Effect of neurolytic nerve block on systemic carrageenan-induced inflammatory response in mice

T. A. Pham-Marcou1, M. Gentili1,2, K. Asehnoune1, D. Fletcher3 and J.-X. Mazoit1,*

1 Laboratoire d'Anesthésie, UPRES EA 3540, Faculté de Médecine, Université de Paris-Sud, 94276 Le Kremlin Bicêtre Cedex, France. 2 Département d'Anesthésie, Hôpital Ponchaillou, Rennes, France. 3 Département d'Anesthésie, Hôpital Raimond Poincarré, Garches, France

* Corresponding author. E-mail: jean-xavier.mazoit{at}kb.u-psud.fr

Accepted for publication March 3, 2005.


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Local tissue inflammation caused by injury or inflammatory processes may be reduced by section or local anaesthetic block of the nerve innervating the inflamed area.

Methods. Using a neurolytic nerve block, we assessed the systemic inflammatory response elicited by a local injection of carrageenan in mice. Sixty mice received a unilateral sciatic nerve block with 70% ethanol followed 5 days later by an injection of carrageenan on the ipsilateral or contralateral hind paw. Whole blood was sampled 15 h after carrageenan injection.

Results. Fifty-six animals had a complete nerve block. Tumour necrosis factor-alpha (TNF-{alpha}), interleukin 1ß (IL-1ß) and interleukin 10 (IL-10) concentrations were measured in the plasma of 20 mice (10 in the ispilateral group and 10 in the contralateral group). In the remaining 36 mice (18 in each group), blood was cultured for 24 h in the presence of lipopolysaccharide or Staphylococcus aureus extract. TNF-{alpha} and IL-1ß production in the supernatant were, on average, 30% lower in the ispilateral group than in the contralateral group.

Conclusion. A prolonged nerve block decreases the systemic consequences of the local inflammatory reaction elicited by carrageenan injection.

Keywords: complications, tissue inflammation ; model, mouse ; nerve block ; pharmacology, cytokines


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Small-diameter primary afferent fibres not only transmit nociception, but also release neuropeptides into innervated tissues within their immediate vicinity through an axon reflex mechanism.1 The release of neurogenic substances, such as substance P, calcitonin gene related peptide or neurokinin A, may actively contribute to the development of peripheral inflammation in combination with the other mediators released by cell lysis and leucocytes. The evidence of neurogenic inflammation has led to its experimental prevention by deafferentation. Indeed, experimental nerve sections and sciatic nerve blocks have been partially effective in peripheral inflammation.2 3 Moreover, carrageenan-induced inflammation of localized tissues has been shown to affect systemic blood cell reactivity. Carrageenan pretreatment appeared to sensitize leucocytes to lipopolysaccharide challenge and enhance production of tumour necrosis factor-{alpha} (TNF-{alpha}).4 In a previous experiment, we showed that bupivacaine injected intramuscularly before carrageenan injection in the hind paws of mice, was able to inhibit leucocyte sensitization to in vitro stimulation induced by carrageenan.5

The aim of the present study was to assess the effect of a neurolytic nerve block on the systemic carrageenan-induced inflammatory response in mice.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study was conducted according to the official edict of the French Ministry of Agriculture (Paris, France) and the recommendations of the International Association for the Study of Pain.6 Experiments were performed on 60 8-week-old male Balb/c mice (Janvier, Le Genest St Isle, France) (Fig. 1). In the first group of 20 mice, cytokines were measured in plasma. In the second group of 40 mice, cytokine production was measured in whole blood cultured in the presence of stimulants (see below). In both groups, a neurolytic sciatic block was performed on the right hind paw with ethanol 70% (50 µl) (VWR, Fontenay sous Bois, France) after localization of the sciatic nerve just posterior of the great trochanter with a Stimuplex nerve stimulator (B-Braun, Melsungen, Germany). After 5 days recovery, those mice with full blockade of motor function and nociception to mechanical stimulus (tested using a 50 g Von Frey filament applied to the metatarsus) were assigned to two subgroups.7 8 Animals received an intraplantar injection of 0.5 mg {lambda}-carrageenan (Sigma, St Quentin-Fallavier, France) in either the right hind paw (ipsilateral group) or the left hind paw (contralateral group). Fifteen hours after carrageenan administration, the animals were killed via cardiac puncture under isoflurane anaesthesia. The collected blood was used for cytokine measurement. In the first group of 20 animals (10 in each subgroup) cytokines were measured in plasma. In the second group (36 mice with complete block, 18 in each subgroup) whole blood was diluted 1:5 in RPMI-1640 medium (Glutamax, Gibco-life Technologies, Paisley, UK) supplemented with antibiotics [penicillin 100 IU ml–1 (Panpharma, Luitré-Fougères, France) and streptomycin 100 µg ml–1 (Sigma, Saint Quentin-Fallavier, France)]. Five hundred microlitres of diluted blood was cultured for 18 h at 37°C in a 5% carbon dioxide incubator under three different conditions: without stimulation (control), stimulated with lipopolysaccharide 1 µg ml–1 (LPS) (Escherichia coli O111:B4, Sigma, Saint Quentin-Fallavier, France) or stimulated with heat-killed Staphylococcus aureus Cowan 100 µg ml–1 (SAC) (Pansorbin Calbiochem, Fontenay sous Bois, France). The supernatant was then harvested and stored at –70°C until assay. The amounts of TNF-{alpha} and interleukin 1ß (IL-1ß) (pro-inflammatory cytokines) and interleukin 10 (IL-10) (anti-inflammatory cytokine) in plasma or in the supernatant of cultured blood were quantified with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Duoset, R&D Systems, UK). The assay detection limit was 30 pg ml–1 for TNF-{alpha} and IL-10 and 15 pg ml–1 for IL-1ß. The coefficient of variation between assays was <10 % and there was no cross-reactivity with other cytokines. Differences between groups were assessed using Student t-test after Shapiro–Wilk testing. Data are reported as mean (SEM).



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Fig 1 (A) Mice received a right sciatic nerve block with 70% alcohol followed 5 days later by an injection of carrageenan in the right hindpaw (ipsilateral group) or the left hindpaw (contralateral group). (B) Blood was sampled for measurement of cytokines in plasma or in whole blood cultured in the presence of stimulants (lipopolysaccharide and S.aureus extract) 15 h after carrageenan injection (blood drawn from one animal was subjected to all three conditions).

 

    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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The concentration of cytokines in plasma (first group) after treatment for 15 h was below the limit of detection (i.e. <30 pg ml–1 for TNF-{alpha} and IL-10 and <15 pg ml–1 for IL-1ß).

In the second group, LPS and SAC induced an increase in TNF-{alpha} and IL-1ß production. In the controls (i.e. without LPS or SAC challenge) cytokine production remained below the limit of detection, indicating the absence of spontaneous activation. TNF-{alpha} production in the ipsilateral group decreased by 30% compared with the contralateral group after LPS and SAC challenges (P<0.05), and IL-1ß production decreased by 14% after LPS challenge (NS) and by 36% after SAC challenge (P<0.05) (Table 1 and Fig. 2). IL-10 production was higher after stimulation by SAC than after stimulation by LPS. Indeed, IL-10 concentration remained below the limit of detection in the contralateral group after LPS and increased to just above this limit in the ispilateral group.


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Table 1 Immunoreactive cytokine concentrations in the supernatant of whole blood culture. N=18 animals in each group. BLD, below the limit of detection. Data [mean (SEM)] are given to three significant figures

 


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Fig 2 (A) TNF-{alpha}, (B) IL-1ß and (C) IL-10 production 15 h after carrageenan injection plus 18 h ex vitro whole blood challenge. Whole blood was cultured in three different situations: baseline; with lipopolysaccharide stimulation; with S.aureus extract stimulation. *P<0.05, **P<0.01 ipsilateral group vs. contralateral group. (IL-10 concentration was below the detection limit in 17/18 mice in the contralateral group.)

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previous studies showed that a prolonged block was required to decrease the local inflammation induced by carrageenan.3 We performed a neurolytic nerve block with ethanol because this technique induces a prolonged nerve block with the advantage of reducing tissue injury due to surgery or to repeated injections. Because the contralateral group served as a control for the ipsilateral group, we did not include a saline or intramuscular ethanol group. The block, which was performed 5 days before injection of carrageenan in the same limb, affected the systemic response induced by peripheral inflammation. Although we did not measure the degree of local inflammation precisely, we hypothesize that the systemic effect could be attributed to a direct effect of the block on the peripheral carrageenan-induced inflammation, and that the decreased peripheral inflammatory response led to a decrease in leucocyte priming. Peripheral nerve section or capsaicin pretreatment has already been proved to reduce the symptoms of neurogenic inflammation.1 9 In particular, capsaicin pretreatment abolished the release of pro-inflammatory cytokines and nerve growth factor induced by a further injection of capsaicin. Similarly, prolonged, but not short, nerve blocks have been shown to limit oedema and mechanical allodynia in rats injected with carrageenan.2 3

The local inflammation produced by carrageenan injection not only induces a local release of pro-inflammatory cytokines, but also sensitizes the circulating leucocytes.4 5 Because this sensitization of leucocytes by carrageenan appears to be delayed, we decided to sample the animals 15 h after carrageenan injection.4 5 Whole-blood cultures were stimulated with LPS and SAC which act on the intracellular inflammatory signalling through Toll-like receptors 4 and 2, respectively.10 11 Two major pro-inflammatory cytokines (TNF-{alpha} and IL-1ß), which are mainly produced by monocytes, and the anti-inflammatory cytokine IL-10 were measured. The neurolytic nerve block was associated with less leucocyte sensitization after ex vivo LPS and SAC stimulation in the ipsilateral group. This effect was obvious since we observed a decrease of >30% in TNF-{alpha} production after LPS or SAC challenge. In a previous study, we showed that bupivacaine injected intramuscularly far from the carrageenan injection was able to decrease the leucocyte sensitization to ex vivo stimulation by LPS or by SAC.5

Increased production of anti-inflammatory cytokines such as IL-10 may counterbalance the production of pro-inflammatory cytokines.12 Just as in our previous experiment showing that bupivacaine was able to limit the sensitization induced by carrageenan, an increased production of IL-10 by the stimulated leucocytes seems to accompany the decreased secretion of TNF-{alpha} and IL-1ß (Table 1 and Fig. 2). However, this production did not appear to be closely associated with the decreased TNF-{alpha} and IL-1ß production. Indeed, IL-10 has been shown to have a limiting effect on the hyperalgesia induced by carrageenan injection in rats,13 but in this experiment the cytokine was injected directly into the hind paw, unlike the conditions of our experiment. Clearly, further experiments are needed to assess the role of IL-10 in the modulation of the systemic inflammatory response after carrageenan injection.

It is well known that leucocytes act at the site of inflammation to modulate nociception by releasing endogenous opioids,14 15 cytokines and other pro- or anti-inflammatory mediators.16 However, the inverse relationship, i.e. the effect of local inflammation on the general inflammatory status, has not been extensively studied in the context of nociception. In this setting, we speculate that a modulation of a local inflammatory reaction induces a critical effect on the systemic inflammatory response.

In conclusion, we have shown that a neurolytic nerve block can limit the systemic inflammatory response observed in mice after intraplantar injection of carrageenan. This study emphasized the evidence for neurogenic inflammation and its relationship with the general inflammatory response. A prolonged nerve block decreases not only the local inflammatory reaction observed after carrageenan injection,3 but also its systemic consequences.


    Acknowledgments
 
The authors are grateful to Mrs Régine le Guen for technical assistance. This work was supported by the Ministère de la Recherche et de la Technologie, Paris, France, and by the Association Mises au Point en Anesthésie et Réanimation, Le Kremlin-Bicêtre, France.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Jancso N, Jancso-Gabor A, Szolcsanyi J. Direct evidence for neurogenic inflammation and its prevention by denervation and by pretreatment with capsaicin. Br J Pharmacol 1967; 31: 138–51[Medline]

2 Kissin I, Lee SS, Bradley EL Jr. Effect of prolonged nerve block on inflammatory hyperalgesia in rats: prevention of late hyperalgesia. Anesthesiology 1998; 88: 224–32[CrossRef][ISI][Medline]

3 Gentili ME, Mazoit JX, Samii K, Fletcher D. The effect of a sciatic nerve block on the development of inflammation in carrageenan injected rats. Anesth Analg 1999; 89: 979–84[Abstract/Free Full Text]

4 Ogata M, Matsui T, Kita T, Shigematsu A. Carrageenan primes leukocytes to enhance lipopolysaccharide-induced tumor necrosis factor alpha production. Infect Immun 1999; 67: 3284–9[Abstract/Free Full Text]

5 Beloeil H, Asehnoune K, Moine P, Benhamou D, Mazoit JX. Bupivacaine action on the carrageenan-induced inflammatory response in mice: cytokine production by leukocytes after ex-vivo stimulation. Anesth Analg 2005; 100: 1081–6[Abstract/Free Full Text]

6 Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983; 16: 109–10[CrossRef][ISI][Medline]

7 Thalhammer JG, Vladimirova M, Bershadsky B, Strichartz GR. Neurologic evaluation of the rat during sciatic nerve block with lidocaine. Anesthesiology 1995; 82: 1013–25[CrossRef][ISI][Medline]

8 Hu D, Hu R, Berde CB. Neurologic evaluation of infant and adult rats before and after sciatic nerve blockade. Anesthesiology 1997; 86: 957–65[CrossRef][ISI][Medline]

9 Saadé NE, Massaad CA, Ochoa-Chaar CI, Jabbur SJ, Safieh-Garabedian B, Atweh SF. Upregulation of proinflammatory cytokines and nerve growth factor by intraplantar injection of capsaicin in rats. J Physiol 2002; 545: 241–53[Abstract/Free Full Text]

10 Rabehi L, Irinopoulou T, Cholley B, Haeffner-Cavaillon N, Carreno MP. Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways. Infect Immun 2001; 69: 4590–9[Abstract/Free Full Text]

11 Lehner MD, Morath S, Michelsen KS, Schumann RR, Hartung T. Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Toll-like receptors independent of paracrine mediators. J Immunol 2001; 166: 5161–7[Abstract/Free Full Text]

12 Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Ann Rev Immunol 2001; 19: 683–765[CrossRef][ISI][Medline]

13 Poole S, Cunha FQ, Selkirk S, Lorenzetti BB, Ferreira SHl. Cytokine-mediated inflammatory hyperalgesia limited by interleukin-10. Br J Pharmacol 1995; 115: 684–8[ISI][Medline]

14 Stein C, Hassan AH, Przewlocki R, Gramsch C, Peter K, Herz A. Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc Natl Acad Sci USA 1990; 87: 5935–9[Abstract/Free Full Text]

15 Brack A, Labuz D, Schiltz A, et al. Tissue monocytes/macrophages in inflammation: hyperalgesia versus opioid-mediated peripheral antinociception. Anesthesiology 2004; 101: 204–11[ISI][Medline]

16 Watkins LR, Maier SF. Beyond neurons: evidence that immune and glial cells contribute to pathological pain states. Physiol Rev 2002; 82: 981–1011[Abstract/Free Full Text]





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