Experiences and reflections about behavioral pain assays in laboratory animals.

Pharmacological assays based on the measurement of nociceptive responses in laboratory animals are a fundamental tool to assess analgesic strategies. During our experience with this type of experiments, we have been repeatedly challenged by different concerns related to their interpretation or relevance. Although these subjects are frequently discussed in our lab, they do not usually find a place in research articles with original data, in which the focus on results seems mandatory. In the present manuscript we try to discuss as central issues some of these aspects that often cross transversally our research. We have gathered them in five topics inspired by the results obtained in our laboratory. The two initial sections are devoted to the influence of the behavioral method used to assess nociception on the results achieved, as well as to the possibility that data may be more easily accepted when obtained with standard methods than with alternative ones. The third topic is related to the difficulties encountered when working with a molecule that may evoke dual effects, acting as pronociceptive or antinociceptive depending on the dose. The fourth point deals with the situation in which a particular hyperalgesic reaction is related to several molecules but the single inhibition of only one of them can completely prevent it. Finally, the last issue is addressed to comment the impact in the progress of pain research of experiments performed in animal models of pathological settings.

Involvement of CD4+ and CD8+ T-lymphocytes in the modulation of nociceptive processing evoked by CCL4 in mice.

AIMS: To explore the mechanisms involved in the transformation of analgesia produced by low doses of CCL4 (pg/kg) to hyperalgesia when higher doses (ng/kg) are administered to mice. MAIN METHODS: The unilateral hot plate test was used to assess thermal nociception. CD3+, CD4+ or CD8+ blood cells were depleted with selective antibodies. Expression of CCR5 and IL-16 in lymphocytes was studied by flow cytometry and IL-16 blood levels were measured by ELISA. IL-16 and CD8 were detected by immunofluorescence. KEY FINDINGS: IL-16 and CCR5 expression were demonstrated in CD4+ and CD8+ T-lymphocytes by flow cytometry. Furthermore, CCL4-induced hyperalgesia was abolished by reducing circulating T-lymphocyte levels or by selectively depleting CD4+ lymphocytes. In contrast, when the anti-CD4 antibody was acutely administered, CCL4 induced analgesia instead of hyperalgesia. A similar response was obtained when administering A-770041, that prevents CD4-mediated CCR5 desensitization by inhibiting p56lck kinase. As occurred with the analgesic effect evoked by low doses of CCL4, analgesia evoked by combining CCL4 and A-770041 was reverted by naloxone, naltrindole or an anti-met-enk antibody. Interestingly, flow cytometry assays showed that the number of CD8+, but not CD4+, T-cells expressing IL-16 is reduced after the acute administration of CCL4, a result compatible with the description that CD8+-lymphocytes can rapidly release preformed IL-16. Accordingly, the rise in IL-16 blood concentration evoked by CCL4 was prevented after CD8+ lymphocyte depletion. SIGNIFICANCE: CCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4+ T-cells and to the release of IL-16 from CD8+ lymphocytes.

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target.

The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

BACKGROUND: The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. OBJECTIVES: Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. METHODS: Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. RESULTS: The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. CONCLUSIONS: Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

A low pKa ligand inhibits cancer-associated pain in mice by activating peripheral mu-opioid receptors.

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC50 of NFEPP to displace bound [3H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30-100 nmol/kg) or fentanyl (17-30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

Dual dose-related effects evoked by CCL4 on thermal nociception after gene delivery or exogenous administration in mice.

As recently described, the administration of extremely low doses (pg/kg) of CCL4 (Macrophage inflammatory protein 1ß, MIP-1ß) can induce antinociceptive effects in mice (García-Domínguez et al., 2019b). We describe here that hydrodynamic delivery of a plasmid containing CCL4 cDNA provokes a biphasic response consisting in an initial thermal hyperalgesic reaction for 8 days followed by analgesia at days 10-12, being both responses blocked after the administration of the CCR5 antagonist DAPTA. Both the luminiscence evoked in liver after the administration of a plasmid containing CCL4 and luciferase cDNAs and the hepatic concentration of CCL4 measured by ELISA were maximal 4 days after plasmid administration and markedly diminished at day 10. A dose-effect curve including a wide dose range of exogenous CCL4 revealed thermal analgesia after the administration of 10-100 pg/kg whereas 1000 times higher doses (30-100 ng/kg) induced, instead, thermal hyperalgesia inhibited by DAPTA. This hyperalgesia was absent in mice with reduced white blood cells after cyclophosphamide treatment, thus supporting the involvement of circulating leukocytes. A multiarray bioluminescent assay revealed increased plasma levels of IL-1α, CCL2, CXCL1, CXCL13, IL-16 and TIMP-1 in mice treated with 100 ng/kg of CCL4. The hyperalgesic response evoked by CCL4 was prevented by IL-1R, CXCR2 or CCR2 antagonists or by the neutralization of CXCL13 or IL-16, but not TIMP-1, with selective antibodies. The administration of the anti-IL-16 antibody was the unique treatment able to convert hyperalgesia evoked by 100 ng/kg of CCL4 in an analgesic effect. The ability of IL-16 to evoke hypernociception was confirmed by studying the response to its exogenous administration (10-30 ng/kg). In summary, the present results demonstrate that CCL4 induces a dual modulation of nociception and describe some mechanisms involved in the hyperalgesic response evoked by this chemokine.

The Systemic Administration of the Chemokine CCL1 Evokes Thermal Analgesia in Mice Through the Activation of the Endocannabinoid System.

Apart from its involvement in immune functions, the chemokine CCL1 can participate in the modulation of nociceptive processing. Previous studies have demonstrated the hypernociceptive effect produced by CCL1 in the spinal cord, but its possible action on peripheral nociception has not yet been characterized. We describe here that the subcutaneous administration of CCL1 (1-10 µg/kg) produces dose-dependent and long-lasting increases in thermal withdrawal latencies measured by the unilateral hot plate test in mice. The antinociceptive nature of this effect is further supported by the reduction of spinal neurons expressing Fos protein in response to a noxious thermal stimulus observed after the administration of 10 µg/kg of CCL1. CCL1-induced antinociception was inhibited after systemic, but not spinal administration of the selective antagonist R243 (0.1-1 mg/kg), demonstrating the participation of peripheral CCR8 receptors. The absence of this analgesic effect in mice treated with a dose of cyclophosphamide that produces a drastic depletion of leukocytes suggests its dependency on white blood cells. Furthermore, whereas the antinociceptive effect of CCL1 was unaffected after the treatment with either the antagonist of opioid receptors naloxone or the cannabinoid type 1 receptor blocker AM251, it was dose-dependently inhibited after the administration of the CB2 receptor antagonist SR144528 (0.1-1 mg/kg). The detection by ELISA of an increased presence of the endocannabinoid 2-arachidonoylglycerol after the administration of an analgesic dose of CCL1 supports the notion that CCL1 can evoke thermal analgesia through the release of this endocannabinoid from circulating leukocytes.

The Chemokine CCL4 (MIP-1ß) Evokes Antinociceptive Effects in Mice: a Role for CD4+ Lymphocytes and Met-Enkephalin.

In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not ß-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4+ than in CD8+ T lymphocytes. CCL4-induced antinociception almost disappeared upon CD4+, but not CD8+, lymphocyte depletion with selective antibodies, thus supporting that the release of met-enk from CD4+ lymphocytes underlies the opioid antinociceptive response evoked by CCL4.

Synergistic combinations of the dual enkephalinase inhibitor PL265 given orally with various analgesic compounds acting on different targets, in a murine model of cancer-induced bone pain.

BACKGROUND: The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 (P2X3 receptor antagonist), ACEA (CB1 receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). METHODS: Experiments. Experiments were performed in 5-6 weeks old (26-33g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2×106 cells/100µL. Then 105 cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49±0.2°C. Mechanical threshold values were obtained by performing the von Frey test using the "up and down" method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. RESULTS: The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENKs, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X3 receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Nav1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. CONCLUSIONS: These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. IMPLICATIONS: This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain.

Hyperalgesic and hypoalgesic mechanisms evoked by the acute administration of CCL5 in mice.

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3-10ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01-0.1µg) and DAPTA (0.3-3µg), respectively. The prevention of this hyperalgesia by diclofenac (1-10µg), the inhibitors of COX-1 SC-560 (0.1-1µg) or COX-2 celecoxib (1-5µg), the TRPV1 antagonist capsazepine (0.03-0.3µg) or the TRPA1 antagonist HC030031 (10-50µg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17µg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5µg), nor-binaltorphimine (10mg/kg) or an anti-dynorphin A antibody (0.62-2.5ng). The administration of 30ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.

Analgesic effects evoked by a CCR2 antagonist or an anti-CCL2 antibody in inflamed mice.

Chemokine CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), is a molecule that in addition to its well-established role in chemotaxis can also act as nociceptor sensitizer. The upregulation of this chemokine in inflamed tissues could suggest its involvement in inflammatory hypernociception. Thus, we have measured CCL2 levels in mice with acute or chronic inflammation due to the intraplantar (i.pl.) injection of carrageenan or complete Freund's adjuvant (CFA), respectively, and we have studied whether inflammatory hyperalgesia or allodynia could be attenuated by blocking CCR2 receptors or neutralizing CCL2 with an anti-CCL2 antibody. A remarkable increase in CCL2 concentration was detected by ELISA in paw homogenates coming from carrageenan- or CFA-inflamed mice, being its expression mainly localized in macrophages, as shown by immunohistochemical assays. The s.c. (0.3-3 mg/kg) or i.pl. (0.3-3 µg) administration of the CCR2 antagonist, RS 504393, dose dependently inhibited thermal hyperalgesia measured in acutely or chronically inflamed mice, whereas s.c. administration of this drug did not reduce inflammatory mechanical allodynia. Furthermore, the inhibition of inflammatory hyperalgesia after the administration of an anti-CCL2 antibody (0.1-1 µg; i.pl.) suggests that CCL2 could be the endogenous chemokine responsible for CCR2-mediated hyperalgesic effects. Besides, the acute administration of the highest antihyperalgesic dose of RS 504393 assayed did not reduce paw tumefaction or modify the presence of inflammatory cells. These results indicate that the blockade of the CCL2/CCR2 system can counteract inflammatory hyperalgesia, being this antinociceptive effect unrelated to a decrease in the inflammatory reaction.

Involvement of CC Chemokine Receptor 1 and CCL3 in Acute and Chronic Inflammatory Pain in Mice.

Chemokines are chemotactic cytokines whose involvement in nociceptive processing is being increasingly recognized. Based on the previous description of the involvement of CC chemokine receptor type 1 (CCR1) in pathological pain, we have assessed the participation of CCR1 and its endogenous ligands CCL3 and CCL5 in hyperalgesia and allodynia in mice after acute inflammation with carrageenan and chronic inflammation with complete Freund's adjuvant (CFA). The subcutaneous administration of the CCR1 antagonist J113863 (3-30 mg/kg; 30 min. before) dose dependently inhibited carrageenan- and CFA-evoked thermal hyperalgesia and mechanical allodynia produced by CFA, but not by carrageenan. The maximal dose of J113863 did not modify the increase in paw thickness induced by carrageenan or CFA. An almost ten times augmentation of CCL3 levels was detected by ELISA assays in both carrageenan and CFA paws, but not in spinal cords of inflamed mice, whereas CCL5 concentrations remained unaltered. Accordingly, a marked increase of CCL3 mRNA expression was observed in inflamed paws, with CCL3 protein detected in neutrophils and macrophages by immunohistochemical experiments. The intraplantar administration of an anti-CCL3 antibody (0.3-3 µg) blocked thermal hyperalgesia in carrageenan- and CFA-inflamed mice as well as CFA-evoked mechanical allodynia. Our data suggest that the increased concentrations of CCL3 present in inflamed tissues can be involved in acute and chronic inflammatory hyperalgesia as well as in chronic mechanical allodynia, and that these hypernociceptive symptoms can be counteracted by its neutralization with an antibody or by the blockade of CCR1 receptors.

Hypernociceptive responses following the intratibial inoculation of RM1 prostate cancer cells in mice.

BACKGROUND: Pain due to bone metastases of prostatic origin is a relevant clinical issue. We study here the nociceptive responses obtained in mice receiving the intratibial inoculation of RM1 prostate cancer cells. METHODS: 10(2) -10(5) RM1 cells were inoculated to C57BL/6 mice and tumor development was analysed histologically and with luciferase-expressing RM1 cells. Spinal astroglial (GFAP) or microglial (Iba-1) expression was assessed with immunohistochemical methods and hypernociception was measured by the unilateral hot plate, the paw pressure and the von Frey tests. The analgesic effect of morphine, zoledronic acid or the CCR2 antagonist RS504393 was measured. Levels of the chemokines CCL2, CCL3, and CCL5 were determined by ELISA. RESULTS: The inoculation of 10(3) RM1 cells induced tumoral growth in bone with a mixed osteoclastic/osteoblastic pattern and evoked astroglial, but not microglial, activation in the spinal cord. Hyperalgesia and allodynia were already established four days after inoculation and dose-dependently inhibited by the s.c. administration of morphine (1-5 mg/kg) or zoledronic acid (1-3 mg/kg). CCL2 and CCL5, but not CCL3, were released by RM1 cells in culture whereas only an increased presence of CCL2 was found in bone tumor homogenates. The administration of the CCR2 antagonist RS504393 (0.3-3 mg/kg) inhibited RM1 induced thermal hyperalgesia without modifying mechanical allodynia. CONCLUSION: The intratibial inoculation of RM1 cells in immunocompetent mice induces hypernociceptive responses and can be useful to perform studies of bone cancer induced pain related to androgen-independent prostate cancer. The antinociceptive role derived from the blockade of the CCR2 chemokine receptors is further envisaged.

Involvement of spinal chemokine CCL2 in the hyperalgesia evoked by bone cancer in mice: a role for astroglia and microglia.

The hypernociceptive role played by the chemokine CCL2, and its main receptor, CCR2, in pathological settings is being increasingly recognized. We aimed to characterize the involvement of spinal CCL2 in the hyperalgesia due to the intratibial inoculation of fibrosarcoma NCTC 2472 cells in mice. The intrathecal (i.t.) administration of the CCR2 antagonist RS 504393 (1­3 µg) or an anti-CCL2 antibody inhibited tumoral hyperalgesia. No change in the expression of spinal CCR2 was detected by western blot, whereas immunohistochemical experiments demonstrated increased CCL2 staining at the superficial laminae of the spinal cord ipsilateral to the tumor. This spinal CCL2 does not seem to be released from nociceptors since CCL2 mRNA and CCL2 levels in DRGs, as measured by RT-PCR and ELISA, remain unmodified in tumor-bearing mice. In contrast, immunohistochemical assays demonstrated the spinal up-regulations of GFAP and Iba-1, respective markers of astroglia and microglia, and the expression of CCL2 in both types of glial cells at the superficial laminae of the spinal cord of tumor-bearing mice. Finally, since CCL2 could induce astroglial or microglial activation, we studied whether the blockade of CCR2 could inhibit the increased spinal glial expression. GFAP, but not Iba-1, up-regulation was reduced in tumor-bearing mice treated for 3 days with i.t. RS 504393, indicating that spinal CCL2 acts as an astroglial activator in this setting. The participation at spinal level of CCL2/CCR2 in tumoral hypernociception, together with its previously described involvement at periphery, makes attractive the modulation of this system for the alleviation of neoplastic pain.

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.

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.

An inhibitor of neuronal exocytosis (DD04107) displays long-lasting in vivo activity against chronic inflammatory and neuropathic pain.

Small peptides patterned after the N terminus of the synaptosomal protein of 25 kDa, a member of the protein complex implicated in Ca(2+)-dependent neuronal exocytosis, inhibit in vitro the release of neuromodulators involved in pain signaling, suggesting an in vivo analgesic activity. Here, we report that compound DD04107 (palmitoyl-EEMQRR-NH(2)), a 6-mer palmitoylated peptide that blocks the inflammatory recruitment of ion channels to the plasma membrane of nociceptors and the release of calcitonin gene-related peptide from primary sensory neurons, displays potent and long-lasting in vivo antihyperalgesia and antiallodynia in chronic models of inflammatory and neuropathic pain, such as the complete Freund's adjuvant, osteosarcoma, chemotherapy, and diabetic neuropathic models. Subcutaneous administration of the peptide produced a dose-dependent antihyperalgesic and antiallodynic activity that lasted ≥24 h. The compound showed a systemic distribution, characterized by a bicompartmental pharmacokinetic profile. Safety pharmacology studies indicated that the peptide is largely devoid of side effects and substantiated that the in vivo activity is not caused by locomotor impairment. Therefore, DD04107 is a potent and long-lasting antinociceptive compound that displays a safe pharmacological profile. These findings support the notion that neuronal exocytosis of receptors and neuronal algogens pivotally contribute to chronic inflammatory and neuropathic pain and imply a central role of peptidergic nociceptor sensitization to the pathogenesis of pain.

Potentiation of acute morphine-induced analgesia measured by a thermal test in bone cancer-bearing mice.

Agonists of µ-opioid receptors are currently used in the management of cancer pain. However, several data suggest that the analgesic effect of morphine can diminish during the development of experimental tumors. By using a thermal test, we have studied whether the analgesic effect evoked by morphine is altered in mice bearing two painful bone tumors. The analgesic effect evoked by systemic morphine remained unaltered after the intratibial inoculation of B16-F10 melanoma cells and was potentiated after the inoculation of NCTC 2472 osteosarcoma cells. Although the number of spinal µ-opioid receptors measured by western blot studies was not augmented in osteosarcoma-bearing mice, the analgesia evoked by intrathecal (i.t.) morphine was also enhanced. The analgesic response produced by the spinal administration of the Gi/o protein activator mastoparan was amplified, whereas the analgesic response evoked by the i.t. administration of the N-type calcium channel blocker ω-conotoxin remained unaltered. The efficacy of the GIRK channel blocker tertiapin-Q to antagonize the analgesic effect produced by a maximal dose of morphine was also increased in osteosarcoma-bearing mice. Our results seem to indicate that the analgesic effect of morphine on thermal nociception can be enhanced in response to the development of particular bone tumors in mice, being this potentiation probably related to a greater efficacy of the transduction system driven by Gi/o proteins and GIRK channels.