Monoclonal antibodies against GFRα3 are efficacious against evoked hyperalgesic and allodynic responses in mouse join pain models but, one of these, REGN5069, was not effective against pain in a randomized, placebo-controlled clinical trial in patients with osteoarthritis pain.

The artemin-GFRα3 signaling pathway has been implicated in various painful conditions including migraine, cold allodynia, hyperalgesia, inflammatory bone pain, and mouse knees contain GFRα3-immunoreactive nerve endings. We developed high affinity mouse (REGN1967) and human (REGN5069) GFRα3-blocking monoclonal antibodies and, following in vivo evaluations in mouse models of chronic joint pain (osteoarthritic-like and inflammatory), conducted a first-in-human phase 1 pharmacokinetics (PK) and safety trial of REGN5069 (NCT03645746) in healthy volunteers, and a phase 2 randomized placebo-controlled efficacy and safety trial of REGN5069 (NCT03956550) in patients with knee osteoarthritis (OA) pain. In three commonly used mouse models of chronic joint pain (destabilization of the medial meniscus, intra-articular monoiodoacetate, or Complete Freund's Adjuvant), REGN1967 and REGN5069 attenuated evoked behaviors including tactile allodynia and thermal hyperalgesia without discernably impacting joint pathology or inflammation, prompting us to further evaluate REGN5069 in humans. In the phase 1 study in healthy subjects, the safety profiles of single doses of REGN5069 up to 3000 mg (intravenous) or 600 mg (subcutaneous) were comparable to placebo; PK were consistent with a monoclonal antibody exhibiting target-mediated disposition. In the phase 2 study in patients with OA knee pain, two doses of REGN5069 (100 mg or 1000 mg intravenous every 4 weeks) for 8 weeks failed to achieve the 12-week primary and secondary efficacy endpoints relative to placebo. In addition to possible differences in GFRα3 biology between mice and humans, we highlight here differences in experimental parameters that could have contributed to a different profile of efficacy in mouse models versus human OA pain. Additional research is required to more fully evaluate any potential role of GFRα3 in human pain.

Evaluating instruments for assessing healthspan: a multi-center cross-sectional study on health-related quality of life (HRQL) and frailty in the companion dog.

Developing valid tools that assess key determinants of canine healthspan such as frailty and health-related quality of life (HRQL) is essential to characterizing and understanding aging in dogs. Additionally, because the companion dog is an excellent translational model for humans, such tools can be applied to evaluate gerotherapeutics and investigate mechanisms underlying longevity in both dogs and humans. In this multi-center, cross-sectional study, we investigated the use of a clinical questionnaire (Canine Frailty Index; CFI; Banzato et al., 2019) to assess frailty and an owner assessment tool (VetMetrica HRQL) to evaluate HRQL in 451 adult companion dogs. Results demonstrated validity of the tools by confirming expectations that frailty score increases and HRQL scores deteriorate with age. CFI scores were significantly higher (higher frailty) and HRQL scores significantly lower (worse HRQL) in old dogs (≥ 7 years of age) compared to young dogs (≥ 2 and < 6 years of age). Body size (small < 11.3 kg (25 lbs) or large > 22.7 kg (50 lbs)) was not associated with CFI or total HRQL score. However, older, larger dogs showed faster age-related decline in HRQL scores specific to owner-reported activity and comfort. Findings suggest that the clinician-assessed CFI and owner-reported VetMetrica HRQL are useful tools to evaluate two determinants of healthspan in dogs: the accumulation of frailty and the progressive decline in quality of life. Establishing tools that operationalize the assessment of canine healthspan is critical for the advancement of geroscience and the development of gerotherapeutics that benefit both human and veterinary medicine. Graphical summary of the design, results, and conclusions of the study.

The phenotype of aging in the dog: how aging impacts the health and well-being of dogs and their caregivers.

Aging is the single most important cause of disease, disability, and death in adult dogs. Contrary to the common view of aging as a mysterious and inevitable natural event, it is more usefully understood as a set of complex but comprehensible biological processes that are highly conserved across species. Although the phenotypic expression of these processes is variable, there are consistent patterns both within and between species. The purpose of this feature is to describe the patterns currently recognized in the physical and behavioral manifestations of aging in the dog and how these impact the health and welfare of companion dogs and their human caregivers. Important gaps in our knowledge of the canine aging phenotype will be identified, and current research efforts to better characterize aging in the dog will be discussed. This will help set the context for future efforts to develop clinical assessments and treatments to mitigate the negative impact of aging on dogs and humans.

AdRoit is an accurate and robust method to infer complex transcriptome composition.

Bulk RNA sequencing provides the opportunity to understand biology at the whole transcriptome level without the prohibitive cost of single cell profiling. Advances in spatial transcriptomics enable to dissect tissue organization and function by genome-wide gene expressions. However, the readout of both technologies is the overall gene expression across potentially many cell types without directly providing the information of cell type constitution. Although several in-silico approaches have been proposed to deconvolute RNA-Seq data composed of multiple cell types, many suffer a deterioration of performance in complex tissues. Here we present AdRoit, an accurate and robust method to infer the cell composition from transcriptome data of mixed cell types. AdRoit uses gene expression profiles obtained from single cell RNA sequencing as a reference. It employs an adaptive learning approach to alleviate the sequencing technique difference between the single cell and the bulk (or spatial) transcriptome data, enhancing cross-platform readout comparability. Our systematic benchmarking and applications, which include deconvoluting complex mixtures that encompass 30 cell types, demonstrate its preferable sensitivity and specificity compared to many existing methods as well as its utilities. In addition, AdRoit is computationally efficient and runs orders of magnitude faster than most methods.

C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.

The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.

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

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.

Patterns of pain: meta-analysis of microarray studies of pain.

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.

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.

The beta3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test.

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 beta3 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, beta3 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 beta3 subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the beta3 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.

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.

Sex-specific pain modulation: the growth factor, neuregulin-1, as a pro-nociceptive cytokine.

An increasing amount of evidence indicates that there are significant sex differences in clinical and experimental pain sensitivity in men and women. While it is now clear that the endogenous sex steroids are involved in mediating these sex differences, the cellular and molecular mechanisms that underlie their effects on nociceptive sensitivity remain elusive. Recent studies have shown that sex steroids are potent regulators of gene expression in glial cells, particularly astrocytes. This review specifically highlights some of the evidence of sex steroid regulation of growth factor expression. Growth factors have been shown to be potent pro-nociceptive mediators in rodents. Thus, regulation of their expression by sex steroids may be a general mechanism by which sex steroids exert their effect on pain sensitivity. One such mechanism, the progesterone specific regulation of the growth factor, neuregulin-1, following nerve root injury in the rat, is described in detail. Neuregulin-1 expression is increased in spinal cord astrocytes only in female rats with circulating progesterone. Neuregulin-1 has also been shown to produce transient tactile allodynia when delivered intrathecally in rats. Our understanding of growth factor regulation by sex steroids promises to open up new avenues of investigation into the mechanisms that drive sex differences in pain sensitivity.

Neuregulin 1 is a pronociceptive cytokine that is regulated by progesterone in the spinal cord: implications for sex specific pain modulation.

Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Females typically display lower threshold responses to painful stimuli as compared to males. We have previously observed sexually dimorphic expression of the growth factor, neuregulin 1 (NRG1) following L5 nerve root ligation (LR) in male and female rats. In the present study, we sought to determine which gonadal hormones were involved in regulating NRG1 expression following L5 nerve root ligation. We observed that expression of NRG1 mRNA and the neuregulin receptors, ErbB2 and ErbB4 in the lumbar spinal cord was facilitated by the presence of progesterone in female rats following L5 nerve root ligation. An increase in NRG1 protein and NRG1 immunoreactivity was also observed in the ipsilateral spinal cord of progesterone treated female rats as compared to ovariectomized female rats and male rats at day 14 following LR. NRG1 immunoreactivity was equally colocalized with either the astrocytic marker, GFAP, and with NeuN labeled neurons 14days following L5 nerve root ligation. Intrathecal administration of recombinant NRG1-beta1 protein significantly decreased the hindpaw tactile withdrawal threshold in male rats, ovariectomized female rats, and progesterone treated female rats. These results demonstrate a role for progesterone-dependent regulation of glial and/or neuronal neuregulin 1 in female rats in mediating sex differences in nociception. Furthermore, our results suggest that NRG1 may be involved in central sensitization during the maintenance phase, but not in the initiation of persistent pain in female rats.

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

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").

Reprint of "efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury" [Brain Behav. Immun. 21 (2007) 238-246].

Increasing evidence points to a role for spinal neuroimmune dysregulation (glial cell activation and cytokine expression) in the pathogenesis of chronic pain. Suppression of astrocytic and microglial activation with the methylxanthine derivative, propentofylline, pre-emptively attenuates the development of nerve injury-induced allodynia. Currently, we investigated the ability of systemic propentofylline to reverse existing, long-term allodynia after nerve injury-a clinically relevant paradigm. Rats received L5 spinal nerve transection or sham surgery and the development of mechanical allodynia was assessed daily for 2 weeks, at which time injured rats exhibited robust responses to non-noxious von Frey filaments. On days 14-27, rats received either saline or 101 mg/kg propentofylline by intraperitoneal (i.p.) injection. On day 28 or 42 (after a 14-day drug washout period), lumbar spinal cord sections were processed for assessment of astrocytic glial fibrillary acidic protein (GFAP) and microglial OX-42 (antibody against CR3/CD11b). Propentofylline treatment to nerve injured rats resulted in significant reversal of allodynia that lasted throughout the 14-day washout period. Spinal microglial activation was observed at days 28 and 42 post-injury at the protein level, in the absence of mRNA level changes. Less robust increases in GFAP immunoreactivity were observed at days 28 and 42 post-transection. Interestingly, propentofylline treatment suppressed microglial activation at both time points in this paradigm. Taken together, our results highlight the clinical potential of the glial modulating agent, propentofylline, for the treatment of neuropathic pain as well as a role for microglia in the long-term maintenance of allodynia.

Efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury.

Increasing evidence points to a role for spinal neuroimmune dysregulation (glial cell activation and cytokine expression) in the pathogenesis of chronic pain. Suppression of astrocytic and microglial activation with the methylxanthine derivative, propentofylline, pre-emptively attenuates the development of nerve injury-induced allodynia. Currently, we investigated the ability of systemic propentofylline to reverse existing, long-term allodynia after nerve injury--a clinically relevant paradigm. Rats received L5 spinal nerve transection or sham surgery and the development of mechanical allodynia was assessed daily for 2 weeks, at which time injured rats exhibited robust responses to non-noxious von Frey filaments. On days 14-27, rats received either saline or 101 mg/kg propentofylline by intraperitoneal (i.p.) injection. On day 28 or 42 (after a 14-day drug washout period), lumbar spinal cord sections were processed for assessment of astrocytic glial fibrillary acidic protein (GFAP) and microglial OX-42 (antibody against CR3/CD11b). Propentofylline treatment to nerve injured rats resulted in significant reversal of allodynia that lasted throughout the 14-day washout period. Spinal microglial activation was observed at days 28 and 42 post-injury at the protein level, in the absence of mRNA level changes. Less robust increases in GFAP immunoreactivity were observed at days 28 and 42 post-transection. Interestingly, propentofylline treatment suppressed microglial activation at both time points in this paradigm. Taken together, our results highlight the clinical potential of the glial modulating agent, propentofylline, for the treatment of neuropathic pain as well as a role for microglia in the long-term maintenance of allodynia.

Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: heterogeneity of the quiescent phenotype.

Reactive astrocytes display decreased glutamate transporters, such as GLT-1, and as a result synaptic glutamate clearance is impaired. In addition, these activated astrocytes are immunocompetent and release algesic mediators that can sensitize neurons in the spinal cord. Currently, we evaluated the effect of propentofylline (PPF), an experimental antiallodynic agent, on the phenotype and glutamate transporter expression of astrocytes. Primary astrocyte cultures, which represent an activated phenotype with a polygonal morphology and low GLT-1 expression, were treated for 3 or 7 days with 10, 100, or 1,000 microM PPF or dibutyryl-cAMP (db-cAMP), a known inducer of GLT-1 expression. PPF dose-dependently induced astrocytes to display a mature phenotype, with elongated processes and a stellate shape, as well as increased GLT-1 and GLAST immunoreactivity, similar to that seen with db-cAMP. Real time RT-PCR and Western blot analysis clearly demonstrated that PPF caused a potent dose-dependent induction of GLT-1 and GLAST mRNA and protein in these astrocytes. Importantly, the observed increase in glutamate transporters was found to have a functional effect, with significantly enhanced glutamate uptake in astrocytes treated with 100 or 1,000 microM PPF that was sensitive to dihydrokainate inhibition, suggesting it is GLT-1 mediated. Finally, the effect of PPF on lipopolysaccharide-induced chemokine release was investigated. Interestingly, PPF was able to dampen both MCP-1 (CCL2) and MIP-2 (CXCL2) release from astrocytes while db-cAMP significantly enhanced this chemokine expression. These findings suggest that PPF is capable of differentiating astrocytes to a homeostatic, mature phenotype, competent for glutamate clearance and distinct from that induced by db-cAMP.

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(R) (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.