Luis Menéndez

Unilateral hot plate test: a simple and sensitive method for detecting central and peripheral hyperalgesia in mice.

The application of thermal noxious stimuli forms the basis of some widely used tests to detect either hyperalgesic or analgesic reactions. In the classical hot plate test, mice react by licking their paws and/or jumping. However, tests relying on the unilateral application of thermal radiant heat to the plantar side of the hindpaw, have become popular in recent years since unilateral changes in nociceptive sensitivity can be detected. Based on the aforementioned tests, we developed a testing procedure in mice, the unilateral hot plate (UHP): the plantar side of one hindpaw is placed on a hot plate surface and, thus, the withdrawal latency of each paw can be measured separately. The effectiveness of several analgesic and hyperalgesic drugs measured by the UHP was compared with that measured by a method based on the application of radiant heat (RH) stimuli. In the UHP method, morphine (1-10 mg/kg) increases latencies, while spinal NMDA (0.001-1 ng) or PGE2 (30-300 ng), intraplantar carrageenan (2 - 4%) or PGE2 (30-300 ng) decrease latencies. In all cases, the UHP method detected changes in pain reactivity at lower doses than the RH test. The sensitivity and usefulness of the UHP test for performing pain studies in mice is described.

Initial thermal heat hypoalgesia and delayed hyperalgesia in a murine model of bone cancer pain.

The recent development of rodent models of bone cancer pain has started to provide the basis for demonstrating the particular neurochemical and behavioral entity of cancer pain. Behaviourally, both spontaneous pain and hyperalgesia related to mechanical, but not thermal, noxious stimuli have been described in cancer-bearing animals. We have carried out a histological and behavioural study focused on the reactivity to noxious heat in C3H/HeJ mice receiving an intratibial injection of 10(5) NCTC 2472 cells. These cells, able to induce an osteosarcoma, break through bone into soft tissues 2 weeks after cell inoculation, producing a macroscopical increase of the limb size from the fourth week. Thermal reactivity is diminished during the first 2 weeks after cell implantation, this hypoalgesia being reversed by the administration of naloxone (10 mg/kg). In contrast, during the fourth and fifth weeks after NCTC 2472 cell implantation, an increased nociceptive heat reactivity, instead of hypoalgesia, was obtained. This thermal hyperalgesia was prevented by the systemic administration of morphine (15 mg/kg). Throughout the whole period studied, mice showed signs of spontaneous pain behaviour that reached its maximum 3 weeks after inoculation. In conclusion, we show that the presence of thermal heat hyperalgesia is preceded by an initial opioid-mediated hypoalgesic state, in this murine model of bone cancer pain.

Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain

Background and purpose:  The activation of CB2 receptors induces analgesia in experimental models of chronic pain. The present experiments were designed to study whether the activation of peripheral or spinal CB2 receptors relieves thermal hyperalgesia and mechanical allodynia in two models of bone cancer pain.

Analgesic effects of capsazepine and resiniferatoxin on bone cancer pain in mice

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 Freunds 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.

Metalloproteinase MT5-MMP is an essential modulator of neuro-immune interactions in thermal pain stimulation

Peripheral interactions between nociceptive fibers and mast cells contribute to inflammatory pain, but little is known about mechanisms mediating neuro-immune communication. Here we show that metalloproteinase MT5-MMP (MMP-24) is an essential mediator of peripheral thermal nociception and inflammatory hyperalgesia. We report that MT5-MMP is expressed by CGRP-containing peptidergic nociceptors in dorsal root ganglia and that Mmp24-deficient mice display enhanced sensitivity to noxious thermal stimuli under basal conditions. Consistently, mutant peptidergic sensory neurons hyperinnervate the skin, a phenotype that correlates with changes in the regulated cleavage of the cell-cell adhesion molecule N-cadherin. In contrast to basal nociception, Mmp24−/− mice do not develop thermal hyperalgesia during inflammation, a phenotype that appears associated with alterations in N-cadherin-mediated cell-cell interactions between mast cells and sensory fibers. Collectively, our findings demonstrate an essential role of MT5-MMP in the development of dermal neuro-immune synapses and suggest that this metalloproteinase may be a target for pain control.

Analgesic effects of loperamide in bone cancer pain in mice

The intratibial inoculation of NCTC 2472 cells induces an osteosarcoma in C3H/HeJ mice. These mice show thermal hyperalgesic responses which may be blocked by the local administration of opiates over the tibial tumoral mass (Menéndez L, Lastra A, Hidalgo A, Meana A, Garcia E, Baamonde A. Peripheral opioids act as analgesics in bone cancer pain in mice. NeuroReport 2003b; 14:867-9). The aim of this report was to characterize the analgesic responses obtained by activating peripheral opioid receptors in bone cancer pain. Here, we initially describe that this osteosarcoma induces mechanical as well as thermal hyperalgesia. Loperamide, an opioid agonist unable to cross the blood-brain barrier, inhibits both thermal and mechanical hyperalgesia when s.c. injected, locally over the tibial tumoral mass (7.5-75 microg) or distantly, under the fur of the neck (4 mg/kg). These analgesic effects seem peripherally mediated since they are reverted by the administration of naloxone methiodide (10 mg/kg) and because the withdrawal latencies of the contralateral, non-affected, paws remain unaltered. Furthermore, only cyprodime (1 mg/kg) but not naltrindole (0.1 mg/kg) or nor-binaltorphimine (10 mg/kg) blocked these effects, showing the involvement of gamma-opioid receptors in the peripheral analgesia induced by loperamide on thermal and mechanical hyperalgesia. The advantages of using peripheral acting opiates -- devoid of central colateral effects -- for the treatment of cancer related pain are suggested.

Spinal CCL2 and microglial activation are involved in paclitaxel-evoked cold hyperalgesia.

The antineoplastic paclitaxel induces a sensory neuropathy that involves the spinal release of neuroinflammatory mediators and activation of glial cells. Although the chemokine CCL2 can evoke glial activation and its participation in neuropathic pain has been demonstrated in other models, its involvement in paclitaxel-evoked neuropathy has not been previously explored. Paclitaxel-evoked cold hypernociception was assessed in mice by the unilateral cold plate test and the effects on cold hyperalgesia of the CCR2 antagonist RS 504393, the CCR1 antagonist J113863, the microglial inhibitor minocycline or an anti-CCL2 antibody were tested. Furthermore, ELISA measurements of CCL2 concentration and immunohistochemical assays of Iba-1 and GFAP, markers of microglial and astroglial cells respectively, were performed in the lumbar spinal cord. Cold hypernociception measured 3 days after the administration of paclitaxel (10mg/kg) was inhibited by the s.c. (0.3-3mg/kg) or i.t. (1-10 μg) administration of RS 504393 but not of J113863 (3-30 mg/kg). CCL2 levels measured by ELISA in the lumbar spinal cord were augmented in mice treated with paclitaxel and the i.t. administration of an anti-CCL2 antibody completely suppressed paclitaxel-evoked cold hyperalgesia, strongly suggesting that CCL2 is involved in the hypernociception evoked by this taxane. Besides, the implication of microglial activation is supported by the increase in the immunolabelling of Iba-1, but not GFAP, in the spinal cord of paclitaxel-treated mice and by the inhibition of cold hyperalgesia produced by the i.t. administration of the microglial inhibitor minocycline (1-10 nmol). Finally, the neutralization of spinal CCL2 by the i.t. administration of a selective antibody for 3 days almost totally inhibited paclitaxel-evoked microglial activation. In conclusion, our results indicate that paclitaxel-evoked cold hypernociception depends on the activation of CCR2 due to the spinal release of CCL2 and the subsequent microglial activation.

Involvement of spinal κ opioid receptors in a type of footshock induced analgesia in mice

We have studied the effects of several opioid antagonists on a type of footshock stress-induced analgesia (FSIA) measured by the tail-flick test in male mice. Naloxone injected either subcutaneously (0.1-10 mg/kg) or intrathecally (1-20 micrograms) antagonized FSIA at higher doses than those that blocked a similar degree of analgesia induced by morphine. Intracerebroventricular (i.c.v.) naloxone (1-20 micrograms) did not modify the FSIA while antagonizing the i.c.v. morphine-induced analgesia. As a consequence, the antagonism of the FSIA by naloxone probably occurs at the level of the spinal cord and through receptors different than mu. The delta selective antagonist naltrindole (0.1-3 mg/kg s.c.) did not antagonize the analgesic effects of the stress. Nor-binaltorphimine, a kappa selective antagonist, blocked the FSIA when administered systemically (1-4 mg/kg i.p.) or locally (0.1-1 microgram i.t.). These results strongly suggest that spinal kappa opioid receptors are responsible for this type of endogenous analgesia.

Endogenous β-endorphin induces thermal analgesia at the initial stages of a murine osteosarcoma

Transient thermal, but not mechanical, hypoalgesia appears at the early stages of the development of an hyperalgesic murine osteosarcoma. This hypoalgesia is suppressed by the administration of naloxone, its peripherally acting analog naloxone methiodide, the mu- and delta-opioid receptor antagonists cyprodime and naltrindole, or the CRF receptor antagonist, alpha-helical CRF (9-41). When immunohistochemical assays were performed with an anti-beta-endorphin antibody, whose in vivo administration suppressed the analgesia, labeled mononuclear immune cells appeared both inside and surrounding the tumoral tissue. In conclusion, the peripheral action of beta-endorphin, released in response to the osteosarcoma seems responsible for the observed thermal analgesia.

Involvement of enkephalins in the inhibition of osteosarcoma-induced thermal hyperalgesia evoked by the blockade of peripheral P2X3 receptors

Although previous studies describe the up-regulation of purinergic P2X(3) receptors expressed at peripheral nociceptive fibers in experimental painful neoplastic processes, the analgesic efficacy of P2X(3) receptor antagonists has not been tested in these settings. We study here the effect of the P2X(3) receptor antagonist, A-317491, on thermal hyperalgesia produced by the intratibial inoculation of NCTC 2472 fibrosarcoma cells to C3H/HeJ mice. The peritumoral administration of A-317491 (10-100 microg) dose-dependently attenuated osteosarcoma-induced thermal hyperalgesia without modifying thermal latencies measured in the contralateral paws. This antihyperalgesic effect was inhibited by the coadministration of naloxone-methiodide (0.1-1 microg) or the systemic injection of the selective mu-opioid receptor antagonist cyprodime (1 mg/kg), demonstrating the involvement of peripheral mu-opioid receptors. Furthermore, the antihyperalgesic effect induced by A-317491, was antagonised by the coadministration of an anti-enkephalin antibody supporting the participation of endogenous enkephalins. Consistent with this result, the antihyperalgesic effect induced by A-317491 was dramatically enhanced by the administration of an enkephalin-degrading inhibitor, Debio 0827, as demonstrated by isobolographic analysis. This synergism opens the theoretical possibility that the combination of both types of drugs could be useful to counteract some nociceptive symptoms derived from tumor development.