Ultra-low dose naltrexone attenuates chronic morphine-induced gliosis in rats
© Mattioli et al; licensee BioMed Central Ltd. 2010
Received: 10 February 2010
Accepted: 16 April 2010
Published: 16 April 2010
The development of analgesic tolerance following chronic morphine administration can be a significant clinical problem. Preclinical studies demonstrate that chronic morphine administration induces spinal gliosis and that inhibition of gliosis prevents the development of analgesic tolerance to opioids. Many studies have also demonstrated that ultra-low doses of naltrexone inhibit the development of spinal morphine antinociceptive tolerance and clinical studies demonstrate that it has opioid sparing effects. In this study we demonstrate that ultra-low dose naltrexone attenuates glial activation, which may contribute to its effects on attenuating tolerance.
Spinal cord sections from rats administered chronic morphine showed significantly increased immuno-labelling of astrocytes and microglia compared to saline controls, consistent with activation. 3-D images of astrocytes from animals administered chronic morphine had significantly larger volumes compared to saline controls. Co-injection of ultra-low dose naltrexone attenuated this increase in volume, but the mean volume differed from saline-treated and naltrexone-treated controls. Astrocyte and microglial immuno-labelling was attenuated in rats co-administered ultra-low dose naltrexone compared to morphine-treated rats and did not differ from controls. Glial activation, as characterized by immunohistochemical labelling and cell size, was positively correlated with the extent of tolerance developed. Morphine-induced glial activation was not due to cell proliferation as there was no difference observed in the total number of glial cells following chronic morphine treatment compared to controls. Furthermore, using 5-bromo-2-deoxyuridine, no increase in spinal cord cell proliferation was observed following chronic morphine administration.
Taken together, we demonstrate a positive correlation between the prevention of analgesic tolerance and the inhibition of spinal gliosis by treatment with ultra-low dose naltrexone. This research provides further validation for using ultra-low dose opioid receptor antagonists in the treatment of various pain syndromes.
Opioid drugs, such as morphine, are widely used for the management of moderate to severe pain. Unfortunately, the usefulness of morphine and other opioid analgesics in the management of pain is limited due to the development of tolerance to the analgesic effects of these drugs with repeated exposure . Clinically, the onset of tolerance necessitates increasing doses of opioids, which in turn typically increases the number and severity of adverse effects and compliance .
Morphine acts to inhibit nociception predominately via Gi protein-coupled μ-opioid receptors [3, 4] located in nociceptive pathways throughout the central nervous system including the dorsal spinal cord. Within the spinal cord, μ-opioid receptors are well recognized to localize on pre- and post-synaptic nociceptive neurons, but they are also present on astrocytes and microglia [5–10], however the function of μ-opioid receptors on glial cells remains elusive.
A number of factors appear to contribute to the development of analgesic tolerance. In general, the development of tolerance is thought to involve cellular adaptation/modulation that results in decreased analgesic potency. The precise mechanism(s) of action is not known; however, investigators have been able to attenuate or reverse established analgesic tolerance to morphine by inhibiting either the release of neurotransmitters and/or inhibition of their receptors [11–16]. Within the last decade, activation of spinal glia has emerged as a novel mechanism underlying analgesic tolerance [17–19]. Relevant to the current study, the administration of sub-therapeutic (ultra-low) doses of opioid specific antagonists (e.g. naloxone, naltrexone) augmented opioid-induced analgesia and inhibited and/or reversed the development of tolerance and physical dependence . Although this relationship was studied intensively in various in vitro and in vivo models [20–22], only recently have clinical trials been undertaken to investigate the improved therapeutic benefit of combining opioid analgesics with ultra-low dose opioid receptor antagonists. To date, clinical trials have confirmed that combinations of opioids and ultra-low dose antagonists both enhance and prolong opioid-induced analgesia, and prevent analgesic tolerance and physical dependence [23, 24]. Precisely how ultra-low dose antagonists prevent/reverse tolerance to opioid analgesics is not fully understood, but spinal glia may play a crucial role. We demonstrate that one contributing mechanism is that ultra-low dose naltrexone blocks opioid-induced activation of spinal glial cells.
Ultra-low dose naltrexone attenuated the development of tolerance to morphine antinociception
Ultra-low dose naltrexone attenuated morphine-induced increases in the expression of glial fibrillary acidic protein (GFAP) and CD3/CD11B (OX42)
Ultra-low dose naltrexone attenuated morphine-induced astrocyte hypertrophy
Chronic morphine does not induce cell proliferation
Glial cell counts.
6.767 ± 0.502
8.295 ± 0.558
8.083 ± 0.911
8.886 ± 0.499
The current study has provided additional evidence that ultra-low dose naltrexone attenuates the development of tolerance to the antinociceptive effects of morphine as previously demonstrated by Powell et al . As the mechanism by which this phenomenon occurs is unknown, this study sought to investigate the contribution of glia in the actions of ultra-low dose opioid antagonists. Intrathecal catheterization has been shown to induce gliosis , therefore the current study used lumbar puncture drug delivery to reproduce the original behavioural findings of Powell et al . Preliminary experiments investigated the effects of different ultra-low doses of naltrexone on morphine tolerance. In this study, the dose of naltrexone (0.05 ng) used in experiments by Powell et al  did not attenuate the loss in antinociception observed with chronic morphine administration. However, a hundred-fold greater dose (5 ng) preserved the analgesic effects of morphine throughout the treatment period, and thus was used to determine the effects on morphine-induced gliosis. In addition to the use of intrathecal catheters for drug delivery, another important difference in experimental protocol in the present study were the housing conditions; animals used in this study were housed in a room on a reverse light-dark cycle (lights off at 7:00 am), with all behavioural testing conducted during the animals' active (dark) phase. It is well accepted that pain responsiveness and endogenous opioids have circadian fluctuations in rats  and that morphine-induced antinociception is greater during the active phase compared to during the inactive light phase. These fluctuations may account for the greater dose of opioid antagonist required to attenuate tolerance in the present study compared to what has been previously published.
Current research has demonstrated that spinal glia are not merely support cells within the CNS as previously hypothesized (i.e. responsible for the maintenance of neurons and CNS homeostasis); they also actively communicate with neurons, are involved in the modulation of synaptic signalling and may be involved in the development of opioid tolerance. Chronic, but not acute, morphine administration, induces gliosis characterized by cell hypertrophy, and is associated with increased expression of GFAP [19, 27–30] in astrocytes and CD3/CD11B (OX42) in microglia . Reactive glial cells (microglia and astrocytes) can release a variety of pro-nociceptive and neuroexcitatory substances (e.g. prostaglandins, excitatory amino acids, interleukins, nitrogen oxide species, ATP, glutamate etc.), which may enhance pain transmission by nociceptive neurons [19, 28, 32–34].
This is the first report to demonstrate that co-administration of ultra-low dose naltrexone prevents morphine-induced gliosis, demonstrated by normalization of GFAP and CD3/CD11B expression and attenuation of increased astrocyte cell volume. The observed increases in GFAP/CD3/CD11B expression and astrocyte cell volume in spinal cord sections from animals chronically administered intrathecal morphine are consistent with gliosis and are in agreement with previous findings of astrocyte and microglial activation by chronic morphine administration [19, 28–30]. As no significant difference was found in the number of immuno-positive cells or in the number of newly generated cells between morphine treated and saline controls, glial proliferation likely contributes very little to the observed increases in GFAP and CD3/CD11B expression. This finding is in agreement with that of Song and Zhao , in which chronic morphine resulted in increased astrocyte immunoreactivity with no difference in the number of cells from saline treated controls. In contrast, Narita et al  reported that astrocyte proliferation was induced by chronic morphine administration; however, no quantification of the number of GFAP-positive cells was reported. Agents that modify  or inhibit  activation of astrocytes and microglia prevent the development of morphine tolerance; thus inhibition of gliosis by ultra-low dose naltrexone may prevent the development of analgesic tolerance. This evidence, taken in concert with the findings of the current study, supports the hypothesis that spinal glia are involved in the development of morphine analgesic tolerance and in the mediation of nociception. It has also been reported that ultra-low dose naltrexone augments morphine antinociception in a model of pertussis toxin induced hyperalgesia .
While the present study provides strong support for the role of glia in the ultra-low dose effect, various molecular studies indicate that ultra-low dose antagonists may prevent opioid receptor coupling to stimulatory G-proteins (Gs). Classically, opioid activation of μ-opioid receptors results in coupling to inhibitory G-protein subunits (Gi/Go) and produces analgesia; however, following chronic opioid administration, increased coupling of μ-opioid receptors to Gs has been observed . Therefore, increased excitatory stimulation via Gs-coupled μ-opioid receptors may oppose the analgesic effects mediated via Gi/Go signalling, and manifest as tolerance [21, 36]. Wang et al  demonstrated that the switch in G-protein coupling to μ-opioid receptors induced by chronic morphine could be prevented by co-administering an ultra-low dose of naloxone, further supporting this hypothesis. Despite these advances, the switch in G-protein coupling to μ-opioid receptors induced by chronic morphine treatment has not been localized to a specific cell population within the spinal cord, and therefore, may occur in glia or nociceptive neurons. On the contrary, the effects of ultra-low dose antagonists may not be mediated by μ-opioid receptors but through a novel mechanism such as an interaction with filamin A  or Toll-like receptors . Thus, future studies will aim to identify the mechanism by which ultra-low dose naltrexone alters gliosis.
The results of this study may have a significant impact on the clinical management of moderate to severe pain. Patients currently treated with chronic opioid therapy may benefit not only from increased efficacy of combined opioid treatment [23, 39], but may also experience fewer and less severe adverse effects [24, 40], as sufficient analgesia can be achieved and maintained at lower opioid doses. Additionally, an understanding of the mechanism of action of opioid drugs will provide insight toward the development of more selective and efficacious pharmacological treatments for pain management. Not the least of which could be for improving treatment of chronic pain conditions such as neuropathic pain where glial activation is also evident, with reactive gliosis being a key contributor to the painful neuropathy [41–44]. Additionally, reduced opioid analgesic efficacy has also been reported in patients with neuropathic pain [45, 46], however, co-administration of ultra-low dose antagonists with opioid agonists increased analgesic efficacy in animal models of neuropathic pain  and in clinical trials [23, 24]. Future research will be required to determine if ultra-low dose naltrexone is able to alleviate established chronic pain.
Adult male Sprague-Dawley rats (180-200 g; Charles River, Québec, Canada), were housed in groups of two with ad libitum access to food and water, and maintained on a reverse 12/12 h light/dark cycle. All behavioural experiments were performed during the dark phase of the cycle, and animals were handled prior to experimentation in order to reduce stress-related analgesia. All experimental protocols were approved by the Queen's University Animal Care Committee, and complied with the policies and directives of the Canadian Council on Animal Care and the International Association for the Study of Pain.
Morphine was purchased from Sabex, Kingston General Hospital, Kingston, Ontario, Canada. Naltrexone and 5-bromo-2-deoxyuridine (BrdU) were purchased from Sigma (St. Louis, MO, USA). Animals were separated into one of five groups receiving i) morphine (15 μg; n = 18), ii) morphine and naltrexone (5 ng; n = 19), iii) morphine and naltrexone (0.05 ng; n = 3), iv) naltrexone (5 ng) alone (n = 8), or v) saline (n = 15). Intrathecal (i.t.) administration of all drugs (diluted in saline to 30 μl volume) was accomplished by way of lumbar puncture between the L4 and L5 vertebrae under brief isofluorane anesthesia. Successful drug placement was confirmed by a vigorous tail flick upon injection.
To determine if chronic morphine treatment induced cell proliferation, animals received 5-bromo-2-deoxyuridine (BrdU, 100 mg/kg; prepared in a concentration of 25 mg/ml in 0.007 N NaOH and saline), injected intraperitoneally (i.p.) on days 1, 3, and 5. Animals were separated into two groups receiving intrathecal morphine (15 μg/15 μl; n = 3) or saline (15 μl; n = 3) by lumbar puncture under brief isoflurane anaesthesia for 5 days. Saline or morphine was injected 30 minutes after BrdU injections.
Behavioural tail flick assay
The effects of drug administration on thermal nociceptive responses were assessed on Days 1, 3 and 5 of the study using the tail flick assay. In brief, a beam of radiant light was applied to a spot marked 5 cm from the tip of the tail, and the latency to a vigorous tail flick was measured. Three baseline latencies were measured prior to drug injection to determine the normal nociceptive responses of the animals. A cut-off time of three times the animal's average baseline was imposed to avoid tissue damage in the event that the animal became unresponsive following drug injection. Rats were then injected intrathecally with their respective treatments, and the thermal latency measured at 30 minutes post-injection, as previous studies have found that the peak antinociceptive effects of morphine occur at this time point . Tail-flick values were converted to a maximum possible effect (% MPE): (post-drug latency - baseline) ÷ (cut-off latency - baseline) × 100. Statistical analyses were performed using a two-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc multiple comparisons test to determine between group differences. P values less than 0.05 were considered significant. All behavioural testing was performed by the experimenter blind to drug treatment.
On day 6, 24 h after the last injection, rats (n = 3 per drug treatment) were deeply anesthetized with sodium pentobarbital (75 mg/kg, i.p.; MTC Pharmaceuticals, Cambridge, Ontario, Canada) and transaortically perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; 500 ml, pH 7.4). The spinal cords were removed by spinal ejection and post-fixed in the above fixative for 1 hour on ice and cryoprotected in 30% sucrose in 0.1 M PB for 48 hours at 4°C. Lumbar segments were isolated and cut into 40 μm transverse sections on a freezing sledge microtome and collected in 0.1 M Tris buffered saline (TBS; pH 7.4).
Free-floating sections were incubated in a blocking solution containing 5% NGS in TBS-T (TBS and 0.2% Triton X-100), followed by incubation with a rabbit polyclonal antisera recognizing glial fibrillary acidic protein (GFAP; 1:2500 working dilution; DakoCytomation, Ontario, Canada) to label astrocytes and a mouse monoclonal antisera recognizing OX42 (1:1000 working dilution; Serote, NC, USA) to label CD3/CD11B receptors on microglia. Spinal cord sections were incubated overnight at 4°C with both primary antibodies, followed by incubation with goat anti-rabbit and goat anti-mouse secondary antibodies (1:200 working dilution; Molecular Probes, Invitrogen, Ontario, Canada) conjugated to Alexa 488 and Alexa 594 fluorophores, respectively. To assess non-specific labelling, control sections were processed in the absence of primary antibody. Sections were mounted on glass slides, air-dried and cover-slipped using Aquamount (Fisher Scientific, Ontario, Canada).
BrdU immuno-labelling was performed as described by Suter et al . Briefly, spinal cord sections were heated in solution containing 50% formamide, 50% 2× saline sodium citrate (SSC) for 2 h at 65°C. Sections were further incubated at 37°C for 30 min in 2N HCL then placed in 0.1 M borate buffer (pH 8.5). Sections were incubated in blocking solution, followed by incubation with a mouse monoclonal antibody against BrdU (1:500, Chemicon, Temecula, CA). To identify the phenotype of newly formed cells, sections were double labelled with one of the following antibodies: rabbit anti-GFAP (for astrocytes, 1:2500), rabbit anti-Iba1 polyclonal antibody (ionizing calcium-binding adaptor molecule, for microglia and macrophages, 1:1000; Wako, Richmond, VA), or rabbit anti-MAP-2 polyclonal antibody (microtubule-associated protein 2, for neurons, 1:1000; Chemicon, Temecula, CA). Sections were then incubated with goat anti-rabbit and goat anti-mouse secondary antibodies (1:200) conjugated to Alexa 488 and Alexa 594 fluorophores, respectively, and mounted as described above.
Imaging of immunoreactive cells was performed as previously described . In brief, immunoreactive cells were imaged using the Leica TCS SP2 multi photon confocal microscope (Leica Microsystems Inc, Ontario, Canada). Images were taken within the dorsal horn (lamina III-V) at 63× magnification for quantification of intensity. Serial images (twenty-five to thirty-five) were captured at 100× magnification, at 0.75 μm increments throughout the z plane in the deep and superficial dorsal horn (4 series per section, 3 sections per animal).
For quantification of the intensity of antibody labelling, images were converted to gray scale using Adobe Photoshop 7.0. Using Image J (NIH), the mean gray values were measured and the average within each treatment group calculated and expressed as mean ± s.e.m. For quantification of GFAP, OX42 and BrdU-positive cells, immunolabelled cell bodies were counted for each section (150 μm × 150 μm) and the average within each treatment group calculated and expressed as mean ± s.e.m. To quantify astrocyte volume, images taken at 100× magnification were stacked and reconstructed in three-dimensions using ImagePro Plus v5.0 software (MediaCybernetics, MD, USA). Total cell volume was calculated for each reconstructed cell. The average volume for cells within each treatment group was calculated and expressed as mean ± s.e.m. Mean intensities and 3D volumes were analyzed by one-way ANOVA followed by Tukey's post-hoc multiple comparison test. Differences in cell numbers were analyzed by unpaired T-tests. P values less than 0.05 were considered significant. All quantification data was collected by experimenter blind to drug treatment.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Canadian Foundation for Innovation awarded to CMC. CMC is a Canadian Research Chair in chronic pain.
- Cox BM: Molecular and cellular mechanisms in opioid tolerance. In Towards a New Pharmacotherapy of Pain. Edited by: Bausbaum AI, Besson JM. John Wiley; 1999:137–156.
- Benyamin R, Trescot AM, Datta S, Buenaventura R, Adlaka R, Sehgal N, Glaser SE, Vallejo R: Opioid complications and side effects. Pain Physician 2008,11(2-Suppl):S105-S120.PubMed
- Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dollé P, Tzavara E, Hanoune J, Roques BP, Kieffer BL: Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 1996, 383: 819–823. 10.1038/383819a0PubMedView Article
- Koski G, Klee WA: Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis. Proc Natl Acad Sci USA 1981,78(7):4185–4189. 10.1073/pnas.78.7.4185PubMed CentralPubMedView Article
- Eriksson PS, Hansson E, Ronnback L: Delta and kappa opiate receptors in primary astroglial cultures from rat cerebral cortex. Neurochem Res 1990,15(11):1123–1126. 10.1007/BF01101714PubMedView Article
- Eriksson PS, Hansson E, Ronnback L: Mu and Delta opiate receptors in neuronal and astroglial primary cultures from various regions of the brain - coupling with adenylate cyclase, localization on the same neurones and association with dopamine (D1) receptors adenylate cyclase. Neuropharmacology 1991,30(11):1233–1239. 10.1016/0028-3908(91)90170-GPubMedView Article
- Dobrenis K, Makman MH, Stefano GB: Occurrence of the opiate alkaloid-selective mu3 receptor in mammalian microglia, astrocytes and Kupffer cells. Brain Res 1995,686(2):239–248. 10.1016/0006-8993(95)00452-VPubMedView Article
- Ruzicka BB, Fox CA, Thompson RC, Meng F, Watson SJ, Akil H: Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA. Brain Res Mol Brain Res 1995,34(2):209–220. 10.1016/0169-328X(95)00165-OPubMedView Article
- Stiene-Martin A, Zhou R, Hauser KF: Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa-opioid receptor expression among cultured mouse astrocytes. Glia 1998,22(3):249–259. 10.1002/(SICI)1098-1136(199803)22:3<249::AID-GLIA4>3.0.CO;2-0PubMed CentralPubMedView Article
- Cheng PY, Liu-Chen LY, Pickel VM: Dual ultrastructural immunocytochemical labelling of μ and δ opioid receptors in the superficial layers of the rat cervical spinal cord. Brain Res 1997,778(2):367–380. 10.1016/S0006-8993(97)00891-3PubMedView Article
- Powell KJ, Ma W, Sutak M, Doods H, Quirion R, Jhamandas K: Blockade and reversal of spinal morphine tolerance by peptide and non-peptide calcitonin gene-related peptide receptor antagonists. Br J Pharmacol 2000, 131: 875–884. 10.1038/sj.bjp.0703655PubMed CentralPubMedView Article
- Powell KJ, Quirion R, Jhamandas K: Inhibition of neurokinin-1-substance P receptor and prostanoid activity prevents and reverses the development of morphine tolerance in vivo and morphine-induced increase in CGFP expression in cultured dorsal root ganglion neurons. Eur J Neurosci 2003,18(6):1572–1583. 10.1046/j.1460-9568.2003.02887.xPubMedView Article
- Menard DP, van Rossum D, Kar S, Jolicoeur B, Jhamandas K, Quirion R: Tolerance to the antinociceptive properties of morphine in the rat spinal cord: alteration of calcitonin gene-related peptide-like immunostaining and receptor binding sites. J Pharmacol Exp Ther 1995,273(2):887–894.PubMed
- Price DD, Mayer DJ, Mao J, Caruso FS: NMDA-receptor antagonists and opioid receptor interactions as related to analgesia and tolerance. J Pain Symptom Manage 2000,19(1):S7-S11. 10.1016/S0885-3924(99)00121-9PubMedView Article
- Mao J, Price DD, Mayer DJ: Thermal hyperalgesia in association with the development of morphine tolerance in rats: roles of excitatory amino acid receptors and protein kinase C. J Neurosci 1994,14(4):2301–2312.PubMed
- Trujillo KA, Akil H: Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-901. Science 1991, 251: 85–87. 10.1126/science.1824728PubMedView Article
- Raghavendra V, Tanga F, Rutkowski MD, DeLeo JA: Anti-hyperalgesic and morphine-sparing actions of propentofylline following peripheral nerve injury in rats: mechanistic implications of spinal glia and proinflammatory cytokines. Pain 2003, 104: 655–664. 10.1016/S0304-3959(03)00138-6PubMedView Article
- Raghavendra V, Tanga FY, DeLeo JA: Attenuation of morphine tolerance, withdrawal-induced hyperalgesia, and associated spinal inflammatory immune responses by propentofylline in rats. Neuropsychopharmacology 2004, 29: 327–334. 10.1038/sj.npp.1300315PubMedView Article
- Song P, Zhao Z: The involvement of glial cells in the development of morphine tolerance. Neurosci Res 2001, 39: 281–286. 10.1016/S0168-0102(00)00226-1PubMedView Article
- Powell KJ, Abul-Husn NS, Jhamandas A, Olmstead MC, Beninger RJ, Jhamandas K: Paradoxical effects of the opioid antagonist naltrexone on morphine analgesia, tolerance, and reward in rats. J Pharmacol Exp Ther 2002,300(2):588–596. 10.1124/jpet.300.2.588PubMedView Article
- Wang HY, Friedman E, Olmstead MC, Burns LH: Ultra-low-dose naloxone suppresses opioid tolerance, dependence and associated changes in mu opioid receptor-G protein coupling and Gβγ signaling. Neuroscience 2005, 135: 247–261. 10.1016/j.neuroscience.2005.06.003PubMedView Article
- Shen KF, Crain SM: Ultra-low doses of naltrexone or etorphine increase morphine's antinociceptive potency and attenuate tolerance/dependence in mice. Brain Res 1997, 757: 176–190. 10.1016/S0006-8993(97)00197-2PubMedView Article
- Chindalore VL, Craven RA, Peony Yu K, Butera PG, Burns LH, Friedmann N: Adding ultra-low dose naltrexone to oxycodone enhances and prolongs analgesia: a randomized, controlled trial of oxytrex. J Pain 2005,6(6):392–399. 10.1016/j.jpain.2005.01.356PubMedView Article
- Webster LR, Butera PG, Moran LV, Wu N, Burns LH, Friedmann N: Oxytrex minimizes physical dependence while providing effective analgesia: a randomized controlled trial in low back pain. J Pain 2006,7(12):937–946. 10.1016/j.jpain.2006.05.005PubMedView Article
- DeLeo JA, Colburn RW, Rickman AJ, Yeager MP: Intrathecal catheterization alone induces neuroimmune activation in the rat. Eur J Pain 1997, 1: 115–122. 10.1016/S1090-3801(97)90069-0PubMedView Article
- Kurumaji A, Takashima M, Ohi K, Takahashi K: Circadian fluctuations in pain responsiveness and brain Met-enkephalin-like immunoreactivity in the rat. Pharmacol Biochem Behav 1988,29(3):595–599. 10.1016/0091-3057(88)90025-1PubMedView Article
- O'Callaghan JP, Miller DB: Nervous system-specific proteins as biochemical indicators of neurotoxicity. Trends Pharmacol Sci 1983, 4: 388–390. 10.1016/0165-6147(83)90457-1View Article
- Raghavendra V, Rutkowski MD, DeLeo JA: The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham-operated rats. J Neurosci 2002,22(22):9980–9989.PubMed
- Tawfik VL, LaCroix-Fralish ML, Nutile-McMenemy N, DeLeo JA: Transcriptional and translational regulation of glial activation by morphine in a rodent model of neuropathic pain. J Pharmacol Exp Ther 2005,313(3):1239–1247. 10.1124/jpet.104.082420PubMedView Article
- Narita M, Suzuki M, Narita M, Yajima Y, Suzuki R, Shioda S, Suzuki T: Neuronal protein kinase Cγ-dependent proliferation and hypertrophy of spinal cord astrocytes following repeated in vivo administration of morphine. Eur J Neurosci 2004, 19: 479–484. 10.1111/j.0953-816X.2003.03119.xPubMedView Article
- Cui Y, Chen Y, Zhi JL, Guo RX, Feng JQ, Chen PX: Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance. Brain Res 2006,1069(1):235–243. 10.1016/j.brainres.2005.11.066PubMedView Article
- Raghavendra V, Tanga F, DeLeo JA: Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther 2003,306(2):624–630. 10.1124/jpet.103.052407PubMedView Article
- Sweitzer SM, Colburn RW, Rutkowski M, DeLeo JA: Acute peripheral inflammation induces moderate glial activation and spinal IL-1beta expression that correlates with pain behaviour in the rat. Brain Res 1999,829(1–2):209–221. 10.1016/S0006-8993(99)01326-8PubMedView Article
- Kreutzberg GW: Microglia: a sensor for pathological events in the CNS. Trends NeuroscI 1996,19(8):312–318. 10.1016/0166-2236(96)10049-7PubMedView Article
- Tsai RY, Tai YH, Tzeng JI, Lin SL, Shen CH, Yang CP, Hsin ST, Wang CB, Wong CS: Ultra-low dose naloxone restores the antinociceptive effect of morphine in pertussis toxin-treated rats and prevents glutamate transporter downregulation by suppressing the p38 mitogen-activated protein kinase signaling pathway. Neuroscience 2009, 159: 1244–1256. 10.1016/j.neuroscience.2009.01.058PubMedView Article
- Crain SM, Shen KF: Opioids can evoke direct receptor-mediated excitatory effects on sensory neurons. Trends Pharmacol Sci 1990, 11: 77–81. 10.1016/0165-6147(90)90322-YPubMedView Article
- Wang HY, Frankfurt M, Burns LH: High-affinity naloxone binding to filamin A prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence. PLoS ONE 2008,3(2):e1554. 10.1371/journal.pone.0001554PubMed CentralPubMedView Article
- Hutchinson MR, Bland ST, Johnson KW, Rice KC, Maier SF, Watkins LR: Opioid-induced glial activation: mechanisms of activation and implications for opioid analgesia, dependence, and reward. ScientificWorldJournal 2007, 7: 98–111. 10.1100/tsw.2007.57PubMedView Article
- La Vincente SF, White JM, Somogyi AA, Bochner F, Chapleo CB: Enhanced buprenorphine analgesia with the addition of ultra-low dose naloxone in healthy subjects. Clin Pharmacol Ther 2008,83(1):144–152. 10.1038/sj.clpt.6100262PubMedView Article
- Imasogie NN, Singh S, Watson JT, Hurly D, Morley-Forster P: Ultra low dose naloxone and tramadol/acetaminophen in elderly patients undergoing joint replacement surgery: A pilot study. Pain Res Manage 2009,14(2):103–108.
- Garrison CJ, Dougherty PM, Kajander KC, Carlton SM: Staining of glial fibrillary acidic protein (GFAP) in lumbar spinal cord increases following a sciatic nerve constriction injury. Brain Res 1991, 565: 1–7. 10.1016/0006-8993(91)91729-KPubMedView Article
- Sweitzer SM, Schubert P, DeLeo JA: Propentofylline, a glial modulating agent, exhibits antiallodynic properties in a rat model of neuropathic pan. J Pharmacol Exp Ther 2001, 297: 1210–1217.PubMed
- Jin SX, Zhuang ZY, Woolf CJ, Ji RR: P38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neuroscience 2003, 23: 4017–4022.PubMed
- Tsuda M, Mizokoshi A, Shigemoto-Mogami Y, Koizumi S, Inoue K: Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia 2004, 45: 89–95. 10.1002/glia.10308PubMedView Article
- Arner S, Meyerson BA: Lack of analgesic effect of opioids on neuropathic and idiopathic forms of pain. Pain 1988, 33: 11–23. 10.1016/0304-3959(88)90198-4PubMedView Article
- Watkins LR, Hutchinson MR, Ledeboer A, Wieseler-Frank J, Milligan ED, Maier SF: Glial as the "bad guys": implications for improving clinical pain control and the clinical utility of opioids. Brain Behav Immun 2007,21(2):131–146. 10.1016/j.bbi.2006.10.011PubMed CentralPubMedView Article
- Largent-Milnes TM, Guo W, Wang HY, Burns LH, Vanderah TW: Oxycodone Plus Ultra-Low-Dose Naltrexone Attenuates Neuropathic Pain and Associated μ-Opioid Receptor-Gs Coupling. J Pain 2008,9(8):700–713. 10.1016/j.jpain.2008.03.005PubMedView Article
- Gouarderes C, Sutak M, Zajac JM, Jhamandas K: Role of adenosine in the spinal antinociceptive and morphine modulatory actions of neuropeptide FF analogs. Eur J Pharmacol 2000, 406: 391–401. 10.1016/S0014-2999(00)00716-0PubMedView Article
- Suter MR, Berta T, Gao YJ, Decosterd I, Ji RR: Large A-fiber activity is required for microglial proliferation and p38 MAPK activation in the spinal cord: different effects of resiniferatoxin and bupivacaine on spinal microglial changes after spared nerve injury. Mol Pain 2009, 5: 53. 10.1186/1744-8069-5-53PubMed CentralPubMedView Article
- Holdridge SV, Armstrong SA, Taylor AMW, Cahill CM: Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance. Mol Pain 2007, 3: 7. 10.1186/1744-8069-3-7PubMed CentralPubMedView Article
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