndromes, the CeA-vlPAG pathway may play a facilitatory (and not inhibitory) role in pain modulation.

The E3 ubiquitin ligase RNF216/TRIAD3 is a key coordinator of the hypothalamic-pituitary-gonadal axis.

Recessive mutations in RNF216/TRIAD3 cause Gordon Holmes syndrome (GHS), in which dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis and neurodegeneration are thought to be core phenotypes. We knocked out Rnf216/Triad3 in a gonadotropin-releasing hormone (GnRH) hypothalamic cell line. Rnf216/Triad3 knockout (KO) cells had decreased steady-state GnRH and calcium transients. Rnf216/Triad3 KO adult mice had reductions in GnRH neuron soma size and GnRH production without changes in neuron densities. In addition, KO male mice had smaller testicular volumes that were accompanied by an abnormal release of inhibin B and follicle-stimulating hormone, whereas KO females exhibited irregular estrous cycling. KO males, but not females, had reactive microglia in the hypothalamus. Conditional deletion of Rnf216/Triad3 in neural stem cells caused abnormal microglia expression in males, but reproductive function remained unaffected. Our findings show that dysfunction of RNF216/TRIAD3 affects the HPG axis and microglia in a region- and sex-dependent manner, implicating sex-specific therapeutic interventions for GHS.

Considering sex as a biological variable will require a global shift in science culture.

For over half a century, male rodents have been the default model organism in preclinical neuroscience research, a convention that has likely contributed to higher rates of misdiagnosis and adverse side effects from drug treatment in women. Studying both sexes could help to rectify these public health problems, but incentive structures in publishing and career advancement deter many researchers from doing so. Moreover, funding agency directives to include male and female animals and human participants in grant proposals lack mechanisms to hold recipients accountable. In this Perspective, we highlight areas of behavioral, cellular and systems neuroscience in which fundamental sex differences have been identified, demonstrating that truly rigorous science must include males and females. We call for a cultural and structural change in how we conduct research and evaluate scientific progress, realigning our professional reward systems and experimental standards to produce a more equitable, representative and therefore translational body of knowledge.

Advanced age attenuates the antihyperalgesic effect of morphine and decreases µ-opioid receptor expression and binding in the rat midbrain periaqueductal gray in male and female rats.

The present study investigated the impact of advanced age on morphine modulation of persistent inflammatory pain in male and female rats. The impact of age, sex, and pain on µ-opioid receptor (MOR) expression and binding in the ventrolateral periaqueductal gray (vlPAG) was also examined using immunohistochemistry and receptor autoradiography. Intraplantar administration of complete Freund's adjuvant induced comparable levels of edema and hyperalgesia in adult (2-3 mos) and aged (16-18 mos) male and female rats. Morphine potency was highest in adult males, with a greater than two-fold increase in morphine EC50 observed in adult versus aged males (3.83 mg/kg vs. 10.16 mg/kg). Adult and aged female rats also exhibited significantly higher EC50 values (7.76 mg/kg and 8.74 mg/kg, respectively) than adult males. The upward shift in EC50 from adult to aged males was paralleled by a reduction in vlPAG MOR expression and binding. The observed age-related reductions in morphine potency and vlPAG MOR expression and binding have significant implications in pain management in the aged population.

Alterations in SUMOylation of the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 during persistent inflammation.

BACKGROUND: Unilateral injection of Complete Freund's Adjuvant (CFA) into the intra-plantar surface of the rodent hindpaw elicits chronic inflammation and hyperalgesia in the ipsilateral hindlimb. Mechanisms contributing to this hyperalgesia may act over multiple time courses and can include changes in ion channel expression and post-translational SUMOylation. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current, Ih . An HCN2-mediated increase in C-nociceptor Ih contributes to mechanical hyperalgesia in the CFA model of inflammatory pain. Changes in HCN2 post-translational SUMOylation and protein expression have not been systematically documented for a given dorsal root ganglia (DRG) throughout the time course of inflammation. METHODS: This study examined HCN2 protein expression and post-translational SUMOylation in a rat model of CFA-induced hindpaw inflammation. L5 DRG cryosections were used in immunohistochemistry experiments and proximity ligation assays to investigate HCN2 expression and SUMOylation, respectively, on days 1 and 3 post-CFA. RESULTS: Unilateral CFA injection elicited a significant bilateral increase in HCN2 staining intensity in small diameter DRG neurons on day 1 post-CFA, and a significant bilateral increase in the number of small neurons expressing HCN2 but not staining intensity on day 3 post-CFA. HCN2 channels were hyper-SUMOylated in small diameter neurons of ipsilateral relative to contralateral DRG on days 1 and 3 post-CFA. CONCLUSIONS: Unilateral CFA injection elicits unilateral mechanical hyperalgesia, a bilateral increase in HCN2 expression and a unilateral increase in post-translational SUMOylation. This suggests that enhanced HCN2 expression in L5 DRG is not sufficient for mechanical hyperalgesia in the early stages of inflammation and that hyper-SUMOylation of HCN2 channels may also be necessary. SIGNIFICANCE: Nociceptor HCN2 channels mediate an increase in Ih that is necessary for mechanical hyperalgesia in a CFA model of chronic pain, but the mechanisms producing the increase in nociceptor Ih have not been resolved. The data presented here suggest that the increase in Ih during the early stages of inflammation may be mediated by an increase in HCN2 protein expression and post-translational SUMOylation.

Early life opioid exposure and potential long-term effects.

The long-term consequences of perinatal opioid exposure and subsequent development of neonatal opioid withdrawal syndrome is largely unknown and likely dependent on a multitude of factors, including co-morbid drug use, pre- and post-natal care, and individual factors including the maternal-infant relationship and home environment. This review summarizes the current literature from clinical and preclinical studies on perinatal opioid exposure, focusing on the consequences in the offspring. Although a large number of preclinical studies have been conducted examining the impact of prenatal opioid exposure, the models employed are not necessarily representative of clinical use patterns, making it challenging to translate these results to the impacted population. Use of more clinically-relevant models of perinatal opioid exposure are requisite for the development of improved pharmacological and behavioral treatment strategies to improve quality of life for this vulnerable population.

Inflammatory mediators of opioid tolerance: Implications for dependency and addiction.

Each year, over 50 million Americans suffer from persistent pain, including debilitating headaches, joint pain, and severe back pain. Although morphine is amongst the most effective analgesics available for the management of severe pain, prolonged morphine treatment results in decreased analgesic efficacy (i.e., tolerance). Despite significant headway in the field, the mechanisms underlying the development of morphine tolerance are not well understood. The midbrain ventrolateral periaqueductal gray (vlPAG) is a primary neural substrate for the analgesic effects of morphine, as well as for the development of morphine tolerance. A growing body of literature indicates that activated glia (i.e., microglia and astrocytes) facilitate pain transmission and oppose morphine analgesia, making these cells important potential targets in the treatment of chronic pain. Morphine affects glia by binding to the innate immune receptor toll-like receptor 4 (TLR4), leading to the release of proinflammatory cytokines and opposition of morphine analgesia. Despite the established role of the vlPAG as an integral locus for the development of morphine tolerance, most studies have examined the role of glia activation within the spinal cord. Additionally, the role of TLR4 in the development of tolerance has not been elucidated. This review attempts to summarize what is known regarding the role of vlPAG glia and TLR4 in the development of morphine tolerance. These data, together, provide information about the mechanism by which central nervous system glia regulate morphine tolerance, and identify a potential therapeutic target for the enhancement of analgesic efficacy in the clinical treatment of chronic pain.

Neuronal and glial factors contributing to sex differences in opioid modulation of pain.

Morphine remains one of the most widely prescribed opioids for alleviation of persistent and/or severe pain; however, multiple preclinical and clinical studies report that morphine is less efficacious in females compared to males. Morphine primarily binds to the mu opioid receptor, a prototypical G-protein coupled receptor densely localized in the midbrain periaqueductal gray. Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray, and its descending projections to the rostral ventromedial medulla and spinal cord, as an essential descending inhibitory circuit mediating opioid-based analgesia. Remarkably, the majority of studies published over the following 30 years were conducted in males with the implicit assumption that the anatomical and physiological characteristics of this descending inhibitory circuit were comparable in females; not surprisingly, this is not the case. Several factors have since been identified as contributing to the dimorphic effects of opioids, including sex differences in the neuroanatomical and neurophysiological characteristics of the descending inhibitory circuit and its modulation by gonadal steroids. Recent data also implicate sex differences in opioid metabolism and neuroimmune signaling as additional contributing factors. Here we cohesively present these lines of evidence demonstrating a neural basis for sex differences in opioid modulation of pain, with a focus on the PAG as a sexually dimorphic core of descending opioid-induced inhibition and argue for the development of sex-specific pain therapeutics.

Impact of sex on pain and opioid analgesia: a review.

Chronic pain is a debilitating condition that impacts tens of millions each year, resulting in lost wages for workers and exacting considerable costs in health care and rehabilitation. A thorough understanding of the neural mechanisms underlying pain and analgesia is critical to facilitate the development of therapeutic strategies and personalized medicine. Clinical and epidemiological studies report that women experience greater levels of pain than men and have higher rates of pain-related disorders. Studies in both rodents and humans report sex differences in the anatomical and physiologic properties of the descending antinociceptive circuit, mu opioid receptor (MOR) expression and binding, morphine metabolism, and immune system activation, all of which likely contribute to the observed sex differences in pain and opioid analgesia. Although more research is needed to elucidate the underlying mechanisms, these sex differences present potential therapeutic targets to optimize pain management strategies for both sexes.