Spontaneous painful disease in companion animals can facilitate the development of chronic pain therapies for humans.

OBJECTIVE: To outline the role that spontaneous osteoarthritis (OA) in companion animals can play in translational research and therapeutic pharmacological development. OUTLINE: Narrative review summarizing the opportunities and limitations of naturally occurring, spontaneous OA as models of human OA pain, with a focus on companion animal pets. The background leading to considering inserting spontaneous disease models in the translational paradigm is provided. The utility of this model is discussed in terms of outcome measures that have been validated as being related to pain, and in terms of the potential for target discovery is outlined. The limitations to using companion animal pets as models of human disease are discussed. CONCLUSIONS: Although many steps along the translational drug development pathway have been identified as needing improvement, spontaneous painful OA in companion animals offers translational potential. Such 'models' may better reflect the complex genetic, environmental, temporal and physiological influences present in humans and current data suggests the predictive validity of the models are good. The opportunity for target discovery exists but is, as yet, unproven.

Systems genetic and pharmacological analysis identifies candidate genes underlying mechanosensation in the von Frey test.

Mechanical sensitivity is commonly affected in chronic pain and other neurological disorders. To discover mechanisms of individual differences in punctate mechanosensation, we performed quantitative trait locus (QTL) mapping of the response to von Frey monofilament stimulation in BXD recombinant inbred (BXD) mice. Significant loci were detected on mouse chromosome (Chr) 5 and 15, indicating the location of underlying polymorphisms that cause heritable variation in von Frey response. Convergent evidence from public gene expression data implicates candidate genes within the loci: von Frey thresholds were strongly correlated with baseline expression of Cacna2d1, Ift27 and Csnk1e in multiple brain regions of BXD strains. Systemic gabapentin and PF-670462, which target the protein products of Cacna2d1 and Csnk1e, respectively, significantly increased von Frey thresholds in a genotype-dependent manner in progenitors and BXD strains. Real-time polymerase chain reaction confirmed differential expression of Cacna2d1 and Csnk1e in multiple brain regions in progenitors and showed differential expression of Cacna2d1 and Csnk1e in the dorsal root ganglia of the progenitors and BXD strains grouped by QTL genotype. Thus, linkage mapping, transcript covariance and pharmacological testing suggest that genetic variation affecting Cacna2d1 and Csnk1e may contribute to individual differences in von Frey filament response. This study implicates Cacna2d1 and Ift27 in basal mechanosensation in line with their previously suspected role in mechanical hypersensitivity. Csnk1e is implicated for von Frey response for the first time. Further investigation is warranted to identify the specific polymorphisms involved and assess the relevance of these findings to clinical conditions of disturbed mechanosensation.

Constitutive µ-opioid receptor activity leads to long-term endogenous analgesia and dependence.

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced µ-opioid receptor (MOR) constitutive activity (MOR(CA)) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3',5'-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MOR(CA) initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MOR(CA) suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

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 Nav 1.8-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.

Positional cloning of a quantitative trait locus contributing to pain sensitivity: possible mediation by Tyrp1.

To identify novel pain-relevant genes, a set of 35 recombinant congenic strains derived from the sensitive C57BL/6 and resistant A/J strains were tested for their sensitivity to noxious heat on the radiant heat paw-withdrawal test. Nine strains were found to display differential sensitivity, and the two most extreme responders were used to generate independent secondary crosses for quantitative trait locus (QTL) mapping. From these genetic analyses, a QTL, which we call Tpnr5, was mapped to a 14-Mb interval of mouse chromosome 4 containing 39 genes. In addition to the paw-withdrawal test phenotype, Tpnr5 may be relevant to mechanical and inflammatory nociception. A series of strategies - including in silico analyses, reverse transcriptase polymerase chain reaction (RT-PCR) in multiple tissues and exonic DNA sequencing - were used to generate a list of six prioritized candidate genes. One of these, tyrosinase-related protein 1 (Tyrp1), displayed enriched expression in the dorsal root ganglia, an inactivating (C110Y) mutation in the resistant A/J strain, and a null mutant found to be more resistant to thermal nociception compared to its wild-type counterpart. Although other genes cannot be definitively ruled out, existing data are supportive of the candidacy of Tyrp1 as representing the Tpnr5 QTL. Tyrosinase-related protein 1 is the rate-limiting enzyme in the production of eumelanin, and possible relationships between eumelanin-expressing cells and thermal nociception are discussed. The positional cloning of Tpnr5 is also considered in light of the heuristic value but continuing challenges of QTL mapping in the mouse.

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.

Gnao1 (G alphaO 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.

Melanocortin-1 receptor gene variants affect pain and mu-opioid analgesia in mice and humans.

BACKGROUND: A recent genetic study in mice and humans revealed the modulatory effect of MC1R (melanocortin-1 receptor) gene variants on kappa-opioid receptor mediated analgesia. It is unclear whether this gene affects basal pain sensitivity or the efficacy of analgesics acting at the more clinically relevant mu-opioid receptor. OBJECTIVE: To characterise sensitivity to pain and mu-opioid analgesia in mice and humans with non-functional melanocortin-1 receptors. METHODS: Comparisons of spontaneous mutant C57BL/6-Mc1r(e/e) mice to C57BL/6 wildtype mice, followed by a gene dosage study of pain and morphine-6-glucuronide (M6G) analgesia in humans with MC1R variants. RESULTS: C57BL/6-Mc1r(e/e) mutant mice and human redheads--both with non-functional MC1Rs--display reduced sensitivity to noxious stimuli and increased analgesic responsiveness to the mu-opioid selective morphine metabolite, M6G. In both species the differential analgesia is likely due to pharmacodynamic factors, as plasma levels of M6G are similar across genotype. CONCLUSIONS: Genotype at MC1R similarly affects pain sensitivity and M6G analgesia in mice and humans. These findings confirm the utility of cross species translational strategies in pharmacogenetics.

Serotonin-GABA interactions in the modulation of mu- and kappa-opioid analgesia.

In the present study, we studied the interaction between serotonergic (5-HTergic) and gamma-aminobutyric acid (GABA)-ergic systems in the modulation of analgesia from morphine, a mu-opioid agonist, and U50,488, a kappa-opioid agonist. All experiments were performed in mice using the 49 degrees C tail-withdrawal assay. The benzodiazepine receptor agonist, diazepam, the serotonin synthesis inhibitor, para-chlorophenylalanine (p-CPA), and the 5-HT(1A) receptor agonist, 8-OH-DPAT, were all found to attenuate morphine and U50,488 analgesia. In each case, the attenuation was itself blocked by treatment with L-5-HTP, a serotonin precursor, bicuculline, a GABA(A) receptor antagonist or picrotoxin, a GABA(A)-gated chloride channel blocker. Neither L-5-HTP nor the GABA(A) receptor antagonists were found to affect morphine or U50,488 analgesia per se. Thus, these findings indicate that a benzodiazepine-GABAergic agent (diazepam) attenuates opioid analgesia through the serotonergic system, and antiserotonergic agents (8-OH-DPAT, p-CPA) attenuate opioid analgesia through the GABAergic system. The intimate interactions between GABA and serotonin in the present study further suggest that these neurotransmitters work in complex ways together rather than alone in the modulation of opioid analgesia.

Naloxone-precipitated withdrawal jumping in 11 inbred mouse strains: evidence for common genetic mechanisms in acute and chronic morphine physical dependence.

Physical dependence is a widely known consequence of morphine intake. Although commonly associated with prolonged or repeated morphine administration, withdrawal symptoms can be elicited even after a single prior morphine exposure. What remains contentious is the extent to which physical dependence following acute and chronic morphine treatment is mediated by common physiological substrates and, accordingly, represent distinct syndromes. The genetic relationship between acute and chronic morphine dependence was thus presently studied by comparing mice of 11 inbred strains (129P3, A, AKR, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and SWR) for naloxone-precipitated withdrawal jumping responses using three subcutaneous morphine administration paradigms: acute (single injection) or chronic (three daily morphine injections for 4 days) injection, or chronic infusion (7 days via implanted osmotic minipumps). Although there were differences in the magnitude of withdrawal jumping between the three different morphine administration paradigms, large and significant strain differences were observed for each. In addition, the same strains were unusually sensitive or, conversely, altogether refractory to withdrawal jumping across all morphine treatment conditions. Overall, strain jumping means between acute and chronic dependence paradigms displayed a high degree of genetic correlation (r=0.87-0.95). The significant correlation between chronic morphine injection and continuous morphine infusion discounts the possible confounding effect of contextual learning and spontaneous withdrawal between chronic injections on the assessment of naloxone-precipitated withdrawal. Substantial heritability was also observed for acute and both paradigms of chronic dependence, with estimates ranging from h(2)=0.53 to 0.70. The present demonstration of a strong genetic correlation between physical dependence to morphine following acute and chronic treatment implies that genes associated with variable sensitivity in the two traits are the same, and is suggestive of shared physiological substrates. The data also demonstrate that the differential genetic liability to morphine physical dependence begins with, and is predicted by, the first morphine exposure.

Measuring pain in the (knockout) mouse: big challenges in a small mammal.

The increasing popularity of the mouse as a subject in basic science studies of pain can largely be attributed to the development of transgenic "knockout" technology in this species only. To take advantage of this biological technique, many investigators are rushing to adapt to the mouse experimental protocols that were designed for the rat. However, the myriad physiological and behavioral differences between these two rodent species render such adaptations non-trivial and in many cases seriously problematic. In this article we review the basic nociceptive assays used in behavioral pain research (thermal, mechanical, electrical and chemical), and highlight how species differences affect their proper application. In addition, some of the issues specifically pertaining to the interpretation of such data in knockout studies are addressed.

Endogenous nociceptin signaling and stress-induced analgesia.

Nociceptin/orphanin FQ (NC) and its receptor (OP4) have been implicated in pain transmission. The aim of the present study was to investigate the role of the NC/OP4 system in stress-induced analgesia (SIA). The tail-withdrawal assay was performed in mice stressed by forced swimming in water at 15 degrees C (high severity swims) or 32 degrees C (low severity swims). High severity swims produced a naloxone-insensitive antinociceptive effect which was blocked by supraspinal NC (1 nmol). The selective OP4 receptor antagonist, [Nphe1]NC(-13)NH2 (30 nmol), was inactive by itself, but prevented the effect of NC. Low severity swims produced a milder analgesic effect that was partially antagonized by naloxone, completely blocked by NC and potentiated by [Nphe1]NC(-13)NH2. These findings confirm the anti-analgesic role of supraspinal NC and suggest that endogenous NC signaling counteracts the opioid component of SIA.

The pharmacogenetics of analgesia: toward a genetically-based approach to pain management.

Interindividual differences in the experience of pain have been appreciated clinically for over a century. More recently, there has been a growing body of evidence demonstrating differences in analgesic response to various pharmacotherapies, although the source of this variability largely remains to be explained. To this end, basic science research is beginning to identify the allelic variants that underlie such antinociceptive variability using a multiplicity of animal models, and powerful genetic approaches are being exploited to accelerate this process. Although the vast majority of these studies have focused on the pharmacogenetics of opioids, owing to their prominent status as analgesics, the number of pharmacotherapies evincing genetically-based variability is rapidly expanding. In addition, analogous studies have been undertaken in humans, as a small but growing number of clinical trials have begun to evaluate prospectively the existence, if oftentimes not the origin, of interindividual differences in analgesic drug response. Importantly, with a few notable exceptions, such efforts have primarily identified differences in analgesic efficacy and/or potency between male and female human subjects. Looking toward the future development of one or more widely utilised, pharmacogenetic screens that would lead to modifications in treatment planning, at least with respect to the pharmacologic management of pain, this review will document the breadth of genetically-based variability in drug-mediated antinociception in animals. Specific examples in which the gene or genes underlying such variability have been postulated or identified will be given, while highlighting the effect of sex and its interactions with other genetic backgrounds. Finally, we will summarise and evaluate the literature on pharmacogenetic differences in human analgesic drug response, for which the influence of sex has served as one of the better studied and heuristically insightful examples.

The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family.

The isolation of an opioid receptor-related clone soon led to the isolation and characterization of a new neuropeptide, termed orphanin FQ or nociceptin (OFQ/N). This heptadecapeptide binds to the NOP(1) (previously termed ORL1) receptor with exceedingly high affinity, but does not interact directly with classical opioid receptors. Functionally, the actions of OFQ/N are diverse and intriguing. Most work has focused upon pain mechanisms, where OFQ/N has potent anti-analgesic actions supraspinally and analgesic actions spinally. Other OFQ/N activities are less clear. The diversity of responses might reflect NOP(1) receptor heterogeneity, but this remains to be established. The actions of this neurochemical system may also be uniquely dependent on contextual factors, both genetic and environmental. This review will address the molecular biology and behavioral pharmacology of OFQ/N and its receptor.

Quantitative trait loci influencing morphine antinociception in four mapping populations.

Analgesia (pain reduction, or antinociception) is a classical and clinically important effect of morphine administration, and in rodent models sensitivity to morphine has been shown to be strongly influenced by genotype. For example, several studies have reported marked differences in morphine antinociception between the insensitive C57BL/6 (B6) and sensitive DBA/2 (D2) inbred mouse strains on the hot-plate assay. This prompted the present genome-wide search for quantitative trait loci (QTLs) that are chromosomal sites influencing the magnitude of antinociception, by using four mapping populations derived from the B6 and D2 progenitor inbred strains. These four were the BXD recombinant inbred (RI) strain set, an F2 (B6D2F2) population, short-term selective breeding for antinociception from a B6D2F2 founding population, and incipient or completed congenic strains. In the BXD RI set and in the B6D2F2, a genome-wide search identified 10-12 provisional QTLs at a nominal p <.05. The other populations were subsequently used as confirmation steps to test each of the provisional QTL regions. Based on all available mapping populations, four QTLs emerged as significant (p <.00005) on proximal Chromosome (Chr) 1 (females only), proximal Chr 9 (females only), mid Chr 9, and proximal Chr 10. The Chr 10 QTL comaps to the same region as the micro-opioid receptor gene (Oprm); this receptor is a known mediator of morphine's antinociceptive effects. The Chr 1 QTL was evident only in females and comapped with the kappa-opioid receptor gene, Oprk.

Morphine tolerance and dependence in nociceptin/orphanin FQ transgenic knock-out mice.

It has been hypothesized that morphine tolerance and dependence in mice following chronic exposure may reflect increased compensatory activity of antiopioid systems. The endogenous peptide nociceptin/orphanin FQ has been shown to have anti-opioid effects, for example antagonizing morphine analgesia. Moreover, chronic morphine administration increases synthesis of the peptide, and morphine tolerance and dependence can be attenuated or reversed by antagonists and agonists of the nociceptin/orphanin FQ receptor, respectively. The present study seeks to confirm a role for nociceptin/orphanin FQ in opioid tolerance and dependence by comparing morphine ED(50) values and naloxone-precipitated withdrawal jumping in mice homozygous (knock-out) and heterozygous for a null mutation of the Npnc1 gene encoding the nociceptin/orphanin FQ propeptide, and their wild type littermates, following chronic morphine exposure. Relative to morphine-naive control mice, significant rightward shifts in the morphine dose-response curve, resulting in increased morphine ED(50) values (approximately two to three-fold), was observed for all genotypes following three days of repeated systemic morphine injections. However, no differences between genotypes in the magnitude of tolerance were observed. In contrast, knock-out mice displayed significantly increased naloxone-precipitated withdrawal jumping relative to heterozygous and wild-type mice following implantation with a morphine pellet (25mg) for 72h. Use of nociception/orphaninFQ transgenic knock-out mice thus demonstrate the differential involvement of nociceptin/orphanin FQ in morphine tolerance and dependence.

Transgenic studies of pain and analgesia: mutation or background genotype?

The application of transgenic (knockout) technology to the study of pain is rapidly expanding. Despite its power, this technique has several shortcomings that complicate the interpretation of the data obtained. Although compensation by other genes is a well recognized problem, issues related to the background genotype of the mutant mice are less well appreciated. This review describes these confounds as they apply to studies of pain and pain inhibition. We show that the 129 and C57BL/6 mouse strains, which provide the default genetic background on which null mutants are constructed, display significant and sometimes extreme phenotypic differences in many assays of nociception, hypersensitivity, and analgesia. Although problems related to the differential responsiveness of the two strains are minimized by placing knockouts onto "pure" 129 and/or C57BL/6 backgrounds, we also illustrate that neither of these strains are particularly representative of inbred mice in general. Procedures to reduce confounds and converging evidence must be used to accurately determine the functions of the targeted genes in pain-related phenomena.

The effect of genotype on sensitivity to electroacupuncture analgesia.

Individual differences in sensitivity to pain and analgesia are well appreciated, and increasing evidence has pointed towards a role of inherited genetic factors in explaining some proportion of such variability. It has long been known by practitioners of acupuncture, an ancient modality of analgesia, that some patients are 'responders' and others 'non-responders.' The present research was aimed at defining the inherited genetic influence on acupuncture analgesia in the mouse, using 10 common inbred strains. Two pairs of metallic needles were inserted into acupoints ST 36 and SP 6, fixed in situ and then connected to the output channel of an electric pulse generator. Electroacupuncture (EA) parameters were set as constant current output (intensity: 1.0-1.5-2.0 mA, 10 min each; frequency: 2 or 100 Hz) with alteration of a positive and negative square wave, 0.3 ms in pulse width. Tail-flick latencies evoked by radiant heat were measured before, during and after EA stimulation. Narrow-sense heritability estimates of 2 and 100 Hz EA were 0.37 and 0.16, respectively. We found that the C57BL/10 strain was the most sensitive, and the SM strain was the least sensitive to both 2 and 100 Hz EA. However, the relative sensitivities of other strains to these two EA frequencies suggested some genetic dissociation between them as well. These results demonstrate a role of inherited genetic factors in EA sensitivity in the mouse, although the low-to-moderate heritability estimates suggest that environmental factors may be of greater importance in predicting who will benefit from this analgesic modality.