Agustín Hidalgo

Sex-related differences in the effects of morphine and stress on visceral pain

The effect of morphine (in the absence of stress and after a type of naloxone-sensitive swim-stress) on the painful contractions induced by intraperitoneal injection of hypertonic saline was studied in four groups of rats: normally cycling females in oestrus, males, ovariectomized females and females subjected to constant light, to induce a hyperoestrogenemic state. In the absence of swim-stress, the sensitivity to morphine was maximum for males (i.e. analgesia was produced with the smallest doses). After swim-stress, both males and ovariectomized rats showed less sensitivity to the analgesic effect of morphine than normal females in oestrus, while females subjected to constant light were less sensitive. In females in natural oestrus, but not in the other groups, small doses (0.05 mg/kg) of morphine, paradoxically reduced the analgesic effect of swim-stress considerably. These results show the importance of sex-related factors in the sensitivity of visceral pain to morphine and stress. A selective role for endogenous androgens and oestrogens is likely.

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.

Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl2.

1. The effects of the flavonoids genistein (3-60 microM), kaempferol (3-60 microM) and quercetin (1-100 microM) on KCl (60 mM)-induced tonic contraction in rat uterus and their modifications with the inhibitor of cAMP-dependent protein kinases (TPCK, 3 microM), the inhibitor of ornithine decarboxylase [alpha-difluoromethyl ornithine (DFMO), 10 mM] and the polyamine spermine (1 mM) have been assayed. The effects of the three flavonoids were also studied on the contraction elicited by CaCl2 (30 microM to 10 mM) on rat uterus incubated in medium lacking calcium and supplemented with 33, 60 or 90 mM of KCl. For comparison, the effects of the calcium channel blockers nifedipine and verapamil and the activator of adenylyl cyclase forskolin were assayed on contractions induced by KCl and CaCl2. 2. Genistein (IC50: 20.2 +/- 1.0 microM, n = 11), kaempferol (IC50: 10.1 +/- 0.8 microM, n = 8) and quercetin (IC50: 13.2 +/- 0.5 microM, n = 8) relaxed the tonic contraction induced by KCl (60 mM) in a concentration-dependent way. Verapamil (IC50: 70.1 +/- 5.8 nM, n = 7), nifedipine (IC50: 8.4 +/- 0.7 nM, n = 6) and forskolin (IC50: 0.62 +/- 0.08 microM, n = 14) also relaxed the KCl-induced contraction. TPCK (3 microM) significantly antagonized the effect of quercetin, kaempferol and forskolin (P < 0.01) but did not modify the effect of genistein. 3. Spermine (1 mM) increased the effects of genistein and verapamil and antagonized the effect of quercetin but did not modify those of kaempferol and forskolin. DFMO (10 mM) did not modify the effect of quercetin but increased that of genistein and antagonized those of kaempferol and forskolin. The addition of spermine (1 mM) plus DFMO (10 mM) antagonized the effect of quercetin. Spermine counteracted the effect of DFMO on forskolin but not on genistein. 4. KCl (33, 60 or 90 mM) did not produce contraction in calcium-free solution, but CaCl2 (30 microM to 10 mM) induced concentration-dependent contraction after depolarizing with KCl. The EC50 values for CaCl2 were: 0.74 +/- 0.08 (n = 12), 0.34 +/- 0.03 (n = 14) and 0.48 +/- 0.02 (n = 12) mM in a medium with 33, 60 or 90 mM of KCl, respectively. 5. Genistein (20 microM), kaempferol (10 microM), quercetin (15 microM), verapamil (70 nM), nifedipine (10 nM) and forskolin (0.5 microM) inhibited the concentration-response curve to CaCl2 in medium supplemented with 33, 60 or 90 mM of KCl. The effect of kaempferol was independent of the concentration of KCl in the incubation medium. However, the inhibitory effect of genistein on CaCl2-induced contraction was inversely related to the concentration of KCl in the medium. On the contrary, the effect of quercetin was directly related to the concentration of KCl in the medium. 6. The antagonism of verapamil, nifedipine and forskolin on CaCl2-induced contraction seems to be related to the degree of depolarization because increasing the KCl in the medium counteracted their effects. 7. Our results suggest that (1) cAMP contributes to the relaxant effects of quercetin and kaempferol on KCl (60 mM)-induced tonic contraction; (2) polyamines are involved in the relaxant effects of forskolin and kaempferol on KCl-induced tonic contraction but not on CaCl2-induced contraction in the depolarized uterus, and (3) the flavonoids assayed also possess a calcium antagonist action but show a different behavior toward the calcium channel blockers and the cAMP enhancer forskolin.

A Role for Tachykinins in Female Mouse and Rat Reproductive Function

Abstract Tachykinins may be involved in reproduction. A reverse transcription-polymerase chain reaction assay was used to analyze the expression of tachykinins and tachykinin receptors in different types of reproductive cells from mice. The preprotachykinin (PPT) genes, PPT-A, PPT-B and PPT-C, that encode substance P/neurokinin A, neurokinin B, and hemokinin-1, respectively, and the genes that encode the tachykinin NK1, NK2, and NK3 receptors were all expressed, at different levels, in the uterus of superovulated, unfertilized mice. The mRNA of neprilysin (NEP), the main enzyme involved in tachykinin metabolism, was also expressed in the uterus. Isolated cumulus granulosa cells expressed PPT-A, PPT-B, PPT-C, and NEP and low levels of the tachykinin NK1 and NK2 receptors. Mouse oocytes expressed PPT-A and -B mRNA transcripts. A low expression of the three tachykinin receptors was observed but PPT-C and NEP were undetectable. Two- and 8- to 16-cell mouse embryos expressed only a low-abundance transcript corresponding to the NK1 receptor. However, the mRNAs of PPT-B, PPT-C and NEP appeared in blastocyst-stage embryos. A low-abundance transcript corresponding to the NK2 receptor was the only target gene detected in mice sperm. Female mice or rats treated neonatally with capsaicin showed a reduced fertility. A reduction in litter size was observed in female rats treated in vivo with the tachykinin NK3 receptor antagonist SR 142801. These data show that tachykinins of both neuronal and nonneuronal origin are differentially expressed in various types of reproductive cells and may play a role in female reproductive function.

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.

Hepatotoxicity Induced by Antiandrogens: A Review of the Literature

Introduction: Hepatotoxicity is a serious adverse reaction potentially induced by all antiandrogens. We have reviewed here the published cases of hepatotoxicity induced by steroidal and nonsteroidal antiandrogens, and compared the type and characteristics of liver damage. Methods: Using two different databases: MEDLINE and IDIS (Iowa Drug Information Service), we searched for published cases of liver injury induced by antiandrogens. Analysis was made of the type of hepatotoxicity, therapeutic indication, other pharmacological treatments and evolution. Mean values of latency and recovery periods of the adverse reactions and liver function tests were also evaluated. Results: Hepatitis was the most common type of hepatotoxicity reported, and was associated with all antiandrogens. This adverse reaction does not seem to be dependent on the patients age, therapeutic indication or the dose prescribed. Hepatitis showed a longer latency period for cyproterone acetate than for flutamide. Some transaminase levels were significantly higher for flutamide than for cyproterone acetate, although the evolution was no worse in the cases reported for flutamide. We also found occasional reports of hepatocellular carcinoma and hepatic cirrhosis suspected of being induced by cyproterone acetate. Conclusion: Although there are differences in the clinical features of hepatotoxicity induced by steroidal and nonsteroidal antiandrogens, these do not predict which patients will develop hepatotoxicity during treatment or evolution. Serial liver function tests are required for early detection of liver damage.

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.

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.