Innovations and advances in modelling and measuring pain in animals.

Best practices in preclinical algesiometry (pain behaviour testing) have shifted over the past decade as a result of technological advancements, the continued dearth of translational progress and the emphasis that funding institutions and journals have placed on rigour and reproducibility. Here we describe the changing trends in research methods by analysing the methods reported in preclinical pain publications from the past 40 years, with a focus on the last 5 years. We also discuss how the status quo may be hampering translational success. This discussion is centred on four fundamental decisions that apply to every pain behaviour experiment: choice of subject (model organism), choice of assay (pain-inducing injury), laboratory environment and choice of outcome measures. Finally, we discuss how human tissues, which are increasingly accessible, can be used to validate the translatability of targets and mechanisms identified in animal pain models.

Gabapentin alleviates chronic spontaneous pain and acute hypoxia-related pain in a mouse model of sickle cell disease.

Pain is the main complication of sickle cell disease (SCD). Individuals with SCD experience acute pain episodes and chronic daily pain, both of which are managed with opioids. Opioids have deleterious side effects and use-associated stigma that make them less than ideal for SCD pain management. After recognizing the neuropathic qualities of SCD pain, clinically-approved therapies for neuropathic pain, including gabapentin, now present unique non-opioid based therapies for SCD pain management. These experiments explored the efficacy of gabapentin in relieving evoked and spontaneous chronic pain, and hypoxia/reoxygenation (H/R)-induced acute pain in mouse models of SCD. When administered following H/R, a single dose of gabapentin alleviated mechanical hypersensitivity in SCD mice by decreasing peripheral fibre activity. Gabapentin treatment also alleviated spontaneous ongoing pain in SCD mice. Longitudinal daily administration of gabapentin failed to alleviate H/R-induced pain or chronic evoked mechanical, cold or deep tissue hypersensitivity in SCD mice. Consistent with this observation, voltage-gated calcium channel (VGCC) α2 δ1 subunit expression was similar in sciatic nerve, dorsal root ganglia and lumbar spinal cord tissue from SCD and control mice. Based on these data, gabapentin may be an effective opioid alternative for the treatment of chronic spontaneous and acute H/R pain in SCD.

Urine Trouble: Alterations in Brain Function Associated with Bladder Pain.

PURPOSE: Chronic bladder pain is a debilitating condition often accompanied by alterations in affective and autonomic function. Many symptoms associated with chronic bladder pain are mediated by the central nervous system. In this review data from preclinical animal models and human neuroimaging studies were analyzed and a theoretical supraspinal bladder pain network was generated. MATERIALS AND METHODS: We comprehensively reviewed the literature using PubMed® and Google Scholar™. Relevant reviews and original research articles, and the cited references were summarized and then organized on a neuroanatomical basis. RESULTS: The brain loci the most predominant in the bladder pain literature are the thalamus, parabrachial nucleus, cerebral cortex, amygdala, hypothalamus, periaqueductal gray and rostral ventromedial medulla. This review highlights each of these regions, discussing the molecular and physiological changes that occur in each in the context of bladder pain. CONCLUSIONS: A complex network of brain loci is involved in bladder pain modulation. Studying these brain regions and the changes that they undergo during the transition from acute to chronic bladder pain will provide novel therapeutic strategies for patients with chronic bladder pain diseases such as interstitial cystitis/bladder pain syndrome and chronic prostatitis/chronic pelvic pain syndrome.

Microbiome contributions to pain: a review of the preclinical literature.

ABSTRACT: Over the past 2 decades, the microbiome has received increasing attention for the role that it plays in health and disease. Historically, the gut microbiome was of particular interest to pain scientists studying nociplastic visceral pain conditions given the anatomical juxtaposition of these microorganisms and the neuroimmune networks that drive pain in such diseases. More recently, microbiomes both inside and across the surface of the body have been recognized for driving sensory symptoms in a broader set of diseases. Microbiomes have never been a more popular topic in pain research, but to date, there has not been a systematic review of the preclinical microbiome pain literature. In this article, we identified all animal studies in which both the microbiome was manipulated and pain behaviors were measured. Our analysis included 303 unique experiments across 97 articles. Microbiome manipulation methods and behavioral outcomes were recorded for each experiment so that field-wide trends could be quantified and reported. This review specifically details the animal species, injury models, behavior measures, and microbiome manipulations used in preclinical pain research. From this analysis, we were also able to conclude how manipulations of the microbiome alter pain thresholds in naïve animals and persistent pain intensity and duration in cutaneous and visceral pain models. This review summarizes by identifying existing gaps in the literature and providing recommendations for how to best plan, implement, and interpret data collected in preclinical microbiome pain experiments.

High-speed imaging of evoked rodent mechanical behaviors yields variable results that are not predictive of inflammatory injury.

ABSTRACT: Few analgesics identified using preclinical models have successfully translated to clinical use. These translational limitations may be due to the unidimensional nature of behavioral response measures used to assess rodent nociception. Advances in high-speed videography for pain behavior allow for objective quantification of nuanced aspects of evoked paw withdrawal responses. However, whether videography-based assessments of mechanical hypersensitivity outperform traditional measurement reproducibility is unknown. First, we determined whether high-speed videography of paw withdrawal was reproducible across experimenters. Second, we examined whether this method distinguishes behavioral responses exhibited by naive mice and mice with complete Freund's adjuvant (CFA)-induced inflammation. Twelve experimenters stimulated naive C57BL/6 mice with varying mechanical stimuli. Paw withdrawal responses were recorded with high-speed videography and scored offline by one individual. Our group was unable to replicate the original findings produced by high-speed videography analysis. Surprisingly, ∼80% of variation was not accounted for by variables previously reported to distinguish between responses to innocuous and noxious stimuli (paw height, paw velocity, and pain score), or by additional variables (experimenter, time-of-day, and animal), but rather by unidentified factors. Similar high-speed videography assessments were performed in CFA- and vehicle-treated animals, and the cumulative data failed to reveal an effect of CFA injection on withdrawal as measured by high-speed videography. This study does not support using paw height, velocity, or pain score measurements from high-speed recordings to delineate behavioral responses to innocuous and noxious stimuli. Our group encourages the continued use of traditional mechanical withdrawal assessments until additional high-speed withdrawal measures are validated in established pain models.

Central amygdala metabotropic glutamate receptor 5 in the modulation of visceral pain.

Painful bladder syndrome is a debilitating condition that affects 3-6% of women in the United States. Multiple lines of evidence suggest that changes in CNS processing are key to the development of chronic bladder pain conditions but little is known regarding the underlying cellular, molecular, and neuronal mechanisms. Using a mouse model of distention-induced bladder pain, we found that the central nucleus of the amygdala (CeA) is a critical site of neuromodulation for processing of bladder nociception. Furthermore, we demonstrate that metabotropic glutamate receptor 5 (mGluR5) activation in the CeA induces bladder pain sensitization by increasing CeA output. Thus, pharmacological activation of mGluR5 in the CeA is sufficient to increase the response to bladder distention. Additionally, pharmacological blockade or virally mediated conditional deletion of mGluR5 in the CeA reduced responses to bladder distention suggesting that mGluR5 in the CeA is also necessary for these responses. Finally, we used optogenetic activation of the CeA and demonstrated that this caused a robust increase in the visceral pain response. The CeA-localized effects on responses to bladder distention are associated with changes in extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation in the spinal cord. Overall, these data demonstrate that mGluR5 activation leads to increased CeA output that drives bladder pain sensitization.

Peripheral transient receptor potential vanilloid type 4 hypersensitivity contributes to chronic sickle cell disease pain.

ABSTRACT: Debilitating pain affects the lives of patients with sickle cell disease (SCD). Current pain treatment for patients with SCD fail to completely resolve acute or chronic SCD pain. Previous research indicates that the cation channel transient receptor potential vanilloid type 4 (TRPV4) mediates peripheral hypersensitivity in various inflammatory and neuropathic pain conditions that may share similar pathophysiology with SCD, but this channel's role in chronic SCD pain remains unknown. Thus, the current experiments examined whether TRPV4 regulates hyperalgesia in transgenic mouse models of SCD. Acute blockade of TRPV4 alleviated evoked behavioral hypersensitivity to punctate, but not dynamic, mechanical stimuli in mice with SCD. TRPV4 blockade also reduced the mechanical sensitivity of small, but not large, dorsal root ganglia neurons from mice with SCD. Furthermore, keratinocytes from mice with SCD showed sensitized TRPV4-dependent calcium responses. These results shed new light on the role of TRPV4 in SCD chronic pain and are the first to suggest a role for epidermal keratinocytes in the heightened sensitivity observed in SCD.

Gut microbiota and metabolites drive chronic sickle cell disease pain.

Pain is a debilitating symptom and leading reason for hospitalization of individuals with sickle cell disease. Chronic sickle cell pain is poorly managed because the biological basis is not fully understood. Using transgenic sickle cell mice and fecal material transplant, we determined that the gut microbiome drives persistent sickle cell pain. In parallel patient and mouse analyses, we identified bilirubin as one metabolite that induces sickle cell pain by altering vagus nerve activity. Furthermore, we determined that decreased abundance of the gut bacteria Akkermansia mucinophila is a critical driver of chronic sickle cell pain. These experiments demonstrate that the sickle cell gut microbiome drives chronic widespread pain and identify bacterial species and metabolites that should be targeted for chronic sickle cell disease pain management.

Keratinocyte Piezo1 drives paclitaxel-induced mechanical hypersensitivity.

Recent work demonstrates that epidermal keratinocytes are critical for normal touch sensation. However, it is unknown if keratinocytes contribute to touch evoked pain and hypersensitivity following tissue injury. Here, we used inhibitory optogenetic and chemogenetic techniques to determine the extent to which keratinocyte activity contributes to the severe neuropathic pain that accompanies chemotherapeutic treatment. We found that keratinocyte inhibition largely alleviates paclitaxel-induced mechanical hypersensitivity. Furthermore, we found that paclitaxel exposure sensitizes mouse and human keratinocytes to mechanical stimulation through the keratinocyte mechanotransducer Piezo1. These findings demonstrate the contribution of non-neuronal cutaneous cells to neuropathic pain and pave the way for the development of new pain-relief strategies that target epidermal keratinocytes and Piezo1.

Contextual control of conditioned pain tolerance and endogenous analgesic systems.

The mechanisms underlying the transition from acute to chronic pain are unclear but may involve the persistence or strengthening of pain memories acquired in part through associative learning. Contextual cues, which comprise the environment in which events occur, were recently described as a critical regulator of pain memory; both male rodents and humans exhibit increased pain sensitivity in environments recently associated with a single painful experience. It is unknown, however, how repeated exposure to an acute painful unconditioned stimulus in a distinct context modifies pain sensitivity or the expectation of pain in that environment. To answer this question, we conditioned mice to associate distinct contexts with either repeated administration of a mild visceral pain stimulus (intraperitoneal injection of acetic acid) or vehicle injection over the course of 3 days. On the final day of experiments, animals received either an acid injection or vehicle injection prior to being placed into both contexts. In this way, contextual control of pain sensitivity and pain expectation could be tested respectively. When re-exposed to the noxious stimulus in a familiar environment, both male and female mice exhibited context-dependent conditioned analgesia, a phenomenon mediated by endogenous opioid signaling. However, when expecting the presentation of a painful stimulus in a given context, males exhibited conditioned hypersensitivity whereas females exhibited endogenous opioid-mediated conditioned analgesia. These results are evidence that pain perception and engagement of endogenous opioid systems can be modified through their psychological association with environmental cues. Successful determination of the brain circuits involved in this sexually dimorphic anticipatory response may allow for the manipulation of pain memories, which may contribute to the development of chronic pain states.

Keratinocyte PIEZO1 modulates cutaneous mechanosensation.

Epidermal keratinocytes mediate touch sensation by detecting and encoding tactile information to sensory neurons. However, the specific mechanotransducers that enable keratinocytes to respond to mechanical stimulation are unknown. Here, we found that the mechanically-gated ion channel PIEZO1 is a key keratinocyte mechanotransducer. Keratinocyte expression of PIEZO1 is critical for normal sensory afferent firing and behavioral responses to mechanical stimuli in mice.

Sensory-specific peripheral nerve pathology in a rat model of Fabry disease.

Fabry disease (FD) causes life-long pain, the mechanisms of which are unclear. Patients with FD have chronic pain that mirrors symptoms of other painful peripheral neuropathies. However, it is unclear what underlying damage occurs in FD peripheral nerves that may contribute to chronic pain. Here, we characterized myelinated and unmyelinated fiber pathology in peripheral nerves of a rat model of FD. Decreased nerve fiber density and increased nerve fiber pathology were noted in unmyelinated and myelinated fibers from FD rats; both observations were dependent on sampled nerve fiber modality and anatomical location. FD myelinated axons exhibited lipid accumulations that were determined to be the FD-associated lipid globotriaosylceramide (Gb3), and to a lesser extent lysosomes. These findings suggest that axonal Gb3 accumulation may drive peripheral neuron dysfunction and subsequent pain in FD.

Transient receptor potential canonical 5 mediates inflammatory mechanical and spontaneous pain in mice.

Tactile and spontaneous pains are poorly managed symptoms of inflammatory and neuropathic injury. Here, we found that transient receptor potential canonical 5 (TRPC5) is a chief contributor to both of these sensations in multiple rodent pain models. Use of TRPC5 knockout mice and inhibitors revealed that TRPC5 selectively contributes to the mechanical hypersensitivity associated with CFA injection, skin incision, chemotherapy induced peripheral neuropathy, sickle cell disease, and migraine, all of which were characterized by elevated concentrations of lysophosphatidylcholine (LPC). Accordingly, exogenous application of LPC induced TRPC5-dependent behavioral mechanical allodynia, neuronal mechanical hypersensitivity, and spontaneous pain in naïve mice. Lastly, we found that 75% of human sensory neurons express TRPC5, the activity of which is directly modulated by LPC. On the basis of these results, TRPC5 inhibitors might effectively treat spontaneous and tactile pain in conditions characterized by elevated LPC.

Keratinocytes contribute to normal cold and heat sensation.

Keratinocytes are the most abundant cell type in the epidermis, the most superficial layer of skin. Historically, epidermal-innervating sensory neurons were thought to be the exclusive detectors and transmitters of environmental stimuli. However, recent work from our lab (Moehring et al., 2018) and others (Baumbauer et al., 2015) has demonstrated that keratinocytes are also critical for normal mechanotransduction and mechanically-evoked behavioral responses in mice. Here, we asked whether keratinocyte activity is also required for normal cold and heat sensation. Using calcium imaging, we determined that keratinocyte cold activity is conserved across mammalian species and requires the release of intracellular calcium through one or more unknown cold-sensitive proteins. Both epidermal cell optogenetic inhibition and interruption of ATP-P2X4 signaling reduced reflexive behavioral responses to cold and heat stimuli. Based on these data and our previous findings, keratinocyte purinergic signaling is a modality-conserved amplification system that is required for normal somatosensation in vivo.

Neuronal transient receptor potential (TRP) channels and noxious sensory detection in sickle cell disease.

Pain is the leading cause for hospitalization in patients with sickle cell disease (SCD). While the characteristics of SCD pain can vary widely between patients and between phases of the disease (e.g. vasoocclusive crisis pain vs. chronic pain), similar neuronal mechanisms likely underlie the various aspects of nociceptive processing. In the peripheral nervous system, small unmyelinated C fibers and lightly-myelinated Aδ fibers detect and transmit noxious stimuli. Both classes of neurons express members of the transient receptor potential (TRP) family, a group of ligand gated ion-channels that are activated by thermal, chemical, and mechanical stimuli. Promiscuous TRP channel family members are activated by a wide range of stimuli, many of which are dysregulated in patients with SCD and transgenic SCD mouse models. In 2011, our lab published the first report of TRP channel contributions to rodent SCD pain. Since that time, additional basic and clinical research efforts have investigated the genetic and biochemical status of TRP channels in SCD, placing particular focus on TRPV1. This review will discuss these advances and highlight the clinical SCD presentations that have not yet been studied, but which may be mediated by TRP channel activity.

Peripheral nerve pathology in sickle cell disease mice.

INTRODUCTION: Many patients with sickle cell disease (SCD) suffer from chronic pain, which is often described as neuropathic in nature. Although vascular and inflammatory pathology undoubtedly contribute to the SCD pain experience, the nociceptive signals that ultimately drive symptoms are detected and transmitted by peripheral sensory neurons. To date, no systematic histological examination of peripheral nerves has been completed in patients or mouse models of SCD to diagnose disease-related neuropathy. OBJECTIVES: In this brief report, we compared peripheral nerve morphology in tissues obtained from Berkeley transgenic SCD mice and control animals. METHODS: Sciatic nerves were visualized using light and transmission electron microscopy. Myelin basic protein expression was assessed through Western blot. Blood-nerve barrier permeability was measured using Evan's blue plasma extravasation. RESULTS: Peripheral fibers from SCD mice have thinner myelin sheaths than control mice and widespread myelin instability as evidenced by myelin sheath infolding and unwrapping. Deficits are also observed in nonmyelinating Schwann cell structures; Remak bundles from SCD nerves contain fewer C fibers, some of which are not fully ensheathed by the corresponding Schwann cell. Increased blood-nerve barrier permeability and expression of myelin basic protein are noted in SCD tissue. CONCLUSIONS: These data are the first to characterize Berkeley SCD mice as a naturally occurring model of peripheral neuropathy. Widespread myelin instability is observed in nerves from SCD mice. This pathology may be explained by increased permeability of the blood-nerve barrier and, thus, increased access to circulating demyelinating agents at the level of primary sensory afferents.

Chemokine (c-c motif) receptor 2 mediates mechanical and cold hypersensitivity in sickle cell disease mice.

Approximately one-third of individuals with sickle cell disease (SCD) develop chronic pain. This debilitating pain is inadequately treated because the underlying mechanisms driving the pain are poorly understood. In addition to persistent pain, patients with SCD are also in a tonically proinflammatory state. Previous studies have revealed that there are elevated plasma levels of many inflammatory mediators including chemokine (c-c motif) ligand 2 (CCL2) in individuals with SCD. Using a transgenic mouse model of SCD, we investigated the contributions of CCL2 signaling to SCD-related pain. Inhibition of chemokine receptor 2 (CCR2), but not CCR4, alleviated the behavioral mechanical and cold hypersensitivity in SCD. Furthermore, acute CCR2 blockade reversed both the behavioral and the in vitro responsiveness of sensory neurons to an agonist of TRPV1, a neuronal ion channel previously implicated in SCD pain. These results provide insight into the immune-mediated regulation of hypersensitivity in SCD and could inform future development of analgesics or therapeutic measures to prevent chronic pain.