Journal of the American Association for Laboratory Animal Science Copyright 2014 by the American Association for Laboratory Animal Science

Vol 53, No 3 May 2014 Pages 290–300

Effects of Anesthesia with Isoflurane, Ketamine, or Propofol on Physiologic Parameters in Neonatal Rhesus Macaques (Macaca mulatta) Lauren D Martin,1,* Gregory A Dissen,2 Matthew J McPike,1 and Ansgar M Brambrink3 Isoflurane, ketamine, and propofol are common anesthetics in human and nonhuman primate medicine. However, scant normative data exist regarding the response of neonatal macaques to these anesthetics. We compared the effects of isoflurane, ketamine, and propofol anesthesia on physiologic parameters in neonatal rhesus macaques. Neonatal rhesus macaques (age, 5 to 7 d) were exposed to isoflurane (n = 5), ketamine (n = 4), propofol (n = 4) or no anesthesia (n = 5) for 5 h. The anesthetics were titrated to achieve a moderate anesthetic plane, and heart rate, blood pressure, respiratory rate, end tidal carbon dioxide, oxygen saturation, and temperature were measured every 15 min. Venous blood samples were collected to determine blood gases and metabolic status at baseline, 0.5, 2.5, and 4.5 h after induction and at 3 h after the end of anesthesia. Compared with ketamine, isoflurane caused more hypotensive events and necessitated the administration of increased volumes of intravenous fluids to support blood pressure throughout anesthesia; no significant differences were observed between the isoflurane and propofol groups for these parameters. In addition, isoflurane resulted in a significantly shorter average time to extubation, compared with both ketamine and propofol. Due to supportive care, other physiologic variables remained stable between anesthetic regimens and throughout the 5-h exposure. These data improve our understanding of the effects of these 3 anesthetics in neonatal rhesus macaques and will aid veterinarians and researchers as they consider the risks and benefits of and resources required during general anesthesia in these animals. Abbreviations: pvCO2, pCO2 in venous blood; pvO2, pO2 in venous blood.

Neonatal rhesus macaques (Macaca mulatta) have been used in biomedical research as animal models for studies addressing infectious diseases (including those involving SIV1,45-49,51 and SHIV39), cognitive and behavioral development,12,15,23,27 toxicology,29,43 nutrition,5,14,32,33,40 respiratory distress in preterm neonates,41 and the neuroapoptotic effects of various anesthetic agents.8,9,13 Within nonhuman primate breeding programs, neonates often present with diarrhea, dehydration, and trauma necessitating clinical treatment. Anesthesia of neonatal macaques frequently is required both in the context of research protocols and in the clinical setting; however there is a paucity of normative data, especially physiologic data, that describe the response of this age subset of macaques to general anesthetics used routinely in humans and adult nonhuman primates. Isoflurane, ketamine and propofol are well-established pediatric anesthetics7-9,13 in human medicine and routinely are used in nonhuman primate medicine.6,19,20,30,37,42 Isoflurane is a halogenated ether, like desflurane and sevoflurane, that is used as an inhalational anesthetic.16,17 In the brain, isoflurane acts predominantly at GABAA receptors (agonist) and to a lesser degree on NMDA receptors (antagonist), which is thought to explain its anesthetic action, although the precise mechanism of action is not fully understood. Systemically, isoflurane causes direct vasodilation through inhibition of calcium mobilization and myofilament calcium sensitivity in vascular smooth muscle.2,3

Received: 29 Aug 2013. Revision requested: 30 Sep 2013. Accepted: 05 Nov 2013. 1Division of Comparative Medicine and 2Division of Neuroscience, Oregon National Primate Research Center, Beaverton, Oregon; 3Anesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland, Oregon. *Corresponding author. Email: [email protected]

Clinically, isoflurane has been associated with dose-dependent hypotension in human pediatric medicine.52 Isoflurane is eliminated predominately by direct exhalation; only a small percentage of the inhalant is metabolized in the liver, thus isoflurane is rarely associated with hepatotoxicity.19 Ketamine is a phencyclidine analog that produces ‘dissociative anesthesia’ characterized by a cataleptoid state with unconsciousness and some somatic analgesia.7 Ketamine acts as a noncompetitive antagonist of NMDA receptors in the brain and spinal cord and as an agonist at opioid sigma and α- and β-adrenergic receptors, and it blocks reuptake of catecholamines.7 In humans, ketamine undergoes biotransformation in the liver, via the cytochrome p450 system, with norketamine as a major metabolite, and is excreted by the kidneys.7,25 Propofol (2,6-diisopropylphenol) is a phenolic derivative with sedative and hypnotic properties.44 It acts through positive modulation of the inhibitory function of the neurotransmitter GABA through GABAA receptors44 and sodium channels.26 In humans, including young children, propofol has been associated with transient dose-dependent hypotension and decreased heart rate, particularly after bolus administration.28 Propofol undergoes hepatic metabolism to inactive metabolites; however some extrahepatic metabolism is likely, and these metabolites are excreted by the kidneys.4 The purpose of the current study was to determine the effects of isoflurane, ketamine, and propofol anesthesia on diverse physiologic parameters in neonatal rhesus macaques. According to evidence from human clinical literature, we hypothesized that (1) general anesthesia maintained with isoflurane, titrated to a moderate surgical plane, in neonatal macaques would be associated with more pronounced systemic hypotension than that maintained by using ketamine or propofol at equipotent

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doses and that (2) neonatal macaques would recover faster after a 5-h general anesthesia using isoflurane compared with ketamine or propofol. Additional physiologic parameters were monitored continuously and recorded throughout anesthesia to ensure animal wellbeing and the maintenance of physiologic homeostasis.

Materials and Methods

Animals. All animals were born and housed at the Oregon National Primate Research Center, an AAALAC-accredited facility. All animal procedures were conducted at this facility in accordance with the Public Health Services Policy on Humane Care and Use of Laboratory Animals and were approved by the IACUC. This study used 18 (10 male, 8 female) healthy Indianorigin neonatal rhesus macaques (Macaca mulatta; age, 5 to 7 d; weight, 411 g to 680 g; gestational days, 156 to 176 d) that were randomly assigned to either an isoflurane (n = 5), ketamine (n = 4), propofol (n = 4) or no anesthesia (n = 5) treatment group. SPF (SIV, Macacine herpesvirus 1, simian T lymphotropic virus, simian type D retrovirus) and nonSPF neonates were used for this study, because viral status was not a limiting factor for animal assignment. Neonates were born naturally to time-mated breeding program dams or outdoor group-housed dams that had not received any sedatives or anesthetics after the midsecond trimester of gestation. Neonates were not sedated between birth and the initiation of the experimental anesthesia exposure. Anesthesia protocol. Dams were sedated with an intramuscular injection of ketamine (5 to 10 mg/kg; Ketathesia, Butler Schein Animal Health, Dublin, OH). Unsedated neonates were removed from the dam and transported to the Surgical Services Unit. Intravenous access was established by placement of a 22gauge catheter (Introcan Safety, B Braun Medical, Melsungen, Germany) in the cephalic or saphenous vein prior to induction of anesthesia. Venous blood (0.3 mL) was collected through the catheter for baseline point-of-care analysis (CG4+ and EC8+ cartridges, iSTAT, Abbott Point of Care, Princeton, NJ). Parameters measured included sodium, potassium, chloride, total CO2, BUN, glucose, hematocrit, pH, pvCO2, bicarbonate, base excess, anion gap, hemoglobin, pvO2, oxygen saturation, and lactate. Isoflurane (Piramal Healthcare, Andhra, India) anesthesia was induced at 4% to 5% in 100% oxygen administered via mask until the animal was sedated sufficiently (absence of withdrawal reflexes) to allow endotracheal intubation. Ketamine anesthesia was induced by using a 20 mg/kg IV bolus followed by additional intravenous boluses of ketamine until the animal was sedated sufficiently to allow endotracheal intubation. A maintenance infusion rate of 20 to 55 mg/kg/h was initiated after successful airway management. Propofol (APP Pharmaceuticals, Schaumburg, IL) anesthesia was induced with a 2 mg/kg IV bolus followed by additional intravenous boluses of propofol to allow endotracheal intubation. A maintenance infusion rate of 24 to 37 mg/kg/h was initiated after intubation. Injectable maintenance anesthetics were administered via a constant-rate infusion pump (Medfusion 3500, Smiths Medical MD, St Paul, MN). Time 0 for the experiment was defined as the initial exposure to the anesthetic (that is, initial application of mask isoflurane, intravenous injection of ketamine or propofol or 2 mL IV saline bolus in the control group). Animals were intubated via direct visualization using a bulb laryngoscope (Welch Allyn, Skaneateles Falls, NY) with a Miller 0 blade (Welch Allyn, Skaneateles Falls, NY) or assisted by a semirigid fiber-optic endoscope (Karl Storz America, El Segundo, CA) with a 2.0-mm (inner diameter)

noncuffed endotracheal tube (Mallinckrodt, Hazelwood, MO). Time to intubation was defined as the period from time 0 until successful intubation of the macaque. Successful intubation was confirmed by visualizing an end-tidal CO2 waveform on the capnograph (SurgiVet Monitor; Smith Medical, Wankesha, WI and Capnomac; Datex Ohmeda, Madison, WI), auscultation of the lungs, and adequate bilateral chest rise. Additional evidence was provided by the oxygen saturation monitor (peripheral oxygen saturation, greater than 98%; SurgiVet Monitor; Smith Medical). After intubation, macaques were maintained on either isoflurane at 1.5% to 3.0 % (n = 5) or continuous IV infusions of ketamine (n = 4) or propofol (n = 4) respectively. Initially, all macaques received a 50:50 oxygen:air mix, which subsequently was titrated to an inspired O2 concentration of 35%. Anesthetic doses were titrated to provide a moderate plane of surgical anesthesia, which was defined as no movement and not more than a 10% increase in heart rate or blood pressure after a deep, pain-inducing hemostat pinch (hand or foot; checked every 15 to 30 min). Figure 1 displays the experimental timeline for neonatal anesthesia and control exposures. Monitoring and supportive care. Macaques in anesthetized treatment groups were placed on mechanical ventilation (Small Animal Ventilator; Harvard Apparatus, Holliston, MA). Ventilator settings included 20 to 30 breaths per minute, a tidal volume of 6 to 7 mL, and one full-tidal–volume sigh every 10 breaths to counteract the tendency for developing atelectasis with positive-pressure ventilation. Neonates were positioned in lateral recumbency and turned to the contralateral side every 2 h. A second intravenous catheter was placed after the induction of anesthesia, and a second venous blood sample (0.3 mL) for point-of-care analysis (parameters listed previously) was collected about 0.5 h after time 0. All macaques received warmed Lactated Ringers Solution (10 mL/h IV initially; Baxter Healthcare, Deerfield, IL) for fluid replacement and maintenance during anesthesia; fluid rates were modified and fluid boluses (2 mL per bolus) were administered as directed by the veterinarian and investigator to treat for hypotension. In addition, macaques received 5% dextrose (1 mL/h IV); this rate was modified based on hourly (or more frequent) glucose measurements. Animals were maintained on a recirculating warm-water blanket (Gaymar Industries, Orchard Park, NY) and covered with a forced-air heating device (Bair Hugger, Arizant Healthcare, Eden Prairie, MN) that was regulated to maintain normothermia. Animal vital signs were monitored continuously (SurgiVet Monitor, Smith Medical, and Capnomac, Datex Ohmeda) and recorded every 15 min. Physiologic parameters recorded included noninvasive blood pressure, heart rate, oxygen saturation, respiratory rate, end-tidal CO2, rectal temperature (every 30 min; handheld rectal thermometer, model V965F; Vicks; KAZ, Proctor, and Gamble, Cincinnati, OH), inspired O2, and end-tidal anesthetic gas concentrations (isoflurane group only). Additional venous blood samples (0.3 mL at each time point) were collected at 2.5 and 4.5 h after time 0. After 5 h of anesthesia, the anesthetics were discontinued, and the macaques were allowed to recover. Animals were extubated after regaining protective airway reflexes including a consistent swallowing reflex. Time to extubation was defined as the period from discontinuation of gas or injectable anesthesia to successful extubation, including return to normal spontaneous respiration, pink mucous membranes, and a peripheral oxygen saturation consistently greater than 98%. Animals then were transferred to an ICU cage (Snyder Manufacturing, Centennial, CO), which supplied additional temperature and oxygen support as needed for a 3-h observation period. At 8 h after time 0, 291

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Figure 1. Flowchart for experimental anesthesia exposure of neonatal rhesus macaques (postnatal days 5 through 7).

venous blood samples (0.3 mL) for blood gas and metabolic analysis were collected from each unsedated neonate. Subsequently, the animals were sedated with ketamine (20 mg/kg IV) and transported to an adjacent lab, where basic morphometric measurements were taken. Intravenous injection of 0.25 mg/kg pentobarbital was administered to induce a barbiturate coma followed by exsanguination and in vivo transcardial perfusion–fixation with saline followed by 4% paraformaldehyde to prepare the brain for histopathologic analysis. Although histopathology was not an endpoint of the present study, tissues were harvested at necropsy for a concurrent study evaluating neuroapoptosis.8,9,13 Animals randomized to the control group (n = 5) had an intravenous catheter placed for collection of a blood sample (0.3 mL) at time 0 and to allow intravenous administration of saline (2 mL). The catheter then was removed, and the neonate was returned to the dam. At 8 h after time 0, the dam was sedated, and the awake neonate was removed and transported to the surgical suite. An intravenous catheter was placed for collection of a final blood sample (0.3 mL), followed by sedation with 20 mg/kg ketamine IV. The macaques then were transferred to an adjacent lab for transcardial perfusion as previously described. Surgical facilities, anesthetic monitoring equipment, and personnel were consistent throughout the study. Statistical analysis. Analyses were performed by using Excel (Microsoft, Redmond, WA) and STATA (StataCorp, College Station, TX). One-way ANOVA followed by Bonferroni posthoc testing was used to compare primary study endpoints, including the effect of anesthetics on hemodynamic parameters, number of hypotensive events, total fluid administration, and number of tachycardic events throughout anesthesia, time to intubation, and time to extubation. In addition, one-way ANOVA with Bonferroni posthoc testing was used to evaluate secondary study endpoints, including data from blood sample analysis, for possible differences between experimental groups. Repeated-measures ANOVA was used to evaluate change in blood pressure and heart rate between different time points within anesthetic groups. Data were reported as mean ± 1 SD. A P value of less than 0.05 was considered significant.

Results

Table 1 summarizes the demographics of the neonatal rhesus macaques used in this study and the mean doses used for each experimental anesthetic group. Animal age, weight, and estimated length of gestation were relatively consistent across groups. All neonates tolerated the procedures well and recovered without incident after the 5-h anesthesia.

Parameters reflective of hemodynamic response were analyzed to test the hypothesized increased systemic hypotension induced by isoflurane compared with ketamine and propofol. Table 2 summarizes the physiologic data for each anesthetic group at 0.5, 2.5, and 4.5 h after time 0 and the average over the entire anesthetic exposure. Differences in systemic blood pressure parameters occurred between anesthetics, especially during the early anesthetic time points. At 0.5 h after initiation of anesthesia, systolic, diastolic, and mean blood pressures were significantly lower in the isoflurane group compared with the ketamine group (systolic P = 0.029; diastolic P = 0.003; mean P = 0.007). In contrast, diastolic blood pressure values at 0.5 h differed between isoflurane and propofol-exposed neonates (P = 0.029), but systolic (P = 0.361) and mean (P = 0.150) blood pressure values did not differ. Blood pressure values did not differ between ketamine and propofol-exposed neonates at 0.5 h (systolic P = 0.571; diastolic P = 0.554; mean P = 0.365). At 2.5 h after the induction of anesthesia, the systolic blood pressure of the isoflurane group was significantly (P = 0.034) lower than that of the ketamine group, but no difference was noted between the isoflurane and propofol groups (P = 1.000) or the ketamine and propofol groups (P = 0.120). The diastolic (P = 0.930) and mean (P = 0.684) blood pressure values did not differ between anesthetic groups at 2.5 h. Finally, at 4.5 h after the induction of anesthesia, blood pressure was similar between the 3 anesthetic regimens (systolic P = 0.350; diastolic P = 0.982; mean P = 0.690). Overall (average across all recorded anesthetic time points), systolic and mean blood pressure values were lower (P = 0.043 and P = 0.042, respectively) in the isoflurane-exposed macaques compared with the ketamine-exposed animals, but no differences were noted between isoflurane and propofol (P = 0.104 and P = 0.109, respectively) or ketamine and propofol (P = 1.000 and P = 1.000, respectively) groups. No difference in overall diastolic blood pressure (P = 0.061) was noted between anesthetics. Figure 2 displays the average mean blood pressure (mm Hg) values recorded every 15 min over the 5-h isoflurane, ketamine, and propofol anesthesia exposures in the neonatal macaques. In animals receiving isoflurane, blood pressure values did not significantly change over time during the anesthetic exposure (systolic: 0.5 to 2.5 h, P = 0.833; 0.5 to 4.5 h, P = 0.317; 2.5 to 4.5 h, P = 0.429; diastolic: 0.5 to 2.5 h, P = 0.800; 0.5 to 4.5 h, P = 0.673; 2.5 to 4.5 h, P = 0.500; and mean: 0.5 to 2.5 h, P = 0.660; 0.5 to 4.5 h, P = 0.507; and 2.5 to 4.5 h, P = 0.825). In contrast, during the course of the 5-h ketamine anesthesia, systolic, diastolic, and mean blood pressure values significantly decreased between 0.5 and 2.5 h (P = 0.016; P = 0.0001; and P = 0.0001, respectively)

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Table 1. Demographic and anesthetic dose data for neonatal rhesus macaques undergoing 5 h of isoflurane (n = 5), ketamine (n = 4), propofol (n = 4), or no (control; n = 5) anesthesia Age (d)

Weight (g)

Estimated gestation (d)

Induction dose (% or mg/kg)

Total dose (mg/kg)

Anesthetic rate (% or mg/kg/min)

Isoflurane

5.8 ± 0.5

533.7 ± 52.8

167 ± 1

4.5%

—

Ketamine

5.8 ± 0.5

530.9 ± 102.6

164 ± 6

25.5 ± 5.3

193.6 ± 88.1

2.30 ± 0.42% 0.65 ± 0.29

Propofol

6.0 ± 0.0

509.7 ± 99.9

168 ± 5

17.7 ± 9.9

162.4 ± 31.8

0.54 ± 0.11

Control

5.6 ± 1.0

511.0 ± 42.5

169 ± 5

—

—

—

Table 2. Physiologic parameters monitored during 5 h of isoflurane (n = 5), ketamine (n = 4) or propofol (n = 4) anesthesia in neonatal rhesus macaques Time after induction of anesthesia Group Heart rate (bpm)

Systolic blood pressure (mm Hg)

Diastolic blood pressure (mm Hg)

Mean blood pressure (mm Hg)

Respiratory rate (bpm)

End-tidal CO2 (mm Hg)

SpO2 (%)

Rectal temperature (°C)

0.5 h

2.5 h

4.5 h

Overalla

Isoflurane

175 ± 14

173 ± 18

184 ± 23

176 ± 14

Ketamine

164 ± 25

166 ± 30

164 ± 43

167 ± 33

Propofol

176 ± 5

166 ± 27

160 ± 26

171 ± 18

Isoflurane

59 ± 15b

61 ± 7b

63 ± 6

60 ± 9b

Ketamine

86 ± 10d,e

75 ± 9

72 ± 13

74 ± 5

Propofol

73 ± 11d,e

64 ± 4

62 ± 9

71 ± 5

Isoflurane

27 ± 3b,c

30 ± 10

28 ± 3

28 ± 5

Ketamine

55 ±

11d,e

31 ± 13

28 ± 7

37 ± 8

Propofol

46 ± 11d,e

29 ± 4

27 ± 8

36 ± 2

Isoflurane

38 ± 6b

41 ± 9

40 ± 3

38 ± 6b

Ketamine

64 ± 11d,e

44 ± 5

42 ± 5

49 ± 6

Propofol

52 ± 11d,e

41 ± 2

39 ± 6

47 ± 3

Isoflurane

23 ± 3

24 ± 9

23 ± 6

22 ± 2

Ketamine

29 ± 5

25 ± 4

24 ± 4

26 ± 2

Propofol

27 ± 8

22 ± 3

22 ± 2

24 ± 5

Isoflurane

27 ± 5

29 ± 6

28 ± 4

28 ± 3

Ketamine

27 ± 2

26 ± 2

28 ± 3

27 ± 1

Propofol

27 ± 7

25 ± 3

28 ± 8

27 ± 2

Isoflurane

95 ± 5

98 ± 3b

97 ± 3

97 ± 3

Ketamine

98 ± 3

90 ± 6

93 ± 4

94 ± 4

Propofol

100 ± 1

98 ± 2b

99 ± 2

98 ± 1

Isoflurane

37.0 ± 1.0

37.9 ± 0.4

37.8 ± 0.6

37.5 ± 0.4

Ketamine

37.6 ± 0.9

38.3 ± 0.5

38.5 ± 0.7

37.6 ± 0.5

Propofol

36.7 ± 0.7

38.1 ± 0.3

37.7 ± 0.5

37.5 ± 0.2

All data are expressed as mean ± 1 SD. aAverage over the entire 5-h anesthetic exposure. bValue significantly (P < 0.05) different from that of ketamine group. cValue significantly (P < 0.05) different from that of propofol group. dValue significantly (P < 0.05) different from that of 2.5-h time point. eValue significantly (P < 0.05) different from that of 4.5-h time point.

and between 0.5 and 4.5 h (P = 0.001; P = 0.0001; and P = 0.0001, respectively) but not between 2.5 and 4.5 h (P = 0.377; P = 0.540; and P = 0.536, respectively). Similarly, blood pressure values decreased early during the propofol anesthesia between 0.5 and 2.5 h (systolic P = 0.029; diastolic P = 0.001; mean P = 0.004) and between 0.5 and 4.5 h (systolic P = 0.010; diastolic P = 0.0001; mean P = 0.0001) but not between 2.5 and 4.5 h (systolic P = 0.680; diastolic P = 0.770; mean P = 0.621).

The frequency of hypotensive events during each anesthesia was analyzed. Hypotensive events were defined as anesthesia time points (every 15 min) for individual macaques at which mean blood pressure dropped below 40 mm Hg. There were significantly (P = 0.047) more hypotensive events on average during isoflurane anesthesia (13.0 ± 7.0 events) compared with ketamine anesthesia (3.0 ± 3.8 events). In contrast, no difference was observed between the isoflurane and propofol groups 293

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Figure 2. Average mean blood pressure (mm Hg) in neonatal rhesus macaques during 5 h of anesthesia with isoflurane, ketamine, or propofol. At 0.5 h after initiation of anesthesia, mean blood pressure was significantly (P < 0.05) lower in the isoflurane group compared with the ketamine group. Mean blood pressure did not differ between isoflurane- and propofol-exposed neonates. At 2.5 and 4.5 h after induction of anesthesia, no difference was noted in mean blood pressure between anesthetic groups. Overall mean blood pressure was significantly (P < 0.05) lower in the isoflurane group compared with the ketamine group but did not differ from that in the propofol group. Overall mean blood pressure values for the ketamine and propofol groups did not differ. Mean blood pressure did not significantly change over time during isoflurane anesthesia. During ketamine and propofol anesthesia, mean blood pressure significantly (P < 0.05) decreased between 0.5 and 2.5 h and between 0.5 and 4.5 h of anesthesia but not between 2.5 and 4.5 h.

(4.5 ± 2.6 events, P = 0.099) and the ketamine and propofol groups (P = 1.000). Intravenous fluid therapy was provided throughout anesthesia and served as another surrogate parameter for analysis. Fluid rates were increased and fluid boluses were administered as the primary means of treating hypotension during anesthesia. The total number of fluid boluses (isoflurane, 4.8 ± 3.0 boluses; ketamine, 1.5 ± 1.0 boluses; propofol, 2.0 ± 2.3 boluses) as well as the total volume of fluid boluses (11.6 ± 7.8 mL; 3.8 ± 2.5 mL; 5.0 ± 5.8 mL) administered did not differ among anesthetics (no. of boluses, P = 0.123; total volume of boluses, P = 0.153). However, the total fluid volume administered over the entire anesthesia period (continuous IV infusion plus individual IV boluses) was significantly higher in the isoflurane (80.6 ± 15.5 mL, P = 0.001) and propofol (77.1 ± 16.1 mL, P = 0.004) groups compared with the ketamine group (34.3 ± 5.4 mL). No significant difference (P = 1.000) was noted between the isoflurane and propofol groups. Figure 3 provides the mean blood pressure curves (mm Hg) for each of the 5 isoflurane-treated animals during the 5-h exposure. The panels illustrate the time sequence of hypotensive events and associated intravenous fluid boluses that were administered to stabilize the mean blood pressure above the predefined lower limit of 40 mm Hg. No significant differences in heart rate were observed between isoflurane, ketamine, and propofol groups at 0.5 h (P = 0.500), 2.5 h (P = 0.878), and 4.5 h (P = 0.468) during anesthesia (Table 2). Figure 4 displays the average heart rate (bpm) values recorded every 15 min over the isoflurane, ketamine, and propofol anesthesia exposures in the neonatal macaques. Heart rate did not significantly change over time during the isoflurane (0.5 to 2.5 h, P = 0.868; 0.5 to 4.5 h, P = 0.183; 2.5 to 4.5 h, P = 0.134), ketamine (0.5 to 2.5 h, P = 0.770; 0.5 to 4.5 h, P = 1.00; 2.5 to 4.5 h, P = 0.770) or propofol (0.5 to 2.5 h, 0.5 to 4.5 h, P = 0.287; P = 0.079; 2.5 to 4.5 h, P = 0.489) anesthesia exposures. Tachycardia events were defined as anesthesia time points (every 15 min) for individual animals at which heart rate was greater than

200 bpm. No statistical difference (P = 0.610) in the number of tachychardic events was noted between the isoflurane (2.0 ± 2.5 events), ketamine (4.3 ± 3.8 events), and propofol (2.0 ± 3.3 events) groups. Time to extubation was significantly shorter with isoflurane anesthesia (14.2 ± 4.3 min), compared with both ketamine (46.0 ± 11.2 min, P = 0.033) and propofol (75.0 ± 24.9 min, P < 0.001) anesthesia. In contrast, time to intubation did not differ significantly between groups (isoflurane, 25.0 ± 12.7 min; ketamine, 32.3 ± 38.8 min; propofol, 23.8 ± 13.8 min; P = 0.862). Additional physiologic data were recorded as secondary endpoints (Tables 2 and 3). Minimal differences in these parameters occurred between anesthetic groups, given that supportive care was provided throughout the anesthesia exposures to maintain physiologic homeostasis. At 3 h after recovery from anesthesia, Hct and Hgb were higher in the control group compared with the isoflurane (Hct, P = 0.012; Hgb, P = 0.012) and propofol (Hct, P = 0.016; Hgb, P = 0.017) groups. In addition, pvO2 was lower in the propofol group compared with the isoflurane (P = 0.048) and control (P = 0.012) groups.

Discussion

Neonatal macaques are used as animal models in infectious disease, nutrition, toxicology, neurodevelopmental, and behavioral studies, which frequently involve sedation and anesthesia. However, systematic evidence is scarce regarding these animals’ response to general anesthetics established in pediatric anesthesiology and commonly used in adult nonhuman primates. In the current study, we evaluated the effects of isoflurane, ketamine, and propofol as single anesthetics on physiologic variables of neonatal rhesus macaques undergoing anesthesia for 5 h. On the basis of strong evidence from human medicine and anecdotal evidence from nonhuman primate clinical experience, we expected that exposure to isoflurane would be associated with a greater incidence of decreased blood pressure but more rapid recovery, compared with ketamine and propofol. To our knowledge, this study is the first systematic comparison of the effects of these 3 anesthetic regimens on physiologic homeostasis in neonatal macaques. This study was a component of a larger research program evaluating the effects of general anesthetics on the developing brain, including assessment of potential neuro- and glial apoptosis secondary to the exposure.8,9,13 Each anesthetic procedure was approached with the goal of maintaining physiologic homeostasis in the individual animal subject throughout the exposure to minimize potentially confounding effects on primary endpoints of the overarching investigation. With this goal in mind, supportive care including intravenous fluids with dextrose supplementation, mechanical ventilation, and supplemental heat, was provided to ensure physiologic stability. Primary physiologic variables guiding supportive care included endpoints suggestive of the animals’ hemodynamic status, gas exchange, metabolic status, and body temperature. Physiologic values considered outside of the normal range for neonatal human patients were treated immediately to return neonatal macaques to a presumed homeostatic state. Frequent assessment of vital signs, point-of-care blood analysis, and respective therapeutic interventions allowed the assessment of the differential effects of the 3 anesthetics on physiologic variables. In addition, surrogate parameters were identified for additional analysis. Blood pressure values were lower early during isoflurane anesthesia compared with ketamine use. Although basic fluid therapy was started with the induction of anesthesia in all

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Figure 3. Mean blood pressure (mm Hg) curves during the 5-h isoflurane anesthesia for each animal (n = 5; panels A through E). The study protocol defined the threshold for therapeutic intervention at a mean blood pressure of less than 40 mm Hg (black line). Arrows indicate the time of individual fluid-therapy interventions (2-mL IV bolus of lactated Ringers solution) for each animal. Asterisks (*) indicate missing data; hash marks (#) indicate spurious values.

groups, this provision apparently was not sufficient to compensate for the vasodilatory effects of isoflurane. Furthermore, the same neonates had more single hypotensive events than those receiving ketamine, particularly during the early phase of anesthesia. Accordingly, neonates receiving isoflurane needed frequent boluses of crystalloid fluid during the early phase of anesthesia to maintain the mean blood pressure above the target of 40 mm Hg (Figure 3). Similar hypotensive effects have been reported in adult macaques anesthetized with isoflurane

compared with ketamine–midazolam6 and in children undergoing anesthesia using volatile anesthetics like isoflurane.22,35,52 Blood pressure variation under isoflurane anesthesia was minimal compared with ketamine and propofol anesthesia. This effect likely is explained by the continuous titration of the anesthetic drugs to achieve the targeted moderate plane of anesthesia. The dose required for titration by using isoflurane was within a narrower range than those for the other 2 drugs. 295

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Figure 4. Average heart rate (bpm) in neonatal rhesus macaques during a 5-h isoflurane, ketamine or propofol anesthesia exposure. No significant differences in heart rate occurred between isoflurane, ketamine, and propofol cohorts at 0.5, 2.5, and 4.5 h during anesthesia. Heart rate did not significantly change over time during exposure to isoflurane, ketamine, or propofol anesthesia.

Hypotension was managed actively with fluid rate increases and boluses throughout the 5-h anesthesia. Because fluid therapy was used to stabilize blood pressure within physiologic ranges, the amount of fluid administered was considered a potential surrogate parameter for the systemic hypotensive effect of the different anesthetics and analyzed. Although the number of fluid boluses and total volume of fluid boluses administered were not different between experimental groups, the total volume of fluids administered (total constant rate infusion plus fluid bolus volume) over the entire anesthesia was higher in the isoflurane and propofol groups compared with the ketamine group; there was no difference between the isoflurane and propofol groups. These observations support the initial hypothesis that isoflurane has a greater systemic hypotensive effect than ketamine in neonatal macaques. Similarly, propofol resulted in a hemodynamic profile that triggered the provision of more fluids than were administered to animals under ketamine anesthesia at a comparable anesthetic plane. This situation echoes clinical data in human medicine, where transient systemic hypotension occurs frequently when propofol is administered to pediatric patients.28,31 According to our data, hypotension induced by anesthetics such as isoflurane and propofol in neonatal macaques is amendable to therapy using crystalloid fluids. However, alternative strategies to stabilize blood pressure during anesthesia in this animal model, including therapeutic catecholamines (pressors; for example, dopamine) and a balance of fluid and pressor treatment, have been used successfully in subsequent experiments. Neonates maintained under isoflurane anesthesia for 5 h recovered faster than animals maintained with ketamine or propofol for the same duration. In addition, the macaques recovering from isoflurane anesthesia were more alert and responsive to observers earlier compared with those receiving ketamine or propofol and were transferred to the ICU cage within 10 to 15 min after extubation. This difference is likely explained by the fundamental differences in clearance of the 3 anesthetics following prolonged exposure. Isoflurane present in an animal at the end of anesthesia is cleared rapidly via exhalation, and in a neonate with limited fat and muscle mass, residual amounts of third-spaced drug are small. In contrast, clearance of ketamine and propofol is dependent on liver and kidney function,7,36 which explains the longer half-life of both drugs compared with isoflurane. Similar to the reported observations in neonatal macaques, differences in recovery time between injectable and inhalant anesthetics have been reported in adult macaques6 and

humans of all ages.17,24 Moreover, the hepatic enzyme systems responsible for drug metabolism in neonatal mammals are not developed to an adult level.11,24 Similarly, the kidney function in this age group is immature. These physiologic factors retard the elimination of drugs dependent on hepatic metabolism and renal elimination11,24 and explain the prolonged recovery after ketamine and propofol anesthesia that occurred in the neonatal macaques in this study. No significant difference in time to intubation was noted between anesthetic groups. Intubation of neonatal rhesus macaques is technically challenging due to the small size of their trachea, relatively long epiglottis, largely anterior position of the glottal opening, and propensity for laryngospasm. In addition, neuromuscular blocking agents were not used to reduce the number of possible confounders for the overarching study objectives. In the absence of neuromuscular blockers, airway reflexes and muscle tone are better maintained under ketamine anesthesia in both nonhuman primates and humans7 leading theoretically to prolonged intubation times compared with isoflurane and propofol anesthesia. However, the general technical difficulty of endotracheal intubation in macaques at this age likely eliminated any potential subtle effects of ketamine, and explains the equivalent intubation times across the 3 anesthetic groups in this study. No significant differences in heart rate or number of tachycardic events (heart rate greater than 200 bpm) were seen between the isoflurane, ketamine, and propofol groups. Heart rate remained stable during anesthesia for each agent, with no significant difference between early, mid-, and late time points despite fluctuations in blood pressure. Although tachycardia is an expected compensatory response to hypotension in adults mammals, clinical literature in human medicine suggests that in infants, especially preterm neonates, tachycardia is not always associated with hypotension.18 Recent studies also have shown that systemic blood flow and heart rate are not significantly correlated in preterm neonates.18,34 It is therefore likely that the observed absence of an elevated heart rate in the presence of low blood pressure in neonatal macaques is reflective of their immature hemodynamic physiology, which prevents a fully developed sympathetic response, apparently similar to the physiology of human neonates. Respiratory muscle fatigue is more common in neonatal mammals compared with fully developed adults.21 All neonates in this study were mechanically ventilated throughout the 5-h anesthesia to control for possible respiratory muscle fatigue,21 and end-tidal CO2 was used as a monitor to avoid accidental iatrogenic apnea. The end-tidal CO2 readings were low throughout but not different between the 3 anesthetic regimes. This finding was likely a result of mixing of inspired and expired air at the CO2 sampling site (Y-piece of the breathing circuit) and the relatively large fresh-gas flow (approximately 1 L/min; 700 to 800 mL/min room air with 200 to 300 mL/min oxygen). Venous blood gases were used to titrate ventilation to a presumed physiologic homeostasis (PvCO2 = 35 to 45 mm Hg). Statistical analysis of venous blood-gas parameters resulted in sporadic differences (Table 3) between anesthetics that were not consistently identified across anesthetic time points. These differences are difficult to explain, given that they are likely multifactorial in etiology and associated with the continual manipulation of anesthetic and fluid delivery in response to blood-gas analyses. Blood-gas results remained otherwise stable between anesthetic regimens and across time points throughout the 5-h anesthesia exposures.

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Table 3. Characteristics of venous blood samples collected before, during, and after the 5-h isoflurane (n = 5), ketamine (n = 4), propofol (n = 4), or no (control; n =5) anesthesia Time after induction of anesthesia 0h

0.5 h

2.5 h

4.5 h

At 3 h after recovery

Isoflurane

7.30 ± 0.04

7.36 ± 0.08

7.36 ± 0.03

7.37 ± 0.03

7.38 ± 0.05

Ketamine

7.29 ± 0.05

7.43 ± 0.04

7.33 ± 0.09

7.34 ± 0.04

7.27 ± 0.14

Propofol

7.31 ± 0.04

7.41 ± 0.06

7.48 ± 0.07b

7.41 ± 0.04

7.35 ± 0.04

Control

7.29 ± 0.03

—

—

—

7.31 ± 0.04

Isoflurane

43.5 ± 4.6

42.5 ± 12.0

37.2 ± 3.8a

38.0 ± 7.7

37.9 ± 7.6

Group pH

pvCO2 (mm Hg)

pvO2 (mm Hg)

O2 saturation (%)

HCO3 (mmol/L)

Base excess (mmol/L)

Glucose (mg/dL)

Lactate (mmol/L)

Hematocrit (%)

Hemoglobin (g/dL)

BUN (mg/dL)

Ketamine

41.1 ± 3.9

28.4 ± 5.5

32.8 ± 4.6

36.1 ± 5.3

41.1 ± 6.5

Propofol

36.6 ± 9.1

35.9 ± 7.1

26.9 ± 6.0

31.9 ± 4.2

37.4 ± 8.5

Control

46.5 ± 7.2

Isoflurane

24 ± 3

39.4 ± 3.7 38 ± 6

48 ± 18

43 ± 19

29 ± 5a

Ketamine

28 ± 2

37 ± 5

35 ± 9

33 ± 6

29 ± 2

Propofol

31 ± 15

37 ± 11

65 ± 20

66 ± 26

18 ± 7c

Control

25 ± 6

—

—

—

33 ± 5

Isoflurane

37 ± 8

69 ± 12

76 ± 17

82 ± 16

53 ± 11

Ketamine

46 ± 5

73 ± 6

62 ± 17

58 ± 13

47 ± 12

Propofol

48 ± 26

80 ± 9

92 ± 9

90 ± 7b

35 ± 10

Control

39 ± 12

—

—

—

59 ± 7 22.0 ± 2.9

Isoflurane

21.5 ± 0.6

23.2 ± 3.3

21.0 ± 2.9

22.0 ± 3.4

Ketamine

18.9 ± 3.1

18.5 ± 2.2

17.6 ± 4.0

19.5 ± 2.2

19.3 ± 5.6

Propofol

18.2 ± 3.8

21.9 ± 3.4

20.1 ± 2.2

20.0 ± 0.8

20.9 ± 4.2

Control

23.1 ± 1.7

—

—

—

20.0 ± 1.1 −3 ± 3

Isoflurane

−5 ± 1

−2 ± 3

−4 ± 3

−4 ± 3

Ketamine

−8 ± 4

−6 ± 2

−9 ± 5

−5 ± 3

−8 ± 7

Propofol

−8 ± 4

−3 ± 3

−4 ± 2

−5 ± 1

−5 ± 4

Control

−3 ± 1

—

—

—

−6 ± 1 58 ± 14

Isoflurane

79 ± 16

112 ± 31

102 ± 46

76.6 ± 29

Ketamine

66 ± 9

114 ± 80

131 ± 26

90 ± 44

62 ± 8

Propofol

74 ± 23

117 ± 65

111 ± 66

96 ± 27

66 ± 5

Control

92 ± 47

—

—

—

74 ± 18

Isoflurane

5.51 ± 2.74

3.00 ± 1.57

3.66 ± 0.99

3.71 ± 0.85a,b

3.17 ± 1.37

Ketamine

6.09 ± 2.87

2.91 ± 1.18

3.23 ± 1.78

2.08 ± 0.49

2.48 ± 1.41

Propofol

6.53 ± 2.02

2.81 ± 2.18

2.10 ± 0.48

1.73 ± 0.23

3.82 ± 1.11

Control

3.87 ± 1.41

—

—

—

3.11 ± 1.65

43 ± 5

36 ± 9

28 ± 5

30 ± 5

31 ± 6c

Isoflurane Ketamine

41 ± 4

31 ± 11

33 ± 6

32 ± 3

33 ± 5

Propofol

42 ± 9

36 ± 5

32 ± 2

31 ± 3

31 ± 7c

Control

52 ± 4

—

—

—

44 ± 5

14.6 ± 1.8

12.2 ± 3.2

10.2 ± 0.7

10.1 ± 1.7

10.5 ± 1.9c

Isoflurane Ketamine

13.9 ± 1.5

10.6 ± 3.6

11.1 ± 2.0

10.8 ± 1.1

11.2 ± 1.8

Propofol

14.1 ± 3.1

12.3 ± 1.8

10.7 ± 0.6

10.4 ± 1.2

10.6 ± 2.4c

Control

17.6 ± 1.4

—

—

—

15.0 ± 1.5 7±3

Isoflurane

7±3

8±3

8±4

7±5

Ketamine

9±1

8±2

7±2

7±1

6±1

Propofol

6±1

7±1

6±1

5±2

5±2 297

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Table 3. Continued Time after induction of anesthesia Group Control Sodium (mmol/L)

Potassium (mmol/L)

Chloride (mmol/L)

Anion gap (mmol/L)

Isoflurane

0h 9±7

0.5 h —

2.5 h —

4.5 h —

At 3 h after recovery 10 ± 5

144 ± 2

141 ± 2

140 ± 2b

142 ± 3

147 ± 3 149 ± 5

Ketamine

143 ± 1

141 ± 3

146 ± 4

146 ± 1

Propofol

145 ± 2

141 ± 1

141 ± 1

142 ± 2

144 ± 3

Control

143 ± 0

—

—

—

143 ± 1

Isoflurane

3.7 ± 0.2

3.4 ± 0.3

3.6 ± 0.5

3.6 ± 0.3

4.2 ± 1.5

Ketamine

3.6 ± 0.6

2.0 ± 0.4

3.8 ± 0.4

3.9 ± 2.0

3.2 ± 0.8

Propofol

3.6 ± 0.1

3.0 ± 0.3

3.1 ± 0.3

2.9 ± 0.2

3.4 ± 0.6

Control

3.9 ± 0.5

—

—

—

3.1 ± 0.3

Isoflurane

110 ± 2

106 ± 3

109 ± 4

108 ± 3b

113 ± 3

Ketamine

113 ± 2

111 ± 2

111 ± 6

118 ± 5

111 ± 15

Propofol

112 ± 5

108 ± 3

107 ± 3

105 ± 1b

110 ± 3

Control

108 ± 2

—

—

—

113 ± 3

Isoflurane

16 ± 3

16 ± 4

15 ± 3

17 ± 4

16 ± 5

Ketamine

14 ± 3

16 ± 3

20 ± 7

13 ± 1

10 ± 6

Propofol

16 ± 5

14 ± 2

17 ± 2

17 ± 1

16 ± 4

Control

15 ± 1

—

—

—

13 ± 2

significantly (P < 0.05) different from that of propofol group. bValue significantly (P < 0.05) different from that of ketamine group. cValue significantly (P < 0.05) different from that of control group. aValue

Blood analyses for metabolic status were conducted to identify derangements, particularly of glucose and electrolytes, and to document animal wellbeing. Because the neonatal macaques did not receive any nutrition during the 5-h anesthesia, dextrose was supplemented intravenously to maintain systemic glucose in a homeostatic range. The electrolyte levels remained stable within narrow ranges between anesthetics and throughout the anesthesia exposures. Few differences were observed between anesthetics (Table 3), and they were not consistent and presented no recognizable patterns. More likely, and similar to the blood gas fluctuations, the observed sporadic differences between groups and between time points in metabolic status stem from reactive manipulations in response to periprocedural test results. One set of variables systematically changed over time. Hct and Hgb levels dropped sequentially over the 5-h procedure, reaching the level of significance between isoflurane and propofol anesthesia compared with the control condition at the 3-h recovery time point. The change in these 2 parameters over time was likely a function of the repeated blood draws and continuous intravenous fluid therapy, causing some hemodilution. Specifically, the isoflurane and propofol anesthetic regimens required larger total volumes of fluid therapy during anesthesia to support blood pressure. Nevertheless, the results of the serial blood gas and metabolic status measurements during the 3 anesthetic regimens provide real-life evidence that is a valuable resource for clinicians and investigators working with neonatal macaque models. Neonatal animals are prone to hypothermia because of their large surface area to body weight ratio and poor thermal insulation due to minimal subcutaneous adipose tissue stores.10,50 When neonates are under anesthesia for prolonged periods of time, their ability to thermoregulate is weakened further by an inability to move and generate heat through nonshivering thermogenesis.50 Supplemental heat is crucial to supporting normothermia and

maintaining normal physiologic function. In our study, recirculating warm-water blankets and forced air-heating blankets were used to maintain normothermia and prevent significant metabolic derangements associated with hypothermia. Care was taken to avoid overheating the neonates, which can happen rapidly. Rectal temperature was closely monitored and forced air heat was reduced if neonatal temperatures rose above expected normal human pediatric ranges. Table 2 provides evidence that neonatal temperatures were well controlled across anesthetic groups throughout the 5-h anesthesia. Anesthesia administration was titrated to maintain a moderate depth of anesthesia that would allow for surgical procedures to be conducted in a standard clinical setting. To maintain the intended plane of anesthesia, a mean isoflurane dose of 2.3% was required. Standard maintenance rates for inhalant anesthesia performed on juvenile or adult macaques at this institution generally range between 1% to 1.5 %. Similarly, neonates randomized to the ketamine anesthesia cohort required a mean ketamine dose of 0.647 mg/kg/min to maintain the targeted anesthetic plane. A previous study that sought to maintain a light surgical plane of anesthesia in neonates and pregnant adult macaques reported ketamine doses of 0.33 to 0.83 mg/kg/min30 during the maintenance phase. Finally, neonates randomized to propofol anesthesia required 541 µg/kg/min on average to attain a moderate anesthesia level. Previous reports indicate that propofol doses of 300 to 400 µg/kg/min were needed to maintain general anesthesia in adult nonhuman primates.19 It is well established in human medicine that, for many agents, the doses necessary to induce and maintain anesthesia are higher in young children compared with older children and adults.25,31,38 This difference is attributed to the larger central compartment volume and differences in drug clearance in young compared with adult humans.25,31,38 In addition, the higher doses needed

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to maintain the targeted anesthetic depth were a result of the use of a single anesthetic to achieve the effect, in contrast to the multimodal anesthesia regimens frequently applied during surgical interventions both in humans and nonhuman primates. In these multimodal regimens, several drugs are combined to achieve a given anesthetic depth by capitalizing on synergistic drug effects yet minimize unwanted side effects by applying lower doses of the individual agents. The principle limitations of this study are the small sample size and the limited data available from control (no anesthesia) animals. Although the sample size of each anesthetic cohort was small, these groups met the statistical requirements of the overarching study that aimed at evaluating anesthetic-induced neuroapoptosis for which these experiments primarily were designed. Larger group sizes would not be expected to result in different outcomes than are here, but would have lent additional validity to the results and conclusions. In addition, because nonhuman primates are a valuable resource, the sample size limitation in this model was a function of ethical considerations. Control data were limited to 2 blood-collection time points: baseline and 8 h later (just prior to the end of the experiment). This choice was the result of careful considerations that the least stressful, and therefore best possible, control environment for this study was returning the nonanesthetized neonates to their dam, whereas the other animals would undergo 5 h of anesthesia followed by a 3-h recovery period. In addition, no systematic data regarding baseline vital signs are available. This deficit resulted from an inability to reliably measure vital signs in awake neonatal macaques at the time of this project. For subsequent experiments, modified equipment was obtained and specific gentle restraint techniques were developed that now allow for the collection of a full set of vital signs in unsedated neonates prior to the initiation of anesthesia. In conclusion, the data reported here document the physiologic response of neonatal rhesus macaques to general anesthesia with isoflurane, ketamine, and propofol. The results establish the feasibility of using any of the 3 test agents for this purpose in the neonatal macaque and the safety of the agent under these conditions. The data show that provision of supportive care allowed maintenance of several physiologic variables within homeostatic ranges throughout the 5-h anesthesia. While the 3 anesthetic regimens had different effects on hemodynamics, neonates recovered faster from isoflurane anesthesia as compared with ketamine or propofol. This study is the first report of differential effects in neonatal macaques of long-term anesthesia using agents that are well established in human pediatric medicine, and the results echo, in principle, the effects that are observed in human infants in response to the same anesthetics under similar conditions. Taken together, the reported findings offer evidence-based guidance to clinicians and researchers working with neonatal nonhuman primates for providing general anesthesia using isoflurane, ketamine, or propofol.

Acknowledgments

We thank Nikki Gattuccio (ONPRC) for her outstanding technical support during the project and Kimberly Ray, MS, MPH (ONPRC) for statistical consultation. We also thank Stephanie J Murphy, VMD, DACLAM (Department of Anesthesiology and Perioperative Medicine, OHSU) for her review of the manuscript. This work was supported by NIH grant OD 011092.

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