Michael L. LaCroix-Fralish

Coding of facial expressions of pain in the laboratory mouse

Facial expression is widely used as a measure of pain in infants; whether nonhuman animals display such pain expressions has never been systematically assessed. We developed the mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability; assays involving noxious stimuli of moderate duration are accompanied by facial expressions of pain. This measure of spontaneously emitted pain may provide insight into the subjective pain experience of mice.

Animal models and the prediction of efficacy in clinical trials of analgesic drugs : A critical appraisal and call for uniform reporting standards

a Pain Research, Department of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea & Westminster Hospital Campus, 369 Fulham Road, London SW10 9NH, UK b Mari Lowe Centre for Comparative Oncology Research, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104-6010, USA c Department of Anesthesia, Wake Forest University School of Medicine, Medical Center Boulevard, Winston Salem, NC 27157, USA d Department of Anaesthesiology and Intensive Care Medicine, Helsinki University Central Hospital, Helsinki, Finland e Pfizer Global Research and Development, Sandwich CT13 9NJ, UK f Department of Psychology, McGill University, 1205 Dr. Penfield Avenue, Montreal, Que., Canada H3A 1B1 g Bitop AG, Stockumerstr. 28, 58453 Witten, Germany

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.

Progress in Genetic Studies of Pain and Analgesia

Interindividual variability in pain sensitivity and the response to analgesic manipulations remains a considerable clinical challenge as well as an area of intense scientific investigation. Techniques in this field have matured rapidly so that much relevant data have emerged only in the past few years. Our increasing understanding of the genetic mediation of these biological phenomena have nonetheless revealed their surprising complexity. This review provides a comprehensive picture and critical analysis of the field and its prospects.

The Pain Genes Database: An interactive web browser of pain-related transgenic knockout studies.

Abstract The transgenic knockout mouse is one of the most important tools of modern biology, and commonly employed by pain researchers to examine the function of genes of interest. Over 400 papers, at a current rate of >60 papers per year, have been published to date describing a statistically significant behavioral pain “phenotype” resulting from the null mutation of a single gene. The standard literature review format is incapable of providing a sufficiently broad and up‐to‐date overview of the field. We have therefore constructed the Pain Genes Database, an interactive, web‐based data browser designed to allow easy access to and analysis of the published pain‐related phenotypes of mutant mice (over 200 different mutants at the date of submission). Manuscripts describing results of pain‐relevant knockout studies were identified via Medline search. Manuscripts were included in the database if they described the testing of a spontaneous or genetically engineered mutant mouse with null expression of a single gene on a behavioral assay of acute or tonic nociception, injury‐ or stimulus‐induced hypersensitivity (i.e., allodynia or hyperalgesia), or drug‐ or stress‐induced inhibition of nociception (i.e., analgesia), and reported at least one statistically significant difference between the mutant mice and their simultaneously tested wildtype controls. The database features two levels of exploration, one allowing the identification of genes by name, acronym, genomic position or “summary” phenotype, and the other allowing in‐depth browsing, paper‐by‐paper, of specific phenotypes and test parameters. Links to genetic databases and Medline abstracts are provided for each gene and paper. It is our intention to update the database continually based on weekly Medline searches. This database should provide pain researchers with a useful and easy‐to‐use tool for the generation of novel hypotheses regarding the roles of genes and their protein products in pain processing and modulation. It can be accessed at http://paingeneticslab.ca/4105/06_02_pain_genetics_database.asp (or by visiting paingeneticslab.ca and clicking on the “Pain Genes Db” link under “Resources”).

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.

Pain sensitivity and vasopressin analgesia are mediated by a gene-sex-environment interaction

Quantitative trait locus mapping of chemical/inflammatory pain in the mouse identified the Avpr1a gene, which encodes the vasopressin-1A receptor (V1AR), as being responsible for strain-dependent pain sensitivity to formalin and capsaicin. A genetic association study in humans revealed the influence of a single nucleotide polymorphism (rs10877969) in AVPR1A on capsaicin pain levels, but only in male subjects reporting stress at the time of testing. The analgesic efficacy of the vasopressin analog desmopressin revealed a similar interaction between the drug and acute stress, as desmopressin inhibition of capsaicin pain was only observed in nonstressed subjects. Additional experiments in mice confirmed the male-specific interaction of V1AR and stress, leading to the conclusion that vasopressin activates endogenous analgesia mechanisms unless they have already been activated by stress. These findings represent, to the best of our knowledge, the first explicit demonstration of analgesic efficacy depending on the emotional state of the recipient, and illustrate the heuristic power of a bench-to-bedside-to-bench translational strategy.

Differential Spinal Cord Gene Expression in Rodent Models of Radicular and Neuropathic Pain

Background:Neuropathic pain and radicular low back pain both have a major impact on human health worldwide. Microarray gene analysis on central nervous system tissues holds great promise for discovering novel targets for persistent pain modulation. Methods:Rat models of lumbar radiculopathy (L5 nerve root ligation) and neuropathy (L5 spinal nerve transection) were used for these studies. The authors measured mechanical allodynia followed by analysis of global gene expression in the lumbar spinal cord at two time points (7 days and 14 days) after surgery using the Affymetrix RAE230A GeneChip® (Santa Clara, CA). The expression patterns of several genes of interest were subsequently confirmed using real-time reverse transcriptase polymerase chain reaction. Results:The authors observed similarly robust mechanical allodynia in both models. Second, they observed significant differences in lumbar spinal cord gene expression across chronic pain models. There was little overlap between genes altered in each injury model, suggesting that the site and type of injury produce distinct spinal mechanisms mediating the observed mechanical allodynia. The authors further confirmed a subset of the genes using reverse transcriptase polymerase chain reaction and identified several genes as either neuropathy-associated genes or radiculopathy-associated genes. Conclusions:These two models of persistent pain produce similar allodynic outcomes but produce differential gene expression. These results suggest that diverging mechanisms lead to a common behavioral outcome in these pain models. Furthermore, these distinct pathophysiologic mechanisms in neuropathic versus radicular pain may implicate unique drug therapies for these types of chronic pain syndromes.

The Magnitude of Mechanical Allodynia in a Rodent Model of Lumbar Radiculopathy is Dependent on Strain and Sex

Study Design. This study examined the differences in tactile hypersensitivity across 6 different strains of male mice, and between male and female rats of 3 different strains in a rodent model of low back pain associated with lumbar radiculopathy. Objective. We investigated the possibility that differences in tactile allodynia following the same nerve root injury are affected by genotype and sex in rodents. Summary of Background Data. Low back pain associated with radiculopathy affects countless people throughout the world, encompassing a wide range of individual pain susceptibility. The roles of genetics and sex on differences in nociceptive sensitivities following lumbar nerve root injury have yet to be fully characterized. Methods. Six strains of mice (BALB/cJ, CBA/J, C57BL/6J, 129P3/J, C3H/HeJ, and C58/J; all males) and male and female Sprague Dawley, Holtzman, and Long-Evans rats underwent a lumbar nerve root injury followed by assessment of tactile allodynia. Results. The most sensitive mouse strains following nerve root injury were: 129P3/J, C58/J, and BALB/cJ; and the less sensitive strains were: C57BL/6J, C3H/HeJ, and CBA/J. Female Sprague Dawley and Long-Evans rats displayed increased hypersensitivity following nerve root injury compared to males. No sex differences were observed in Holtzman rats. Conclusions. Different mouse strains, and male and female rats that are exposed to identical nerve root injuries have diverse levels of tactile hypersensitivity, supporting the hypothesis that genetic factors and sex play a key role in radicular pain. Our results correlate with data compiled in identical mouse and rat strains after L5–L6 nerve ligation, suggesting that the precise nature of the injury is not relevant to the inheritance of neuropathic symptom sensitivity.

The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test

ABSTRACT It is widely appreciated that there is significant inter‐individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the β3 subunit of the Na+,K+‐ATPase pump (Atp1b3) contributes to inter‐strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, β3 protein expression, and biophysical properties of the Na+,K+ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the β3 subunit produced strain‐specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the β3 subunit of the Na+,K+‐ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.