λ-carrageenan is a polysaccharide used in animal models of inflammation and hyperalgesia (1). Injected in the plantar surface of the rat or the mouse hindpaw, carrageenan induces inflammation with edema and hyperalgesia. It has been shown to induce inflammatory pain mediated through the production of inflammatory cytokines (2). Inflammation is the most important component of pain resulting from tissue injury. Cytokines, in particular, are important for regulation of the inflammatory response. The local release of cytokines (interleukin [IL] -1, IL-8, and tumor necrosis factor [TNF] -α) plays a major role in the sensory hypersensitivity associated with inflammation. The inflammatory response is essential for structural and functional repair of injured tissue, but excessive generation of proinflammatory signals can aggravate tissue damage because of the products derived from inflammatory cells. Some studies suggested that the treatment of the inflammatory response associated with pain could limit allodynia. Indeed, local anesthetics could modulate the inflammatory response (3). We have previously shown that a prolonged administration of bupivacaine via a sciatic nerve block was able to decrease both inflammation and hyperalgesia induced by carrageenan in the rat (4).
Opioids, which are potent antinociceptive drugs, modulate the immune function (5). Few studies have detailed the action of local anesthetics compared with opioids, and also, the mechanisms underlying these effects remain largely unknown. Of interest, the role of local anesthetics in modulating blood leukocyte response after a local hyperalgesic insult has not been investigated. However, long-lasting local anesthetics (bupivacaine and ropivacaine) are now extensively used for intraoperative anesthesia and also in the postoperative period for providing continuous pain relief. Indeed, these techniques of regional anesthesia were shown to reduce postoperative morbidity and to accelerate recovery (6,7).
In the present experiments, we explored the interactions of bupivacaine and carrageenan in mice on TNF-α, IL-1β, and IL-10 production by whole blood cultured in the presence of lipopolysaccharide (LPS) or heat-killed Staphylococcus Aureus Cowan (SAC).
All procedures were performed in accordance with the International Association for the Study of Pain guidelines for animal studies and after approval of our Regional Animal Use Committee. Male Balb/c mice 8 to 10 wk of age were used. The mice were kept on a 12-h light/dark cycle with free access to food and water.
Mice were assigned to one of four experimental groups (n = 21–24 per group). The control group (cont) consisted of mice receiving a subcutaneous injection of saline in the right hindpaw and of vehicle IM in the left leg. Group carr consisted of mice receiving a subcutaneous injection of carrageenan in the right hindpaw and of vehicle in the left leg. Group bupi consisted of mice receiving an IM injection of bupivacaine-loaded microspheres (BM) in the left leg and saline in the right hindpaw. Group carr + bupi consisted of mice receiving a subcutaneous injection of carrageenan in the right hindpaw and BM in the left leg. Mice were killed 15 h (according to the results of a pilot study) after carrageenan or saline injection, using exsanguination through cardiac puncture under general anesthesia with isoflurane (Baxter, Maurepas, France). The collected blood was kept into a heparinized syringe (heparin, 5 U) from which whole blood cultures were performed. In addition, 40 mice (10 per group) received the same treatment, and the blood was harvested for direct cytokine measurement in plasma.
Carrageenan (50 μL of 1% solution) was injected with a 25-gauge needle 1 h after bupivacaine or vehicle injection (time = zero). BM (0.15 mL of 100 mg/mL BM solution with a 70/30 wt/wt% formulation) were prepared by a solvent evaporation process (8). Bupivacaine content in microspheres was determined in triplicate. Bupivacaine concentration in plasma was measured 1 and 15 h after injection in 2 separate groups of 6 mice each using gas chromatography. The expected prolonged bupivacaine absorption process was achieved because the bupivacaine concentration in mice plasma was 4.65 ± 0.54 μg/mL and 0.19 ± 0.04 μg/mL (mean ± sem) 1 and 15 h after injection, respectively.
Blood (0.5 mL) collected during euthanasia was diluted 1:5 in Roswell Park Memorial Institute (RPMI)-1640 medium (Glutamax; Gibco-life Technologies, Paisley, United Kingdom) supplemented with antibiotics (penicillin, 100 UI/mL; Panpharma, Luitré-Fougères, France; and streptomycin, 100 μg/mL; Sigma, Saint Quentin-Fallavier, France). Five-hundred microliters of diluted blood was cultured in 20 4-well plates according to 3 different conditions: (a) without any stimulation (baseline), (b) with LPS (Escherichia coli O111 :B4), 10 μg/mL; Sigma), or (c) with SAC (Staphylococcus aureus Cowan, 100 μg/mL; Pansorbin Calbiochem, Fontenay sous Bois, France) in a 5% CO2 incubator for 24 h at 37°C. The supernatant was then harvested and kept at −70°C until assayed. Using Romanowsky stain, a blood count was performed in a subset of four animals in each group, and the viability of the cells was determined before culture by trypan blue exclusion.
The amounts of TNF-α, IL-10, and IL-1β in the supernatant were measured with a commercial enzyme-linked immunosorbent assay kit (DuoSet; R&D systems, Abingdon, United Kingdom) according to the manufacturer’s instructions. The assay detection limit was 30 pg/mL.
The distribution of cytokines concentrations in each unit was checked for normality using the Shapiro-Wilk test. The difference among groups (cont, carr, bupi, and carr + bupi) was assessed using a one-way analysis of variance with post hoc analysis via Newman-Keul test. The results are expressed as the mean ± sem. A P value less than 0.05 was considered as the minimum level of statistical significance.
Mice had a similar white cell count in all groups (6520 ± 210 cells/mm3 for the pooled value). The type of cells was also similar in the 4 groups: 75%–88% lymphocytes, 10%–16% neutrophils, and 1%–3% monocytes. The concentration of cytokines in plasma after the 15-h treatment was less than the limit of detection (i.e., less than 30 pg/mL for TNF-α and IL-10 and less than 15 pg/mL for IL-1β. The cell viability was more than 94% before the culture.
The concentration of TNF-α, IL-1β, and IL-10 in cultured blood in the absence of stimulation (baseline) was always small and not statistically different among groups (Table 1), indicating that the culture plates did not significantly stimulate cultured cells. After stimulation by LPS or SAC, TNF-α, IL-1β, and IL-10 levels were significantly increased as compared with baseline values.
After stimulation with LPS or SAC, TNF-α production was enhanced as expected (Group cont) (Fig. 1). A significant increase in TNF-α production after LPS stimulation was observed 15 h after carrageenan injection (Group carr) as compared with the saline injection (Group cont). In the Group bupi, the TNF-α production was not significantly different from the production observed in Group cont but significantly less than in the Group carr. In the Group carr + bupi, TNF-α production was not different than the production observed in Group cont or in Group Bupi but significantly less than in the Group carr, showing that bupivacaine may limit the increased production of TNF-α observed with carrageenan. After stimulation with SAC, TNF-α production was significantly increased in the Group carr, as compared with the Group cont. In the Group bupi, the TNF-α production was not significantly different from the production observed in the Group cont. In the Group carr + bupi, TNF-α production was significantly more than in the Groups cont and bupi and significantly less than in the Group carr.
After 24 h of stimulation with LPS or SAC, IL-1β production was enhanced as expected (Group cont) (Fig. 2). LPS-induced IL-1β production was significantly more in Group carr than in the other three groups. IL-1β concentration after stimulation with LPS in Groups bupi and carr + bupi was not different from that in the Group cont. The stimulation with SAC induced a significant increase in IL-1β production. This production was not different in Group cont and in Group bupi. In the Groups carr and carr + bupi, the production was significantly more than in the two other groups that did not receive carrageenan but was not significantly different between the two groups.
Twenty-four hours after stimulation with LPS or SAC, IL-10 production was enhanced as expected (Group cont) (Fig. 3). LPS and SAC induced an increase in IL-10 production in the two groups receiving carrageenan as compared with Groups cont and bupi. The latter two groups (cont and bupi) were not significantly different in their production. Also, no difference was observed between the two groups receiving carrageenan. It should be noticed that IL-10 production was not larger in Group carr + bupi, as compared with the Group carr, thus demonstrating that the significant reduction in TNF-α and IL-1β production observed in Group carr + bupi was not related to an increase in IL-10 production.
The current results show that the injection of carrageenan in the paw of mice stimulates the production of proinflammatory cytokines in cultured whole blood challenged by LPS or SAC. When the mice were pretreated with bupivacaine, the increased production of TNF-α and IL-1β observed in Group carr mice, as compared to group cont mice, was reversed in the case of LPS-stimulated cultures and only partially reversed in the case of SAC-stimulated cultures. The antiinflammatory cytokine IL-10 was not involved in the decreased LPS- or SAC-induced TNF-α and IL-1β production after bupivacaine administration. Finally, the effect of bupivacaine on the inflammatory response does not seem to be a ubiquitous phenomenon. Indeed, the modulation of the inflammatory response was different after LPS or SAC stimulation.
The carrageenan-induced hindpaw inflammation model has been extensively studied (1,2,4). Several experiments performed in rats and mice have shown that after an injection of carrageenan, edema rapidly develops followed by a period of allodynia, which lasts 24–96 hours (1,9). In our experiments, the carrageenan-induced hindpaw inflammation model had an effect on the general immune status and particularly on blood cell reactivity to LPS. Carrageenan sensitization to LPS challenge has been shown in vivo in mice (10). These authors and others (11,12) also reported that, in vitro, carrageenan primes mice for TNF-α production in response to LPS. In the latter experiments, it was suggested that not only monocytes, but also neutrophils, were responsible for such an increased immune response. To reduce the confounding factors associated with the isolation of monocytes or neutrophils, such as adherence-induced activation or altered expression of cell-surface receptors, we induced cytokine production in whole blood culture (13).
To provide a prolonged effect without the need of reinjection, we used BM (8). This technique allows continuous delivery at concentrations similar to those encountered in the clinical situation. Indeed, we did not observe any sign of toxicity, and the concentrations observed after 1 and 15 hours of administration were not in the toxic range. Therefore, contrary to numerous in vitro studies in which supratherapeutic concentrations are used, our experiment used small, nontoxic bupivacaine concentrations similar to those measured in patients. When injected in the legs of the mice, bupivacaine alone did not modify blood reactivity to LPS or SAC. Local anesthetics have antiinflammatory properties, which may have clinical implications (3). However, these properties are not considered to impair the immune response, and the intensity of the modulation, if any, does not seem to be as important with local anesthetics as with opioids. Until now, it was shown that lidocaine has an action on neutrophils at small concentrations (14) and on activated murine macrophages at supratherapeutic concentrations (15).
Carrageenan was shown to induce a general inflammatory response, and our experiments show that this inflammatory response is partly inhibited by bupivacaine when injected IM before carrageenan injection. The inhibition of inflammatory response elicited by immuno-competent cells might be the result of the action on targets different from Na+ channels (16). Gentili et al. (4) described a significant decrease of carrageenan-induced edema in rats related to a prolonged (more than six hours) peripheral nerve block. This antiinflammatory effect was associated with a persistent antinociceptive effect. However, the antiinflammatory properties of local anesthetics were mainly studied in animal models on hindpaw inflammation, and the immunomodulatory effects of these drugs on the blood compartment remain unsolved. In our study, IM injection of bupivacaine far from the inflamed hindpaw had a systemic effect on inflammatory response, as shown by the blood cell reactivity modification. Indeed, bupivacaine fully restored the blood cells’ reactivity to LPS and partly restored the blood cell reactivity to SAC. One could hypothesize that the observed effect on edema described by Gentili et al. (4) was not only because of a prolonged interruption of neuronal efferent activity, but also of the direct antiinflammatory effect of bupivacaine. Sinclair et al. (17) have shown in vitro that local anesthetics inhibit IL-1β release in LPS-stimulated human peripheral blood mononuclear cells. The present results confirm this inhibition of LPS-induced IL-1β and TNF-α release ex vivo. Leduc et al. (18) reported that bupivacaine (plain or encapsulated) showed antioxidant properties toward lipid peroxidation induced by carrageenan inflammation when administered via a sciatic nerve block at clinically relevant concentrations. Of interest, oxidative stress has been thought to enhance the inflammatory response and the production of proinflammatory cytokines (19). Thus, it can be hypothesized that local anesthetics, like other antioxidants (20), may exhibit antiinflammatory properties through an inhibition of the oxidative stress. Also, Hollmann et al. (14) showed that clinically relevant local anesthetic concentrations were able to inhibit superoxide production by neutrophils by a mechanism involving the G-protein signaling pathway.
IL-10 inhibits LPS-induced production of proinflammatory cytokines by mononuclear cells during in vitro and in vivo conditions (21). Production of IL-10 has therefore been considered to be part of a host-protective mechanism during excessive inflammatory processes. IL-10 was previously shown to inhibit the production of proinflammatory cytokines when injected before, but not after, carrageenan (2). In our experiments, both challenges (LPS and SAC) increased the production of IL-10 by the cultured cells, and carrageenan enhanced this production. However, IL-10 does not seem to be a factor for the antiinflammatory effects of bupivacaine.
The family of Toll-like receptors (TLRs) plays a crucial role in immunity in mammals. LPS and SAC action on mononuclear cells leading to TNF-α and IL-1β release involves different TLRs and different signaling pathways (22). This difference has clinical consequences. LPS activates principally TLR4, which is involved in the innate reaction to Gram-negative bacteria, whereas SAC activates principally TLR2, which is involved in the innate reaction to Gram-positive bacteria (23). In humans, McCall et al. (24) reported that neutrophils from septic patients were hyporesponsive to LPS but remained fully responsive to SAC. The same observation was made in trauma patients (25,26) and in patients resuscitated after cardiac arrest (27). These observations illustrate that blood cell leukocyte responsiveness to LPS or to SAC is not a ubiquitous phenomenon after various insults. This is consistent with our observations of a different effect of bupivacaine on the inflammatory profiles elicited by LPS or SAC in the mice receiving carrageenan.
We provide evidence ex vivo, in a murine model of local inflammation, that bupivacaine regulates the systemic inflammatory response elicited by carrageenan. Furthermore, IL-10 is not a factor in the modulation of the inflammatory response induced by bupivacaine. The precise mechanism underlying this effect of bupivacaine on the blood inflammatory response remains to be clarified.
The authors thank Severine Beloeil for technical advice in microspheres preparation and Régine Le Guen for technical assistance.
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