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Supplemental Digital Content is Available in the Text., Endometriosis (ENDO) and interstitial cystitis/bladder pain syndrome (IC/BPS) are chronic pain conditions for which better treatments are urgently needed. Development of new therapies with proven clinical benefit has been slow. We have conducted a review of existing preclinical in vivo models for ENDO and IC/BPS in rodents, discussed to what extent they replicate the phenotype and pain experience of patients, as well as their relevance for translational research. In 1009 publications detailing ENDO models, 41% used autologous, 26% syngeneic, 18% xenograft, and 11% allogeneic tissue in transplantation models. Intraperitoneal injection of endometrial tissue was the subcategory with the highest construct validity score for translational research. From 1055 IC/BPS publications, most interventions were bladder centric (85%), followed by complex mechanisms (8%) and stress-induced models (7%). Within these categories, the most frequently used models were instillation of irritants (92%), autoimmune (43%), and water avoidance stress (39%), respectively. Notably, although pelvic pain is a hallmark of both conditions and a key endpoint for development of novel therapies, only a small proportion of the studies (models of ENDO: 0.5%-12% and models of IC/BPS: 20%-44%) examined endpoints associated with pain. Moreover, only 2% and 3% of publications using models of ENDO and IC/BPS investigated nonevoked pain endpoints. This analysis highlights the wide variety of models used, limiting reproducibility and translation of results. We recommend refining models so that they better reflect clinical reality, sharing protocols, and using standardized endpoints to improve reproducibility. We are addressing this in our project Innovative Medicines Initiative-PainCare/Translational Research in Pelvic Pain.

Supplemental Digital Content is Available in the Text. We here report on the use of in vivo optical imaging to study lamina I projection neurons and their response to cutaneous stimuli., Lamina I of the dorsal horn, together with its main output pathway, lamina I projection neurons, has long been implicated in the processing of nociceptive stimuli, as well as the development of chronic pain conditions. However, the study of lamina I projection neurons is hampered by technical challenges, including the low throughput and selection biases of traditional electrophysiological techniques. Here we report on a technique that uses anatomical labelling strategies and in vivo imaging to simultaneously study a network of lamina I projection neurons in response to electrical and natural stimuli. Although we were able to confirm the nociceptive involvement of this group of cells, we also describe an unexpected preference for innocuous cooling stimuli. We were able to characterize the thermal responsiveness of these cells in detail and found cooling responses decline when exposed to stable cold temperatures maintained for more than a few seconds, as well as to encode the intensity of the end temperature, while heating responses showed an unexpected reliance on adaptation temperatures.

Bladder pain syndrome (BPS) is associated with breakdown of the protective uroepithelial barrier of the urinary bladder allowing urinary constituents access to bladder sensory neurons. Although there are several animal models of cystitis, none specifically relates to BPS. Here, we aimed to create such a model using enzymatic digestion of the barrier proteoglycans (PGs) in the rat. Twenty female Wistar rats were anaesthetized and transurethrally catheterized. Ten animals were treated with 0.25IU of intravesical chondroitinase ABC and heparanase III to digest chondroitin sulphate and heparin sulphate PGs, respectively. Ten animals received saline. Following PG deglycosylation, bladders showed irregular loss of the apical uroplakin and a significant increase in neutrophils, not evident in the control group. Spinal cord sections were also collected for c-fos analysis. A large and significant increase in fos immunoreactivity in the L6/S1 segments in the treatment vs control bladders was observed. Cystometry was performed on 5 treatment and 5 control animals. Analysis revealed a significant increase in micturition reflex excitability postdeglycosylation. On a further group of 10 animals, von Frey mechanical withdrawal thresholds were tested on abdominal skin before and after PG digestions. There was a significant decrease in abdominal mechanical withdrawal threshold postdeglycosylation compared with controls. The results of this animal study suggest that many of the clinical features of BPS are seen after PG digestion from the bladder lumen. This model can be used to further understand mechanisms of pain in patients with BPS and to test new therapeutic strategies.

Supplemental Digital Content is Available in the Text. Mast cells do not play a role as a direct nociceptor sensitiser during acute inflammatory pain and in mechanical hypersensitivity triggered by local nerve growth factor., The immune and sensory systems are known for their close proximity and interaction. Indeed, in a variety of pain states, a myriad of different immune cells are activated and recruited, playing a key role in neuronal sensitisation. During inflammatory pain it is thought that mast cells (MC) are one of the immune cell types involved in this process, but so far the evidence outlining their direct effect on neuronal cells remains unclear. To clarify whether MC are involved in inflammatory pain states, we used a transgenic mouse line (Mctp5Cre-iDTR) in which MC could be depleted in an inducible manner by administration of diphtheria toxin. Our results show that ablation of MC in male mice did not result in any change in mechanical and thermal hypersensitivity in the CFA model of inflammatory pain. Similarly, edema and temperature triggered by CFA inflammation at the injection site remained identical in MC depleted mice compared with their littermate controls. In addition, we show that Mctp5Cre-iDTR mice display normal levels of mechanical hypersensitivity after local injection of nerve growth factor (NGF), a factor well characterised to produce peripheral sensitisation and for being upregulated upon injury and inflammation. We also demonstrate that NGF treatment in vitro does not lead to an increased level of tumor necrosis factor-α in bone marrow-derived MC. Furthermore, our qRT-PCR data reveal that MC express negligible levels of NGF receptors, thereby explaining the lack of response to NGF. Together, our data suggest that MC do not play a direct role in peripheral sensitisation during inflammatory conditions.

Supplemental Digital Content is Available in the Text. Deletion of the potassium channel Kcns1 from peripheral sensory neurons alters acute and neuropathic pain processing. Compounds that enhance Kcns1 activity may provide analgesia., Voltage-gated potassium (Kv) channels are increasingly recognised as key regulators of nociceptive excitability. Kcns1 is one of the first potassium channels to be associated with neuronal hyperexcitability and mechanical sensitivity in the rat, as well as pain intensity and risk of developing chronic pain in humans. Here, we show that in mice, Kcns1 is predominantly expressed in the cell body and axons of myelinated sensory neurons positive for neurofilament-200, including Aδ-fiber nociceptors and low-threshold Aβ mechanoreceptors. In the spinal cord, Kcns1 was detected in laminae III to V of the dorsal horn where most sensory A fibers terminate, as well as large motoneurons of the ventral horn. To investigate Kcns1 function specifically in the periphery, we generated transgenic mice in which the gene is deleted in all sensory neurons but retained in the central nervous system. Kcns1 ablation resulted in a modest increase in basal mechanical pain, with no change in thermal pain processing. After neuropathic injury, Kcns1 KO mice exhibited exaggerated mechanical pain responses and hypersensitivity to both noxious and innocuous cold, consistent with increased A-fiber activity. Interestingly, Kcns1 deletion also improved locomotor performance in the rotarod test, indicative of augmented proprioceptive signalling. Our results suggest that restoring Kcns1 function in the periphery may be of some use in ameliorating mechanical and cold pain in chronic states.

A painful neuropathy is frequently observed in people living with human immunodeficiency virus type 1 (HIV-1). The HIV coat protein, glycoprotein 120 (gp120), implicated in the pathogenesis of neurological disorders associated with HIV, is capable of initiating neurotoxic cascades via an interaction with the CXCR4 and/or CCR5 chemokine receptors, which may underlie the pathogenesis of HIV-associated peripheral neuropathic pain. In order to elucidate the mechanisms underlying HIV-induced painful peripheral neuropathy, we have characterised pathological events in the peripheral and central nervous system following application of HIV-1 gp120 to the rat sciatic nerve. Perineural HIV-1 gp120 treatment induced a persistent mechanical hypersensitivity (44% decrease from baseline), but no alterations in sensitivity to thermal or cold stimuli, and thigmotactic (anxiety-like) behaviour in the open field. The mechanical hypersensitivity was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. Immunohistochemical studies reveal: decreased intraepidermal nerve fibre density, macrophage infiltration into the peripheral nerve at the site of perineural HIV-1 gp120; changes in sensory neuron phenotype including expression of activating transcription factor 3 (ATF3) in 27% of cells, caspase-3 in 25% of cells, neuropeptide Y (NPY) in 12% of cells and galanin in 13% of cells and a spinal gliosis. These novel findings suggest that this model is not only useful for the elucidation of mechanisms underlying HIV-1-related peripheral neuropathy but may prove useful for preclinical assessment of drugs for the treatment of HIV-1 related peripheral neuropathic pain.

Hundreds of genes are proposed to contribute to nociception and pain perception. Historically, most studies of pain-related genes have examined them in isolation or alongside a handful of other genes. More recently the use of systems biology techniques has enabled us to study genes in the context of the biological pathways and networks in which they operate. Here we describe a Web-based resource, available at http://www.PainNetworks.org. It integrates interaction data from various public databases with information on known pain genes taken from several sources (eg, The Pain Genes Database) and allows the user to examine a gene (or set of genes) of interest alongside known interaction partners. This information is displayed by the resource in the form of a network. The user can enrich these networks by using data from pain-focused gene expression studies to highlight genes that change expression in a given experiment or pairs of genes showing correlated expression patterns across different experiments. Genes in the networks are annotated in several ways including biological function and drug binding. The Web site can be used to find out more about a gene of interest by looking at the function of its interaction partners. It can also be used to interpret the results of a functional genomics experiment by revealing putative novel pain-related genes that have similar expression patterns to known pain-related genes and by ranking genes according to their network connections with known pain genes. We expect this resource to grow over time and become a valuable asset to the pain community. © 2013 International Association for the Study of Pain.

Summary Intrathecal delivery of histone deacetylase inhibitors ameliorates hypersensitivity in models of neuropathic pain. This effect may be mediated at the level of the spinal cord through inhibition of HDAC1 function., Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug–induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.

Previous evidence suggests that, in adult animals, nerve growth factor (NGF) can induce hyperalgesia, and may be an endogenous mediator in some persistent pain states. Here we have studied the effects of single intradermal injections of 50-500 ng of human recombinant NGF into the plantar skin of adult rat hindpaws. We found that doses of 250 ng and more produced a prolonged and stable thermal hyperalgesia to radiant heat. NGF did not produce overt pain behaviour as judged by the absence of paw licking or guarding of the injected paw. In animals subjected to surgical or chemical sympathectomy, by repeated systemic guanethidine treatments, the hyperalgesic effects of NGF were markedly reduced. We also found that NGF produced plasma extravasation in rat skin, using the Evan's blue method, with a dose dependency similar to that determined for hyperalgesia. Together, these findings suggest that NGF can lead to a rapid activation and sensitization of cutaneous nociceptors. However, these actions appear at least partly indirect, requiring the presence of normal sympathetic post-ganglionic terminals.

Bradykinin (BK) is an inflammatory mediator that can evoke oedema and vasodilatation, and is a potent algogen signalling via the B1 and B2 G-protein coupled receptors. In naïve skin, BK is effective via constitutively expressed B2 receptors (B2R), while B1 receptors (B1R) are purported to be upregulated by inflammation. The aim of this investigation was to optimise BK delivery to investigate the algesic effects of BK and how these are modulated by inflammation. BK iontophoresis evoked dose- and temperature-dependent pain and neurogenic erythema, as well as thermal and mechanical hyperalgesia (P0.001 vs saline control). To differentiate the direct effects of BK from indirect effects mediated by histamine released from mast cells (MCs), skin was pretreated with compound 4880 to degranulate the MCs prior to BK challenge. The early phase of BK-evoked pain was reduced in degranulated skin (P0.001), while thermal and mechanical sensitisation, wheal, and flare were still evident. In contrast to BK, the B1R selective agonist des-Arg9-BK failed to induce pain or sensitise naïve skin. However, following skin inflammation induced by ultraviolet B irradiation, this compound produced a robust pain response. We have optimised a versatile experimental model by which BK and its analogues can be administered to human skin. We have found that there is an early phase of BK-induced pain which partly depends on the release of inflammatory mediators by MCs; however, subsequent hyperalgesia is not dependent on MC degranulation. In naïve skin, B2R signaling predominates, however, cutaneous inflammation results in enhanced B1R responses.

Dorsal horn sensitization following somatic noxious stimuli is partly mediated by the N- methyl - d - aspartate (NMDA) sub-type of glutamate receptor. This phenomenon has been comparatively sparsely investigated in the area of visceral pain. We have therefore investigated the role of spinal NMDA receptors in central sensitization in an animal model of persistent visceral pain. In anaesthetized rats the lumbosacral spinal cord was exposed by laminectomy and the pre-emptive effect of intrathecal AP-5 upon the hyper-reflexia associated with chemical inflammation of the bladder was investigated. The effect of intrathecal AP-5 (an NMDA receptor antagonist) upon the normal cystometrogram (CMG) was also measured. AP-5 (125–1000 μg) prevented the hyper-flexia associated with bladder inflammation in a dose-dependant fashion. In general, within the dose range 62.5–1000 μg, AP-5 had no significant effect upon the normal micturition reflex. However, at the top of this dose range a minor non-significant depression of this reflex was noted. NMDA receptors do not appear to mediate the micturition reflex at a spinal cord level. However, they are involved in the induction of hyper-reflexia following urinary bladder inflammation, this hyper-reflexia can be prevented by pre-emptive intrathecal administration of AP-5.

Capsaicin has been used extensively as an experimental tool and in traditional and proprietary topical medications for acute soft tissue injuries. More recently it has been prescribed for several chronic pain conditions where it is usually administered topically for periods of several weeks. Here we have studied the consequences of this mode of application in the rat. Capsaicin cream (0.075% or 0.75%). or a vehicle cream, was applied twice daily to the hind paws of rats for a continuous period of 10 weeks. The hind paws treated with 0.75% capsaicin (but not 0.075%) became transiently hyperalgesic, but there were no signs of discomfort or distress associated with the treatment. After 10 weeks of capsaicin application, the ability of C fibres to produce neurogenic extravasation was markedly reduced. After 4 weeks of recovery this ability returned to normal in 0.075% capsaicin-treated animals, but remained impaired in the 0.75% group. This latter group showed a partial recovery 12 weeks after the end of treatment. The levels of substance P and CGRP in the sural nerve supplying the treated skin area were unchanged after both the 0.075% and 0.75% capsaicin treatments. The results suggest that the topical application of capsaicin at low concentration produces a reversible impairment of the terminals of C fibres in the skin without greatly exciting those fibres and without affecting the properties of cell soma. The number of afferent neurones in the L5 dorsal root ganglion projecting through the sural nerve was unchanged after 0.75% capsaicin treatment, suggesting that the topical capsaicin treatment does not produce any cell death in the adult animal.

In humans, the acute inflammatory reaction caused by ultraviolet (UV) radiation is well studied and the sensory changes that are found have been used as a model of cutaneous hyperalgesia. Similar paradigms are now emerging as rodent models of inflammatory pain. Using a narrowband UVB source, we irradiated the plantar surface of rat hind paws. This produced the classical feature of inflammation, erythema, and a significant dose-dependent reduction in both thermal and mechanical paw withdrawal thresholds. These sensory changes peaked 48 h after irradiation. At this time there is a graded facilitation of noxious heat evoked (but not basal) c-fos-like immunoreactivity in the L4/5 segments of the spinal cord. We also studied the effects of established analgesic compounds on the UVB-induced hyperalgesia. Systemic as well as topical application of ibuprofen significantly reduced both thermal and mechanical hyperalgesia. Systemic morphine produced a dose-dependent and naloxone sensitive reversal of sensory changes. Similarly, the peripherally restricted opioid loperamide also had a dose-dependent anti-hyperalgesic effect, again reversed by naloxone methiodide. Sequestration of NGF, starting at the time of UVB irradiation, significantly reduced sensory changes. We conclude that UVB inflammation produces a dose-dependent hyperalgesic state sensitive to established analgesics. This suggests that UVB inflammation in the rat may represent a useful translational tool in the study of pain and the testing of analgesic agents.

Since there is growing evidence that nerve growth factor (NGF) acts as a mediator of persistent pain states, here we have studied its acute effects on the properties of primary afferent neurones innervating adult rat urinary bladder. Single A delta and C fibres were dissected from the L6 or S1 dorsal roots of urethane anaesthetized rats. The stimulus-response function of these afferents was evaluated with a series of isotonic distensions of the bladder (0-60 cm H2O). The afferents were then studied after filling the bladder with a vehicle solution of 10% DMSO for 30 min, and then again after filling the bladder with a 10 micrograms/ml solution of human recombinant NGF in 10% DMSO. In the control state, and after filling with 10% DMSO, the myelinated and about one-half of the unmyelinated afferents were mechanosensitive with pressure thresholds in the innocuous range and responsiveness extending into the supra physiological, presumed noxious range. The remaining one-half of unmyelinated afferents showed no mechanosensitivity. After filling with NGF, the large majority of units, both myelinated and unmyelinated, sensitised, evidenced by the development of ongoing activity and a leftward shift of stimulus-response functions. Some of the initially nonmechanosensitive units developed a novel mechanosensitivity. The sensitisation began within 30 min of exposure to NGF, and persisted for the period studied (up to the 3 h). In separate experiments, intravesical NGF at concentrations greater than 1 microgram/ml was found to elicit a dose-dependent extravasation of Evan's blue into the bladder. These data support the notion that NGF may be an endogenous mediator in some persistent pain states.

We have studied experimentally induced itch (using histamine iontophoresis) and pain (using topical mustard oil) in healthy human volunteers, measured using visual analogue scale (VAS) ratings. The effects of the following counterstimuli were evaluated: innocuous vibration; innocuous transcutaneous electrical nerve stimulation (TENS); innocuous warming of skin; noxious heating of skin; noxious chemical skin stimulation (using mustard oil); mildly noxious constant current transdermal electrical stimulation. Inncuous stimuli applied 2 min after histamine or mustard oil challenge produced a moded reduction of itch and pain ratings (20–30%), which did not persist for more than 20 sec when the counterstimuli were removed. These changes may well be due to distraction. The same stimuli (in some cases applied for periods of up to 1 h) given prior to the histamine or mustard oil, produced no significant reduction in evoked itch or pain. Noxious stimuli of all types applied 2 after histamine or mustard oil challenge produced a consistent and significant inhibition of itch (22.8–52.7%) which outlasted the period of counterstimulation application. In contrast, these stimuli had no significant effects on chemically induced pain. The anti-pruritic state produced by brief noxious stimuli were effective for periods of more than 30 min. The differential effects of noxious counterstimuli on itch and pain do not support the suggestion that itch is a subliminal form of pain. Noxious counterstimuli are likely to act via a central rather than peripheral mechanism. The novel finding that a persistent anti-pruritic state can be induced by transdermal constant current may be useful in conditions of clinical itch.

The consequences of microneurography were assessed, using tungsten and coaxial electrodes in rat and man. Firstly, the sequelae of microneurography were examined for up to 1 month in a rat sciatic nerve model, using 3 techniques. Expression of the injury-associated protein GAP-43 was measured by immunofluorescence in sciatic nerve and dorsal root ganglia 3 and 28 days following sciatic nerve injury by microneurography electrodes. No increase in GAP-43 was observed in sciatic nerve 3 days following injury, but 28 days after injury the coaxial electrode was associated with an increase in GAP-43 expression. All electrodes were associated with an increase in GAP-43 in dorsal root ganglia 28 days after injury. The capacity of unmyelinated afferent fibres to induce neurogenic oedema was examined up to the 28th post-experimental day, as measured by Evan's Blue extravasation. The tungsten electrode induced a decrease in extravasation, which persisted for 28 days. In contrast, the decrease in plasma extravasation associated with lesions induced by the coaxial electrode returned to baseline within 7 days. The hind-limb withdrawal time from a noxious stimulus was also measured up to 28 days following injury. Both types of electrode induced a significant change in the immediate post-experimental period, but this returned to normal within 1 week. The direction of change differed between the tungsten and coaxial electrodes. In humans, symptomatology questionnaires were collected after microneurography experiments with the coaxial electrode. Thirty-two volunteers were studied. Two subjects reported mild paraesthesiae, which fully resolved within 24 h. This compares favourably with similar studies of the tungsten electrode.

Normal healthy visceral tissue is largely insensitive to many kinds of tissue damaging stimuli, such as cutting, crushing or burning. When inflamed, however, visceral tissue frequently and easily elicits pain. Here we have produced inflammatory responses in urinary bladders of chronically decerebrated rats with irritant chemicals, as a model of a visceral pain state. The irritants used were 25% turpentine, 2.5% mustard oil, 2% croton oil. All produced a strong inflammatory response, as judged by protein extravasation and oedema, and invasion of the tissue with leucocytes, which started within hours of treatment. The inflammatory states were associated with hyperexcitable bladder reflexes; baseline pressures were larger at given volumes, and large micturition contractions occurred at lower volumes. These changes persisted for up to 48 h after a single treatment. The behaviour of the animals and their responsiveness to noxious stimulation were also assessed. In general animals were hypersensitive to noxious stimuli applied to the tail or caudal abdomen, with small or no changes in responses to hind limb or rostral abdominal stimulation. These irritants, particularly turpentine, applied to chronic decerebrate rats may thus provide useful models for studies of the neuronal processes which contribute to visceral nociception.

The effects of punctate burns to the skin on the response properties of a group of lamina 1 projection cells in the dorsal horn of the spinal cord has been studied in decerebrate rats. The burns were made 5-15 mm outside the boundaries of the receptive fields (RF) of the cells and they produced a slow increase in the excitability of the cells as seen by an enlargement of RF, towards the lesion, a decreased mechanical threshold and an increased responsiveness to transcutaneous electrical stimulation applied with needle electrodes both inside and outside the RF. These changes were first seen 10-15 min after burning and were not reversed during the time course of the investigation. Local anaesthetic injected at the site of injury produced only a partial and slow reversal of the excitability increases. Since responses to transcutaneous electrical stimulation were increased, injury-induced changes were unlikely to be due entirely to alterations in the properties of primary afferent fibres, but this was directly tested by attempting to mimic the afferent barrage produced by the burn by electrical stimulation of the central cut ends of either the sural nerve or a dorsal rootlet. In both cases a single 10 min period of stimulation (0.5 Hz supramaximal intensity) was found to induce a slow and progressive increase in RF size, confirming the existence of a central component. The changes seen with peripheral burns were probably triggered at least in part by a discharge in primary afferent C fibres since units studied in capsaicin treated animals (which have an impairment of C fibre function) did not show equivalent changes. These results suggest that mechanisms of sensory processing, especially nociception, are subject to reorganization in the face of altered input. The slow time course of the changes suggests that the underlying mechanism may not be the rapid post-synaptic potentials of traditional neurophysiology.