Chronic postoperative pain occurs with an alarmingly high frequency after many surgical procedures, and knowledge of its mechanisms remains limited.1–3 Peripheral factors released by damaged tissue are important for the induction of acute postincisional pain,4–7 and a role for spinal glutamate receptors,8,9 TRPV1,10 and activated p38 MAPKinases,11,12 has been shown for acute postincisional and persistent pain after soft-tissue retraction.13 However, there have been virtually no studies on the factors that drive chronic pain that persists for months after surgery.
As many as half of the patients undergoing thoracotomy experience pain lasting for >6 months.2,14,15 Pain occurs at rest, that is, with shallow breathing, and also shows a pronounced movement-related component during coughing, stretching, or twisting, an example of mechanohyperalgesia.16,17
A variety of local anesthetic procedures have been shown to reduce postthoracotomy pain,18–20 but the mechanisms of effect here are not obvious because such drugs are known to act on several different targets at their clinical concentrations.21 The cellular and molecular mechanisms that cause persistent postoperative pain, for the most part assigned to the central nervous system,2,22,23 remain obscure.
Understanding the sources of post-thoracotomy pain was facilitated by the development of a model in the rat by Buvanendran et al.24 This first report described the surgical procedure, characterized the resulting mechanical hypersensitivity and cold allodynia, and showed evidence of peripheral nerve injury at the surgical site.
Changes in the sensitivity of local responses, such as simple nocifensive withdrawals, to local stimulation are not necessarily indications of perceived pain.25,26 To address this possible difference, our laboratory developed a Qualitative Hyperalgesia Profile (QHP) that characterizes pain behavior not by the threshold to elicit a response but by the behavioral characteristics of that response.27 The behaviors that comprise the QHP are scored among 4 grades that range from no response to a complete turn away from the tactile stimulus, shuddering of the entire body, and, often, audible (to humans) vocalization. Postthoracotomy rats demonstrate these higher grade behaviors and also show a transient shift back to the baseline preoperative lower grades in response to administration (intraperitoneal) of the known analgesic, morphine, thus validating the equation of these complex behaviors with actual perceived pain.27
In previous studies of microspheres that slowly release bupivacaine, we reported a reduction of postoperative pain from skin incision, sometimes accompanied by blunt dissection, that lasted for 4 to 7 days.28–30 These temporary antihyperalgesic actions were substantially better than those from preoperative infiltration of the wound area with bupivacaine solutions but did not have an enduring effect that supported a possible reduction of chronic postoperative pain. Nevertheless, because the mechanisms of chronic postthoracotomy pain are likely to differ from those arising from skin incision alone, we undertook the present study.
All procedures were approved by the Harvard Medical Area Standing Committee on Animals (Boston, MA) and are in keeping with international standards for the care and treatment of laboratory animals.31 Male Sprague-Dawley rats were purchased from Charles River Laboratory (Wilmington, MA) and kept in the animal housing facilities at Brigham and Women’s Hospital, with controlled relative humidity (20%–30%), at room temperature (24°C), and under a 12 to 12 hour light-dark cycle, with free access to food and water. They were handled for 5 to 7 days before the procedure to familiarize them with the experimental environment, so as to minimize the stress-induced analgesia and to establish baseline behavioral parameters for each individual animal. At the time of surgery, animals weighed 280 to 310 g.
Three groups of 8 rats each were studied under 3 respective conditions. Each group was the sum of 2 cohorts of 4 rats each, with each cohort being received from the supplier on the same day and being familiarized by handling and having surgery at the same time. Data from all 8 rats of a group are analyzed together, without regard for cohort identification.
All rats underwent a thoracotomy (see next section); 1 group was injected with MS-Bupi at the incision site before the surgery (MS-Bupi-L), a second group with MS-Placebo at the same location and time, and a third group with MS-Bupi at a distant caudal location (MS-Bupi-D). All surgery and injections and behavioral analyses were conducted by the same investigator (JC-FW), an experienced researcher who is adept at all these procedures. In scoring the behavioral results, he was not blinded to the treatment group.
Rats were briefly anesthetized with 4% to 5% sevoflurane (Sevorane; Abbott Laboratory, North Chicago, IL) before receiving intraperitoneal pentobarbital sodium (60 mg/kg, Nembutal; Akorn, Inc., Lake Forest, IL). Animals were then tracheally intubated by a method modified from Weksler et al.32 The anesthetized rat was placed in the supine position with a small pillow under the neck. An otoscope (Welch Allyn, Inc., Skaneateles Falls, NY) with a number 3 speculum was introduced into the oropharynx of the animal, and the tongue was gently retracted and fixed above the speculum by left index-finger compression. A guide wire (spring-wire guide: 0.46 mm diameter × 25 cm; Arrow® International, Inc., Reading, PA) was introduced through the epiglottis, vocal cords, and then into the trachea. The otoscope removed over the wire, a 16G polyethylene catheter (Angiocath, 1.7 × 51 mm, Insyte™ Autoguard™ winged; BD Infusion Therapy Systems Inc., Sandy, UT) was glided over the wire to its full length. The wire was removed, and the catheter was connected to a Y-connector attached to tubing from a small animal pressure-controlled ventilator (model TOPO220; Kent Scientific Corporation, Torrington, CT), which was set at a respiratory rate of 65 to 80/min. An isoflurane vaporizer (SurgiVet, Smiths Medical, Norwell, MA) was connected to the intake of the ventilator to deliver a concentration 1.0% to 1.5% isoflurane in oxygen when necessary. A CO2 analyzer (CapStar-100; IITC Inc., Woodland Hills, CA) was connected to the expiratory end to monitor end-tidal CO2, which was maintained at 25 to 40 mm Hg for the entire surgical procedure.
Thoracotomy and Rib Retraction
Consistent with the procedure of Buvanendran et al.,24 the anesthetized rats were placed in the left decubitus position with a pillow under the contralateral armpit to elevate the surgical field. The skin below the ear line and above the superior iliac crest was shaved on both sides. A 3-cm-long incision was made in the skin of the right lateral chest wall along the fourth intercostal line, beginning from 1 cm lateral to the midline and 1 cm below the inferior angle of right scapula. The superficial and deep lateral thoracic muscles covering the ribs were incised and retracted to expose the intercostal muscles. A 1.0-cm incision was made through the intercostal muscle and pleura along the cranial border of the fifth rib. The blunt tines of a small retractor (model 17003-03, Goldstein 3 × 3 sharp teeth with depth 4.5 mm, teeth width 6.5 mm; FST, Inc., Foster City, CA) were placed under the fourth and fifth ribs. The retractor was opened to separate the ribs by 1 cm and was left in place for 60 minutes, as previously described.33,34 During this time, the open wound was covered with wet-dressing gauze kept moist with sterile phosphate-buffered saline. After 1 hour, the retractor was closed and removed, and the fourth and fifth ribs were approximated and ligated tightly with 4-0 Vicryl sutures (Ethicon, Somerville, NJ). Air was aspirated from the pleural cavity with a 5-mL syringe attached to the polyethylene tubing to restore the normal intrapleural pressure. The superficial muscle covering the ribs was then apposed with 4-0 Vicryl sutures (MYCO Medical, Cary, NC), and the skin was closed with 3-0 prolene sutures (Ethicon). The animals were allowed to recover, and the endotracheal catheter was removed once spontaneous breathing was reestablished.
Thresholds were assayed daily or every other day during the entire testing period of 31 days (3 preoperative and 28 postoperative). Tests were always done at the same time of day (9:00 AM to 12:00 PM or 2:00 PM to 5:00 PM) for each separate cohort (n = 4). The threshold force necessary to elicit any response from a rat was determined by pressing against the shaved thoracolumbar dorsal area of the rat with a series of single von Frey filaments (VFHs). Each rat was tested alone, probed with a VFH while loosely constrained in a holding chamber that allowed it to reorient the body, and thus escape the potentially painful stimulus, without running away.27,33,34 Individual VFH was applied starting from the lightest force (1–2 g) and progressing in a series of increasing thickness/force until a response was elicited, but limited to 15 g. Rats that did not respond even to this highest force were assigned a threshold of 15 g because stronger forces caused local swelling/inflammation of the skin after repeated stimulation.
Once any response was elicited (see response characteristics in QHP, below), the next stimulation was by the preceding ineffective force VFH and then again by the effective force. Threshold was defined as the weakest force that could reliably elicit a response, for example, 3 responses from 3 pokes.
Rats were acclimated for 5 to 7 days before the surgery, being handled by the investigator, placed in the restraining chamber, and exposed to the overall environment (light, smells, and noises) of the testing room. Such familiarization abolishes stress-induced analgesia and allows the establishment of a stable preoperative baseline threshold. This baseline threshold was calculated from the average of thresholds measured on each of the 3 days preceding the operation.
Qualitative Hyperalgesic Profile
The nature of the response at threshold changes after surgery was characterized by the QHP.27 Behavior is classified according to the following scoring system of grades:
- Grade Behavioral response to tactile stimulation
- 0 No response
- I Brief contractions of the local subcutaneous muscles
- II Grade I + brisk lateral escape movement and/or a 180° rotation of the trunk
- III Grade 2 + whole-body shuddering, scratching, and squealing
These patterns of behavior are characteristic of different stages of preoperative and postoperative pain. Grades 0 and I are the only responses that occur in the intact preoperative skin and are elicited by VFH forces of 15 g. After surgery, grades II and III appear in response to the much lower threshold stimulation, for example, 3 g.
The sustained-release bupivacaine microsphere (MS-Bupi) formulation used as part of this study was the same as that reported in previously published studies.28–30 Microspheres containing no drug (MS-Placebo) were used as a negative control.
Subcutaneous injections of the microspheres were made through a 21G, thin-walled, beveled needle (Becton-Dickinson, Franklin Lakes, NJ) while the rat was under brief general anesthesia from muzzle-inspired sevoflurane (Fig. 1). Two injections of 0.3 mL each were made parallel to and about 0.5 cm away from an inked line marking the incision site. Previous studies had shown that the subcutaneous injection of MS-Bupi as well as bupivacaine solutions (0.5%) produced an effective analgesia in the intact skin above the injected zone, shown by the cross-hatched circles in Figure 1, but not far beyond its margins.29
Two boluses totaling 0.6 mL, dissolving 100 mg of MS-Bupi containing 60 mg of bupivacaine base, were injected 2 hours before the surgery, locally (MS-Bupi-L) alongside the location of the intended incision and retraction. Previous studies in intact rats showed that this period was adequate to allow full anesthesia to develop when only 40 mg equivalent of bupivacaine-free base in MS-Bupi had been injected. The larger volume used here was required to anesthetize a roughly circular area with a diameter of approximately 2 cm, the length of the incision for the thoracotomy.
Drug control experiments were conducted using MS-Placebo injected with the same volume and in the same location. MS-Placebo 40 mg was injected per animal to make it equivalent to the amount of polymer injected per animal in the MS-Bupi-administered animals.
To control for possible effects of systemic bupivacaine, released from microspheres and resorbed into the circulation, the same volume of MS-Bupi was injected at a more posterior location, approximately 6 cm caudal from the incision site (MS-Bupi-D), where the anesthetized skin did not extend to the surgical field but from which systemic uptake would be very similar.
Withdrawal thresholds were compared among treatment groups for the period of constant, chronic pain, 14 to 28 days postoperative, using the Kruskal-Wallis test, followed by pairwise comparisons of groups using a Wilcoxon-Mann-Whitney odds (WMWodds) determination with Bonferroni-corrected 95% confidence intervals for multiple groups.35,36 Analysis of the area under the curve (AUC), as the displacement of the threshold from the preoperative baseline level integrated over time, was also assessed for significance between treatment groups using the same statistical method as for threshold changes (above). AUC was calculated using the trapezoidal rule with 14, 18, 22, and 28 days as cut-offs. Population distributions among the different QHP grades were compared using Jonckheere-Terpstra analyses. All tests were performed with SAS (Cary, NC) statistics software.
Preoperative MS-Bupi at the Surgical Site Lessens Postoperative Mechanical Hypersensitivity
After a brief initial delay, the tactile response threshold in control MS-Placebo-treated rats fell from the baseline preoperative value of 15 g to approximately 3 to 4 g, where it remained for up to 4 weeks, after which the animals were killed (Fig. 2). This pattern is identical to the change observed in previous studies from our laboratory after thoracotomy alone.27 In contrast, when local MS-Bupi was injected preoperatively, the initial drop in threshold was arrested, at approximately 10 g, after postoperative day (POD) 4 and remained near this level for the next 24 days of testing (Fig. 2). Comparison of threshold values among MS-Placebo, MS-Bupi-L, and MS-Bupi-D groups on the individual PODs revealed significance on PODs 14 to 28 (except on POD21) as shown in Table 1. Subsequent post hoc analysis of WMWodds showed that MS-Bupi-L suppressed the threshold more than MS-Placebo (or previously determined treatment-free controls)27,33 as shown in Table 2.
The possibility that this antihyperalgesic effect could result from systemically distributed bupivacaine, released from the microspheres and resorbed by the local circulation, was tested by experiments where the same dose, that is, 0.6 mL dissolving 100 mg of MS-Bupi containing 60 mg of bupivacaine-free base, was injected subcutaneously at the same preoperative time but at a more caudal location (MS-Bupi-D). The pattern of mean threshold change with this distant treatment was very similar to that with MS-Placebo at the wound site for PODs 14 to 28 (Fig. 2), as shown by WMWodds analysis (Table 2), a finding that rules out a contribution of systemic bupivacaine to postoperative antihyperalgesia.
The integrated effects of the treatments were analyzed by comparisons of AUC for reductions of threshold forces from the baseline preoperative values. Such AUC analysis was conducted for each rat in a group, and the distributions compared among the 3 groups, for the assessed steady-state period of chronic pain, PODs 14 to 28. The mean AUCs (in g/days) were MS-Bupi-L, 66.4 ± 48.0; MS-Bupi-D, 132.5 ± 42.7; and MS-Placebo, 142.6 ± 36.6 (means ± SD).
Preoperative infiltration with MS-Bupi-L at the surgical site significantly reduced the AUC from the MS-Placebo control and MS-Bupi-D group (P = 0.0235, Kruskal-Wallis, Table 1). Pairwise comparisons of the AUCs between local MS-Bupi and the other 2 treatment groups showed the same relations as the day-to-day ones (Table 2) with local delivery being more effective than either MS-Bupi-D or MS-Placebo. The latter 2 did not differ significantly.
The Time Course of Threshold Changes Among Individual Rats Is Affected by MS-Bupi
The changes in threshold reported above were composites of the responses of individual animals, which are shown in Figure 3. Each of the 8 control rats that received MS-Placebo shows a pattern of threshold changes after surgery that can be classified among 3 time courses: course 1: no drop in threshold: zero (0) control rats showed this course; course 2: threshold falls to values of approximately 8 g, intermediate between baseline and the lower values of <4 g: 2 control rats (1 and 6) showed this; and course 3: threshold falls to ≤4 g and remains there through day 28: 6 control rats (2, 3, 4, 5, 7, and 8) had this pattern (Fig. 3A).
When local MS-Bupi was injected locally, the distribution of time courses was different (Fig. 3B). Course 1: 3 rats (5, 6, and 7) had threshold remaining at the preoperative baseline of 15 g; course 2: 3 rats (1, 2, and 3) had threshold fall to approximately 8 g by PODs 5 to 6 and remained there; and course 3: 1 rat (8) had threshold fall to a low value <4 g and remained there.
Injection of distant subcutaneous MS-Bupi in 6 rats resulted in course 1: 0 rats; course 2: 2 rats; and course 3: 4 rats (individual patterns of these 6 rats are not shown). This distribution is indistinguishable from that of MS-Placebo rats.
Preoperative MS-Bupi Lessens Pain Indicated by the Qualitative Hyperalgesia Profile
The nature of the behavioral responses to stimulation at threshold changes after thoracotomy. The QHP grades of the individual rats at different postoperative stages are listed next to the traces of Figure 3. In the preoperative baseline period, all of the rats in any of the 3 groups showed either grade I or grade 0 responses; that is, either they had no response or only a contraction of the local back muscles.37 After thoracotomy, however, grades II and III appeared at the much lower thresholds and became the predominant behavioral expression.27 This same shift in QHP occurred when MS-Placebo was injected preoperatively (Fig. 4A); by 14 and 28 days after surgery, these 2 grades were observed in 7 of the 8 rats in this group, with only 1 rat showing grade 1 and none showing grade 0. In contrast, when MS-Bupi was injected, 3 of the 8 rats showed grade 1 behavior and the remaining 5 showed grade II and III behaviors (Fig. 4B). Of note, the 3 rats that retained their QHP grade of 1 are the same rats that showed no drop in threshold after surgery (see QHP notations on Fig. 2A). The QHP distribution at day 28 in the group that received MS-Bupi at the distant site (Fig. 4C) was almost identical to that of the MS-Placebo group (P > 0.05, Jonkheere-Terpstra).
Some of the individual animals in the MS-Bupi-L treatment group had no hypersensitivity, measured by threshold change, and, correspondingly, did not show nocifensive behavior consistent with intense pain in contrast to control animals that all showed hypersensitivity by threshold measures and, correspondingly, developed intense pain behaviors.
The major finding of this investigation is that the local release of bupivacaine from microspheres injected preoperatively, which in intact skin reduces the response to pin-prick for approximately 24 hours,29 results in the lessening of the chronic postoperative tactile allodynia for ≥4 weeks. This was an unexpected result, given that previous reports on postoperative pain for less injurious operations, for example, skin incision and blunt dissection, found only 4 to 7 days of antihyperalgesic effect of local MS-Bupi.28–30
Mechanohypersensitivity is a well-documented symptom after thoracotomy.14,38 Coughs, movement of the thorax, and even deep breathing can cause pain for many days after surgery, and spontaneous pain also is often present. The hypersensitivity that is quantitated by the fall in threshold force to elicit a nocifensive response in the model used here is also paralleled by the appearance of very different behaviors at threshold, as scored by the QHP grades, and by spontaneous pain, as indicated by the Conditioned Place Preference test (unpublished results). Normalization of the threshold back toward preoperative levels can be accomplished by systemic morphine or gabapentin, 2 known analgesics, and is accompanied by a shift of QHP profiles back toward lower grades of the preoperative distribution,27 behaviors that are indicative of only local reflexes.37
In the present article, prevention of any threshold drop after thoracotomy in individual local MS-Bupi-treated rats was accompanied by a QHP profile similar to that in the preoperative period (Fig. 4). Therefore, the tactile hypersensitivity measured by a drop in threshold for response by these rats appears to parallel the pain experienced by rats after thoracotomy and supports the use of this system to model clinical postthoracotomy pain. Previous studies with this model showed that tactile hypersensitivity persists for ≥9 weeks after thoracotomy,24 and the constancy of the suppression of such hypersensitivity after local MS-Bupi suggests that the effect could last well beyond the 28 days of observation, although additional experiments are required to definitively establish the complete time course of effect.
Previous publications from our laboratory with the same MS-Bupi formulation, using preclinical models of acute postoperative pain (including the lateral paw incision model and the back incision model in rats), report the mitigation of allodynia and hyperalgesia in the acute postoperative period.28–30 As in the present study, contralateral administration of the MS-Bupi formulation had no effect on postoperative pain. The collective body of data on this MS-Bupi formulation therefore suggests that the systemically distributed local anesthetic appears to yield no therapeutic effect.
Others have used this thoracotomy model to assess the ability of intercostal or systemically (intraperitoneally) injected bupivacaine (0.2 mL, 0.5%, 1 mg dose) to suppress the postoperative hypersensitivity.33 They found that the nocifensive force threshold fell continuously for 21 PODs in the control, uninjected rats, but that this decline was reduced, and similarly so, in the rats receiving either intercostal or systemic bupivacaine solutions. Previous studies using a simple incision model on the rat’s back skin had also concluded that systemic bupivacaine, resulting from a distant subcutaneous injection of 0.4 mL, 0.25% (1 mg dose), could almost completely prevent acute secondary postincisional hypersensitivity measured at a distance from the incision site although not the immediately occurring primary hypersensitivity measured near the incision.39 For this primary hypersensitivity, local bupivacaine injections were effective inhibitors of both the immediate (0–4 hours) and the later (1–4 days) change. Those authors concluded that bupivacaine was acting both locally and at the central nervous system, in the latter case suppressing the central sensitization that underlies the secondary hypersensitivity that is measured at distant, for example, contralateral, sites from the surgery.40
Because subcutaneous injections of bupivacaine solutions have effects attributable to systemic distributions, as detailed above, and because these effects have already been reported for thoracotomy in rats,33 we chose not to include a group that had received only bupivacaine.
Systemic bupivacaine is usually avoided in clinical situations because of its central nervous system and, especially, cardiac toxicity.41 In those articles, mentioned above, where an analgesic effect of systemic bupivacaine on postoperative pain was claimed, no blood levels were measured. However, we have recently reported serum levels of bupivacaine after subcutaneous injection into the back, at the same location as the present study, of 0.4 mL of 0.5% bupivacaine solution, containing 1.78 mg bupivacaine.42 Peak levels of approximately 400 ng/mL were detected at 30 minutes after the injection, and 8 hours later, serum bupivacaine was undetectable (<3.2 ng/mL). Assuming that the pharmacokinetics of bupivacaine do not differ greatly between intraperitoneal and subcutaneous delivery,43 it is likely that the systemic concentrations over time that occurred in the previous study of effect of intraperitoneal bupivacaine on post-thoracotomy pain33 were not very different from those reported in our recent article42 and that the temporal window of effectiveness here is thus within a few hours of the administration of bupivacaine solution.
In contrast, in the present article, and in our previous study of postincisional pain after local MS-Bupi,29 we found no evidence of any postoperative pain relief from systemic bupivacaine that might be resorbed from a subcutaneous injection of the bupivacaine microspheres. Serum levels after such an injection, delivering 0.4 mL containing 40 mg bupivacaine base, have maximal values of approximately 220 ng/mL, appear more slowly than that from bupivacaine solution, and remain at detectable levels, approximately 40 ng/mL, for ≥144 hours after injection.42 The lack of any reduction of postoperative hypersensitivity from systemic bupivacaine that results from this subcutaneous infiltration, in the incision–extension model29,30 as well as in the thoracotomy model, when contrasted with the effectiveness of systemic bupivacaine resulting from subcutaneous or intraperitoneal injections of bupivacaine solutions demonstrated in other studies,33,39 suggests that both an early transient presence of systemic bupivacaine44 and a later persistent presence of bupivacaine restricted to the wound site are effective in reducing the postoperative hypersensitivity although different mechanisms might account for these 2 actions.
What specific mechanisms might explain the long duration (28 days) of antihypersensitivity that is affected by the local administration of MS-Bupi in this rat model? Systemic drug, acting at spinal cord or brain, cannot account for the effects because the distally injected MS-Bupi is without effect. Local actions in the acute phase after surgery might blunt the neuroplasticity that underlies long-term hyperalgesia and explain the observed results. Early effects of insoluble precipitates of bupivacaine applied directly to nerve have been shown to reduce both long-lasting hyperalgesia and increased neuronal excitability after nerve injury.45 Both the generation46 and the propagation47 of action potentials in peripheral nerves are inhibited by local anesthetics although in the 10−3 mol/L range.48 Impulse activity is triggered acutely by nerve and tissue injury during surgery, and spontaneous activity may persist for days or longer after surgery as a result of long-term changes in neuronal excitability induced by factors released from such injury.4–6 Neural blockade is a well-established perioperative mode of reducing postoperative pain,49 and the inhibition of impulses, whether stimulated or occurring spontaneously after nerve injury, will prevent the release of local substances from injured and noninjured nerve endings (neurogenic inflammation) as well as the generation of afferent impulses that can lead to central (spinal) sensitization.
Other local actions are also possible, including the actions on substances released in the injured tissue and known to sensitize neurons. Among these are endothelin-1 and substance P, both implicated in incision-related hypersensitivity6,7 and both known to be inhibited at the receptor or its downstream pathway by local anesthetics.50,51 In addition, local inflammation that accompanies virtually every injury, including skin incision, is directly inhibited by local anesthetics, sometimes at remarkably low concentrations.52,53 However, the relative contributions of these possible mechanisms involving local bupivacaine released from the MS-Bupi formulation and the anticipated duration of effect are unknown and will require additional experimental investigation.
In contrast to the present work, reports of other sustained-release formulations of bupivacaine that have been developed, whether polymer encapsulation based54,55 or liposome based,56,57 did not report an impact on chronic pain. Although clinical data suggest that effective mitigation of acute pain can, in turn, positively affect chronic pain outcomes,2,17,58 there is no obvious way to predict which sustained-release formulation of bupivacaine will influence chronic pain outcomes. Drug load, release rate,59 and actions on the local vasculature that removes the released local anesthetic60 will all contribute to the overall status of nerve block, and there may be optimal formulations for different anatomical sites/routes of delivery.
In summary, chronic postthoracotomy pain, which is well matched to the clinical syndrome by the rat preparation used here, is substantially reduced by the preoperative infiltration of the wound locus with a novel slow-release formulation of bupivacaine. This therapeutic action is not present when the formulation is deposited at a distant site, and the previously reported systemic concentrations of bupivacaine after such infiltrations are well below the reported toxic concentrations. It thus appears that this material may provide a safe and effective means of reducing chronic postoperative pain.
Name: Gary R. Strichartz, PhD.
Contribution: This author designed the studies, helped analyze the data, and wrote most of the manuscript.
Attestation: Gary R. Strichartz is the archival author. He has read and reviewed the final manuscripts, attests to the veracity of the original data and the correctness of its analysis, and approves the final manuscript.
Conflicts of Interest: Gary R. Strichartz is a member of a partnership that has formed for the purpose of licensing the entity reported here and of carrying the entity forward into clinical trials and eventual commercialization. Rachit Ohri, PhD, and Gary R. Strichartz, PhD, are members of a partnership formed for the purpose of advancing the formulation studied here to clinical application. As such they stand to benefit monetarily from the results reported here.
Name: Jeffrey Chi-Fei Wang, MD.
Contribution: This author conducted the surgical and behavioral testing, collected and did the initial analysis of the original data, and also read and commented on the manuscript.
Attestation: Jeffrey Chi-Fei Wang attests to the veracity of the original data and its analysis and has read and approves the manuscript.
Conflicts of Interest: This author has no conflicts of interest to declare.
Name: Phillip Blaskovich, BS.
Contribution: This author synthesized all the drug formulations used in this paper, reviewed the original data, and read the manuscript.
Attestation: Phillip Blaskovich attests that he has read the manuscript and attests to the accuracy of the data reported in this manuscript.
Conflicts of Interest: Phillip Blaskovich is an employee of Covidien, which holds the patent on the entity investigated here.
Name: Rachit Ohri, PhD.
Contribution: Rachit Ohri helped to design the experiments, reviewed all the original data, and contributed to the writing of the manuscript.
Attestation: This author attests to the accuracy of the data and to the veracity of the analysis and the interpretation in the manuscript.
Conflicts of Interest: Rachit Ohri is a member of a partnership that has formed for the purpose of licensing the formulation reported here and of carrying the entity forward into clinical trials and eventual commercialization. Rachit Ohri, PhD, and Gary R. Strichartz, PhD, are members of a partnership formed for the purpose of advancing the entity studied here to clinical application. As such they stand to benefit monetarily from the results reported here.
This manuscript was handled by: Spencer S. Liu, MD.
The authors thank Mr. James Bell, Department of Anesthesiology, Perioperative, and Pain Medicine, Brigham and Women’s Hospital, for assistance with the figures, and Drs. Alla Khodorova, Pain Research Center, Brigham and Women’s Hospital. Special gratitude is due to Chuan-Chin Huang, PhD, Department of Anesthesiology, for assistance with the statistical analysis.
1. Dworkin RH, McDermott MP, Raja SN. Preventing chronic postsurgical pain: how much of a difference makes a difference? Anesthesiology. 2010;112:516–8
2. Kehlet H, Jensen TS, Woolf CJ. Persistent postsurgical pain: risk factors and prevention. Lancet. 2006;367:1618–25
3. Kissin I, Gelman S. Chronic postsurgical pain: still a neglected topic? J Pain Res. 2012;5:473–89
4. Banik RK, Subieta AR, Wu C, Brennan TJ. Increased nerve growth factor after rat plantar incision contributes to guarding behavior and heat hyperalgesia. Pain. 2005;117:68–76
5. Clark JD, Qiao Y, Li X, Shi X, Angst MS, Yeomans DC. Blockade of the complement C5a receptor reduces incisional allodynia, edema, and cytokine expression. Anesthesiology. 2006;104:1274–82
6. Mujenda FH, Duarte AM, Reilly EK, Strichartz GR. Cutaneous endothelin—a receptors elevate post-incisional pain. Pain. 2007;133:161–73
7. Sahbaie P, Shi X, Guo TZ, Qiao Y, Yeomans DC, Kingery WS, Clark JD. Role of substance P signaling in enhanced nociceptive sensitization and local cytokine production after incision. Pain. 2009;145:341–9
8. Zahn PK, Pogatzki-Zahn EM, Brennan TJ. Spinal administration of MK-801 and NBQX demonstrates NMDA-independent dorsal horn sensitization in incisional pain. Pain. 2005;114:499–510
9. Zahn PK, Sluka KA, Brennan TJ. Excitatory amino acid release in the spinal cord caused by plantar incision in the rat. Pain. 2002;100:65–76
10. Banik RK, Brennan TJ. Trpv1 mediates spontaneous firing and heat sensitization of cutaneous primary afferents after plantar incision. Pain. 2009;141:41–51
11. Wen YR, Suter MR, Ji RR, Yeh GC, Wu YS, Wang KC, Kohno T, Sun WZ, Wang CC. Activation of p38 mitogen-activated protein kinase in spinal microglia contributes to incision-induced mechanical allodynia. Anesthesiology. 2009;110:155–65
12. Gao YJ, Ji RR. Activation of JNK pathway in persistent pain. Neurosci Lett. 2008;437:180–3
13. Huang L, Gao YJ, Wang J, Strichartz G. Shifts in cell-type expression accompany a diminishing role of spinal p38-mapkinase activation over time during prolonged postoperative pain. Anesthesiology. 2011;115:1281–90
14. Gottschalk A, Ochroch EA. Clinical and demographic characteristics of patients with chronic pain after major thoracotomy. Clin J Pain. 2008;24:708–16
15. Bayman EO, Brennan TJ. Incidence and severity of chronic pain at 3 and 6 months after thoracotomy: meta-analysis. J Pain. 2014;15:887–97
16. Wang HT, Liu W, Luo AL, Ma C, Huang YG. Prevalence and risk factors of chronic post-thoracotomy pain in Chinese patients from Peking Union Medical College Hospital. Chin Med J (Engl). 2012;125:3033–8
17. Katz J, Jackson M, Kavanagh BP, Sandler AN. Acute pain after thoracic surgery predicts long-term post-thoracotomy pain. Clin J Pain. 1996;12:50–5
18. Obata H, Saito S, Fujita N, Fuse Y, Ishizaki K, Goto F. Epidural block with mepivacaine before surgery reduces long-term post-thoracotomy pain. Can J Anaesth. 1999;46:1127–32
19. Bong CL, Samuel M, Ng JM, Ip-Yam C. Effects of preemptive epidural analgesia on post-thoracotomy pain. J Cardiothorac Vasc Anesth. 2005;19:786–93
20. Sentürk M, Ozcan PE, Talu GK, Kiyan E, Camci E, Ozyalçin S, Dilege S, Pembeci K. The effects of three different analgesia techniques on long-term postthoracotomy pain. Anesth Analg. 2002;94:11–5
21. Strichartz G, Pastijn E, Sugimoto KCousins MJ, Carr DB, Horlocker TT, Bridenbaugh PO. Neural physiology and local anesthetic action. Neural Blockade in Clinical Anesthesia and Pain Medicine. 2009 Philadelphia, PA Wolters Kluwer-Lippincott Williams & Wilkins:26–47 In:
22. Wildgaard K, Ravn J, Kehlet H. Chronic post-thoracotomy pain: a critical review of pathogenic mechanisms and strategies for prevention. Eur J Cardiothorac Surg. 2009;36:170–80
23. Ito N, Obata H, Saito S. Spinal microglial expression and mechanical hypersensitivity in a postoperative pain model: comparison with a neuropathic pain model. Anesthesiology. 2009;111:640–8
24. Buvanendran A, Kroin JS, Kerns JM, Nagalla SN, Tuman KJ. Characterization of a new animal model for evaluation of persistent postthoracotomy pain. Anesth Analg. 2004;99:1453–60
25. Mogil JS, Davis KD, Derbyshire SW. The necessity of animal models in pain research. Pain. 2010;151:12–7
26. Rice AS, Cimino-Brown D, Eisenach JC, Kontinen VK, Lacroix-Fralish ML, Machin I, Mogil JS, Stöhr TPreclinical Pain Consortium. . Animal models and the prediction of efficacy in clinical trials of analgesic drugs: a critical appraisal and call for uniform reporting standards. Pain. 2008;139:243–7
27. Chi-Fei Wang J, Hung CH, Gerner P, Ji RR, Strichartz GR. The qualitative hyperalgesia profile: a new metric to assess chronic post-thoracotomy pain. Open Pain J. 2013;6:190–8
28. Ohri R, Blaskovich P, Wang JC, Pham L, Nichols G, Hildebrand W, Costa D, Scarborough N, Herman C, Strichartz G. Prolonged nerve block by microencapsulated bupivacaine prevents acute postoperative pain in rats. Reg Anesth Pain Med. 2012;37:607–15
29. Ohri R, Wang JC, Blaskovich PD, Pham LN, Costa DS, Nichols GA, Hildebrand WP, Scarborough NL, Herman CJ, Strichartz GR. Inhibition by local bupivacaine-releasing microspheres of acute postoperative pain from hairy skin incision. Anesth Analg. 2013;117:717–30
30. Ohri R, Wang JC, Pham L, Blaskovich PD, Costa D, Nichols G, Hildebrand W, Scarborough N, Herman C, Strichartz GR. Prolonged amelioration of experimental postoperative pain by bupivacaine released from microsphere-coated hernia mesh. Reg Anesth Pain Med. 2014;39:97–107
31. Guide for the Care and Use of Laboratory Animals. National Research Council of the National Academies, the National Academies Press. 20118th ed. Washington D.C.
32. Weksler B, Ng B, Lenert J, Burt M. A simplified method for endotracheal intubation in the rat. J Appl Physiol (1985). 1994;76:1823–5
33. Shin JW, Pancaro C, Wang CF, Gerner P. Low-dose systemic bupivacaine prevents the development of allodynia after thoracotomy in rats. Anesth Analg. 2008;107:1587–91
34. Shin JW, Pancaro C, Wang CF, Gerner P. The effects of resiniferatoxin in an experimental rat thoracotomy model. Anesth Analg. 2010;110:228–32
35. Dexter F. Wilcoxon-Mann-Whitney test used for data that are not normally distributed. Anesth Analg. 2013;117:537–8
36. Divine G, Norton HJ, Hunt R, Dienemann J. A review of analysis and sample size calculation considerations for Wilcoxon tests. Anesth Analg. 2013;117:699–710
37. Theriault E, Diamond J. Nociceptive cutaneous stimuli evoke localized contractions in a skeletal muscle. J Neurophysiol. 1988;60:446–62
38. Guastella V, Mick G, Soriano C, Vallet L, Escande G, Dubray C, Eschalier A. A prospective study of neuropathic pain induced by thoracotomy: incidence, clinical description, and diagnosis. Pain. 2011;152:74–81
39. Duarte AM, Pospisilova E, Reilly E, Mujenda F, Hamaya Y, Strichartz GR. Reduction of postincisional allodynia by subcutaneous bupivacaine: findings with a new model in the hairy skin of the rat. Anesthesiology. 2005;103:113–25
40. Raja SN, Campbell JN, Meyer RA. Evidence for different mechanisms of primary and secondary hyperalgesia following heat injury to the glabrous skin. Brain. 1984;107 (pt 4):1179–88
41. Knudsen K, Suurkula NB, Blomberg S, Sjövall J, Edvardsson N. Central nervous system and cardiovascular effects of IV infusions of ropivacaine, bupivacaine, and placebo in volunteers. Brit J Anaesth. 1997;78:507–14
42. Schmidt B, Ohri R, Wang JC, Blaskovich P, Kesselring A, Scarborough N, Herman C, Strichartz G. Local pathology and systemic serum bupivacaine after subcutaneous delivery of slow-releasing bupivacaine microspheres. Anesth Analg. 2015;120:36–44
43. Covino BG, Vassalo HG Local Anesthetics: Mechanisms of Action and Clinical Use. 1965 New York, NY Grune and Stratton:97
44. Haller Y, Gantenbein AR, Willimann P, Spahn DR, Maurer K. Systemic ropivacaine diminishes pain sensitization processes: a randomized, double-blinded, placebo-controlled, crossover study in healthy volunteers. Pain Ther. 2014;3:45–58
45. Xie W, Strong JA, Meij JT, Zhang JM, Yu L. Neuropathic pain: early spontaneous afferent activity is the trigger. Pain. 2005;116:243–56
46. Raymond SA. Subblocking concentrations of local anesthetics: effects on impulse generation and conduction in single myelinated sciatic nerve axons in frog. Anesth Analg. 1992;75:906–21
47. Gokin AP, Philip B, Strichartz GR. Preferential block of small myelinated sensory and motor fibers by lidocaine: in vivo electrophysiology in the rat sciatic nerve. Anesthesiology. 2001;95:1441–54
48. Huang JH, Thalhammer JG, Raymond SA, Strichartz GR. Susceptibility to lidocaine of impulses in different somatosensory afferent fibers of rat sciatic nerve. J Pharmacol Exp Ther. 1997;282:802–11
49. Barreveld A, Witte J, Chahal H, Durieux ME, Strichartz G. Preventive analgesia by local anesthetics: the reduction of postoperative pain by peripheral nerve blocks and intravenous drugs. Anesth Analg. 2013;116:1141–61
50. Makdessi M, Barr TP, Xue W, Strichartz GR. Bupivacaine inhibits Endothelin-1 evoked increases in intracellular calcium in model sensory neurons. Acta Anaesthesiol Scand. 2015 Feb 15. doi:10.1111/aas.12481. [Epub ahead of print]
51. Li YM, Wingrove DE, Too HP, Marnerakis M, Stimson ER, Strichartz GR, Maggio JE. Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+
. Anesthesiology. 1995;82:166–73
52. Hollmann MW, Durieux ME. Local anesthetics and the inflammatory response: a new therapeutic indication? Anesthesiology. 2000;93:858–75
53. Ploppa A, Kiefer RT, Haverstick DM, Groves DS, Unertl KE, Durieux ME. Local anesthetic effects on human neutrophil priming and activation. Reg Anesth Pain Med. 2010;35:45–50
54. Castillo J, Curley J, Hotz J, Uezono M, Tigner J, Chasin M, Wilder R, Langer R, Berde C. Glucocorticoids prolong rat sciatic nerve blockade in vivo from bupivacaine microspheres. Anesthesiology. 1996;85:1157–66
55. Curley J, Castillo J, Hotz J, Uezono M, Hernandez S, Lim JO, Tigner J, Chasin M, Langer R, Berde C. Prolonged regional nerve blockade. Injectable biodegradable bupivacaine/polyester microspheres. Anesthesiology. 1996;84:1401–10
56. Chahar P, Cummings KC 3rd. Liposomal bupivacaine: a review of a new bupivacaine formulation. J Pain Res. 2012;5:257–64
57. Bagsby DT, Ireland PH, Meneghini RM. Liposomal bupivacaine versus traditional periarticular injection for pain control after total knee arthroplasty. J Arthroplast. 2014;29:1687–90
58. Perkins FM, Kehlet H. Chronic pain as an outcome of surgery. A review of predictive factors. Anesthesiology. 2000;93:1123–33
59. Gerner P, Wang CF, Lee BS, Suzuki S, Degirolami U, Gandhi A, Knaack D, Strichartz G. The relationship between functional sciatic nerve block duration and the rate of release of lidocaine from a controlled-release matrix. Anesth Analg. 2010;111:221–9
60. Newton DJ, Burke D, Khan F, McLeod GA, Belch JJ, McKenzie M, Bannister J. Skin blood flow changes in response to intradermal injection of bupivacaine and levobupivacaine, assessed by laser Doppler imaging. Reg Anesth Pain Med. 2000;25:626–31