resiniferatoxin and Pain

Resiniferatoxin (RTX) is an ultrapotent capsaicin analog with a unique spectrum of pharmacological actions. The therapeutic window of RTX is broad, allowing for the full desensitization of pain perception and neurogenic inflammation without causing unacceptable side effects. Intravesical RTX was shown to restore continence in a subset of patients with idiopathic and neurogenic detrusor overactivity. RTX can also ablate sensory neurons as a "molecular scalpel" to achieve permanent analgesia. This targeted (intrathecal or epidural) RTX therapy holds great promise in cancer pain management. Intra-articular RTX is undergoing clinical trials to treat moderate-to-severe knee pain in patients with osteoarthritis. Similar targeted approaches may be useful in the management of post-operative pain or pain associated with severe burn injuries. The current state of this field is reviewed, from preclinical studies through veterinary medicine to clinical trials.

Topics: Diterpenes; Humans; Pain; Precision Medicine; TRPV Cation Channels; Urinary Bladder, Overactive

With 336 reviews, the capsaicin receptor TRPV1 arguably represent today's most extensively reviewed analgesic target. TRPV1 is strategically located at the peripheral terminals of primary sensory neurons where pain is generated. TRPV1 as a target for analgesic drugs has been validated in preclinical studies.. The therapeutic potential of targeting TRPV1 by agonists and antagonists for pain relief is discussed based on our experience and a critical review of the literature. Strategies to overcome adverse effects are explored.. Since its discovery in 1997, TRPV1 has run the gamut from excitement to disappointment to cautious optimism. Topical capsaicin has been disappointing for pain relief. By contrast, intrathecal resiniferatoxin is currently undergoing clinical trials in patients with intractable cancer pain. Some of the small-molecule TRPV1 antagonists have successfully passed Phase I safety and tolerability studies in healthy volunteers into Phase II studies to access efficacy in patients. Others showed worrisome unforeseen adverse effects, most important, hyperthermia and impaired noxious heat sensation. We conclude that TRPV1 blockade and desensitization are two promising, complimentary approaches for pain relief. Despite the roadblocks, TRPV1 remains a powerful tool in pain research and a promising therapeutic target.

Topics: Analgesics; Animals; Capsaicin; Diterpenes; Drug Delivery Systems; Drug Design; Humans; Pain; TRPV Cation Channels

In primary sensory neurons, the capsaicin receptor TRPV1 functions as a molecular integrator for a broad range of seemingly unrelated chemical and physical noxious stimuli, including heat and altered pH. Indeed, TRPV1 is thought to be a major transducer of the thermal hyperalgesia that follows inflammation and tissue injury as this response is impaired in TRPV1-deficient mice. Following the molecular cloning of TRPV1 in 1997, over a dozen companies embarked on efforts to find clinically useful TRPV1 antagonists, but side-effects and limited efficacy have thus far prevented any compounds from progressing beyond phase II. This has rekindled interest in desensitization of nociceptive neurons to TRPV1 agonists (e.g. capsaicin and its ultrapotent analog resiniferatoxin) as an alternative pharmacological approach to block pain in the periphery where it is generated. The clinical value of capsaicin is, however, limited by its unfavorable irritancy to desensitization ratio. In animal experiments, resiniferatoxin treatment is a powerful approach to achieve long-lasting analgesia. In patients with overactive bladder, intravesical resiniferatoxin improves bladder function (or even restores continence) without significant irritancy and/or toxicity. In this review, we argue that resiniferatoxin is an attractive alternative to capsaicin in that it achieves lasting desensitization without the side effects that complicate capsaicin therapy.

Topics: Animals; Capsaicin; Clinical Trials as Topic; Diterpenes; Drug Evaluation, Preclinical; Humans; Pain; Structure-Activity Relationship; TRPV Cation Channels

The idea of selectively targeting nociceptive transmission at the level of the peripheral nervous system is attractive from multiple perspectives, particularly the potential lack of non-specific (non-targeted) CNS side effects. Out of the multiple TRP channels involved in nociception, TRPV1 is a strong candidate based on its biophysical conductance properties and its expression in inflammation-sensitive dorsal root ganglion neurons and their axons and central and peripheral nerve terminals. While TRPV1 antagonists have undergone extensive medicinal chemical and pharmacological investigation, for TRPV1 agonists nature has provided an optimized compound in RTX. RTX is not suitable for systemic administration, but it is highly adaptable to a variety of pain problems when used by local administration. This can include routes as diverse as subcutaneous, intraganglionic or intrathecal (CSF space around the spinal cord). The present review focuses on the molecular and preclinical animal experiments that form the underpinnings of our clinical trial of intrathecal RTX for pain in advanced cancer. As such this represents a new approach to pain control that emerges from a long line of research on capsaicin and other vanilloids, their physiological actions, and the molecular biology of the capsaicin receptor TRPV1.

Topics: Animals; Capsaicin; Diterpenes; Humans; Pain; Structure-Activity Relationship; TRPV Cation Channels

Transient receptor potential V1 (TRPV1) is specifically expressed in the nociceptive receptors and can detect a variety of noxious stimuli, thus potentiating pain sensitization. While peripheral delivery of capsaicin causes the desensitization of sensory neurons, thus alleviating pain. Therefore capsaicin is used in the clinical treatment of various types of pain; however, these treatments will bring many side effects, such as a strong burning pain in the early stages of treatment which hampers the further use of capsaicin. Thus, the studies of the functional regulation of TRPV1 are mainly focused on two aspects: to develop more potent analogues of capsaicin with less side effects; or to elucidate the mechanisms of TRPV1 in pain sensitivity, especially of that TRPV1 as a target of various protein kinases such as PKD1 and Cdk5 is involved pain hypersensitivity. Thus we would summarize the progress of these two aspects in this mini review.

Topics: Animals; Capsaicin; Diterpenes; Pain; Protein Kinases; TRPV Cation Channels

Members of the transient receptor potential (TRP) family of nonselective cation channels are involved in several pathological and physiological conditions. The search for the molecular targets for naturally occurring substances, especially from plants, allowed the characterization of many TRP channels. In fact, attempts to understand the hot and painful action of the vanillyl group containing compounds capsaicin (from Capsicum sp.) and its ultrapotent analogue resiniferatoxin (RTX, from Euphorbia sp.) led to the cloning of the vanilloid receptor (TRPV1) 7 years ago. TRPV1 is found in sensory fibers and functions as a molecular integrator of several painful stimuli, being especially stimulated during inflammation. Since TRPV1 is involved in several pathological conditions, selective ligands or modulators of this channel are substances of potential interest to treat such diseases. Once again, natural products seem to be also interesting sources of compounds that might be prototype TRPV1 ligands. The cloning of TRPV1 also enabled the discovery of other members of the TRPV family of channels. Similar to TRPV1, these receptors function as molecular detectors of physical and chemical stimuli, such as innocuous and noxious heat, as well as mechanical force. Recently, novel TRP channels sensitive to low temperatures also have been cloned, namely, TRPM8 and TRPA1. Such channels are also activated by naturally occurring substances but knowledge of their involvement in health and disease is in its infancy. In the present review, we focused on the contribution of natural products to the discovery of TRP channels and to the development of novel drugs to treat pathological conditions in which these channels are involved.

Topics: Animals; Biological Products; Calcium Channels; Capsaicin; Diterpenes; Humans; Nociceptors; Pain; Receptors, Drug; Structure-Activity Relationship; TRPC Cation Channels

C-fiber sensory afferent neurons, which contain neuropeptides such as calcitonin-gene related peptide and substance P, mediate a wide variety of physiologic responses, including chemogenic pain, thermoregulation, and neurogenic inflammation. Capsaicin, the pungent constituent in red pepper, functions to activate and then, at higher doses and longer times, desensitize this class of neurons. This latter response provides the basis for the therapeutic application of capsaicin. A major advance in the field has been the identification of resiniferatoxin, a phorbol-related diterpene, as an analog of capsaicin that is ultrapotent but with differential selectivity. In particular, resiniferatoxin is only similar in potency for induction of pain but is much more effective for desensitization. Structure-activity analysis in whole animal experiments provides further evidence for dissociation of biologic endpoints, strongly arguing for the existence of vanilloid receptor subclasses. Using resiniferatoxin, we have been able to define specific, high-affinity receptors for capsaicin both in animal models such as rats and in man. Of great importance, the pharmacologic characterization in cultured dorsal root ganglion cells of the high-affinity resiniferatoxin-binding site and of the physiologic response believed to be directly coupled to the receptor, viz. calcium uptake, differed in structure-activity and in cooperativity. We conclude that multiple high-affinity vanilloid receptor subclasses mediate vanilloid response; moreover, the resiniferatoxin-selective subclass of vanilloid receptors is not the voltage-independent, cation-nonselective ion channel as previously believed. Optimization of ligands for the individual vanilloid receptor subclasses should revolutionize this therapeutic area.

Topics: Animals; Capsaicin; Dermatitis; Diterpenes; Ganglia, Spinal; Humans; Neurotoxins; Pain; Radioligand Assay; Receptors, Drug

Capsaicin, the pungent constituent of chili peppers, represents the paradigm for the capsaicinoids or vanilloids, a family of compounds shown to stimulate and then desensitize specific subpopulations of sensory receptors, including C-polymodal nociceptors, A-delta mechanoheat nociceptors and warm receptors of the skin, as well as enteroceptors of thin afferent fibers. An exciting recent advance in the field has been the finding that resiniferatoxin (RTX), a naturally occurring diterpene containing a homovanillic acid ester, a key structural motif of capsaicin, functions as an ultrapotent capsaicin analog. For most of the responses characteristic of capsaicin, RTX is 100-10,000 fold more potent. Structure/activity analysis indicates, however, that RTX and related homovanillyl-diterpene esters display distinct spectra of activity. Specific [3H]RTX binding provides the first direct proof for the existence of vanilloid receptors. We expect that the RTX class of vanilloids will promote rapid progress in understanding of vanilloid structure/activity requirements and mechanism.

Topics: Animals; Body Temperature Regulation; Capsaicin; Diterpenes; Nervous System; Nervous System Physiological Phenomena; Neurons, Afferent; Pain; Receptors, Cell Surface; Structure-Activity Relationship

Resiniferatoxin (RTX) is the most potent among all known endogenous and synthetic agonists for the transient receptor potential vanilloid 1 (TRPV1) receptor, which is a calcium-permeable nonselective cation channel, expressed on the peripheral and central terminals of small-diameter sensory neurons. Prolonged calcium influx induced by RTX causes cytotoxicity and death of only those sensory neurons that express the TRPV1 ion channel leading to selective targeting and permanent deletion of the TRPV1-expressing C-fiber neuronal cell bodies in the dorsal root ganglia. The goal of this project was to provide preclinical efficacy data, that intrathecal RTX could provide effective pain relief and improve function in dogs with bone cancer without significant long-term side effects. In a single-blind, controlled study, 72 companion dogs with bone cancer pain were randomized to standard of care analgesic therapy alone (control, n = 36) or 1.2 μg/kg intrathecal RTX in addition to standard of care analgesic therapy (treated, n = 36). Significantly more dogs in the control group (78%) required unblinding and adjustment in analgesic protocol or euthanasia within 6 weeks of randomization, than dogs that were treated with RTX (50%; P < 0.03); and overall, dogs in the control group required unblinding significantly sooner than dogs that had been treated with RTX (P < 0.02). The analgesic effect was documented in these dogs without any evidence of development of deafferentation pain syndrome that can be seen with neurolytic therapies.

Topics: Analgesics; Animals; Bone Neoplasms; Disease Models, Animal; Diterpenes; Dogs; Female; Injections, Spinal; Male; Pain; Pain Measurement; Single-Blind Method; Time Factors; Treatment Outcome

Painful diabetic peripheral neuropathy (PDPN) is one of the major complications of diabetes. Currently, centrally acting drugs and topical analgesics are used for treating PDPN. These drugs have adverse effects; some are ineffective, and treatment with opioids is associated with use dependence and addiction. Recent research indicates that transient receptor potential vanilloid 1 (TRPV1) expressed in the peripheral sensory nerve terminals is an emerging target to treat pain associated with PDPN. Block of TRPV1 ion channel with specific antagonists, although effective as an analgesic, induced hyperthermia in clinical trials. However, TRPV1 agonists are useful to treat pain by virtue of their ability to cause Ca 2+ influx and subsequently leading to nerve terminal desensitization. Here, we report the effectiveness of an ultrapotent TRPV1 agonist, resiniferatoxin (RTX) nanoparticle, in a topical formulation (RTX-cream; RESINIZIN) that alleviates pain associated with DPN in animal models of diabetes. Resiniferatoxin causes nerve terminal depolarization block in the short term, which prevents pain during application and leading to nerve terminal desensitization/depletion in the long term resulting in long-lasting pain relief. Application of RTX cream to the hind limbs suppresses thermal hyperalgesia in streptozotocin-induced diabetic rats and mini pigs without any adverse effects as compared with capsaicin at therapeutic doses, which induces intense pain during application. Resiniferatoxin cream also decreases the expression of TRPV1 in the peripheral nerve endings and suppresses TRPV1-mediated calcitonin gene-related peptide release in the skin samples of diabetic rats and mini pigs. Our preclinical data confirm that RTX topical formulation is an effective treatment option for PDPN.

Topics: Analgesics; Animals; Capsaicin; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Diterpenes; Pain; Rats; Swine; Swine, Miniature; TRPV Cation Channels

Topics: Diterpenes; Humans; Nociception; Pain; Spine

Topics: Diterpenes; Humans; Nociception; Pain; Spine

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

Topics: Animals; Cerebral Ventricles; Diterpenes; Mice; Pain; Pain Measurement; TRPV Cation Channels

Topics: Analgesics; Animals; Calcium; Capsaicin; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Flavonoids; Inflammation; Male; Mice; Neuralgia; Pain; Pain Measurement; Spinal Cord; Synaptosomes; Toxicity Tests; TRPV Cation Channels

Electroacupuncture (EA) is an effective treatment to relieve pain in patients with postherpetic neuralgia. However, the mechanisms of EA involved therein are still unknown. We first injected resiniferatoxin (RTX) into Sprague Dawley rats to construct the neuralgia model. One week after injection, the rats were treated with EA at the "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints for 5 weeks. Nociceptive behavioral tests were performed to analyze the changes in thermal sensitivity and mechanical allodynia after RTX induction and EA treatment. Deep sequencing was performed to identify differentially expressed miRNAs in the spinal cord of RTX-induced rats in response to EA treatment. The nociceptive behavioral tests showed that EA at the left GB30 and GB34 acupoints significantly reduced RTX-induced tactile sensitivity and increased RTX-inhibited thermal sensitivity. The sequencing data indicated that RTX resulted in one upregulated and five downregulated miRNAs, and EA treatment resulted in two upregulated miRNAs. Furthermore, seven upregulated and two downregulated miRNAs were found between rats subjected to EA and sham operation. Functional analysis suggested that the targets of differentially expressed miRNAs were enriched in many nervous system-related pathways. The pathway-gene-miRNA net analysis showed that miR-7a-5p had the most target genes. Moreover, miR-233-3p was downregulated after RTX injection and upregulated by EA treatment. We speculated that the upregulation of miR-7a-5p and miR-233-3p is involved in the analgesic effects of EA. Our analysis on the EA-induced differential expression of miRNAs provides novel insights into the mechanisms of EA analgesia in postherpetic neuralgia.

Topics: Animals; Diterpenes; Electroacupuncture; Gene Expression; Gene Regulatory Networks; High-Throughput Nucleotide Sequencing; Male; MicroRNAs; Neurotoxins; Pain; Pain Management; Pain Measurement; Rats; Rats, Sprague-Dawley; Spinal Cord

The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.

Topics: Animals; Bacterial Proteins; Botulinum Toxins, Type A; Calcitonin Gene-Related Peptide; Caspase 1; Diterpenes; Fasciitis, Necrotizing; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Neutrophils; Pain; Signal Transduction; Skin; Streptococcal Infections; Streptococcus pyogenes; Streptolysins; TRPV Cation Channels

The translational potential of analgesic approaches emerging from basic research can be augmented by client-owned dog trials. We report on a peripheral interventional approach that uses intra-articular injection of the ultrapotent TRPV1 agonist resiniferatoxin (RTX) to produce a selective long-term chemoinactivation of nociceptive primary afferent nerve endings for pain control in naturally occurring canine osteoarthritis. A single injection of 10 µg of RTX, produced suppression of pain, improvement in gait, weight bearing, and improvement in the dog's activities of daily living lasting 4 months or longer. Two to 3 years after the injection, there are no alterations to suggest that removal of inflammatory pain caused accelerated joint degeneration (Charcot joint) in any of the dogs. To amplify the effective use of canine subjects in translational analgesia research, we report a high-quality canine dorsal root ganglion transcriptome. Some targets for analgesia are highly conserved both in protein sequence and level of expression within a target tissue while others diverge substantially from the human. This knowledge is especially important for development of analgesics aimed at peripheral molecular targets and provides a template for informed translational research. The peripheral site of action, long duration of analgesia, apparent safety, and retention of coordination, all resulting from a single dose suggest that intra-articular RTX may be an effective intervention for osteoarthritis pain with few or no side effects and lead to an improved quality of life.

Topics: Analgesics; Animals; Cohort Studies; Diterpenes; Dogs; Gait Analysis; Injections, Intra-Articular; Osteoarthritis; Pain; Pain Measurement; Phylogeny; Receptors, Opioid, kappa; Transcriptome; TRPV Cation Channels

The transient receptor potential vanilloid type 1 (TRPV1) is a thermosensitive cation channel that triggers heat pain in the periphery. Long-term desensitization of TRPV1, which can be induced by excess amounts of agonists, has been a method for investigating the physiological relevance of TRPV1-containing neuronal circuits, and desensitization induced by various routes of administration, including systemic, intrathecal and intraganglionic, has been demonstrated in rodents. In the present study, we examined the effect of intracerebroventricular (i.c.v.) treatment with an ultrapotent TRPV1 agonist, resiniferatoxin (RTX), on nociception and the analgesic effect of acetaminophen, which is known to mediate the activation of central TRPV1. I.c.v. administration of RTX a week before the test did not affect the licking/biting response to intraplantar injection of RTX (RTX test), suggesting that such i.c.v. treatment spares the function of TRPV1 at the hindpaw. Mice that had been i.c.v.-administered RTX also exhibited normal nociceptive responses in the formalin test and the tail pressure test, but acetaminophen failed to induce analgesia in those mice in any of the tests. These results suggest that i.c.v. administration of RTX leads to brain-selective TRPV1 desensitization in mice.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Behavior, Animal; Diterpenes; Gene Expression; Hyperalgesia; Injections, Intraventricular; Male; Mice; Neurotoxins; Nociception; Pain; Pain Measurement; TRPV Cation Channels

There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons.

Topics: Binding Sites; Calmodulin; Capsaicin; Diterpenes; Humans; Pain; Protein Binding; Protein Conformation; Protein Interaction Maps; Sensory Receptor Cells; TRPV Cation Channels

Neural cross-sensitization has been postulated as a mechanism underlying overlaps of chronic pelvic pain disorders such as bladder pain syndrome/interstitial cystitis (BPS/IC) and irritable bowel syndrome (IBS). Animals with experimental colitis have been used to study the underlying mechanisms for overlapped pelvic pain symptoms, and shown to exhibit bladder overactivity evidenced by frequent voiding; however, it has not directly been evaluated whether pain sensation derived from the lower urinary tract is enhanced in colitis models. Also, the cross-sensitization between the colon and urethra has not been studied previously. In the present study, we therefore investigated pain behaviors induced by nociceptive stimuli in the lower urinary tract and the involvement of C-fiber afferent pathways using rats with colitis induced by intracolonic application of 2,4,6-trinitrobenzenesulfonic acid (TNBS). In TNBS-induced colitis rats at 10 days, intravesical application of resiniferatoxin (RTx) induced a significantly greater number of episodes of both licking and freezing behaviors, which were reduced by capsaicin-sensitive C-fiber afferent desensitization. Histochemical studies using fluorescent dye tracers injected into the colon, bladder or urethra showed that dichotomized afferent neurons comprised 6.9-14.5% of L1, L6 and S1 dorsal root ganglion (DRG) neurons innervating the colon or the lower urinary tract. Transient receptor potential vanilloid 1 (TRPV1) mRNA expression was significantly increased in, the bladder, urethra and S1 DRG in colitis rats. An increase in myeloperoxidase (MPO) activity was found in the colon, but not in the bladder or urethra after intracolonic TNBS treatment. These results indicate that TNBS-induced colitis increased pain sensitivity in the bladder and urethra via activation of C-fiber afferent pathways due to colon-to-bladder and colon-to-urethral cross-sensitization, suggesting the contribution of pelvic organ cross-sensitization mechanisms to overlapped pain symptoms in BPS/IC and IBS.

Topics: Animals; Colitis; Colon; Disease Models, Animal; Diterpenes; Female; Freezing Reaction, Cataleptic; Ganglia, Spinal; Grooming; Neurons, Afferent; Pain; Peroxidase; Rats, Sprague-Dawley; RNA, Messenger; Trinitrobenzenesulfonic Acid; TRPV Cation Channels; Urethra; Urinary Bladder

One approach to analgesia is to block pain at the site of origin or along the peripheral pathway by selectively ablating pain-transmitting neurons or nerve terminals directly. The heat/capsaicin receptor (TRPV1) expressed by nociceptive neurons is a compelling target for selective interventional analgesia because it leaves somatosensory and proprioceptive neurons intact. Resiniferatoxin (RTX), like capsaicin, is a TRPV1 agonist but has greater potency. We combine RTX-mediated inactivation with the precision of computed tomography (CT)-guided delivery to ablate peripheral pain fibers in swine. Under CT guidance, RTX was delivered unilaterally around the lumbar dorsal root ganglia (DRG), and vehicle only was administered to the contralateral side. During a 4-week observation period, animals demonstrated delayed or absent withdrawal responses to infrared laser heat stimuli delivered to sensory dermatomes corresponding to DRG receiving RTX treatment. Motor function was unimpaired as assessed by disability scoring and gait analysis. In treated DRG, TRPV1 mRNA expression was reduced, as were nociceptive neuronal perikarya in ganglia and their nerve terminals in the ipsilateral dorsal horn. CT guidance to precisely deliver RTX to sites of peripheral pain transmission in swine may be an approach that could be tailored to block an array of clinical pain conditions in patients.

Topics: Animals; Behavior, Animal; Diterpenes; Female; Ganglia; Ganglia, Spinal; Gene Expression Profiling; Hot Temperature; Lasers; Lumbar Vertebrae; Microscopy, Fluorescence; Motor Skills; Neurons; Pain; RNA, Messenger; Swine; Tomography, X-Ray Computed; TRPV Cation Channels

We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH.. Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals.. Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of TRPV1 receptors via a PKC-dependent pathway, which leads to the development of TH mimicking what occurs in chronic ischemic patients with severe acidosis. More importantly, peripheral P2Y1 receptors play a pivotal role in this process, suggesting a novel peripheral mechanism underlying the development of TH in these patients.

Topics: Acids; Acrylamides; Adenosine Diphosphate; Animals; Benzophenanthridines; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Diterpenes; Hindlimb; Hot Temperature; Hyperalgesia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Injections; Ion Channels; Ischemia; Pain; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2Y1; Sodium Chloride; Thrombosis; Tissue Extracts; TRPV Cation Channels

We developed a "chemical-pain sensor" that could recognize chemical pain stimuli such as capsaicin and resiniferatoxin just like mammalian chemical pain sensory systems. Here, we first prepared nanovesicles containing rat pain sensory receptor, rat transient receptor potential vanilloid 1 (rTRPV1), which is activated by noxious heat and capsaicin. And the nanovesicles were immobilized on a single-walled carbon nanotube-based field effect transistor. The chemical-pain sensor could selectively detect chemical pain stimuli with a high sensitivity of a 1 pM detection limit. It also responded to different chemical pain stimuli in a manner similar as to that of mammalian chemical pain sensory systems. This sensor platform can be utilized for various practical applications such as food screening tools and artificial somesthetic sensors. Moreover, TRP families have been suggested as potential drug targets related to nerve and circulation disorders. Thus, the capability of monitoring TRP responses using our sensor platforms should provide a powerful means for the development of new drugs as well as the basic research about nerve and circulation systems.

Topics: Animals; Biosensing Techniques; Capsaicin; Cloning, Molecular; Diterpenes; HEK293 Cells; Hot Temperature; Humans; Immobilized Proteins; Limit of Detection; Nanostructures; Nanotubes, Carbon; Pain; Rats; Sensitivity and Specificity; Sensory System Agents; TRPV Cation Channels

Glycine is a major inhibitory neurotransmitter in the spinal cord, the concentration of which is regulated by two types of glycine transporters (GlyTs): GlyT1 and GlyT2. We hypothesized that the inhibition of GlyTs could ameliorate bladder overactivity and/or pain sensation in the lower urinary tract.. Investigate the effects of GlyT inhibitors on bladder overactivity and pain behavior in rats.. Cystometry was performed under urethane anesthesia in cyclophosphamide (CYP)-treated rats. In behavioral studies using conscious rats, nociceptive responses were induced by intravesical administration of resiniferatoxin (3μM). Selective GlyT1 or GlyT2 inhibitors were administered intrathecally to evaluate their effects.. Cystometric parameters, nociceptive behaviors (licking and freezing), and messenger RNA (mRNA) levels of GlyTs and glycine receptor (GlyR) subunits in the dorsal spinal cord (L6-S1) were measured.. During cystometry in CYP-treated rats, significant increases in intercontraction interval and micturition pressure threshold were elicited by ALX-1393, a selective GlyT2 inhibitor, but not by sarcosine, a GlyT1 inhibitor. These effects were completely reversed by strychnine, a GlyR antagonist. ALX-1393 also significantly suppressed nociceptive behaviors in a dose-dependent manner. In sham rats, GlyT2 mRNA was expressed at a much higher level (23-fold) in the dorsal spinal cord than GlyT1 mRNA. In CYP-treated rats, mRNA levels of GlyT2 and the GlyR α1 and β subunits were significantly reduced.. These results indicate that GlyT2 plays a major role in the clearance of extracellular glycine in the spinal cord and that GlyT2 inhibition leads to amelioration of CYP-induced bladder overactivity and pain behavior. GlyT2 may be a novel therapeutic target for the treatment of overactive bladder and/or bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Topics: Animals; Diterpenes; Female; Freezing Reaction, Cataleptic; Glycine Agents; Glycine Plasma Membrane Transport Proteins; Nociceptive Pain; Pain; Rats; Rats, Sprague-Dawley; Sarcosine; Serine; Spinal Cord; Strychnine; Urinary Bladder, Overactive; Urination

The transient receptor potential vanilloid 1 (TRPV1) receptor is relevant to the perception of noxious information and has been studied as a therapeutic target for the development of new analgesics. The goal of this study was to perform in vivo and in vitro screens to identify novel, efficacious, and safe TRPV1 antagonists isolated from leaves of the medicinal plant Vernonia tweedieana Baker. All of the fractions and the hydroalcoholic extract produced antinociception in mice during the capsaicin test, but the dichloromethane fraction also had antioedematogenic effect. Among the compounds isolated from the dichloromethane fraction, only α-spinasterol reduced the nociception and edema induced by capsaicin injection. Moreover, α-spinasterol demonstrated good oral absorption and high penetration into the brain and spinal cord of mice. α-Spinasterol was able to displace [3H]resiniferatoxin binding and diminish calcium influx mediated by capsaicin. Oral administration of the dichloromethane fraction and α-spinasterol also produced antinociceptive effect in the noxious heat-induced nociception test; however, they did not change the mechanical threshold of naive mice. The treatment with α-spinasterol did not produce antinociceptive effect in mice systemically pretreated with resiniferatoxin. In addition, α-spinasterol and the dichloromethane fraction reduced the edema, mechanical, and heat hyperalgesia elicited by complete Freund's adjuvant paw injection. The dichloromethane fraction and α-spinasterol did not affect body temperature or locomotor activity. In conclusion, α-spinasterol is a novel efficacious and safe antagonist of the TRPV1 receptor with antinociceptive effect.

Topics: Analgesics; Animals; Binding, Competitive; Body Temperature; Calcium; Capsaicin; Chromatography, High Pressure Liquid; Diterpenes; Edema; Freund's Adjuvant; Hot Temperature; Male; Mice; Nociceptors; Pain; Pain Measurement; Plant Extracts; Plant Leaves; Stigmasterol; Tissue Distribution; TRPV Cation Channels; Vernonia

Despite success in treating many forms of cancer, pain associated with malignancy remains a serious clinical issue with a poorly understood etiology. This study determined if certain sarcoma cell lines produced a soluble factor that activates the TRPV1 ion channel expressed on nociceptive sensory neurons, thereby activating a major pain transduction system.. Trigeminal ganglia were harvested from rats and cultured. A rhabdomyosarcoma (CRL1598) and osteosarcoma (CRL 1543) cell line were grown to 75% confluency. Conditioned media (CM) was collected after 24 h of exposure and subjected to reverse phase chromatography. Neuronal activation in the presence of CM was measured using iCGRP RIA and calcium imaging after treatment with vehicle or I-RTX, a potent TRPV1 antagonist. Data were analyzed by ANOVA/Bonferroni or t test.. The rhabdomyosarcoma CM produced a 4-fold increase in iCGRP release compared with control media (P < 0.001). The osteosarcoma cell line CM produced a 7-fold increase in iCGRP release compared with control media (P < 0.001). This evoked iCGRP release was via TRPV1 activation since the effect was blocked by the antagonist I-RTX. The application of rhabdomyosarcoma CM produced about a 4-fold increase in [Ca(2+)]I levels (P < 0.001), and this effect was blocked by pretreatment with the TRPV1 antagonist, I-RTX.. We have shown that certain sarcoma cell lines produce a soluble, lipophilic factor that activates the peripheral nociceptor transduction system via TRPV1 activation, thereby contributing to cancer pain. Further investigations are needed to develop tumor-specific analgesics that do not produce unwanted or harmful side-effects.

Topics: Animals; Bone Neoplasms; Calcitonin Gene-Related Peptide; Calcium; Culture Media, Conditioned; Diterpenes; Humans; Male; Nociceptors; Osteosarcoma; Pain; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Rhabdomyosarcoma; TRPV Cation Channels; Tumor Cells, Cultured

Tissue injury elicits both hypersensitivity to evoked stimuli and ongoing, stimulus-independent pain. We previously demonstrated that pain relief elicits reward in nerve-injured rats. This approach was used to evaluate the temporal and mechanistic features of inflammation-induced ongoing pain.. Intraplantar Complete Freund's Adjuvant (CFA) produced thermal hyperalgesia and guarding behavior that was reliably observed within 24 hrs and maintained, albeit diminished, 4 days post-administration. Spinal clonidine produced robust conditioned place preference (CPP) in CFA treated rats 1 day, but not 4 days following CFA administration. However, spinal clonidine blocked CFA-induced thermal hyperalgesia at both post-CFA days 1 and 4, indicating different time-courses of ongoing and evoked pain. Peripheral nerve block by lidocaine administration into the popliteal fossa 1 day following intraplantar CFA produced a robust preference for the lidocaine paired chamber, indicating that injury-induced ongoing pain is driven by afferent fibers innervating the site of injury. Pretreatment with resiniferatoxin (RTX), an ultrapotent capsaicin analogue known to produce long-lasting desensitization of TRPV1 positive afferents, fully blocked CFA-induced thermal hypersensitivity and abolished the CPP elicited by administration of popliteal fossa lidocaine 24 hrs post-CFA. In addition, RTX pretreatment blocked guarding behavior observed 1 day following intraplantar CFA. In contrast, administration of the selective TRPV1 receptor antagonist, AMG9810, at a dose that reversed CFA-induced thermal hyperalgesia failed to reduce CFA-induced ongoing pain or guarding behavior.. These data demonstrate that inflammation induces both ongoing pain and evoked hypersensitivity that can be differentiated on the basis of time course. Ongoing pain (a) is transient, (b) driven by peripheral input resulting from the injury, (c) dependent on TRPV1 positive fibers and (d) not blocked by TRPV1 receptor antagonism. Mechanisms underlying excitation of these afferent fibers in the early post-injury period will offer insights for development of novel pain relieving strategies in the early post-traumatic period.

Topics: Animals; Diterpenes; Freund's Adjuvant; Inflammation; Male; Nerve Fibers; Neurons, Afferent; Nociceptors; Pain; Rats; Rats, Sprague-Dawley; Reaction Time; TRPV Cation Channels

Gout is characterized by the deposition of monosodium urate (MSU) crystals. Despite being one of the most painful forms of arthritis, gout and the mechanisms responsible for its acute attacks are poorly understood. In the present study, we found that MSU caused dose-related nociception (ED(50) [ie, the necessary dose of MSU to elicit 50% of the response relative to the control value]=0.04 [95% confidence interval 0.01-0.11]mg/paw) and edema (ED(50)=0.08 [95% confidence interval 0.04-0.16]mg/paw) when injected into the hind paw of rats. Treatment with the selective TRPV1 receptor (also known as capsaicin receptor and vanilloid receptor-1) antagonists SB366791 or AMG9810 largely prevented nociceptive and edematogenic responses to MSU. Moreover, the desensitization of capsaicin-sensitive afferent fibers as well as pretreatment with the tachykinin NK(1) receptor antagonist RP 67580 also significantly prevented MSU-induced nociception and edema. Once MSU was found to induce mast cell stimulation, we investigated the participation of these cells on MSU effects. Prior degranulation of mast cells by repeated treatment with the compound 48/80 decreased MSU-induced nociception and edema or histamine and serotonin levels in the injected tissue. Moreover, pretreatment with the mast cell membrane stabilizer cromolyn effectively prevented nociceptive and edematogenic responses to MSU. MSU induced a release of histamine, serotonin, and tryptase in the injected tissue, confirming mast cell degranulation. Furthermore, the antagonism of histaminergic H1 and serotoninergic receptors decreased the edema, but not the nociception of MSU. Finally, the prevention of the tryptase activity was capable of largely reducing both MSU-induced nociception and edema. Collectively, the present findings demonstrate that MSU produces nociceptive and edematogenic responses mediated by TRPV1 receptor activation and mast cell degranulation.

Topics: Acrylamides; Anilides; Animals; Anti-Allergic Agents; Anti-Asthmatic Agents; Antioxidants; Bridged Bicyclo Compounds, Heterocyclic; Capsaicin; Cinnamates; Cromolyn Sodium; Disease Models, Animal; Diterpenes; Edema; Gabexate; Histamine; Male; Mast Cells; Methysergide; Pain; Promethazine; Prostaglandin-Endoperoxide Synthases; Protein Binding; Rats; Rats, Wistar; Serine Proteinase Inhibitors; Serotonin; Serotonin Antagonists; Tritium; TRPV Cation Channels; Uric Acid

Polyamines (putrescine, spermidine and spermine) are important endogenous regulators of ion channels, such as vanilloid (TRPV1), glutamatergic (NMDA or AMPA/kainate) and acid-sensitive (ASIC) receptors. In the present study, we have investigated the possible nociceptive effect induced by polyamines and the mechanisms involved in this nociception in vivo. The subcutaneous (s.c.) injection of capsaicin (as positive control), spermine, spermidine or putrescine produced nociception with ED(50) of 0.16 (0.07-0.39)nmol/paw, 0.4 (0.2-0.7) μmol/paw, 0.3 (0.1-0.9) μmol/paw and 3.2 (0.9-11.5) μmol/paw, respectively. The antagonists of NMDA (MK801, 1 nmol/paw), AMPA/kainate (DNQX, 1 nmol/paw) or ASIC receptors (amiloride, 100 nmol/paw) failed to reduce the spermine-trigged nociception. However, the TRPV1 antagonists capsazepine or SB366791 (1 nmol/paw) reduced spermine-induced nociception, with inhibition of 81 ± 10 and 68 ± 9%, respectively. The previous desensitization with resiniferatoxin (RTX) largely reduced the spermine-induced nociception and TRPV1 expression in the sciatic nerve, with reductions of 82 ± 9% and 67 ± 11%, respectively. Furthermore, the combination of spermine (100 nmol/paw) and RTX (0.005 fmol/paw), in doses which alone were not capable of inducing nociception, produced nociceptive behaviors. Moreover, different concentrations of spermine (3-300 μM) enhanced the specific binding of [(3)H]-RTX to TRPV1 receptor. Altogether, polyamines produce spontaneous nociceptive effect through the stimulation of TRPV1, but not of ionotropic glutamate or ASIC receptors.

Topics: Acid Sensing Ion Channels; Animals; Biogenic Polyamines; Blotting, Western; Diterpenes; Glutamic Acid; Male; Mice; Nerve Fibers; Nerve Tissue Proteins; Nociceptors; Pain; Pain Measurement; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium Channels; Spermine; TRPV Cation Channels

Streptozotocin (STZ) is used as a common tool to induce diabetes and to study diabetes-induced complications including diabetic peripheral neuropathy (DPN). Previously, we have reported that STZ induces a direct effect on neurons through expression and function of the Transient receptor potential vanilloid 1 (TRPV1) channel in sensory neurons resulting in thermal hyperalgesia, even in non-diabetic STZ-treated mice. In the present study, we investigated the role of expression and function of TRPV1 in the central sensory nerve terminals in the spinal cord in STZ-induced hyperalgesia in rats.. We found that a proportion of STZ-treated rats were normoglycemic but still exhibited thermal hyperalgesia and mechanical allodynia. Immunohistochemical data show that STZ treatment, irrespective of glycemic state of the animal, caused microglial activation and increased expression of TRPV1 in spinal dorsal horn. Further, there was a significant increase in the levels of pro-inflammatory mediators (IL-1β, IL-6 and TNF-α) in spinal cord tissue, irrespective of the glycemic state. Capsaicin-stimulated release of calcitonin gene related peptide (CGRP) was significantly higher in the spinal cord of STZ-treated animals. Intrathecal administration of resiniferatoxin (RTX), a potent TRPV1 agonist, significantly attenuated STZ-induced thermal hyperalgesia, but not mechanical allodynia. RTX treatment also prevented the increase in TRPV1-mediated neuropeptide release in the spinal cord tissue.. From these results, it is concluded that TRPV1 is an integral component of initiating and maintaining inflammatory thermal hyperalgesia, which can be alleviated by intrathecal administration of RTX. Further, the results suggest that enhanced expression and inflammation-induced sensitization of TRPV1 at the spinal cord may play a role in central sensitization in STZ-induced neuropathy.

Topics: Animals; Blood Glucose; Body Weight; Calcitonin Gene-Related Peptide; Capsaicin; Cytokines; Diterpenes; Glucose Tolerance Test; Hyperalgesia; Hyperglycemia; Inflammation Mediators; Injections, Intraperitoneal; Injections, Spinal; Insulin; Microglia; Pain; Posterior Horn Cells; Rats; Streptozocin; TRPV Cation Channels

Here we make use of neural ablation to investigate the properties of the TrpV1-expressing neurons in the trigeminal and dorsal root ganglia of mice. Resiniferotoxin (RTX), a potent TrpV1 agonist, administered either by direct injection in the ganglion or intrathecally killed approximately 70% of TrpV1 cells and resulted in modest thermal analgesia. Interestingly, after carageenan injection in the hind paw, the analgesic effects of RTX were dramatically increased with mice now paradoxically showing far less response to heat applied at sites of inflammation. This additional carageenan and RTX-induced analgesia was transient, lasting less than 2 days, and likely resulted from deafferentation of remaining TrpV1 neurons. Remarkably, although RTX affected sensitivity to heat, mechanical sensitivity (both of normal and inflamed tissue) was completely unaltered by toxin-mediated silencing of the TrpV1 sensory input. Thus, our data demonstrate that TrpV1 neurons are selectively tuned nociceptors that mediate responses to thermal but not mechanical pain and insinuate a labeled line model for somatosensory coding.

Topics: Analgesics; Animals; Capsaicin; Diterpenes; Male; Mechanoreceptors; Mice; Mice, Inbred C57BL; Mice, Knockout; Mustard Plant; Nociceptors; Pain; Pain Measurement; Plant Oils; Sensory System Agents; Stress, Mechanical; Thermosensing; Trigeminal Nerve; TRPV Cation Channels

Analgesics currently available for the treatment of pain following ophthalmic surgery or injury are limited by transient effectiveness and undesirable or adverse side effects. The cornea is primarily innervated by small-diameter C-fiber sensory neurons expressing TRPV1 (transient receptor potential channel, subfamily V, member 1), a sodium/calcium cation channel expressed abundantly by nociceptive neurons and consequently a target for pain control. Resiniferatoxin (RTX), a potent TRPV1 agonist, produces transient analgesia when injected peripherally by inactivating TRPV1-expressing nerve terminals through excessive calcium influx. The aim of the present study was to evaluate topical RTX as a corneal analgesic. In rat cornea, a single application of RTX dose dependently eliminated or reduced the capsaicin eye wipe response for 3-5 days, with normal nociceptive responses returning by 5-7 days. RTX alone produced a brief but intense noxious response, similar to capsaicin, necessitating pretreatment of the cornea with a local anesthetic. Topical lidocaine, applied prior to RTX, blocks acute nociceptive responses to RTX without impairing the subsequent analgesic effect. Importantly, RTX analgesia (a) did not impair epithelial wound healing, (b) left the blink reflex intact and (c) occurred without detectable histological damage to the cornea. Immunohistochemistry showed that loss of CGRP immunoreactivity, a surrogate marker for TRPV1-expressing fibers, extended at least to the corneal-scleral boundary and displayed a progressive return, coincident with the return of capsaicin sensitivity. These data suggest that RTX may be a safe and effective treatment for post-operative or post-injury ophthalmic pain.

Topics: Administration, Topical; Analgesics; Animals; Cornea; Disease Models, Animal; Diterpenes; Male; Nociceptors; Pain; Rats; Rats, Sprague-Dawley; Reaction Time; Sensory Receptor Cells; Treatment Outcome; TRPV Cation Channels

The transient receptor potential vanilloid 1 (TRPV1) receptor is activated by noxious heat, various endogenous mediators and exogenous irritants. The aim of the present study was to compare three TRPV1 receptor antagonists (SB705498, BCTC and AMG9810) in rat models of heat hyperalgesia. The behavioural noxious heat threshold, defined as the lowest temperature evoking nocifensive reaction, was measured with an increasing-temperature water bath. The effects of TRPV1 receptor antagonists were assessed in thermal hyperalgesia induced by the TRPV1 agonist resiniferatoxin (RTX), mild heat injury (51 degrees C, 20s) or plantar incision in rats. The control heat threshold was 43.2+/-0.4 degrees C. RTX induced an 8-10 degrees C decrease in heat threshold which was dose-dependently inhibited by oral pre-treatment with any of the TRPV1 receptor antagonists with a minimum effective dose of 1mg/kg. The mild heat injury-evoked 7-8 degrees C heat threshold drop was significantly reversed by all three antagonists injected i.p. as post-treatment. The minimum effective doses were as follows: SB705498 10, BCTC 3 and AMG9810 1mg/kg. Plantar incision-induced heat threshold drop (7-8 degrees C) was dose-dependently diminished by an oral post-treatment with any of the antagonists with minimum effective doses of 10, 3 and 3mg/kg, respectively. Assessment of RTX hyperalgesia by measurement of the paw withdrawal latency with a plantar test apparatus yielded 30 mg/kg minimum effective dose for each antagonist. In conclusion, measurement of the noxious heat threshold with the increasing-temperature water bath is suitable to sensitively detect the effects of TRPV1 receptor antagonists in thermal hyperalgesia models.

Topics: Acrylamides; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cold Temperature; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Female; Hot Temperature; Hyperalgesia; Pain; Pyrazines; Pyridines; Pyrrolidines; Rats; Rats, Sprague-Dawley; TRPV Cation Channels; Urea

TRPV1 (transient receptor potential vanilloid 1) is a ligand-gated ion channel expressed predominantly in nociceptive primary afferents that plays a key role in pain processing. In vivo activation of TRPV1 receptors by natural agonists like capsaicin is associated with a sharp and burning pain, frequently described as pungency. To elucidate the mechanisms underlying pungency we investigated a series of TRPV1 agonists that included both pungent and non-pungent compounds covering a large range of potencies. Pungency of capsaicin, piperine, arvanil, olvanil, RTX (resiniferatoxin) and SDZ-249665 was evaluated in vivo, by determining the increase in the number of eye wipes caused by direct instillation of agonist solutions into the eye. Agonist-induced calcium fluxes were recorded using the FLIPR technique in a recombinant, TRPV1-expressing cell line. Current-clamp recordings were performed in rat DRG (dorsal root ganglia) neurons in order to assess the consequences of TRPV1 activation on neuronal excitability. Using the eye wipe assay the following rank of pungency was obtained: capsaicin>piperine>RTX>arvanil>olvanil>SDZ-249665. We found a strong correlation between kinetics of calcium flux, pungency and lipophilicity of TRPV1 agonists. Current-clamp recordings confirmed that the rate of receptor activation translates in the ability of agonists to generate action potentials in sensory neurons. We have demonstrated that the lipophilicity of the compounds is directly related to the kinetics of TRPV1 activation and that the latter influences their ability to trigger action potentials in sensory neurons and, ultimately, pungency.

Topics: Action Potentials; Alkaloids; Animals; Benzodioxoles; Capsaicin; Diterpenes; Ganglia, Spinal; Kinetics; Lipid Metabolism; Male; Neurons, Afferent; Pain; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sensory Receptor Cells; Solubility; TRPV Cation Channels; Urea

Pulsed radiofrequency (PRF) is a popular pain treatment modality. The effect of PRF current on neuropathic pain has not been examined in detail. We investigated the effect of PRF current on mechanical allodynia induced with resiniferatoxin (RTX) in rats, especially regarding the influence of the duration of allodynia before PRF procedures and that of exposure time to PRF.. Adult male Sprague-Dawley rats (weighing 250-400 g) received a single intraperitoneal injection of RTX (200 microg/kg) under 2 to 3% sevoflurane anesthesia. Rats in group S(2) (n = 5) were assigned to receive PRF current to the right sciatic nerve for 2 minutes 1 week after RTX treatment; rats in group M(2) (n = 6), PRF current for 2 minutes 3 weeks after RTX treatment; rats in group L(2) (n = 7), PRF current for 2 minutes 5 weeks after RTX treatment; rats in group S(4) (n = 5), PRF current for 4 minutes 1 week after RTX treatment; rats in group S(6) (n = 5), PRF current for 6 minutes 1 week after RTX treatment; and rats in group S(0) (n = 3), no PRF current was delivered. Instead, the needle and electrode were inserted at proper points for 6 minutes 1 week after RTX treatment. All rats were evaluated for sensitivity to mechanical stimulation with von Frey filaments and to thermal stimulation with a thermal testing apparatus and for motor function using placing and grasping reflexes before injection of RTX, every week after injection of RTX, and 1, 2, 3, 4, and 5 weeks after PRF treatment.. The paw withdrawal thresholds of both hindpaws 1 week after RTX treatment were significantly lower than the pre-RTX baseline in all groups. In groups S(2), S(4), S(6), and M(2), after PRF procedures, the ipsilateral paw withdrawal thresholds significantly increased. A statistically significant difference was detected between the PRF-treated and PRF-untreated hindpaws. The ipsilateral-contralateral paw withdrawal thresholds after PRF procedures in group S(2) were significantly higher than those in groups M(2) and L(2). Between groups M(2) and L(2), significant differences were found 1, 2, 4, and 5 weeks after PRF procedures. The ipsilateral-contralateral paw withdrawal thresholds in group S(6) were significantly higher than those in groups S(2) and S(4) 5 weeks after PRF procedures. No significant difference was found between groups S(2) and S(4) at any time. After PRF procedures, no difference in the withdrawal latency after heat stimulation and no motor disturbance were observed at any time in all groups.. PRF treatment was more effective when applied in the early stages of mechanical allodynia (1 week) in rats. Increased exposure time to PRF current from 2 to 6 minutes showed a significant antiallodynic effect without motor impairment. We propose the application of PRF current for 6 minutes adjacent to the nerve as soon as possible when allodynia appears.

Topics: Animals; Behavior, Animal; Diterpenes; Electric Stimulation; Functional Laterality; Hand Strength; Hot Temperature; Male; Neurotoxins; Pain; Pain Measurement; Physical Stimulation; Radio Waves; Rats; Rats, Sprague-Dawley; Reflex

Ablation of TRPV1-expressing nociceptive fibers with the potent capsaicin analog resiniferatoxin (RTX) results in long lasting pain relief. RTX is particularly adaptable to focal application, and the induced chemical axonopathy leads to analgesia with a duration that is influenced by dose, route of administration, and the rate of fiber regeneration. TRPV1 is expressed in a subpopulation of unmyelinated C- and lightly myelinated Adelta fibers that detect changes in skin temperature at low and high rates of noxious heating, respectively. Here we investigate fiber-type specific behaviors, their time course of recovery and molecular correlates of axon damage and nociception using infrared laser stimuli following an RTX-induced peripheral axonopathy.. RTX was injected into rat hind paws (mid-plantar) to produce thermal hypoalgesia. An infrared diode laser was used to stimulate Adelta fibers in the paw with a small-diameter (1.6 mm), high-energy, 100 msec pulse, or C-fibers with a wide-diameter (5 mm), long-duration, low-energy pulse. We monitored behavioral responses to indicate loss and regeneration of fibers. At the site of injection, responses to C-fiber stimuli were significantly attenuated for two weeks after 5 or 50 ng RTX. Responses to Adelta stimuli were significantly attenuated for two weeks at the highest intensity stimulus, and for 5 weeks to a less intense Adelta stimulus. Stimulation on the toe, a site distal to the injection, showed significant attenuation of Adelta responses for 7- 8 weeks after 5 ng, or 9-10 weeks after 50 ng RTX. In contrast, responses to C-fiber stimuli exhibited basically normal responses at 5 weeks after RTX. During the period of fiber loss and recovery, molecular markers for nerve regeneration (ATF3 and galanin) are upregulated in the dorsal root ganglia (DRG) when behavior is maximally attenuated, but markers of nociceptive activity (c-Fos in spinal cord and MCP-1 in DRG), although induced immediately after RTX treatment, returned to normal.. Behavioral recovery following peripheral RTX treatment is linked to regeneration of TRPV1-expressing Adelta and C-fibers and sustained expression of molecular markers. Infrared laser stimulation is a potentially valuable tool for evaluating the behavioral role of Adelta fibers in pain and pain control.

Topics: Ablation Techniques; Animals; Behavior, Animal; Diterpenes; Electric Stimulation; Foot; Hot Temperature; Lasers; Male; Nerve Fibers, Myelinated; Nerve Fibers, Unmyelinated; Nerve Regeneration; Neurotoxins; Pain; Rats; Rats, Sprague-Dawley; TRPV Cation Channels

Resiniferatoxin, the most potent agonist of inflammatory pain/vanilloid receptor/cation channel (TRPV1) can be used for neuron subtype specific ablation of pain generating cells at the level of the peripheral nervous system by Ca(2+)-excytotoxicity. Molecular neurosurgery is an emerging technology either to alleviate severe pain in cancer or treat/prevent different local neuropathies. Our aim was determining sensory modalities that may be lost after resiniferatoxin treatment.. Newborn or adult mice were treated with resiniferatoxin, then changes in chemical and heat sensitivity were correlated with alterations of the cell composition of sensory ganglions.. Only mice treated at adult age became less sensitive to heat stimuli, while both treatment groups lost sensitivity to specific vanilloid agonists of TRPV1 and, interestingly, to allyl-isothiocyanate, a selective agonist of TRPA1. Our in vivo and post mortem analytical results confirmed that TRPV1 and TRPA1 function together and resiniferatoxin-mediated neurosurgery removes both sensor molecules.. In adult mice resiniferatoxin causes: i) desensitization to heat and ii) sensitization to cold. Cold hyperalgesia, an imbalance in thermosensation, might be conferred by a prominent cold receptor that is expressed in surviving resiniferatoxin-resistant sensory neurons and compensates for pain signals lost with TRPA1 and TRPV1 double positive cells in the peripheral nervous system.

Topics: Animals; Blotting, Western; Cold Temperature; Diterpenes; Hot Temperature; Immunohistochemistry; Mice; Pain; Pain Threshold; Sensory Receptor Cells; Transient Receptor Potential Channels; Trigeminal Ganglion; TRPA1 Cation Channel; TRPV Cation Channels

Chronic pain is a major clinical problem and opiates are often the only treatment, but they cause significant problems ranging from sedation to deadly respiratory depression. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1), causes a slow, sustained and irreversible activation of TRPV1 and increases the frequency of spontaneous excitatory postsynaptic currents, but causes significant depression of evoked EPSCs due to nerve terminal depolarization block. Intrathecal administration of RTX to rats in the short-term inhibits nociceptive synaptic transmission, and in the long-term causes a localized, selective ablation of TRPV1-expressing central sensory nerve terminals leading to long lasting analgesia in behavioral models. Since RTX actions are selective for central sensory nerve terminals, other efferent functions of dorsal root ganglion neurons can be preserved. Preventing nociceptive transmission at the level of the spinal cord can be a useful strategy to treat chronic, debilitating and intractable pain.

Topics: Analgesia; Animals; Capsaicin; Diterpenes; Excitatory Postsynaptic Potentials; Ganglia, Spinal; Male; Neurons; Pain; Rats; Rats, Sprague-Dawley; Spinal Cord; Synaptic Transmission; TRPV Cation Channels

After peripheral nerve injury, spontaneous ectopic activity arising from the peripheral axons plays an important role in inducing central sensitization and neuropathic pain. Recent evidence indicates that activation of spinal cord microglia also contributes to the development of neuropathic pain. In particular, activation of p38 mitogen-activated protein kinase (MAPK) in spinal microglia is required for the development of mechanical allodynia. However, activity-dependent activation of microglia after nerve injury has not been fully addressed. To determine whether spontaneous activity from C- or A-fibers is required for microglial activation, we used resiniferatoxin (RTX) to block the conduction of transient receptor potential vanilloid subtype 1 (TRPV1) positive fibers (mostly C- and Adelta-fibers) and bupivacaine microspheres to block all fibers of the sciatic nerve in rats before spared nerve injury (SNI), and observed spinal microglial changes 2 days later.. SNI induced robust mechanical allodynia and p38 activation in spinal microglia. SNI also induced marked cell proliferation in the spinal cord, and all the proliferating cells (BrdU+) were microglia (Iba1+). Bupivacaine induced a complete sensory and motor blockade and also significantly inhibited p38 activation and microglial proliferation in the spinal cord. In contrast, and although it produced an efficient nociceptive block, RTX failed to inhibit p38 activation and microglial proliferation in the spinal cord.. (1) Blocking peripheral input in TRPV1-positive fibers (presumably C-fibers) is not enough to prevent nerve injury-induced spinal microglial activation. (2) Peripheral input from large myelinated fibers is important for microglial activation. (3) Microglial activation is associated with mechanical allodynia.

Topics: Animals; Bromodeoxyuridine; Bupivacaine; Diterpenes; Enzyme Activation; Hot Temperature; Microglia; Microspheres; Motor Activity; Nerve Block; Nerve Fibers; Nociceptors; p38 Mitogen-Activated Protein Kinases; Pain; Phosphorylation; Proprioception; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Nerves

The role of capsaicin-sensitive primary afferent fibers in rat pain-related behaviors and paw edema induced by scorpion Buthus martensi Karch (BmK) venom was investigated in this study. It was found that functional depletion of capsaicin-sensitive primary afferent fibers with a single systemic injection of resiniferatoxin (RTX) dramatically decreased spontaneous nociceptive behaviors, prevented the development of primary mechanical and thermal hyperalgesia as well as mirror-image mechanical hyperalgesia. RTX treatment significantly attenuated BmK venom-induced c-Fos expression in all laminaes of bilateral L4-L5 lumbar spinal cord, especially in superficial laminaes. Moreover, RTX treatment markedly reduced the early paw edema induced by BmK venom. Thus, the results indicate that capsaicin-sensitive primary afferent fibers play a critical role in various pain-related behaviors and paw edema induced by BmK venom in rats.

Topics: Animals; Capsaicin; Diterpenes; Edema; Hindlimb; Hyperalgesia; Immunohistochemistry; Neurons, Afferent; Nociceptors; Pain; Pain Threshold; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Scorpion Venoms; Spinal Cord

Cold allodynia is a frequent clinical symptom of patients with neuropathic pain. Despite numerous studies of cold allodynia, using animal models of neuropathic pain, little is known about its underlying mechanisms. This study was performed to establish a method for the pharmacologic evaluation of cold allodynia using several analgesics in a chronic constriction injury (CCI) rat model of neuropathic pain. Compared with the results obtained before the CCI operation, the CCI rats placed on a cork plate at 20 degrees C exhibited a slight change in the paw withdrawal latency because of the mechanical stimulus mediated by the injured paw touching the plate. By contrast, there was a significant reduction in the paw withdrawal latency on a cold metal plate compared with that on the cork plate after the CCI surgery, with the maximum decrease occurring on postoperative day 7. This reduction is thought to specifically reflect cold-induced pain behavior. In addition, both naïve and CCI rats showed behavioral changes at 5 and 0 degrees C, but not at 10 degrees C or higher. Interestingly, a subcutaneous morphine dose of 6 mg/kg completely inhibited cold allodynia induced at 10 degrees C on postoperative day 7. Under this condition, both the sodium channel blocker mexiletine (10 and 30 mg/kg, subcutaneously) and the calcium channel alpha2delta subunit blocker pregabalin (30 and 100 mg/kg, orally) significantly suppressed cold allodynia. Additionally, both resiniferatoxin (0.3 mg/kg, subcutaneously), an ultrapotent analog of capsaicin that desensitizes C fibers, and the VR1 channel antagonist N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (10 and 30 mg/kg, orally) significantly prolonged the paw withdrawal latency. In conclusion, our data suggest that the activation of C fibers mediates cold allodynia.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Calcium Channel Blockers; Chronic Disease; Cold Temperature; Constriction, Pathologic; Disease Models, Animal; Diterpenes; gamma-Aminobutyric Acid; Male; Mexiletine; Morphine; Nerve Fibers, Unmyelinated; Pain; Pain Measurement; Peripheral Nervous System Diseases; Pregabalin; Pyrazines; Pyridines; Rats; Rats, Sprague-Dawley; Reaction Time; Sciatic Nerve; Sodium Channel Blockers; TRPV Cation Channels

A common complication associated with diabetes is painful or painless diabetic peripheral neuropathy (DPN). The mechanisms and determinants responsible for these peripheral neuropathies are poorly understood. Using both streptozotocin (STZ)-induced and transgene-mediated murine models of type 1 diabetes (T1D), we demonstrate that Transient Receptor Potential Vanilloid 1 (TRPV1) expression varies with the neuropathic phenotype. We have found that both STZ- and transgene-mediated T1D are associated with two distinct phases of thermal pain sensitivity that parallel changes in TRPV1 as determined by paw withdrawal latency (PWL). An early phase of hyperalgesia and a late phase of hypoalgesia are evident. TRPV1-mediated whole cell currents are larger and smaller in dorsal root ganglion (DRG) neurons collected from hyperalgesic and hypoalgesic mice. Resiniferatoxin (RTX) binding, a measure of TRPV1 expression is increased and decreased in DRG and paw skin of hyperalgesic and hypoalgesic mice, respectively. Immunohistochemical labeling of spinal cord lamina I and II, dorsal root ganglion (DRG), and paw skin from hyperalgesic and hypoalgesic mice reveal increased and decreased TRPV1 expression, respectively. A role for TRPV1 in thermal DPN is further suggested by the failure of STZ treatment to influence thermal nociception in TRPV1 deficient mice. These findings demonstrate that altered TRPV1 expression and function contribute to diabetes-induced changes in thermal perception.

Topics: Animals; Blood Glucose; Body Weight; Diabetes Mellitus, Experimental; Diterpenes; Ganglia, Spinal; Hot Temperature; Immunohistochemistry; Injections, Intraperitoneal; Ion Channel Gating; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Pain; Streptozocin; Tritium; TRPV Cation Channels

Omega-3 (n-3) fatty acids are essential for proper neuronal function, and they possess prominent analgesic properties, yet their underlying signalling mechanisms are unclear. Here we show that n-3 fatty acids interact directly with TRPV1, an ion channel expressed in nociceptive neurones and brain. These fatty acids activate TRPV1 in a phosphorylation-dependent manner, enhance responses to extracellular protons, and displace binding of the ultrapotent TRPV1 ligand [3H]resiniferatoxin. In contrast to their agonistic properties, n-3 fatty acids competitively inhibit the responses of vanilloid agonists. These actions occur in mammalian cells in the physiological concentration range of 1-10 mum. Significantly, docosahexaenoic acid exhibits the greatest efficacy as an agonist, whereas eicosapentaenoic acid and linolenic acid are markedly more effective inhibitors. Similarly, eicosapentaenoic acid but not docosahexaenoic acid profoundly reduces capsaicin-evoked pain-related behaviour in mice. These effects are independent of alterations in membrane elasticity because the micelle-forming detergent Triton X-100 only minimally affects TRPV1 properties. Thus, n-3 fatty acids differentially regulate TRPV1 and this form of signalling may contribute to their biological effects. Further, these results suggest that dietary supplementation with selective n-3 fatty acids would be most beneficial for the treatment of pain.

Topics: Animals; Binding, Competitive; Calcium; Capsaicin; Cell Line; Cell Membrane; Diterpenes; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Female; Humans; Hydrogen-Ion Concentration; Linoleic Acid; Male; Membrane Fluidity; Membrane Potentials; Mice; Mice, Inbred C57BL; Oocytes; Pain; Rats; RNA, Complementary; TRPV Cation Channels; Xenopus laevis

Deletion of transient receptor potential vanilloid type 1 (TRPV1)-expressing afferent neurons reduces presynaptic mu opioid receptors but paradoxically potentiates the analgesic efficacy of mu opioid agonists. In this study, we determined if removal of TRPV1-expressing afferent neurons by resiniferatoxin (RTX), an ultrapotent capsaicin analog, influences the development of opioid analgesic tolerance. Morphine tolerance was induced by daily intrathecal injections of 10 microg of morphine for 14 consecutive days or by daily i.p. injections of 10 mg/kg of morphine for 10 days. In vehicle-treated rats, the effect of intrathecal or systemic morphine on the mechanical withdrawal threshold was gradually diminished within 7 days. However, the analgesic effect of intrathecal and systemic morphine was sustained in RTX-treated rats at the time the morphine effect was lost in the vehicle group. Furthermore, the mu opioid receptor-G protein coupling in the spinal cord was significantly decreased ( approximately 22%) in vehicle-treated morphine tolerant rats, but was not significantly altered in RTX-treated rats receiving the same treatment with morphine. Additionally, there was a large reduction in protein kinase Cgamma-immunoreactive afferent terminals in the spinal dorsal horn of RTX-treated rats. These findings suggest that loss of TRPV1-expressing sensory neurons attenuates the development of morphine analgesic tolerance possibly by reducing mu opioid receptor desensitization through protein kinase Cgamma in the spinal cord. These data also suggest that the function of presynaptic mu opioid receptors on TRPV1-expressing sensory neurons is particularly sensitive to down-regulation by mu opioid agonists during opioid tolerance development.

Topics: Analgesics, Opioid; Animals; Diterpenes; Down-Regulation; Drug Tolerance; Male; Morphine; Nerve Degeneration; Neurons, Afferent; Nociceptors; Pain; Pain Threshold; Posterior Horn Cells; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; TRPV Cation Channels

Success of chemotherapy and alleviation of pain are frequently less than optimal in pancreatic cancer patients, leading to increasing interest in new pharmacological substances, such as vanilloids. Our study addressed the question of whether vanilloids influence pancreatic cancer cell growth, and if vanilloids could be used for pain treatment via the vanilloid 1 receptor (VR1) in pancreatic cancer patients.. In vitro, the effect of resiniferatoxin (vanilloid analogue) on apoptosis and cell growth in pancreatic cancer cells--either alone, combined with 5-fluorouracil (5-FU), or combined with gemcitabine--was determined by annexin V staining, FACS analysis, and MTT assay, respectively. VR1 expression was evaluated on RNA and protein level by quantitative polymerase chain reaction and immunohistochemistry in human pancreatic cancer and chronic pancreatitis. Patient characteristics--especially pain levels--were registered in a prospective database and correlated with VR1 expression.. Resiniferatoxin induced apoptosis by targeting mitochondrial respiration and decreased cell growth in pancreatic cancer cells without showing synergistic effects with 5-FU or gemcitabine. Expression of VR1 was significantly upregulated in human pancreatic cancer and chronic pancreatitis. VR1 expression was related to the intensity of pain reported by cancer patients but not to the intensity of pain reported by patients with chronic pancreatitis.. Resiniferatoxin induced apoptosis in pancreatic cancer cells indicates that vanilloids may be useful in the treatment of human pancreatic cancer. Furthermore, vanilloid might be a novel and effective treatment option for neurogenic pain in patients with pancreatic cancer.

Topics: Adult; Aged; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Tumor; Chronic Disease; Deoxycytidine; Diterpenes; Drug Synergism; Drug Therapy, Combination; Female; Fluorouracil; Gemcitabine; Humans; Immunohistochemistry; Male; Middle Aged; Mitochondria; Oxidative Stress; Pain; Pancreas; Pancreatic Neoplasms; Pancreatitis; Prospective Studies; TRPV Cation Channels

In the present paper, we describe the analgesic effects induced by the transient receptor potential vanilloid type 1 (TRPV1) antagonist, capsazepine, and the TRPV1 agonist, resiniferatoxin, on the thermal hyperalgesia induced by the presence of a tibial osteosarcoma or an inflammatory process in mice. The administration of capsazepine abolished the osteosarcoma-induced hyperalgesia at a dose range (3-10 mg/kg; s.c.) ineffective to inhibit the hyperalgesia elicited by the intraplantar administration of complete Freund's adjuvant (CFA). In contrast, the administration of resiniferatoxin (0.01-0.1 mg/kg; s.c.) inhibited both the osteosarcoma- and the CFA-induced hyperalgesia. Remarkably, a single dose of resiniferatoxin abolished the osteosarcoma-induced hyperalgesia for several days and completely prevented the instauration of thermal hyperalgesia when administered at the initial stages of osteosarcoma development. The potential of drugs acting through TRPV1 for the management of some types of bone cancer pain is proposed.

Topics: Analgesics; Analysis of Variance; Animals; Bone Neoplasms; Capsaicin; Cell Line; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Freund's Adjuvant; Functional Laterality; Inflammation; Mice; Mice, Inbred C3H; Osteosarcoma; Pain; Pain Measurement; Reaction Time; Time Factors

Nociceptive signals are transmitted to the spinal dorsal horn via primary afferent fibers, and the signals induce withdrawal reflexes by activating spinal motoneurons in the ventral horn. Therefore, nociceptive stimuli increase motoneuronal firing and ventral root discharges. This study was aimed to develop a method for the study of pain mechanisms and analgesics by recording ventral root discharges. Spinalized rats were laminectomized in the lumbo-sacral region. The fifth lumbar ventral root was sectioned and placed on a pair of wire electrodes. Multi unit efferent discharges from the ventral root were increased by mechanical stimulation using a von Frey hair applied to the plantar surface of the hindpaw. The low-intensity mechanical stimuli increased the discharges during stimulation (during-discharges) without increasing the discharges after cessation of stimulation (after-discharges), and the high-intensity mechanical stimuli increased both during- and after-discharges. Pretreatment with resiniferatoxin, an ultrapotent analogue of capsaicin, halved during-discharges and eliminated after-discharges, suggesting that after-discharges are generated by heat- and mechanosensitive polymodal nociceptors. Ezlopitant, a neurokinin-1 (NK-1) receptor antagonist, but not its inactive enantiomer, selectively reduced the after-discharges. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, preferentially reduced the after-discharges, demonstrating that NK-1 and NMDA receptors mediate the after-discharges. Morphine reduced the after-discharges without affecting during-discharges. By contrast, mephenesin, a centrally acting muscle relaxant, reduced both during- and after-discharges. There results suggest that simultaneous recordings of during- and after-discharges are useful to study pain mechanisms and analgesics as well as to discriminate the analgesic effects from the side effects such as muscle relaxant effects.

Topics: Action Potentials; Analysis of Variance; Animals; Benzylamines; Bridged Bicyclo Compounds, Heterocyclic; Diterpenes; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Excitatory Amino Acid Antagonists; Ketamine; Male; Mephenesin; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Neurotoxins; Pain; Pain Measurement; Physical Stimulation; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Receptors, Neurokinin-1; Spinal Nerve Roots; Time Factors

The function of the transient receptor potential vanilloid type 1 capsaicin receptor is subject to modulation by phosphorylation catalyzed by various enzymes including protein kinase C and cAMP-dependent protein kinase. The aim of this study was to compare the significance of the basal and stimulated activity of protein kinase C and cAMP-dependent protein kinase in transient receptor potential vanilloid type 1 receptor responsiveness in the rat in vitro by measurement of the intracellular calcium concentration in cultured trigeminal ganglion neurons and in vivo by determination of the behavioral noxious heat threshold. KT5720, a selective inhibitor of cAMP-dependent protein kinase, reduced the calcium transients induced by capsaicin or the other, much more potent transient receptor potential vanilloid type 1 receptor agonist resiniferatoxin in trigeminal sensory neurons and diminished the drop of the noxious heat threshold (heat allodynia) evoked by intraplantar resiniferatoxin injection. Chelerythrine chloride, a selective inhibitor of protein kinase C, failed to alter either of these responses, although it inhibited the effect of phorbol 12-myristate 13-acetate in the in vitro assay. Staurosporine, a rather nonselective protein kinase inhibitor, failed to reduce the capsaicin- and resiniferatoxin-induced calcium transients but inhibited the resiniferatoxin-evoked heat allodynia. Dibutyryl-cAMP and phorbol 12-myristate 13-acetate, activator(s) of cAMP-dependent protein kinase and protein kinase C, respectively, enhanced the effect of capsaicin in the calcium uptake assay while forskolin, an activator of adenylyl cyclase, augmented that of resiniferatoxin in the heat allodynia model. None of the protein kinase inhibitors or activators altered the calcium transients evoked by high potassium, a nonspecific depolarizing stimulus. It is concluded that basal activity of cAMP-dependent protein kinase, unlike protein kinase C, is involved in the maintenance of transient receptor potential vanilloid type 1 receptor function in somata of trigeminal sensory neurons but stimulation of either cAMP-dependent protein kinase or protein kinase C above the resting level can lead to an enhanced transient receptor potential vanilloid type 1 receptor responsiveness. Similar mechanisms are likely to operate in vivo in peripheral terminals of nociceptive dorsal root ganglion neurons.

Topics: Adenylyl Cyclases; Animals; Animals, Newborn; Bucladesine; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Diterpenes; Enzyme Activation; Enzyme Inhibitors; Hyperalgesia; Neurons, Afferent; Nociceptors; Pain; Pain Threshold; Phosphorylation; Protein Kinase C; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate; Trigeminal Ganglion; TRPV Cation Channels

Peripheral bee venom (BV) administration produces 2 contrasting effects, nociception and antinociception. This study was designed to evaluate whether the initial nociceptive effect induced by BV injection into the Zusanli acupoint is involved in producing the more prolonged antinociceptive effect observed in the mouse formalin test, and whether capsaicin-sensitive primary afferents are involved in these effects. BV injection into the Zusanli point increased spinal Fos expression but not spontaneous nociceptive behavior. BV pretreatment 10 minutes before intraplantar formalin injection dose-dependently attenuated nociceptive behavior associated with the second phase of the formalin test. The destruction of capsaicin-sensitive primary afferents by resiniferatoxin (RTX) pretreatment selectively decreased BV-induced spinal Fos expression but did not affect BV-induced antinociception. Furthermore, BV injection increased Fos expression in tyrosine hydroxylase immunoreactive neurons in the locus caeruleus, and this expression was unaltered by RTX pretreatment. Finally, BV's antinociception was blocked by intrathecal injection of 10 microg idazoxan, and this effect was not modified by RTX pretreatment. These findings suggest that subcutaneous BV stimulation of the Zusanli point activates central catecholaminergic neurons via capsaicin-insensitive afferent fibers without induction of nociceptive behavior. This in turn leads to the activation of spinal alpha2-adrenoceptors, which ultimately reduces formalin-evoked nociceptive behaviors.. This study demonstrates that BV acupuncture produces a significant antinociception without nociceptive behavior in rodents, which is mediated by capsaicin-insensitive afferents and involves activation of central adrenergic circuits. These results further suggest that BV stimulation into this acupuncture point might be a valuable alternative to traditional electrical or mechanical acupoint stimulation.

Topics: Acupuncture Analgesia; Acupuncture Points; Afferent Pathways; Analgesics; Animals; Bee Venoms; Capsaicin; Disease Models, Animal; Diterpenes; Drug Resistance; Locus Coeruleus; Male; Mice; Mice, Inbred ICR; Neurons, Afferent; Neurotoxins; Nociceptors; Norepinephrine; Pain; Pain Measurement; Proto-Oncogene Proteins c-fos; Receptors, Adrenergic, alpha-2; Spinal Cord; Tyrosine 3-Monooxygenase

The long-lasting imprint of acute pain in the central nervous system may contribute to the transition of acute pain to chronicity. The long-term potentiation (which is proposed as a mechanism of memory) and central sensitization were each reported as a form of synaptic plasticity, and both can be initiated by stimulation of C fibers. In the current study, we assessed nociceptive memory regarding hyperalgesia by measuring distant hyperalgesia after repeated carrageenan-induced inflammation. This approach was used to determine whether selective blockade of C fibers can prevent the development of a long-lasting imprint of hyperalgesia. In rat experiments, resiniferatoxin was administered percutaneously at the sciatic and saphenous nerves, and two crossover intraplantar injections of carrageenan into the hindpaws were performed 2 wk apart. Responses to noxious pressure and heat and changes in paw volumes were measured at various intervals during two carrageenan-induced inflammations. The experiments demonstrated that after recovery of hyperalgesia induced by the initial inflammation, repeated inflammation led to the development of a distant hyperalgesia that was absent during the initial inflammation. The maximum of distant hyperalgesia (decrease of noxious pressure threshold in the contralateral hindpaw from 141 +/- 23 g to 96 +/- 19 g; P < 0.0001) was reached 24 h after the second injection of carrageenan. The development of distant hyperalgesia during the repeated inflammation was completely prevented (P < 0.0002) by perineural resiniferatoxin (0.001%) administered before the initial injection of carrageenan. These results indicate that selective blockade of nociceptive fibers prevents formation of long-term hyperalgesia-related imprint in the central nervous system. Thus, pain memory can be preempted by selective and prolonged blockade of C-fibers.

Topics: Animals; Carrageenan; Central Nervous System; Diterpenes; Humans; Hyperalgesia; Inflammation; Male; Memory; Nerve Block; Pain; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Saphenous Vein; Sciatic Nerve

The mixture of the two pentacyclic triterpenes alpha-amyrin and beta-amyrin, isolated from the resin of Protium kleinii and given by intraperitoneal (i.p.) or oral (p.o.) routes, caused dose-related and significant antinociception against the visceral pain in mice produced by i.p. injection of acetic acid. Moreover, i.p., p.o., intracerebroventricular (i.c.v.), or intrathecal (i.t.) administration of alpha,beta-amyrin inhibited both neurogenic and inflammatory phases of the overt nociception caused by intraplantar (i.pl.) injection of formalin. Likewise, alpha,beta-amyrin given by i.p., p.o., i.t., or i.c.v. routes inhibits the neurogenic nociception induced by capsaicin. Moreover, i.p. treatment with alpha,beta-amyrin was able to reduce the nociception produced by 8-bromo-cAMP (8-Br-cAMP) and by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the hyperalgesia caused by glutamate. On the other hand, in contrast to morphine, alpha,beta-amyrin failed to cause analgesia in thermal models of pain. The antinociception caused by the mixture of compounds seems to involve mechanisms independent of opioid, alpha-adrenergic, serotoninergic, and nitrergic system mediation, since it was not affected by naloxone, prazosin, yohimbine, DL-p-chlorophenylalanine methyl ester, or L-arginine. Interestingly, the i.p. administration of alpha,beta-amyrin reduced the mechanical hyperalgesia produced by i.pl. injection of carrageenan, capsaicin, bradykinin, substance P, prostaglandin E2, 8-Br-cAMP, and TPA in rats. However, the mixture of compounds failed to alter the binding sites of [3H]bradykinin, [3H]resiniferatoxin, or [3H]glutamate in vitro. It is concluded that the mixture of triterpene alpha-amyrin and beta-amyrin produced consistent peripheral, spinal, and supraspinal antinociception in rodents, especially when assessed in inflammatory models of pain. The mechanisms involved in their action are not completely understood but seem to involve the inhibition of protein kinase A- and protein kinase C-sensitive pathways.

Topics: Acetic Acid; Analgesics; Animals; Bradykinin; Cyclic AMP-Dependent Protein Kinases; Diterpenes; Dose-Response Relationship, Drug; Drug Combinations; Formaldehyde; Glutamic Acid; Heart; Male; Mice; Oleanolic Acid; Pain; Pain Measurement; Peripheral Nervous System; Physical Stimulation; Protein Kinase C; Psychomotor Performance; Rats; Rats, Wistar; Reaction Time; Spinal Cord; Stimulation, Chemical; Triterpenes

Resiniferatoxin is a potent capsaicin analog. Intrathecal administration leads to selective, prolonged opening of the transient receptor potential V1 ion channel, which is localized mainly to C-fiber primary afferent nociceptive sensory neurons. Following work in laboratory animals, the authors explored the use of intrathecal resiniferatoxin to control spontaneous bone cancer pain in companion (pet) dogs.. Normal canine population: Behavioral testing was performed to establish baseline paw withdrawal latency; subsequently, general anesthesia was induced and resiniferatoxin was administered intrathecally while hemodynamic parameters were recorded. Behavior testing was repeated for 12 days after administration of resiniferatoxin. Clinical canine population: Twenty companion dogs with bone cancer pain were recruited. The animal's baseline level of discomfort and analgesic use were recorded. Resiniferatoxin was administered intrathecally and hemodynamic parameters were monitored while the dogs were under general anesthesia. Dogs were reevaluated up to 14 weeks after resiniferatoxin administration.. Normal canine population: In the first minutes after resiniferatoxin injection, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate from baseline. Two days after injection, limb withdrawal latencies increased to the point of cutoff in the dogs that received at least 1.2 microg/kg resiniferatoxin. Clinical canine population: From baseline, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate after resiniferatoxin injection. Comfort scores were significantly improved at 2, 6, 10, and 14 weeks after resiniferatoxin administration (P < 0.0001). There was decreased or discontinued use of supplemental analgesics in 67% of the dogs 2 weeks after resiniferatoxin administration.. Intrathecal resiniferatoxin elicits transient hemodynamic effects. In controls, a profound and sustained blockade of thermal stimuli is produced in a dose-dependent fashion. Similar administration in dogs with bone cancer produces a prolonged antinociceptive response.

Topics: Analgesics; Animals; Behavior, Animal; Bone Neoplasms; Catheterization; Cohort Studies; Diterpenes; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Hemodynamics; Injections, Spinal; Pain; Pain Measurement

Topics: Analgesics; Chronic Disease; Diterpenes; Euphorbiaceae; Humans; Pain; Phytotherapy; Plants, Medicinal

Somatostatin released from capsaicin-sensitive sensory nerves exerts systemic anti-inflammatory and antinociceptive actions. TT-232 is a stable, peripherally acting heptapeptide (D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH2) somatostatin analogue with highest binding affinity for somatostatin sst4 receptors. It has been shown to inhibit acute and chronic inflammatory responses and sensory neuropeptide release from capsaicin-sensitive nociceptors. In the present study the antinociceptive effects of TT-232 were analysed using both acute and chronic models of nociception. Formalin-induced pain behaviour, noxious heat threshold and streptozotocin-induced diabetic neuropathic mechanical allodynia were examined in rats and phenylquinone-evoked abdominal constrictions were tested in mice. TT-232 (80 microg/kg i.p.) inhibited both early (0-5 min) and late phases (25-45 min) of formalin-induced nociception as revealed by determination of the composite pain score. The minimum effective dose to elevate the noxious heat threshold and diminish the heat threshold drop (heat allodynia) evoked by resiniferatoxin (0.05 nmol intraplantarly) was 20 and 10 microg/kg i.p., respectively, as measured by an increasing-temperature hot plate. TT-232 (10-200 microg/kg s.c.) significantly inhibited phenylquinone-evoked writhing movements in mice, but within this dose range no clear dose-response correlation was found. Five weeks after streptozotocin administration (50 mg/kg i.v.) the diabetes-induced decrease in the mechanonociceptive threshold was inhibited by 10-100 microg/kg i.p. TT-232. These findings show that TT-232 potently inhibits acute chemical somatic/visceral and thermal nociception and diminishes chronic mechanical allodynia associated with diabetic neuropathy, thereby it could open new perspectives in the treatment of various pain syndromes.

Topics: Acute Disease; Analgesics; Animals; Behavior, Animal; Benzoquinones; Diabetes Mellitus, Experimental; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Female; Formaldehyde; Male; Mice; Mice, Inbred BALB C; Pain; Pain Measurement; Peptides, Cyclic; Rats; Rats, Wistar; Somatostatin

The epsilon-isozyme of protein kinase C (PKCepsilon) and the vanilloid receptor 1 (VR1) are both expressed in dorsal root ganglion (DRG) neurons and are reported to be predominantly and specifically involved in nociceptive function. Using phosphospecific antibody against the C-terminal hydrophobic site Ser729 of PKCepsilon as a marker of enzyme activation, the state-dependent activation of PKCepsilon, as well as the expression of VR1 in rat DRG neurons, was evaluated in different experimental pain models in vivo. Quantitative analysis showed that phosphorylation of PKCepsilon in DRG neurons was significantly up-regulated after carrageen- and Complete Freund's Adjuvant-induced inflammation, while it was markedly down-regulated after chronic constriction injury. A double-labeling study showed that phosphorylation of PKCepsilon was expressed predominantly in VR1 immunoreactivity positive small diameter DRG neurons mediating the nociceptive information from peripheral tissue to spinal cord. The VR1 protein expression showed no significant changes after either inflammation or chronic constriction injury. These data indicate that functional activation of PKCepsilon has a close relationship with the production of inflammatory hyperalgesia and the sensitization of the nociceptors. Inflammatory mediator-induced activation of PKCepsilon and subsequent sensitization of VR1 to noxious stimuli by PKCepsilon may be involved in nociceptor sensitization.

Topics: Animals; Behavior, Animal; Capsaicin; Carrageenan; Disease Models, Animal; Diterpenes; Freund's Adjuvant; Ganglia, Spinal; Hindlimb; Hyperalgesia; Inflammation; Ligation; Male; Neurons; Nociceptors; Pain; Pain Measurement; Peripheral Nervous System Diseases; Phosphorylation; Protein Kinase C; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptors, Drug

1. An increasing-temperature hot plate (ITHP) was introduced to measure the noxious heat threshold (45.3+/-0.3 degrees C) of unrestrained rats, which was reproducible upon repeated determinations at intervals of 5 or 30 min or 1 day. 2. Morphine, diclofenac and paracetamol caused an elevation of the noxious heat threshold following i.p. pretreatment, the minimum effective doses being 3, 10 and 200 mg kg(-1), respectively. 3. Unilateral intraplantar injection of the VR1 receptor agonist resiniferatoxin (RTX, 0.048 nmol) induced a profound drop of heat threshold to the innocuous range with a maximal effect (8-10 degrees C drop) 5 min after RTX administration. This heat allodynia was inhibited by pretreatment with morphine, diclofenac and paracetamol, the minimum effective doses being 1, 1 and 100 mg kg(-1) i.p., respectively. 4. The long-term sensory desensitizing effect of RTX was examined by bilateral intraplantar injection (0.048 nmol per paw) which produced, after an initial threshold drop, an elevation (up to 2.9+/-0.5 degrees C) of heat threshold lasting for 5 days. 5. The VR1 receptor antagonist iodo-resiniferatoxin (I-RTX, 0.05 nmol intraplantarly) inhibited by 51% the heat threshold-lowering effect of intraplantar RTX but not alpha,beta-methylene-ATP (0.3 micromol per paw). I-RTX (0.1 or 1 nmol per paw) failed to alter the heat threshold either acutely (5-60 min) or on the long-term (5 days). The heat threshold of VR1 receptor knockout mice was not different from that of wild-type animals (45.6+/-0.5 vs 45.2+/-0.4 degrees C). 6. In conclusion, the RTX-induced drop of heat threshold measured by the ITHP is a novel heat allodynia model exhibiting a high sensitivity to analgesics.

Topics: Acetaminophen; Analgesics; Animals; Diclofenac; Diterpenes; Dose-Response Relationship, Drug; Female; Hindlimb; Hot Temperature; Mice; Mice, Knockout; Morphine; Nociceptors; Pain; Pain Measurement; Physical Stimulation; Rats; Rats, Wistar; Receptors, Drug; Reproducibility of Results; Sensory Thresholds; Thermosensing; Time Factors

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain.

Topics: Animals; Animals, Newborn; Arachidonate 12-Lipoxygenase; Bradykinin; Cell Line; Cells, Cultured; Diterpenes; Ganglia, Spinal; Humans; Hyperalgesia; Inflammation; Leukotrienes; Neurons; Neurons, Afferent; Neurotoxins; Pain; Rats; Receptors, Bradykinin; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.

Topics: Analgesics; Animals; Binding, Competitive; Capsaicin; Cells, Cultured; Diterpenes; Drug Interactions; Humans; Iodine Radioisotopes; Mice; Oocytes; Pain; Pain Measurement; Radioligand Assay; Receptors, Drug; Transfection; TRPV Cation Channels; Xenopus laevis

Topics: Afferent Pathways; Animals; Arousal; Cats; Cerebral Cortex; Cystitis, Interstitial; Diterpenes; Efferent Pathways; Heart Rate; Neurotoxins; Pain; Reflex; Respiration; Tail; Urinary Bladder

Propofol (2,6-diisopropylphenol) is an intravenous anesthetic agent structurally unrelated to any other intravenous anesthetics. We examined the effect of propofol on a rat vanilloid receptor that was expressed in the human embryonic kidney (HEK) 293 cells by using calcium imaging method. Propofol caused a concentration-dependent increase in [Ca(2+)](i) in the HEK293 cells with the receptor. These responses were inhibited by removing extracellular calcium ions. The propofol-evoked increase in [Ca(2+)](i) in the HEK293 cells with the receptor was partially inhibited by capsazepine, a competitive antagonist of capsaicin. We conclude that propofol acts as an agonist for the receptor.

Topics: Anesthetics, Intravenous; Binding Sites; Calcium; Calcium Signaling; Capsaicin; Cells, Cultured; Diterpenes; Dose-Response Relationship, Drug; Humans; Membrane Potentials; Nervous System; Pain; Patch-Clamp Techniques; Propofol; Receptors, Drug

The present study assessed the effect of a single subcutaneous injection of resiniferatoxin (RTX), an ultrapotent capsaicin analogue, on the activity of spinal cholecystokinin (CCK) systems, by using electrophysiological and in situ hybridization techniques. Subcutaneous RTX at 0.3 mg/kg, but not vehicle, produced marked thermal hypoalgesia in rats on the hot plate and tail flick tests. Partial recovery from hypoalgesia occurred in some (<50%), but not all, RTX-treated rats after 2 weeks. The flexor reflex in response to activation of high threshold afferents was recorded 15-35 days after RTX- or vehicle-treatment. There was no obvious difference between RTX- and vehicle-treated rats in the baseline flexor reflex. Intravenous morphine at 1 mg/kg caused a depression of the flexor reflex in vehicle- and in RTX-treated rats. The reflex depressive effect of morphine was significantly briefer in RTX-treated, non-recovered rats than vehicle-treated rats. Furthermore, CI-988, a high affinity antagonist of CCKB receptors, caused a minor depression of the reflex in vehicle- and RTX-treated rats that had partially recovered, whereas the reflex depressive effect of CI-988 was significantly enhanced in RTX-treated, non-recovered rats. In situ hybridization showed that RTX treatment caused a marked and significant increase in the number of dorsal root ganglion (DRG) neurone profiles expressing CCKB receptor mRNA, whereas only a small increase was observed for CCKA receptor mRNA expressing neurone profiles. Significantly more DRG neurone profiles expressed CCKB receptor mRNA in RTX-treated, non-recovered rats compared to partially recovered rats. RTX-treatment did not influence the expression of CCK mRNA in DRGs. Since CCK functions as a physiological antagonist of morphine, it is suggested that RTX treatment enhances the activity of spinal CCK systems, leading to the reduced effect of morphine and increased effect of the CCKB receptor antagonist CI-988. This may mainly be due to upregulation of CCKB receptors in DRG neurones.

Topics: Animals; Cholecystokinin; Decerebrate State; Diterpenes; Ganglia, Spinal; Hormone Antagonists; Indoles; Injections, Subcutaneous; Male; Meglumine; Morphine; Neurons; Neurotoxins; Pain; Rats; Rats, Sprague-Dawley; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Reflex; RNA, Messenger; Spinal Cord; Time Factors; Transcription, Genetic

The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the "pain" pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43 degrees C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Here, we demonstrate that sensory neurons from mice lacking VR1 are severely deficient in their responses to each of these noxious stimuli. VR1-/- mice showed normal responses to noxious mechanical stimuli but exhibited no vanilloid-evoked pain behavior, were impaired in the detection of painful heat, and showed little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia.

Topics: Animals; Body Temperature; Calcium; Capsaicin; Cells, Cultured; Diterpenes; Ganglia, Spinal; Gene Targeting; Hot Temperature; Hydrogen-Ion Concentration; Inflammation; Mice; Mice, Knockout; Nerve Fibers; Neurons; Neurons, Afferent; Nociceptors; Pain; Pain Threshold; Receptors, Drug; Spinal Cord; TRPV Cation Channels

Tactile allodynia and thermal hyperalgesia, two robust signs of neuropathic pain associated with experimental nerve injury, have been hypothesized to be mechanistically distinguished based on (a) fiber types which may be involved in the afferent input, (b) participation of spinal and supraspinal circuitry in these responses, and (c) sensitivity of these endpoints to pharmacological agents. Here, the possibility that nerve-injury induced tactile allodynia and thermal hyperalgesia may be mediated via different afferent fiber input was tested by evaluating these responses in sham-operated or nerve-injured (L5/L6) rats before or after a single systemic injection of resiniferatoxin (RTX), an ultrapotent analogue of the C-fiber specific neurotoxin, capsaicin. Tactile allodynia, and three measures of thermal nociception, tail-flick, paw-flick and hot-plate responses, were determined before and at various intervals for at least 40 days after RTX injection. Nerve-injured, but not sham-operated, rats showed a long-lasting tactile allodynia and thermal hyperalgesia (paw-flick) within 2-3 days after surgery; responses to other noxious thermal stimuli (i.e., tail-flick and hot-plate tests) did not distinguish the two groups at the stimulus intensities employed. RTX treatment resulted in a significant and long-lasting (i.e. essentially irreversible) decrease in sensitivity to thermal noxious stimuli in both sham-operated and nerve-injured rats; thermal hyperalgesia was abolished and antinociception produced by RTX. In contrast, RTX treatment did not affect the tactile allodynia seen in the same nerve-injured rats. These data support the concept that thermal hyperalgesia seen after nerve ligation, as well as noxious thermal stimuli, are likely to be mediated by capsaicin-sensitive C-fiber afferents. In contrast, nerve-injury related tactile allodynia is insensitive to RTX treatment which clearly desensitizes C-fibers and, therefore such responses are not likely to be mediated through C-fiber afferents. The hypothesis that tactile allodynia may be due to inputs from large (i.e. A beta) afferents offers a mechanistic basis for the observed insensitivity of this endpoint to intrathecal morphine in this nerve-injury model. Further, these data suggest that clinical treatment of neuropathic pains with C-fiber specific agents such as capsaicin are unlikely to offer significant therapeutic benefit against mechanical allodynia.

Topics: Animals; Capsaicin; Diterpenes; Hyperalgesia; Ligation; Male; Neurons, Afferent; Pain; Pain Measurement; Peripheral Nervous System; Physical Stimulation; Rats; Rats, Sprague-Dawley; Reaction Time; Skin; Time Factors

Adequate treatment of cancer pain remains a significant clinical problem. To reduce side effects of treatment, intrathecal and epidural routes of administration have been used where appropriate to reduce the total dose of agent administered while achieving regional control. Resiniferatoxin (RTX), an ultrapotent capsaicin analog, gives long-term desensitization of nociception via C-fiber sensory neurons. We evaluate here the analgesic effect on rats of epidurally administered RTX, using latency of response to a thermal stimulus in unrestrained animals. Results were compared with those for systemically administered RTX. Vehicle or graded doses of RTX were injected subcutaneously (s.c.) or through an indwelling lumbar (L4) epidural catheter as a single dose. Both routes of application of RTX produced profound thermal analgesia, reaching a plateau within 4-6 h and showing no restoration of pain sensitivity over 7 days. Vehicle was without effect. For the epidural route, the effect was selective as expected for the targeted spinal cord region, whereas the subcutaneous administration of RTX had a generalized analgesic effect. At doses yielding a tripling of back paw withdrawal latency, epidural treatment was 25-fold more effective than the subcutaneous route of application. Consistent with the regional selectivity of the lumbar epidural route, the front paws showed no more effect than by systemic RTX treatment. Binding experiments with [3H]RTX provided further evidence of the segmental desensitization induced by epidural RTX. We conclude that epidural administration of RTX at the lumbar spinal level produces profound, long-lasting, segmental analgesia to C-fiber mediated pain in the rat.

Topics: Analgesia, Epidural; Animals; Diterpenes; Dose-Response Relationship, Drug; Hot Temperature; Injections, Epidural; Injections, Subcutaneous; Lumbosacral Region; Male; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Reaction Time; Time Factors

Antinociceptive effects of systemically or locally administered opioid mu, kappa and delta agonists were evaluated in a rat model of visceral pain. Resiniferatoxin (RTX, 3 nmol), a capsaicin-like mutant, produced abdominally directed grooming behavior after direct administration into the urinary bladder (intravesical, Lves.) by indwelling cannula. Systemic (s.c. or i.p.) pretreatment with the mu agonists morphine or [D-Ala2, NMePhe4, Gly-ol]enkephalin (Damgo), the kappa agonists trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488) or [5R-(5,7,8-beta)]-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro[4,5]dec - 8-yl]-4-benzofuranacetamide (CI-977), or the nonpeptidic delta agonist (+/-)-4-((alpha-R*)-alpha-((2S*,5R(*)-4-Allyl-2,5-dimethyl-1- piperazinyl)-3-hydroxybenzyl)-N, N-diethylbenzamide (BW373U86) dose-dependently decreased RTX-induced abdominal licking; such antinociception was selectively blocked by the appropriate receptor-selective antagonists beta-funaltrexamine (mu), nor-binaltorphimine (kappa) and naltrindole (delta). Local (i.ves.) BW373U86, [D-Ala2,Glu4]deltorphin (DELT II) and Cl-977 also significantly decreased RTX-induced licking. Intracerabroventricular quaternary naloxone partially blocked the effects of systemic morphine, but not that of CI-977 or BW373U86. Intraperitoneal quaternary naloxone blocked the effect of local and systemic BW373U86 but not that of local or systemic CI-977; systemic morphine was partially blocked. Thus, systemic mu, kappa and delta agonists all produced antinociception against a novel visceral chemical stimulus in the rat. Local CI-977 also produced antinociception, but the only compound clearly acting at peripheral opioid receptors was BW373U86, a delta agonist. This study suggests that opioid delta receptors may be present on bladder nociceptive afferents and may be activated for production of peripheral analgesia.

Topics: Analgesics, Opioid; Animals; Diterpenes; Drug Administration Routes; Female; Grooming; Naloxone; Narcotic Antagonists; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Viscera

Topics: Action Potentials; Analgesics; Animals; Biphenyl Compounds; Capsaicin; Diterpenes; Hot Temperature; Injections, Spinal; Pain; Peripheral Nerves; Rats; Ruthenium Red; Spinal Cord; Spinal Nerve Roots; Stereoisomerism; Substance P

Whole cell currents evoked by pain-inducing agents--bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (INa) was 100 microA/cm2 and the INa was abolished by 10(-6) M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient ICa(t) and a high threshold sustained ICa(l). In addition, another high threshold transient Ca current, presumably ICa(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of -70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10(-6) M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10(-6) M Bk in the majority of the cells. Similarly, both 10(-6) M Cap and 10(-9) M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (Gi).

Topics: Action Potentials; Animals; Bradykinin; Calcium Channel Blockers; Calcium Channels; Capsaicin; Cell Line; Diterpenes; Electrophysiology; Ganglia, Spinal; Mice; Neurons; omega-Conotoxins; Pain; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Receptors, Bradykinin; Receptors, Neurotransmitter; Tetrodotoxin

A dose-dependent loss of vanilloid receptors (specific [3H]resiniferatoxin binding sites) was found in sensory ganglia of rats 24 h after s.c. administration of resiniferatoxin (RTX), an ultrapotent capsaicin analog. This receptor loss displayed an ED50 of 30 micrograms/kg both in dorsal root and trigeminal ganglia; the ED50 was 6-fold higher than the ED50 for loss of the neurogenic inflammatory response and 30-60-fold higher than the ED50 for desensitization in the standard eye-wiping (chemogenic pain) response. The receptor loss appeared later (24 h) than the loss of the physiological responses (6 h) and showed modest recovery (to 20-30% of control levels) over the following 4 weeks. This vanilloid receptor loss may represent a novel, specific mechanism for vanilloid-induced chronic desensitization.

Topics: Animals; Binding Sites; Diterpenes; Dose-Response Relationship, Drug; Female; Ganglia, Spinal; Inflammation; Kinetics; Pain; Rats; Rats, Wistar; Receptors, Drug; Time Factors; Trigeminal Ganglion

Topics: Afferent Pathways; Animals; Capsaicin; Diterpenes; Dose-Response Relationship, Drug; Heart Rate; Hot Temperature; Neurons; Neurotoxins; Pain; Rats

Resiniferatoxin is an extremely irritant diterpene present in the latex of several members of the genus Euphorbia. Its mechanism of action has been shown to be clearly distinct from that of the structurally related phorbol esters. Since resiniferatoxin possesses a 4-hydroxy-3-methoxyphenyl substituent, a key feature of capsaicin, the major pungent ingredient of plants of the genus Capsicum, we examined the ability of resiniferatoxin to induce typical capsaicin responses. We report here that treatment of rats with resiniferatoxin, like treatment with capsaicin, caused hypothermia, neurogenic inflammation, and pain. These responses were followed by loss of thermoregulation, by desensitization to neurogenic inflammation, and by chemical and thermal analgesia, with cross-tolerance between resiniferatoxin and capsaicin. Resiniferatoxin was 3 4 orders of magnitude more potent than capsaicin for the effects on thermoregulation and neurogenic inflammation. Resiniferatoxin was only comparable in potency to capsaicin, however, in the assay for induction of acute pain, and the desensitization to acute pain appeared to require less resiniferatoxin than did desensitization for the other responses. We conclude that resiniferatoxin acts as an ultrapotent capsaicin analog and hypothesize that it may distinguish between subclasses of capsaicin response.

Topics: Animals; Capsaicin; Diterpenes; Female; Hypothermia; Inflammation; Pain; Rats; Rats, Inbred Strains