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.

Sickle cell disease iPSC-derived sensory neurons exhibit increased excitability and sensitization to patient plasma.

ABSTRACT: Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. Challenges to gaining mechanistic insight into pathogenic SCD pain processes include differential gene expression and function of sensory neurons between humans and mice with SCD, and extremely limited availability of neuronal tissues from patients with SCD. Here, we used induced pluripotent stem cells (iPSCs), derived from patients with SCD, differentiated into sensory neurons (SCD iSNs) to begin to overcome these challenges. We characterize key gene expression and function of SCD iSNs to establish a model to investigate intrinsic and extrinsic factors that may contribute to SCD pain. Despite similarities in receptor gene expression, SCD iSNs show pronounced excitability using patch clamp electrophysiology. Furthermore, we find that plasma taken from patients with SCD during acute pain associated with a vaso-occlusive event increases the calcium responses to the nociceptive stimulus capsaicin in SCD iSNs compared with those treated with paired plasma from patients with SCD at steady state baseline or healthy control plasma samples. We identified high levels of the polyamine spermine in baseline and acute pain states of plasma from patients with SCD, which sensitizes SCD iSNs to subthreshold concentrations of capsaicin. Together, these data identify potential intrinsic mechanisms within SCD iSNs that may extend beyond a blood-based pathology.

Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

The anthelmintic drug praziquantel activates a schistosome transient receptor potential channel.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca2+ influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use. Here, we demonstrate that PZQ activates a schistosome transient receptor potential (TRP) channel, christened SmTRPMPZQ, present in parasitic schistosomes and other PZQ-sensitive parasites. Several properties of SmTRPMPZQ were consistent with known effects of PZQ on schistosomes, including (i) nanomolar sensitivity to PZQ; (ii) stereoselectivity toward (R)-PZQ; (iii) mediation of sustained Ca2+ signals in response to PZQ; and (iv) a pharmacological profile that mirrors the well-known effects of PZQ on muscle contraction and tegumental disruption. We anticipate that these findings will spur development of novel therapeutic interventions to manage schistosome infections and broader interest in PZQ, which is finally unmasked as a potent flatworm TRP channel activator.

Satellite glial cells in sensory ganglia express functional transient receptor potential ankyrin 1 that is sensitized in neuropathic and inflammatory pain.

Transient receptor potential ankyrin 1 (TRPA1) is well documented as an important molecule in pain hypersensitivity following inflammation and nerve injury and in many other cellular biological processes. Here, we show that TRPA1 is expressed not only by sensory neurons of the dorsal root ganglia (DRG) but also in their adjacent satellite glial cells (SGCs), as well as nonmyelinating Schwann cells. TRPA1 immunoreactivity is also detected in various cutaneous structures of sensory neuronal terminals, including small and large caliber cutaneous sensory fibers and endings. The SGC-expressed TRPA1 is functional. Like DRG neurons, dissociated SGCs exhibit a robust response to the TRPA1-selective agonist allyl isothiocyanate (AITC) by an increase of intracellular Ca2+ concentration ([Ca2+]i). These responses are abolished by the TRPA1 antagonist HC030031 and are absent in SGCs and neurons from global TRPA1 null mice. SGCs and neurons harvested from DRG proximal to painful tissue inflammation induced by plantar injection of complete Freund's adjuvant show greater AITC-evoked elevation of [Ca2+]i and slower recovery compared to sham controls. Similar TRPA1 sensitization occurs in both SGCs and neurons during neuropathic pain induced by spared nerve injury. Together, these results show that functional TRPA1 is expressed by sensory ganglia SGCs, and TRPA1 function in SGCs is enhanced after both peripheral inflammation and nerve injury, and suggest that TRPA1 in SGCs may contribute to inflammatory and neuropathic pain.

Piezo2 is required for Merkel-cell mechanotransduction.

How we sense touch remains fundamentally unknown. The Merkel cell-neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aß sensory fibres to encode fine details of objects. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown. Synapse-like junctions are observed between Merkel cells and associated afferents, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell-neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus.

Optogenetic Inhibition of CGRPα Sensory Neurons Reveals Their Distinct Roles in Neuropathic and Incisional Pain.

Cutaneous somatosensory neurons convey innocuous and noxious mechanical, thermal, and chemical stimuli from peripheral tissues to the CNS. Among these are nociceptive neurons that express calcitonin gene-related peptide-α (CGRPα). The role of peripheral CGRPα neurons (CANs) in acute and injury-induced pain has been studied using diphtheria toxin ablation, but their functional roles remain controversial. Because ablation permanently deletes a neuronal population, compensatory changes may ensue that mask the physiological or pathophysiological roles of CANs, particularly for injuries that occur after ablation. Therefore, we sought to define the role of intact CANs in vivo under baseline and injury conditions by using noninvasive transient optogenetic inhibition. We assessed pain behavior longitudinally from acute to chronic time points. We generated adult male and female mice that selectively express the outward rectifying proton pump archaerhodopsin-3 (Arch) in CANs, and inhibited their peripheral cutaneous terminals in models of neuropathic (spared nerve injury) and inflammatory (skin-muscle incision) pain using transdermal light activation of Arch. After nerve injury, brief activation of Arch reversed the chronic mechanical, cold, and heat hypersensitivity, alleviated the spontaneous pain, and reversed the sensitized mechanical currents in primary afferent somata. In contrast, Arch inhibition of CANs did not alter incision-induced hypersensitivity. Instead, incision-induced mechanical and heat hypersensitivity was alleviated by peripheral blockade of CGRPα peptide-receptor signaling. These results reveal that CANs have distinct roles in the time course of pain during neuropathic and incisional injuries and suggest that targeting peripheral CANs or CGRPα peptide-receptor signaling could selectively treat neuropathic or postoperative pain, respectively.SIGNIFICANCE STATEMENT The contribution of sensory afferent CGRPα neurons (CANs) to neuropathic and inflammatory pain is controversial. Here, we left CANs intact during neuropathic and perioperative incision injury by using transient transdermal optogenetic inhibition of CANs. We found that peripheral CANs are required for neuropathic mechanical, cold, and heat hypersensitivity, spontaneous pain, and sensitization of mechanical currents in afferent somata. However, they are dispensable for incisional pain transmission. In contrast, peripheral pharmacological inhibition of CGRPα peptide-receptor signaling alleviated the incisional mechanical and heat hypersensitivity, but had no effect on neuropathic pain. These results show that CANs have distinct roles in neuropathic and incisional pain and suggest that their targeting via novel peripheral treatments may selectively alleviate neuropathic versus incisional pain.

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.

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step toward identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top-down mass spectrometry to identify proteoforms released during 1 min of mechanical stimulation of mouse skin from naïve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top-down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.

Characterization of a mouse model of sickle cell trait: parallels to human trait and a novel finding of cutaneous sensitization.

Sickle cell trait (SCT) has classically been categorized as a benign condition except in rare cases or upon exposure to severe physical conditions. However, several lines of evidence indicate that individuals with SCT are not always asymptomatic, and additional physiological changes and risks may remain unexplored. Here, we utilized mice harbouring one copy of normal human ß globin and one copy of sickle human ß globin as a model of SCT to assess haematological, histopathological and somatosensory outcomes. We observed that SCT mice displayed renal and hepatic vascular congestion after exposure to hypoxia. Further, we observed that SCT mice displayed increased cold aversion as well as mechanical and heat sensitivity, though to a lesser degree than homozygous sickle cell disease mice. Notably, mechanical hypersensitivity increased following hypoxia and reoxygenation. Overall our findings suggest that SCT is not entirely benign, and further assessment of pain and cutaneous sensitization is warranted both in animal models and in clinical populations.

Amplified Mechanically Gated Currents in Distinct Subsets of Myelinated Sensory Neurons following In Vivo Inflammation of Skin and Muscle.

Primary afferents are sensitized to mechanical stimuli following in vivo inflammation, but whether sensitization of mechanically gated ion channels contributes to this phenomenon is unknown. Here we identified two populations of murine A fiber-type sensory neurons that display markedly different responses to focal mechanical stimuli of the membrane based on their expression of calcitonin gene-related peptide (CGRP). Following inflammation of the hindpaw, myelinated, CGRP-positive neurons projecting to the paw skin displayed elevated mechanical currents in response to mechanical stimuli. Conversely, muscle inflammation markedly amplified mechanical currents in myelinated, CGRP-negative neurons projecting to muscle. These data show, for the first time, that mechanically gated currents are amplified following in vivo tissue inflammation, and also suggest that mechanical sensitization can occur in myelinated neurons after inflammation.

Selective antagonism of TRPA1 produces limited efficacy in models of inflammatory- and neuropathic-induced mechanical hypersensitivity in rats.

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC90 of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund's adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC90 concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.

Substance P is increased in patients with sickle cell disease and associated with haemolysis and hydroxycarbamide use.

Sickle cell disease (SCD) pain transitions from acute to chronic for unknown reasons. Chronic elevation of the pain neurotransmitter substance P (SP) sensitizes pain nociceptors. We evaluated SP levels in controls and SCD patients during baseline and acute pain and investigated associations between SP and age, gender, pain history, haemolysis and hydroxycarbamide (also termed hydroxyurea) use. Plasma SP levels were measured using enzyme-linked immunosorbent assay. Independent samples t-test compared SP levels between: (i) SCD baseline and controls, and (ii) SCD baseline and acute pain. Multivariate linear regression determined associations between SP and age, gender, pain history and hydroxycarbamide use. Spearman correlation determined an association between SP and haemolysis. We enrolled 35 African American controls, 25 SCD baseline and 12 SCD pain patients. SCD patients were 7-19 years old. Mean ± standard deviation SP level (pg/ml) in SCD baseline was higher than controls (32·4 ± 11·6 vs. 22·9 ± 7·6, P = 0·0009). SP in SCD pain was higher than baseline (78·1 ± 43·4 vs. 32·4 ± 11·6, P = 0·004). Haemolysis correlated with increased SP: Hb (r = -0·7, P = 0·0002), reticulocyte count (r = 0·61, P = 0·0016), bilirubin (r = 0·68, P = 0·0216), lactate dehydrogenase (r = 0·62, P = 0·0332), aspartate aminotransferase (r = 0·68, P = 0·003). Patients taking hydroxycarbamide had increased SP (ß = 29·2, P = 0·007). SP could be a mediator of or marker for pain sensitization in SCD and a biomarker and/or target for novel pain treatment.

Chemical structure and morphology of dorsal root ganglion neurons from naive and inflamed mice.

Fourier transform infrared spectromicroscopy provides label-free imaging to detect the spatial distribution of the characteristic functional groups in proteins, lipids, phosphates, and carbohydrates simultaneously in individual DRG neurons. We have identified ring-shaped distributions of lipid and/or carbohydrate enrichment in subpopulations of neurons which has never before been reported. These distributions are ring-shaped within the cytoplasm and are likely representative of the endoplasmic reticulum. The prevalence of chemical ring subtypes differs between large- and small-diameter neurons. Peripheral inflammation increased the relative lipid content specifically in small-diameter neurons, many of which are nociceptive. Because many small-diameter neurons express an ion channel involved in inflammatory pain, transient receptor potential ankyrin 1 (TRPA1), we asked whether this increase in lipid content occurs in TRPA1-deficient (knock-out) neurons. No statistically significant change in lipid content occurred in TRPA1-deficient neurons, indicating that the inflammation-mediated increase in lipid content is largely dependent on TRPA1. Because TRPA1 is known to mediate mechanical and cold sensitization that accompanies peripheral inflammation, our findings may have important implications for a potential role of lipids in inflammatory pain.

Correction: Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows.

Correction for 'Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows' by Ebrahim Aboualizadeh et al., Analyst, 2015, 140, 4046-4056.

Cold shock induces apoptosis of dorsal root ganglion neurons plated on infrared windows.

The chemical status of live sensory neurons is accessible with infrared microspectroscopy of appropriately prepared cells. In this paper, individual dorsal root ganglion (DRG) neurons have been prepared with two different protocols, and plated on glass cover slips, BaF2 and CaF2 substrates. The first protocol exposes the intact DRGs to 4 °C for between 20-30 minutes before dissociating individual neurons and plating 2 hours later. The second protocol maintains the neurons at 23 °C for the entire duration of the sample preparation. The visual appearance of the neurons is similar. The viability was assessed by means of trypan blue exclusion method to determine the viability of the neurons. The neurons prepared under the first protocol (cold exposure) and plated on BaF2 reveal a distinct chemical signature and chemical distribution that is different from the other sample preparations described in the paper. Importantly, results for other sample preparation methods, using various substrates and temperature protocols, when compared across the overlapping spectral bandwidth, present normal chemical distribution within the neurons. The unusual chemically specific spatial variation is dominated by a lack of protein and carbohydrates in the center of the neurons and signatures of unraveling DNA are detected. We suggest that cold shock leads to apoptosis of DRGs, followed by osmotic stress originating from ion gradients across the cell membrane leading to cell lysis.

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.

Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.

A 'toothache tree' alkylamide inhibits Aδ mechanonociceptors to alleviate mechanical pain.

In traditional medicine, the 'toothache tree' and other plants of the Zanthoxylum genus have been used to treat inflammatory pain conditions, such as toothache and rheumatoid arthritis. Here we examined the cellular and molecular mechanisms underlying the analgesic properties of hydroxy-α-sanshool, the active alkylamide produced by Zanthoxylum plants. Consistent with its analgesic effects in humans, sanshool treatment in mice caused a selective attenuation of mechanical sensitivity under naïve and inflammatory conditions, with no effect on thermal sensitivity. To elucidate the molecular mechanisms by which sanshool attenuates mechanical pain, we performed single fibre recordings, calcium imaging and whole-cell electrophysiology of cultured sensory neurons. We found that: (1) sanshool potently inhibits Aδ mechanonociceptors that mediate both sharp acute pain and inflammatory pain; (2) sanshool inhibits action potential firing by blocking voltage-gated sodium currents in a subset of somatosensory neurons, which express a unique combination of voltage-gated sodium channels; and (3) heterologously expressed Nav1.7 is most strongly inhibited by sanshool as compared to other sodium channels expressed in sensory neurons. These results suggest that sanshool targets voltage-gated sodium channels on Aδ mechanosensory nociceptors to dampen excitability and thus induce 'fast pain' analgesia.