Neural Selective Cryoneurolysis with Ice Slurry Injection in a Rat Model.

BACKGROUND: Postoperative pain caused by trauma to nerves and tissue around the surgical site is a major problem. Perioperative steps to reduce postoperative pain include local anesthetics and opioids, the latter of which are addictive and have contributed to the opioid epidemic. Cryoneurolysis is a nonopioid and long-lasting treatment for reducing postoperative pain. However, current methods of cryoneurolysis are invasive, technically demanding, and are not tissue-selective. This project aims to determine whether ice slurry can be used as a novel, injectable, drug-free, and tissue-selective method of cryoneurolysis and resulting analgesia. METHODS: The authors developed an injectable and selective method of cryoneurolysis using biocompatible ice slurry, using rat sciatic nerve to investigate the effect of slurry injection on the structure and function of the nerve. Sixty-two naïve, male Sprague-Dawley rats were used in this study. Advanced Coherent anti-Stokes Raman Scattering microscopy, light, and fluorescent microscopy imaging were used at baseline and at various time points after treatment for evaluation and quantification of myelin sheath and axon structural integrity. Validated motor and sensory testing were used for evaluating the sciatic nerve function in response to ice slurry treatment. RESULTS: Ice slurry injection can selectively target the rat sciatic nerve. Being injectable, it can infiltrate around the nerve. The authors demonstrate that a single injection is safe and selective for reversibly disrupting the myelin sheaths and axon density, with complete structural recovery by day 112. This leads to decreased nocifensive function for up to 60 days, with complete recovery by day 112. There was up to median [interquartile range]: 68% [60 to 94%] reduction in mechanical pain response after treatment. CONCLUSIONS: Ice slurry injection selectively targets the rat sciatic nerve, causing no damage to surrounding tissue. Injection of ice slurry around the rat sciatic nerve induced decreased nociceptive response from the baseline through neural selective cryoneurolysis.

Contralateral Hyperalgesia from Injection of Endothelin-1 into the Ipsilateral Paw Requires Efferent Conduction into the Contralateral Paw.

BACKGROUND: Contralateral hyperalgesia, occurring after unilateral injury, is usually explained by central sensitization in spinal cord and brain. We previously reported that injection of endothelin-1 (ET-1) into one rat hindpaw induces prolonged mechanical and chemical sensitization of the contralateral hindpaw. Here, we examined the role of contralateral efferent activity in this process. METHODS: ET-1 (2 nmol, 10 µL) was injected subcutaneously into the plantar surface of right (ipsilateral) hindpaw (ILP), and the thermal response latency and mechanical threshold for nocifensive withdrawal were determined by the use of, respectively, plantar radiant heating and von Frey filaments, for both ILP and contralateral hindpaws (CLP). Either paw was anesthetized for 60 minutes by direct injection of bupivacaine (0.25%, 40 µL), 30 minutes before ET-1. Alternatively, the contralateral sciatic nerve was blocked for 6 to 12 hours by percutaneous injection of bupivacaine-releasing microspheres 30 minutes before injection of ET-1. Systemic actions of these bupivacaine formulations were simulated by subcutaneous injection at the nuchal midline. RESULTS: After the injection of ET-1, the mechanical threshold of both ILP and CLP decreased by 2 hours, appeared to be lowest around 24 hours, and recovered through 48 hours to preinjection baseline at 72 hours. These hypersensitive responses were suppressed by bupivacaine injected into the ipsilateral paw before ET-1. Injection of the CLP by bupivacaine also suppressed the hypersensitivity of the CLP at all test times, and that of the ILP, except at 2 hours when it increased the sensitivity. This same pattern of change occurred when the contralateral sciatic nerve was blocked by bupivacaine-releasing microspheres. The systemic actions of these bupivacaine formulations were much smaller and only reached significance at 24 hours post-ET-1. Thermal hypersensitivity after ET-1 injection also occurred in both ILP and CLP and showed the same pattern in response to the 2 contralateral anesthetic procedures. CONCLUSIONS: These results show that efferent transmission through the contralateral innervation into the paw is necessary for contralateral sensitization by ET-1, suggesting that the release of substances by distal nerve endings is involved. The release of substances in the periphery is essential for contralateral sensitization by ET-1 and may also contribute to secondary hyperalgesia, occurring at loci distant from the primary injury, that occurs after surgery or nerve damage.

Cryoneurolysis with Injectable Ice Slurry Modulates Mechanical Skin Pain.

Cutaneous pain is a common symptom of skin disease, and available therapies are inadequate. We developed a neural selective and injectable method of cryoneurolysis with ice slurry, which leads to a long-lasting decrease in mechanical pain. The aim of this study is to determine whether slurry injection reduces cutaneous pain without inducing the side effects associated with conventional cryoneurolysis. Using the rat sciatic nerve, we examined the effects of slurry on nerve structure and function in comparison with the effects of a Food and Drug Administration‒approved cryoneurolysis device (Iovera). Coherent anti-Stokes Raman scattering microscopy and immunofluorescence staining were used to investigate histological effects on the sciatic nerve and on downstream cutaneous nerve fibers. Complete Freund's Adjuvant model of cutaneous pain was used to study the effect of the slurry on reducing pain. Structural changes in myelin induced by slurry were comparable with those induced by Iovera, which uses much colder temperatures. Compared with that of Iovera, the decrease in mechanical pain due to slurry was less profound but lasted longer without signs of dysesthesia. Slurry did not cause a reduction of epidermal nerve fibers or a change in thermal pain sensitivity. Slurry-treated rats showed reduced cutaneous mechanical pain in response to Complete Freund's Adjuvant. Slurry injection can be used to successfully reduce cutaneous pain without causing dysesthesia.

Fast-acting and injectable cryoneurolysis device.

Cryoneurolysis is an opioid-sparing therapy for long-lasting and reversible reduction of pain. We developed a nerve-selective method for cryoneurolysis by local injection of ice-slurry (- 5 to - 6 °C) that induced decrease in nocifensive response starting from about a week after treatment and lasting up to 8 weeks. In this study, we test the hypothesis that injection of colder slurry leads to faster onset of analgesia. Colder slurry (- 9ºC) was injected around the rat sciatic nerve to induce cryoneurolysis. Hematoxylin and Eosin (H&E) staining was used to examine histologic effects on surrounding tissues. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to study effects on myelin sheaths. Functional tests were used to assess changes in sensory and motor function in the treated hind paw. No inflammation or scarring was detected in surrounding skin and muscle tissues at day 7 post slurry injection. Functional tests showed rapid onset reduction in mechanical pain sensitivity starting from day 1 and lasting up to day 98. CARS imaging demonstrated disintegration of myelin sheaths post treatment followed by complete recovery of nerve structure by day 140. In this study we showed that colder slurry (- 9 °C) produces more rapid onset and longer duration of analgesia, while remaining nerve-selective.

Endothelin-B receptor activation triggers an endogenous analgesic cascade at sites of peripheral injury.

Endothelin-1 (ET-1) is a newly described pain mediator that is involved in the pathogenesis of pain states ranging from trauma to cancer. ET-1 is synthesized by keratinocytes in normal skin and is locally released after cutaneous injury. While it is able to trigger pain through its actions on endothelin-A (ET(A)) receptors of local nociceptors, it can coincidentally produce analgesia through endothelin-B (ET(B)) receptors. Here we map a new endogenous analgesic circuit, in which ET(B) receptor activation induces the release of beta-endorphin from keratinocytes and the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs, also named Kir-3) linked to opioid receptors on nociceptors. These results indicate the existence of an intrinsic feedback mechanism to control peripheral pain in skin, and establish keratinocytes as an ET(B) receptor-operated opioid pool.

Remarkably long-lasting tachyphylaxis of pain responses to ET-1: evidence against central nervous system involvement.

A profound tachyphylaxis of the acute nocifensive flinching (pain) response to subcutaneous injection of endothelin-1 (ET-1) into the hind paw footpad is shown by the reduced response to a second injection. Flinching from the second injection was 20% +/- 5%, 57% +/- 18%, 79% +/- 35%, and 100% +/- 17% of that from the first injection (both 200 micromol/L, 2 nmol) at respective intervals of 24, 30, 48, and 72 h. Inhibition of afferent impulses by local anesthesia of the sciatic nerve, reducing initial flinching to 6%-13% of control, did not affect the tachyphylaxis for the second injection at 24 h. There was no cross-desensitization between formalin and ET-1 injected sequentially into the same paw. Suppression of descending inhibitory effects from endogenous opiates by naloxone (5-8 mg/kg, i.p.), given 30 min before the second ET-1 injection, did not prevent tachyphylaxis. Diffuse effects caused by an initial subcutaneous ET-1 injection into the tail or forepaw resulted in sensitization of the response to ET-1 in the hind paw, rather than tachyphylaxis. In contrast, selective inhibition of local ETA receptors during the initial administration of ET-1, by the antagonist BQ-123 (3.2 mmol/L), reduced tachyphylaxis of nocifensive flinching. Therefore, prolonged pain tachyphylaxis is not due to reduced responsiveness of the CNS, but rather depends on the functional sensitivity or availability of peripheral ET(A) receptors.

The TrkA receptor mediates experimental thermal hyperalgesia produced by nerve growth factor: Modulation by the p75 neurotrophin receptor.

The p75 neurotrophin receptor (p75NTR) and its activation of the sphingomyelin signaling cascade are essential for mechanical hypersensitivity resulting from locally injected nerve growth factor (NGF). Here the roles of the same effectors, and of the tropomyosin receptor kinase A (TrkA) receptor, are evaluated for thermal hyperalgesia from NGF. Sensitivity of rat hind paw plantar skin to thermal stimulation after local sub-cutaneous injection of NGF (500ng) was measured by the latency for paw withdrawal (PWL) from a radiant heat source. PWL was reduced from baseline values at 0.5-22h by ∼40% from that in naïve or vehicle-injected rats, and recovered to pre-injection levels by 48h. Local pre-injection with a p75NTR blocking antibody did not affect the acute thermal hyperalgesia (0.5-3.5h) but hastened its recovery so that it had reversed to baseline by 22h. In addition, GW4869 (2mM), an inhibitor of the neutral sphingomyelinase (nSMase) that is an enzyme in the p75NTR pathway, also failed to prevent thermal hyperalgesia. However, C2-ceramide, an analog of the ceramide produced by sphingomyelinase, did cause thermal hyperalgesia. Injection of an anti-TrkA antibody known to promote dimerization and activation of that receptor, independent of NGF, also caused thermal hyperalgesia, and prevented the further reduction of PWL from subsequently injected NGF. A non-specific inhibitor of tropomyosin receptor kinases, K252a, prevented thermal hyperalgesia from NGF, but not that from the anti-TrkA antibody. These findings suggest that the TrkA receptor has a predominant role in thermal hypersensitivity induced by NGF, while p75NTR and its pathway intermediates serve a modulatory role.

Tactile allodynia initiated by local subcutaneous endothelin-1 is prolonged by activation of TRPV-1 receptors.

Subcutaneous endothelin-1 (ET-1; 200 microM, 2 nmoles/paw) injected into the rat hind paw, has been shown to cause robust hind paw flinching (HPF) and paw licking, and to induce impulses selectively in primary nociceptors. Here we report that a much lower [ET-1] sensitizes the paw to a nocifensive withdrawal response to tactile stimulation (by von Frey hairs, VFH), a sensitization that involves local TRPV1 receptors. Injection of 10 microM ET-1 (0.1 nmole/paw) causes only marginal HPF but rapidly (20 mins after injection) lowers the force threshold for paw withdrawal (PWT) to VFH, to approximately 30% of pre-injection baseline. Such tactile allodynia persists for 3 hrs. In rats pre-injected with the TRPV1-antagonists capsazepine (CPZ; 1.33 mM) or 5'-iodoresiniferatoxin (I-RTX; 0.13 microM), 15 min before ET-1, a fast initial drop in PWT, as with ET-1 alone, occurs (to 40% or to 19% of baseline, respectively), but this earliest reduction then regresses back to the pre-injection PWT value more rapidly than with ET-1 alone. The recovery of allodynia from the maximum value is about two times faster for ET-1+CPZ and about 4 times faster for ET-1+ I-RTX, compared with that from ET-1 +vehicle (t(1/2) = 130, 60, and 250 mins, respectively). In contrast, spontaneous pain indicated by overt HPF from ET-1 is not attenuated by TRPV1 antagonists. Tactile allodynia is similarly abbreviated by antagonists of both ET(A) (BQ-123, 32 nmoles/paw) and ET(B) (BQ-788, 30 nmoles/paw) receptors, whereas HPF is abolished by this ET(A) antagonist but enhanced by the ET(B) antagonist. We conclude that low ET-1 causes tactile allodynia, which is characterized by a different time-course and pharmacology than ET-1-induced nociception, and that local TRPV1 receptors are involved in the maintenance of this ET-1-induced allodynia but not in the overt algesic action of ET-1.

Local injection of a selective endothelin-B receptor agonist inhibits endothelin-1-induced pain-like behavior and excitation of nociceptors in a naloxone-sensitive manner.

We showed previously that subcutaneous injection of the injury-associated peptide mediator endothelin-1 (ET-1) into the rat plantar hindpaw produces pain behavior and selective excitation of nociceptors, both through activation of ET(A) receptors likely on nociceptive terminals. The potential role of ET(B) receptor activation in these actions of ET-1-has not been examined. Therefore, in these experiments, we studied the effect of blocking or activating ET(B) receptors on ET-1-induced hindpaw flinching and excitation of nociceptors in rats. An ET(B) receptor-selective antagonist, BQ-788 (3 mm), coinjected with ET-1 (200 microm) reduced the time-to-peak of flinching and significantly enhanced the average maximal flinch frequency (MFF). In contrast, coinjection of an ET(B) receptor selective agonist, IRL-1620 (100 or 200 microm), with ET-1 reduced the average MFF and the average total number of flinches. Interestingly, this unexpected inhibitory effect of IRL-1620 was prevented by the nonselective opioid receptor antagonist naloxone (2.75 mm). To confirm these inhibitory actions, we studied the effects of IRL-1620 on ET-1-induced spike responses in single, physiologically characterized nociceptive C-fibers. IRL-1620 suppressed spike responses to ET-1 in all (n = 12) C-units, with mean and maximum response frequencies of 0.08 +/- 0.02 and 1.5 +/- 0.4 impulses/sec versus 0.32 +/- 0.07 and 4.17 +/- 0.17 impulses/sec for ET-1 alone. In additional support of the behavioral results, coinjection of naloxone (2.75 mm) completely prevented this inhibitory action of IRL-1620. These results establish that ET(B) receptor activation inhibits ET-1-induced pain behavior and nociception in a naloxone-sensitive manner and point to a previously unrecognized dual modulation of acute nociceptive signaling by ET(A) and ET(B) receptors in cutaneous tissues.

Sensory fibers resistant to the actions of tetrodotoxin mediate nocifensive responses to local administration of endothelin-1 in rats.

Endothelin-1 (ET-1) applied to the sciatic nerve or injected into the plantar hindpaw of rats induces pain behavior (ipsilateral hindpaw flinching) and selective excitation of nociceptors by activation of endothelin-A (ET(A)) receptors. To determine the pharmacological profile of the sensory fibers that mediate this pain behavior, we administered lidocaine (LID, a non-selective conduction blocker) or tetrodotoxin (TTX) prior to ET-1. LID (1 or 2%, 0.1 ml) was injected percutaneously into the sciatic notch, or TTX (10 microM, 4 microl) was injected into the sciatic nerve prior to the more distal application of ET-1 (400 microM, 40 microl) onto the sciatic nerve or subcutaneously into the plantar hindpaw (400 microM, 10 microl). LID inhibited ET-1-induced flinching in a dose-dependent manner; the mean total number of flinches was reduced by 39% for 1% LID and by 87% for 2% LID. In contrast, TTX failed to inhibit flinching behavior induced by sciatic nerve application of ET-1 despite a similar magnitude of motor and sensory blockade as that observed with 2% LID. Partial blockade of flinching behavior by intraneural TTX (mean total flinches were reduced by 51%) was observed after subcutaneous injection of ET-1. Unexpectedly, ET-1 prolonged the actions of 1% LID and, even when applied alone, produced clear signs of motor and sensory conduction block. These results are evidence that ET-1-induced pain is transmitted to the central nervous system via sensory fibers using tetrodotoxin-resistant sodium channels, and that ET-1 has analgesic actions that exist despite the activation of local pain pathways.

Therapeutic concentrations of local anaesthetics unveil the potential role of sodium channels in neuropathic pain.

Neuropathic pain is frequently associated with hyperexcitability of primary afferents, characterized by spontaneous impulses and repetitive firing. Electrophysiology and molecular biology reveal changes in dorsal root ganglion Na+ channels under conditions of neuropathic pain, but the manner by which these changes alter the physiology of sensory afferents remains unknown. Equally mysterious is the mechanism by which i.v. local anaesthetic-like Na+ channel blockers suppress neuropathic pain behaviour at concentrations well below those reported for channel inhibition. We have compared the anti-allodynic actions of i.v. lidocaine (L) and stereoisomers of mexiletine (R-M, S-M), in rats after spinal nerve ligation, with their ability: (1) to inhibit fast, tetrodotoxin-sensitive neuronal Na+ currents, elicited by brief (1 ms) pulses, at 10 Hz, from 'resting' potentials (-80, -60 mV) and (2) to suppress the seconds long plateau and the repetitive firing produced in axons by slowing of Na+ channel inactivation (e.g. using scorpion alpha-toxins). Both L and R-M at 5-10 microM relieved allodynia; S-M was ineffective. Na+ currents also were inhibited by M, with affinities that were increased by both repetitive 'firing' (K(R,S) = 5 microM) and depolarization of the 'resting' membrane (K(R) = 15 microM; K(S) = 30 microM). Stereopotency ratios depended on the manner in which different states of the channel were inducted. Both L and M shortened the action potential's 'plateau' in alpha-toxin treated axons, without reducing the spike, and suppressed repetitive firing with IC(50)s = 5 microM, and no stereoselectivity. These findings together demonstrate that Na+ channel blockers, at 'therapeutic' concentrations, can inhibit neuronal hyperexcitability.

Sensitization of cutaneous neuronal purinergic receptors contributes to endothelin-1-induced mechanical hypersensitivity.

Endothelin (ET-1), an endogenous peptide with a prominent role in cutaneous pain, causes mechanical hypersensitivity in the rat hind paw, partly through mechanisms involving local release of algogenic molecules in the skin. The present study investigated involvement of cutaneous ATP, which contributes to pain in numerous animal models. Pre-exposure of ND7/104 immortalized sensory neurons to ET-1 (30nM) for 10min increased the proportion of cells responding to ATP (2µM) with an increase in intracellular calcium, an effect prevented by the ETA receptor-selective antagonist BQ-123. ET-1 (3nM) pre-exposure also increased the proportion of isolated mouse dorsal root ganglion neurons responding to ATP (0.2-0.4µM). Blocking ET-1-evoked increases in intracellular calcium with the IP3 receptor antagonist 2-APB did not inhibit sensitization to ATP, indicating a mechanism independent of ET-1-mediated intracellular calcium increases. ET-1-sensitized ATP calcium responses were largely abolished in the absence of extracellular calcium, implicating ionotropic P2X receptors. Experiments using quantitative polymerase chain reaction and receptor-selective ligands in ND7/104 showed that ET-1-induced sensitization most likely involves the P2X4 receptor subtype. ET-1-sensitized calcium responses to ATP were strongly inhibited by broad-spectrum (TNP-ATP) and P2X4-selective (5-BDBD) antagonists, but not antagonists for other P2X subtypes. TNP-ATP and 5-BDBD also significantly inhibited ET-1-induced mechanical sensitization in the rat hind paw, supporting a role for purinergic receptor sensitization in vivo. These data provide evidence that mechanical hypersensitivity caused by cutaneous ET-1 involves an increase in the neuronal sensitivity to ATP in the skin, possibly due to sensitization of P2X4 receptors.

Endothelin receptors and pain.

UNLABELLED: The endogenous endothelin (ET) peptides participate in a remarkable variety of pain-relatedprocesses. Pain that is elevated by inflammation, by skin incision, by cancer, during a Sickle Cell Disease crisis and by treatments that mimic neuropathic and inflammatory pain and are all reduced by local administration of antagonists of endothelin receptors. Many effects of endogenously released endothelin are simulated by acute, local subcutaneous administration of endothelin, which at very high concentrations causes pain and at lower concentrations sensitizes the nocifensive reactions to mechanical, thermal and chemical stimuli. PERSPECTIVE: In this paper we review the biochemistry, second messenger pathways and hetero-receptor coupling that are activated by ET receptors, the cellular physiological responses to ET receptor activation, and the contribution to pain of such mechanisms occurring in the periphery and the CNS. Our goal is to frame the subject of endothelin and pain for a broad readership, and to present the generally accepted as well as the disputed concepts, including important unanswered questions.

Early and late contributions of glutamate and CGRP to mechanical sensitization by endothelin-1.

UNLABELLED: Intraplantar injection of endothelin-1 (ET-1) (1.5-10 muM) in the rat produces mechanical allodynia. Here we identify the receptor subtypes for ET-1, glutamate and CGRP critical to such allodynia. Antagonism of ET(A) or ET(B) receptors alone, by BQ123 or BQ788, respectively, only partially suppressed allodynia; the combined antagonists prevented allodynia, showing the involvement of both receptor subtypes. Co-injection of NMDA receptor antagonists, (+)MK-801 or D-AP5, with ET-1 also prevented allodynia. In contrast, co-injection of the CGRP1 antagonist CGRP(8-37) attenuated only the later phase of allodynia (>30 min). A mechanistic basis for these effects is shown by ET-1's ability to enhance basal release from cultured sensory neurons of glutamate and CGRP (2.4-fold and 5.7-fold, respectively, for 10 nM ET-1). ET(A) blockade reduced ET-1's enhancement of basal CGRP release by approximately 80%, but basal glutamate release by only approximately 30%. ET-1 also enhanced the capsaicin-stimulated release of CGRP (up to 2-fold for 0.3 nM ET-1), but did not change capsaicin-stimulated glutamate release. Release stimulated by elevated K+ was not altered by ET(A) blockade, nor did blockade of ET(B) reduce any type of release. Thus, ET-1 may induce release of glutamate and CGRP from nerve terminals innervating skin, thereby sensitizing primary afferents, accounting for ET-1-dependent tactile allodynia. PERSPECTIVE: The endogenous endothelin peptides participate in a remarkable variety of pain-related processes. The present results provide evidence for the participation of ionotropic glutamatergic receptors and CGRP receptors in the hyperalgesic responses to exogenous ET-1 and suggest clinically relevant targets for further study of elevated pain caused by release of endogenous ET-1.

Dual Roles for Endothelin-B Receptors in Modulating Adjuvant-Induced Inflammatory Hyperalgesia in Rats.

Injection of endothelin-1 (ET-1) into the plantar rat hindpaw causes acute pain at high concentrations and tactile sensitization at low concentrations. The pro-nociceptive actions are driven through ET(A) receptors for both levels of [ET-1], but the ET(B) receptors are only pro-nociceptive for allodynia from low [ET-1] and anti-nociceptive for pain from high [ET-1]. The goal of the present work was to discriminate the roles of the ET receptors in the acute hyperalgesia from inflammation by complete Freund's adjuvant (CFA, 20 mg/paw) into the rat hindpaw. Selective antagonists were injected l0 min before and then together with CFA. An ET(A) receptor antagonist, BQ-123, reduced CFA-induced thermal hyperalgesia (by up to 50%), as did an ET(B) receptor antagonist, BQ-788 (by up to 66%). BQ-123 and BQ-788 also delayed the onset (by 1.5 - 2 h) but insignificantly reduced the maximum degree of CFA-induced allodynia (~10%). Surprisingly, an ET(B) receptor agonist, IRL-1620, also reduced maximum thermal hyperalgesia induced by CFA, suppressed peak allodynia and delayed its occurrence by ~ 3 h. The latter actions of IRL-1620 were reversed by co-administration of BQ-788, naloxone hydrochloride and the peripherally restricted opiate receptor antagonist naloxone methiodide, and by antiserum against ß-endorphin. These findings demonstrate an important role for endogenous ET-1 in acute inflammatory pain and a dual action of ET(B) receptors, including a pro-algesic action along with the important activation of a local analgesic pathway, implying that at least two different ET(B) receptors contribute to modulation of inflammatory pain.

Local antinociception induced by endothelin-1 in the hairy skin of the rat's back.

UNLABELLED: Subcutaneous injection of endothelin-1 (ET-1) into the glabrous skin of the rat's hind paw is known to produce impulses in nociceptors and acute nocifensive behavioral responses, such as hind paw flinching, and to sensitize the skin to mechanical and thermal stimulation. In this report, we show that in contrast to the responses in glabrous skin, ET-1 injected subcutaneously into rat hairy skin causes transient antinociception. Concentrations of 1 to 50 microM ET-1 (in 0.05 mL) depress the local nocifensive response to noxious tactile probing at the injection site with von Frey filaments for 30 to 180 minutes; distant injections have no effect at this site, showing that the response is local. Selective inhibition of ET(A) but not of ET(B) receptors inhibits this antinociception, as does coinjection with nimodipine (40 muM), a blocker of L-type Ca(2+) channels. Local subcutaneous injection of epinephrine (45 microM) also causes antinociception through alpha-1 adrenoreceptors, but such receptors are not involved in the ET-1-induced effect. Both epinephrine and ET-1, at antinociceptive concentrations, reduce blood flow in the skin; the effect from ET-1 is largely prevented by subcutaneous nimodipine. These data suggest that ET-1-induced antinociception in the hairy skin of the rat involves cutaneous vasoconstriction, presumably through neural ischemia, resulting in conduction block. PERSPECTIVE: The pain-inducing effects of ET-1 have been well documented in glabrous skin of the rat, a frequently used test site. The opposite behavioral effect, antinociception, occurs from ET-1 in hairy skin and is correlated with a reduction in blood flow. Vasoactive effects are important in assessing mechanisms of peripherally acting agents.

CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids.

CB(2) cannabinoid receptor-selective agonists are promising candidates for the treatment of pain. CB(2) receptor activation inhibits acute, inflammatory, and neuropathic pain responses but does not cause central nervous system (CNS) effects, consistent with the lack of CB(2) receptors in the normal CNS. To date, there has been virtually no information regarding the mechanism of CB(2) receptor-mediated inhibition of pain responses. Here, we test the hypothesis that CB(2) receptor activation stimulates release from keratinocytes of the endogenous opioid beta-endorphin, which then acts at opioid receptors on primary afferent neurons to inhibit nociception. The antinociceptive effects of the CB(2) receptor-selective agonist AM1241 were prevented in rats when naloxone or antiserum to beta-endorphin was injected in the hindpaw where the noxious thermal stimulus was applied, suggesting that beta-endorphin is necessary for CB(2) receptor-mediated antinociception. Further, AM1241 did not inhibit nociception in mu-opioid receptor-deficient mice. Hindpaw injection of beta-endorphin was sufficient to produce antinociception. AM1241 stimulated beta-endorphin release from rat skin tissue and from cultured human keratinocytes. This stimulation was prevented by AM630, a CB(2) cannabinoid receptor-selective antagonist and was not observed in skin from CB(2) cannabinoid receptor-deficient mice. These data suggest that CB(2) receptor activation stimulates release from keratinocytes of beta-endorphin, which acts at local neuronal mu-opioid receptors to inhibit nociception. Supporting this possibility, CB(2) immunolabeling was detected on beta-endorphin-containing keratinocytes in stratum granulosum throughout the epidermis of the hindpaw. This mechanism allows for the local release of beta-endorphin, where CB(2) receptors are present, leading to anatomical specificity of opioid effects.