Sex differences in peripheral immune cell activation: Implications for pain and pain resolution.

Decades of research into chronic pain has deepened our understanding of the cellular mechanisms behind this process. However, a failure to consider the biological variable of sex has limited the application of these breakthroughs into clinical application. In the present study, we investigate fundamental differences in chronic pain between male and female mice resulting from inflammatory activation of the innate immune system. We provide evidence that female mice are more sensitive to the effects of macrophages. Injecting small volumes of media conditioned by either unstimulated macrophages or macrophages stimulated by the inflammatory molecule TNFα lead to increased pain sensitivity only in females. Interestingly, we find that TNFα conditioned media leads to a more rapid resolution of mechanical hypersensitivity and altered immune cell recruitment to sites of injury. Furthermore, male and female macrophages exhibit differential polarization characteristics and motility after TNFα stimulation, as well as a different profile of cytokine secretions. Finally, we find that the X-linked gene Tlr7 is critical in the facilitating the adaptive resolution of pain in models of acute and chronic inflammation in both sexes. Altogether, these findings suggest that although the cellular mechanisms of pain resolution may differ between the sexes, the study of these differences may yield more targeted approaches with clinical applications.

Astrocytes show increased levels of Ero1α in multiple sclerosis and its experimental autoimmune encephalomyelitis animal model.

Multiple sclerosis (MS) and its animal models are characterized by cellular inflammation within the central nervous system (CNS). The sources and consequences of this inflammation are currently not completely understood. Critical signs and mediators of CNS inflammation are reactive oxygen species (ROS) that promote inflammation. ROS originate from a variety of redox-reactive enzymes, one class of which catalyses oxidative protein folding within the endoplasmic reticulum (ER). Here, the unfolded protein response and other signalling mechanisms maintain a balance between ROS producers such as ER oxidoreductin 1α (Ero1α) and antioxidants such as glutathione peroxidase 8 (GPx8). The role of ROS production within the ER has so far not been examined in the context of MS. In this manuscript, we examined how components of the ER redox network change upon MS and experimental autoimmune encephalomyelitis (EAE). We found that unlike GPx8, Ero1α increases within both MS and EAE astrocytes, in parallel with an imbalance of other oxidases such of GPx7, and that no change was observed within neurons. This imbalance of ER redox enzymes can reduce the lifespan of astrocytes, while neurons are not affected. Therefore, Ero1α induction makes astrocytes vulnerable to oxidative stress in the MS and EAE pathologies.

Regulation of microglia population dynamics throughout development, health, and disease.

The dynamic expansions and contractions of the microglia population in the central nervous system (CNS) to achieve homeostasis are likely vital for their function. Microglia respond to injury or disease but also help guide neurodevelopment, modulate neural circuitry throughout life, and direct regeneration. Throughout these processes, microglia density changes, as does the volume of area that each microglia surveys. Given that microglia are responsible for sensing subtle alterations to their environment, a change in their density could affect their capacity to mobilize rapidly. In this review, we attempt to synthesize the current literature on the ligands and conditions that promote microglial proliferation across development, adulthood, and neurodegenerative conditions. Microglia display an impressive proliferative capacity during development and in neurodegenerative diseases that is almost completely absent at homeostasis. However, the appropriate function of microglia in each state is critically dependent on density fluctuations that are primarily induced by proliferation. Proliferation is a natural microglial response to insult and often serves neuroprotective functions. In contrast, inappropriate microglial proliferation, whether too much or too little, often precipitates undesirable consequences for nervous system health. Thus, fluctuations in the microglia population are tightly regulated to ensure these immune cells can execute their diverse functions.

Endoplasmic reticulum stress in the dorsal root ganglia regulates large-conductance potassium channels and contributes to pain in a model of multiple sclerosis.

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca2+ -sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.

Endoplasmic reticulum-mitochondria interplay in chronic pain: The calcium connection.

Chronic pain is a debilitating condition that affects roughly a third to a half of the world's population. Despite its substantial effect on society, treatment for chronic pain is modest, at best, notwithstanding its side effects. Hence, novel therapeutics are direly needed. Emerging evidence suggests that calcium plays an integral role in mediating neuronal plasticity that underlies sensitization observed in chronic pain states. The endoplasmic reticulum and the mitochondria are the largest calcium repositories in a cell. Here, we review how stressors, like accumulation of misfolded proteins and oxidative stress, influence endoplasmic reticulum and mitochondria function and contribute to chronic pain. We further examine the shuttling of calcium across the mitochondrial-associated membrane as a mechanism of cross-talk between the endoplasmic reticulum and the mitochondria. In addition, we discuss how endoplasmic reticulum stress, mitochondrial impairment, and calcium dyshomeostasis are implicated in various models of neuropathic pain. We propose a novel framework of endoplasmic reticulum-mitochondria signaling in mediating pain hypersensitivity. These observations require further investigation in order to develop novel therapies for chronic pain.

Linguistic Predictors of Problematic Drinking in Alcohol-related Facebook Posts.

Emerging adults often increase problematic drinking during college. Although they generally do not seek help for problematic drinking, college students discuss their drinking on social media. This study followed college students' Facebook profiles from the inception of their attendance at a university and identified alcohol-related posts. Within 28 days of their first alcohol-related Facebook post, participants were interviewed to assess problematic drinking (binge drinking episodes and number of drinks). Linguistic analysis of alcohol-related Facebook posts found that use of negative emotion language and swear words were related to problematic drinking, in support of proposed hypotheses. Results are situated within alcohol use disorder and health research examining the link between problematic drinking and anxiety, deviant behavior, and negative emotions.

Profiling the microRNA signature of the peripheral sensory ganglia in experimental autoimmune encephalomyelitis (EAE).

BACKGROUND: Multiple sclerosis is an autoimmune disease with a distinct female bias, as well as a high prevalence of neuropathic pain in both sexes. The dorsal root ganglia (DRG) contain the primary sensory neurons that give rise to pain, and damage to these neurons may lead to neuropathic pain. Here, we investigate the sex differences of the DRG transcriptome in a mouse model of MS. METHODS: Next-generation sequencing was used to establish RNA and microRNA profiles from the DRG of mice with MOG35-55-induced EAE, a model of CNS inflammation that mimics aspects of MS. Differential expression and multiple meta-analytic approaches were used to compare expression profiles in immunized female and male mice. Differential expression of relevant genes and microRNAs were confirmed by qPCR. RESULTS: Three thousand five hundred twenty genes and 29 microRNAs were differentially expressed in the DRG of female mice with MOG35-55-EAE, while only 189 genes and 3 microRNAs were differentially expressed in males with MOG35-55-EAE. Genes related to the immune system were uniquely regulated in immunized female mice. Direct comparison of sex within disease indicates significant differences in interferon and phagosomal pathways between the sexes. miR-21a-5p is the primary dysregulated microRNA in both sexes, with females having additional dysregulated microRNAs, including miR-122-5p. CONCLUSIONS: This study provides evidence that females are uniquely affected by MOG35-55-EAE and that this difference may result from additional signaling not present in the male. The altered transcriptome of females correlates with other studies finding hyperactivity of pain-sensing neurons and suggests underlying sex-specific pathways for neuropathic pain.

Metabolism and disposition of lesinurad, a uric acid reabsorption inhibitor, in humans.

The objectives of this study were to determine the absolute bioavailability of lesinurad and to characterized its disposition in humans. The oral bioavailability assessment was performed using a clinical design of simultaneous dosing of a therapeutic oral dose of lesinurad with an intravenous infusion of [14C]lesinurad microdose. The bioavailability of lesinurad was determined to be 100%. The disposition of lesinurad in humans involves hepatic oxidation and renal elimination following administration of oral [14C]lesinurad dose. Metabolism of lesinurad occurred post-systemically with low circulating levels of metabolites <3% of total radioactivity as 74.2% of total radioactivity was attributed to lesinurad. In vitro metabolism studies identified CYP2C9 as the predominant isoform, and summation of metabolites indicated that it was responsible for ∼50% of metabolism.

Testing Young Adults' Reactions to Facebook Cues and Their Associations with Alcohol Use.

Background: Alcohol cues on social media may influence young adults' drinking patterns, these cues may be pro-social or pro-alcohol in nature. The influence of individual Facebook cues on young adults' drinking intentions and behaviors remains unknown. Objectives: The purpose of this study was to assess how Facebook cues influence intention to drink, and how intention was associated with Theory of Reasoned Action constructs including alcohol-related attitudes and norms, and future behavior. Methods: Incoming university students completed a pre-college and a 2-year follow-up phone interview. A vignette presented individual Facebook cues representing "pro-social" or "pro-alcohol" sentiments. Participants indicated intention to drink alcohol and their rationale for this intention after each cue. Additional measures included TRA constructs of alcohol-related attitudes and norms, and problem alcohol use. Analyses included a qualitative approach to examine rationales for intention to drink in response to Facebook cues, and linear mixed effects models. Results: Of 338 participants, 56.1% were female, 74.8% were Caucasian. Alcohol-related attitudes and norms were positively associated with intention to drink in response to pro-social and pro-alcohol Facebook cues. Participants' intention to drink in response to pro-alcohol cues was positively associated with problem alcohol use two years later. Conclusions/importance: Findings illuminate the influence of social media on alcohol-related behaviors and highlight potential future screening approaches.

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.

BACKGROUND: Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction. METHODS: We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression. RESULTS: We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines. CONCLUSIONS: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.

Pain in autoimmune disorders.

Most autoimmune diseases are associated with pathological pain development. Autoimmune diseases with pathological pain include complex regional pain syndrome, rheumatoid arthritis, and Guillian-Barré syndrome to name a few. The present Review explores research linking the immune system to the development of pathological pain in autoimmune diseases. Pathological pain has been linked to T-cell activation and the release of cytokines from activated microglia in the dorsal horn of the spinal cord. New research on the role of autoantibodies in autoimmunity has generated insights into potential mechanisms of pain associated with autoimmune disease. Autoantibodies may act through various mechanisms in autoimmune disorders. These include the alteration of neuronal excitability via specific antigens such as the voltage-gated potassium channel complexes or by mediating bone destruction in rheumatoid arthritis. Although more research must be done to understand better the role of autoantibodies in autoimmune disease related pain, this may be a promising area of research for new analgesic therapeutic targets. © 2016 Wiley Periodicals, Inc.

Altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis.

BACKGROUND: Chronic neuropathic pain is a common symptom of multiple sclerosis (MS). MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) has been used as an animal model to investigate the mechanisms of pain in MS. Previous studies have implicated sensitization of spinal nociceptive networks in the pathogenesis of pain in EAE. However, the involvement of supraspinal sites of nociceptive integration, such as the primary somatosensory cortex (S1), has not been defined. We therefore examined functional, structural, and immunological alterations in S1 during the early stages of EAE, when pain behaviors first appear. We also assessed the effects of the antidepressant phenelzine (PLZ) on S1 alterations and nociceptive (mechanical) sensitivity in early EAE. PLZ has been shown to restore central nervous system (CNS) tissue concentrations of GABA and the monoamines (5-HT, NA) in EAE. We hypothesized that PLZ treatment would also normalize nociceptive sensitivity in EAE by restoring the balance of excitation and inhibition (E-I) in the CNS. METHODS: We used in vivo flavoprotein autofluorescence imaging (FAI) to assess neural ensemble responses in S1 to vibrotactile stimulation of the limbs in early EAE. We also used immunohistochemistry (IHC), and Golgi-Cox staining, to examine synaptic changes and neuroinflammation in S1. Mechanical sensitivity was assessed at the clinical onset of EAE with Von Frey hairs. RESULTS: Mice with early EAE exhibited significantly intensified and expanded FAI responses in S1 compared to controls. IHC revealed increased vesicular glutamate transporter (VGLUT1) expression and disrupted parvalbumin+ (PV+) interneuron connectivity in S1 of EAE mice. Furthermore, peri-neuronal nets (PNNs) were significantly reduced in S1. Morphological analysis of excitatory neurons in S1 revealed increased dendritic spine densities. Iba-1+ cortical microglia were significantly elevated early in the disease. Chronic PLZ treatment was found to normalize mechanical thresholds in EAE. PLZ also normalized S1 FAI responses, neuronal morphologies, and cortical microglia numbers and attenuated VGLUT1 reactivity-but did not significantly attenuate the loss of PNNs. CONCLUSIONS: These findings implicate a pro-excitatory shift in the E-I balance of the somatosensory CNS, arising early in the pathogenesis EAE and leading to large-scale functional and structural plasticity in S1. They also suggest a novel antinociceptive effect of PLZ treatment.

Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n. METHODS: We used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n. RESULTS: In the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 µg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4(+) and CD8(+) T cells) into the CNS. CONCLUSION: Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.

Pharmacodynamic, pharmacokinetic and tolerability evaluation of concomitant administration of lesinurad and febuxostat in gout patients with hyperuricaemia.

OBJECTIVE: The aim of this study was to evaluate the pharmacodynamics (PDs), pharmacokinetics (PKs) and safety of lesinurad (selective uric acid reabsorption inhibitor) in combination with febuxostat (xanthine oxidase inhibitor) in patients with gout. METHODS: This study was a phase IB, multicentre, open-label, multiple-dose study of gout patients with serum uric acid (sUA) >8 mg/dl following washout of urate-lowering therapy with colchicine flare prophylaxis. Febuxostat 40 or 80 mg/day was administered on days 1-21, lesinurad 400 mg/day was added on days 8-14 and then lesinurad was increased to 600 mg/day on days 15-21. sUA, urine uric acid and PK profiles were evaluated at the end of each week. Safety was assessed by adverse events, laboratory tests and physical examinations. RESULTS: Initial treatment with febuxostat 40 or 80 mg/day monotherapy resulted in 67% and 56% of subjects, respectively, achieving a sUA level <6 mg/dl. Febuxostat 40 or 80 mg/day plus lesinurad 400 or 600 mg/day resulted in 100% of subjects achieving sUA <6 mg/dl and up to 100% achieving sUA <5 mg/dl. No clinically relevant changes in the PKs of either drug were noted. The combination was well tolerated. CONCLUSION: The clinically important targets of sUA <6 mg/dl and <5 mg/dl are achievable in 100% of patients when combining lesinurad and febuxostat.

The transition from acute to chronic pain: understanding how different biological systems interact.

PURPOSE: Although pain is an adaptive sensory experience necessary to prevent further bodily harm, the transition from acute to chronic pain is not adaptive and results in the development of a chronic clinical condition. How this transition occurs has been the focus of intense study for some time. The focus of the current review is on changes in neuronal plasticity as well as the role of immune cells and glia in the development of chronic pain from acute tissue injury and pain. PRINCIPAL FINDINGS: Our understanding of the complex pathways that mediate the transition from acute to chronic pain continues to increase. Work in this area has already revealed the complex interactions between the nervous and immune system that result in both peripheral and central sensitization, essential components to the development of chronic pain. Taken together, a thorough characterization of the cellular mechanisms that generate chronic pain states is essential for the development of new therapies and treatments. Basic research leading to the development of new therapeutic targets is promising with the development of chloride extrusion enhancers. It is hoped that one day they will provide relief to patients with chronic pain. CONCLUSIONS: A better understanding of how chronic pain develops at a mechanistic level can aid clinicians in treating their patients by showing how the underlying biology of chronic pain contributes to the clinical manifestations of pain. A thorough understanding of how chronic pain develops may also help identify new targets for future analgesic drugs.

Problematic Internet Use and Technology Interactions Among Transgender Adolescents.

Purpose: To compare risk of problematic internet use (PIU) and importance of digital media interactions for transgender and cisgender adolescents. Methods: A nationally representative group of adolescents took an online survey that included a measure of PIU (Problematic and Risky Internet Use Screening Scale-3 [PRIUSS-3]) and technology interactions (Adolescent Digital Technology Interactions and Importance scale). We compared mean scores for these scales and their subscales and rates of positive screens for PIU for transgender and cisgender adolescents. Results: Of 4575 adolescents participating, 53 (1.2%) were transgender, nonbinary, and gender-diverse (TNG) adolescents. TNG adolescents had higher PRIUSS-3 scores and higher mean scores for importance of technology to explore identity/go outside their offline environment. Conclusions: TNG adolescents report higher PIU risk, which may relate to differences in technology importance for this group.

Reducing agents facilitate membrane patch seal integrity and longevity.

The patch clamp method is a widely applied electrophysiological technique used to understand ion channel activity and cellular excitation. The formation of a high resistance giga-ohm seal is required to obtain high-quality recordings but can be challenging due to variables including operator experience and cell preparation. Therefore, the identification of methods to promote the formation and longevity of giga-ohm seals may be beneficial. In this report, we describe our observation that the application of reducing agents (DTT and TCEP) to the external bath solution during whole-cell patch clamp recordings of heterologous cells (HEK and LM) and cultured primary cells (DRG neurons) enhanced the success of giga-ohm seal formation. Reducing agents also maintained the integrity of the seal for longer periods of time at strong hyperpolarizing voltages, whereas an oxidizing agent (H2O2) appeared to have the opposite effect. In summary, we report a useful tool to improve the quality of patch clamp recordings that may be helpful in certain experimental contexts.

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

Family stress during the COVID-19 pandemic: a qualitative analysis.

OBJECTIVE: This study aimed to understand experiences with stress and coping strategies used among families in the COVID-19 pandemic. DESIGN/SETTING: This qualitative study took place in the paediatric outpatient clinics of a large academic medical centre in the USA between March and July of 2021. PARTICIPANTS: Parents (over the age of 18 years) of children under the age of 18 years were invited to complete a 30-minute semistructured interview. PRIMARY AND SECONDARY OUTCOME MEASURES: Participants were asked about types of stressors experienced during the COVID-19 pandemic and coping strategies used. All interviews were audio recorded and transcribed. In the grounded theory tradition, transcripts underwent thematic analysis. RESULTS: A total of 26 participants completed interviews, including 88% (n=23) women, 85% (n=22) reported having children under the age of 10 years and 65% (n=17) were 30-50 years of age. Themes that emerged included the compounding effect of COVID-19 stressors, in which participants described multiple, intersecting sources of stress. One parent noted, "I worked two different jobs, since the other job I had counted on working, I lost because of COVID. And so, working from home, also with the kids, was stressful." The second theme reflected the challenges for children with virtual schooling due to decreased educational support. The third theme was the need for parental self-care. The fourth theme was finding the silver lining in which parents noted unforeseen opportunities for resilience by spending time in nature and activities promoting family bonding. CONCLUSIONS: Parents indicated need for self-care, connecting with their child(ren) and spending time in nature. Future work should develop approaches to support families in these areas when facing complex stressors, especially during a pandemic or other times of crisis.