A large and rapidly increasing body of evidence indicates that microglia-neuron signaling is essential for chronic pain hypersensitivity. Here we show using multiple approaches that microglia are not required for mechanical pain hypersensitivity in female mice; female mice achieve 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.

The innate immune system is increasingly appreciated to play an important role in the mediation of chronic pain, and one molecule implicated in this process is the Toll-like receptor 4 (TLR4). Here using pharmacological and genetic manipulations we found that activating TLR4 in the spinal cord, with the agonist lipopolysaccharide (LPS), causes robust mechanical allodynia but only in male mice. Spinal LPS had no pain-producing effect in female mice. TLR4 also has a sex-specific role in inflammatory (complete Freund’s adjuvant) and neuropathic (spared nerve injury) pain: pain behaviors were TLR4-dependent in males but TLR4-independent in females. The sex differences appear to be specific to the spinal cord, as LPS administered to the brain or the hindpaw produces equivalent allodynia in both sexes, and specific to pain, as intrathecal LPS produces equivalent hypothermia in both sexes. The involvement of TLR4 in pain behaviors in male mice is dependent on testosterone, as shown by gonadectomy and hormone replacement. We found no sex differences in spinal Tlr4 expression at baseline or after LPS, suggesting the existence of parallel spinal pain processing circuitry in female mice not involving TLR4s.

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary1. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da2,3. Using genome-wide linkage analyses, we discovered an association between nerve-injury–induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.

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Joint pathology following DMM surgery in mice is associated with progressive mechanical allodynia. ADAMTS-5 null mice are resistant to DMM-induced OA-like lesions and to the associated mechanical allodynia.

that involves TRPV1, the PI3K/AKT/mTOR/4E-BP1 signaling pathway, and MMP-9, a molecule known to be involved in inflammation and the early stages of chronic pain (18).

Heat sensitivity shows considerable functional variability in humans and laboratory animals, and is fundamental to inflammatory and possibly neuropathic pain. In the mouse, at least, much of this variability is genetic because inbred strains differ robustly in their behavioral sensitivity to noxious heat. These strain differences are shown here to reflect differential responsiveness of primary afferent thermal nociceptors to heat stimuli. We further present convergent behavioral and electrophysiological evidence that the variable responses to noxious heat are due to strain-dependence of CGRP expression and sensitivity. Strain differences in behavioral response to noxious heat could be abolished by peripheral injection of CGRP, blockade of cutaneous and spinal CGRP receptors, or long-term inactivation of CGRP with a CGRP-binding Spiegelmer. Linkage mapping supports the contention that the genetic variant determining variable heat pain sensitivity across mouse strains affects the expression of the Calca gene that codes for CGRP␣.calcitonin gene-related peptide ͉ genetic ͉ Calca ͉ nociceptors ͉ pain H umans display wide individual variability in sensitivity to pain.Although the relative importance of genes versus experience in human pain perception is unclear, recent studies have shown that mouse strains display large differences in behavioral pain sensitivity that are heritable (1). These same studies revealed genetic correlations between baseline thermal nociception and the hypersensitivity states associated with inflammatory and neuropathic pain (2). Of the strains examined, AKR and C57BL͞6 mice displayed the largest and most consistent differences in several different assays of thermal nociception, with AKR being much less sensitive than C57BL͞6. In contrast, AKR mice exhibit more robust heat hyperalgesia after inflammatory or nerve injury (1). Despite our considerable knowledge of the behavioral ''phenomics'' of baseline heat pain and hyperalgesia, there are almost no published data regarding the underlying cellular or molecular mechanisms. Here we show that the observed strain differences in response to thermal stimulation are caused by corresponding differences in the functioning of primary afferent nociceptors. The differences in nociceptor sensitivity, in turn, are caused by the presence of and sensitivity to the neuropeptide calcitonin gene-related polypeptide (CGRP). Finally, linkage mapping revealed a candidate gene likely responsible for the strain difference: Calca, the gene encoding CGRP␣.CGRP␣ is a secretory neuropeptide released from thin nerve fibers at their peripheral and central terminals, which is thought to contribute importantly to neurogenic inflammation in the skin and to central sensitization in the spinal cord (3-6). CGRP acts through a G s protein-coupled receptor complex to activate cAMPdependent protein kinase (PKA). PKA, via the transcription factor cAMP response element-binding protein, enhances expression of pronociceptive genes including the Calca gene itself (7-9). Moreover, many...

BackgroundSpontaneous (non-evoked) pain is a major clinical symptom of neuropathic syndromes, one that is understudied in basic pain research for practical reasons and because of a lack of consensus over precisely which behaviors reflect spontaneous pain in laboratory animals. It is commonly asserted that rodents experiencing pain in a hind limb exhibit hypolocomotion and decreased rearing, engage in both reflexive and organized limb directed behaviors, and avoid supporting their body weight on the affected side. Furthermore, it is assumed that the extent of these positive or negative behaviors can be used as a dependent measure of spontaneous chronic pain severity in such animals. In the present study, we tested these assumptions via blinded, systematic observation of digital video of mice with nerve injuries (chronic constriction or spared nerve injury), and automated assessment of locomotor behavior using photocell detection and dynamic weight bearing (i.e., gait) using the CatWalk® system.ResultsWe found no deficits in locomotor activity or rearing associated with neuropathic injury. The frequency of asymmetric (ipsilaterally directed) behaviors were too rare to be seriously considered as representing spontaneous pain, and in any case did not statistically exceed what was blindly observed on the contralateral hind paw and in control (sham operated and unoperated) mice. Changes in dynamic weight bearing, on the other hand, were robust and ipsilateral after spared nerve injury (but not chronic constriction injury). However, we observed timing, pharmacological, and genetic dissociation of mechanical allodynia and gait alterations.ConclusionsWe conclude that spontaneous neuropathic pain in mice cannot be assessed using any of these measures, and thus caution is warranted in making such assertions.