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Spontaneous Pain-Like Behaviors Are More Sensitive to Morphine and Buprenorphine Than Mechanically Evoked Behaviors in a Rat Model of Acute Postoperative Pain

Kabadi, Rajiv BE*†; Kouya, Francois PhD; Cohen, Hillel W. DrPH, MPH; Banik, Ratan K. MD, PhD*†

doi: 10.1213/ANE.0000000000000571
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Nonevoked spontaneous pain is most problematic for postoperative patients. Physicians assess this form of pain using the human visual analog scale or verbal numeric rating scale. Recent studies have proposed that spontaneous foot-lifting (SFL) behaviors are an expression of spontaneous pain in animals after spinal nerve injury or adjuvant-induced inflammation. In the current study, we characterize SFL behaviors in a rat model of acute postoperative pain, which includes comparisons with evoked behaviors to analgesic treatments.

METHODS: SFL was manually recorded over four 5-minute periods with 10-minute intervals between each testing session. Paw-withdrawal thresholds were subsequently measured with an electronic von Frey esthesiometer. To evaluate the effects of age, rats were tested in different age groups: 2, 7, and >26 months. The effects of buprenorphine and morphine were tested in a separate group of animals, which received intraperitoneal injections of saline, morphine (0.01, 0.1, 1, or 2 mg/kg), or buprenorphine (0.001, 0.01, or 0.1 mg/kg) before testing.

RESULTS: SFL behaviors peaked at 3 hours after incision and significantly recovered by the 3rd or 4th postoperative day (P < 0.0001). The presentation of these behaviors did not significantly vary with animal age (2, 7, and >26 months old; P = 0.30). SFL behaviors, with the exception of rapid SFL at 3 hours after incision, did not show significant correlation with paw-withdrawal threshold behaviors. The median effective dose of buprenorphine for reversal of mechanical hyperalgesia (0.0452 mg/kg; 95% CI, 0.0259–0.0787) was significantly larger than for reversing rapid (0.0027 mg/kg; 95% CI, 0.0009–0.0083; P < 0.0001) and prolonged (0.0004 mg/kg, 95% CI, 0.0000, 0.0035; P = 0.001) SFL at 3 hours after incision. Similarly, the median effective dose of morphine for reversal of mechanical hypersensitivity behaviors (2.901 mg/kg; 95% CI, 1.132–7.436) was larger than for SFL count (0.4044 mg/kg; 95% CI, 0.1048–1.561; P = 0.0103) and SFL duration (0.0309 mg/kg; 95% CI, 0.0095–0.0998; P < 0.0001) at 3 hours after incision.

CONCLUSIONS: The present study demonstrates that a hindpaw plantar incision induces SFL behaviors in rats and that these behaviors have higher bioassay sensitivity to analgesic interventions with morphine and buprenorphine compared with mechanically evoked behaviors.

From the *Department of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York; Pain Research Program, NJ Neuroscience Institute and Seton Hall University School of Graduate Medical Education, JFK Medical Center, Edison, New Jersey; and Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York.

Accepted for publication September 29, 2014.

Funding: This work is supported in part through a grant from the American Federation for Aging Research and the grants AG030352 and AG030352-02S2 from the National Institutes of Health (NIH), Bethesda, MD (to RKB). In addition, this work was supported in part by the CTSA Grant 1 UL1 TR001073-01, 1 TL1 TR001072-01, and 1 KL2 TR001071-01 from the National Center for Advancing Translational Sciences (NCATS), a component of the NIH, Bethesda, MD (to HWC).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Ratan K. Banik, MD, PhD, Department of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314. Address e-mail to talktoratan@gmail.com.

Pain from a surgical incision occurs in the absence of external stimuli and is exacerbated by locomotion or mechanical stimulation. This form of pain is most problematic for postoperative patients.1 Accordingly, clinical studies involving postoperative patients routinely evaluate spontaneous, nonevoked pain using the human visual analog scale or verbal numerical rating scale.2 Preclinical studies have proposed that spontaneous foot-lifting (SFL) behaviors are an expression of spontaneous pain from nerve injury or adjuvant-induced inflammation models. These behaviors, which involve an animal rapidly withdrawing an injured limb, and, occasionally, maintaining elevation for a brief period of time, can be observed immediately after a formalin injection,3 an injection of complete Freund adjuvant,4 an injection of capsaicin,5 a chronic constriction injury to the sciatic nerve,6 an L4 or L5 spinal nerve ligation,7 or an L5 spinal nerve axotomy.4 Moreover, recordings from peripheral nerve fibers innervating the areas of formalin-induced injury exhibited dramatic increases in their frequencies of afferent firing.8 In addition, counts of SFL in animals that received an L5 axotomy were associated with rates of ectopic discharge recorded from afferent neurons.4

The objective of the current study was 2-fold: first, to characterize SFL behaviors in the rat model of incisional pain and, second, to compare the effects of analgesics on evoked mechanosensitivity behaviors and SFL behaviors.

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METHODS

Animals and Ethics

The studies were approved by the Institutional Animal Care and Use Committee of Seton Hall University. Male Sprague-Dawley rats from Harlan Laboratories, Inc. (Somerville, NJ) weighing 250 to 300 g were used throughout the study with the exception of the age-related experiments, which used Fischer-344 rats from Taconic Farms, Inc. (Hudson, NY) because of the limited availability of other strains. Two or 3 rats were housed together on soft bedding (Carefresh Ultra, W.B. Fisher, NJ) in a 14.3 × 21.5 × 25.5-cm Plexiglas cage and kept on a 12-hour light/dark cycle. Food and water were available ad libitum. Animals were given an acclimation period of at least 5 days onsite before entry into the study. All animals were euthanized after the completion of testing.

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Anesthesia

Anesthesia was induced by placing each preoperative animal within a Plexiglas chamber containing room air with 5% isoflurane. Once loss of righting reflex was observed, the animal was moved to the operating stage where 2% to 3% isoflurane with room air was provided through a tightly fitting nose cone throughout the surgery.

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Plantar Skin and Muscle Incision

As previously described by Brennan et al.,9 a 1-cm longitudinal incision was made through the skin and fascia of the midplantar surface of the right hindpaw of each anesthetized animal. The plantaris muscle was then elevated, stressed, and incised longitudinally with the origin and insertion of the muscle remaining intact. The skin was closed with 2 mattress 5-0 silk sutures, and the animals were returned to their cages to recover.

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Measurement of Spontaneous Foot-Lifting Behaviors

Rats were placed within individual Plexiglas compartments (12 × 20 × 17 cm) on a shared, elevated aluminum mesh floor (IITC Life Sciences, Woodland Hills, CA) and given approximately 10 minutes to acclimate. Testing began once exploratory locomotion diminished and the rats rested on the mesh floor only exhibiting intermittent grooming behaviors. A mirror was placed under the mesh floor at an angle of 45° for easier viewing of both hindpaws.

Spontaneous, rapid lifting of the incised paw was manually recorded with a hand tally counter, whereas, simultaneously, the total duration of persistent (>1 seconds) paw elevation was recorded with a stopwatch. These behaviors were discrete and manually quantifiable by a single investigator (see video, http://www.youtube.com/watch?v=yoKmuYyDj8g). Behaviors associated with normal locomotion were excluded from measurements. Testing took place with 3 individually compartmentalized rats on the mesh floor. SFL was recorded for each animal over 4.5-minute periods with 10-minute intervals between tests, followed by measurement summation. The predominant SFL behaviors observed within this model were (1) spontaneous and rapid lifting of the incised paw, (2) rapid withdrawal followed by licking of the incised paw, and (3) rapid withdrawal with sustained elevation of the incised paw for a discernible period of time.

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Mechanosensitivity Testing with Electronic von Frey Esthesiometer

After SFL measurements, the paw-withdrawal threshold (PWT) to mechanical stimulation was measured keeping animals in the same testing environment. The electronic von Frey esthesiometer consists of a handheld force transducer with attachable, rigidity-graded 0.8-mm polypropylene von Frey hair filaments (IITC Life Sciences). The esthesiometer was used to transversely apply an ascending series of forces, increasing with each successive filament, to the ipsilateral hindpaw midplantar surface of each rat, adjacent to any surgical injury. The stimulation begins with the least rigid filament and increases stepwise if a withdrawal response is not elicited. The force transducer displays measurement of the exact force delivered during each application. The criterion for the stimulation end point was a flinching response accompanied by rapid paw withdrawal. The results are expressed as the mean PWT force (in grams) for mechanical nociceptive behaviors from 3 trials.

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Experimental Protocol

Eighty-four rats were used. Data from 5 rats were excluded for wound dehiscence. Rats were tested three at a time. Unless stated otherwise, experimental variables such as animal age and gender, room temperature, time of day for behavioral testing, and drug preparations and injections were consistent between groups. On each day of testing, SFL was assessed before PWT. SFL behaviors were not observed in any animals in the absence of acute injury (data not shown) and were therefore not measured before surgery. Differences between consecutive time points were evaluated. A single investigator randomly assigned animals to testing groups and kept experimenters blinded to the specific aims of each study and treatment assignments throughout data collection.

In the time-course study, 32 rats were tested: 14 Sprague-Dawley rats aged 2 months and 18 Fischer-344 rats aged 2, 7, and >26 months. A hindpaw plantar skin and muscle incision was performed on each rat. SFL and PWT testing was performed at 3 hours, 1 day, 2 days, 3 days, and 4 days after surgery.

The effects of buprenorphine and morphine on SFL behaviors were tested on 47 rats. Rats received intraperitoneal injections of saline, morphine (0.01, 0.1, 1, or 2 mg/kg), or buprenorphine (0.001, 0.01, or 0.1 mg/kg) at 3 hours or 1 day after plantar incision. Different doses of morphine were used at 3 hours (0.1, 1, and 2 mg/kg) and 1 day (0.01, 0.1, or 1 mg/kg) after incision. Although 0.1 mg/kg morphine had minimal effect on SFL count at 3 hours after incision, it had significantly attenuated SFL behaviors at 1 day after incision. Therefore, to depict a dose–response curve, a 10× lower dose of morphine was used as a starting dose for 1 day after incision.

Time points were chosen based on results of the time-course study showing SFL behaviors to be most prominent at 3 hours and 1 day after incision. In addition, previous studies used these time points to record cutaneous primary afferents from animals after plantar incision surgeries, which may have facilitated comparisons between behavioral and electrophysiological observations.10–12 SFL and PWT behaviors were evaluated 30 minutes after each injection. Testing was completed within 2 hours of drug administration. All drugs were diluted to their final concentrations with saline (0.9%) and warmed immediately before administration.

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Data Analysis and Statistics

The data for SFL count and PWT have normal distributions (Kolmogorov-Smirnov test, P > 0.10, n = 32) and are presented as mean ± SEM. Data for SFL duration are not normally distributed (P < 0.0001) and are presented as median and interquartile range (IQR, 25th–75th percentile). To compare the effects of drugs on SFL and PWT behaviors at 3 hours and 1 day after incision, data were normalized by calculating percentages of the maximal possible effect (%MPE) for each drug and assay. The %MPEs were calculated as follows: [(SFLsaline − SFLpostdrug)/SFLsaline] × 100; [(PWTpostdrug − PWTsaline)/(PWTcontrol − PWTsaline)] × 100. The %MPE data are normal and continuous from 0% to 100% and are presented as mean ± SE. Median effective doses (ED50) were calculated with nonlinear regression analyses between the log10(dose) and normalized %MPE for each drug on SFL and PWT.

Time-dependent changes in SFL behaviors were discerned using either a 1-way analysis of variance (ANOVA) followed by the Newman-Keuls and Tukey post hoc multiple comparison tests or a Kruskal-Wallis test followed by Dunn multiple comparison test. To determine whether SFL and mechanosensitivity behaviors were correlated, Spearman correlation analyses were performed on the SFL count or SFL duration and PWT data. Interaction between animal age and SFL behavior was evaluated using 1-way ANOVA or nonparametric Friedman ANOVA. Mann-Whitney U tests were conducted to examine interstrain variations in SFL. One-way ANOVA with Dunnett multiple comparison post hoc tests were used to compare %MPE on SFL and PWT. ED50 values were compared with extra sum-of-squares F tests. Analyses were performed using Prism 5 (GraphPad Software, San Diego, CA). The specific P values of the statistical analyses are reported.13P < 0.05 is considered significant for all analyses.

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RESULTS

Plantar Incision Induces Spontaneous Foot-Lifting Behaviors

Figure 1 displays scatter plots representing the after incision time courses of the SFL count and duration recorded for 20 minutes over 1 hour (see Methods) and PWT to punctate mechanical stimulation. All incised rats (100%) used for time-course experiments (n =32) displayed SFL behaviors at 3 hours after incision. The count of SFL was most pronounced at 3 hours after incision (39.8 ± 3.6, mean ± SE) and significantly decreased on the 1st postoperative day (POD; 19.7 ± 3.0; P < 0.0001; Fig. 1A) but remained stable through the 2nd POD (20.2 ± 2.5; P > 0.99). On the 3rd POD, the SFL count significantly decreased from the previous day (8.9 ± 1.4; P = 0.007; Fig. 1A) and remained stable through the 4th POD (6.3±1.0; P > 0.99). When the above analyses performed with Newman-Keuls post hoc tests were repeated with Tukey post hoc tests, the results were almost identical.

Figure 1

Figure 1

The duration of prolonged (>1 second) SFL was highest at 3 hours after incision and steadily diminished through the 3rd POD. The median duration of prolonged SFL was 27.0 seconds (IQR, 10.0–58.0) at 3 hours after incision and 3.5 seconds (IQR, 0.0–13.3) on the 1st POD (P = 0.0063; Fig. 1B). There was no significant change in the median duration between the 1st and 2nd POD. The prolonged SFL largely subsided from the 2nd to 3rd POD (P = 0.001; Fig. 1B).

Before incision, the mean PWT was 35.4 ± 1.0 g (mean ± SEM; data not shown). After plantar incision, PWT decreased to 7.7 ± 0.7, 10.2 ± 1.1, 11.3 ± 0.6, 17.7 ± 1.1, and 20.5 ± 1.3 g, respectively, measured at 3 hours, 1 day, 2 days, 3 days, and 4 days after incision (P < 0.0001; Fig. 1C).

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The Relationship Between SFL and Mechanosensitivity Behaviors

Spearman correlation analysis between the count of SFL and PWT behaviors found a significant correlation at 3 hours (Spearman r = −0.477; 95% CI, −0.713 to −0.144; P = 0.006; Fig. 2A) but not at 1 day (r = 0.089; 95% CI, −0.278 to 0.433; P = 0.6294; Fig. 2C) after incision. No significant correlation was observed between the duration of prolonged SFL and PWT behaviors at 3 hours (r = −0.099; 95% CI, −0.442 to 0.269; P = 0.59; Fig. 2B) or 1 day (r = −0.147; 95% CI, −0.480 to 0.223; P = 0.42; Fig. 2D) after incision.

Figure 2

Figure 2

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Effects of Animal Age and Strain on SFL Behaviors

Animal age did not influence the expression of SFL behaviors at 3 hours (Fig. 3, A and B) and 1 day (Fig. 3, C and D) after incision, and throughout the testing period (Fig. 3, E and F). Compared with the Sprague-Dawley rats (n = 14) used for the remaining studies, Fischer-344 rats demonstrated higher counts of SFL at 3 hours (P < 0.0001) and 1 day (P = 0.004) after incision (data not shown). The duration of prolonged SFL did not significantly vary between strains at 3 hours (P = 0.88) or 1 day (P = 0.11) after incision. However, we cannot exclude that the lack of statistical significance was attributed to insufficient statistical power.

Figure 3

Figure 3

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Comparison of the Sensitivities of SFL and PWT Behaviors to Morphine and Buprenorphine

At 3 hours after incision, buprenorphine dose dependently attenuated the SFL count (ED50 = 0.0027 mg/kg; 95% CI, 0.0009–0.0083), SFL duration (ED50 = 0.0004 mg/kg; 95% CI, 4.8e-5 to 0.0035), and PWT behaviors (ED50 = 0.0452 mg/kg; 95% CI, 0.0259–0.0787; Fig. 4A; Table 1). The ED50 values to reverse PWT behaviors were significantly higher than those to reverse SFL count (P < 0.0001, F1,32 = 20.23) and duration (P = 0.001, F1,32 = 13.13; Fig. 4A). Significantly higher %MPEs on SFL count (99.2% ± 0.5%; P = 0.0008) and SFL duration (100.0% ± 0.0%; P = 0.0007) were achieved in comparison with PWT (70.7% ± 11.0%) at the highest dose (0.1 mg/kg) of buprenorphine (Fig. 5A, Table 1).

Figure 4

Figure 4

Table 1

Table 1

Figure 5

Figure 5

In a separate group of rats at 3 hours after incision, morphine dose dependently decreased the SFL count (ED50 = 0.4044 mg/kg; 95% CI, 0.1048–1.561) and SFL duration (ED50 = 0.0309 mg/kg; 95% CI, 0.0095–0.0998; Fig. 4B). No statistically significant dose–response was found for the effect of morphine on PWT behaviors. The lowest doses of morphine (0.1 mg/kg) significantly attenuated (78.1% ± 10.8%) prolonged SFL with no obvious recovery (3.4% ± 3.8%) of PWT behaviors (Fig. 5B; P = 0.009; Table 1).

On the 1st POD, buprenorphine dose dependently attenuated the SFL count (ED50 = 0.0030 mg/kg; 95% CI, 0.0014–0.0061), SFL duration (ED50 = 0.0015 mg/kg; 95% CI, 0.0005–0.0044), and PWT behaviors (ED50 = 0.0138 mg/kg, 95% CI, 0.0048–0.0400; Fig. 4C). The ED50 to reverse PWT behaviors were significantly higher than those required to reverse SFL count (P = 0.02, F1,29 = 6.60) and duration (P = 0.004, F1,29 = 9.57; Fig. 4C). The %MPE elicited by buprenorphine at 0.01 mg/kg was significantly higher on prolonged SFL (96.0% ± 4.0%) in comparison with that on PWT (47.6% ± 12.9%; P = 0.007; Fig. 5C, Table 2).

Table 2

Table 2

In a separate group of rats, 1 day after plantar incision, morphine dose dependently attenuated the SFL count (ED50 = 0.0141 mg/kg; 95% CI, 0.0033–0.0592) and SFL duration (ED50 = 0.0082 mg/kg; 95% CI, 0.0041–0.0164; Fig. 4D). At all administered doses of morphine, higher %MPEs were achieved on the SFL count and SFL duration in comparison with the PWT (Fig. 5D, Table 2).

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DISCUSSION

Plantar Incision Induces Spontaneous Foot-Lifting Behaviors

The present study supports the observations by Zhu et al.14 that skin and muscle incision in the plantar hindpaw induces quantifiable SFL. Most studies evaluating SFL in other models have considered SFL behaviors to be the expressions of spontaneous pain in rodents.4,6,7 In contrast, others speculated that these behaviors represent touch-evoked pain, that is, animals are withdrawing their injured paws in response to pain from touching the metal mesh floor.11 Our findings suggest that SFL partly reflects spontaneous pain-like behaviors. In our study, linear regression analysis between mechano-allodynia and SFL behaviors shows that SFL behaviors at 3 hours after incision have a component related to mechanical allodynia because animals with lower withdrawal thresholds to von Frey stimulation displayed greater SFL at 3 hours after incision and vice versa (Fig. 2). No significant correlation was found between SFL and mechanical allodynia at 1 day after incision. In addition, the duration of prolonged SFL displayed no significant correlation with mechanosensitivity behaviors at both 3 hours and 1 day after incision (Fig. 2), suggesting that this behavior may be purely nonevoked. However, with our modest sample size, we cannot exclude that the lack of statistical significance was not attributable to low statistical power. However, it should be noted that Djouhri et al.4 suggested SFL as a behavioral sign of spontaneous pain because they found no correlation between SFL and evoked pain behaviors (allodynia or hyperalgesia). These authors showed that rats after L4 spinal nerve injury demonstrate increased SFL and also have an increased percentage of L4 C-fiber neurons with spontaneous discharges. Moreover, they observed that SFL after local inflammation or loose spinal nerve ligation in rats was associated with higher mean rates of spontaneous activity recorded from C-fiber neurons.4

An interesting finding of our study is the complete elimination of SFL behaviors by morphine (2 mg/kg) and buprenorphine (0.01 mg/kg; Fig. 4). This observation is consistent with clinical observations that these drugs provide a period of complete pain relief in postoperative patients.15

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Difference in Bioassay Sensitivity Between SFL and Mechanically Evoked Behaviors

Both SFL and mechanical hypersensitivity behaviors demonstrated reversals by morphine and buprenorphine. However, these 2 methods differ in their sensitivity when tested in the same group of animals. The doses of morphine and buprenorphine required to reduce SFL behaviors were substantially lower than those required to attenuate mechanical hyperalgesia. For example, the lowest administered dose of buprenorphine (0.001 mg/kg) almost abolished (80%–85%) the SFL duration at 3 hours after incision, but it had no observable effect on mechanical hyperalgesia. Similarly, morphine (0.1 mg/kg) was sufficient to significantly reduce the SFL duration at 3 hours after incision, but it had no observable effect on mechanical hyperalgesia.

The greater bioassay sensitivity of SFL behaviors in comparison with evoked behaviors is in agreement with at least 1 previous study.14 In that study, the authors have shown that ED50s of morphine to attenuate mechanical (1.5 mg/kg, subcutaneous) and heat (1.8 mg/kg, subcutaneous) hyperalgesia were significantly higher than those (0.7 mg/kg, subcutaneous) to attenuate nonevoked SFL behaviors.14

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CONCLUSIONS

Several preclinical studies proposed SFL as an expression of spontaneous pain after nerve injury or adjuvant-induced inflammation in rats. In this study, we show that plantar incision induces SFL behaviors, which are entirely eliminated by morphine and buprenorphine at doses that have minimal effects on mechanical hypersensitivity behaviors. Our results suggest that the SFL model of nonevoked pain may be advantageous over measures of mechanical hyperalgesia in reflecting the efficacies of analgesic drugs to attenuate spontaneous pain.

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DISCLOSURES

Name: Rajiv Kabadi, BE.

Contribution: This author conducted part of the experiments, and helped to prepare the manuscript.

Attestation: Rajiv Kabadi approved the final manuscript.

Name: Francois Kouya, PhD.

Contribution: This author conducted part of the experiments.

Attestation: Francois Kouya approved the final manuscript.

Name: Hillel W. Cohen, DrPH, MPH.

Contribution: This author conducted statistical analyses.

Attestation: Hillel W. Cohen approved the final manuscript.

Name: Ratan K. Banik, MD, PhD.

Contribution: This author helped to design and prepare the manuscript.

Attestation: Ratan K. Banik approved the final manuscript.

This manuscript was handled by: Martin S. Angst, MD.

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ACKNOWLEDGMENTS

We thank Martin Angst, MD, and five anonymous reviewers of Anesthesia & Analgesia for comments on our manuscript.

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