Continuous instillation of local anesthetic into surgical wounds is a technique frequently used for the treatment of postoperative pain.1–3 However, instillation of local anesthetics at the site of surgery may not be completely innocuous. For example, recent evidence suggests that local anesthetics instilled into joints after surgery may cause chondrolysis.4,5 The pathogenesis of local anesthetic-induced toxicity is poorly understood. Alteration of the inflammatory response is a potential mechanism for local anesthetic-induced tissue toxicity.
A large body of literature suggests that local anesthetics exert a significant antiinflammatory effect when administered systemically.6–9 Local anesthetics have been found to inhibit immune function and the migration of granulocytes to inflamed tissue, thereby attenuating the release of proinflammatory mediators such as interleukin (IL)-1, IL-8, and tumor necrosis factor α (TNF-α).10–15 However, little is known about the effects of local anesthetics when administered locally rather than systemically. Local administration may result in effects quite different from those observed after systemic administration because resident cells such as keratinocytes are critically involved in local inflammatory processes after tissue injury.16–18 Local neuronal structures are also involved in the inflammatory response after tissue injury by releasing neuropeptides such as substance P (SP) from peripheral nerve endings, which in turn facilitate the release of cytokines from keratinocytes and fibroblasts.17,19 Whereas suppression of leukocyte migration and function may be at the core of the antiinflammatory actions associated with systemic administration of local anesthetics, effects on resident cells and neuronal structures may be more pertinent to their local administration.
The aim of this study was to determine whether continuous wound instillation of a local anesthetic in a clinically used dose would affect the inflammatory response in postsurgical wounds. In particular, the local release profile of cytokines, chemokines, SP, prostaglandin E2 (PGE2), and nerve growth factor (NGF) were studied over a 24-hour postoperative period in surgical wounds of patients undergoing cesarean delivery, using a recently developed human bioassay technique.20,21
After receiving Stanford University Human Subjects IRB approval and written informed consent, 38 healthy ASA physical status I or II women with term (gestational age >37 weeks), singleton pregnancies undergoing elective cesarean delivery under spinal anesthesia were enrolled in this randomized controlled study. Group allocation was done using computer-generated random-number allocation. To maintain blinding, the bupivacaine and saline were prepared and added to the On-Q® PainBuster® Post-Op Pain Relief System (I-Flow, Lake Forest, CA) by an anesthesiologist not involved in the study or any data collection. The patient, investigator, and all study staff remained blinded to the assigned treatment groups throughout the study period.
Patients were excluded from study participation if they met any of the following criteria: twin pregnancy, morbid obesity (body mass index >40 kg/m2), emergency cesarean delivery, postpartum tubal ligation after cesarean, any contraindication to neuraxial anesthesia, hypersensitivity or previous reaction to opioid medications, history of chronic opioid use, or intolerance to nonsteroidal antiinflammatory drugs (NSAIDs).
All patients received 500 mL lactated Ringer solution plus 500 mL hetastarch after obtaining peripheral IV access. Anesthesia for the cesarean delivery consisted of spinal anesthesia with intrathecal hyperbaric bupivacaine 12 mg, intrathecal fentanyl 10 μg, and intrathecal morphine 200 μg. Immediately before wound closure, an On-Q PainBuster Post-Op Pain Relief System (I-Flow) was inserted subcutaneously to facilitate the administration of plain bupivacaine 5 mg/mL or normal saline at 2 mL/h.
Postoperatively, patients received oral ibuprofen 600 mg every 6 hours for the 48-hour study period with the first dose administered in the postanesthetic care unit 30 minutes after the end of surgery. Breakthrough pain was managed with oral oxycodone 5 mg with acetaminophen 325-mg tablets. Patients were offered 1 to 2 tablets every 4 hours as needed with a maximum allowable dose of oxycodone 60 mg or acetaminophen 4 g in a 24-hour period. Intravenous morphine was available for severe pain or pain not responding to oral opioid analgesics. All other NSAIDs, cycloxygenase-2 inhibitors, and opioids were prohibited during the study period.
We determined the total amount of supplemental opioid analgesic medication used by each patient in the first 24-hour and second 24-hour postoperative periods. The time of surgical incision was time zero. For analysis, all doses of oral oxycodone were converted to IV morphine milligram equivalents using a conversion ratio of 0.5.22
Pain intensity was recorded at rest and during activity (sitting up at 90°) using a numerical verbal pain scale (0 to 10 with 0 = no pain to 10 = worst pain imaginable) at 2, 4, 6, 24, and 48 hours postoperatively by study investigators. The requirement for oral or IV postoperative morphine was recorded.
Nociceptive and Inflammatory Biochemical Mediator Collection and Assays
Wound exudate was collected at 1, 3, 5, 7, and 24 hours after cesarean delivery using a technique we recently developed.20,21 The system uses a 3-way stopcock incorporated into an On-Q PainBuster Post-Op Pain Relief System (I-Flow) to withdraw wound exudate at specified time points. One milliliter of wound exudate was withdrawn into a polyethylene cup containing 30 μL proteinase inhibitor (Complete Proteinase Inhibitor; Roche Bioscience, Palo Alto, CA). The samples were put on ice and centrifuged at 3000 rpm for 10 minutes within 1 hour of collection. The supernatant was removed and transferred to a standard microcentrifuge tube (E&K Scientific Inc., Santa Clara, CA) and stored at −20°C until further processing.
All samples were analyzed at the same time after completion of study enrollment. The following cytokines were measured using a17-multiplex bead array immunoassay plate (Bio-Plex™ system; Bio-Rad, Hercules, CA): IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-α, interferon γ (INF-γ), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 (MIP-1β).
NGF assessment was measured in the same multiplexed assay using a commercially available anti-NGF antibody, DY256 (R&D Systems, Minneapolis, MN), and the Bio-Plex amine coupling kit (Bio-Rad). Each measurement was made in duplicate according to the manufacturer's specifications. Standard curves for each analyte were generated by using the reference analytes supplied by the manufacturer at concentrations of 0.20, 0.78, 3.13, 12.5, 50, 200, 800, and 3200 pg/mL plus a zero standard (normal saline only). Standard curves were included in each run and sample concentrations were calculated with Bio-Plex Manager software (Minneapolis, MN). PGE2 and SP were measured in duplicate using additional enzyme-linked immunosorbent assay kits (Assay Designs, Inc., Ann Arbor, MI).
The primary outcome of this study was the local release profile of key biochemical mediators (IL-6, IL-10, IL-1, SP, PGE2) as assessed by the area under the 24-hour concentration versus time curve (AUC). An a priori sample size analysis based on our previous study20 predicted that we required 19 subjects per study arm to detect a 30% change in the AUC for IL-6 release (power 0.8, α = 0.01). Any assay result above the range of detection was included in the analysis by replacing the value with the upper range of detection value; assay results below the range of detection were assumed to have a value of zero.
Demographic, assay, and outcome data are summarized with descriptive statistics. Results are expressed as the mean ± SD, median (interquartile range), and number (percentage) as appropriate. Normal distribution was determined using QQ plots and the Kolmogorov-Smirnov test. Outcome measures of interest between the 2 groups were compared using the Student t test for normally distributed variables and the Mann-Whitney test for nonparametric comparisons. Individual concentrations of the various mediators were expressed as the AUC of concentration versus time measured during the first 24-hour postcesarean period. One-way repeated-measures analysis of variance followed by Student-Newman-Keuls post hoc test was used to test whether changes over time were significant. Correction for multiple comparisons of the biochemical mediators were made by setting the α value at 0.0025. Analyses were performed with SPSS 11.0 for Windows statistical package (Chicago, IL).
All 38 patients enrolled and randomized in this prospective study completed the protocol. There were no patients lost to follow-up or noncompliance. Demographic and obstetric data are outlined in Table 1. All cesarean deliveries were uncomplicated and the mean ± SD of surgical duration was 87 ± 19 and 84 ± 23 minutes in the bupivacaine and normal saline groups, respectively (P = 0.66). The median (interquartile range) duration from end of surgery until discharge from hospital was 92 (76–97) and 76 (74–97) hours in the bupivacaine and normal saline groups, respectively (P = 0.37). There were no study-related complications or adverse events reported during the study period.
The lower limit of detection for all analytes analyzed by the Bio-Plex system varied between 0.1 and 1.1 pg/mL. The upper limit of the detectable range varied between 233 and 4286 pg/mL. The assay sensitivity range for PGE2 was 7.8 to 1000 pg/mL and for SP was 9.8 to 10,000 pg/mL. The median coefficient of variation for the measured concentrations was between 0.1% and 23.1%. High coefficient of variation values (>10%) were for IL-4, IL-7, IL-17, TNF-α, and MCP-1 and were found close to the limit of detection. The percentage of measurements outside the lower limit of detection and above the upper limit of the detectable range were 6% for IL-1β, 17% for IL-2, 12% for IL-4, 50% for IL-6, 15% for IL-7, 20% for IL-8, 12% for IL-10, 14% for IL-12, 7% for IL-13, 64% for IL-17, 17% for TNF-α, 11% for INF-γ, 3% for G-CSF, 24% for GM-CSF, 30% for MCP-1, 4% for MIP-1, 8% for NGF, 20% for PGE2, and 10% for SP. Measurements below the lower limit of detection were replaced with zero and measurements above the upper limit of detection were substituted with the analytes' upper limit concentration detection value.
All biochemical mediators with the exception of IL-5 were detected in wound exudate. The AUC of individual analytes during the first 24-hour postcesarean period are depicted in Figure 1. Bupivacaine infiltration resulted in a significant and sustained decrease in IL-10 and increase in SP AUC in wound exudate compared with saline administration (P < 0.001). No significant differences in the concentration of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-12, IL-13, IL-17, TNF-α, INF-γ, G-CSF, GM-CSF, MCP-1, MIP-1, NGF, or PGE2 were detected between the study groups. The concentrations of IL-10 and SP over the 24-hour time course are shown in Figure 2. Among the cytokines, mediators typically peaked within 6 hours and levels were sustained for 24 hours (Fig. 3).
There were no differences in postoperative pain scores (at rest and during activity) and analgesic consumption during the 48-hour study period (Table 2). Thirty-five of 38 patients required supplemental oral opioid analgesics. The 3 patients not requiring supplemental opioid analgesics were in the normal saline group. Two patients (1 in each group) required IV opioids.
We report the novel and somewhat unexpected finding that continuous instillation of the local anesthetic bupivacaine at clinically used doses markedly decreased IL-10 and increased SP in wound exudates after cesarean delivery. The change in IL-10 is compatible with a disruption of antiinflammatory mechanisms and this result, combined with the release of the proinflammatory mediator SP, may indicate an overall proinflammatory wound response. These findings contrast with the antiinflammatory effects typically observed with the systemic administration of local anesthetics.6–9
IL-10 is produced by both immune (activated peripheral blood T cells, monocytes, and mast cells) and nonimmune (keratinocytes, epithelial) cells, and possesses potent antiinflammatory activity.23,24 Plasma levels of IL-10 increase significantly after major surgery.25 A previous study found that bupivacaine significantly inhibited cytokine production in endotoxin-activated macrophages,26 and such action may account for changes in wound cytokine content. We found that local wound levels of IL-10 increased markedly after surgery but that the increase was significantly suppressed by the local instillation of clinical doses of bupivacaine. The clinical consequences of our findings on wound healing remain speculative. Decreased IL-10 and increased IL-6 and IL-8 have been associated with increased collagen deposition in rabbit mucosa during wound healing,27 and overexpression of IL-10 has been found to decrease the inflammatory response after skin injury in mice. These findings suggest that IL-10 has a key role in wound healing.28
SP supports nociceptive sensitization and is an important inflammatory mediator in peri-incisional tissue.29 SP also promotes peripheral inflammatory responses, mediates neurogenic inflammation, increases inflammatory cell density in wounds, and enhances the response to cutaneous injury.30–32 SP acts predominantly through the Gq protein–coupled neurokinin 1 receptors, and neurokinin receptor expression in inflammatory and parenchymal cells appears essential for wound healing.33,34 Our study showed that wound instillation of bupivacaine resulted in an increased concentration of SP, most likely by facilitating its release from primary afferent nociceptors. Lidocaine has been shown to induce a TRPV1-dependent release of SP in a rodent model.35
Neurotoxicity is a well-recognized side effect of local anesthetics applied in high concentrations close to neuronal structures such as peripheral nerves, nerve plexuses, or the spinal cord.36 Recently, intraarticular bupivacaine has been associated with postoperative chondrolysis after arthroscopic joint surgery.4,5 The pathogenesis of local anesthetic-induced local tissue toxicity is poorly understood. This study suggests an immune-mediated mechanism may be possible. Local instillation of bupivacaine in humans may exert a net proinflammatory effect by altering the composition of wound inflammatory mediators and triggering the release of proinflammatory neuropeptides.
Levels of PGE2 in wound fluid were not affected by the instillation of bupivacaine. However, patients in both groups received regular ibuprofen, which may have decreased wound PGE2 levels. Studies have found conflicting effects of local anesthetics on PGE2. Lidocaine decreased PGE2 concentrations in an in vivo model of skin burn in rats, but had no effect on PGE2 concentrations in a burn model that assayed skin ex vivo.37,38 A study in patients undergoing third molar extraction showed decreased PGE2 concentrations in conjunction with an increased expression of cyclooxygenase 2 if bupivacaine rather than lidocaine was injected.39 The divergent results indicate that the net effect of local anesthetics on nociceptive and inflammatory mediators are context sensitive and may differ depending on the species studied, the injury, and/or the type of local anesthetic used.
This study did not find that bupivacaine instillation decreased postoperative pain and analgesic consumption. Several reasons may account for the negative finding of this secondary end point. First, to facilitate wound exudate collection, the On-Q PainBuster catheter was placed in the subcutaneous space rather than the subfascial space. Subcutaneous placement is less effective for postoperative analgesia.1,40 Second, the local anesthetic was infused at 2 mL/h and higher infusion rates may have improved analgesia.34 Third, patients experienced relatively low levels of postoperative pain, which makes detection of differences difficult. Lastly, changes in IL-10 and/or SP wound levels associated with local anesthetic instillation may have counteracted the local anesthetic analgesic effect.
This study confirms that the method of wound exudate sampling described in our previous study20,21 is sensitive enough to detect biochemical inflammatory mediators, and to describe changes of these mediators over time. We were able to successfully measure various pro- and antiinflammatory cytokines, NGF, PGE2, and SP at serial time points from cesarean delivery wound exudate. We found that release profiles of these biochemical mediators were similar to those previously reported.20
This study has a number of limitations. The study design does not allow for true baseline measurements. The first measurement was taken in the postanesthetic care unit immediately after cesarean delivery approximately 1 hour after starting the surgery. Animal studies indicate that skin neutrophil infiltration and cytokine production can be observed 30 minutes after incision.16 However, absolute baseline levels are not that critical because the primary end point of the study was to detect differences between local anesthetic and saline placebo treatment over the course of 24 hours after surgery. A potential limitation is the percentage of measurements of certain biochemical mediators, in particular IL-6 and IL-17, outside the limit of detection that may have reduced our ability to detect the effects of local anesthetic instillation. Another limitation is the fact that all patients received neuraxial anesthesia and NSAIDs for postoperative pain control. These interventions may have altered the inflammatory response to surgery. However, these interventions would be expected to be antiinflammatory in nature and potentially obscure antiinflammatory rather than proinflammatory actions of local anesthetics.41,42 In addition, systemic exposure to neuraxial anesthesia and NSAIDs was similar in patients treated with bupivacaine or saline placebo, and unlikely explains differences observed between groups. The study may have been underpowered to detect subtle changes of other measured mediators, which raises the possibility that IL-10–induced suppression of such mediators may have gone undetected. This study was also not a comprehensive examination of all relevant aspects of wound biology; in particular, the effects of local anesthetic on immune cell activity were not assessed.
In conclusion, we found that continuous subcutaneous infusion of bupivacaine after cesarean delivery resulted in a significant decrease in IL-10 and increase in SP in wound exudates compared with saline. These observed changes suggest a disruption of antiinflammatory modulation. Whether such modulation combined with the release of the proinflammatory mediator SP results in an overall proinflammatory wound response is uncertain. This study, however, does indicate that local anesthetic wound instillation affects wound biochemistry. The impact of this response requires future investigations including studies on cellular migration, global measures of wound healing and scarring, wound infection, and pain associated with surgical incisions.
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All authors have contributed to the study design, study conduct, data collection, data analysis, and manuscript preparation. BC is the author responsible for archiving the study files. All the authors have approved the final manuscript.