Ana Lastra

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.

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.

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.

TRPV1 desensitisation and endogenous vanilloid involvement in the enhanced analgesia induced by capsaicin in inflamed tissues

The intra-plantar acute administration of 10 microg of capsaicin to mice which had received complete Freunds adjuvant (CFA) 1 week before inhibits the thermal inflammatory hyperalgesia it induces and even produces a long-lasting analgesia for at least 2 weeks. In this study, we show that the administration of capsaicin (10 microg) also reduces the immediate licking behavior evoked by the intra-plantar administration of a lower dose of capsaicin (0.1 microg), the duration of this inhibitory effect being greater in CFA-inflamed mice (at least 2 weeks) than in non-inflamed animals (less than 4 days). Since this reduction of capsaicin-induced licking behavior may be interpreted as a consequence of the transient receptor potential vanilloid 1 receptor (TRPV1) unresponsiveness, we conclude that the administration of 10 microg of capsaicin into inflamed tissues can render the TRPV1 desensitised. We next explored whether endogenous vanilloids released during inflammation contribute to maintain the analgesia triggered by exogenous capsaicin. The acute administration of capsazepine (10 microg; intra-plantarly (i.pl.)) abolished the analgesic effect induced by the injection of capsaicin 1 week before in inflamed mice. From these results, it may be proposed that the maintenance by endovanilloids of the TRPV1 desensitisation induced by capsaicin could contribute to prolonging the analgesic effect induced by this agonist in inflamed tissues.

Implantation of Tumoral XC Cells Induces Chronic, Endothelin-Dependent, Thermal Hyperalgesia in Mice

Abstract1. We describe here the alterations in the nociceptive sensitivity of Swiss CD1 mice receiving an intraplantar (i.pl.) administration of XC Rous sarcoma-virus-transformed rat fibroblasts (XC cells).2. Histological studies reveal that XC cells remain at the injection site 2–3 weeks after implantation, a time at which an inflammatory reaction is also detected. No tumoral growth was found and 5 weeks after inoculation neither XC cells nor inflammatory reaction were observed.3. Measures to different types of noxious stimuli were performed. At week 1 after XC cell inoculation, hyperalgesia to thermal, but not mechanical, stimuli as well as to capsaicin injection is present in the implanted paw. At week 5 after XC cell implantation, only thermal hyperalgesia is present, and this enhanced reactivity persisted for even 25 weeks after the disappearance of XC tumoral cells.4. Pharmacological studies on thermal hyperalgesia were conducted at two different stages, week 1 and week 5 after XC cell inoculation. The systemic administration of morphine (1–10 mg/kg i.p. (intraperitoneal); 30 min before testing) prevents this thermal hyperalgesic reaction both at week 1 and week 5. The endothelin type A (ETA) receptor antagonist BQ-123 (10 nmol; i.pl.; 90 min before testing) abolishes both the early (week 1) and the late (week 5) thermal hyperalgesia. In contrast, the selective endothelin type B (ETB) receptor antagonist, BQ-788 (10 nmol; i.pl.; 90 min before) abolishes thermal hyperalgesia only at week 1, but not at week 5 after XC cell inoculation.5. It might be concluded that endothelins are probably involved in this type of long-term thermal hyperalgesia produced by the transitory presence of the XC tumoral cell line.

The chemokine CCL5 induces CCR1-mediated hyperalgesia in mice inoculated with NCTC 2472 tumoral cells.

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10-30μg) but not spinal (3-5μg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863. The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium. The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

CCL2 released at tumoral level contributes to the hyperalgesia evoked by intratibial inoculation of NCTC 2472 but not B16-F10 cells in mice

The participation of the chemokine CCL2 (monocyte chemoattractant protein-1) in inflammatory and neuropathic pain is well established. Furthermore, the release of CCL2 from a NCTC 2472 cells-evoked tumor and its involvement in the upregulation of calcium channel α2δ1 subunit of nociceptors was demonstrated. In the present experiments, we have tried to determine whether the increase in CCL2 levels is a common property of painful tumors and, in consequence, the administration of a chemokine receptor type 2 (CCR2) antagonist can inhibit tumoral hypernociception. CCL2 levels were measured by ELISA in the tumoral region of mice intratibially inoculated with NCTC 2472 or B16-F10 cells, and the antihyperalgesic and antiallodynic effects evoked by the administration of the selective CCR2 antagonist RS 504393 were assessed. Cultured NCTC 2472 cells release CCL2 and their intratibial inoculation evokes the development of a tumor in which CCL2 levels are increased. Moreover, the systemic or peritumoral administration of RS 504393 inhibited thermal and mechanical hyperalgesia, but not mechanical allodynia evoked after the inoculation of these cells. Thermal hyperalgesia was also inhibited by the peritumoral administration of a neutralizing CCL2 antibody. In contrast, no change in CCL2 levels was observed in mice inoculated with B16-F10 cells, and RS 504393 did not inhibit the hypernociceptive reactions evoked by their intratibial inoculation. The peripheral release of CCL2 is involved in the development of thermal and mechanical hyperalgesia, but not mechanical allodynia evoked by the inoculation of NCTC 2472 cells, whereas this chemokine seems unrelated to the hypernociception induced by B16-F10 cells.