Local anaesthetics can inhibit components of the inflammatory response.1 We previously reported that local anaesthetics via a nerve block could attenuate the inflammatory hind paw oedema and hyperalgesia induced by hind paw injection of carrageenan in rats.2 This effect was associated with an inhibition of the increase in cyclooxygenase-2 (COX-2) induced by peripheral inflammation in dorsal root ganglia (DRG) and cord.3 The subsequent production of prostaglandin E2 (PGE2) in cerebrospinal fluid (CSF) was also impaired. As human and animal data have demonstrated that an increase in spinal PGE2 after peripheral inflammation (including surgery) positively correlated with pain,4 inhibition of PGE2 may prevent hyperalgesia and local inflammation. Actually, it has been previously reported that the prevention of hyperalgesia by analgesic drugs is associated with prevention of the changes in PGE2 concentration in CSF in animals.5 NSAIDs by inhibiting the prostaglandin pathway and, therefore, PGE2 are the traditional anti-inflammatory medication. They have been used for decades for their multiple properties: analgesia, morphine-sparing effect, anti-inflammatory and antioedema.6 The anti-inflammatory properties of local anaesthetics administered via a nerve block have, however, never been compared with a traditional anti-inflammatory drug such as a NSAID.
The purpose of the present study, therefore, was to compare the effects of bupivacaine via sciatic block with those of a systemic NSAID on behavioural measures of oedema and hyperalgesia and on indices of systemic inflammation as measured by cytokine and PGE2 production in a model of peripheral inflammation in rats.
Materials and methods
Young adult male Sprague–Dawley rats weighing 200–250 g were used. Guidelines of the International Association for the Study of Pain were followed.7 Our institutional committee on research in animals approved this study. The animals were kept on a 12 h light/dark cycle with free access to food and water. The rats were handled repeatedly over at least 3 days prior to experiments to habituate them to the investigators and the test conditions.
Carrageenan was prepared fresh before each experiment [0.2 ml of 2% w/v solution of lambda carrageenan in physiological saline (Sigma, Saint Quentin-Fallavier, France)]. Bupivacaine [(Sigma) 0.5% w/v (5 g l−1)] was used as the local anaesthetic. Ketoprofen (50 mg kg−1; Sanofi-Aventis, Antony, France) was used as the NSAID. Lipopolysaccharide (LPS) from Escherichia coli O111:B4 was from Sigma. Doses were chosen according to previous studies on rodents.8,9
Animals were randomly assigned to one of seven experimental groups (n = 8/group) as described in Table 1. Under a brief general anaesthesia (using isoflurane), each animal received three injections at time = 0: a left subcutaneous hind paw injection (0.2 ml with either carrageenan 2% w/v or physiological saline); a left sciatic block (0.2 ml with either bupivacaine 0.5% or physiological saline); and a systemic injection (0.2 ml subcutaneous interscapular, with NSAID or physiological saline). The sciatic block was performed as previously described.2 The efficacy of the block was tested by measuring paw withdrawal latency in response to a radiant thermal stimulus applied using a Hargreaves-type testing device (see Behavioural measurements) 30 min after the injection. Failure to remove the hind paw after 22 s was regarded as dense thermal nociceptive blockade. All animals receiving a sciatic blockade with bupivacaine had a dense blockade 30 min after the injection of bupivacaine. Behavioural measurements as well as blood or CSF sampling were carried out 6 h after the initial injections.
A single blinded investigator performed all behavioural tests. Nociceptive responses to a thermal stimulus were evaluated by measuring paw withdrawal latency in response to a radiant thermal stimulus applied using a Hargreaves-type testing device (Ugo Basile, Milan, Italy). It consists of a movable infrared source, which the operator glides below a glass pane, upon which a three-compartment enclosure for a rat is positioned. When the rat feels pain and withdraws its paw, the instrument automatically detects the withdrawal latency to the nearest 0.1 s. The paw withdrawal latency was evaluated 6 h after initial injections. This test was repeated three times for each rat.
The development of mechanical hypersensitivity after hind paw inflammation was assessed by the application of calibrated von Frey filaments. Animals were placed on a mesh floor in individual plastic boxes and allowed to become accustomed to their environment. Then, von Frey filaments were applied vertically to the plantar surface of both hind paws. If no response was elicited, a larger diameter filament was applied in the same manner. The filaments were applied in increasing order until brisk withdrawal or paw flinching was elicited, which was considered to be a positive response. This withdrawal threshold was determined three times and the mean withdrawal threshold was used for data analysis.
As a method to evaluate the oedema, we used a technique previously described.10 The paw circumference was measured by a thread, to the nearest millimetre, at the metatarsal level.
Assay for stimulated tumour necrosis factor-α and interleukin 1β production in whole blood cultures
Different animals were used and randomly assigned to one of seven experimental groups (n = 8/group) as described in Table 1. A blood sample (0.5 ml) collected 6 h after drug injections and during euthanasia 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). Five hundred microlitres of diluted blood was cultured in 24-well plates according to two different conditions – without LPS (baseline) and with LPS [E. coli O111:B4, 10 μg ml−1, Sigma] – in a 5% CO2 incubator for 24 h at 37°C. The supernatant was then harvested, centrifuged at 300g and at 4°C for 10 min and kept at −70°C until assayed. The amounts of tumour necrosis factor-α (TNF-α) and of interleukin 1β (IL-1β) in the supernatant were measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit (DuoSet; R&D systems, Abingdon, UK) according to the manufacturer's instructions. The assay detection limit was 30 pg ml−1.
Prostaglandin E2 in cerebrospinal fluid
Different animals were used and randomly assigned to one of seven experimental groups (n = 8/group) as described in Table 1. CSF was collected 6 h after drug injections under general anaesthesia and before euthanasia. The amounts of PGE2 were measured with an ELISA kit (R&D systems). The assay detection limit was 10 pg ml−1. At the end of each experiment, rats were killed with an overdose of pentobarbital (100 mg kg−1 intravenously).
As the behavioural and the paw circumference data were not normally distributed, differences between groups were assessed using nonparametric tests (Kruskal–Wallis test and Mann–Whitney U-test with the Bonferroni correction). The results are expressed as the median with 25th and 75th percentiles. The difference between groups in the distribution of cytokines was assessed using a one-way analysis of variance (ANOVA) with post-hoc analysis via a Fisher's exact test. The results are expressed as the mean ± SD. A P value below 0.05 was considered to be the minimum level of statistical significance.
Evaluation of hind paw oedema by paw circumference
Six hours after the injection of carrageenan, a significantly increased (+84%) paw circumference was observed in the carrageenan group compared with the control group (Fig. 1). A sciatic block with bupivacaine as well as a systemic injection of NSAID significantly decreased the oedema induced by carrageenan by approximately 12%. The combination of a sciatic block and NSAID did not further decrease the carrageenan-induced oedema compared with either injection alone.
Thermal nociceptive withdrawal latencies and von Frey filament mechanical withdrawal thresholds
Six hours after injection of carrageenan, a significant decrease in heat withdrawal latency (approximately six-fold) and in the mechanical withdrawal threshold (approximately two-fold) was observed in the carrageenan group compared with the control group, indicating thermal and mechanical hyperalgesia in the carrageenan group (Figs 2 and 3). A sciatic block with bupivacaine or a systemic injection of NSAID partially prevented the carrageenan-induced thermal (approximately three-fold to four-fold) and mechanical hyperalgesia (approximately 1.5–2-fold). The combination of a sciatic block and NSAID did not further restore the carrageenan-induced thermal latency and mechanical threshold decrease compared with either injection alone. As previously described in studies on rodents,8 the duration of a motor blockade after a single injection of the nerve block was short. Indeed, the motor blockade was not present 6 h later.
Production of prostaglandin E2 in cerebrospinal fluid
Basal PGE2 concentration in CSF was 112 (86–246) pg ml−1 (Fig. 4). In the group receiving carrageenan, PGE2 production rose and was significantly different from that in the control group. A bupivacaine block or systemic NSAID inhibited the increased production of PGE2 induced by a hind paw carrageenan injection.
Production of tumour necrosis factor-α and interleukin 1β by leucocytes after stimulation
Concentrations of TNF-α and IL-1β in cultured blood in the absence of stimulation were always low in all the study groups, indicating that the culture plates did not significantly stimulate cultured cells. Twenty-four hours after stimulation with LPS, TNF-α and IL-1β production was enhanced as expected (Table 2). LPS-induced TNF-α and IL-1β production was significantly higher in the carrageenan group. A sciatic block with bupivacaine or a systemic injection of NSAID prevented the carrageenan-induced TNF-α and IL-1β production.
In the current study, a bupivacaine block was as effective as systemic NSAID in preventing local inflammation, hyperalgesia, CSF PGE2 increase and systemic spread of inflammation, 6 h after the initial inflammatory insult.
There is now enough evidence in the literature to report about the anti-inflammatory effect of a nerve block. As reported previously and in the current study, local anaesthetics via a nerve block attenuated the inflammatory hind paw oedema and hyperalgesia induced by hind paw injection of carrageenan in rats.2,8 Local anaesthetics have indeed been shown in previous studies using carrageenan-induced inflammation to influence systemic as well as local inflammatory responses.1,2 It has been reported that a bupivacaine nerve block could inhibit the inflammatory-induced increase in COX-2 expression in DRG and spinal cord and subsequent PGE2 production in CSF.3,11 A systemic effect of bupivacaine cannot be excluded, but this hypothesis appears very unlikely. The phenomenon observed may be related to the nerve block itself and not the local anaesthetics even if they intrinsically present anti-inflammatory properties12 when administered systemically.13 A prolonged nerve block itself (70% ethanol) can indeed decrease not only the local inflammatory reaction observed after carrageenan injection, but also its systemic consequences.14 This effect could be related to the conduction blockade initiated by the nerve block. Inhibition of axonal transport by local anaesthetics was, however, only reported in vitro.15 Inhibition of axonal transport by a nerve block in the context of peripheral inflammation has never been reported so far, though this phenomenon could explain the effects of a nerve block on inflammation. As previously described,2,14 a bupivacaine block was effective in suppressing the systemic inflammatory markers. We previously showed that a nerve block may influence systemic inflammatory responses by a mechanism not involving sodium channel blockade.2 Ketoprofen in our study also suppressed the systemic cytokine response. Effects of NSAID on cytokines are not fully understood. NSAIDs were shown to upregulate TNF-α and downregulate IL-6 production in vitro.16
In patients, there are few studies specifically reporting the anti-inflammatory effect of a local anaesthetic nerve block. Martin et al.17 showed that a femoral block could inhibit the local inflammation and oedema after knee surgery. In their study, the local and systemic (i.e. as measured in whole blood) production of inflammatory cytokines was not affected by the femoral block. They, however, did not sensitize their assay by stimulating (e.g. with LPS) the whole blood before measuring the cytokine concentration in vitro. Inflammation primes the leucocytes but they need to be stimulated to produce excess inflammatory biological markers like cytokines. Bagry et al.18 reported a positive correlation between lower levels of inflammatory markers and lower levels of pain in patients receiving continuous lumbar plexus and sciatic nerve blocks with ropivacaine compared with those receiving systemic morphine after knee surgery. In a specific inflammatory disease, the type I complex regional pain syndrome (CRPS I), a local anaesthetic nerve block can treat the inflammatory symptoms.19 To our knowledge, the anti-inflammatory effects of a nerve block have surprisingly never been compared with a reference anti-inflammatory treatment such as a NSAID even if the combination of a nerve block and a NSAID injection is very often administered in postoperative patients. Studies20,21 comparing different analgesic regimens, including a NSAID and a nerve block, were not designed to compare the anti-inflammatory effect and showed very few differences. Interestingly, in our study, the combination of a sciatic block and a NSAID did not add further benefit compared with either injection alone. One can hypothesize that, as the anti-inflammatory effect of a nerve block does not differ from the effect of a NSAID, the combination of both does not bring any further improvement. This hypothesis needs to be confirmed in patients.
A potential mechanism for the effects of the sciatic blocks would be a systemic effect. Previous results argue against this possibility. Indeed, previous studies comparing this technique of sciatic block with systemic administration of bupivacaine reported that bupivacaine did not exert the same effects when administered systemically.2 However, dosing differs between studies and such a systemic effect of bupivacaine cannot be excluded, though this is very unlikely. Only one dose of each drug was used and the doses of bupivacaine and ketoprofen used in this study were higher than those used in humans. Effective doses are usually higher in rodents than in humans.9 Therefore, additional data are required to confirm that, in humans and at therapeutic doses, bupivacaine and ketoprofen effects are not additive. Finally, another limitation of the study is the timing. We administered one single injection sciatic block and/or one NSAID injection and evaluated the animals 6 h later. We cannot extrapolate our results later in time.
In conclusion, we reported for the first time that a bupivacaine nerve block is as effective as a NSAID injection in preventing local and general inflammatory symptoms 6 h after a painful inflammatory insult in rats. More studies are needed, especially in patients, to determine whether the administration of NSAID is or is not adding a benefit to a nerve block after peripheral surgery.
We thank Mrs. Régine Le Guen for her technical assistance.
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Keywords:© 2010 European Society of Anaesthesiology
bupivacaine; hyperalgesia; nerve block; NSAID; rats