Journal of the American Association for Laboratory Animal Science Copyright 2016 by the American Association for Laboratory Animal Science

Vol 55, No 5 September 2016 Pages 548–557

Intraperitoneal Continuous-Rate Infusion for the Maintenance of Anesthesia in Laboratory Mice (Mus musculus) Rebecca L Erickson,1,2 Matthew C Terzi,3 Samer M Jaber,4 F Claire Hankenson,5 Andrew McKinstry-Wu,6 Max B Kelz,6 and James O Marx1,2,* Intraperitoneal injectable anesthetics are often used to achieve surgical anesthesia in laboratory mice. Because bolus redosing of injectable anesthetics can cause unacceptably high mortality, we evaluated intraperitoneal continuous-rate infusion (CRI) of ketamine with or without xylazine for maintaining surgical anesthesia for an extended period of time. Anesthesia was induced in male C57BL/6J mice by using ketamine (80 mg/kg) and xylazine (8 mg/kg) without or with acepromazine at 0.1 mg/kg or 0.5 mg/kg. At 10 min after induction, CRI for 90 min was initiated and comprised 25%, 50%, or 100% of the initial ketamine dose per hour or 50% of the initial doses of both ketamine and xylazine. Anesthetic regimens were compared on the basis of animal immobility, continuous surgical depth of anesthesia as determined by the absence of a pedal withdrawal reflex, and mortality. Consistent with previous studies, the response to anesthetics was highly variable. Regimens that provided the longest continuous surgical plane of anesthesia with minimal mortality were ketamine–xylazine–acepromazine (0.1 mg/kg) with CRI of 100% of the initial ketamine dose and ketamine–xylazine–acepromazine (0.5 mg/kg) with CRI of 50% of the initial ketamine and xylazine doses. In addition, heart rate and respiratory rate did not increase consistently in response to a noxious stimulus during CRI anesthesia, even when mice exhibited a positive pedal withdrawal reflex, suggesting that these parameters are unreliable indicators of anesthetic depth during ketamine–xylazine anesthesia in mice. We conclude that intraperitoneal CRI anesthesia in mice prolongs injectable anesthesia more consistently and with lower mortality than does bolus redosing. Abbreviations: CRI, continuous rate infusion; KXA, ketamine–xylazine–acepromazine; PWR, pedal withdrawal reflex

Effective and reliable anesthesia of laboratory mice is an essential component of conducting humane biomedical research. For procedures requiring prolonged surgical anesthesia or immobility for imaging, inhalant anesthetics such as isoflurane are recommended due to their safety, reliability, ease in controlling anesthetic depth, and rapid recovery.11 However, in a laboratory setting, some experimental procedures and research aims might preclude the use of inhalant anesthetics owing to their effects on physiologic parameters (for example, blood pressure) or because imaging modalities might be incompatible with anesthetic equipment (for example, MRI).10,11,29,36,40 Ketamine–xylazine (KX) and ketamine–xylazine–acepromazine (KXA) combinations are 2 common injectable anesthetic mixtures delivered to laboratory mice.38 Two separate studies2,3 examined multiple intraperitoneal injectable anesthetic combinations in mice (including ketamine with or without xylazine combined with acepromazine, medetomidine, tiletamine with zolazepam, azaperone, buprenorphine, or carprofen) and concluded that KXA produced the longest duration of surgical anesthesia with the fewest adverse effects.2,3 Ketamine, xylazine, and acepromazine work synergistically to provide

Received: 14 Nov 2015. Revision requested: 11 Dec 2015. Accepted: 11 Feb 2016. 1University Laboratory Animal Resources, 2Department of Pathobiology, 3School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 4Department of Animal Medicine, University of Massachusetts Medical School, Worcester, Massachusetts; 5Campus Animal Resources and College of Veterinary Medicine, Michigan State University, East Lansing, Michigan; and 6Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. *Corresponding author. Email: [email protected]

balanced anesthesia and can provide an average 40 to 50 min of surgical anesthesia in mice.2,3,19 However, this duration is highly variable, and some procedures may require longer durations of anesthesia.2,19 Bolus redosing of ketamine by itself or in combination with xylazine has been the default method of extending the surgical plane of anesthesia beyond that achieved by the initial injection. Previously, our group examined the use of repeated intraperitoneal bolus dosing of K or KX to extend the duration of surgical anesthesia in C57BL/6J mice, with the goal of providing a continuous surgical plane.19 Redosing with ketamine or ketamine and xylazine prior to emergence from surgical anesthesia, confirmed by a lack of response to a pedal-withdrawal stimulus, resulted in unacceptable mortality rates. Redosing with the same drug combination after emergence from a surgical plane improved survivability. However, the interruption in surgical anesthesia levels may have left mice vulnerable to awareness or pain perception during experimental procedures.2,19 Although bolus redosing does not consistently maintain a continuous surgical plane of anesthesia beyond that provided by the initial injection, redosing mice with either 50% of the initial ketamine dose or 25% of the initial doses of both ketamine and xylazine can be used to extend the duration of surgical anesthesia after response to a pedal stimulus.19 Compared with repeated bolus administration of injectable anesthetics in mice, a superior method to maintain an anesthetic state without over- or underdosing is the delivery of anesthetics by intraperitoneal continuous-rate infusion (CRI). By minimizing rapid increases and decreases in plasma concentrations of

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drugs, CRI can provide a more stable plane of anesthesia and can be used alone or as an adjunct to inhalant anesthetics to produce anesthesia, sedation, and analgesia.40,44,48 Although CRI is typically given intravenously in humans and larger veterinary species, intraperitoneal infusion is advantageous in mice due to relative technical ease of administration.11,42 However, the drug combinations of K and KX for CRI anesthesia and their efficacy in maintaining surgical anesthesia have not previously been evaluated.48,49 Therefore, the primary goal of this project was to determine the safety and efficacy of intraperitoneal CRI of K and KX to extend the duration of surgical anesthesia in mice in which anesthesia had been induced by using KX or KXA. These drug combinations were chosen based on their demonstrated efficacy in maintaining anesthesia compared with other injectable drug combinations and their frequent use in research.2,3,19,38 In addition, we sought to examine autonomic reflexes under KX and KXA anesthesia and to evaluate their suitability for anesthetic monitoring. Monitoring the depth of anesthesia is critical to ensuring the safety and efficacy of anesthetic regimens. If anesthesia is insufficient, the animal is vulnerable to awareness or pain perception, whereas excessive depth of anesthesia increases risks for prolonged recovery, adverse events, and potentially death. A surgical plane of anesthesia requires the elimination of movement in response to a noxious or surgical stimulus.21 Autonomic responses to noxious stimuli—for example, increases in heart rate, blood pressure, or respiratory rate following surgical stimulation—are eliminated at very deep anesthetic planes, potentially making these parameters useful indicators of anesthetic depth in humans and veterinary species.2,4,11,21,40 Blunting, that is, reduction in magnitude, of autonomic responses during anesthesia may be a sign of impending anesthetic complications and an indication for intervention. The secondary goal of this project was to evaluate the ability to detect blunted autonomic reflexes—specifically, changes in heart rate and respiratory rate in response to a noxious stimulus—under CRI anesthesia using KX or KXA. Overall, our study was designed to investigate the use of intraperitoneal CRI for the anesthesia of laboratory mice and to identify appropriate anesthetic regimens to maintain immobility for imaging studies or a surgical plane of anesthesia. We hypothesized that induction with KX or KXA followed by CRI of ketamine with or without xylazine would produce a continuous, stable plane of anesthesia with decreased anesthetic mortality compared with repeated bolus dosing.

Materials and Methods

Animals and facility. Male C57BL/6J mice (Mus musculus; n = 68; mean age at time of anesthesia, 9.7 wk [range 7 to 14 wk]; weight, 26.5 ± 0.3 g [range, 20 to 31 g]) were obtained from Jackson Laboratories (Bar Harbor, ME). In addition, a small group of female C57BL/6J mice (Mus musculus; n = 10; mean age at time of anesthesia, 8 wk [range, 7 to 9 wk]; weight, 17.9 ± 0.3 g [range, 16 to 21 g]) was tested to assess potential sex-associated differences. Mice were housed under a 12:12-h light:dark cycle with 4 or 5 animals per cage in static polycarbonate microisolation cages (Max 75, Alternative Design, Siloam Springs, AR) containing disposable bedding (0.12-in. Bed-O-Cobs, The Andersons, Maumee, OH). Mice were fed standard pelleted laboratory rodent chow (5001, LabDiet, St Louis, MO) and received municipal water supplied by bottle. Sentinel mice were tested routinely and found to be free from fur mites and pinworms by cecal exam; sentinels were also negative for antibodies to tested pathogens, including mouse hepatitis virus, mouse parvoviruses, rotavirus, ectromelia virus, Sendai virus, pneumonia virus of mice, Theiler

murine encephalomyelitis virus, reovirus, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus, and polyomavirus. All procedures were approved by the University of Pennsylvania’s IACUC. Mice were allowed at least 1 wk to acclimate to the housing facility and cage environment prior to the start of the study. Each mouse in this study underwent no more than 3 anesthetic events total and no more than 2 anesthetic events using ketamine and xylazine, with at least a 10-d washout period between anesthetic events. There were a total of 124 anesthetic events, comprising 14 using isoflurane and 110 using injectable KX or KXA. Anesthetic induction and monitoring. Mice were weighed individually on a digital scale (US-ACE, US Balance, Vincennes, IN) prior to dosing. Anesthetic drugs used were ketamine (100 mg/mL, Ketathesia, Henry Schein Animal Health, Dublin, OH), xylazine (20 mg/mL, AnaSed, Lloyd Laboratories, Shenandoah, IA), and acepromazine (10 mg/mL, acepromazine maleate, Phoenix Pharmaceuticals, St Joseph, MO). Drugs were combined into a single syringe and diluted with sterile 0.9% NaCl to a concentration that would administer the appropriate doses at 0.1 mL per 10 g of body weight. Mice received 1 of 3 induction doses: ketamine (80 mg/kg) and xylazine (8 mg/kg); ketamine (80 mg/kg), xylazine (8 mg/kg), and acepromazine (0.1 mg/kg); or ketamine (80 mg/kg), xylazine (8 mg/kg), and acepromazine (0.5 mg/kg); these regimens are abbreviated as KX, KXA(0.1), and KXA(0.5), respectively, hereafter. Pilot experiments for CRI anesthesia were performed in male mice (n = 5) by using an induction dose of ketamine, xylazine, and acepromazine at 80, 8, and 1.0 mg/kg, respectively, which resulted in 100% mortality at all CRI doses tested and led us to decrease the dose of acepromazine accordingly. Each mouse was manually restrained for intraperitoneal injection, using a 25-gauge 5/8 in. needle into the right lower quadrant of the abdomen. Artificial tears ointment (Akwa Tears, Akorn, Lake Forest, IL) was applied to the eyes at the beginning of each anesthetic procedure. After injection, mice were monitored for loss of the righting reflex, which was defined as loss of the ability to return to standing or sternal recumbency after being placed in dorsal recumbency. The righting reflex was tested at 30-s intervals after mice stopped ambulating and until loss of the reflex was confirmed. After the righting reflex was lost, mice were placed in dorsal recumbency on a circulating-water heating pad (Gaymar Industries, Orchard Park, NY) to maintain a body temperature of 36 to 37 °C. Body temperature was measured by using a rectal temperature probe (19 mm; RET3, Thermoworks, Lindon, UT) connected to a thermometer (TW2-193, MicroTherma, Thermoworks). Heart rate during anesthesia was monitored by using electrocardiography (ECGenie and eMouse 11 Analysis Software, Mouse Specifics, Quincy, MA). Electrocardiography leads were applied to both forelimbs or to one forelimb and the tail and were secured by using conductive putty (Mouse Specifics) or coupling gel (Aquasonic 100 Ultrasound Transmission Gel, Parker Laboratories, Fairfield, NJ) and standard medical adhesive tape. Respiratory rate was measured visually by counting thoracic excursions. Immobility was defined as the absence of all spontaneous movement, including movement of limbs or vibrissae, except for respiration. A surgical plane of anesthesia was defined by immobility and absence of motor response to a noxious stimulus. The pedal-withdrawal reflex (PWR) was assessed by using a Touch Test Sensory Evaluator (300-g, 6.65-gauge; North Coast Medical, Gilroy, CA) as previously described.18,23 The point of compression was located on the dorsal aspect of the metatarsal region and was alternated between hindlimbs. A positive response was defined as withdrawal of the stimulated limb or 549

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any spontaneous motion of the mouse unassociated with the stimulated limb; because the PWR was tested at 5-min intervals, a single negative PWR was interpreted as 5 min of surgical anesthesia. The time to the loss of the PWR was recorded as the beginning of the longest continuous surgical plane and not necessarily the first absent PWR, although first absent PWR and the beginning of the longest duration of surgical plane coincided for the majority of animals. Heart rate, respiratory rate, PWR, and rectal temperature were recorded every 5 min during anesthesia until the return of spontaneous movement, at which point the monitoring equipment was removed. Times for the loss and return of the righting reflex, spontaneous movement, and PWR were recorded. CRI. Mice received 1 of 4 CRI for 90 min: (1) 25% of the initial ketamine dose hourly (that is, 20 mg/kg/h); (2) 50% of the initial ketamine dose hourly (40 mg/kg/h), (3) 100% of the initial ketamine dose hourly (80 mg/kg/h), or (4) 50% of both the initial ketamine and xylazine doses hourly (ketamine, 40 mg/kg/h; xylazine, 4 mg/kg/h). Because of its long half-life, acepromazine was not included in the CRI regimens.16,17 In light of the results from these initial experimental dosing regimens, an additional group of male mice (n = 4 ) was anesthetized by using the induction regimen of 80 mg/kg ketamine, 8 mg/kg xylazine, and 0.1 mg/kg acepromazine and CRI of 150% initial ketamine dose hourly (that is, 120 mg/kg/h). Male mice were randomly assigned to each combination of induction regimen and CRI dose. To test for sex-associated differences in response to KX-based anesthesia, a group of female mice (n =8) was anesthetized by using an induction regimen of 80 mg/kg ketamine, 8 mg/kg xylazine, and 0.5 mg/kg acepromazine and a CRI dose 40 mg/kg/h ketamine, 4 mg/kg/h xylazine; this dosing regimen was chosen because it achieved the longest duration of surgical anesthesia in male mice with minimal mortality. The anesthetist was blinded to the CRI dose administered. Ketamine and xylazine were combined in a single 3-mL syringe and diluted with sterile 0.9% NaCl to achieve the indicated hourly doses per 1 mL of fluid. An infusion rate of 1 mL/h was selected to deliver a sufficient volume of fluid to support the anesthetized animal without overloading the abdomen with fluid or excessively diluting the anesthetics delivered. A butterfly catheter with a 25-gauge, 3/4 in. needle and 12 in. of tubing (SurFlo Winged Infusion Set, Terumo Medical, Somerset, NJ) was affixed to the syringe, and the catheter was primed with the syringe contents to eliminate air from the tubing. The syringe was loaded into a programmable syringe pump (NE4000, Programmable 2-Channel Syringe Pump, New Era Pump Systems, Farmingdale, NY), and the pump was programmed to continuously deliver the syringe contents at a rate of 1.00 mL/h. The CRI was initiated 10 min after the anesthetic induction dose, with the mice in dorsal recumbency and instrumented for physiologic monitoring (Figure 1). A few mice (3 of the 110 induced) did not lose the righting reflex within 10 min after the induction dose and therefore did not receive the CRI anesthetic, leaving a total of 107 mice that underwent CRI. The butterfly catheter needle was inserted into the right caudal quadrant of the anesthetized mouse’s abdomen and was stabilized by using medical adhesive tape, when needed. The CRI anesthetic was administered for 90 min. When a mouse was immobilized during the CRI but exhibited a return to spontaneous movement before 90 min, the CRI was discontinued at that time and the mouse was allowed to recover. After spontaneous movement returned, mice were disconnected from CRI and remained in dorsal recumbency until the righting reflex returned (that is, mice were able to right themselves from dorsal recumbency 3

Figure 1. Mouse anesthetized with intraperitoneal CRI and instrumented for physiologic monitoring. The syringe pump was programmed to deliver 1.00 mL/h (white arrow) from a syringe containing anesthetic drugs (green arrow) via a butterfly catheter inserted intraperitoneally (red arrow). Mice were warmed on a circulating water heating pad, and body temperature was monitored by using a rectal thermometer probe (blue arrow). Heart rate was monitored by using electrocardiography, with leads applied to the forelimbs and secured by using conductive putty (yellow arrows).

consecutive times within 30 s). Mice were observed continuously until they became fully ambulatory and then were monitored once daily for 2 d for any postanesthetic complications or adverse events. Of the 107 mice that underwent CRI, 8 mice developed agonal breathing under anesthesia and subsequently died. CRI was discontinued when agonal breathing developed; to maintain uniformity of experimental conditions, no other interventions were performed. The time of death was recorded when mice exhibited complete respiratory arrest for 1 min; in addition, cardiac arrest was confirmed by ECG. Postmortem examinations were performed to rule out gross anatomic abnormalities or trauma, such as lacerations or hemorrhage due to intraperitoneal injection or catheter placement. Isoflurane anesthesia. Control groups of male (n = 8) and female (n = 6) mice were anesthetized with isoflurane. Mice were placed in an induction chamber containing isoflurane (Isoflurane USP, Phoenix Pharmaceuticals, St Joseph, MO) at an induction dose of 4% until they were fully anesthetized, as confirmed by loss of the righting reflex. Anesthetized mice were placed in dorsal recumbency, and isoflurane was delivered via a nose cone at a maintenance dose of 2% in O2 at 250 mL/ min for 90 min. Waste anesthetic gas was scavenged by using an activated charcoal canister (Omnicon F/Air Anesthesia Gas Filter Unit, AM Bickford, Wales Center, NY). CRI of 0.9% NaCl was delivered at a rate of 1.00 mL/h by using the syringe pump as described in the previous section. At 90 min after induction, the saline CRI and isoflurane were discontinued, and mice were allowed to breathe room air until fully recovered. Heart and respiratory rates and PWR were recorded every 5 min during anesthesia, and the times of the return of the righting reflex, spontaneous movement, and PWR were recorded as previously described. For a subset of mice (n = 6), the isoflurane concentration was measured by using an anesthetic gas monitor (Poet IQ2 Anesthetic Gas Monitor, Criticare Systems, Waukesha, WI) to verify that the vaporizer setting was consistent with inhaled anesthetic concentrations. Autonomic responses to noxious stimulus. To evaluate autonomic responses to a noxious stimulus, heart and respiratory

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rates were measured within 5 s before and after assessment of the PWR every 5 min while mice were immobilized. Increases of more than 10 bpm or 5 breaths per minute were established as clinically detectable positive responses with routine monitoring techniques used in mice. The differences between the heart and respiratory rates before and after PWR testing were calculated, and the percentages of positive responses were compared according to the induction regimen and whether mice were at a surgical plane of anesthesia (as determined according to the PWR). Statistical analysis. Means ± SE were calculated for all data. For CRI dosing regimens, the duration of recumbency, longest continuous duration of immobility, and longest continuous duration of surgical plane of anesthesia were compared by 2-way ANOVA, with main effects of induction dose and CRI dose. When significant differences were detected, Tukey post hoc analysis was performed. χ2 analyses were used to compare mortality and the ability of CRI anesthetic regimens to maintain continuous immobility or continuous surgical anesthesia for at least 5, 30, 60, and 90 min. The durations of recumbency, continuous immobility, and surgical anesthesia were compared between male and female mice receiving the same anesthetic regimens by using t tests. To determine the responsiveness of the autonomic nervous system to a noxious stimulus, the heart rate and respiratory rate were measured before and immediately after performing the Touch Test for PWR. Increases of more than 10 bpm in heart rate and more than 5 breaths per minute in respiratory rate were considered to be the minimal changes detectable during routine monitoring in a research laboratory setting. For all analyses, a P value of less than 0.05 was considered to be statistically significant. All statistical analysis was performed by using SigmaPlot 12.3 (Systat Software, San Jose, CA).

Results

Animal numbers. There were a total of 124 anesthetic events, including 14 using isoflurane and 110 using injectable KX or KXA. For all 14 isoflurane events, mice were fully anesthetized and recovered from anesthesia. Among the 110 anesthesia sessions achieved by using injectable agents, 107 mice were anesthetized sufficiently to receive CRI anesthesia, of which 99 mice recovered from anesthesia. Induction for CRI. Overall, 3 of 110 mice (one induced with KXA[0.1], 2 induced with KXA[0.5]) did not lose the righting reflex after the initial injection; these mice did not undergo CRI and were excluded from further analysis. All of the remaining mice (n = 107) lost the righting reflex within 10 min, and the CRI was initiated at 10 min after injection of the induction dose. The average times from induction to loss of the righting reflex, spontaneous movement, and the PWR for the 99 mice that recovered from anesthesia are summarized in Table 1. CRI groups. Table 2 summarizes the survival and average durations of recumbency, continuous immobility, and continuous surgical anesthesia for all anesthetic groups. Mice that died during anesthesia and CRI anesthetic regimens that resulted in greater than 50% mortality were excluded from further analysis due to unacceptable mortality rates and insufficient animal numbers per group. Two-way ANOVA showed that the induction and CRI regimens both had significant effects on durations of recumbency (P < 0.001 for both induction and CRI regimens), immobility (P < 0.001 and P = 0.002, respectively), and surgical anesthesia (P < 0.001 for both), but the interaction of induction regimen and CRI dose was nonsignificant. Mortality and durations of recumbency, continuous immobility, and continuous surgical anesthesia (Table 2) were greater in groups that received

acepromazine during induction and were increased at higher CRI doses of ketamine and xylazine. Female mice (Table 2) in which anesthesia was induced by using KXA(0.5) and that received CRI of 40 mg/kg/h ketamine, 4 mg/kg/h xylazine had significantly (P < 0.001) shorter durations of recumbency, immobility, and surgical anesthesia than did male mice anesthetized with the same dosing regimen. Figure 2 illustrates the proportion of mice that remained immobile or at a surgical plane for continuous durations of 5, 30, 60, and 90 min after each intraperitoneal CRI anesthetic regimen (excluding female mice, mortalities, and anesthetic regimens that resulted in 50% mortality or greater) and selected bolus redosing regimens from a previous study.19 χ2 analysis was performed to detect significant differences across all CRI dosing regimens for each indicated duration of immobility or surgical anesthesia; significant differences were detected for immobility durations of 5 min (P < 0.05), 60 min (P < 0.01), and 90 min (P < 0.01), whereas significant differences were detected for surgical-plane durations of 60 min (P < 0.01) and 90 min (P < 0.05). For comparison, Figure 2 also includes previous data19 from male C57BL/6J mice anesthetized with KXA(1.0) for the first period of surgical anesthesia prior to being redosed on emergence from a surgical plane. Mice (37 of 79; 46.8%) that reached a surgical plane of anesthesia demonstrated intermittent positive PWR or alternated between positive and negative PWR during CRI, such that the total duration of surgical anesthesia was longer than the continuous duration. This pattern typically occurred near the beginning and end of an anesthetic event. However some mice regained a positive PWR at several time points within a longer duration of surgical anesthesia; the times at which this response occurred varied across animals and anesthetic regimens. Because the goal of this study was to improve upon bolus redosing by developing a CRI to provide a continuous surgical plane of anesthesia, only the longest continuous duration of surgical anesthesia for each trial was used for further analysis. Anesthetic deaths. A total of 8 mice died during CRI anesthesia (Table 2). The anesthesia regimens of induction with KXA(0.1) followed by CRI of 120 mg/kg/h ketamine and induction with KXA(0.5) followed by CRI of 80 mg/kg/h ketamine were tested in 5 or fewer mice and therefore were not included in the χ2 analysis for mortality or other statistical analyses. Mortality tended to increase with increasing induction dose and increasing CRI dose, although χ2 analysis did not reveal any significant differences in mortality across all CRI dosing regimens. All deaths occurred at least 1 h into the anesthetic event; the average time of death after induction was 85.4 ± 6.9 min (range, 60 to 125; n = 8). Prior to death, all mice exhibited a decreased respiratory rate (less than 100 breaths per minute), abnormal respiratory pattern, and agonal breathing characterized by gasping breaths. Postmortem examinations of deceased mice revealed no gross abnormalities such as splenic or hepatic lacerations from intraperitoneal cannulation. In addition, one mouse anesthetized with KXA(0.5) and CRI 40 mg/kg/h, 4 mg/kg/h xylazine had a precipitous decrease in respiratory rate at 105 min after induction (that is, 5 min after CRI had been discontinued) and then resumed normal breathing and recovered from anesthesia. This bradypnea lasted for approximately 5 min, and the respiratory rate reached as low as 40 breaths per minute before recovering over the next 5 min; no physical or pharmacologic interventions were provided to the mouse. No mortalities or other adverse events occurred after recovery from anesthesia. Isoflurane. Mortality and average durations of recumbency, continuous immobility, and continuous surgical plane for male (n = 8) and female (n = 6) mice anesthetized with isoflurane are 551

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Table 1. Times (mean ± SE [range]) to losses of righting reflex, spontaneous movement, and pedal withdrawal reflex during intraperitoneal CRI in the 99 mice that recovered from anesthesia Time (min) to loss of Induction regimen (n = 33 in each group)

Righting reflex

Spontaneous movement

Pedal withdrawal reflex

Ketamine–xylazine

2.7 ± 0.2 (1.5–6.5, n = 33)

7.8 ± 0.7 (4–17.5, n = 27)

26.6 ± 3.8 (10–85, n = 22)

Ketamine–xylazine–acepromazine (0.1 mg/mL)

2.4 ± 0.1 (0.5–4.8, n = 33)

10.0 ± 2.7 (2.3–85, n = 30)

20.4 ± 1.7 (10–40, n = 28)

Ketamine–xylazine–acepromazine (0.5 mg/mL)

2.3 ± 0.1 (1.8–3.3, n = 33)

6.7 ± 0.4 (4.0–16.0, n = 33)

27.7 ± 2.8 (10–65, n = 28)

Table 2. Survival and duration of continuous recumbency, immobility, and surgical anesthesia among anesthetic regimens Duration (min; mean ± SE [range]) among the 99 mice that recovered Inductiona

CRI (mg/kg/h)

Survivalb

Recumbency

Immobility

Surgical anesthesia

Groups analyzed statistically KX

KXA(0.1)

KXA(0.5)

20 K

8/8

53.3 ± 2.0(43–59)

36.7 ± 2.5 (24–50)

11.5 ± 5.0 (0–41)

40 K

9/9

59.4 ± 6.5 (39–88)

30.5 ± 10.1 (0–68)

19.9 ± 8.2 (0–53)

80 K

8/8

115.6 ± 12.2 (41–154)

78.5 ± 18.4 (0–130)

52.9 ± 14.1 (0–100)

40 K, 4 X

8/8

123.8 ± 7.8 (78–148)

96.2 ± 11.5 (50–135)

52.0 ± 14.0 (0–120)

20 K

8/8

69.4 ± 8.1 (49–122)

52.0 ± 7.6 (27–99)

33.3 ± 9.0 (0–75)

40 K

8/8

103.2 ± 11.3 (41–143)

77.0 ± 13.3 (0–116)

51.9 ± 11.1 (0–93)

80 K

7/8

126.5 ± 14.7 (41–150)

89.3 ± 16.1 (0–122)

69.3 ± 14.4 (0–110)

40 K, 4 X

8/8

115.0 ± 11.8 (50–153)

80.8 ± 18.9 (0–145)

61.9 ± 18.2 (0–125)

20 K

9/9

73.3 ± 3.3 (52–82)

54.2 ± 2.9 (39–67)

23.2 ± 6.3 (0–45)

40 K

7/8

107.1 ± 9.3 (60–134)

79.8 ± 9.4 (33–104)

40 K, 4 X

7/8

145.3 ± 6.7 (125–172)c

126.9 ± 6.5 (107–151)c

35.9 ± 14.8 (0–98) 84.4 ± 14.1 (25–125)c

Groups not included in statistical analysis KXA(0.5)

80 K

2/5

173.4 ± 0.6 (173–174)

146.8 ± 4.8 (142–152)

130.0 ± 0.0 (130–130)

KXA(0.1)

120 K

2/4

185.8 ± 20.5 (165–206)

157.3 ± 22.3 (135–180)

142.5 ± 17.5 (125–160)

Female mice, KXA(0.5)

40 K, 4 X

8/8

80.4 ± 7.9 (49–110)c

62.9 ± 8.6 (35–98)c

29.0 ± 9.9 (0–75)c

Male mice, isoflurane 4%

Isoflurane 2%

8/8

102.4 ± 1.1 (99–108)

95.4 ± 0.7 (94–100)

83.1 ± 3.9 (58–93)

Female mice, isoflurane 4%

Isoflurane 2%

6/6

94.5 ± 1.7 (91–101)

92.5 ± 1.7 (89–99)

83.2 ± 1.0 (81–87)

A, acepromazine; CRI, continuous-rate infusion; K, ketamine; X, xylazine induction, ketamine dose was 80 mg/kg; xylazine dose was 8 mg/kg; and acepromazine dose was 0, 0.1, or 0.5 mg/kg. bNo. of mice that recovered after losing righting reflex/total no in group; overall, n = 107. cDuration differed significantly (P < 0.05; 2-way ANOVA) between male and female mice. aFor

included in Table 2. All isoflurane-anesthetized mice (n = 14) survived anesthesia; 13 of the 14 mice reached surgical anesthesia quickly and remained there until isoflurane was discontinued. The remaining mouse reached a surgical plane of anesthesia, had several positive PWR responses in the middle of the procedure and then returned to a surgical plane until the isoflurane was discontinued; this behavior resulted in a continuous surgical plane of anesthesia of 58 min. Isoflurane was discontinued at 90 min, and all mice recovered quickly afterward, resulting in continuous surgical anesthesia of less than 90 min. Mice took an average of 6.9 ± 1.0 min (range, 3 to 14; n = 14) to regain the righting reflex after the isoflurane was stopped; this duration is consistent with previously published data on emergence from isoflurane anesthesia in C57BL/6J mice.39 Autonomic responses to noxious stimulus. The average heart and respiratory rates of mice at surgical anesthesia under isoflurane were 526 ± 52 (range 383-622, n = 197 time points) and 67 ± 25 (range 28-150, n = 203 time points), respectively. The average heart and respiratory rates of surviving mice at surgical anesthesia

under KXA anesthesia (including all mice induced with KXA[0.1] and KXA[0.5]) were 300 ± 36 (range 186-371, n = 620 time points) and 209 ± 20 (range 150-275, n = 625 time points), respectively. These parameters were recorded before and after a noxious stimulus and then compared to determine whether mice exhibited detectable autonomic responses (Table 3). Mice anesthetized with 2% isoflurane demonstrated a detectable increase in respiratory rate for 79% of time points during surgical anesthesia. Autonomic responses under isoflurane were recorded only while mice were at a surgical plane of anesthesia, given that mice entered a surgical plane very quickly after losing the righting reflex and exited surgical anesthesia shortly prior to the return of this reflex. In comparison, mice anesthetized with KX or KXA showed detectable increases in either heart or respiratory rate at fewer than 45% of time points, regardless of the depth of anesthesia.

Discussion

The primary goal of this study was to investigate potential dosing regimens for intraperitoneal CRI anesthesia by using

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Figure 2. Proportion (%) of mice that maintained continuous immobility and surgical anesthesia for at least (A) 5 min, (B) 30 min, (C) 60 min, or (D) 90 min after each intraperitoneal CRI anesthetic regimen (excluding female mice, mice that died, and anesthetic regimens resulting in 50% mortality or greater) and selected bolus redosing regimens from reference 19. Data for the bolus redosing group are from reference 19 for the recommended protocol (that is, induction with KXA[1.0] mg/kg, with redosing after emergence from surgical anesthesia) and represent data for surgical anesthesia only (immobility data unavailable).

induction and maintenance combinations of ketamine, xylazine, and acepromazine in C57BL/6J mice to safely maintain and extend the duration of immobility or surgical anesthesia. A previous study by our group found that bolus redosing of injectable anesthetics does not safely maintain a continuous surgical plane of anesthesia for prolonged procedures and that mice demonstrate a highly variable response to injectable anesthetics.19 In the present study, we found that, compared with bolus redosing, intraperitoneal CRI maintained a longer duration of continuous surgical anesthesia with comparably lower mortality. These dosing regimens may serve as guidelines for intraperitoneal CRI anesthesia in mice and should be tailored to specific mouse strains, animal models, procedures, and experimental needs.

We identified 2 dosing regimens that were most successful in achieving a continuous surgical plane of anesthesia for an extended period of time with minimal mortality: (1) induction with KXA(0.1) and CRI of 100% of the initial ketamine dose hourly and (2) induction with KXA(0.5) and CRI of 50% of both the initial doses of ketamine and xylazine. Each of these regimens maintained a continuous surgical plane of anesthesia for 30 min in at least 86% of mice and for 60 min in approximately 71% of mice with 12.5% mortality. In addition, induction with KXA(0.5) and CRI of 50% of the initial ketamine and xylazine doses maintained continuous surgical anesthesia for 90 min in 57% of mice. Intraperitoneal CRI dosing regimens maintained a longer duration of continuous surgical anesthesia compared with bolus redosing after reemergence from surgical anesthesia. 553

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Table 3. Percentages of time points at which mice exhibited detectable increases in heart rate (greater than 10 bpm) and respiratory rate (more than 5 breaths per minute) after the noxious stimulus. Nonsurgical plane of anesthesia

Surgical plane of anesthesia

Increased heart rate

Increased respiratory rate

Increased heart rate

Isoflurane

not applicable

not applicable

28% (32/116)

Increased respiratory rate 79% (94/119)

KX

12% (17/140)

33% (50/151)

2% (5/247)

33% (83/251)

KXA(0.1)

11% (14/131)

39% (53/137)

4% (17/436)

41% (188/458)

KXA(0.5)

5% (8/166)

37% (65/177)

6% (17/283)

45% (154/342)

A, acepromazine (0.1 or 0.5 mg/kg); K, ketamine (80 mg/kg); X, xylazine (8 mg/kg). Data are given as % of time points at which rate was increased (no. of points with increased rate/total no. of time points assessed).

For comparison, our group previously found that induction with KXA(1.0) achieved continuous surgical anesthesia for 30 min in only 27% of mice and for 60 min in just 10% of mice; none of the bolus-redosed mice experienced 90 min of continuous surgical anesthesia.19 Four dosing regimens maintained extended periods of continuous immobility and thus might be useful for imaging procedures requiring prolonged immobility but not necessarily the depth of surgical anesthesia: (1) induction with KX and CRI of 50% the initial ketamine and xylazine doses hourly (0% mortality); (2) induction with KX and 100% of the initial ketamine dose hourly (0% mortality); (3) induction with KXA(0.1) and CRI of 50% the initial ketamine dose hourly (0% mortality); and (4) induction with KXA(0.5) and CRI of 50% the initial ketamine hourly (12.5% mortality). In surviving mice, these 4 dosing regimens maintained continuous immobility for 30 min in 100%, 75%, 88%, and 100% of mice, respectively; 60 min in 75%, 75%, 75%, and 100% of mice; and for 90 min in 63%, 63%, 50%, and 100% of mice. Anesthetic regimens with lower CRI doses were unsuccessful in keeping mice anesthetized for extended periods, and many of these CRI sessions were discontinued prior to 90 min because mice regained spontaneous movement and the righting reflex. The lowest dose CRI (20 mg/kg/h ketamine) failed to maintain recumbency in mice throughout CRI regardless of induction dose. Induction with KX without acepromazine was ineffective at maintaining recumbency at CRI doses of 20 or 40 mg/ kg/h ketamine but, at CRI regimens of 40 mg/kg/h ketamine, 4 mg/kg/h xylazine or 80 mg/kg/h ketamine maintained immobility for at least 60 min in 75% of mice and a surgical plane of anesthesia for at least 30 min in 75% of mice. Several reports indicate that KX anesthesia has a narrow therapeutic range: a single injection of ketamine and xylazine at published doses does not achieve surgical anesthesia in mice, whereas higher doses can result in considerable mortality.2,3,5,6,9 In light of the variability and relatively short duration of surgical plane of anesthesia after KX induction, other anesthetic regimens should be used when more than 30 min of surgical anesthesia is needed. Across all of the CRI dosing regimens and for a single mouse anesthetized with isoflurane, some animals demonstrated intermittent positive PWR responses or alternated between positive and negative PWR. This pattern occurred most frequently near the beginning and end of the anesthetic event, consistent with mice entering or leaving a surgical plane of anesthesia. However, some mice briefly regained PWR for several time points in the middle of a long duration of surgical anesthesia, such that total cumulative time at surgical anesthesia was significantly greater than the longest single episode of continuous surgical anesthesia. Such inconsistent reflexes may indicate periods of decreased anesthetic depth, in the case of positive responses at the middle of an anesthetic event, or these time points might

represent nadirs in plasma drug levels after the induction dose had largely been eliminated but before CRI had achieved stable drug levels. Alternatively, the positive responses might reflect the transient destabilization of the anesthetic state by noxious stimuli.34 Either way, these responses highlight the importance of frequent monitoring during procedures to ensure sufficient anesthetic depth. Compared with their lower-dose counterparts, mice anesthetized with higher induction and CRI doses had longer anesthetic recovery times. Induction with KXA(0.5) and CRI of 40 mg/kg/h ketamine, 4 mg/kg/h xylazine was successful in maintaining surgical anesthesia with low mortality, but mice were recumbent for an average of 145 min (that is, the righting reflex returned 45 min after CRI ended). Previous studies of KXA single-injection anesthesia similarly found that mice did not regain ambulation until 2 h after the initial injection, consistent with the recovery times observed after intraperitoneal CRI anesthesia using KXA.3,19,24 For comparison, mice anesthetized with isoflurane all regained the righting reflex within 15 min of discontinuation of isoflurane. Mortality was associated with higher anesthetic induction doses, inclusion of acepromazine in the anesthetic induction, and higher CRI doses. Decreasing the initial dose acepromazine from 1.0 mg/kg to 0.5 or 0.1 mg/kg markedly reduced mortality rates. No deaths occurred after induction with KX or anesthesia with isoflurane, and the mortality rate associated with recommended intraperitoneal CRI dosing regimens was 12.5%. This mortality rate is similar to those that occur during bolus redosing after emergence from surgical anesthesia (that is, interrupted surgical plane) as recommended in our group’s previous study; in comparison, bolus redosing prior to emergence from surgical anesthesia (that is, continuous surgical plane) resulted in 50% mortality.19 Deaths most likely were related to respiratory depression and hypoxia, which are reported complications of KXA anesthesia.2,12,19 CRI was discontinued as soon as mice exhibited agonal breathing. Of those mice that developed abnormal breathing patterns (n = 9), all but one died. This remaining mouse recovered from anesthesia after experiencing a precipitous decrease in respiratory rate, to fewer than 40 breaths per minute, shortly after CRI had been discontinued. We speculate that this mouse experienced respiratory depression under anesthesia but was able to recover because plasma drug levels began decreasing after CRI ended. Other than discontinuing the CRI when agonal breathing was observed, we did not intervene. In a previous study, 43% of agonal mice given atipamezole went on to recover from anesthesia.19 We anticipate that with close anesthetic monitoring, the mortality rate and recovery time associated with CRI anesthetic regimens can be reduced with interventions such as oxygen supplementation and the administration of an α2-adrenergic receptor antagonist like atipamezole.

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A limitation of the current study is that the noxious stimulus likely was less intense than surgical stimuli, and the intensity of the stimulus may affect the response to anesthesia.28,47 A more intense stimulus during intraperitoneal CRI anesthesia might lighten the plane of anesthesia or shorten the duration of surgical anesthesia. As such, when surgical or other painful procedures are to be performed, increased anesthetic doses or additional analgesics may be needed to ensure a sufficient depth of anesthesia. Conversely, by keeping the animal at a lighter plane of anesthesia, a more intense stimulus might improve survival and shorten recovery times for intraperitoneal CRI anesthesia. Therefore these recommended dosing regimens should be used as starting points for future studies and modified as needed to accommodate specific experimental models. In combination, KXA provides multimodal anesthesia sufficient for surgical manipulation. Ketamine is an N-methylD-aspartate receptor antagonist and dissociative anesthetic with hypnotic, sedative, and analgesic properties and a serum halflife of 13 min when administered intraperitoneally in mice.15,24,26 This relatively short duration of action compared with other anesthetics makes ketamine an intriguing drug candidate for CRI infusion in mice, in addition to its use via intravenous CRI in other animal species.44,48 Xylazine is an α2-adrenergic receptor agonist with sedative and analgesic properties.11 The elimination half-life of xylazine in mice is unknown but presumably is similar to or shorter than the half-life in rats, which has been reported as 1 to 2 h.43,45 Xylazine has the potential to be reversed with α2-adrenergic receptor antagonists such as atipamezole. In the current study, induction with KX served both as a loading dose—CRI alone would take 3 to 5 drug half-lives to reach steady-state blood levels44—and achieved sufficient anesthesia to allow placement of the intraperitoneal catheter and physiologic instrumentation. Acepromazine is a phenothiazine derivative sedative that provides additional sedation and retards movement but has no analgesic properties.11,40 The half-life of acepromazine has not been evaluated in mice but is relatively long in other species—for example, 7.1 h in dogs and 2.6 h in horses.16,17 Because of its long half-life, we did not include acepromazine in the CRI regimens. Published doses for inducing anesthesia with KXA in mice vary widely, ranging from 50 to 100 mg/kg ketamine, 3 to 10 mg/kg xylazine, and 1 to 3 mg/kg acepromazine.2,19 Previous studies have reported that a single injection provides an average of 40 to 60 min of surgical anesthesia: 54 min for 100 mg/ kg ketamine, 20 mg/kg xylazine, and 3 mg/kg acepromazine;2 40 min for 100 mg/kg ketamine, mg/kg xylazine, and 3 mg/ kg acepromazine;3 and 45 min for 80 mg/kg ketamine, 8 mg/ kg xylazine, and 1 mg/kg acepromazine.19,24 Average anesthesia times are highly variable, even within a single anesthetic regimen. Across studies, differences in mouse strain, sex, and methods for determining surgical plane also may account for variability in anesthetic response and recommended dosing. Drug pharmacokinetics, efficacy, and doses required to achieve surgical anesthesia may vary significantly with sex, age, strain, genotype, nutrition, health status, environmental factors, and experimental procedure.13,35,43,49 Similar to previous studies of injectable anesthetics in mice, we also found a high degree of interindividual variability in responses to anesthesia, even within treatment groups.2,19 Higher doses may provide longer periods of anesthesia but often result in significant mortality.2,19 During our investigation, we found that lower induction doses, particularly for regimens including acepromazine, may be needed when combined with a CRI.

In the current study, CRI began 10 min after administration of the induction dose; at this time, mice were expected to be in dorsal recumbency and immobilized but not yet at a surgical plane of anesthesia. Initiating the CRI early in the course of the anesthetic event was done in consideration for the needs of the researcher and experimental procedures. The time at which the CRI was initiated represents a potential variable to consider regarding intraperitoneal CRI dosing regimens—for example, a higher induction dose of KXA could be administered or CRI could be started later in the procedure. In addition, the rate of drug infusion during CRI could be adjusted to vary the depth of anesthesia according to the purposes of experimental manipulation or on the basis of assessment of the animal’s physiologic parameters (for example if the depth of anesthesia is determined to be too light or too deep). Modern syringe pumps allow operators to quickly and easily adjust the rate of drug infusion, although changes in anesthetic depth might occur more slowly with intraperitoneal than with intravenous CRI. Other anesthetics that are administered either intravenously or as intraperitoneal boluses are potential candidates for intraperitoneal CRI, either alone or in combination. Previous studies48,49 reported the use of CRI of ketamine, medetomidine, and atropine; fentanyl, fluanisone, and midazolam; and propofol, fentanyl, and midazolam to maintain anesthesia in mice; however, the duration of anesthesia was not evaluated, and bolus redosing of drugs occurred whenever mice exhibited a positive PWR.48,49 A group of female C57BL/6J mice were anesthetized by using KXA(0.5) and CRI of 50% of the initial doses of ketamine and xylazine hourly, one of the intraperitoneal CRI dosing regimens most successful at providing continuous surgical anesthesia in male C57BL/6J mice. In general, recommendations for anesthetic dosing do not differ between male and female mice.11,26,40 Surprisingly, we found significantly shorter durations of recumbency, immobility, and surgical anesthesia in female mice compared with male mice using the same anesthetic regimen. The male and female mice in this study were similar in age, but female mice weighed considerably less (mean, 17.9 g) than did male mice (mean, 26.5 g). These differences in body mass might influence the pharmacokinetics of anesthetic drugs.25 In addition, sex-associated differences in response to anesthetics might be attributed to other sexual dimorphisms, including metabolism, body composition, and hormonal environment.7,8,22,30 Pilot experiments, which were performed in male mice, led us to select lower induction doses of acepromazine for this study, and potential sex predilection in responses to different doses of acepromazine cannot be ruled out. Although further investigation is needed prior to drawing conclusions regarding these unexpected sex-associated differences, the current findings should be considered when developing future intraperitoneal CRI dosing regimens for female mice. We sought to examine whether autonomic changes in the heart or respiratory rate are useful indicators of anesthetic depth in mice during KXA and isoflurane anesthesia. We found that under KXA anesthesia, neither heart rate nor respiratory rate reliably increased in response to a noxious stimulus, even when mice were not at a surgical plane anesthesia, as indicated by a positive PWR. Under isoflurane anesthesia, respiratory rate increased after a noxious stimulus, but heart rate was largely unchanged. Subjectively, some mice demonstrated a brief change in depth of respiration without a change in respiratory rate as measured by thoracic excursions; we interpreted this pattern as an increase in respiratory effort. Approximately one third of mice 555

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demonstrated increased respiratory effort at least once during the anesthetic event, and these observations were consistent across anesthetic regimens. Without the use of a sophisticated method such as anemometry or capnography, we were unable to objectively quantify or characterize this change in respiration to determine whether it represented a physiologic response to a noxious stimulus. Ultimately, counting of chest excursions is more representative of anesthetic monitoring that would be practiced in routine laboratory procedures. Somatic responses to noxious stimuli are mainstays for determining surgical tolerance under anesthesia.2,3,14,21,46 The PWR is accepted as one of most sensitive and useful indicators of surgical anesthesia in rodents and other veterinary species.2,3 More intense noxious stimuli, such as surgical manipulation, were not evaluated in the current study and might prompt different autonomic responses. The depth of surgical anesthesia can be characterized further according to the progressive loss of various reflexes and autonomic functions.14,21,41 Autonomic reflexes, such as transient increases in heart rate or respiratory rate after a surgical or noxious stimulus, become blunted at very deep planes of surgical anesthesia.4 The loss of autonomic responses suggests that the patient has become deeply anesthetized and may be an indication for intervention or adjustment of the anesthetic depth before the patient progresses to anesthetic overdose.4,21,41 One study found that mice under KXA anesthesia and given a surgical stimulus demonstrated the smallest increases in heart rate, respiratory rate, and blood pressure compared with those after other injectable combinations.2 However, that study evaluated physiologic parameters in response to surgical stimuli only when mice were already at a surgical plane of anesthesia. A recent study evaluated pigs premedicated with ketamine, xylazine, and buprenorphine and then anesthetized with isoflurane; changes in heart rate and blood pressure after a noxious stimulus were not clinically detectable and therefore were not sensitive indicators of anesthetic depth.20 According to our findings, autonomic changes in the heart or respiratory rate in response to testing the PWR are not clinically detectable and are unreliable indicators of analgesia and anesthetic depth in KXA-anesthetized mice. Autonomic responses were blunted even when mice were not under surgical anesthesia.4 KXA anesthesia is associated with respiratory depression, cardiovascular depression, hypotension, and consequent compensatory responses, all of which may blunt or mask autonomic changes in physiologic parameters.2,5,12,40 Although somatic responses such as pedal withdrawal are commonly used to gauge anesthetic depth, KXA anesthesia might blunt autonomic increases in the heart or respiratory rate but leave spinal reflexes intact. Furthermore, these animals might be anesthetized sufficiently to eliminate awareness, memory, and pain perception but not a reflex response to a toe pinch.1,31-33 Autonomic responses are often used to monitor anesthetic depth in patients under neuromuscular blockade or paralytics, when somatic responses are inhibited.37 Our findings suggest that our ability to accurately assess anesthetic depth during neuromuscular blockade may be limited even further when combined with KX-based anesthesia, given that neither somatic nor autonomic responses can be relied on to indicate when a mouse is not under surgical anesthesia. As anticipated, isoflurane was more effective at maintaining a continuous surgical plane than was intraperitoneal CRI anesthesia and, owing to its safety, reliability, and rapid control of anesthetic depth, is recommended over injectable anesthetics whenever feasible. Specifically, 2% isoflurane in mice reliably maintains a surgical plane of anesthesia, as defined by the

absence of a somatic response to a noxious stimulus. During isoflurane anesthesia, the heart rate was largely unaffected by the noxious stimulus, but the respiratory rate increased in response to a noxious stimulus approximately 80% of the time, indicating that some autonomic reflexes remain intact during surgical anesthesia. Therefore, autonomic increases in respiratory rate may be a useful indicator of excessive anesthetic depth during isoflurane anesthesia. In conclusion, we found that our recommended dosing regimens for intraperitoneal CRI anesthesia were successful in providing prolonged, continuous surgical anesthesia and immobility in C57BL/6J mice, compared with bolus redosing of KXA injectable anesthesia—although isoflurane still remains the anesthetic of choice. Furthermore, we found that physiologic changes in the heart and respiratory rates in response to a noxious stimulus were unreliable indicators of anesthetic depth during KXA anesthesia. These recommendations provide important starting points for the further development of intraperitoneal CRI anesthesia protocols for mice that can be tailored to specific animal models, procedures, and experimental needs.

Acknowledgments

This work was supported in part by funding from the Office of the Vice Provost for Research at the University of Pennsylvania. MBK was funded by NIH grants GM107117 and GM088156. These contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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