Characterization and comparison of rat monosodium iodoacetate and medial meniscal tear models of osteoarthritic pain.

Osteoarthritis (OA) is a degenerative form of arthritis that can result in loss of joint function and chronic pain. The pathological pain state that develops with OA disease involves plastic changes in the peripheral and central nervous systems, however, the cellular mechanisms underlying OA are not fully understood. We characterized the medial meniscal tear (MMT) surgical model and the intra-articular injection of monosodium iodoacetate (MIA) chemical model of OA in rats. Both models produced histological changes in the knee joint and associated bones consistent with OA pathology. Both models also increased p38 activation in the L3, but not L4 dorsal root ganglia (DRG), increased tyrosine hydroxylase immunostaining in the L3 DRG indicating sympathetic sprouting, and increased phosphorylated (p)CREB in thalamic neurons. In MIA-OA, but not MMT-OA rats, p38 and pERK were increased in the spinal cord, and pCREB was enhanced in the prefrontal cortex. Using in vivo electrophysiology, elevated spontaneous activity and increased responsiveness of wide dynamic range neurons to stimulation of the knee was found in both models. However, a more widespread sensitization was observed in the MIA-OA rats as neurons with paw receptive fields spontaneously fired at a greater rate in MIA-OA than MMT-OA rats. Taken together, the MIA and MMT models of OA share several common features associated with histopathology and sensitization of primary somatosensory pathways, but, observed differences between the models highlights unique consequences of the related specific injuries, and these differences should be considered when choosing an OA model and when interpreting data outcomes. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

TRPV3 modulates nociceptive signaling through peripheral and supraspinal sites in rats.

TRPV3 is a nonselective cation channel activated by temperatures above 33°C and is reported to be localized in keratinocytes and nervous tissue. To investigate a role for TRPV3 in pain modulation, we conducted a series of in vivo electrophysiological studies on spinal and brain nociceptive neurons. Structurally diverse TRPV3 receptor antagonists reduced responses of spinal wide dynamic range (WDR) neurons to low-intensity mechanical stimulation in neuropathic rats, but only CNS-penetrant antagonists decreased elevated spontaneous firing. Injections of an antagonist into the neuronal receptive field, into the L5 dorsal root ganglion, or intracerebroventricularly (ICV) attenuated the evoked firing, but only ICV injections reduced spontaneous activity. Intraspinal injections did not affect either. Spinal transection blocked the effect on spontaneous but not evoked firing after systemic delivery of a TRPV3 antagonist. Systemic administration of an antagonist to neuropathic rats also impacted the firing of On- and Off-cells in the rostral ventromedial medulla in a manner consistent with dampening nociceptive signaling. An assessment of nonevoked "pain," an EEG-measured pain-induced sleep disturbance induced by hind paw injections of CFA, was also improved with CNS-penetrant TRPV3 antagonists but not by an antagonist with poor CNS penetration. Antagonism of TRPV3 receptors modulates activity of key classes of neurons in the pain pathway in a manner consistent with limiting pathological nociceptive signaling and was mediated by receptors in the periphery and brain. Blockade of TRPV3 receptors is likely an effective means to alleviate mechanical allodynia and nonevoked pain. However, the latter will only be obtained by blocking supraspinal TRPV3 receptors.NEW & NOTEWORTHY Recent studies have linked TRPV3 to pain modulation, and much of this work has focused on its role in the skin-primary afferent interface. In this electrophysiological study, we demonstrate that receptor antagonists modulate evoked signals through peripheral mechanisms but blockade of supraspinal TRPV3 receptors contributes to dampening both evoked and nonevoked "pain" through descending modulation. Thus, the full therapeutic potential of TRPV3 antagonists may only be realized with the ability to access receptors in the brain.

Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 Antagonists.

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

Primary afferent neurons express functional delta opioid receptors in inflamed skin.

Peripherally-restricted opiate compounds attenuate hyperalgesia in experimental models of inflammatory pain, but have little discernable effect on nociceptive behavior in normal animals. This suggests that activation of opioid receptors on peripheral sensory axons contributes to decreased afferent activity after injury. Previously, we reported that direct application of morphine to cutaneous receptive fields decreased mechanical and heat-evoked responses in a population of C-fiber nociceptors in inflamed skin. Consistent with reported behavioral studies, direct application of morphine had no effect on fiber activity in control skin. The aim of the present study was to determine whether mechanical responsiveness of nociceptors innervating inflamed skin was attenuated by direct activation of delta opioid receptors (DORs) on peripheral terminals. An ex vivo preparation of rat plantar skin and tibial nerve was used to examine effects of a selective DOR agonist, deltorphin II, on responsiveness of single fibers innervating inflamed skin. Electrical recordings were made eighteen hours after injection of complete Freund's adjuvant into the hindpaw. Deltorphin II produced an inhibition of the mechanical responsiveness of single fibers innervating inflamed skin; an effect blocked by the DOR-selective antagonist, naltrindole. The population of units responsive to deltorphin II was identified as consisting of C fiber mechanical nociceptors.

Biochemical and pharmacological assessment of MAP-kinase signaling along pain pathways in experimental rodent models: a potential tool for the discovery of novel antinociceptive therapeutics.

Injury to the peripheral or central nervous system can induce changes within the nervous tissues that promote a state of sensitization that may underlie conditions of pathological chronic pain. A key biochemical event in the initiation and maintenance of peripheral and central neuronal sensitization associated with chronic pain is the phosphorylation and subsequent activation of mitogen-activated protein kinases (MAPKs) and immediate early gene transcription factors, in particular cAMP-response element binding protein (CREB). In this commentary we review the preclinical data that describe anatomical and mechanistic aspects of nociceptive-induced signaling along nociceptive pathways including peripheral cutaneous axons, the dorsal root ganglia, spinal cord dorsal horn and cerebral cortex. In addition to the regional manifestation of nociceptive signaling, investigations have attempted to elucidate the cellular origin of biochemical nociceptive processing in which communication, i.e. cross-talk between neurons and glia is viewed as an essential component of pathogenic pain development. Here, we outline a research strategy by which nociceptive-induced cellular signaling in experimental pain models, specifically MAPK and CREB phosphorylation can be utilized to provide mechanistic insight into drug-target interaction along the nociceptive pathways. We describe a series of studies using nociceptive inflammatory and neuropathic pain models to investigate the effects of known pain therapeutics on nociceptive-induced biochemical signaling and present this as a complementary research strategy for assessing antinociceptive activity useful in the preclinical development of novel pain therapeutics.

Targeting TRP channels for pain relief.

Preclinical research has recently uncovered new molecular mechanisms underlying the generation and transduction of pain, many of which represent opportunities for pharmacological intervention. Manipulating temperature-sensitive Transient Receptor Potential (TRP) channels (so-called "thermoTRPs") on nociceptive neurons is a particularly attractive strategy in that it targets the beginning of the pain pathway. In the focus of current drug development efforts are the heat-sensitive TRPV1, warm-activated TRPV3, cold-responsive TRPA1, and cool-activated TRPM8 channels. TRPV1 desensitization by topical agonists (e.g. high concentration capsaicin creams and patches) has been in clinical use for decades to alleviate chronic painful conditions like diabetic neuropathy. Currently, site-specific resiniferatoxin (an ultrapotent capsaicin analogue) injections are being evaluated as "molecular scalpels" to achieve permanent analgesia in cancer patients with chronic, intractable pain. In the past few years a number of potent, small molecule TRPV1, TRPV3 and TRPA1 antagonists have been advanced into clinical trials for the treatment of inflammatory, neuropathic and visceral pain. TRPM8 antagonists are following closely behind for cold allodynia. Early TRPV1 antagonists in the clinic, however, showed worrisome adverse effects including hyperthermia and impaired noxious heat sensation. These adverse effects placed the patients at risk for scalding injury and prompted their withdrawal from the clinical trials. Second generation TRPV1 antagonists that do not cause core body temperature elevation have been reported, although the therapeutic utility of this class of compounds is not yet known. This review discusses the promise and challenges of developing TRP channel antagonists as a new generation of pain therapeutics.

PARP inhibitors attenuate chemotherapy-induced painful neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major toxicity of chemotherapy treatment for which no therapy is approved. Poly(ADP-ribose) polymerase (PARP)1/2 are nuclear enzymes activated upon DNA damage, and PARP1/2 inhibition provides resistance against DNA damage. A role for PARP inhibition in sensory neurotransmission has also been established. PARP inhibitors attenuate pain-like behaviors and neuropathy-associated decreased peripheral nerve function in diabetic models. The hypothesis tested was that PARP inhibition protects against painful neuropathy. The objective of this study was to investigate whether the novel, selective PARP1/2 inhibitors (ABT-888 and related analogues) would attenuate development of mechanical allodynia in vincristine-treated rats. PARP inhibitors were dosed for 2 days, and then co-administered with vincristine for 12 days. Mechanical allodynia was observed in rats treated with vincristine. PARP1/2 inhibition significantly attenuated development of mechanical allodynia and reduced poly ADP-ribose (PAR) activation in rat skin. The data presented here show that PARP inhibition attenuates vincristine-induced mechanical allodynia in rats, and supports that PARP inhibition may represent a novel therapeutic approach for CIPN.

The novel calpain inhibitor A-705253 prevents stress-induced tau hyperphosphorylation in vitro and in vivo.

Calcium-mediated pathologic activation of the cysteine protease calpain has been linked to neurodegenerative disorders such as Alzheimer's disease (AD) through the cleavage of proteolytic substrates that negatively affect neuronal function. Hyperphosphorylation of the microtubule-associated protein tau and the subsequent aggregation of tau filaments resulting in the intracellular formation of neurofibrillary tangles are recognized as key etiological factors in AD pathology. Cyclin-dependent kinase 5 (Cdk5), a major kinase responsible for tau hyperphosphorylation in the AD brain, becomes hyperactivated through calpain-mediated cleavage-conversion of the Cdk5 regulatory protein p35 to p25. In the present study, we examined the effects of the novel small-molecule calpain inhibitor A-705253 in acute models of tau hyperphosphorylation in vitro and in vivo. In hippocampal slices in vitro, lowering medium temperature to 33 °C increased tau phosphorylation in which incubation with A-705253 blocked low temperature-induced tau phosphorylation as measured by Western blot analysis. Pentobarbital-induced hypothermia or acute systemic LPS treatment in normal mice increased tau phosphorylation in hippocampal CA3 mossy fibers, as measured by immunohistochemistry, whereas acute A-705253 pretreatment prevented the stress-induced tau hyperphosphorylation in both models. In support of a Cdk5-mediated mechanism, A-705253 administered for two weeks in the drinking water of six month-old prepathogenic 3x Tg-AD mice resulted in decreased expression of the calpain proteolytic p25 fragment. Taken together, results of these studies suggest that calpain inhibition has potential utility in reducing tau hyperphosphorylation and may represent a novel disease-modifying approach in the treatment of AD.

Spontaneous firing and evoked responses of spinal nociceptive neurons are attenuated by blockade of P2X3 and P2X2/3 receptors in inflamed rats.

P2X3 and P2X2/3 receptors are selectively expressed on primary afferent nociceptors and have been implicated in modulating nociception in different models of pathological pain, including inflammatory pain. In an effort to delineate further the role of P2X3 receptors (homomeric and heteromeric) in the modulation of nociceptive transmission after a chronic inflammation injury, A-317491, a potent and selective P2X3-P2X2/3 antagonist, was administered to CFA-inflamed rats in order to examine its effects on responses of spinal dorsal horn neurons to mechanical and thermal stimulation. Systemic injection of A-317491 (30 µmol/kg, i.v.) reduced the responses of wide-dynamic-range (WDR) and nociceptive specific (NS) neurons to both high-intensity mechanical (pinch) and heat (49°C) stimulation. A-317491 also decreased low-intensity (10 g von Frey hair) mechanically evoked activity of WDR neurons but did not alter WDR neuronal responses to cold stimulation (5°C). Spontaneous firing of WDR neurons in CFA-inflamed rats was also significantly attenuated by A-317491 injection. By using immunohistochemistry, P2X3 receptors were demonstrated to be enhanced in lamina II of the spinal dorsal horn after inflammation. In summary, blockade of P2X3 and P2X2/3 receptors dampens mechanical- and heat-related signaling, as well as nonevoked activity of key classes of spinal nociceptive neurons in inflamed animals. These data suggest that P2X3 and/or P2X2/3 receptors have a broad contribution to somatosensory/nociceptive transmission in rats with a chronic inflammatory injury and are consistent with previous behavioral data demonstrating antiallodynic and antihyperalgesic effects of receptor antagonists.

TRPV1 antagonist, A-889425, inhibits mechanotransmission in a subclass of rat primary afferent neurons following peripheral inflammation.

TRPV1 (transient receptor potential vanilloid family type 1) is a nonselective cation channel that is activated and/or sensitized by noxious heat, protons, and other endogenous molecules released following tissue injury. In addition, a role for TRPV1 in mechanotransmission is emerging. We have recently reported that a selective TRPV1 receptor antagonist, A-889425, reduces mechanical allodynia and spinal neuron responses to mechanical stimulation of complete Freund's adjuvant (CFA)-inflamed rat hind paws. The population of peripheral nerve fibers through which TRPV1 antagonists mediate their effect on mechanotransmission have not yet been described. The objective of this study was to characterize TRPV1-mediated modulation of mechanically evoked activity in sensory axons innervating rat hind paws. We used an in vitro skin-nerve preparation to record neural activity from single axons isolated from rat tibial nerve. Single fibers were classified by conduction velocity, mechanical threshold, and stimulus-response relationships. We used A-889425 to investigate uninjured and inflamed skin afferent neuron populations to evoked mechanical stimulation. Application of A-889425 had no effect on the mechanical responsiveness of Aδ and C-fiber units innervating uninjured skin. In contrast, A-889425 inhibited responses of slowly conducting Aδ fiber units to noxious mechanical stimulation in a population of axons innervating CFA-inflamed hind paws. These data support a role for TRPV1 in mechanotransmission following peripheral inflammation, and highlight the importance of a distinct subclass of primary afferent neurons in mediating this effect.

Monosodium iodoacetate-induced joint pain is associated with increased phosphorylation of mitogen activated protein kinases in the rat spinal cord.

BACKGROUND: Intra-articular injection of monosodium iodoacetate (MIA) in the knee joint of rats disrupts chondrocyte metabolism resulting in cartilage degeneration and subsequent nociceptive behavior that has been described as a model of osteoarthritis (OA) pain. Central sensitization through activation of mitogen activated protein kinases (MAPKs) is recognized as a pathogenic mechanism in chronic pain. In the present studies, induction of central sensitization as indicated by spinal dorsal horn MAPK activation, specifically ERK and p38 phosphorylation, was assessed in the MIA-OA model. RESULTS: Behaviorally, MIA-injected rats displayed reduced hind limb grip force 1, 2, and 3 weeks post-MIA treatment. In the same animals, activation of phospho ERK1/2 was gradually increased, reaching a significant level at post injection week 3. Conversely, phosphorylation of p38 MAPK was enhanced maximally at post injection week 1 and decreased, but remained elevated, thereafter. Double labeling from 3-wk MIA rats demonstrated spinal pERK1/2 expression in neurons, but not glia. In contrast, p-p38 was expressed by microglia and a subpopulation of neurons, but not astrocytes. Additionally, there was increased ipsilateral expression of microglia, but not astrocytes, in 3-wk MIA-OA rats. Consistent with increased MAPK immunoreactivity in the contralateral dorsal horn, mechanical allodynia to the contralateral hind-limb was observed 3-wk following MIA. Finally, intrathecal injection of the MEK1 inhibitor PD98059 blocked both reduced hind-limb grip force and pERK1/2 induction in MIA-OA rats. CONCLUSION: Results of these studies support the role of MAPK activation in the progression and maintenance of central sensitization in the MIA-OA experimental pain model.

Fibromyalgia: mechanisms, current treatment and animal models.

Fibromyalgia syndrome (FMS) is a chronic pain syndrome characterized by diffuse musculoskeletal pain. In quantitative sensory testing studies, FMS patients display alterations in heat, cold, and mechanical sensitivity. Genetic studies support a key role for the biogenic amine system, and single nucleotide polymorphisms have been identified in serotonin and dopamine transporter and receptor genes of FMS patients. The pathophysiology of fibromyalgia includes contributions from both the ascending and descending somatosensory systems, and decreased central nervous system inhibition of peripheral nociceptive signalling. Three drugs have been approved for the treatment of FMS: Lyrica® (pregablin), Cymbalta® (duloxetine), and Savella® (milnacipran). These drugs were originally developed for indications other than FMS, and were later approved for FMS after successful clinical trials. One hurdle in the development of drugs specifically for FMS is the availability of preclinical animal models of the disease. Recently, several rodent models have been described with potential for translation to the human pain syndrome. In this review, we discuss recent developments toward understanding the pathophysiology of FMS, currently available pharmacologic therapy, ongoing clinical trials, and potential animal models of FMS.

Homomeric and heteromeric P2X3 receptors in peripheral sensory neurons.

ATP contributes to nociceptive sensory processing by activating a family of ligand-gated ion channels, the P2X receptors. One of these, the P2X3 receptor, is highly localized on primary afferent neurons. In sensory neurons, P2X3 receptors function as homomeric (P2X3) and heteromeric (P2X2/3) channels. Exogenous application of ATP and related agonists excites peripheral and central nerves, and increases sensitivity to noxious stimuli. Specific targeting of P2X3 receptors by gene deletion and knockdown results in a hypoalgesic phenotype. In animal models of pain, pharmacological blockade of P2X3 receptors fully blocked specific types of chronic inflammatory and neuropathic pain. Peripheral nerve injury differentially alters functional expression of P2X3 receptors on small and large diameter primary afferent neurons. These data have delineated discrete roles for homomeric P2X3 and heteromeric P2X2/3 receptor activation in acute and chronic pain. Similar findings have also been generated from extensive research of the bladder urothelial-sensory neuron system. The urinary bladder is richly innervated by P2X3 receptor-containing neurons. Heteromeric P2X2/3 channels in the bladder contribute to both mechanosensitivity and nociceptive responses. Thus, both genetic and pharmacological approaches have provided converging evidence that activation of P2X3-containing channels is an important mediator of acute and persistent nociceptive signaling in the peripheral nervous system.

Morphine directly inhibits nociceptors in inflamed skin.

Peripherally delivered opiates attenuate mechanical and thermal hyperalgesia in experimental models of inflammation, suggesting that activation of peripheral opioid receptors decreases the excitability of nociceptors in inflamed tissues. The current study examines the effects of peripheral morphine sulfate on response properties of sensory neurons in healthy and inflamed skin. Afferent units (185) were isolated from tibial nerve of rats using an in vitro glabrous skin-nerve teased-fiber preparation. Of these, 107 units were from normal healthy skin, and 78 were from inflamed skin 18 h after intraplantar injection of complete Freund's adjuvant. As a population, C-fiber units innervating inflamed skin exhibited properties characteristic of sensitization when compared with units innervating healthy control skin. Mechanical thresholds were lowered, responses to noxious mechanical and thermal stimuli were elevated, a greater proportion of units was spontaneously active, and the average rate of spontaneous discharge was higher. Response properties in other conduction velocity groups remained unchanged. Fifty-eight percent of C and C/Adelta nociceptors innervating inflamed skin were opiate-sensitive, and their excitability was attenuated by direct application of morphine to their receptive fields. All morphine-sensitive units were nociceptors from inflamed skin with conduction velocities <1.3 m/s. Morphine effects were concentration-dependent and naloxone-sensitive, indicating that the effects were receptor-mediated. These findings provide direct evidence that morphine acts through peripheral opioid receptors to inhibit the activity of cutaneous nociceptors under conditions of inflammation.