Behavioral Measurement (n = 11 to 12 Animals/Group)
All behavioral tests were performed by a single investigator (T.K.) who was blinded to the study groups. Rats were isolated in individual cages. Mechanical and visceral thresholds after stimulation were evaluated 3 times during the 48 hours after surgery at 6 (day 0), 24 (day 1), and 48 (day 2) hours. For each test, the thresholds were determined 3 times, with testing separated by 10 minutes, and the mean withdrawal threshold was used for data analysis.
Visceral sensitivity was evaluated via colorectal distension (CRD). A CRD balloon was prepared according to the model described by Yang et al.14 The balloon covered with lubricant was totally inserted into the descending colon and rectum under isoflurane anesthesia. Rats were evaluated 30 minutes after they had fully recovered from anesthesia. The tube of the balloon was connected via a Y connector to an air pump and a sphygmomanometer. The balloon was progressively inflated with air under the control of the inside pressure that was continuously monitored. The test was stopped when the investigator observed a response (see below) or at the cutoff pressure of 80 mm Hg. The balloon was inflated with air for 20 seconds for each trial, and the pressure inside was continuously monitored. The pressure was determine 3 times, with testing separated by 10 minutes, and the mean pressure was used for data analysis.
Five abdominal withdrawal reflex (AWR) scores (AWR 0 to AWR 4) were used to assess the intensity of noxious visceral stimuli: AWR 0 = no remarkable behavior changes; AWR 1 = immobility of the rat body or occasional clinches of the head; AWR 2 = mild abdominal muscle contraction; AWR 3 = lifting the abdomen off the box platform or flattening of abdomen; AWR 4 = body arching or lifting pelvic structures off the platform.15 The threshold was defined as the minimal pressure inside the balloon when the rat showed flattening of abdomen (AWR 3) during the CRD.
Nociceptive mechanical threshold at the periphery of the abdominal scar was assessed by application of calibrated von Frey filaments. Animals were placed on a plastic mesh floor in individual plastic boxes and allowed to acclimate to their environment. Von Frey filaments were then applied vertically to the abdominal incision on 3 different points. Testing began with a small (10 g · mm−2) filament. Filaments were applied 3 times over 2 seconds. If no response was elicited, a larger-diameter filament was applied in the same manner. The filaments were applied in increasing order until a positive response was elicited. The next filament was applied after the animal fully returned to its basal behavior. The maximum force applied was 137 g · mm−2. The threshold was determined 3 times, with testing separated by 10 minutes, and the mean threshold was used for data analysis. Abdominal withdrawal was considered a positive response.
A nonvalidated global activity score of 4 points was established and assigned to the rats. This score evaluated the following items: the activity of rats in their cage, their grooming behavior, and the condition of the hair. The score was established as follows:
- The rat is moving in his cage only to eat. The hair bristles are dull and pale.
- The rat is moving by lifting its abdomen off the cage platform. The hair bristles are dull and pale.
- The rat is moving normally in its cage but it does not rear. The hair is smooth, shiny, and white.
- The rat has a normal activity and can rear.
This score was assessed 3 times during the 48 hours after surgery just before pain evaluation: at day 0, after awakening from anesthesia for surgery, and at days 1 and 2. Rats were observed in their cages for 3 minutes without any stimulation. The highest observed score was recorded.
Cytokine Production in Stimulated Whole Blood Culture and in Stimulated Peritoneal Macrophages (n = 8 to 10 Animals/Groups/Time Point)
Blood (0.5 mL in heparin-coated syringe) was collected to measure inflammatory cytokines after stimulation of whole blood in isoflurane-anesthetized animals.
A blood sample (0.5 mL) was diluted 1:5 in Roswell Park Memorial Institute (RPMI)–1640 medium (PAA, Les Mureaux, France) supplemented with antibiotics (penicillin–streptomycin; Sigma, Saint Quentin Fallavier, France). Five hundred microliter aliquots of diluted blood were cultured in 24-well plates under 2 different conditions1: without lipopolysaccharide (LPS) (Escherichia coli O111:B4, 10 μg/mL; Sigma) (baseline)2 and with LPS in a 5% CO2 incubator for 24 hours at 37°C. The supernatant was then harvested and stored at −80°C until assayed. The concentrations of tumor necrosis factor (TNF)–α and interleukin (IL)–1β in the supernatants were measured with a commercial enzyme-linked immunosorbent assay kit (ELISA) (DuoSet; R&D Systems Europe, Lille, France) according to the manufacturer's instructions. The assay detection limits were 30 pg/mL for TNF-α and 15 pg/mL for IL-1β.
To harvest peritoneal macrophages, we washed the peritoneal cavity with 10 mL of cold Dulbecco's phosphate buffered saline (PBS) (PAA, Les Mureaux, France), which was aspirated soon thereafter. The PBS solution was centrifuged for 5 minutes at 750 revolutions per minute (rpm) and the cells were suspended in RPMI solution. The RPMI solution was cultured in 6-well cell culture plates and incubated for 3 hours at 37°C and 5% CO2 to allow the macrophages to adhere to the plastic. The wells were then vigorously washed 3 times with PBS, and the adherent macrophages were harvested using a sterile cell scraper (Sarsted, Marnay, France). Macrophages were then suspended in RPMI at the concentration of 106 cells/mL. The cells were then stimulated with 1 μg · mL−1 LPS (Sigma) and incubated overnight at 37°C and 5% of CO2. The supernatant was collected and stored at −80°C until assayed using an ELISA specific for rat TNF-α and IL-1β (R&D Systems Europe, Lille, France) as described above. Blood sampling and peritoneal wash were done 3 times: at the operation time just after peritoneal incision (day 0), 24 hours (day 1), and 48 hours (day 2) after surgery. The animals were killed after these destructive samplings.
Ropivacaine Plasma Concentration
Blood (0.5 mL) was sampled for ropivacaine assay in plasma in 7 animals per group 5 and 20 minutes (day 0) and 24 hours after the first IM injection (day 1) in the ropi IM group, and 4 hours (day 0), 24 hours (day 1), and 48 hours (day 2) after surgery in the ropi 2 and ropi 7.5 groups. The plasma was then collected and stored at −20°C until assayed by gas chromatography.
Because the data were not normally distributed, statistical analysis was performed using nonparametric tests. In addition, to avoid a type 1 error, the use of global significance tests was preferred, and the Dunn test after the Kruskall–Wallis test was used only when necessary. The time evolution of CRD threshold, parietal pain threshold, and global activity in all sham-operated groups was tested using the Friedman test. To avoid unnecessary comparisons between groups of laparotomized rats, we tested only the time evolution in the saline group using the Friedman test. The comparison across the groups (in the sham-operated and laparotomy groups) at each time was performed using the Kruskall–Wallis test. Post hoc analysis was done using the Dunn test on ranks when necessary. Because measurement of cytokine production by circulating leukocytes (in the sham and laparotomy groups) and peritoneal macrophages (in the laparotomy groups) was done on the basis of destructive sampling, the time evolution in the control and saline groups was assessed by the Kruskall–Wallis test. The time evolution was not tested in the other laparotomy groups. The comparison among groups was assessed using the Kruskall–Wallis test followed by the Dunn test as appropriate. A P value <0.05 was considered the threshold level for statistical significance. Data are reported as the median and range. Statistical analysis was performed with Statview 5.0 and R.
Visceral Stimulation: CRD Thresholds (Fig. 2)
In the nonoperated control group, CRD pressure reached the threshold (80 mm Hg) in all but 1 animal. This was significantly different from the value recorded in all sham-operated groups at day 0 (the first measurement after surgery). The other groups were not different from each other at any time. Only in group ropi IM was recovery complete with time (day 0 vs day 1 vs day 2).
In the saline group, CRD threshold was higher after laparotomy on day 1 and on day 2 than was the threshold observed on day 0. Six hours after laparotomy (day 0), CRD thresholds were significantly higher in the ropi 7.5 and the ropi IM groups in comparison with the saline and the ropi 2 groups. No significant difference was observed between the ropi 2 and the saline groups and between the ropi IM and the ropi 7.5 groups. On day 1, the ropi 7.5 and the ropi IM groups were different from the saline group. On day 2, no difference was observed among groups when all groups had a threshold close to the limit (80 mm Hg).
Parietal Stimulation: Von Frey Filament Mechanical Withdrawal Thresholds (Fig. 3)
All animals in the control group reached the threshold of 110 g. However, the difference between the control group and the 4 laparotomized groups at day 0 did not reach statistical significance. The other groups were not different from each other at day 1 and at day 2. The sham ropi 7.5 group significantly recovered from day 0 to day 2.
Parietal sensitivity evolved similarly to visceral sensitivity in the saline group: the threshold was higher on day 1 and on day 2 than was the threshold observed on day 0. Six hours after laparotomy (day 0), the thresholds were significantly higher in the ropi 7.5 and the ropi IM groups than they were in the saline and the ropi 2 groups. No significant difference was observed between the ropi 2 and the saline and ropi IM groups and between the ropi IM and the ropi 7.5 groups. On day 1, treated groups were different from the saline group. On day 2, treated groups were still different from the saline group. No difference among the 3 treated groups was observed on days 1 and 2.
Global Activity Score
In the control group, all animals had a score of 4, significantly different from sham-operated groups (P < 0.0001). The other groups were not different from each other at any time. All operated groups significantly recovered with time (day 0 vs day 1 vs day 2).
Global activity evolved similarly to parietal and visceral stimulation in the saline group; the score was higher on day 1 and on day 2 than was the score observed on day 0. Six hours after laparotomy (day 0) the score was higher in all treated groups than in the saline group, assessing a better immediate functional postoperative recovery. On day 1 and day 2, the 3 treated groups were different from the saline group, and no difference was observed among treated groups.
Production of TNF-α and IL-1-β in Cultures of Circulating Blood Cells After LPS Stimulation (Fig. 4)
Concentrations of TNF-α and IL-1β in cultured blood in the absence of LPS stimulation were always low in all of the study groups, indicating that incubation in the culture plates per se did not significantly stimulate cytokines.
In the sham-operated groups, TNF-α and IL1-β secreted by stimulated circulating leukocytes was not different between groups and between times (day 0, day 1, day 2).
In the saline group, TNF-α but not IL1-β production significantly increased with time. TNF-α and IL-1β production were similar among groups on day 0. TNF-α production on days 1 and 2 and IL-1β production on day 2 were significantly lower in the ropi IM group than in the saline group, indicating a preventive effect of IM ropivacaine on systemic inflammation.
Production of TNF-α and IL-1-β by Macrophages in Peritoneal Liquid After LPS Stimulation (Fig. 5)
In the sham-operated controls, the quantity of cells obtained after peritoneal wash was too low (<1 million/mL) to stimulate them.
In the saline group, TNF-α and IL1-β significantly increased with time.
TNF-α and IL-1β production were similar between groups on day 0. TNF-α production on day 2 was significantly lower in the ropi 7.5 and the ropi IM groups than in the saline group. IL-1β production on day 2 was significantly lower in the ropi IM group than in the saline group, indicating a preventing effect of IM ropivacaine on local inflammation.
Ropivacaine Plasma Concentrations
Ropivacaine was not detected at any time in the saline group. As usual in rodents, the ropivacaine plasma concentration was always low. Four hours after surgery the ropivacaine concentration was 0.06 ± 0.06 mg · L−1 and 0.16 ± 0.11 mg · L−1 in the ropi 2 and ropi 7.5 groups, respectively. At the end of preperitoneal infusion (H + 24 hours), the ropivacaine concentration was 0.03 ± 0.03 mg · L−1 and 0.05 ± 0.08 mg · L−1 in the ropi 2 and ropi 7.5 groups, respectively. In the ropi IM group, the ropivacaine plasma concentration was 0.42 ± 0.49 mg · L−1 and 0.44 ± 0.27 mg · L−1 5 and 20 minutes, respectively, after the first injection. The concentration was below the quantification limit 8 hours after the last injection for the ropi IM group and 24 hours after cessation of infusion for the ropi 2 and 7.5 groups.
Despite the increasing use of wound infiltration of local anesthetics as part of multimodal analgesia after major surgery, the mechanisms involved in the effect of a direct application of local anesthetics to the wound are not fully established. In the current study, high-dose ropivacaine administered via a preperitoneal infusion or systemic boluses leading to plasma concentration peaks had the same effect on mechanical and visceral sensitivity. Moreover, systemic administration was associated with a significant anti-inflammatory effect.
High-dose preperitoneal infusion and systemic ropivacaine prevented mechanical and visceral sensitivity alterations starting at day 0. The functional recovery measured by an activity score was simultaneously improved. The mechanisms involved in the efficacy of a preperitoneal infusion have not been clearly elucidated. First, it is not clear in which anatomic layer wound infusion has the best effect on postoperative pain; infusion above the superficial abdominal fascia was shown to provide better analgesia than was infusion below the fascia after hysterectomy.16 After a laparotomy, it seems that wound infusion limited to the subcutaneous layers does not provide any benefit.10 Local anesthetics could directly affect the peritoneal membrane, which could explain the inhibition of visceral pain. Studies have indeed suggested that the afferent limb of the reflex leading to a postoperative ileus originates primarily from the peritoneum. A possible explanation of the analgesic effect of local anesthetic wound infiltration would be that blocking the transmission of pain from nociceptive afferents from the wound surface would allow an inhibition of visceral pain and therefore reduce the central sensitization phenomena. The influence of parietal and visceral pain in the global pain experienced after laparotomy and the links between these 2 components of pain have never been clearly evaluated, mostly because it is very difficult to distinguish between the 2 in clinical practice. The poor localization, diffuse character, and referral of visceral pain are explained in large part by the anatomical organization of the visceral innervation and the convergence of visceral and nonvisceral inputs onto second-order spinal neurons.17 It has moreover been shown that noxious cutaneous stimuli can inhibit visceral nociceptive neurons and reflexes.18 For example, a plantar incision of the hindpaw resulted in decreased plantar mechanical and thermal hyperalgesia and in a significant increase in the visceromotor response to CRD.19 In our study, the inhibition of parietal sensitivity via a preperitoneal infusion was associated with an inhibition of visceral sensitivity, leading to the conclusion that both could be linked. However, the mechanistic hypothesis that local anesthetic wound infiltration could inhibit parietal pain and therefore visceral pain cannot be confirmed by our study. Indeed, the plasma concentration of ropivacaine in the ropi 7.5 group was not negligible even if lower than in the ropi IM group. We thus cannot exclude a systemic effect of ropivacaine administered via a catheter. The benefits of the administration of systemic local anesthetics (i.e., lidocaine) during laparotomy have been clearly demonstrated in patients.12
This prevention of mechanical and visceral sensitivity alterations was associated with an anti-inflammatory effect of local anesthetics. In the current study, increased production of TNF-α and IL-1β in cultures of circulating blood cells and in peritoneal macrophages after LPS stimulation in rats after a laparotomy was completely prevented by systemic ropivacaine treatment. A nonsignificant tendency towards an anti-inflammatory effect was observed after a high-dose preperitoneal infusion. Inflammation is the most important component of postoperative pain and can sensitize nociceptive receptors and contribute to pain and hyperalgesia. Local anesthetics can inhibit the local and systemic inflammatory response to injury when administered via a nerve block.9 The mechanisms involved probably imply an inhibition of the axonal transport of inflammatory mediators.20 When administered systemically, local anesthetics did not show any benefit after orthopedic surgery in patients.21 However, continuous IV administration of lidocaine during and after abdominal surgery improves patient rehabilitation and shortens hospital stay.12 Kuo et al.22 showed that the improvement in pain and bowel function observed after intraoperative administration of systemic lidocaine is associated with a decrease in cytokines release. The effects on cytokines were observed until 72 hours after the last administration of lidocaine. The potential mechanisms explaining the benefit of systemic administration of local anesthetics could involve an inhibition of central hyperalgesia23 and of inflammation. Indeed systemic lidocaine can specifically produce inhibition of neuronal and reflex responses to CRD.24 As previously shown,25 the analgesic and functional benefit of systemic administration of local anesthetic was associated with an anti-inflammatory effect in the current study. This could be explained by the ropivacaine plasma concentrations. Indeed, despite a much lower total dose received by ropi IM animals, the peak concentration (Cmax) after each injection was remarkably higher than was the steady concentration observed in the 2 other groups. If we consider that the time to Cmax occurred between 5 and 20 minutes after the IM injection, Cmax was higher than 0.44 mg · L−1, and a peak effect may explain why the animals in this group had similar results to those in the ropi 7.5 group. The very low concentrations observed in the groups receiving a preperitoneal infusion could probably not lead to an anti-inflammatory effect.
The current study had some limitations. First, we arbitrarily chose a small dose of ropivacaine administered systemically to obtain an analgesic efficacy without any side effects. The toxic plasma level in rats is >10 mg · mL−1.26 The systemic route is not used for the administration of ropivacaine in clinical practice. To evaluate the action of systemic ropivacaine as one of the possible mechanisms of the analgesic affect of preperitoneal ropivacaine, we chose to keep ropivacaine as the local anesthetic administered systemically in our study. Second, for technical reasons, we chose not to use continuous systemic administration of local anesthetic. It would have been technically difficult in rats. Finally, we did not directly assess pain but rather the behavioral responses to visceral and parietal stimulation. However, a recent article showed that postoperative signs of pain (persistent tachycardia, increased heart rate variability, and loss of mobility) were associated over time with mechanical hyperalgesia measured with von Frey filaments in rats after laparotomy.27
In conclusion, we did not find any difference between high-dose ropivacaine administered via a preperitoneal infusion or systemic boluses after laparotomy in rats. The merits of the comparable benefit of systemic and high-dose preperitoneal infusion of ropivacaine need to be confirmed with further studies. Infusion catheters are expensive, and no data are available on the safety of high doses in patients. However, side effects are very scarce. On the other hand, systemic administration of local anesthetics (lidocaine in patients) is inexpensive but causes higher plasma levels of local anesthetic and thus potential side effects. The choice between the 2 techniques needs to be evaluated in patients.
Name: Toni Kfoury, MD.
Contribution: This author helped conduct the study.
Attestation: Toni Kfoury has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Jean-Xavier Mazoit, MD, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Jean-Xavier Mazoit has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Michael Schumacher, PhD.
Contribution: This author helped review the manuscript.
Attestation: Michael Schumacher has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Dan Benhamou, MD.
Contribution: This author helped review the manuscript.
Attestation: Helene Beloeil has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Helene Beloeil, MD, PhD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Helene Beloeil has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
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© 2011 International Anesthesia Research Society
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