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PAIN MEDICINE

The Effect of Local Anesthetics and Amitriptyline on Peroxidation In Vivo in an Inflammatory Rat Model: Preliminary Reports

Christine Leduc, MSc^*, Marc E. Gentili, MD MSc, Jean-Pierre Estèbe, MD PhD, Pascal Le Corre, PharmD PhD, Jacques-Philippe Moulinoux, MD PhD^*, and Claude Ecoffey, MD

*GRETAC, Laboratoire de Biologie Cellulaire, and Laboratoire de Pharmacologie Galénique et de Biopharmacie, Université Rennes I; Département d’anesthésie-réanimation II, Clinique de la douleur, Hôpital Universitaire Hôtel-Dieu; and Département d’anesthésie-réanimation II, Hôpital Universitaire Pontchaillou, Rennes, France

Address correspondence and reprint requests to Marc Gentili, MD, MSc, Département d’anesthésie-réanimation II, Clinique de la douleur, Hôpital Universitaire Hôtel-Dieu, 35 000 Rennes, France. Address e-mail to marc.gentili{at}wanadoo.fr

	Abstract

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We studied the inhibition of peroxidation by local anesthetics in an inflammatory animal model. Inflammatory lipid peroxidation was assessed by the thiobarbituric assay in plasma from rats injected or not injected with carrageenan (Carra) and killed 1, 2, 4, 6, 12, and 24 h thereafter. Thiobarbituric acid reactive substances (TBARS) values in inflammatory animals were maximal 6 h after Carra administration. This result, in accordance with the evolution of paw edema width during time, supports that TBARS reflect the intensity of inflammation. Local anesthetics (bupivacaine, lidocaine, ropivacaine, or bupivacaine-loaded microspheres) or amitriptyline were injected in clinically relevant concentrations as a sciatic nerve block or intraperitoneally in inflamed animals. Ropivacaine did not exhibit any protective effect on Carra-induced lipid peroxidation in rats. With all the other drugs administered as a sciatic nerve block, the maximal TBARS increase was not observed at 6 h. Our conclusion is that bupivacaine (plain or encapsulated), lidocaine, and amitriptyline in clinically relevant concentrations administered via the sciatic nerve showed antioxidant properties toward lipid peroxidation induced by Carra inflammation. Intraperitoneal injection of those drugs gave the same effect as nerve block; this result suggests that their mechanism of action is not strictly limited to the nerve.

IMPLICATIONS. We investigated the antioxidant effects of local anesthetics and amitriptyline in an inflammatory rat model. Amitriptyline exhibits antioxidant properties per se, whereas lidocaine and bupivacaine (plain or encapsulated) seem to inhibit the peroxidation process. This may have future application in limiting toxic oxygen metabolite production during the inflammatory process.

	Introduction

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Pain attributed to tissue injury is mainly caused by inflammation: the mechanism of peripheral inflammation includes local liberation of mediators released by cell lysis, inflammatory cells, and nerve endings (1). Local anesthetics (LAs) modulate the inflammatory response in in vivo and in vitro models (2,3). In animal models, prolonged LA effect inhibited edema and allodynia in carrageenan (Carra)-induced inflammation (4,5). The mechanism behind this effect may not be completely related to sodium channel blockade on the nerve fiber and remains largely unknown (6). A hypothesis is that toxic oxygen metabolites generated by leukocytes during the inflammatory response could be one of the LA targets (6): recent reports suggest that LAs may affect the production of superoxide anion in human neutrophils (7). Considering that lipid peroxidation is consecutive to inflammatory response and reflects the production of toxic oxygen metabolites, we used the thiobarbituric acid reactive substances (TBARS) assay to study the antioxidant activity of four LAs and amitriptyline, an antidepressant frequently used orally for the management of chronic pain and which is a blocker of neuronal Na+ channels (8). Moreover, the TBARS assay has been used recently to assess the tissue antioxidant capacity of propofol during anesthesia in an animal model (9).

	Materials and Methods

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This animal study was conducted in accordance with the guidelines of the Ethical Standards of the International Association for the Study of Pain. One hundred thirty male Sprague-Dawley rats (250–300 g) were included in the study. All procedures (injections in hindpaw, LA injections, killing of the animals) were performed under general anesthesia (2%–3% halothane). Blood was collected into citrate-coated tubes, by decapitation. All hindpaw injections were performed in the medial area of the hindpaw. Carra solution was obtained by dissolution of 100 mg of lambda Carra (Sigma, St Louis, MO) in 1 mL of physiologic saline solution (NaCl 0.9%).

In the first step of the experiment, we determined TBARS in plasma from nontreated rats (control group, n = 15) and from rats receiving 0.2 mL of saline solution in the hindpaw (placebo group, n = 5).

Six other groups were set up, corresponding to different times of action of a 0.2-mL injection of Carra solution in the hindpaw: Carra 1 h: rats injected with Carra and killed 1 h later (n = 5); Carra 2 h: rats killed 2 h after Carra injection (n = 5); Carra 4 h: rats killed 4 h after Carra injection (n = 5); Carra 6 h: rats killed 6 h after Carra injection (n = 5); Carra 12 h: rats killed 12 h after Carra injection (n = 5); and Carra 24 h: rats killed 24 h after Carra injection (n = 5).

We repeated the experiment, using a new control group (n = 5). Inflamed animals were killed respectively 3, 6, 9, and 24 h (n = 5 for each group) after Carra injection in the hindpaw. For each group, TBARS were determined and the circumference of ankle and paw was estimated by measurement with a thread, to the nearest millimeter.

In a second step, Carra injection (0.2 mL in hindpaw) was immediately followed by an LA administration (0.2 mL) injected either intraperitoneally (i.p.) or as a sciatic block performed with an insulated needle linked to a nerve stimulator (HNS 111; B. Braun, Melsungen, Germany) when a distal motor response at 0.6 mA of the hindpaw was obtained. LAs were 0.5% bupivacaine (1 mg), 2% lidocaine (4 mg), 0.75% ropivacaine (1.5 mg), and bupivacaine-loaded microspheres (BMs) (12.5 mg of bupivacaine) (10). We also injected amitriptyline (6.25 mg) (8). The motor block evaluation was performed by an examination of the rat’s ability to hop and to place weight on its hind leg: animals that had no motor block in the hindpaw after 1 h were not included in the study. Six hours thereafter the included animals were killed.

Groups are identified as follows: Carra + bupi block: Carra and sciatic block with bupivacaine (n = 5); Carra + lido block: Carra and sciatic block with lidocaine (n = 5); Carra + ropi block: Carra and sciatic block with ropivacaine (n = 5); Carra + BMs block: Carra and sciatic block with bupivacaine-loaded microspheres (n = 5); Carra + ami block: Carra and sciatic block with amitriptyline (n = 5); Carra + bupi i.p.: Carra and i.p. injection of bupivacaine (n = 5); Carra + lido i.p.: Carra and i.p. injection of lidocaine (n = 5); Carra + ropi i.p.: Carra and i.p. injection of ropivacaine (n = 5); Carra + BMs i.p.: Carra and i.p. injection of bupivacaine-loaded microspheres (n = 5); Carra + ami i.p.: Carra and i.p. injection of amitriptyline (n = 5); bupi block: bupivacaine sciatic block alone (n = 5); lido block: lidocaine sciatic block alone (n = 5); ropi block: ropivacaine sciatic block alone (n = 5); BMs block: bupivacaine-loaded microspheres sciatic block alone (n = 5); and ami block: amitriptyline sciatic block alone (n = 5).

For each rat, sampled blood was centrifuged, and plasma isolated by this way was used for the TBARS assay, to estimate lipid peroxidation. The procedure is widely inspired from the method of Jentzsch et al. (11). A 50-µM malondialdehyde (MDA) stock solution was prepared by acidic hydrolysis of tetrahydroxy propane, and diluted with water to obtain 0.2–2 µM MDA, constituting the standard solutions. Two hundred microliters of plasma or standard solution was mixed with 25 µL of a 60-mM ethanol solution of butylated hydroxy toluene and 200 µL of 0.2 M orthophosphoric acid in test tubes and mixed with 25 µL of a 0.11 M alkaline thiobarbituric acid solution. After an incubation at 85°C for 50 min in a water bath, the tubes were put in ice to stop the reaction, and TBARS were extracted by 500 µL of butanol. Two hundred microliters of the butanoic phase was transferred into a Fluoronunc 96-well microplate and fluorescence was measured in a Spectra Max Gemini (Molecular Devices) microplate reader (excitation 500 nm, cutoff 530 nm, emission 550 nm). These fluorescence values were used to calculate the MDA equivalents (TBARS) of plasmas according to the calibration curve.

Results were expressed as mean ± SD; for statistics, the nonparametric Mann-Whitney test was used. Statistical significance was defined as P < 0.05.

	Results

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There was no difference between TBARS values for nontreated (control group) and saline-injected rats (placebo group). To avoid pain to the animals, we used nontreated rats as the control group for the following steps of the experiment.

The maximal TBARS increase induced by Carra-induced inflammation occurred 6 h after injection in the hindpaw (Fig. 1A): values at 6 h were twice that of control values (P < 0.01), but the TBARS increase was not gradual. This isolated increase of TBARS 6 h after Carra injection was found again (P < 0.01) when we repeated the experiment (Fig. 1B). TBARS values are in concordance with the evolution of paw width during time (Fig. 1B).

View larger version (28K): [in this window] [in a new window]   Figure 1. Evolution of plasmatic thiobarbituric acid reactive substances (TBARS) values after carrageenan injection. Rats were injected in the hindpaw with 0.2 mL of carrageenan solution (10 mg/mL) and killed 1, 2, 4, 6, 12, and 24 h later (A) or 3, 6, 9, and 12 h later (B). Controls are nontreated rats. Blood collected during killing was centrifuged to separate plasma from erythrocytes. Plasma such obtained for each rat was assayed for TBARS to quantitate the inflammation-induced lipid peroxidation. Paw and ankle circumference (B) were estimated by measurement with a thread, to the nearest millimeter. **Different from control, P < 0.01. MDA = malondialdehyde.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effects of anesthesia on carrageenan (Carra)-induced inflammation expressed as thiobarbituric acid reactive substances (TBARS) values. Rats received a Carra injection and immediately after an injection (block or intraperitoneal [i.p.]) of local anesthetic: plain lidocaine (lido), bupivacaine (bupi), ropivacaine (ropi), bupivacaine-loaded microspheres (bupiMS), or amitriptyline (ami). The local anesthetics were also administrated by block without Carra injection. Control animals were not treated and Carra 6 h animals were injected with Carra (0.2 mL of a 10 mg/mL solution) without anesthetic. All the animals were killed 6 h after treatment. Blood collected during killing was centrifuged to separate plasma from erythrocytes. Plasma such obtained for each rat was assayed for TBARS to quantitate the inflammation-induced lipid peroxidation. *Different from control, P < 0.05; **different from control, P < 0.01.

With BMs, results for TBARS values were the same as for plain bupivacaine.

Amitriptyline injected in control animals entailed a TBARS reduction in the same proportion as ropivacaine. In contrast with ropivacaine when injected in inflamed animals, amitriptyline inhibited the TBARS increase at 6 h, whatever mode of administration was chosen.

	Discussion

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The fact that TBARS values are in line with the evolution of paw width during time in the inflammatory Carra model supports the idea that TBARS might reflect the magnitude of inflammation.

Bupivacaine or lidocaine blocks performed on control rats do not provide any direct antioxidant effect per se, but those anesthetics (administered via the sciatic nerve or i.p.) are able to inhibit TBARS increase consecutive to Carra inflammation. These results support the idea that bupivacaine and lidocaine own indirect antioxidant properties aimed against lipid peroxidation. BMs results were the same as for plain bupivacaine: the microspheres used as a drug delivery system do not enhance the bupivacaine antioxidant effect, but do not have any prooxydant effect.

Ropivacaine seems to be a direct antioxidant (control animals treated by ropivacaine have TBARS values smaller than control animals), but too weak to protect rats from Carra-induced peroxidation.

Amitriptyline injected in control animals entails a TBARS reduction in the same proportion as ropivacaine by a direct antioxidant mechanism. In contrast with ropivacaine, when injected in inflamed animals, amitriptyline has a strong antioxidant effect, reducing TBARS values at a score lower than for control animals.

In the current study, we have shown that, in clinically relevant doses, bupivacaine (plain or loaded into microspheres), lidocaine, and amitriptyline, but not ropivacaine, may exhibit tissue antioxidant capacity by inhibiting induced peroxidation in an inflammatory animal model. LAs have multiple activities in addition to Na+ channel blockade and are widely used in regional anesthesia or antiarrhythmic treatments: more recent clinical and laboratory investigations support the notion that they might modulate the inflammatory response. Inflammatory disorders seem critical, not only in many clinical situations such as postoperative pain, but also in adult respiratory distress or multiorgan failure (2). Moreover, some of these effects occur at concentrations much smaller than those inducing sodium channel blockade, and the inhibition of inflammatory response via the inflammatory cells is caused by targets different from Na+ channel blockade (3,7). The Carra-induced hindpaw inflammation model has been extensively used as an experimental inflammatory pain model (12). Furthermore, recent studies demonstrate that prolonged bupivacaine block suppresses allodynia through a possible inhibition of the neurogenic inflammation, but none of these studies address the antioxidant activity of LAs. This experiment is thus of particular interest because it aims to compare drugs in vivo with a well described inflammatory model. Experimental studies dedicated to the antioxidant effects of LAs have been progressively entertained in the past years (7,13,14). Bupivacaine and lidocaine were tested in vitr. for their ability to avoid the fluorescence loss of ß-phycoerythrin, an oxidative stress sensible protein (14). In this experiment, oxidative stress was induced by 2,2'azobis (2-amidinopropane) dihydrochloride (AAPH), a generator of peroxyl radicals. Lenfant et al. (14) found that lidocaine alone exhibited weak antioxidant properties, and that bupivacaine did not provide a protective effect against free radicals.

The antioxidant properties of lidocaine have been supported by another test using AAPH (14): lidocaine inhibits the red blood cell lysis induced by AAPH. More recently, Hollman et al. (7) demonstrated that lidocaine in clinical concentrations was able to inhibit the superoxide anion production in human neutrophils primed by the platelet activating factor, a representative inflammatory mediator that has a strong role in critical human clinical disorders. The authors hypothesized that the target of the inhibitory effect of LAs is within a signaling pathway involved in platelet activating factor priming, and obviously different from the Na+ channel.

We hypothesize that bupivacaine and lidocaine do not present per se any direct antioxidant properties in an inflammatory animal model, but have equal capacities against lipid peroxidation, whatever route of administration is chosen (sciatic block or i.p. injection), thus indicating a mechanism of action that seems to be located at the level of the inflammation-related systems, at a systemic-accessible point of junction. By contrast, ropivacaine, which is a weak direct antioxidant, seems to be devoid of such antiinflammatory properties. Such a difference in in vivo properties could be attributable to a dose-response effect.

We also tested LAs with longer LA action. Microspheres do not improve the antioxidant capacity power of bupivacaine. This result may be particularly interesting in helping to determine the safety of these delivery systems. Amitriptyline, an antidepressant considered as a new local analgesic drug and as a strong Na+ channel blocker (8), has both direct and indirect antioxidant properties, and gives the best results, because we obtained TBARS values weaker than controls for every case tested (injection to control or to inflamed animals). Here again, i.p. and sciatic block administration gave similar results, indicating a mechanism of action not strictly located at the nerve level.

Further studies are required to determine the exact target of action for each LA; however, our results sustain the recent interest for research in antioxidant effects of LAs and may be relevant in promoting their use in human therapy, not only for their properties as sodium channel blockers, but also as antiinflammatory or antioxidant drugs. TBARS values could be an interesting tool for evaluating the antioxidant properties of different LAs clinically used or for upgraded drugs. Some differences seem to be revealed by this technique.

	Acknowledgments

 
This work was funded by special departmental funds (Département d’anesthésie-réanimation II, Clinique de la douleur, Hôpital Universitaire Hôtel-Dieu, 35 000 Rennes, France); CL is a recipient for a research fellowship from the Britain Regional Conceal (Conseil Regional de Bretagne, 283 avenue Patton, 35000 Rennes, France).

For their help in animal experiments, we thank Dominique Desury and Myriam Guerrois, GRETAC, Laboratory of Cellular Biology, University Rennes I, Rennes, France.

	Footnotes

 
Presented in part in abstract form in American Society of Anesthesiologists Congress 2001, New Orleans, LA.

	References

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