Postoperative Models of Nociception

Timothy J. Brennan, M.D., Ph.D., is Associate Professor in the Department of Anesthesia, University of Iowa College of Medicine, Iowa City, Iowa. Postoperative pain is a common form of acute pain, yet it is a costly, poorly understood problem ( Kehlet 1994 ). It is generally thought that postoperative pain can be adequately treated by administration of sufficient parenteral opioids. However, studies of patients after major surgeries demonstrate that postsurgical pain induced by activities is poorly responsive to opioids compared with pain at rest ( Gould and others 1992 ). In particular, pain measures during ambulation and with coughing and movement (mechanical stimuli) may be severe even with sufficient parenteral opioid use in patients after major surgery. Additional drug dosing can be limited by respiratory depression and sedation; other side effects such as nausea and vomiting, delayed gastric emptying, and prolonged ileus are common. Better pain control with activity can be achieved with combinations of drugs administered epidurally ( Dahl and others 1992 ); however, this therapy is labor intensive, is not without risk, and has not yet proven to be cost effective. Thus, despite the simple nature of pain caused by tissue injury after incision, uncomplicated, effective, inexpensive treatments are not yet available ( Kehlet 1994 ). Even though pain research has resulted in many remarkable discoveries in the last 15 yr, a small proportionate effort has been made toward understanding pain caused by an incision ( Kalso and Rosenberg 1995 ). If we learn more about mechanisms of acute incisional pain and the sensory processes that intensify pain after surgery, new treatment methods can be advanced. It has been suggested that it is necessary to study the efficacy of postoperative analgesic treatments using pain scores during evoked responses such as movement and coughing if outcome is to improve ( Kehlet 1994 ). Thus, mechanical hyperalgesia--a decreased pain threshold and an increase in pain response to suprathreshold stimuli--is a most important property of incisional pain. Research on mechanical hyperalgesia caused by an incision is important for developing therapies for postoperative pain. Previously, the experimental pain community has lacked an animal model for incision-induced pain. To understand mechanisms of sensitization caused by surgery and investigate new therapies for postoperative pain in humans, colleagues and I developed and characterized a rat model for human postoperative incisional pain ( Brennan and others 1996 ). This new model has several unique properties compared with other animal models of pain. Importantly, it is caused by an incision, is profound and persistent, is characterized by reduced withdrawal thresholds suggesting that mechanical hyperalgesia is present, and results in behaviors that are timed similarly to pain measures in postoperative patients. Colleagues and I have performed our experiments on adult male rats. Anesthesia is induced with 1.5 to 2% halothane in 100% oxygen in a sealed box; then after induction, the same halothane concentration is delivered via a nose cone. Each animal subsequently receives an intramuscular injection of penicillin (30,000 IU of Flocillin) in the triceps muscle. The plantar aspect of either hind paw is prepared in a sterile manner with a 10% povidone-iodine solution, and the foot is placed through a hole in a sterile cloth drape. A 1-cm longitudinal incision is made with a number 11 blade through skin and fascia of the plantar aspect of the foot, starting 0.5 cm from the proximal edge of the heel and extending toward the toes. In most animals, the plantaris muscle is elevated and incised longitudinally ( Figure 1A ). The muscle origin and insertion remain intact. After hemostasis with gentle pressure, the skin is apposed with two mattress sutures of 5-0 nylon on an FS-2 needle ( Figure 1B ). The wound site is covered with a mixture of polymixin B, neomycin, and bacitracin ointment. The sutures are removed under halothane anesthesia at the end of postoperative day 2. Typically the wounds are well healed within 5 to 6 days. Photographs of the different stages of the surgery of the rat foot. (Left) A 1-cm longitudinal incision is made through the skin and fascia starting 0.5 cm from the proximal edge of the heel and extending toward the middle of the foot. The plantaris muscle is elevated and incised longitudinally. (Right) After hemostasis, the wound is apposed with two mattress sutures. Photographs of the different stages of the surgery of the rat foot. (Left) A 1-cm longitudinal incision is made through the skin and fascia starting 0.5 cm from the proximal edge of the heel and extending toward the middle of the foot. The plantaris muscle is elevated and incised longitudinally. (Right) After hemostasis, the wound is apposed with two mattress sutures. After surgery, the rats are housed individually in clean bedding consisting of organic cellulose fiber such as Cellu-Dri (Shepherd Specialty Papers, Inc, Kalamazoo, Michigan) or Tek-Fresh (Harland, Madison, Wisconsin). The incisions are checked daily for any sign of wound infection, dehiscence, or hematoma that would exclude rats from further study. We make every attempt to avoid infection because of the potential for increased inflammation at the incision. Although this has not occurred, it could confound the assessment of pain behaviors. Dehiscence can occur if the rats remove the suture during emergence or in the early postoperative period. Poor apposition of the wound could prolong the time course or intensify pain behaviors. Approximately one in 12 animals is excluded for wound dehiscence. Hematoma formation is rare. There appears little evidence for severe pain and distress since spontaneous pain behaviors like vocalization or persistent flinching behavior have not been observed postoperatively. We have observed limping and some guarding during the recovery period. Typically the animal licks the antibiotic ointment that has been applied at the end of the surgery. It is not known whether this is a pain-related licking behavior as in other pain models. We allow 2 hr for recovery before we observe the rats' behavior, although in some cases, pain-related behaviors have been examined as soon as 1 hr after the incision and termination of anesthesia. After emergence and recovery from anesthesia, unrestrained rats are placed beneath a clear plastic chamber (21 x 27 x 15 cm) on an elevated plastic mesh floor (ADPI Enterprises, Philadelphia, Pennsylvania) and allowed to acclimate until they are resting undisturbed. Withdrawal responses to mechanical stimulation are determined using calibrated Semmes Weinstein von Frey filaments (Stoelting, Wood Dale, Illinois). These filaments are nylon monofilaments with similar lengths and varying diameters; as the diameter of the filament increases, the force necessary to bend the filament increases. These monofilaments are applied individually from under the cage through openings in the plastic mesh floor (12 x 12 mm grid) to an area adjacent to the wound ( Figure 2 ). Most often they are applied medial to the incision near the heel. Each filament is applied once starting with 15 milliNewtons (mN) and continuing in an ascending order until a withdrawal response occurs or 522 mN (the cutoff value) is reached. When these filaments are applied to the unincised normal rat foot near the heel, withdrawal is usually not elicited until the cut-off filament is applied. The withdrawal responses are brisk after the punctate filament application and are not accompanied by vocalization. The rats can be more active after the withdrawal and require a few minutes to acclimate themselves before further testing is possible. Typically, after a 5-min test-free period, each filament is again applied once starting with 15 mN until a withdrawal response is elicited. This is repeated a third time 5 min later. The lowest force from the three tests producing a response is considered the withdrawal threshold. The cutoff value--522 mN--is recorded even if there had been no withdrawal response to this force. Typically the withdrawal threshold in the incised foot decreases after incision to less than 100 mN for 2 to 3 days ( Figure 2 ). A gradual return toward preincision withdrawal thresholds occurs over the next 5 to 6 days. A reduced withdrawal threshold to punctate mechanical stimuli occurs at other sites around the wound as well. A similar time course has been shown in a number of studies and demonstrates that this incisional injury causes reproducible, quantifiable, reduced withdrawal thresholds to mechanical stimuli, indicating that mechanical hyperalgesia occurs. This mechanical hyperalgesia persists for several days after the incision and then gradually decreases. Similar filaments have been used to characterize the area of mechanical hyperalgesia in patients after surgery ( Richmond and others 1993 ; Stubhaug and others 1997 ; Wilk and others 1996 ). Time course of withdrawal thresholds to punctate mechanical stimulation after plantar incision. Results are expressed as medians (horizontal line) with 1st and 3rd quartiles (boxes), and 10th and 90th percentiles (vertical lines). (A) Diagram of the plantar aspect of the rat foot showing the incision and the site of application of von Frey filament (small darkened circle). (B) Withdrawal thresholds after incision in vehicle-treated rats. Pre represents time before foot incision. *p < 0.05 versus preincision values by Friedman and Dunnett's test. Reprinted from Anesthesiology, Vol. 87, T.J. Brennan, E. Umali, and P.K. Zahn, “Comparison of pre- versus post-incision administration of intrathecal bupivacaine and intrathecal morphine in a rat model of postoperative pain,” p 1517-1528, 1997, with permission from Lippincott Williams & Wilkins. Time course of withdrawal thresholds to punctate mechanical stimulation after plantar incision. Results are expressed as medians (horizontal line) with 1st and 3rd quartiles (boxes), and 10th and 90th percentiles (vertical lines). (A) Diagram of the plantar aspect of the rat foot showing the incision and the site of application of von Frey filament (small darkened circle). (B) Withdrawal thresholds after incision in vehicle-treated rats. Pre represents time before foot incision. *p < 0.05 versus preincision values by Friedman and Dunnett's test. Reprinted from Anesthesiology, Vol. 87, T.J. Brennan, E. Umali, and P.K. Zahn, “Comparison of pre- versus post-incision administration of intrathecal bupivacaine and intrathecal morphine in a rat model of postoperative pain,” p 1517-1528, 1997, with permission from Lippincott Williams & Wilkins. In additional studies, another mechanical stimulus has been used to characterize the pain behaviors after incision ( Zahn and others 1997 ). We have observed that after incision, rats do not allow a blunt mechanical stimulus (a plastic disk attached to a nylon monofilament) to be applied to the wound ( Figure 3 ). When this plastic disk is applied directly on the wound, the rats withdraw as with the punctate von Frey filaments, or they lift the foot, not allowing the 400-mN filament to bend. This blunt mechanical stimulus evokes no response in normal rats; blunt mechanical stimuli applied to an incision have also been used to measure hyperalgesia in patients after surgery ( Inagaki and others 1993 ; Moiniche and others 1997 ; Tverskoy and others 1990 ). Time course of responses to a blunt mechanical stimulation (plastic disk attached to avon Frey filament) after plantar incision. (A) Diagram of the plantar aspect of the rat foot showing site of application of the plastic disk (larger, darkened circle). (B) Response frequency after incision in vehicle-treated rats. Pre represents time before foot incision. *p < 0.05 versus preincision values by Friedman and Dunnett's test. Reprinted from Anesthesiology, Vol. 87, T.J. Brennan, E. Umali, and P.K. Zahn, “Comparison of pre- versus post-incision administration of intrathecal bupivacaine and intrathecal morphine in a rat model of postoperative pain,” p 1517-1528, 1997, with permission from Lippincott Williams & Wilkins. Time course of responses to a blunt mechanical stimulation (plastic disk attached to avon Frey filament) after plantar incision. (A) Diagram of the plantar aspect of the rat foot showing site of application of the plastic disk (larger, darkened circle). (B) Response frequency after incision in vehicle-treated rats. Pre represents time before foot incision. *p < 0.05 versus preincision values by Friedman and Dunnett's test. Reprinted from Anesthesiology, Vol. 87, T.J. Brennan, E. Umali, and P.K. Zahn, “Comparison of pre- versus post-incision administration of intrathecal bupivacaine and intrathecal morphine in a rat model of postoperative pain,” p 1517-1528, 1997, with permission from Lippincott Williams & Wilkins. In this model, no measure of spontaneous pain behaviors like biting, scratching, licking, or vocalization have been utilized because these activities have not been observed. We have observed that rats do not bear weight on the incision in the postoperative period and have developed a cumulative pain scale that relies on the position in which the foot is placed during a 1-min period. This nonevoked weight-beating pain behavior may be another measure of mechanical sensitivity since our scoring is based on distortion of the wound by the mesh cage floor and likely represents nonevoked rather than spontaneous pain behavior ( Zahn and others 1997 ). Again, after emergence and recovery from anesthesia, unrestrained rats are placed beneath a clear plastic chamber (21 x 27 x 15 cm) on a smaller elevated plastic mesh floor (8 x 8 mm grid; ADPI Enterprises). Using an angled magnifying mirror, the incised and nonincised feet are viewed. Both feet of each animal are closely observed during a 1-min period repeated every 5 min for 1 hr. Depending on the position in which each foot is found during the majority of the 1-min scoring period, a 0, 1, or 2 is given. Full weight bearing of the foot (when the wound has been blanched or distorted by the mesh) is assigned a score of 0. If the foot is completely off the mesh, a score of 2 is recorded. If the area of the wound touches the mesh without blanching or distorting, a I is given. The 12 scores (0 to 24) obtained during the 1-hr session for each foot are summed, and the difference between scores from the incised and nonincised feet is taken as the cumulative pain score for that 1-hr period. This pain behavior is increased after incision, and a gradual return toward preincision values occurs over the next few days ( Figure 4 ). Summary of the cumulative weight-bearing scores before and after skin, fascia, and muscle incision. Results are expressed as medians with 1st and 3rd quartiles, and 10th and 90th percentiles. *p < 0.05 versus preincision values (Oh) by Friedman and Dunnett's test for nonparametric analyses. Reprinted from Pain, Vol. 64, T.J. Brennan, E. Vandermeulen, and G.F. Gebhart, “Characterization of a rat model of incisional pain,” p 493-501, 1996, with permission from Elsevier Press. We have followed the time course of pain-related behaviors and have observed the rats for 5 to 6 days in a number of studies. Throughout the experimental period, the animals remain well groomed and maintain normal food and water intake. The weights of rats undergoing incision were recorded throughout a study period (n = 6 per group). The weights of a sham group and a group undergoing skin, fascia, and muscle incision before surgery were 346 ± 5 and 355 ± 18 g ± There a small in weight during the 3 days after surgery sham no difference between the two ( Figure 5 ). The weights of the sham and the incision at the end of the were ± and ± g ( Brennan and others 1996 ). for weight bearing on the area of the incision early in the postoperative period, appears Summary of the cumulative weight-bearing scores before and after skin, fascia, and muscle incision. Results are expressed as medians with 1st and 3rd quartiles, and 10th and 90th percentiles. *p < 0.05 versus preincision values (Oh) by Friedman and Dunnett's test for nonparametric analyses. Reprinted from Pain, Vol. 64, T.J. Brennan, E. Vandermeulen, and G.F. Gebhart, “Characterization of a rat model of incisional pain,” p 493-501, 1996, with permission from Elsevier Press. We have followed the time course of pain-related behaviors and have observed the rats for 5 to 6 days in a number of studies. Throughout the experimental period, the animals remain well groomed and maintain normal food and water intake. The weights of rats undergoing incision were recorded throughout a study period (n = 6 per group). The weights of a sham group and a group undergoing skin, fascia, and muscle incision before surgery were 346 ± 5 and 355 ± 18 g ± There a small in weight during the 3 days after surgery sham no difference between the two ( Figure 5 ). The weights of the sham and the incision at the end of the were ± and ± g ( Brennan and others 1996 ). for weight bearing on the area of the incision early in the postoperative period, appears of rats before surgery and on postoperative days 1 through There were no between the incision and the sham of rats before surgery and on postoperative days 1 through There were no between the incision and the sham research has from the of and mechanical in and normal animals and to the of hyperalgesia and the to the we a understanding of pain mechanisms and models for pain be models for postoperative pain application of this to postoperative pain. A incision tissue injury that is different from with inflammation by or injury by few models a incision as the incision is a tissue injury of and The mechanisms for and of pain after incision likely a of and however, it be that the of each of these is not a may the of little or no may be observed in an incision the of incisional pain may be different from pain, or the responses to treatments the and of pain behaviors may also The tests of pain-related behaviors in animals be to the measures of pain in patients. at rest and mechanical hyperalgesia are the most pain measures to in a postoperative pain Although reduced withdrawal to occur after plantar incision in the rat ( and others the of hyperalgesia in postoperative pain is not There is a in drugs and treatments responses to and mechanical stimuli after ( and others 1997 ; Zahn and Brennan ; Zahn and others ). even though a treatment may of responses to mechanical stimuli may not and the of is The of intrathecal and for postoperative pain in has been examined in this rat model for postoperative pain. of either to or intrathecal to morphine increased the withdrawal threshold to punctate mechanical stimulation ( Figure 6 ). In the response to the blunt mechanical stimulus and the pain scores were also decreased by and intrathecal morphine ( Zahn and others 1997 ). We have also observed that of the incision with bupivacaine decreased pain behaviors for at 2 hr after incision ( and others 1994 of morphine on punctate withdrawal thresholds after plantar incision. (A) Withdrawal threshold after incision in rats treated with 3 of morphine on the day of surgery. (B) Withdrawal threshold after incision in rats treated with 5 of intrathecal morphine on the day of surgery. administered at the *p < 0.05 versus min by Friedman and Dunnett's test. The and are in the Figure 1 Reprinted from Anesthesiology, Vol. P.K. Zahn, and T.J. Brennan, of and intrathecal morphine in a rat model of postoperative pain,” 1997, p with permission from Lippincott Williams & Wilkins. of morphine on punctate withdrawal thresholds after plantar incision. (A) Withdrawal threshold after incision in rats treated with 3 of morphine on the day of surgery. (B) Withdrawal threshold after incision in rats treated with 5 of intrathecal morphine on the day of surgery. administered at the *p < 0.05 versus min by Friedman and Dunnett's test. The and are in the Figure 1 Reprinted from Anesthesiology, Vol. P.K. Zahn, and T.J. Brennan, of and intrathecal morphine in a rat model of postoperative pain,” 1997, p with permission from Lippincott Williams & Wilkins. The and of persistent pain behaviors in animal models of inflammation and and pain and hyperalgesia in postoperative patients are In the plantar incision model, the incision and require 15 min to is profound after surgery and on the postoperative gradually decreases persists for about 5 and then is similar to sham and The of pain in other animal models from minutes after injection of to after injection of or to days after This incisional model in the rat with pain behaviors occurs on a time scale similar to most postoperative patients pain and mechanical hyperalgesia are usually the after surgery, severe for several days and then gradually over to days ( Moiniche and others 1997 ). There are to the use of this animal model of postoperative pain to studies. studies a to two pain and opioid analgesic treatments can in a in opioid with the same pain scores or a reduced pain score and the same opioid In this rat model for postoperative pain, a assessment of wound is and treatments and measures are has been used as an animal model for postoperative pain in rats ( and others 1995 ). After anesthesia is a is thresholds and mechanical thresholds are examined at several after surgery to study the of the paw test indicating mechanical hyperalgesia after This model has to a common and the injury is different from a skin and muscle incision. Additional and are likely with and may intensify pain behaviors. The of hyperalgesia after is not this not occur in patients ( and others 1997 ). The same group of has for ( and others 1997 ). A is performed under anesthesia. a used to a is applied to such as the or of the as well as directly to the The thresholds to or guarding are These results in also after occurs and have the of a measure of wound measures are reduced by parenteral administration of an opioid In this model is unique because a assessment scoring has been developed for pain at at rest is in postoperative patients. Postoperative injury may not skin and muscle also experimental model for the mechanisms pain has been by and others ). In rats with a hole is through the or a and guarding of the are observed. to and mechanical stimuli are also a hole through the or could be a experimental model for additional studies on and postoperative pain. the of postoperative pain on the surgery the activity during which the pain is and the analgesic It has been suggested that pain after surgeries is to parenteral opioids compared with other surgeries ( Kalso and Rosenberg 1995 ). the time of this the mechanisms for postoperative pain are not understood even after a simple incision. We a understanding of the unique of postoperative pain caused by because postoperative pain after incision, muscle surgery, and have unique testing to understand the sensitization processes be utilized that more treatments for postoperative pain can be advanced. In the treatment that the of postoperative pain be possible. A in and This performed in the Department of Anesthesia at the University of Iowa and by a from the of Anesthesia for Anesthesia the Research and of to on the by and G.F. are