Surgery-associated tissue damage sets off a cascade of events, including pain and inflammatory responses, mutually influencing each other. Inflammation is accompanied by increased levels of proinflammatory cytokines, including interleukin-1β (IL-1β), which plays an important role in sickness behavior symptoms such as fever, decreased food and water intake, and decreased body weight (BW), and in hyperalgesia (1).
It is generally believed that effective perioperative pain management may result in a more rapid recovery; however, the effects of pain relief on postoperative outcome are inconsistent because of numerous variables, including type and duration of surgery, recovery measures, analgesics used, their side effects, and their route of administration (2).
BW and food consumption (FC) may serve as useful indices of pain in rats because they are related to the severity of the surgical procedure performed (3). Pain relief significantly reduced postsurgical decrease in FC, suggesting that the behavioral suppression was related to pain (4). In the present study, we examined whether specific analgesic procedures may accelerate surgery recovery, as measured by FC and BW after laparotomy in rats. Two perioperative pain management techniques were studied, both consisting of an intrathecal (i.t.) mixture of morphine plus bupivacaine (MIX) before the incision. This preoperative injection was combined with one of two treatments: In the first experiment, the analgesia was extended by slow-release (SR) morphine administered at the end of the surgery. In the second experiment, an antiinflammatory drug, IL-1 receptor antagonist (IL-1ra), was combined with the preoperative i.t. mixture.
Fischer 344 male rats (Harlan; aged 3–4 mo and weighing 270–410 g) were housed 2–3 per cage in a temperature of 23°C ± 1°C, with a light/dark cycle of 12 h (light off at 8:00 am). Experiments were run in the dark phase. Water and food were available ad libitum. Rats were individually housed 1 wk before the beginning of each experiment and were handled for 2 min daily. The experiments were approved by the Hebrew University Committee on Animal Care and Use.
Pain threshold was assessed using the paw-flick latency test (Hargreaves test), as previously described (5). Baseline measures of paw-flick, BW, and FC were taken a few days before surgery.
On surgery day (Day 1), rats were anesthetized by inhaling a mixture of halothane and oxygen and were randomly assigned to one of four groups: Group 1 (laparotomy [LAP]+MIX+SR; n = 12) received i.t. (6) MIX (50 μL) of morphine (30 μg) + bupivacaine (30 μg) diluted in saline. Twenty minutes later, LAP was performed, as previously described (7), and the small intestine was externalized for 10 min. Fifty minutes after the first injection (after surgery completion), rats received an injection of 1 mL of morphine (1.25 mg/mL, s.c.) prepared in SR emulsion, as previously described (8). The average weight of the rats was 310 g, yielding a SR morphine injection of approximately 4 mg/kg. Group 2 (LAP+saline [SAL]+vehicle [VEH]; n = 11) received SAL in the first injection and SAL mixed with the emulsion vehicle in the second injection; this group was operated on, as perviously described. Group 3 (NoLAP+MIX+SR; n = 14) received the same injections as Group 1 but was non-operated. Group 4 (NoLAP+SAL+VEH; n = 10) received the same injections as Group 2 and was non-operated. After the second injection, rats were allowed 5 min of recovery under a heating lamp and were then placed in the paw-flick cages for 30 min of habituation. Paw-flick measures were taken at 85, 100, 130, 160, and 340 min after the first injection. Rats were kept in the paw-flick cages for 105 min (beginning 30 min before the first measure at 85 min up to 160 min postinjection), were then placed back in their home cages (with food and water ad libitum), and returned for 30 min habituation before the last measure on the surgery day (340 min). The following measures were taken on the days after the surgery: paw-flick latency taken 30 min after habituation (Days 2–3) and BW and FC in 24-h periods (Days 2–5).
The procedure of the second experiment was similar. Rats were assigned to the following groups: Group 1 (LAP+MIX/IL-1ra; n = 7) received a combined i.t. injection (50 μL) consisting of morphine (30 μg) + bupivacaine (30 μg) + IL-1ra (100 μg), and 20 min later, LAP was performed. Group 2 (LAP+SAL; n = 10) received a SAL i.t. injection and LAP. Group 3 (NoLAP+MIX/IL-1ra; n = 8) received the same combined treatment as Group 1 but was non-operated. Group 4 (NoLAP+SAL; n = 7) received the same injections as Group 2 and was non-operated. No injection of SR morphine was given in Experiment 2.
Data are presented as mean ± sem. For statistical comparisons, analysis of variance (ANOVA) with repeated measures was performed. Planned contrasts were performed to compare the two operated groups in each experiment.
Both groups receiving analgesics, whether operated or non-operated, exhibited significantly increased paw-flick latency up to 340 min postinjection (Fig. 1). ANOVA with repeated measures revealed significant (P < 0.001) group effect, time effect, and group × time interaction. Averaged over all time points, rats of the LAP+MIX+SR group exhibited a paw-flick latency of 7.0 s (±0.22) versus 5.6 s (±0.16) in the LAP+SAL+VEH group. Comparing these two groups by planned contrasts revealed a significant interaction (P < 0.0001).
The weight of the remaining food in each cage was determined daily. FC was calculated as the difference between food weight on a certain day and the previous one. ANOVA revealed significant (P < 0.001) group effect, time effect, and group × time interaction (Fig. 2a). Planned contrasts between the two operated groups revealed significant interaction (P < 0.0001), demonstrating a better recovery of FC in the analgesic-treated group (LAP+MIX+SR) on postoperative Days 4 and 5.
BW change was calculated as the difference between BW measured on a certain day minus the previous day. ANOVA revealed significant (P < 0.001) group effect, time effect, and group × time interaction (Fig. 2b). Planned contrasts between the two operated groups revealed significant group effect (P < 0.04) and interaction (P < 0.0002). Operated rats receiving analgesics lost significantly (P < 0.001) more BW on the first postoperative day and gained significantly (P < 0.01) more BW on postoperative Days 4 and 5, compared with rats receiving SAL who continued to lose BW throughout the observation period. Analyzing the data in terms of absolute BW reveals significant interactions between the two operated groups (P < 0.01). Absolute BW of the two operated groups did not significantly differ on Day 5; however, on this day, rats of the SAL-treated operated group still lost BW, whereas rats of the analgesic-treated operated group already gained BW beginning on Day 4. The BW results were corroborated by the results of the FC, where the analgesic-treated groups exhibit larger FC on postoperative Days 4 and 5. These findings reflect an earlier return to normal BW in analgesic-treated operated rats.
Both groups receiving the combined treatment, operated or not, exhibited significantly higher paw-flick latency for at least 90 min postinjection (Fig. 3). ANOVA revealed significant (P < 0.006) group effect, time effect, and group × time interaction. Comparing the analgesic response in the two operated groups by planned contrasts revealed a significant interaction (P < 0.0001) and significant differences at 90 and 150 min.
ANOVA of the FC revealed significant (P < 0.0001) group effect, time effect, and group × time interaction (Fig. 4a). Planned contrasts between the two operated groups revealed significant interaction (P < 0.03), indicating better FC recovery on postoperative Days 3 and 4 in rats receiving the analgesic treatment (LAP+MIX/IL-1ra), as compared with the SAL-treated rats (LAP+SAL).
ANOVA of the BW change revealed significant (P < 0.001) group effect, time effect, and group × time interaction (Fig. 4b). Planned contrasts between the two operated groups revealed a significant interaction (P < 0.0002). Operated rats receiving analgesics lost significantly (P < 0.008) more BW on the first postoperative day and gained significantly (P < 0.05) more BW on postoperative Days 3 and 4, suggesting earlier return to normal BW in analgesic-treated operated rats. Similar to Experiment 1, absolute BW of the two operated groups did not significantly differ on Day 4; however, on this day, rats of the SAL-treated operated group still lost BW, whereas rats of the analgesic-treated operated group already gained BW beginning on Day 3.
A significant analgesia was achieved in both experiments; the analgesia produced by the Experiment 1 regimen (i.t. MIX + SR morphine) was more intense and lasted longer (up to approximately six hours), compared with the analgesia produced by the Experiment 2 regimen (i.t. MIX combined with IL-1ra; approximately 150 minutes).
LAP significantly decreased FC and BW, confirming earlier findings (9). Operated rats receiving analgesics lost more BW on postoperative Day 1 but exhibited faster recovery of FC and BW, noticeably, on postoperative Days 4 or 3 (Experiment 1 or 2, respectively). These findings may explain why, in terms of absolute BW, the two groups of operated rats did not differ on the last day of measurements and suggest that these variables should be measured for an extended postoperative period. Decreased FC and BW were also observed in the non-operated groups (especially on Day 1) and were more pronounced in the analgesics-treated groups. Administration of SR morphine (Experiment 1) seems to prolong the suppression of FC and BW in accordance with pharmacological side effects of opiates (10).
Reports regarding the effects of postoperative analgesia on FC and BW in rodents vary widely and often conflict. At the same time, it is widely accepted that effective preemptive analgesia should be extended to cover both the incisional and inflammatory phases of postoperative pain (11,12). In the present experiments, operated rats receiving analgesics demonstrated better recovery compared with those receiving SAL. All analgesic-treated rats received a preoperative i.t. mixture of opiate and local anesthetic. Local anesthetics, in addition to the analgesic effect, also have an antiinflammatory effect of their own (13). Furthermore, the preoperative analgesic MIX was extended by either postoperative SR morphine (Experiment 1) or by coadministration of IL-1ra (Experiment 2). We have recently shown that IL-1ra can enhance and prolong morphine analgesia (14), and it has recently been reported that preventive (preemptive) epidural analgesia in patients was associated with reduced pain disability three weeks after surgery (15). Also, a recent report has shown that a single preincisional IV administration of a proinflammatory cytokine inhibitor (pentoxifylline) improved recovery of bowel function almost three days (70 hours) after colorectal cancer surgery (16). These reports indicate that short-term effective perioperative analgesia can have long lasting beneficial effects on recovery.
The design of the present study does not provide for detailed comparison between the two experiments. Nevertheless, it seems that the recovery of FC and BW began a day earlier in Experiment 2 compared with Experiment 1. It is possible that the large dose of SR morphine adversely induced postoperative slowing of intestinal motility (17,18), which may be evident by the larger decrease in FC and BW in the non-operated SR-treated rats. Alternatively, IL-1 induces inflammatory pain hyperalgesia (1), hypermetabolic state BW loss and anorexia (19), elevated adrenocortical response to surgical stress (20), and increases the risk of postoperative ileus (21). IL-1ra, can antagonize the above-mentioned IL-1-mediated effects (22–25), including BW loss and anorexia (26); thus, the antiinflammatory effects of IL-1ra may contribute to a better postoperative recovery. This hypothesis should be further investigated.
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