Quantification of stimulus-evoked tactile allodynia in free moving mice by the chainmail sensitivity test.

Chronic pain occurs at epidemic levels throughout the population. Hypersensitivity to touch, is a cardinal symptom of chronic pain. Despite dedicated research for over a century, quantifying this hypersensitivity has remained impossible at scale. To address these issues, we developed the Chainmail Sensitivity Test (CST). Our results show that control mice spend significantly more time on the chainmail portion of the device than mice subject to neuropathy. Treatment with gabapentin abolishes this difference. CST-derived data correlate well with von Frey measurements and quantify hypersensitivity due to inflammation. Our study demonstrates the potential of the CST as a standardized tool for assessing mechanical hypersensitivity in mice with minimal operator input.

Peripheralized sepiapterin reductase inhibition as a safe analgesic therapy.

The development of novel analgesics for chronic pain in the last 2 decades has proven virtually intractable, typically failing due to lack of efficacy and dose-limiting side effects. Identified through unbiased gene expression profiling experiments in rats and confirmed by human genome-wide association studies, the role of excessive tetrahydrobiopterin (BH4) in chronic pain has been validated by numerous clinical and preclinical studies. BH4 is an essential cofactor for aromatic amino acid hydroxylases, nitric oxide synthases, and alkylglycerol monooxygenase so a lack of BH4 leads to a range of symptoms in the periphery and central nervous system (CNS). An ideal therapeutic goal therefore would be to block excessive BH4 production, while preventing potential BH4 rundown. In this review, we make the case that sepiapterin reductase (SPR) inhibition restricted to the periphery (i.e., excluded from the spinal cord and brain), is an efficacious and safe target to alleviate chronic pain. First, we describe how different cell types that engage in BH4 overproduction and contribute to pain hypersensitivity, are themselves restricted to peripheral tissues and show their blockade is sufficient to alleviate pain. We discuss the likely safety profile of peripherally restricted SPR inhibition based on human genetic data, the biochemical alternate routes of BH4 production in various tissues and species, and the potential pitfalls to predictive translation when using rodents. Finally, we propose and discuss possible formulation and molecular strategies to achieve peripherally restricted, potent SPR inhibition to treat not only chronic pain but other conditions where excessive BH4 has been demonstrated to be pathological.

Optimizing and Accelerating the Development of Precision Pain Treatments for Chronic Pain: IMMPACT Review and Recommendations.

Large variability in the individual response to even the most-efficacious pain treatments is observed clinically, which has led to calls for a more personalized, tailored approach to treating patients with pain (ie, "precision pain medicine"). Precision pain medicine, currently an aspirational goal, would consist of empirically based algorithms that determine the optimal treatments, or treatment combinations, for specific patients (ie, targeting the right treatment, in the right dose, to the right patient, at the right time). Answering this question of "what works for whom" will certainly improve the clinical care of patients with pain. It may also support the success of novel drug development in pain, making it easier to identify novel treatments that work for certain patients and more accurately identify the magnitude of the treatment effect for those subgroups. Significant preliminary work has been done in this area, and analgesic trials are beginning to utilize precision pain medicine approaches such as stratified allocation on the basis of prespecified patient phenotypes using assessment methodologies such as quantitative sensory testing. Current major challenges within the field include: 1) identifying optimal measurement approaches to assessing patient characteristics that are most robustly and consistently predictive of inter-patient variation in specific analgesic treatment outcomes, 2) designing clinical trials that can identify treatment-by-phenotype interactions, and 3) selecting the most promising therapeutics to be tested in this way. This review surveys the current state of precision pain medicine, with a focus on drug treatments (which have been most-studied in a precision pain medicine context). It further presents a set of evidence-based recommendations for accelerating the application of precision pain methods in chronic pain research. PERSPECTIVE: Given the considerable variability in treatment outcomes for chronic pain, progress in precision pain treatment is critical for the field. An array of phenotypes and mechanisms contribute to chronic pain; this review summarizes current knowledge regarding which treatments are most effective for patients with specific biopsychosocial characteristics.

Automated preclinical detection of mechanical pain hypersensitivity and analgesia.

ABSTRACT: The lack of sensitive and robust behavioral assessments of pain in preclinical models has been a major limitation for both pain research and the development of novel analgesics. Here, we demonstrate a novel data acquisition and analysis platform that provides automated, quantitative, and objective measures of naturalistic rodent behavior in an observer-independent and unbiased fashion. The technology records freely behaving mice, in the dark, over extended periods for continuous acquisition of 2 parallel video data streams: (1) near-infrared frustrated total internal reflection for detecting the degree, force, and timing of surface contact and (2) simultaneous ongoing video graphing of whole-body pose. Using machine vision and machine learning, we automatically extract and quantify behavioral features from these data to reveal moment-by-moment changes that capture the internal pain state of rodents in multiple pain models. We show that these voluntary pain-related behaviors are reversible by analgesics and that analgesia can be automatically and objectively differentiated from sedation. Finally, we used this approach to generate a paw luminance ratio measure that is sensitive in capturing dynamic mechanical hypersensitivity over a period and scalable for high-throughput preclinical analgesic efficacy assessment.

Impaired dopamine metabolism is linked to fatigability in mice and fatigue in Parkinson's disease patients.

Fatigue is a common symptom of Parkinson's disease that compromises significantly the patients' quality of life. Despite that, fatigue has been under-recognized as symptom, its pathophysiology remains poorly understood, and there is no adequate treatment so far. Parkinson's disease is characterized by the progressive loss of midbrain dopaminergic neurons, eliciting the classical motor symptoms including slowing of movements, muscular rigidity and resting tremor. The dopamine synthesis is mediated by the rate-limiting enzyme tyrosine hydroxylase, which requires tetrahydrobiopterin as a mandatory cofactor. Here, we showed that reserpine administration (1 mg/kg, two intraperitoneal injections with an interval of 48 h) in adult Swiss male mice (8-10 weeks; 35-45 g) provoked striatal depletion of dopamine and tetrahydrobiopterin, and intolerance to exercise. The poor exercise performance of reserpinized mice was not influenced by emotional or anhedonic factors, mechanical nociceptive thresholds, electrocardiogram pattern alterations or muscle-impaired bioenergetics. The administration of levodopa (100 mg/kg; i.p.) plus benserazide (50 mg/kg; i.p.) rescued reserpine-induced fatigability-like symptoms and restored striatal dopamine and tetrahydrobiopterin levels. Remarkably, it was observed, for the first time, that impaired blood dopamine metabolism inversely and idependently correlated with fatigue scores in eighteen idiopathic Parkinson's disease patients (male n = 13; female n = 5; age 61.3 ± 9.59 years). Altogether, this study provides new experimental and clinical evidence that fatigue symptoms might be caused by the impaired striatal dopaminergic neurotransmission, pointing to a central origin of fatigue in Parkinson's disease.

Mesenchymal stromal cell-derived small extracellular vesicles restore lung architecture and improve exercise capacity in a model of neonatal hyperoxia-induced lung injury.

Early administration of mesenchymal stromal cell (MSC)-derived small extracellular vesicles (MEx) has shown considerable promise in experimental models of bronchopulmonary dysplasia (BPD). However, the ability of MEx to reverse the long-term pulmonary complications associated with established BPD remains unknown. In this study, MEx were isolated from media conditioned by human Wharton's Jelly-derived MSC cultures. Newborn mice (FVB strain) were exposed to hyperoxia (HYRX (75% O2)) before returning to room air at postnatal day 14 (PN14). Following prolonged HYRX-exposure, animals received a single MEx dose at PN18 or serial MEx treatments at PN18-39 ("late" intervention). This group was compared to animals that received an early single MEx dose at PN4 ("early" intervention). Animals were harvested at PN28 or 60 for assessment of pulmonary parameters. We found that early and late MEx interventions effectively ameliorated core features of HYRX-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. Exercise capacity testing and assessment of pulmonary hypertension (PH) showed functional improvements following both early and late MEx interventions. In conclusion, delivery of MEx following prolonged HYRX-exposure improves core features of experimental BPD, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating PH and improving exercise capacity. Taken together, delivery of MEx may not only be effective in the immediate neonatal period to prevent the development of BPD but may provide beneficial effects for the management and potentially the reversal of cardiorespiratory complications in infants and children with established BPD.

The Stage of the Estrus Cycle Is Critical for Interpretation of Female Mouse Social Interaction Behavior.

Female animals in biomedical research have traditionally been excluded from research studies due to the perceived added complexity caused by the estrus cycle. However, given the importance of sex differences in a variety of neurological disorders, testing female mice is critical to identifying sex-linked effects in diseases. To determine the susceptibility of simple behaviors to hormonal fluctuations in the estrus cycle, we studied the effects of sex and the estrus cycle on a variety of behavioral tasks commonly used in mouse phenotyping laboratories. Male and female C57BL/6J mice were tested in a small battery of short duration tests and, immediately on completion of each test, females were classified using cytology of vaginal lavages as sexually-receptive (proestrus and estrus) or non-receptive (NR; metestrus and diestrus). We showed that there was a significant difference in 3-chamber social interaction (SI) between female mice at different stages of their estrus cycle, with sexually-receptive mice showing no preferential interest in a novel female mouse compared with an empty chamber. NR female mice showed the same level of preference for a novel female mouse as male mice did for a novel male mouse. No differences between or within sexes were found for tests of anxiety elevated plus maze (EPM; Hole board), working memory [Novel object recognition (NOR)], and motor learning (repeated tests on rotarod). We conclude that the stage of the estrus cycle may impact SI between same-sex conspecifics, and does not impact performance in the elevated plus-maze, hole board, NOR, and rotarod.

Sepiapterin Reductase Inhibition Leading to Selective Reduction of Inflammatory Joint Pain in Mice and Increased Urinary Sepiapterin Levels in Humans and Mice.

OBJECTIVE: To evaluate the antiinflammatory and analgesic effects of sepiapterin reductase (SPR) inhibition in a mouse model of inflammatory joint disease, and to determine whether urinary sepiapterin levels, as measured in mice and healthy human volunteers, could be useful as a noninvasive, translational biomarker of SPR inhibition/target engagement. METHODS: The collagen antibody-induced arthritis (CAIA) model was used to induce joint inflammation in mice. The effects of pharmacologic inhibition of SPR on thresholds of heat-, cold-, and mechanical-evoked pain sensitivity and on signs of inflammation were tested in mice with CAIA. In addition, mice and healthy human volunteers were treated with SPR inhibitors, and changes in urinary sepiapterin levels were analyzed by high-performance liquid chromatography. RESULTS: CAIA in mice was characterized by 2 phases: in the acute inflammation (early) phase, joint inflammation and heat-, mechanical-, and cold-induced pain hypersensitivity were present, while in the postinflammation (late) phase, no joint inflammation was observed but heat- and mechanical-induced hypersensitivity, but not cold hypersensitivity, were present. Inhibition of SPR in mice with CAIA significantly attenuated the heat-induced hyperalgesia in both phases, and the mechanical allodynia in the late phase. Signs of inflammation were unaffected by SPR inhibition. Urinary tetrahydrobiopterin levels, as a marker of inflammatory pain, were increased during inflammation in mice with CAIA (2-fold increase over controls; P < 0.05) and significantly reduced by SPR inhibition (P < 0.05 versus vehicle-treated mice). Increased urinary sepiapterin levels in the presence of SPR inhibition in both mice and healthy human volunteers were associated with high sensitivity (70-85%) and high specificity (82-88%) for the prediction of SPR inhibition/target engagement. CONCLUSION: SPR inhibition reduces the pain associated with joint inflammation, thus showing its potential utility as an analgesic strategy for inflammatory joint pain. In addition, SPR inhibition increases urinary sepiapterin levels, indicating the potential of this measurement as a noninvasive biomarker of target engagement of SPR inhibitors, such as sulfasalazine, a disease-modifying antirheumatic drug that is currently used as a first-line treatment for rheumatoid arthritis.

Novel charged sodium and calcium channel inhibitor active against neurogenic inflammation.

Voltage-dependent sodium and calcium channels in pain-initiating nociceptor neurons are attractive targets for new analgesics. We made a permanently charged cationic derivative of an N-type calcium channel-inhibitor. Unlike cationic derivatives of local anesthetic sodium channel blockers like QX-314, this cationic compound inhibited N-type calcium channels more effectively with extracellular than intracellular application. Surprisingly, the compound is also a highly effective sodium channel inhibitor when applied extracellularly, producing more potent inhibition than lidocaine or bupivacaine. The charged inhibitor produced potent and long-lasting analgesia in mouse models of incisional wound and inflammatory pain, inhibited release of the neuropeptide calcitonin gene-related peptide (CGRP) from dorsal root ganglion neurons, and reduced inflammation in a mouse model of allergic asthma, which has a strong neurogenic component. The results show that some cationic molecules applied extracellularly can powerfully inhibit both sodium channels and calcium channels, thereby blocking both nociceptor excitability and pro-inflammatory peptide release.

Composite Pain Biomarker Signatures for Objective Assessment and Effective Treatment.

Pain is a subjective sensory experience that can, mostly, be reported but cannot be directly measured or quantified. Nevertheless, a suite of biomarkers related to mechanisms, neural activity, and susceptibility offer the possibility-especially when used in combination-to produce objective pain-related indicators with the specificity and sensitivity required for diagnosis and for evaluation of risk of developing pain and of analgesic efficacy. Such composite biomarkers will also provide improved understanding of pain pathophysiology.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

Diltiazem Promotes Regenerative Axon Growth.

Axotomy results in permanent loss of function after brain and spinal cord injuries due to the minimal regenerative propensity of the adult central nervous system (CNS). To identify pharmacological enhancers of axon regeneration, 960 compounds were screened for cortical neuron axonal regrowth using an in vitro cortical scrape assay. Diltiazem, verapamil, and bromopride were discovered to facilitate axon regeneration in rat cortical cultures, in the presence of chondroitin sulfate proteoglycans (CSPGs). Diltiazem, an L-type calcium channel blocker (L-CCB), also promotes axon outgrowth in adult primary mouse dorsal root ganglion (DRG) and induced human sensory (iSensory) neurons.

Neuropathic pain drives anxiety behavior in mice, results consistent with anxiety levels in diabetic neuropathy patients.

BACKGROUND: Epidemiological studies in patients with neuropathic pain demonstrate a strong association with psychiatric conditions such as anxiety; however, the precipitating pathology between these symptoms remains unclear. To investigate this, we studied the effects of lifelong stress on levels of neuropathic pain-like behavior and conversely, the effects of chronic neuropathic injury on anxiety-like status in male and female mice. In addition, we assayed this link in painful and painless diabetic peripheral neuropathy patients. METHODS: Male and female mice were subject to ongoing life-stress or control living conditions. Baseline sensitivity and anxiety tests were measured followed by spared nerve injury (SNI) to the sciatic nerve. Subsequent sensory testing occurred until 3 weeks after SNI followed by anxiety tests between 4 and 6 weeks after SNI. RESULTS: Levels of tactile or cold allodynia did not differ between adult mice subject to lifelong chronic stress, relative to nonstressed controls, for at least 3 weeks after SNI. By contrast, longer-term neuropathic mice of both sexes displayed pronounced anxiety-like behavior, regardless of exposure to stress. If sex differences were present, females usually exhibited more pronounced anxiety-like behavior. These ongoing anxiety behaviors were corroborated with plasma corticosterone levels in distinct animal groups. In addition, data from patients with painful and nonpainful diabetic neuropathy showed a clear relationship between ongoing pain and anxiety, with females generally more affected than males. DISCUSSION: Taken together, these data demonstrate a strong link between chronic neuropathic pain and chronic anxiety, with the driver of this comorbidity being neuropathic pain as opposed to on-going stress.

Mechanistic Differences in Neuropathic Pain Modalities Revealed by Correlating Behavior with Global Expression Profiling.

Chronic neuropathic pain is a major morbidity of neural injury, yet its mechanisms are incompletely understood. Hypersensitivity to previously non-noxious stimuli (allodynia) is a common symptom. Here, we demonstrate that the onset of cold hypersensitivity precedes tactile allodynia in a model of partial nerve injury, and this temporal divergence was associated with major differences in global gene expression in innervating dorsal root ganglia. Transcripts whose expression change correlates with the onset of cold allodynia were nociceptor related, whereas those correlating with tactile hypersensitivity were immune cell centric. Ablation of TrpV1 lineage nociceptors resulted in mice that did not acquire cold allodynia but developed normal tactile hypersensitivity, whereas depletion of macrophages or T cells reduced neuropathic tactile allodynia but not cold hypersensitivity. We conclude that neuropathic pain incorporates reactive processes of sensory neurons and immune cells, each leading to distinct forms of hypersensitivity, potentially allowing drug development targeted to each pain type.

Ensuring transparency and minimization of methodologic bias in preclinical pain research: PPRECISE considerations.

There is growing concern about lack of scientific rigor and transparent reporting across many preclinical fields of biological research. Poor experimental design and lack of transparent reporting can result in conscious or unconscious experimental bias, producing results that are not replicable. The Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public-private partnership with the U.S. Food and Drug Administration sponsored a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and Efficacy (PPRECISE) Working Group. International participants from universities, funding agencies, government agencies, industry, and a patient advocacy organization attended. Reduction of publication bias, increasing the ability of others to faithfully repeat experimental methods, and increased transparency of data reporting were specifically discussed. Parameters deemed essential to increase confidence in the published literature were clear, specific reporting of an a priori hypothesis and definition of primary outcome measure. Power calculations and whether measurement of minimal meaningful effect size to determine these should be a core component of the preclinical research effort provoked considerable discussion, with many but not all agreeing. Greater transparency of reporting should be driven by scientists, journal editors, reviewers, and grant funders. The conduct of high-quality science that is fully reported should not preclude novelty and innovation in preclinical pain research, and indeed, any efforts that curtail such innovation would be misguided. We believe that to achieve the goal of finding effective new treatments for patients with pain, the pain field needs to deal with these challenging issues.

Identification and functional significance of N-glycosylation of the 5-ht5A receptor.

The presence and roles of N-glycosylation of the human (h) 5-ht(5A) receptor were investigated using a heterologous expression system. Following transient transfection of COS-7 cells with h5-ht(5A) receptor cDNA, SDS-PAGE/Western blot analysis of immunoreactivity demonstrated two protein species; a predominant species with a molecular weight of approximately 35-45 kDa and a minor species of approximately 45-55 kDa. Transfected cells grown in the presence of the N-glycosylation inhibitor tunicamycin, failed to express the minor immunoreactive species indicating this represented the N-glycosylated form of the h5-ht(5A) receptor. Comparison of the molecular weights of immunoreactive bands arising from the wild-type and each of the mutant 5-ht(5A) receptors with disruption of the predicted N-glycosylation sites (N6S and N21S) demonstrated that both identified asparagines were N-glycosylated. Immunocytochemical and ELISA studies demonstrated that the [N6S]h5-ht(5A) receptor mutation, but not the [N21S]h5-ht(5A) receptor mutation, reduced protein expression in the cell membrane, indicating that N-glycosylation of the N6 residue is important for the membrane expression of this neurotransmitter receptor; a requirement for receptor function.