Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain (original) (raw)
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PLoS ONE, 2014
The analgesic effect of delta-opioid receptor (DOR) ligands in neuropathic pain is not diminished in contrast to other opioid receptor ligands, which lose their effectiveness as analgesics. In this study, we examine whether this effect is related to nerve injury-induced microglial activation. We therefore investigated the influence of minocycline-induced inhibition of microglial activation on the analgesic effects of opioid receptor agonists: morphine, DAMGO, U50,488H, DPDPE, Deltorphin II and SNC80 after chronic constriction injury (CCI) to the sciatic nerve in rats. Pre-emptive and repeated administration of minocycline (30 mg/kg, i.p.) over 7 days significantly reduced allodynia and hyperalgesia as measured on day 7 after CCI. The antiallodynic and antihyperalgesic effects of intrathecally (i.t.) administered morphine (10-20 mg), DAMGO (1-2 mg) and U50,488H (25-50 mg) were significantly potentiated in rats after minocycline, but no such changes were observed after DPDPE (10-20 mg), deltorphin II (1.5-15 mg) and SNC80 (10-20 mg) administration. Additionally, nerve injury-induced downregulation of all types of opioid receptors in the spinal cord and dorsal root ganglia was not influenced by minocycline, which indicates that the effects of opioid ligands are dependent on other changes, presumably neuroimmune interactions. Our study of rat primary microglial cell culture using qRT-PCR, Western blotting and immunocytochemistry confirmed the presence of mu-opioid receptors (MOR) and kappa-opioid receptors (KOR), further we provide the first evidence for the lack of DOR on microglial cells. In summary, DOR analgesia is different from analgesia induced by MOR and KOR receptors because it does not dependent on injury-induced microglial activation. DOR agonists appear to be the best candidates for new drugs to treat neuropathic pain.
Changes in spinal and supraspinal endocannabinoid levels in neuropathic rats
Neuropharmacology, 2007
Recent studies have shown that activation of the cannabinoid CB 1 receptor by synthetic agonists, and pharmacological elevation of endocannabinoid levels, suppress hyperalgesia and allodynia in animal models of neuropathic pain. However, the concentrations of endocannabinoids in the nervous tissues involved in pain transmission during neuropathic pain have never been measured. Here we have determined the levels of anandamide and 2-arachidonoylglycerol (2-AG), as well as of the analgesic anandamide congener, palmitoylethanolamide (PEA), in three brain areas involved in nociception, i.e. the dorsal raphe (DR), periaqueductal grey (PAG) and rostral ventral medulla (RVM), as well as in the spinal cord (SC), following chronic constriction injury (CCI) of the sciatic nerve in the rat, in comparison with sham-operated rats. After 3 days from CCI, anandamide or 2-AG levels were significantly enhanced only in the SC or PAG, respectively. After 7 days from CCI, when thermal hyperalgesia and mechanical allodynia are maximal, a strong (1.3e3-fold) increase of both anandamide and 2-AG levels was observed in the PAG, RVM and SC. At this time point, anandamide, but not 2-AG, levels were also enhanced in the DR. PEA levels were significantly decreased in the SC after 3 days, and in the DR and RVM after 7 days from CCI. These data indicate that anandamide and 2-AG, operating at both spinal and supra-spinal levels, are up-regulated during CCI of the sciatic nerve, possibly to inhibit pain. Yet to be developed substances that inhibit both endocannabinoid and PEA inactivation might be useful for the treatment of neuropathic pain.
Endocannabinoid regulation of spinal nociceptive processing in a model of neuropathic pain
European Journal of Neuroscience, 2010
Models of neuropathic pain are associated with elevated spinal levels of endocannabinoids (ECs) and altered expression of cannabinoid receptors on primary sensory afferents and post-synaptic cells in the spinal cord. We investigated the impact of these changes on the spinal processing of sensory inputs in a model of neuropathic pain. Extracellular single-unit recordings of spinal neurones were made in anaesthetized neuropathic and sham-operated rats. The effects of spinal administration of the cannabinoid CB 1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and the cannabinoid receptor type 2 (CB 2) receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicycloheptan-2-yl]-5-(4-chloro-3methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) on mechanically-evoked responses of spinal neurones were determined. The effects of spinal administration of (5Z,8Z11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707), which binds to CB 2 receptors and alters transport of ECs, on evoked responses of spinal neurones and spinal levels of ECs were also determined. The cannabinoid CB 1 receptor antagonist AM251, but not the CB 2 receptor antagonist, significantly facilitated 10-g-evoked responses of spinal neurones in neuropathic, but not sham-operated, rats. Spinal administration of UCM707 did not alter spinal levels of ECs but did significantly inhibit mechanically-evoked responses of neurones in neuropathic, but not sham-operated, rats. Pharmacological studies indicated that the selective inhibitory effects of spinal UCM707 in neuropathic rats were mediated by activation of spinal CB 2 receptors, as well as a contribution from transient receptor potential vanilloid 1 (TRPV1) channels. This work demonstrates that changes in the EC receptor system in the spinal cord of neuropathic rats influence the processing of sensory inputs, in particular low-weight inputs that drive allodynia, and indicates novel effects of drugs acting via multiple elements of this receptor system.
European Journal of Neuroscience, 2005
Activation of p38 mitogen-activated protein kinase (p38) in spinal microglia is implicated in spinal nociceptive processing. Minocycline, a tetracycline derivative, displays selective inhibition of microglial activation, a function that is distinct from its antibiotic activity. In the present study we examined antinociceptive effects of intrathecal (IT) administration of minocycline in experimental models of inflammation-evoked hyperalgesia in addition to the effect of minocycline on stimulation-induced activation of p38 in spinal microglia. Intrathecal minocycline produced a dose-dependent reduction of formalin-evoked second-phase flinching behaviour in rats, and prevented thermal hyperalgesia induced by carrageenan injection into the paw. In contrast, systemic delivery (intraperitoneally) of minocycline inhibited the first but not the second phase of formalin-induced flinching, and it had no effect on carrageenan-induced hyperalgesia. Centrally mediated hyperalgesia induced by IT delivery of N-methyl-d-aspartate was completely blocked by IT minocycline. An increase in phosphorylation (activation) of p38 (P-p38) was observed in the dorsal spinal cord after carrageenan paw injection, assessed by both Western blotting and immunohistochemistry. The increased P-p38 immunoreactivity was seen primarily in microglia but also in a small population of neurons. Minocycline, at the IT dose that blocked carrageenaninduced hyperalgesia, also attenuated the increased P-p38 in microglia. In addition, minocycline suppressed lipopolysaccharideevoked P-p38 in cultured spinal microglial cells. Taken together, these findings show that minocycline given IT produces a potent and consistent antinociception in models of tissue injury and inflammation-evoked pain, and they provide strong support for the idea that this effect is mediated by direct inhibition of spinal microglia and subsequent activation of p38 in these cells.
Molecular Pain
Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.
International Journal of Molecular Sciences
Pain is a worldwide public health problem and its treatment is still a challenge since clinically available drugs do not completely reverse chronic painful states or induce undesirable effects. Crotalphine is a 14 amino acids synthetic peptide that induces a potent and long-lasting analgesic effect on acute and chronic pain models, peripherally mediated by the endogenous release of dynorphin A and the desensitization of the transient receptor potential ankyrin 1 (TRPA1) receptor. However, the effects of crotalphine on the central nervous system (CNS) and the signaling pathway have not been investigated. Thus, the central effect of crotalphine was evaluated on the partial sciatic nerve ligation (PSNL)-induced chronic neuropathic pain model. Crotalphine (100 µg/kg, p.o.)-induced analgesia on the 14th day after surgery lasting up to 24 h after administration. This effect was prevented by intrathecal administration of CB1 (AM251) or CB2 (AM630) cannabinoid receptor antagonists. Besides ...
Molecular Pain, 2010
Background: Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/ antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.
European Journal of Pharmacology, 2009
The pharmacological attenuation of glial activation represents a novel approach for controlling neuropathic pain, but the role of microglial and astroglial cells is not well established. To better understand the potential role of two types of glial cells, microglia and astrocytes, in the pathogenesis of neuropathic pain, we examined markers associated with them by quantitative RT-PCR, western blot and immunohistochemical analyses in the dorsal horn of the lumbar spinal cord 7 days after chronic constriction injury (CCI) to the sciatic nerve in mice. The mRNA and protein of microglial cells were labeled with C1q and OX42(CD11b/c), respectively. The mRNA and protein of astrocytes were labeled with GFAP. The RT-PCR results indicated an increase in C1q mRNA that was more pronounced than the increased expression of GFAP mRNA ipsilateral to the injury in the dorsal spinal cord. Similarly, western blot and immunohistochemical analyses demonstrated an ipsilateral upregulation of OX42-positive cells (72 and 20%, respectively) and no or little (8% upregulation) change in GFAP-positive cells in the ipsilateral dorsal lumbar spinal cord. We also found that chronic intraperitoneal injection of the minocycline (microglial inhibitor) and pentoxifylline (cytokine inhibitor) attenuated CCI-induced activation of microglia, and both, but not fluorocitrate (astroglial inhibitor), diminished neuropathic pain symptoms and tactile and cold sensitivity. Our findings indicate that spinal microglia are more activated than astrocytes in peripheral injury-induced neuropathic pain. These findings implicate a glial regulation of the pain response and suggest that pharmacologically targeting microglia could effectively prevent clinical pain syndromes in programmed and/or anticipated injury.
Neuroscience, 2010
A role of neuropeptides in neuropathic pain development has been implicated; however, the neuroimmune interactions that are involved in the underlying mechanisms may be more important than previously thought. To examine a potential role of relations between glia cells and neuropeptides in neuropathic pain, we performed competitive reverse-transcription polymerase chain reaction (RT-PCR) from the dorsal lumbar spinal cord and the dorsal root ganglion (DRG) after chronic constriction injury (CCI) in the rat sciatic nerve. The RT-PCR results indicated that complement component 1, q subcomponent (C1q) mRNA expression was higher than glial fibrillary acidic protein (GFAP) in the spinal cord 3 and 7 days post-CCI, suggesting that spinal microglia and perivascular macrophages are more activated than astrocytes. In parallel, we observed a strong upregulation of prodynorphin mRNA in the spinal cord after CCI, with no changes in the expression of proenkephalin or pronociceptin. Conversely, the expression of GFAP mRNA in the DRG was higher than C1q, which suggests that the satellite cells are activated shortly after injury, followed by the macrophages and polymorphonuclear leukocytes infiltrating the DRG. In the DRG, we also observed a very strong upregulation of prodynorphin (1387%) as well as pronociceptin (122%) and a downregulation of proenkephalin (47%) mRNAs. Interestingly, preemptive and repeated i.p. injection of minocycline reversed the activation of microglia/macrophages in the spinal cord and the trafficking of peripheral immune cells into the DRG, and markedly diminished the upregulation of prodynorphin and pronociceptin in the DRG. We thus provide novel findings that inhibition of C1q-positive cells by minocycline can diminish injury-induced neuropeptide changes in the DRG. This suggests that immune cells-derived pronociceptive factors may influence opioid peptide expression. Therefore, the injuryinduced activation of microglia and leukocytes and the subsequent activation of neuropeptides involved in nociception processes are potential targets for the attenuation of neuropathic pain.
Arthritis & Rheumatism, 2010
Objective. To investigate the impact of an experimental model of osteoarthritis (OA) on spinal nociceptive processing and the role of the inhibitory endocannabinoid system in regulating sensory processing at the spinal level. Methods. Experimental OA was induced in rats by intraarticular injection of sodium mono-iodoacetate (MIA), and the development of pain behavior was assessed. Extracellular single-unit recordings of wide dynamic range (WDR) neurons in the dorsal horn were obtained in MIA-treated rats and saline-treated rats. The levels of endocannabinoids and the protein and messenger RNA levels of the main synthetic enzymes for the endocannabinoids (N-acyl phosphatidylethanolamine phospholipase D [NAPE-PLD] and diacylglycerol lipase ␣ [DAGL␣]) in the spinal cord were measured. Results. Low-weight (10 gm) mechanically evoked responses of WDR neurons were significantly (P < 0.05) facilitated 28 days after MIA injection compared with the responses in saline-treated rats, and spinal cord levels of anandamide and 2-arachidonoyl glycerol (2-AG) were increased in MIA-treated rats. Protein levels of NAPE-PLD and DAGL␣, which synthesize anandamide and 2-AG, respectively, were elevated in the spinal cords of MIA-treated rats. The functional role of endocannabinoids in the spinal cords of MIA-treated rats was increased via activation of cannabinoid 1 (CB 1 ) and CB 2 receptors, and blockade of the catabolism of anandamide had significantly greater inhibitory effects in MIA-treated rats compared with control rats. Conclusion. Our findings provide new evidence for altered spinal nociceptive processing indicative of central sensitization and for adaptive changes in the spinal cord endocannabinoid system in an experimental model of OA. The novel control of spinal cord neuronal responses by spinal cord CB 2 receptors suggests that this receptor system may be an important target for the modulation of pain in OA.