Jean-Sebastien Austin

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.

Spinal Cord Toll-Like Receptor 4 Mediates Inflammatory and Neuropathic Hypersensitivity in Male But Not Female Mice

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 Freunds 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 gene expression at baseline or after LPS, suggesting the existence of parallel spinal pain-processing circuitry in female mice not involving TLR4.

Patterns of pain: Meta-analysis of microarray studies of pain

&NA; Existing microarray gene expression profiling studies of tonic/chronic pain were subjected to meta‐analysis to identify genes found to be regulated by these pain states in multiple, independent experiments. Twenty studies published from 2002 to 2008 were identified, describing the statistically significant regulation of 2254 genes. Of those, a total of 79 genes were found to be statistically significant “hits” in 4 or more independent microarray experiments, corresponding to a conservative P < 0.01 overall. Gene ontology‐based functional annotation clustering analyses revealed strong evidence for regulation of immune‐related genes in pain states. A multi‐gene quantitative real‐time polymerase chain reaction experiment was run on dorsal root ganglion (DRG) and spinal cord tissue from rats and mice given nerve (sciatic chronic constriction; CCI) or inflammatory (complete Freund’s adjuvant) injury. We independently confirmed the regulation of 43 of these genes in the rat‐CCI‐DRG condition; the genetic correlates in all other conditions were largely and, in some cases, strikingly, independent. However, a handful of genes were identified whose regulation bridged etiology, anatomical locus, and/or species. Most notable among these were Reg3b (regenerating islet‐derived 3 beta; pancreatitis‐associated protein) and Ccl2 (chemokine [C–C motif] ligand 2), which were significantly upregulated in every condition in the rat. Gene expression profiling (microarray) studies of chronic pain were subjected to meta‐analysis. Two genes were identified that are consistently upregulated in chronic pain states.

ADAMTS-5 deficient mice do not develop mechanical allodynia associated with osteoarthritis following medial meniscal destabilization

OBJECTIVE To characterize pain-related behavior during the course of knee osteoarthritis (OA) induced by destabilization of the medial meniscus (DMM) in wild type (WT) and in ADAMTS-5 null mice. METHODS DMM surgery was performed in the right knee of CD-1 mice. At regular intervals up to 8 weeks after surgery, mice were assessed for the following parameters: mechanical allodynia (via withdrawal thresholds to von Frey filaments applied to the plantar surface of both hind paws or to the tail), thermal hyperalgesia, locomotor activity and gait analysis. In addition, mechanical allodynia was tested in C57BL/6 WT or ADAMTS-5 null mice following DMM surgery. RESULTS In CD-1 mice, a robust and progressive decrease in withdrawal threshold was observed in both hind paws after DMM but not sham surgery. Allodynia was apparent as early as 14 days postoperatively. Both sexes developed OA changes after surgery with concurrent mechanical allodynia. No other pain-related behavioral changes were detected up to 8 weeks post-surgery. In C57BL/6 mice, a genetic background in which only males develop OA changes after DMM, males but not females developed allodynia in the ipsilateral hind paw. In contrast, C57BL/6 ADAMTS-5 null mice did not develop OA changes or mechanical allodynia up to 8 weeks post-surgery. CONCLUSION 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.

Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice.

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.

Role of Central Calcitonin Gene-Related Peptide (CGRP) in Locomotor and Anxiety- and Depression-Like Behaviors in Two Mouse Strains Exhibiting a CGRP-Dependent Difference in Thermal Pain Sensitivity

We have previously shown that, in AKR and C57BL/6 mice, a genetic polymorphism results in differential expression of the peptide, calcitonin gene-related polypeptide (CGRP), explaining a strain difference in thermal pain sensitivity. Although CGRP is widely distributed in the brain, little is known about the effects of supraspinal CGRP. We used AKR and C57BL/6 mice as a model to explore the effects of centrally (intracerebroventricular) injected CGRP and the CGRP receptor antagonists, CGRP8–37 and BIBN4096BS, in a series of behavioral assays. Locomotor activity was significantly increased in C57BL/6 mice following the injection of BIBN4096BS and in both strains after the administration of CGRP8–37 into the third ventricle. CGRP increased paw-withdrawal latencies in C57BL/6 mice only, while decreasing depression-like behaviors in both strains in the forced-swimming test. CGRP and CGRP receptor antagonists failed to modulate activity in the elevated plus maze, a model of anxiety. Taken together, these results suggest a complex role for supraspinal CGRP systems in the regulation of locomotion, nociception, and depression-like behaviors.

Gnao1 (GαO protein) is a likely genetic contributor to variation in physical dependence on opioids in mice

Chronic exposure to opioids leads to physical dependence, which manifests as the symptoms of drug withdrawal. Interindividual differences in withdrawal symptom severity are well known, and at least partially due to genetic variation. To identify genes contributing to variation in withdrawal severity, we chronically treated 30 strains of the AcB/BcA recombinant congenic mouse strain set, including their A/J and C57BL/6J (B6) progenitors, with morphine for seven days and compared jumping frequencies--a sensitive and widely used index of withdrawal magnitude--during naloxone-precipitated withdrawal (NPW). Jumping frequencies of B6 mice were more than threefold greater than values obtained in A/J mice. Visual inspection of the genomic distribution of parental haplotypes in the AcB/BcA strains identified a putative quantitative trait locus (QTL) localized to chromosome 8 (90-117 Mb), and this QTL was confirmed in a B6AF2 intercross. The most salient candidate gene within this QTL, Gnao1 (guanine nucleotide binding protein, alpha(o); G alpha(o); 96.3 Mb), was tested for functional relevance using quantitative PCR and an antisense oligodeoxynucleotide strategy. The expression of Gnao1 in the locus coeruleus was found to be upregulated in morphine-dependent B6 but not A/J mice. Antisense knockdown of Gnao1 reduced NPW jumping in B6, but not A/J, mice rendered dependent on either morphine or heroin, largely rescuing the original strain difference. These data strongly implicate the G alpha(o) protein in the locus coeruleus as contributing to interindividual variability in physical dependence on opioids in mice.

Social propinquity in rodents as measured by tube cooccupancy differs between inbred and outbred genotypes

Significance We developed an assay of social behavior, the tube cooccupancy test. This assay is able to identify deficits in social behavior of known “autistic-like” mouse strains. Using the test, we revealed a major difference in social behavior between inbred and outbred strains, with only the latter behaving similar to wild mice. Existing assays of social interaction are suboptimal, and none measures propinquity, the tendency of rodents to maintain close physical proximity. These assays are ubiquitously performed using inbred mouse strains and mutations placed on inbred genetic backgrounds. We developed the automatable tube cooccupancy test (TCOT) based on propinquity, the tendency of freely mobile rodents to maintain close physical proximity, and assessed TCOT behavior on a variety of genotypes and social and environmental conditions. In outbred mice and rats, familiarity determined willingness to cooccupy the tube, with siblings and/or cagemates of both sexes exhibiting higher cooccupancy behavior than strangers. Subsequent testing using multiple genotypes revealed that inbred strain siblings do not cooccupy at higher rates than strangers, in marked contrast to both outbred and rederived wild mice. Mutant mouse strains with “autistic-like” phenotypes (Fmr1−/y and Eif4e Ser209Ala) displayed significantly decreased cooccupancy.

T-Cell Mediation of Pregnancy Analgesia Affecting Chronic Pain in Mice

It has been reported consistently that many female chronic pain sufferers have an attenuation of symptoms during pregnancy. Rats display increased pain tolerance during pregnancy due to an increase in opioid receptors in the spinal cord. Past studies did not consider the role of non-neuronal cells, which are now known to play an important role in chronic pain processing. Using an inflammatory (complete Freunds adjuvant) or neuropathic (spared nerve injury) model of persistent pain, we observed that young adult female mice in early pregnancy switch from a microglia-independent to a microglia-dependent pain hypersensitivity mechanism. During late pregnancy, female mice show no evidence of chronic pain whatsoever. This pregnancy-related analgesia is reversible by intrathecal administration of naloxone, suggesting an opioid-mediated mechanism; pharmacological and genetic data suggest the importance of δ-opioid receptors. We also observe that T-cell-deficient (nude and Rag1-null mutant) pregnant mice do not exhibit pregnancy analgesia, which can be rescued with the adoptive transfer of CD4+ or CD8+ T cells from late-pregnant wild-type mice. These results suggest that T cells are a mediator of the opioid analgesia exhibited during pregnancy. SIGNIFICANCE STATEMENT Chronic pain symptoms often subside during pregnancy. This pregnancy-related analgesia has been demonstrated for acute pain in rats. Here, we show that pregnancy analgesia can produce a complete cessation of chronic pain behaviors in mice. We show that the phenomenon is dependent on pregnancy hormones (estrogen and progesterone), δ-opioid receptors, and T cells of the adaptive immune system. These findings add to the recent but growing evidence of sex-specific T-cell involvement in chronic pain processing.

The Yin and Yang of pain: variability in formalin test nociception and morphine analgesia produced by the Yin Yang 1 transcription factor gene.

We recently observed a reliable phenotypic difference in the inflammatory pain sensitivity of a congenic mouse strain compared to its background strain. By constructing and testing subcongenic strains combined with gene‐expression assays, we provide evidence for the candidacy of the Yy1 gene – encoding the ubiquitously expressed and multifunctional Yin Yang 1 transcription factor – as responsible. To confirm this hypothesis, we used a Cre/lox strategy to produce mutant mice in which Yy1 expression was ablated in Nav1.8‐positive neurons of the dorsal root ganglion. These mutants also displayed reduced inflammatory pain sensitivity on the formalin test. Further testing of pain‐related phenotypes in these mutants revealed robustly increased sensitivity to systemic and spinal (but not supraspinal) morphine analgesia, and greatly increased endogenous (swim stress‐induced) opioid analgesia. None of the known biological roles of Yin Yang 1 were suggestive of such a phenotype, and thus a novel player in pain modulatory systems has been identified.