Chloride Dysregulation through Downregulation of KCC2 Mediates Neuropathic Pain in Both Sexes.

The behavioral features of neuropathic pain are not sexually dimorphic despite sex differences in the underlying neuroimmune signaling. This raises questions about whether neural processing is comparably altered. Here, we test whether the K+-Cl- co-transporter KCC2, which regulates synaptic inhibition, plays an equally important role in development of neuropathic pain in male and female rodents. Past studies on KCC2 tested only males. We find that inhibiting KCC2 in uninjured animals reproduces behavioral and electrophysiological features of neuropathic pain in both sexes and, consistent with equivalent injury-induced downregulation of KCC2, that counteracting chloride dysregulation reverses injury-induced behavioral and electrophysiological changes in both sexes. These findings demonstrate that KCC2 downregulation contributes equally to pain hypersensitivity in males and females. Whereas diverse (and sexually dimorphic) mechanisms regulate KCC2, regulation of intracellular chloride relies almost exclusively on KCC2. Directly targeting KCC2 thus remains a promising strategy for treatment of neuropathic pain in both sexes.

Microglial P2X4R-evoked pain hypersensitivity is sexually dimorphic in rats.

Microglia-neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in mice: spinal mechanisms underlying nerve injury-induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury-induced hypersensitivity in males differ from those in females, with microglial P2X4 receptors (P2X4Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X4Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X4Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X4Rs drove intracellular signalling through p38 mitogen-activated protein kinase. Furthermore, chromatin immunoprecipitation-qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X4R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species.

Pain in ankylosing spondylitis: a neuro-immune collaboration.

Clinicians have commonly differentiated chronic back pain into two broad subsets: namely, non-inflammatory (or mechanical) back pain and inflammatory back pain. As the terminology suggests, the latter category, in which ankylosing spondylitis (AS) is prominent, presupposes a close link between pain and inflammation. Advances in research into the genetics and immunology of AS have improved our understanding of the inflammatory processes involved in this disease, and have led to the development of potent anti-inflammatory biologic therapeutic agents. However, evidence from clinical trials and from biomarker and imaging studies in patients with AS indicate that pain and inflammation are not always correlated. Thus, the assumption that pain in AS is a reliable surrogate marker for inflammation might be an over-simplification. This Review provides an overview of current concepts relating to neuro-immune interactions in AS and summarizes research that reveals an increasingly complex interplay between the activation of the immune system and pain pathways in the nervous system. The different types of pain experienced by patients with AS, insights from brain imaging studies, neurological mechanisms of pain, sex bias in pain and how the immune system can modify pain in patients with AS are also discussed.

Molecules in pain and sex: a developing story.

Microglia are dynamic immune cells with diverse roles in maintaining homeostasis of the central nervous system. Dysregulation of microglia has been critically implicated in the genesis of neuropathic pain. Peripheral nerve injury, a common cause of neuropathic pain, engages microglia-neuronal signalling which causes disinhibition and facilitated excitation of spinal nociceptive pathways. However, recent literature indicates that the role of microglia in neuropathic pain is sexually dimorphic, and that female pain processing appears to be independent of microglia, depending rather on T cells. Despite this sex difference, pain signalling in the spinal cord converges downstream of microglia, as NMDAR-mediated facilitated excitation in pain transmitting neurons is consistent between males and females. Determining whether pain signalling is sexually dimorphic in humans and, further, addressing the sex bias in pain research will increase the translational relevance of preclinical findings and advance our understanding of chronic pain in women.

Inhibition of the kinase WNK1/HSN2 ameliorates neuropathic pain by restoring GABA inhibition.

HSN2is a nervous system predominant exon of the gene encoding the kinase WNK1 and is mutated in an autosomal recessive, inherited form of congenital pain insensitivity. The HSN2-containing splice variant is referred to as WNK1/HSN2. We created a knockout mouse specifically lacking theHsn2exon ofWnk1 Although these mice had normal spinal neuron and peripheral sensory neuron morphology and distribution, the mice were less susceptible to hypersensitivity to cold and mechanical stimuli after peripheral nerve injury. In contrast, thermal and mechanical nociceptive responses were similar to control mice in an inflammation-induced pain model. In the nerve injury model of neuropathic pain, WNK1/HSN2 contributed to a maladaptive decrease in the activity of the K(+)-Cl(-)cotransporter KCC2 by increasing its inhibitory phosphorylation at Thr(906)and Thr(1007), resulting in an associated loss of GABA (γ-aminobutyric acid)-mediated inhibition of spinal pain-transmitting nerves. Electrophysiological analysis showed that WNK1/HSN2 shifted the concentration of Cl(-)such that GABA signaling resulted in a less hyperpolarized state (increased neuronal activity) rather than a more hyperpolarized state (decreased neuronal activity) in mouse spinal nerves. Pharmacologically antagonizing WNK activity reduced cold allodynia and mechanical hyperalgesia, decreased KCC2 Thr(906)and Thr(1007)phosphorylation, and restored GABA-mediated inhibition (hyperpolarization) of injured spinal cord lamina II neurons. These data provide mechanistic insight into, and a compelling therapeutic target for treating, neuropathic pain after nerve injury.

Sex differences in pain: a tale of two immune cells.

Substantial evidence has implicated microglia in neuropathic pain. After peripheral nerve injury, microglia in the spinal cord proliferate and increase cell-surface expression of the purinergic receptor P2X4. Activation of P2X4 receptors results in release of brain-derived neurotrophic factor, which acts on neurons to produce disinhibition of dorsal horn neurons which transmit nociceptive information to the brain. Disinhibition of these neurons produces pain hypersensitivity, a hallmark symptom of neuropathic pain. However, elucidating this microglia-neuronal signalling pathway was based on studies using only male rodents. Recent evidence has shown that the role of microglia in pain is sexually dimorphic. Despite similar microglia proliferation in the dorsal horn in both sexes, females do not upregulate P2X4Rs and use a microglia-independent pathway to mediate pain hypersensitivity. Instead, adaptive immune cells, possibly T cells, may mediate pain hypersensitivity in female mice. This profound sex difference highlights the importance of including subjects of both sexes in preclinical pain research.

Different immune cells mediate mechanical pain hypersensitivity in male and female mice.

A large and rapidly increasing body of evidence indicates that microglia-to-neuron signaling is essential for chronic pain hypersensitivity. Using multiple approaches, we found that microglia are not required for mechanical pain hypersensitivity in female mice; female mice achieved similar levels of pain hypersensitivity using adaptive immune cells, likely T lymphocytes. This sexual dimorphism suggests that male mice cannot be used as proxies for females in pain research.

Olfactory exposure to males, including men, causes stress and related analgesia in rodents.

We found that exposure of mice and rats to male but not female experimenters produces pain inhibition. Male-related stimuli induced a robust physiological stress response that results in stress-induced analgesia. This effect could be replicated with T-shirts worn by men, bedding material from gonadally intact and unfamiliar male mammals, and presentation of compounds secreted from the human axilla. Experimenter sex can thus affect apparent baseline responses in behavioral testing.

Pain reduces sexual motivation in female but not male mice.

Chronic pain is often associated with sexual dysfunction, suggesting that pain can reduce libido. We find that inflammatory pain reduces sexual motivation, measured via mounting behavior and/or proximity in a paced mating paradigm, in female but not male laboratory mice. Pain was produced by injection of inflammogens zymosan A (0.5 mg/ml) or λ-carrageenan (2%) into genital or nongenital (hind paw, tail, cheek) regions. Sexual behavior was significantly reduced in female mice experiencing pain (in all combinations); male mice similarly treated displayed unimpeded sexual motivation. Pain-induced reductions in female sexual behavior were observed in the absence of sex differences in pain-related behavior, and could be rescued by the analgesic, pregabalin, and the libido-enhancing drugs, apomorphine and melanotan-II. These findings suggest that the well known context sensitivity of the human female libido can be explained by evolutionary rather than sociocultural factors, as female mice can be similarly affected.

The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions.

We recently demonstrated the utility of quantifying spontaneous pain in mice via the blinded coding of facial expressions. As the majority of preclinical pain research is in fact performed in the laboratory rat, we attempted to modify the scale for use in this species. We present herein the Rat Grimace Scale, and show its reliability, accuracy, and ability to quantify the time course of spontaneous pain in the intraplantar complete Freund's adjuvant, intraarticular kaolin-carrageenan, and laparotomy (post-operative pain) assays. The scale's ability to demonstrate the dose-dependent analgesic efficacy of morphine is also shown. In addition, we have developed software, Rodent Face Finder®, which successfully automates the most labor-intensive step in the process. Given the known mechanistic dissociations between spontaneous and evoked pain, and the primacy of the former as a clinical problem, we believe that widespread adoption of spontaneous pain measures such as the Rat Grimace Scale might lead to more successful translation of basic science findings into clinical application.