clinical techniques Victoria Hampshire, VMD, Column Editor
Postoperative multimodal pain management Sarah Kuchinsky, BS & Jill Ascher, DVM Pain management is essential to animal welfare and scientific reliability, as pain in research animals may confound experimental data. Recently, multimodal approaches to pain management have been developed and proposed to offer optimal pain control. This column explores the effects of a multimodal approach versus administration of a single analgesic on postoperative recovery in mice. In laboratory animal science, pain m anagement for invasive procedures is a n ecessary and important refinement. As a general rule, what is deemed painful for humans is considered to be painful for animals, and so it is i mportant to use anesthetics and analgesics in a nimals u ndergoing surgery or other p otentially p ainful study anagement is essential procedures1,2. Pain m from the s tandpoints of both animal welfare and scientific reliability. Research results may be affected if a study animal is in poor health or experiencing pain and distress. Recent controlled studies suggest that use of a m ultimodal approach to t arget d ifferent pain receptors offers optimal pain control3–5. We investigated whether a combination of pain medications achieved a quicker return to normal activity level after surgery in BALB/c mice. Multimodal pain management Pain comes in various forms and degrees. Physiologic pain stems from recognition by specialized skin receptors of a change in t emperature, pressure or chemical stimulus. This information is then sent as a signal along the spinal cord and intercepted by neurotransmitters that identify the signal as physiologic pain6. A multimodal approach to pain management combines multiple drug classes that target different US Food and Drug Administration, Silver Spring, MD. Correspondence should be addressed to S.K. ([email protected]).
pain receptors along the pain pathway7. This method involves using more than one type of medication, such as an opioid, a nonsteroidal anti-inflammatory drug (NSAID) and a local anesthetic. Together, the combination of therapies can increase the effects of pain management and can potentially reduce the effective dose of each drug. Methods The protocol under which these mice were used was approved by the Food and Drug Administration Center for Biologics Evaluation and Research IACUC (Silver Spring, MD). For this study, we purchased 25 female BALB/cAnNCr mice (age 5–6 weeks) from Charles River Laboratories (Frederick, MD), an approved vendor for our animal program. Mice were housed in groups of either two or three per cage (microisolator caging; Allentown Caging, Allentown, PA) on white soft cellulose bedding (Comfort Bedding; Biofresh, Ferndale, WA). Mice were kept in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International on a 12-h:12-h light:dark cycle with an a verage room temperature of 74.6 ºF and average room humidity of 30.5%. Mice received five pellets per day of rodent chow (Prolab Isopro RMH 3000, Lab Diet, St. Louis, MO) and drinking water in a 9-ounce water b ottle. We provided mice with a water-based nutritional supplement used to support compromised rodents (Dietgel Recovery Gel; Clear H2O, Framingham, MA) for 4 d before surgery and for 4 d after surgery. We gathered baseline measurements of body weight, activity level, food and water intake, supplement consumption, ethogram score (assessed by two individual observers) and vertical raises (the number of times each mouse stood upright on its hind paws during a 2-min time period8) from each mouse at six time points during a 2-week period before surgery. We weighed
132 Volume 44, No. 4 | APRIL 2015
the food pellets and water bottles each day to c alculate daily consumption. Ethogram score was based on coat a ppearance, eye appearance, coordination and posture, and overall condition6. We also calculated daily consumption of the nutritional supplement during the 4 d before and the 4 d after surgery by weighing the s upplement each day. We randomly assigned five mice to each of the experimental and control groups and used differently colored ear tags to identify the five groups. Three groups underwent sham surgery and received different postoperative pain medication: the NSAID meloxicam (5 mg per kg body weight; Boehringer Ingelheim GmbH, Ingelheim, Germany), the opioid buprenorphine (Buprenorphine SR Lab, 1 mg per kg body weight; Veterinary Technologies, Windsor, CO) or both medications. One group underwent surgery and received neither analgesic, and one group did not undergo surgery or receive any a nalgesic. Mice from different study groups were housed in each cage in order to eliminate the ‘cage effect’ variable, whereby animals from the same group experience a unique set of variables particular to their specific cage. This study was blinded, as observers did not know which color ear tag corresponded with which treatment group. Sham kidney capsule implantation surgery was carried out under isoflurane anesthesia using aseptic technique. An incision was made into the muscle and tissue layers, exposing the kidney. The k idney remained outside of the body wall while the surgeon incised the kidney capsule. The kidney was then returned to the abdominal cavity. Warmed lactated Ringer’s solution (1 ml; Hospira Worldwide Inc., San Jose, CA) was administered to each mouse by intraperitoneal injection. The abdominal cavity was closed with absorbable sutures, and the dermal layer was closed with onitored s urgical staples. Mice were m continuously by a d edicated technician www.labanimal.com
© 2015 Macmillan Publishers Limited. All rights reserved
clinical techniques
20.5 20.0 19.5
Buprenorphine
Weight (g)
19.0 18.5
No surgery
18.0
Meloxicam + buprenorphine
17.5 No analgesia
17.0
Meloxicam
16.5 16.0
4
3
2
1
0
Ba
se
lin
e
15.5
Time after surgery (d)
FIGURE 1 | Body weight at baseline and during 4 d after surgery in mice receiving meloxicam alone, buprenorphine alone, meloxicam and buprenorphine together or no analgesic and in control mice that did not undergo surgery.
mouse about 4 h after surgery and then each day for the next 4 d. Mice were euthanized at the study endpoint, 4 d after surgery, in a chamber by exposure to carbon dioxide introduced from a cylinder source at the rate of at least 20% of the chamber volume per min. Mice were observed for 10 min to verify that death had occurred. Results and discussion The most notable differences occurred within the first 2 d after surgery. Body weight decreased by 3–5% in all treatment groups during this time period (Fig. 1). Mice t reated with buprenorphine alone lost the most body weight after surgery (5.46%), whereas mice treated with meloxicam alone lost the least body weight after surgery (3.21%).
10 9 8 7 6
Buprenorphine
5
No surgery
4
Meloxicam + buprenorphine
3
No analgesia
2
Meloxicam
Conclusions The results suggest that, compared with administration of a single analgesic, a multimodal approach to pain m anagement did not decrease the time needed for a mouse to recover after surgery. Ethogram scores and vertical raises did not differ substantially among the treatment groups. We are in favor
1
4
3
2
1
Ba
se
lin
0
0
e
Number of vertical raises in 2-min intervals
t hroughout the s urgical p rocedure and recovery period. The procedure was carried out by a licensed veterinarian. During recovery, mice were placed in a dedicated cage that was resting on top of an isothermal heating pad (Deltaphase Isothermal Pad, Braintree Scientific, Braintree, MA) and were monitored continuously until they moved freely about the cage. Indirect heat from a heat lamp was also provided. Meloxicam and buprenorphine were each a dministered subcutaneously while the mouse was under isoflurane anesthesia, before the surgical incision was made. We measured body weight, activity level, food and water intake, supplement consumption, ethogram score (assessed by two i ndividual observers) and vertical raises for each
Time after surgery (d)
FIGURE 2 | Number of vertical raises in 2-min intervals at baseline and during 4 d after surgery in mice receiving meloxicam alone, buprenorphine alone, meloxicam and buprenorphine together or no analgesic and in control mice that did not undergo surgery. LAB ANIMAL
Supplement consumption and food and water intake correlated with one another. Water intake initially rose after surgery and then fell between 1 d and 2 d after surgery in all treatment groups (data not shown), suggesting that mice experienced a decrease in hydration during or immediately after surgery. When supplement consumption was greater, food and water intake were lower (data not shown), suggesting that the gel served as a source of hydration and sustenance. Supplement c onsumption and food and water intake returned to normal levels in mice in all treatment groups by 3 d after surgery (data not shown), s uggesting that multimodal pain m anagement did not result in quicker r ecovery than a dministration of meloxicam or buprenorphine alone. Mice treated with buprenorphine (alone or in combination with meloxicam) had reduced activity levels after surgery (data not shown), indicative of p otential adverse effects, such as sedation. Respiratory depression and sedation are known adverse effects of buprenorphine HCl, an a nalgesic commonly used in small animals 9 . In c ontrast, mice treated with m eloxicam alone had fairly consistent a ctivity levels p ostoperatively (data not shown), s uggesting that the medication did not inhibit normal activity. Mice in the control group that did not receive analgesia also had consistent activity levels, a result that was not expected. We surmise that mice may have masked pain in order to avoid attention from anticipated predators6. Ethogram scores did not differ substantially between treatment groups (data not shown). Mice treated with buprenorphine (either alone or in c ombination with meloxicam) performed markedly fewer vertical raises 4 h and 1 d after surgery than did mice in other treatment groups (Fig. 2). This may have been attributed to the known sedative effect of buprenorphine HCl 9, which likely also contributed to the reduced activity levels in these mice.
© 2015 Macmillan Publishers Limited. All rights reserved
Volume 44, No. 4 | APRIL 2015 133
clinical techniques
of the use of a multimodal approach for postoperative pain relief, the importance of which is discussed in recent literature3–5. However, the criteria we used to monitor postoperative recovery did not substantiate this conclusion. There are several questions to c onsider in interpreting these results. Did b uprenorphine affect metabolism or cause a lasting, mild sedation resulting in decreased food consumption and activity level? Did the nutritional supplement positively affect recovery, such that mice that were given no analgesia were able to maintain normal activity levels? These questions warrant further study.
1. US Department of Agriculture. Animal Care Policy Manual (USDA, Riverdale, MD, 2014). 2. Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals 8th edn. (National Academies Press, Washington, DC, 2011). 3. Bourque, S.L., Adams, M.A., Nakatsu, K. & Winterborn, A. Comparison of Buprenorphine and Meloxicam for postsurgical analgesia in rats: effects on body weight, locomotor activity, and hemodynamic parameters. J. Am. Assoc. Lab. Anim. Sci. 49, 617–622 (2010). 4. Carbone, E.T., Lindstrom, K.E., Diep, S. & Carbone, L. Duration of action of sustainedrelease Buprenorphine in 2 strains of mice. J. Am. Assoc. Lab. Anim. Sci. 51, 815–819 (2012). 5. Clark, M.D. et al. Evaluation of liposome-
134 Volume 44, No. 4 | APRIL 2015
6.
7.
8.
9.
encapsulated oxymorphone hydrochloride in mice after splenectomy. Comp. Med. 84, 558–563 (2004). Stasiak, K.L., Maul, D., French, E., Hellyer, P.W. & Vandewoude, S. Species-specific assessment of pain in laboratory animals. J. Am. Assoc. Lab. Anim. Sci. 42, 13–20 (2003). Lamont, L.A. Multimodal pain management in veterinary medicine: the physiologic basis of pharmacologic therapies. Vet. Clin. North Am. Small Anim. Pract. 38, 1173–1186 (2008). Foley, P.L., Haixaing, L. & Crichlow, A.R. Evaluation of a sustained-release formulation of Buprenorphine for analgesia in rats. J. Am. Assoc. Lab. Anim. Sci. 50, 198–204 (2011). Plumb, D.C. Buprenorphine HCl. in Plumb’s Veterinary Drug Handbook 7th edn. (WileyBlackwell, Ames, IA, 2011).
www.labanimal.com © 2015 Macmillan Publishers Limited. All rights reserved
Postoperative multimodal pain management Sarah Kuchinsky, BS & Jill Ascher, DVM Pain management is essential to animal welfare and scientific reliability, as pain in research animals may confound experimental data. Recently, multimodal approaches to pain management have been developed and proposed to offer optimal pain control. This column explores the effects of a multimodal approach versus administration of a single analgesic on postoperative recovery in mice. In laboratory animal science, pain m anagement for invasive procedures is a n ecessary and important refinement. As a general rule, what is deemed painful for humans is considered to be painful for animals, and so it is i mportant to use anesthetics and analgesics in a nimals u ndergoing surgery or other p otentially p ainful study anagement is essential procedures1,2. Pain m from the s tandpoints of both animal welfare and scientific reliability. Research results may be affected if a study animal is in poor health or experiencing pain and distress. Recent controlled studies suggest that use of a m ultimodal approach to t arget d ifferent pain receptors offers optimal pain control3–5. We investigated whether a combination of pain medications achieved a quicker return to normal activity level after surgery in BALB/c mice. Multimodal pain management Pain comes in various forms and degrees. Physiologic pain stems from recognition by specialized skin receptors of a change in t emperature, pressure or chemical stimulus. This information is then sent as a signal along the spinal cord and intercepted by neurotransmitters that identify the signal as physiologic pain6. A multimodal approach to pain management combines multiple drug classes that target different US Food and Drug Administration, Silver Spring, MD. Correspondence should be addressed to S.K. ([email protected]).
pain receptors along the pain pathway7. This method involves using more than one type of medication, such as an opioid, a nonsteroidal anti-inflammatory drug (NSAID) and a local anesthetic. Together, the combination of therapies can increase the effects of pain management and can potentially reduce the effective dose of each drug. Methods The protocol under which these mice were used was approved by the Food and Drug Administration Center for Biologics Evaluation and Research IACUC (Silver Spring, MD). For this study, we purchased 25 female BALB/cAnNCr mice (age 5–6 weeks) from Charles River Laboratories (Frederick, MD), an approved vendor for our animal program. Mice were housed in groups of either two or three per cage (microisolator caging; Allentown Caging, Allentown, PA) on white soft cellulose bedding (Comfort Bedding; Biofresh, Ferndale, WA). Mice were kept in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International on a 12-h:12-h light:dark cycle with an a verage room temperature of 74.6 ºF and average room humidity of 30.5%. Mice received five pellets per day of rodent chow (Prolab Isopro RMH 3000, Lab Diet, St. Louis, MO) and drinking water in a 9-ounce water b ottle. We provided mice with a water-based nutritional supplement used to support compromised rodents (Dietgel Recovery Gel; Clear H2O, Framingham, MA) for 4 d before surgery and for 4 d after surgery. We gathered baseline measurements of body weight, activity level, food and water intake, supplement consumption, ethogram score (assessed by two individual observers) and vertical raises (the number of times each mouse stood upright on its hind paws during a 2-min time period8) from each mouse at six time points during a 2-week period before surgery. We weighed
132 Volume 44, No. 4 | APRIL 2015
the food pellets and water bottles each day to c alculate daily consumption. Ethogram score was based on coat a ppearance, eye appearance, coordination and posture, and overall condition6. We also calculated daily consumption of the nutritional supplement during the 4 d before and the 4 d after surgery by weighing the s upplement each day. We randomly assigned five mice to each of the experimental and control groups and used differently colored ear tags to identify the five groups. Three groups underwent sham surgery and received different postoperative pain medication: the NSAID meloxicam (5 mg per kg body weight; Boehringer Ingelheim GmbH, Ingelheim, Germany), the opioid buprenorphine (Buprenorphine SR Lab, 1 mg per kg body weight; Veterinary Technologies, Windsor, CO) or both medications. One group underwent surgery and received neither analgesic, and one group did not undergo surgery or receive any a nalgesic. Mice from different study groups were housed in each cage in order to eliminate the ‘cage effect’ variable, whereby animals from the same group experience a unique set of variables particular to their specific cage. This study was blinded, as observers did not know which color ear tag corresponded with which treatment group. Sham kidney capsule implantation surgery was carried out under isoflurane anesthesia using aseptic technique. An incision was made into the muscle and tissue layers, exposing the kidney. The k idney remained outside of the body wall while the surgeon incised the kidney capsule. The kidney was then returned to the abdominal cavity. Warmed lactated Ringer’s solution (1 ml; Hospira Worldwide Inc., San Jose, CA) was administered to each mouse by intraperitoneal injection. The abdominal cavity was closed with absorbable sutures, and the dermal layer was closed with onitored s urgical staples. Mice were m continuously by a d edicated technician www.labanimal.com
© 2015 Macmillan Publishers Limited. All rights reserved
clinical techniques
20.5 20.0 19.5
Buprenorphine
Weight (g)
19.0 18.5
No surgery
18.0
Meloxicam + buprenorphine
17.5 No analgesia
17.0
Meloxicam
16.5 16.0
4
3
2
1
0
Ba
se
lin
e
15.5
Time after surgery (d)
FIGURE 1 | Body weight at baseline and during 4 d after surgery in mice receiving meloxicam alone, buprenorphine alone, meloxicam and buprenorphine together or no analgesic and in control mice that did not undergo surgery.
mouse about 4 h after surgery and then each day for the next 4 d. Mice were euthanized at the study endpoint, 4 d after surgery, in a chamber by exposure to carbon dioxide introduced from a cylinder source at the rate of at least 20% of the chamber volume per min. Mice were observed for 10 min to verify that death had occurred. Results and discussion The most notable differences occurred within the first 2 d after surgery. Body weight decreased by 3–5% in all treatment groups during this time period (Fig. 1). Mice t reated with buprenorphine alone lost the most body weight after surgery (5.46%), whereas mice treated with meloxicam alone lost the least body weight after surgery (3.21%).
10 9 8 7 6
Buprenorphine
5
No surgery
4
Meloxicam + buprenorphine
3
No analgesia
2
Meloxicam
Conclusions The results suggest that, compared with administration of a single analgesic, a multimodal approach to pain m anagement did not decrease the time needed for a mouse to recover after surgery. Ethogram scores and vertical raises did not differ substantially among the treatment groups. We are in favor
1
4
3
2
1
Ba
se
lin
0
0
e
Number of vertical raises in 2-min intervals
t hroughout the s urgical p rocedure and recovery period. The procedure was carried out by a licensed veterinarian. During recovery, mice were placed in a dedicated cage that was resting on top of an isothermal heating pad (Deltaphase Isothermal Pad, Braintree Scientific, Braintree, MA) and were monitored continuously until they moved freely about the cage. Indirect heat from a heat lamp was also provided. Meloxicam and buprenorphine were each a dministered subcutaneously while the mouse was under isoflurane anesthesia, before the surgical incision was made. We measured body weight, activity level, food and water intake, supplement consumption, ethogram score (assessed by two i ndividual observers) and vertical raises for each
Time after surgery (d)
FIGURE 2 | Number of vertical raises in 2-min intervals at baseline and during 4 d after surgery in mice receiving meloxicam alone, buprenorphine alone, meloxicam and buprenorphine together or no analgesic and in control mice that did not undergo surgery. LAB ANIMAL
Supplement consumption and food and water intake correlated with one another. Water intake initially rose after surgery and then fell between 1 d and 2 d after surgery in all treatment groups (data not shown), suggesting that mice experienced a decrease in hydration during or immediately after surgery. When supplement consumption was greater, food and water intake were lower (data not shown), suggesting that the gel served as a source of hydration and sustenance. Supplement c onsumption and food and water intake returned to normal levels in mice in all treatment groups by 3 d after surgery (data not shown), s uggesting that multimodal pain m anagement did not result in quicker r ecovery than a dministration of meloxicam or buprenorphine alone. Mice treated with buprenorphine (alone or in combination with meloxicam) had reduced activity levels after surgery (data not shown), indicative of p otential adverse effects, such as sedation. Respiratory depression and sedation are known adverse effects of buprenorphine HCl, an a nalgesic commonly used in small animals 9 . In c ontrast, mice treated with m eloxicam alone had fairly consistent a ctivity levels p ostoperatively (data not shown), s uggesting that the medication did not inhibit normal activity. Mice in the control group that did not receive analgesia also had consistent activity levels, a result that was not expected. We surmise that mice may have masked pain in order to avoid attention from anticipated predators6. Ethogram scores did not differ substantially between treatment groups (data not shown). Mice treated with buprenorphine (either alone or in c ombination with meloxicam) performed markedly fewer vertical raises 4 h and 1 d after surgery than did mice in other treatment groups (Fig. 2). This may have been attributed to the known sedative effect of buprenorphine HCl 9, which likely also contributed to the reduced activity levels in these mice.
© 2015 Macmillan Publishers Limited. All rights reserved
Volume 44, No. 4 | APRIL 2015 133
clinical techniques
of the use of a multimodal approach for postoperative pain relief, the importance of which is discussed in recent literature3–5. However, the criteria we used to monitor postoperative recovery did not substantiate this conclusion. There are several questions to c onsider in interpreting these results. Did b uprenorphine affect metabolism or cause a lasting, mild sedation resulting in decreased food consumption and activity level? Did the nutritional supplement positively affect recovery, such that mice that were given no analgesia were able to maintain normal activity levels? These questions warrant further study.
1. US Department of Agriculture. Animal Care Policy Manual (USDA, Riverdale, MD, 2014). 2. Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals 8th edn. (National Academies Press, Washington, DC, 2011). 3. Bourque, S.L., Adams, M.A., Nakatsu, K. & Winterborn, A. Comparison of Buprenorphine and Meloxicam for postsurgical analgesia in rats: effects on body weight, locomotor activity, and hemodynamic parameters. J. Am. Assoc. Lab. Anim. Sci. 49, 617–622 (2010). 4. Carbone, E.T., Lindstrom, K.E., Diep, S. & Carbone, L. Duration of action of sustainedrelease Buprenorphine in 2 strains of mice. J. Am. Assoc. Lab. Anim. Sci. 51, 815–819 (2012). 5. Clark, M.D. et al. Evaluation of liposome-
134 Volume 44, No. 4 | APRIL 2015
6.
7.
8.
9.
encapsulated oxymorphone hydrochloride in mice after splenectomy. Comp. Med. 84, 558–563 (2004). Stasiak, K.L., Maul, D., French, E., Hellyer, P.W. & Vandewoude, S. Species-specific assessment of pain in laboratory animals. J. Am. Assoc. Lab. Anim. Sci. 42, 13–20 (2003). Lamont, L.A. Multimodal pain management in veterinary medicine: the physiologic basis of pharmacologic therapies. Vet. Clin. North Am. Small Anim. Pract. 38, 1173–1186 (2008). Foley, P.L., Haixaing, L. & Crichlow, A.R. Evaluation of a sustained-release formulation of Buprenorphine for analgesia in rats. J. Am. Assoc. Lab. Anim. Sci. 50, 198–204 (2011). Plumb, D.C. Buprenorphine HCl. in Plumb’s Veterinary Drug Handbook 7th edn. (WileyBlackwell, Ames, IA, 2011).
www.labanimal.com © 2015 Macmillan Publishers Limited. All rights reserved
