Skip Navigation LinksHome > June 2009 - Volume 110 - Issue 6 > Amitriptyline Suppresses Neuroinflammation-dependent Interle...
doi: 10.1097/ALN.0b013e31819fccd5
Pain Medicine

Amitriptyline Suppresses Neuroinflammation-dependent Interleukin-10-p38 Mitogen-activated Protein Kinase-Heme Oxygenase-1 Signaling Pathway in Chronic Morphine-infused Rats

Tai, Yueh-Hua Ph.D.*; Tsai, Ru-Yin M.S.†; Lin, Shinn-Long M.D.†; Yeh, Chun-Chang M.D.‡; Wang, Jhi-Joung M.D., Ph.D.§; Tao, Pao-Luh Ph.D.∥; Wong, Chih-Shung M.D., Ph.D.#

Free Access
Article Outline
Collapse Box

Author Information

Collapse Box


Background: This study explores the underlying mechanism of the antiinflammatory effect of amitriptyline in chronic morphine-infused rats.
Methods: Male Wistar rats were implanted with two intrathecal catheters. One catheter was for the continuous infusion of saline, amitriptyline (15 μg/h), morphine (15 μg/h), p38 mitogen-activated protein kinase inhibitor SB203580 (0.5 μg/h), morphine plus amitriptyline, or morphine plus amitriptyline plus SB203580 for 5 days. The other catheter was used for daily intrathecal injection of anti–interleukin-10 (IL-10) antibody or heme oxygenase-1 inhibitor zinc protoporphyrin for 5 days.
Results: Amitriptyline/morphine coinfusion upregulated IL-10 protein expression in microglia; this was not observed in morphine-infused rats. Anti–IL-10 antibody effectively neutralized the amitriptyline-induced IL-10 expression in chronic morphine-infused rats. In addition, coinfusion of amitriptyline restored the antinociceptive effect of morphine (a 4.8-fold right-shift of the morphine dose-response curve compared to a 77.8-fold right-shift in its absence), and the injection of anti–IL-10 antibody or coinfusion of SB203580 partially reversed the effect of amitriptyline on the antinociceptive effect of morphine in morphine-infused rats (a 17.9-fold and 15.1-fold right-shift in morphine dose-response curves). Anti–IL-10 antibody and SB203580 significantly inhibited the amitriptyline-induced p38 mitogen-activated protein kinase and heme oxygenase-1 expression and the associated antiinflammatory effect of amitriptyline. Daily injection of zinc protoporphyrin also demonstrated that it reverses the effect of amitriptyline in morphine’s antinociception and antiinflammation in chronic morphine-infused rats.
Conclusions: These results suggest that the antiinflammatory effect of amitriptyline on morphine tolerance, probably acting by increasing IL-10 expression, is mediated by p38 mitogen-activated protein kinase heme oxygenase-1 signal transduction cascade.
MORPHINE is one of the most potent analgesic drugs for clinical pain management. However, its use for chronic pain conditions is limited by the development of tolerance.1 Morphine tolerance is a complex physiologic response; besides opioid receptor uncoupling and downregulation,2 glutamatergic receptor activation and neuroinflammation have also been demonstrated by us and others.3–6 Opioid-induced hyperalgeisa is observed in tolerance; it is similar to the symptom observed in neuropathic pain, where opioids also have with limited analgesic effect. Proinflammatory cytokines and chemokines have been shown to modulate morphine tolerance and associated mechanical allodynia and thermal hyperalgesia.7 Administration of glial metabolic inhibitors, cytokine antagonists, antiinflammatory cytokine interleukin (IL)-10, or IL-1 receptor antagonist IL-1ra has been shown to attenuate the development of opioid tolerance and to prevent hyperalgesia and allodynia.4,8
Tricyclic antidepressants (TCAs) are commonly used for treating major depressive disorders and are also widely used in chronic pain states, such as neuropathic and inflammatory pains.9 TCAs produce analgesia by various mechanisms involving N-methyl-d-aspartate receptors, biogenic amines, opioids, inflammatory mediators, and substance P.10 Studies have shown that intrathecal amitriptyline administration effectively attenuates pain and thermal hyperalgesia in inflammatory and neuropathic pain in rats,11 particularly when combined with opiates and clonidine.12,13 Moreover, in our recent study, we found that morphine tolerance is associated with downregulation of spinal glutamate transporters (GLAST, GLT-1, and EAAC1) and increasing of excitatory amino acids aspartate and glutamate concentration in spinal cerebrospinal fluid dialysates.6 Coadministration of amitriptyline reversed the GLAST and GLT-1 expression and excitatory amino acids elevation, restoring the antinociceptive effect of morphine in chronic morphine-infused rats; this spinal microglia activation and increasing of cytokine tumor necrosis factor-α (TNFα), IL-1β, and IL-6 expression were also suppressed by the amitriptyline coinfusion.6 Furthermore, our recent study demonstrated spinal neuroinflammation, microglia activation, and induction of proinflammatory cytokine expression in peretussis toxin-treated rats.14
IL-10 is a key molecule in controlling inflammation with a wide spectrum of biologic effects on lymphoid and myeloid cells.15 Previous study has shown that antiinflammatory cytokine IL-10 is stimulated by antidepressants.16 An inhibitory effect of IL-10 on lippolysaccharide-induced thermal and mechanical hyperalgesia in mice was attributed to the inhibition of the production of proinflammatory cytokines, including TNF-α, IL-1β, and nerve growth factor.17 IL-10 was also demonstrated to attenuate the thermal hyperalgesia induced by chronic nerve constriction injury.18 In murine macrophages, IL-10 induces expression of heme oxygenase-1 (HO-1), a stress-induced protein with a potent antiinflammatory effect.19 Studies have shown that carbon monoxide and p38 mitogen-activated protein kinase (MAPK)-dependent mechanisms can positively feedback-modulate IL-10 and HO-1 activity, which amplifies the antiinflammatory capacity of IL-10.19 In the current study, therefore, we examined the effect of amitriptyline on inhibition of spinal cord neuroinflammation for its possible mechanisms. We particularly examined the role of IL-10 and its downstream p38MAPK-HO-1 signal transduction pathway in inhibiting proinflammatory cytokine expression in chronic morphine-infused rats. Hopefully, our results will provide the rationale for using of TCAs in clinical pain management, particularly in patients who suffer from neuropathic pain and need long-term opioid treatment.
Back to Top | Article Outline

Materials and Methods

Animal Preparation and Drug Administration
All experiments conformed to the Guiding Principles in the Care and Use of Animals of the American Physiology Society and were approved by the National Defense Medical Center Animal Care and Use Committee (National Defense Medical Center, Taipei, Taiwan). Animal preparation and intrathecal catheter implantation were as described in our previous study.10 Briefly, male Wistar rats (300–350 g) were anesthetized with phenobarbital (50 mg/kg, intra-peritoneal; Sigma, Saint Louis, MO), and two intrathecal catheters were implanted via the atlantooccipital membrane into the intrathecal space at the level of the lumbar enlargement of the spinal cord (L1–L2). Intrathecal catheters were constructed using an 8-cm polyethylene tube (0.008 inch ID, 0.014 inch OD) and a 3.5-cm silastic tube. The silastic tube was inserted into the polyethylene tube and sealed with epoxy resin and silicon rubber. One was connected to a miniosmotic pump for continuous infusion of saline (1 μl/h), amitriptyline (15 μg/h; Sigma), p38 MAPK inhibitor SB203580 (0.5 μg/h; Sigma), morphine (15 μg/h; Sigma), amitriptyline/morphine, or amitriptyline/morphine/SB203580 for 5 days. The other catheter was used for a single daily injection of the anti–IL-10 antibody (10 μg; 1–10 μg as indicated; Pierce-Endogen, Thermo Fisher Scientific Inc, Waltham, MA) or HO-1 antagonist zinc protoporphyrin (24 μg; 6–24 μg as indicated; Tocris Cookson Ltd., Bristol, United Kingdom) for 5 days. Rats received a mouse isotype-matched irrelevant mAb (Pierce-Endogen) as negative control. On day 5, after a discontinuation of drug infusion for 3 h, when tail-flick latency had returned to the baseline level (around 2 s), morphine’s dose-response curves were constructed. All rats were housed individually and maintained on a 12-h light/dark cycle with food and water freely available. Rats with any neurologic deficits were excluded from the study (< 10%).
Back to Top | Article Outline
Behavioral Tests
Tail-flick latency was measured using the hot water immersion test (52 ± 0.5°C) before drug infusion and daily after the start of infusion for 5 days. All rats were placed in plastic restrainers for the antinociceptive test. The average baseline tail-flick test latency was 2 ± 0.5 s in naïve rats, and the cutoff time was 10 s. The percentage of the maximal possible antinociceptive effect was calculated as (maximum latency − baseline latency)/(cutoff latency − baseline latency) × 100. Antinociceptive dose-response curves were constructed for each study group.
Back to Top | Article Outline
Spinal Cord Preparation and Western Blot Analysis
After drug infusion, all rats were sacrificed, and the dorsal portion of the lumbar spinal cord enlargement was immediately removed and homogenized in ice-cold lysis buffer, consisting of 50 mm Tris, pH 7.5, 150 mm NaCl, 2% Triton X-100, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and phosphatase inhibitors. The lysate was centrifuged at 12,000g for 30 min at 4°C, and the supernatant was then used for Western blotting. The protein concentration of the samples was determined by the BCA method (Pierce, Thermo Fisher Scientific Inc, Waltham, MA) using bovine serum albumin as the standard. Equal amounts of total protein (20 μg) were adjusted to a similar volume with loading buffer (10% SDS, 20% glycerin, 125 mm Tris, 1 mm EDTA, 0.002% bromophenol blue, 10% β-mercaptoethanol), denatured by heating at 95°C for 5 min, separated on 10% sodium dodecyl sulfate–polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with polyclonal rabbit anti-p44/p42 MAPK (1:1000), anti-phospho-p44/p42 MAPK (1:500), anti-SAPK/JNK (1:1000), anti-phospho-SAPK/JNK (1:500), anti-p38 MAPK (1:1000), or anti-phosphor-p38 MAPK (1:500) antibodies (all from Cell Signaling, Danvers, MA) or polyclonal rabbit anti-HO-2 (1:2000) or monoclonal mouse anti-HO-1 (1:2000) antibodies (both from Stressgen, Victoria, British Columbia, Canada), and then incubated for 1 h at room temperature with appropriate horse radish peroxidase-conjugated secondary antibodies (1:2500; donkey anti-rabbit or anti-mouse immunoglobulin; Chemicon, Temecula, CA). Membrane-bound secondary antibodies were detected using the Chemiluminescenceplus reagent (PerkinElmer LAS, Boston, MA) and visualized using a chemiluminescence imaging system (Syngene, Cambridge, United Kingdom). Finally, the blots were incubated for 18 min at 56°C in stripping buffer (62.6 mm Tris-HCl, pH 6.7, 2% SDS, 100 mm mercaptoethanol) and reprobed with monoclonal mouse anti-β-actin antibody (1:2500; Sigma) as a loading control. The Western blot analysis was repeated three times. The density of each specific band was measured using a computer-assisted imaging analysis system (Gene Tools Match software, Syngene).
Back to Top | Article Outline
Cytokine Proteins Measurement
Spinal cord lysates were analyzed for four cytokines in a simultaneous multiplexed format using a microbead-based and flow-based protein detection system (Bio-Plex Suspension Array System, Bio-Rad Laboratories Inc., Hercules, CA) based on the Luminex xMAP technology (Luminex Molecular Diagnostics Inc., Toronto, Ontario, Canada). In this quantitative assay system, the surfaces of fluorescence-coded microbeads are conjugated to specific antibodies directed against four cytokines target. Each sample was first incubated with a mixture of all microbead types for 120 min at room temperature. After wash, samples were incubated with a mixture of secondary biotinylated detection antibodies also directed against each target for 30 min at room temperature, then washed again and incubated with a streptavidin-coupled phycoerythrin reporter system for 10 min at room temperature. After a final wash, the samples were resuspended in assay buffer and subjected to flow cytometric analysis. The Bio-Plex system instrumentation incorporates fluidics, laser excitation, fluorescence detection, and digital signal processing in a manner that allows for individual scanning and identification of microbeads. Each bead taken from the samples was identified on the basis of its internal fluorescence signature, and the phycoerythrin reporter signal associated with that bead was quantitated. A minimum 100 microbeads per each of the four targets were analyzed in each spinal cord sample. All spinal cord lysates were assayed in triplicate. Observed concentrations of each target cytokine were determined on the basis of an appropriate set of recombinant rat cytokine internal standard curves.
Back to Top | Article Outline
Fluorescent Immunocytochemistry and Image Analysis
After drug infusion, the rats were sacrificed by exsanguination under isoflurane anesthesia, and the lumbar spinal cord was immediately removed, embedded in optimal cutting temperature compound (Sakura Finetec Inc, Torrance, CA), and frozen at −20°C. Sections (5 μm) were prepared, air-dried on microscope slides for 30 min at room temperature, and fixed by immersion for 10 min in ice-cold acetone/methanol (1:1). After washing three times in ice-cold phosphate-buffered saline, the sections were incubated with blocking solution (0.01% Triton, 4% fetal bovine serums; 1 h). They were then double-labeled by incubation overnight at 4°C with (1) fluorescin isothiocyanate-labeled (green fluorescence) mouse monoclonal anti-rat CD11b/c antibodies (OX42 1:10, for macroglia; Chemicon, Temecula, CA) and unlabeled polyclonal goat anti-IL-10 antibodies (1:50; R&D System, Minneapolis, MN) and then for 1 h at room temperature with rhodamine-labeled guinea pig anti-goat immunoglobulin antibodies (1:200; R&D System), (2) FITC-labeled neuron-specific nuclear protein antibodies (NeuN 1:100; Chemicon) and unlabeled monoclonal mouse anti-HO-1 antibodies (1:500; Stressgen, Ann Arbor, MI) and then for 1 h at room temperature with rhodamine-labeled goat anti-mouse immunoglobulin antibodies (1:200; R&D System), or (3) unlabeled monoclonal mouse anti-HO-1 antibodies and unlabeled polyclonal rabbit anti-phopho-p38 MAPK antibodies (source) and then for 1 h at room temperature with FITC-labeled goat anti-mouse immunoglobulin antibodies and rhodamine-labeled anti-rabbit antibodies. After three phosphate-buffered saline rinses, the sections were incubated in 1% methanol in phosphate-buffered saline containing 20 μg/ml of 4′,6-diamidino-2-phenylindole (Sigma) (blue fluorescence). Images were captured using an Olympus BX 50 fluorescence microscope (Olympus Optical, Tokyo, Japan) and a Delta Vision disconsolation microscopic system operated by SPOT software (Diagnostic Instruments Inc. Sterling Heights, MI).
Back to Top | Article Outline
Data and Statistical Analysis
All values are presented as the mean ± SEM and analyzed using SigmaStat 3.0 (SYSTAT Software Inc., San Jose, CA). Tail-flick latency data were analyzed by using two-way analysis of variance (ANOVA) with post hoc Bonferroni test. Values for the analgesic dose of 50% of the maximal possible antinociceptive effect (AD50) were analyzed using a computer-assisted linear regression program SigmaPlot 10.0 (SYSTAT Software Inc.). The 95% confidence interval (CI) was calculated using pharmacologic calculations system PHARM/PCS version 4.2 (PharmSoft.Net, Wynnewood, PA). For immunoreactivity data, the intensity of each test band was expressed as the relative optical density calculated with respect to the average optical density for the corresponding control band. For statistical analysis, immunoreactivity and cytokine data were analyzed by one-way analysis of variance (ANOVA), followed by multiple comparisons with the Student-Newman-Keuls post hoc test. A significant difference was defined as a P value less than 0.05.
Back to Top | Article Outline


Neutralization of Microglia IL-10 Protein Expression by Anti-IL-10 Antibody in Amitriptyline/morphine Coinfused Rats
Fig. 1
Fig. 1
Image Tools
As shown in figure 1A, analysis of lumbar spinal cord homogenates demonstrated that intrathecal daily injection of anti-IL-10 antibody (10 μg/5 μl) alone did not affect IL-10 expression compared to saline-infused rats. However, coinfusion amitriptyline/morphine significantly increases the antiinflammatory cytokine IL-10 protein levels compared with saline-, amitriptyline-, and morphine-infused rats. Daily injection of anti-IL-10 antibody significantly neutralized the effect of amitriptyline in morphine-infused rats (F(5,18) = 24.35, P < 0.001). Additional experiments in which the rats received an isotype-matched irrelevant Ig show that the IL-10 protein level (956.7 ± 68.35 ng/ml) compared with amitriptyline/morphine–coinfused rats (906.67 ± 52.14 ng/ml) (P = 0.582, NS). As shown in figure 1B, weak immunocytochemistry staining for microglia (OX42-positive cells; green) was distributed throughout the rat spinal cord, and the stained cells had the classic ramified shape of nonactivated microglia in saline-infused, amitriptyline-infused, and anti-IL-10 antibody injection-only rats. In contrast, strong OX42 staining with the amoeboid shape of activated microglia was observed throughout the section, but no IL-10 protein expression was apparent in chronic morphine-infused rats. Overlay of the sections stained for IL-10 and microglia showed colocalization of IL-10 with OX42 immunoreactivity (white arrows) in the spinal cord of amitriptyline plus morphine-infused rats, and additional treatment with anti-IL-10 antibody resulted in a pattern similar to that of morphine-infused rats.
Back to Top | Article Outline
Neutralization of IL-10 Expression or Blockade of p38 MAPK Pathway Partially Reverses the Effect of Amitriptyline on Morphine Tolerance
Fig. 2
Fig. 2
Image Tools
Table 1
Table 1
Image Tools
As shown in figure 2A, measured by the hot water immersion test, daily intrathecal injection of anti-IL-10 antibody, or continuous infusion of amitriptyline (15 μg/h) or SB203580 (0.5 μg/h) alone had no antinociceptive effect throughout the 5 days. As in our previous study,6 the maximal antinociceptive effect of morphine was seen on day 1, and maximal tolerance was observed from day 3 in morphine-infused rats (F(5,235) = 23.447, P < 0.001). Amitriptyline (15 μg/h) prevented the reduction of the antinociceptive effect of morphine seen in chronic morphine-infused rats. Daily injection of anti-IL-10 antibody or coinfusion of SB203580 with amitriptyline plus morphine blocked the maintenance of morphine’s antinociceptive effect by amitriptyline (F(47,235) = 6.231, P < 0.001). As shown in figure 2B and table 1, amitriptyline, anti-IL-10 antibody, and SB203580 alone did not affect the dose-response curve of morphine, with an AD50 of 1.13 μg in saline-infused rats, 1.3 μg in amitriptyline-infused rats, 1.24 μg in anti-IL-10 antibody injection rats, and 1.23 μg in SB203580-infused rats. In morphine-infused rats, the dose–response curve was shifted to the right by 77.8-fold compared to saline-infused rats (AD50, 87.96 μg), amitriptyline/morphine coinfusion shifted the dose-response curve to the left compared to morphine infusion alone (AD50, 5.47 μg), wheras injection of anti-IL-10 antibody or coinfusion of SB203580 produced a rightward shift compared to amitriptyline/morphine-infused rats (AD50, 20.23 μg and 17.03 μg, respectively).
Back to Top | Article Outline
Effect of Neutralizing Anti-IL-10 Antibody and SB203580 on MPAK and HO-1 Expression in Amitriptyline/morphine Coinfused Rats
Fig. 3
Fig. 3
Image Tools
Fig. 4
Fig. 4
Image Tools
Fig. 5
Fig. 5
Image Tools
Figure 3 shows that expression of external signal-regulated kinase (ERK), phospho-ERK (p-ERK), c-Jun N-terminal kinase (JNK), and phospho-JNK (p-JNK) at day 5 in the spinal cord dorsal horn was unaffected by any of the treatments (P = 0.721, 0.632, 0.541, and 0.729, respectively). These results suggest that neither ERK nor JNK mediated amitriptyline-induced IL-10 expression. Figure 4 shows that chronic intrathecal infusion of morphine caused a slight, but significant increase in the level of phosphorylated p38 MAPK (p-p38 MAP) (fig. 4, A and C), but it had no effect on total p38 MAPK, HO-1, or HO-2 levels (fig. 4, B and D). In contrast, amitriptyline/morphine coinfusion significantly increased not only p-p38 MAPK expression (F(4,35) = 820, P < 0.001), but also total p38 MAPK expression (F(4,35) = 25.759, P < 0.001) (fig. 4, A and C) and HO-1 expression (F(4,35) = 84.821, P < 0.001) (fig. 4, B and D), and this injection of anti-IL-10 antibody or infusion of amitriptyline or SB203580 alone had no effect on protein expression (data no shown). As expected, HO-2 expression was not affected by any of the treatments (P = 0.824, NS) (fig. 4, B and D). The effects of amitriptyline/morphine coinfusion on p38 MAPK phosphorylation and HO-1 expression were completely blocked by both daily injection of anti-IL-10 antibody and coinfusion of the p38 MAPK inhibitor SB203580 (fig. 4, A–D). In the current study, we also found that daily intrathecal injection of various doses of anti-IL-10 antibody for 5 days dose-dependently reversed the amitriptyline-induced upregulation of p38 MAPK phosphorylation (F(4,35) = 723.242, P < 0.001) and HO-1 expression (F(4,35) = 39.368, P < 0.001) in morphine-infused rats (fig. 5, A and B). These results suggest that IL-10–induced p38 MAPK phosphorylation is involved in the increase in HO-1 expression in amitriptyline/morphine-infused rats.
Back to Top | Article Outline
Zinc Protoporphyrin Reverses the Effect of Amitriptyline in HO-1 Expression and Morphine’s Antinociception in Chronic Morphine-infused Rats
Fig. 6
Fig. 6
Image Tools
Table 2
Table 2
Image Tools
Daily intrathecal injection of various doses of zinc protoporphyrin for 5 days dose-dependently reversed the upregulation of HO-1 expression (F(4,35) = 41.056, P < 0.001; fig. 6A), and it blocked the maintenance of morphine’s antinociceptive effect by amitriptyline (F(29,145) = 6.682, P < 0.001) in morphine-infused rats (fig. 6B). The HO-2 expression (P = 0.388, NS) was not affected by zinc protoporphyrin injection. In agreement with the above results, the maximal antinociceptive effect of morphine was seen on day 1, and maximal tolerance was observed from day 3 in morphine-infused rats (F(5,145) = 19.902, P < 0.001). Coadministration of HO-1 antagonist zinc protoporphyrin (24 μg/5 μl) with amitriptyline plus morphine blocked the maintenance of morphine’s antinociceptive effect by amitriptyline and produced a rightward shift in dose-response curves (AD50, 22.78 μg; table 2).
Back to Top | Article Outline
Inhibition of IL-10 Expression, p38 MAPK Pathway, and HO-1 Expression Reverses the Suppressive Effect of Amitriptyline on Cytokine Production in Morphine-infused Rats
Fig. 7
Fig. 7
Image Tools
As shown in figure 7, protein levels for the proinflammatory cytokines, TNF-α (F(5,18) = 58.817, P < 0.001), IL-1β (F(5,18) = 20.317, P < 0.001), and IL-6 (F(5,18) = 102.068, P < 0.001), were significantly increased in the morphine-infused rats, and this effect was significantly attenuated by amitriptyline coinfusion. Furthermore, anti-IL-10 antibody injection, SB203580 coinfusion, and HO-1 antagonist zinc protoporphyrin injection significantly abolished the suppressive effect of amitriptyline on proinflammatory cytokine production in morphine-infused rats.
Back to Top | Article Outline
Amitriptyline/morphine Coinfusion Induces p38 MAPK and HO-1 Expression in Spinal Cord Neurons
Fig. 8
Fig. 8
Image Tools
Fluorescence images of spinal cords from amitriptyline/morphine-coinfused rats, single-staining for neurons (green), HO-1 (red), or nuclei (4′,6-diamidino-2-phenylindole, blue) are shown in the upper row in figure 8. As seen in the merged images, HO-1 expression colocalized with neurons (yellow on the merged image in panel D) and was around the nuclear membrane, with some translocated into the nucleus (purple in the merged image). The lower row in figure 8 shows single-staining for HO-1 (green), p-p38 MAPK (red), or 4′,6-diamidino-2-phenylindole (blue); a similar distribution pattern of HO-1 and p-p38 MAPK was seen (yellow in the merged image in panel H). These results indicate a close association among neurons, HO-1, and p-p38 MAPK in the spinal cord dorsal horn.
Back to Top | Article Outline


In the current study, long-term morphine infusion significantly increased protein levels of proinflammatory cytokines, TNF-α, IL-1β, and IL-6, and caused slight but significant phosphorylation of p38 MAPK. Amitriptyline/morphine coinfusion increased the expression of IL-10 in nonactivated microglia, and increasing of p38 MAPK phosphorylation subsequently induced HO-1 expression in neurons, thus further inhibiting the expression of proinflammatory cytokines; however, the protein expression on ERK, p-ERK, JNK, and p-JNK proteins in the rat spinal cord were not affected. Neutralization of IL-10 expression and blockade of p38 MAPK pathway reverses the amitriptyline-induced increase in HO-1 expression and inhibition of proinflammatory cytokines expression, suggesting that the p38 MAPK pathway, but not the ERK or JNK pathways, is involved in the amitriptyline-mediated induction of HO-1 expression. Moreover, inhibition of IL-10 expression, p38 MAPK pathway, or HO-1 expression partially blocked the inhibitory effect of amitriptyline on the morphine’s antinociceptive tolerance, and it shifted the dose-response curve of morphine to the right from an AD50 of 5.47 μg (morphine plus amitriptyline) to 20.23 μg (morphine plus amitriptyline plus anti-IL-10Ab injection), 17.03 μg (morphine plus amitriptyline plus SB203580), and 22.8 μg (morphine plus amitriptyline plus zinc protoporphyrin injection). These results suggest that the suppressive effect of amitriptyline coinfusion on proinflammatory cytokine production in morphine-infused rats may act through increasing IL-10 protein expression in microglia, which activates the neuronal p38 MAPK pathway and increases HO-1 expression.
Studies have shown that TCAs inhibit the release of inflammatory mediators. Imipramine, clomipramine, and citalopram at low micromolar concentrations partially blocked IL-6, IL-1β, and TNF-α release from human blood monocytes and IL-2 and interferon-γ from T cells.20 Similarly, fluoxetine and amitriptyline were shown to inhibit release of nitric oxide and prostaglandin E2 from human synovial cells.21 A previously performed study also showed that production of the antiinflammatory cytokine IL-10 was shown to be stimulated by antidepressants.16 These changes resulted in a decrease of hypersensitization of the nociceptive neurons in the spinal cord, and amitriptyline, when applied peripherally, also prevented c-fos expression in spinal cord in the formalin pain model.22 In the current study, we also demonstrated that coinfusion of amitriptyline increased the expression of antiinflammatory cytokines IL-10 in morphine-infused rats.
A previous study demonstrated that IL-10 inhibited proinflammatory cytokine production.23 Local injection of IL-10 abolishes the mechanical hyperalgesia induced by carrageenan administration in the hind paw of rats.24 IL-10–encoding adenoviral vector not only attenuates the development of tolerance, hyperalgesia, and allodynia; it also potentiates the analgesic effect of morphine.7 Treatment with morphine has been shown to not alter IL-10 messenger RNA level and even reduces lippolysaccharide-induced IL-10 secretion.25,26 In the current study, an increase in IL-10 protein expression was observed in some microglia in the spinal cord of amitriptyline/morphine coinfused rats, these being nonactivated microglia, as opposed to the activated forms seen in morphine-infused rats. Anti-IL-10 antibody neutralized the amitriptyline induced IL-10 in activated microglia. It implies that the suppressive effect of amitriptyline in morphine-infused rats acts by increasing IL-10 protein expression.
A previous study showed that IL-10 exerts its antiinflammatory effect by upregulating HO-1 expression.19 Antiinflammatory effect of HO-1 has been demonstrated in several inflammatory models.27,28 HO-1 overexpression, via carbon monoxide-cyclic guanosine monophosphate (CO-cGMP) pathway, produces a significant antinociceptive effect against formalin pain in mice.29–31 These results show that HO-1 plays a crucial role in the host defense mechanism against inflammation; HO-1 catabolizes heme and produces antioxidants biliverdin and bilirubin, which also play a role in protection against tissue injury-induced inflammation. Carbon monoxide is a key molecule for the protective effect of HO-1.32,33 Scavenging of carbon monoxide by hemoglobin significantly reduces the effect of IL-10 on inhibiting the lippolysaccharide-induced TNF-α and nitric oxide production and increasing MMP-9 expression in macrophages.19 In addition, a recent study indicated that nuclear localization of HO-1 protein causes upregulation of AP-1 gene and promotes cytoprotection against oxidative stress.34 In our current study, we found that amitriptyline/morphine coinfusion significantly increased IL-10 and HO-1 expression, and it decreased the protein level of proinflammatory cytokines TNF-α, IL-1β, and IL-6. In contrast, the inhibition of IL-10 or HO-1 expression significantly downregulated HO-1 expression and increased TNF-α, IL-1β, and IL-6 protein level. These results suggest a role of HO-1 in the antiinflammatory effect of IL-10 in amitriptyline/morphine-coinfused rats. Taken together, carbon monoxide and HO-1 appear to be responsible for the protective effect of IL-10, and the detailed mechanisms need to be determined.
An earlier study revealed that the JAK1/STAT3-dependent pathway is not sufficient for the antiinflammatory effect of IL-10 receptor signaling,35 and the IL-10-activated phosphatidylinositol-3-kinase and p70 S6 kinase pathways are not essential for the antiinflammatory effect of IL-10.36 Moreover, studies have suggested that IL-10 and HO-1 produce a positive feedback circuit to amplify the antiinflammatory effect by upregulation of carbon monoxide-dependent and p38 MAPK-dependent mechanisms.19,37 The transcriptional activation of HO-1 is mediated by the ERK, JNK, and p38 MAPK pathways,38,39 and p38 MAPK is a key mediator of cellular stresses, such as inflammation and apoptosis. Both in vitro and in vivo studies have shown that p38 MAPK regulates the production of proinflammatory cytokines, nitric oxide, and prostaglandin E2 by increasing cytokine release or messenger RNA transcription.40–42 As known, increasing of proinflammatory cytokine expression in the spinal cord is associated with neuropathic pain and morphine tolerance.7,43 p38 MAPK is activated in neuropathic pain by peripheral inflammation and nerve injury.44–46 Morphine tolerance-associated microglia activation is also activated via p38 MAPK signaling pathway.47 Inhibition of p38 MAPK was considered to be a potential therapeutic target for antiinflammatory therapy.48 However, contradictory results were reported. Treatment with a p38 MAPK inhibitor resulted in an enhancement of TNF-α production in mast cells.49 Moreover, inhibition of p38 MAPK resulted in a reduction of cytokine production by mast cells, but increase cytokine release from lippolysaccharide-activated macrophages.50 These results suggest that p38 MAPK possesses both proinflammatory and antiinflammatory characteristics, and plays different roles in different inflammatory conditions, depending on the cell type and applied stimulus.
Communication between neurons and glia involves ion flux, neurotransmitters, cell adhesion molecules, and specialized signaling molecules released from synaptic and nonsynaptic regions of neurons.51 Morphine-induced glia activation and proinflammatory cytokine release are also caused by the induction of neuronal fractalkine release.7 Fractalkine, expressed in spinal neurons, is a diffusible signal that activates nearby microglia and then releases proinflammatory cytokines.52 Theses results suggest that morphine indirectly affects glia via chemokines conducting neuron-glia communication. Activated neuronal protein kinase Cγ (PKCγ) acts as an important factor to modulate the neuron-glia communication to increase astrocyte reactivity after repeated morphine treatment, and this neuron-glia communication may be responsible for the development of morphine tolerance.53 In the current study, we suggest that amitriptyline modulates neuroimmune responses via a two-way communication between glia and neurons. Amitriptyline stimulates microglia release of antiinflammatory cytokine IL-10 via a paracrine-manner and increases neuron phopho-p38 MAPK expression, which induces upregulation of HO-1 via carbon monoxide, exerts negative feedback control of microglia activity, and inhibits proinflammatory cytokine expression.
Activation of the neuroimmune system has been demonstrated to modulate morphine tolerance and consequently develops mechanical allodynia and thermal hyperalgesia.7,54 Administration of glial metabolic inhibitors, cytokine antagonist IL-10, or IL-1 receptor antagonist IL-1ra attenuates opioid tolerance and associated hyperalgesia and allodynia.4,8 Similarly, our previous study also demonstrated that amitriptyline inhibits microglia activation and proinflammatory cytokines expression, thus attenuating morphine tolerance.6 In our current study, we found that injection of anti-IL-10 antibody, or p38MAPK inhibitor SB203580, or HO-1 inhibitor zinc protoporphyrin increased proinflammatory cytokine expression in amitriptyline/morphine-coinfused rats and partially reversed the effect of amitriptyline on the antinociceptive effect of morphine seen in chronic morphine-infused rats.
Currently available medications for the management of chronic pain, particularly those for neuropathic pain, are either inadequate or cause unbearable side effects, and TCAs are known to be effective adjuvant for the treatment of chronic neuropathic pain in clinical practice.55,56 The current study shows that amitriptyline inhibits pro-inflammatory cytokine expression by increasing IL-10 expression; it leads to phosphorylation of p38 MAPK that increases HO-1 expression and thus inhibits the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in morphine-tolerant rats. From our recent studies and the results of current study,5,6 we suggest that amitripyline may be used as an adjuvant in combination with opioids for the treatment of chronic neuropathic pain conditions and patients who need long-term opioid administration for pain management in clinical practice. It explains why TCAs play an important role in the treatment of chronic neuropathic pain via systemic administration. The systemic effect and mechanisms of action of amitryptline in morphine tolerance development and neuropathic pain should require further investigation.
Back to Top | Article Outline


1. Ossipov MH, Lai J, King T, Vanderah TW, Malan TP Jr, Hruby VJ, Porreca F: Antinociceptive and nociceptive actions of opioids. J Neurobiol 2004; 61:126–48

2. Gintzler AR, Chakrabarti S: Opioid tolerance and the emergence of new opioid receptor-coupled signaling. Mol Neurobiol 2000; 21:21–33

3. Hsu MM, Wong CS: The roles of pain facilitatory systems in opioid tolerance. Acta Anaesthesiol Sin 2000; 38:155–66

4. 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; 22:9980–9

5. Tai YH, Wang YH, Tsai RY, Wang JJ, Tao PL, Liu TM, Wang YC, Wong CS: Amitriptyline preserves morphine’s antinociceptive effect by regulating the glutamate transporter GLAST and GLT-1 trafficking and excitatory amino acids concentration in morphine-tolerant rats. Pain 2007; 129:343–54

6. Tai YH, Wang YH, Wang JJ, Tao PL, Tung CS, Wong CS: Amitriptyline suppresses neuroinflammation and up-regulates glutamate transporters in morphine-tolerant rats. Pain 2006; 124:77–86

7. Johnston IN, Milligan ED, Wieseler-Frank J, Frank MG, Zapata V, Campisi J, Langer S, Martin D, Green P, Fleshner M, Leinwand L, Maier SF, Watkins LR: A role for proinflammatory cytokines and fractalkine in analgesia, tolerance, and subsequent pain facilitation induced by chronic intrathecal morphine. J Neurosci 2004; 24:7353–65

8. Song P, Zhao ZQ: The involvement of glial cells in the development of morphine tolerance. Neurosci Res 2001; 39:281–6

9. Onghena P, Van Houdenhove B: Antidepressant-induced analgesia in chronic non-malignant pain: A meta-analysis of 39 placebo-controlled studies. Pain 1992; 49:205–19

10. Sawynok J, Esser MJ, Reid AR: Antidepressants as analgesics: An overview of central and peripheral mechanisms of action. J Psychiatry Neurosci 2001; 26:21–9

11. Esser MJ, Sawynok J: Acute amitriptyline in a rat model of neuropathic pain: Differential symptom and route effects. Pain 1999; 80:643–53

12. Eisenach JC, Gebhart GF: Intrathecal amitriptyline. Antinociceptive interactions with intravenous morphine and intrathecal clonidine, neostigmine, and carbamylcholine in rats. Anesthesiology 1995; 83:1036–45

13. Gray AM, Spencer PS, Sewell RD: The involvement of the opioidergic system in the antinociceptive mechanism of action of antidepressant compounds. Br J Pharmacol 1998; 124:669–74

14. Tsai RY, Jang FL, Tai YH, Lin SL, Shen CH, Wong CS: Ultra-low-dose naloxone restores the antinociceptive effect of morphine and suppresses spinal neuroinflammation in PTX-treated rats. Neuropsychopharmacology 2008; 33:2772–82

15. Howard M, O’Garra A: Biological properties of interleukin 10. Immunol Today 1992; 13:198–200

16. Maes M, Song C, Lin AH, Bonaccorso S, Kenis G, De Jongh R, Bosmans E, Scharpe S: Negative immunoregulatory effects of antidepressants: Inhibition of interferon-gamma and stimulation of interleukin-10 secretion. Neuropsychopharmacology 1999; 20:370–9

17. Kanaan SA, Poole S, Saade NE, Jabbur S, Safieh-Garabedian B: Interleukin-10 reduces the endotoxin-induced hyperalgesia in mice. J Neuroimmunol 1998; 86:142–50

18. Wagner R, Janjigian M, Myers RR: Anti-inflammatory interleukin-10 therapy in CCI neuropathy decreases thermal hyperalgesia, macrophage recruitment, and endoneurial TNF-alpha expression. Pain 1998; 74:35–42

19. Lee TS, Chau LY: Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med 2002; 8:240–6

20. Xia Z, DePierre JW, Nassberger L: Tricyclic antidepressants inhibit IL-6, IL-1 beta and TNF-alpha release in human blood monocytes and IL-2 and interferon-gamma in T cells. Immunopharmacology 1996; 34:27–37

21. Yaron I, Shirazi I, Judovich R, Levartovsky D, Caspi D, Yaron M: Fluoxetine and amitriptyline inhibit nitric oxide, prostaglandin E2, and hyaluronic acid production in human synovial cells and synovial tissue cultures. Arthritis Rheum 1999; 42:2561–8

22. Heughan CE, Allen GV, Chase TD, Sawynok J: Peripheral amitriptyline suppresses formalin-induced Fos expression in the rat spinal cord. Anesth Analg 2002; 94:427–31

23. Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O’Garra A: IL-10 inhibits cytokine production by activated macrophages. J Immunol 1991; 147:3815–22

24. Poole S, Cunha FQ, Selkirk S, Lorenzetti BB, Ferreira SH: Cytokine-mediated inflammatory hyperalgesia limited by interleukin-10. Br J Pharmacol 1995; 115:684–8

25. Messmer D, Hatsukari I, Hitosugi N, Schmidt-Wolf IG, Singhal PC: Morphine reciprocally regulates IL-10 and IL-12 production by monocyte-derived human dendritic cells and enhances T cell activation. Mol Med 2006; 12:284–90

26. Nelson CJ, Lysle DT: Morphine modulation of the contact hypersensitivity response: Characterization of immunological changes. Clin Immunol 2001; 98:370–7

27. Otterbein L, Sylvester SL, Choi AM: Hemoglobin provides protection against lethal endotoxemia in rats: The role of heme oxygenase-1. Am J Respir Cell Mol Biol 1995; 13:595–601

28. Willis D, Moore AR, Frederick R, Willoughby DA: Heme oxygenase: A novel target for the modulation of the inflammatory response. Nat Med 1996; 2:87–90

29. Nascimento CG, Branco LG: Role of the peripheral heme oxygenase-carbon monoxide pathway on the nociceptive response of rats to the formalin test: Evidence for a cGMP signaling pathway. Eur J Pharmacol 2007; 556:55–61

30. Nascimento CG, Branco LG: Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats. Eur J Pharmacol 2008; 581:71–6

31. Rosa AO, Egea J, Lorrio S, Rojo AI, Cuadrado A, Lopez MG: Nrf2-mediated haeme oxygenase-1 up-regulation induced by cobalt protoporphyrin has antinociceptive effects against inflammatory pain in the formalin test in mice. Pain 2008; 137:332–9

32. Otterbein LE, Mantell LL, Choi AM: Carbon monoxide provides protection against hyperoxic lung injury. Am J Physiol 1999; 276:L688–94

33. Sato K, Balla J, Otterbein L, Smith RN, Brouard S, Lin Y, Csizmadia E, Sevigny J, Robson SC, Vercellotti G, Choi AM, Bach FH, Soares MP: Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J Immunol 2001; 166:4185–94

34. Lin Q, Weis S, Yang G, Weng YH, Helston R, Rish K, Smith A, Bordner J, Polte T, Gaunitz F, Dennery PA: Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress. J Biol Chem 2007; 282:20621–33

35. O’Farrell AM, Liu Y, Moore KW, Mui AL: IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: Evidence for Stat3-dependent and -independent pathways. Embo J 1998; 17:1006–18

36. Crawley JB, Williams LM, Mander T, Brennan FM, Foxwell BM: Interleukin-10 stimulation of phosphatidylinositol 3-kinase and p70 S6 kinase is required for the proliferative but not the antiinflammatory effects of the cytokine. J Biol Chem 1996; 271:16357–62

37. Naito Y, Takagi T, Yoshikawa T: Heme oxygenase-1: A new therapeutic target for inflammatory bowel disease. Aliment Pharmacol Ther 2004; 20(Suppl 1):177–84

38. Alam J, Wicks C, Stewart D, Gong P, Touchard C, Otterbein S, Choi AM, Burow ME, Tou J: Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor. J Biol Chem 2000; 275:27694–702

39. Zhang X, Bedard EL, Potter R, Zhong R, Alam J, Choi AM, Lee PJ: Mitogen-activated protein kinases regulate HO-1 gene transcription after ischemia-reperfusion lung injury. Am J Physiol Lung Cell Mol Physiol 2002; 283:L815–29

40. Da Silva J, Pierrat B, Mary JL, Lesslauer W: Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J Biol Chem 1997; 272:28373–80

41. Svensson CI, Hua XY, Protter AA, Powell HC, Yaksh TL: Spinal p38 MAP kinase is necessary for NMDA-induced spinal PGE(2) release and thermal hyperalgesia. Neuroreport 2003; 14:1153–7

42. Svensson CI, Marsala M, Westerlund A, Calcutt NA, Campana WM, Freshwater JD, Catalano R, Feng Y, Protter AA, Scott B, Yaksh TL: Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing. J Neurochem 2003; 86:1534–44

43. Milligan ED, Twining C, Chacur M, Biedenkapp J, O’Connor K, Poole S, Tracey K, Martin D, Maier SF, Watkins LR: Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci 2003; 23:1026–40

44. 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 Neurosci 2003; 23:4017–22

45. Kim SY, Bae JC, Kim JY, Lee HL, Lee KM, Kim DS, Cho HJ: Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury. Neuroreport 2002; 13:2483–6

46. 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

47. 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; 1069:235–43

48. Ono K, Han J: The p38 signal transduction pathway: Activation and function. Cell Signal 2000; 12:1–13

49. Zhang C, Baumgartner RA, Yamada K, Beaven MA: Mitogen-activated protein (MAP) kinase regulates production of tumor necrosis factor-alpha and release of arachidonic acid in mast cells. Indications of communication between p38 and p42 MAP kinases. J Biol Chem 1997; 272:13397–402

50. van den Blink B, Juffermans NP, ten Hove T, Schultz MJ, van Deventer SJ, van der Poll T, Peppelenbosch MP: p38 mitogen-activated protein kinase inhibition increases cytokine release by macrophages in vitro and during infection in vivo. J Immunol 2001; 166:582–7

51. Fields RD, Stevens-Graham B: New insights into neuron-glia communication. Science 2002; 298:556–62

52. Verge GM, Milligan ED, Maier SF, Watkins LR, Naeve GS, Foster AC: Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions. Eur J Neurosci 2004; 20:1150–60

53. Narita M, Suzuki M, Narita M, Yajima Y, Suzuki R, Shioda S, Suzuki T: Neuronal protein kinase C gamma-dependent proliferation and hypertrophy of spinal cord astrocytes following repeated in vivo administration of morphine. Eur J Neurosci 2004; 19:479–84

54. DeLeo JA, Tanga FY, Tawfik VL: Neuroimmune activation and neuroinflammation in chronic pain and opioid tolerance/hyperalgesia. Neuroscientist 2004; 10:40–52

55. Campbell JN, Meyer RA: Mechanisms of neuropathic pain. Neuron 2006; 52:77–92

56. Rintala DH, Holmes SA, Courtade D, Fiess RN, Tastard LV, Loubser PG: Comparison of the effectiveness of amitriptyline and gabapentin on chronic neuropathic pain in persons with spinal cord injury. Arch Phys Med Rehabil 2007; 88:1547–60

Cited By:

This article has been cited 7 time(s).

Naunyn-Schmiedebergs Archives of Pharmacology
Pharmacological activation of heme oxygenase (HO)-1/carbon monoxide pathway prevents the development of peripheral neuropathic pain in Wistar rats
Bijjem, KRV; Padi, SSV; Sharma, PL
Naunyn-Schmiedebergs Archives of Pharmacology, 386(1): 79-90.
A critical review of the mechanism of action for the selective serotonin reuptake inhibitors: Do these drugs possess anti-inflammatory properties and how relevant is this in the treatment of depression?
Walker, FR
Neuropharmacology, 67(): 304-317.
International Journal of Neuropsychopharmacology
Interferon-alpha induces nitric oxide synthase expression and haem oxygenase-1 down-regulation in microglia: implications of cellular mechanism of IFN-alpha-induced depression
Lu, DY; Leung, YM; Su, KP
International Journal of Neuropsychopharmacology, 16(2): 433-444.
Bmc Neurology
Pretreatment with intrathecal amitriptyline potentiates anti-hyperalgesic effects of post-injury intra-peritoneal amitriptyline following spinal nerve ligation
Cheng, KI; Wang, HC; Chang, LL; Wang, FY; Lai, CS; Chou, CW; Tsai, HP; Kwan, AL
Bmc Neurology, 12(): -.
Behavioural Brain Research
Ultra-low dose naloxone upregulates interleukin-10 expression and suppresses neuroinflammation in morphine-tolerant rat spinal cords
Lin, SL; Tsai, RY; Tai, YH; Cherng, CH; Wu, CT; Yeh, CC; Wong, CS
Behavioural Brain Research, 207(1): 30-36.
Pain Medicine
Intrathecal Granuloma Formation in a Patient Receiving Long-Term Spinal Infusion of Tramadol
De Andres, J; Vivo, JT; Palmisani, S; Perez, VLV; Minguez, A
Pain Medicine, 11(7): 1059-1062.

Plos One
Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons
Lin, HY; Yeh, WL; Huang, BR; Lin, CJ; Lai, CH; Lin, H; Lu, DY
Plos One, 7(): -.
ARTN e50138
Back to Top | Article Outline

© 2009 American Society of Anesthesiologists, Inc.

Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.

Article Tools