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Evaluation of the Thermoregulatory Response Between Standard and Downdraft Tables in Anesthetized Rhesus Macaques (Macaca mulatta) and Cynomolgus Macaques (Macaca fascicularis)
Introduction
The use of laboratory animals as experimental models of disease is a vital research tool for scientists in the biomedical field allowing researchers to determine and understand the mechanism of infection and ultimately to develop effective treatment and preventive measures.1 Infectious disease research involves working with infectious agents or animals exposed to them, which can also involve working with select agents.2 Select agents are considered infectious agents and toxins with the potential to pose a severe threat to public health and safety and to animal or plant health, to include animal and plant products.2 Biosafety control measures and precautions protect both workers and the community from accidental exposure to infectious materials, pathogenic organisms and/or toxins, and prevents release into the environment.1,3
New biosafety technologies and guidelines have significantly improved the ways to safely handle biological materials and facilitated our ability to apply appropriate biosafety practices and controls.4 Primary containment barriers provide the first layer of protection usually in direct contact with, or immediately surrounding the biohazardous material or infected animal.1 Secondary barriers are provided by physical and engineering controls and may include physical separation of laboratory areas from public areas, specialized ventilation systems, and airlocks, in order to contain potential hazards.2,7
In a biocontainment laboratory, the ventilation system is particularly designed to provide protection against the unintentional release of infectious agents either within the work environment or outside of the facility itself.8 Downdraft tables are a type of engineering control used for personal protection and to handle hazardous materials that can become airborne.9-11 They protect the worker from airborne particulates produced by the procedure being performed.12 Downdraft tables have grilles on the table surface through which the air and particulates are drawn under negative pressure by a fan through a filter bank, while some may have back and side shields to contain its operation as much as possible.13,14 Although downdraft tables are useful in capturing aerosols, they are not as efficient as a biological safety cabinet (BSC), are not considered primary containment15, and the effective height is usually only up 6 to 8 inches from the surface above the grille.16 During the use of inhalational anesthetics, downdraft tables are of particular importance to scavenge the waste anesthetic gases. They are also commonly used as necropsy tables with the use of 10% formalin to fix tissues17, asThe design of the downdraft table and positioning of the animal or materials on the table can influence the ability to collect particulates because air velocities vary across the surface. The recommended air flow across the table surface is 150-250 cubic feet per minute (cfm) per square foot of table surface area.14 Another consideration when using downdraft tables for animal manipulations is to be careful when placing animals on a cold surface, especially if they are already suffering from hypothermia or during a medical emergency.12 When placing animals on the downdraft table, one must also ensure that the area of the animal being manipulated is located on the work surface of the table in order to help contain potentially contaminated air and aerosols of infected material that could pose a risk to the user.12
Thermoregulation is a basic component in maintaining homeostasis and appropriate physiologic processes in animals.18 In humans, thermoregulatory thresholds not only vary with the circadian rhythm and menstruation, but are also affected by hydration status, exercise, food intake, infection, anesthetic and other drugs; however, the critically ill and the elderly may also have impaired thermoregulation.19 Furthermore, body temperature is one of the fundamental parameters assessed when determining the health status of an animal in both the clinical and research settings.20 In most mammals, the core body temperature is tightly regulated and is protected by a thermoregulatory defense mechanism which can be impaired or overwhelmed, leading to hypothermia.21 Clinical hypothermia is therefore usually diagnosed only after the thermoregulatory defense mechanisms have been depleted or overwhelmed.19 According to the American Medical Association, hypothermia is defined as a core body temperature lower than 35 °C (95 °F), and it occurs when the body loses more heat than it can produce.22 Hypothermia can cause dysfunctions in the heart, kidneys, and the brain. It can also cause liver damage that could cause bleeding disorders as well as breakdown of muscle tissue. When the hypothermia becomes severe, an individual may become unresponsive requiring cardiopulmonary resuscitation.22 Not only can hypothermia adversely affect animal health and welfare, but it can also affect survival and reproducibility of experiments. For this reason, the body temperature should be monitored, and heat should be provided to the animal as required to maintain a normal body temperature during procedures that may lower it.23
In rodents, hypothermia reduces cardiopulmonary and respiratory functions, decreases drug metabolism, prolongs anesthetic recovery, diminishes wound healing and decreases the immunologic responses of T cells and neutrophils.6 It has been shown, that without appropriate warming steps, the heart rate in anesthetized mice can decrease to almost half of the normal heart rate.11 The most frequently observed complication of small animal anesthesia is hypothermia leading to prolonged recovery or death of the animal. Due to the high airflow, the risk of hypothermia is amplified when using downdraft tables, chemical fume hoods, or biosafety cabinets.24 For example, in mice, the body temperature drops very fast after anesthesia, especially in nude mice, if they have not been warmed sufficiently during the anesthetic preparation.24 Mice that feel cool to the touch or have a body temperature of 98 °F (36.5 °C) require prompt attention.25 It is therefore imperative to maintain the body temperature as close as possible to their physiological temperature of 37-38 °C.24 These animals should be provided with a heat source during the pre-, intra-, and post-procedural period to minimize hypothermia and its adverse effects.16 In nonhuman primates, hypothermia is a frequently observed clinical condition with New World monkeys, as well as young, aged, debilitated and anesthetized animals being at a highest risk.26 For example, squirrel monkeys are more susceptible to hypothermia due to their low body fat percentage as well as a relatively high surface area to volume ratio.27 One of the recommendations to minimize further heat loss in nonhuman primates, is that animals should be away from locations with increased drafts such as near supply or exhaust ducts.26
Medical countermeasures (MCM) development is at the forefront of the research conducted at our facility and, under the Food and Drug Administration (FDA) Animal Rule, there is an expectation that the pathophysiologic disease process in the animal model is not only well understood, but that it models the human condition.28 One of the important parameters often studied during infectious disease research, is the clotting cascade and coagulation parameters. Hypothermia can lead to impaired coagulation through an increased prothrombin time and partial thromboplastin time, disrupting the clotting cascade and exacerbating blood loss.19,29,30 Demonstration that the MCM mitigate the disease is also required under the FDA Animal Rule.28 Therefore, immunocompetence and the ability of the body to either mount an immune response or fight an infectious agent is paramount in MCM development such as vaccine research. It has been shown that only 1.9 °C of hypothermia can adversely affect antibody- and cell-mediated immune defense mechanisms such as phagocytosis.19,29
In the absence of any published studies regarding the thermoregulatory effect of the use of downdraft tables on any animal, the objective of the current study is to evaluate the thermoregulatory response, specifically the change in body temperature, in anesthetized rhesus and cynomolgus macaques maintained on a downdraft table versus a standard table for up to 30 minutes. The hypothesis of this study was that the animals maintained on the downdraft table would demonstrate a decrease in body temperature as time progressed as compared to those animals maintained on a standard table as a result of the increased flow of air around the body due to the sustained negative airflow generated by the downdraft table.
Methods
Animals and husbandry
The study population consisted of 10 cynomolgus macaques (Macaca fascicularis) and 10 rhesus macaques (Macaca mulatta), (n=20; age, 4 to 10 y; weight, 3.3 to 9.8 kg; 11 males and 9 females). All animals were of Chinese origin, were housed in the same corridor in four different rooms and were singly or pair-housed. The study animals were deemed to be in good health, had been confirmed seronegative for SIV, STLV-1, B virus, and SRV, and were tested twice annually by tuberculin skin testing and remained negative throughout the study. This study was conducted under an IACUC-approved animal use protocol in an AAALAC-accredited facility and in compliance with the Guide for the Care and Use of Laboratory Animals, Animal Welfare Act and Regulations, Public Health Service Policy, and other federal statutes and regulations relating to experiments involving animals.
The rhesus macaques were housed in two rooms maintained at an average temperature of 70.0 °F with an average relative humidity of 42.2 % during the month leading up to the experiments. The cynomolgus macaques were housed in two rooms maintained at an average temperature of 70.7 °F with an average relative humidity of 42.0 % during the month leading up to the experiments. All rooms were maintained on a 12:12-h light:dark cycle. A commercial diet (2050 Teklad Global 20 % Protein Primate Diet, Envigo, Frederick, MD) was provided to each nonhuman primate (NHP) twice daily, as well as fresh produce once a day. Chlorinated and filtered municipal water was provided ad libitum through an automated watering system (Edstrom Industries, Waterford, WI). A variety of toys and manipulanda were used as part of the environmental enrichment program.
Equipment and Thermometry
Each animal was fasted for a minimum of 6 – 12 hours and anesthetized with Telazol (Zoetis, Parsippany, NJ) 5 mg/kg injected intramuscularly in the cranial or caudal thigh. Once the animal was adequately anesthetized, the animal was weighed and was placed, according to the randomization order, either on a standard table (Metro, Wilkes-Barre, PA) or a downdraft table (Model: FT3048, DualDraw, Denver, CO) which was lined by a disposable absorbent underpad (Henry Schein, Melville, NY) (Figure 1). The standard table used in this study is a type of medical treatment cart constructed of lightweight polymer which provides ample work surface and is the most commonly used at our institution for NHP routine procedures such as physical exams, blood collection, and tuberculin skin testing. The absorbent underpad was placed on the downdraft table in such a way to avoid covering more than 50 % of the total work surface during operations. All temperatures were taken between the period of 0800-1200 to minimize the effect of normal circadian variations on the data using a digital rectal thermometer (SureTemp Plus, Welch-Allyn, Skaneateles Falls, NY), which was covered by using a protective plastic cover and a lubricant. The thermometer has a temperature range of 80 °F (26.7 °C) to 110 °F (43.3 °C), an accuracy of ± 0.2 °F (± 0.1 °C) and a measurement time of approximately 10 seconds. The rectal thermometer was advanced approximately 1 in., and data were recorded by hand from the digital display. Thermometry measurements were collected beginning at time=0 (immediately after the animal was placed on the appropriate table and the rectal probe was placed) and recorded every 5 minutes for the duration of the experiment for a total time of up to 30 minutes. Percent oxygen saturation and heart rate were monitored and recorded every 5 minutes for 30 minutes by using a pulse oximeter (Model: H100, Edan USA, San Diego, CA) attached to the tongue, opposite cheek, or the pinna. In the event that the temperature of an NHP was below 97.0 °C (36.1 °C) at the end of the anesthetic procedure, the NHP was provided thermal support during recovery by using forced-air warming blankets (3M Bair Hugger, St Paul, MN) and/or conductive fabric warming blankets (HotDog, Eden Prairie, MN) until the temperature had reached 99.0 °F or the NHP was awake enough to be safely placed back in the home cage. Each NHP was monitored for appropriate recovery after each anesthetic event in their home cage. Each animal was only anesthetized a total of two times, once while on the standard table and once on the downdraft table. The thermometers, pulse oximeters, and downdraft tables were used according to the manufacturer’s instructions.
Study Design
A 2×2 crossover study design was used to compare the table types. Animals were randomized to receive one of the two possible treatment sequences, stratified by sex and species. Animals were either, maintained and monitored on a standard unventilated table first, followed by the downdraft table, or the animals were maintained and monitored on a downdraft table first, followed by the standard table. A minimum washout period of 5 to 7 days between anesthetic events was allowed. All procedures occurred between 0800 and 1200 hours and were performed under similar conditions to reduce variability.
Statistical Analysis
For the primary analyses of temperatures and heart rates, a two-way repeated measures ANOVA was fit to each time point, taking table type as the within subject, and species as the between subject factor. Subgroup analysis on the effect of sex was carried out by fitting a two-way repeated measures ANOVA separately for each time point and species, taking table type as the within subject, and sex as the between subject factor. To analyze the interaction between age and table type, a repeated measures ANCOVA model was fit and estimates of the effect of table type at 60 and 120 months of age were obtained as single degree of freedom contrasts. No adjustment has been applied for multiple comparisons. For the historical and baseline temperatures, ages and weights, P-values between species were based on t-test or signed rank for comparison to historical controls. The comparison of temperatures to historical control was based on paired t-test. Statistical significance was set at a P value of less than 0.05. Analysis was implemented in SAS Proc mixed, SAS version 9.4 (SAS Institute, Cary, NC).
Results
Summary statistics on historical body temperatures as well as baseline temperatures, age and weight comparison for the study population are presented in Table 1. Historical body temperatures were obtained from the animal medical records by looking at previous routine physical exams during a period of up to 5 years. A statistically significant difference in historical baseline body temperatures between species was observed, with the rhesus macaques (MM) maintaining a higher baseline body temperature, historically, compared to the cynomolgus macaques (MF) under similar conditions (P=0.0011). In our study, MM animals started at a higher baseline temperature (on average) than MF animals on both the standard and the downdraft tables (P=0.0055 and P=0.0196, respectively). Our study population also showed a statistically significant difference in age between MM and MF, with the MF animals having a younger median age of 69.5 months versus 103.5 months for the MM animals (P=0.0443). The median weight was 7.37 kg for MM animals and 6.41 kg for MF animals, such that there was no significant difference between their baseline weights.
Mean body temperatures by time, species and table type are presented in Table 2. The effect of using the downdraft relative to the standard table was larger among MM animals, which also started at a higher baseline temperature. Among rhesus macaques, statistically significant effects of table type on mean body temperatures were observed at 5, 10, and 25 minutes (P=0.0452, P=0.0440, and P=0.0440, respectively), with the downdraft table having mean body temperatures between 0.63 and 0.90 °F lower than the standard table at these time points. In contrast, cynomolgus macaques were not found to have a significant association between table type and body temperatures. Averaging estimates from the MF and MM animals, a significant effect of the downdraft table on body temperature was observed at the 5-minute time point only (P=0.0131), with a mean difference of 0.57 °F. At the end of the study, there were 7 animals on the downdraft table and 9 animals on the standard table that required thermal support during the post-anesthetic recovery because the 30-minute time point temperature fell below 97.0 °F, all of them were cynomolgus macaques.
Figure 2A and 2B summarizes the association of gender and table type with mean body temperatures for both species. In both species, the relative effects of the downdraft table versus the standard table were comparable within each of the genders, however the females had a lower body temperature throughout the study. While the difference between body temperatures of male and females remained relatively constant among MM animals, there was a significant trend toward a greater separation in body temperatures between the sexes as time progressed in the MF animals, with statistically significant differences starting at 15 minutes and continuing through 30 minutes (P<0.05 at each time point by repeated measures ANOVA). MM animals, both males and females, started at a higher temperature and ended at a higher temperature during the study as compared to the MF animals. In MF animals, a larger decrease was observed between mean baseline and final temperatures with many of them ending with mean temperatures below 98 °F, as compared with MM animals, where mean final temperatures were above 98 °F, regardless of gender. Finally, male MF animals maintained higher temperatures while on the downdraft table as compared to the standard table portion of the study.
Figure 2A and 2B summarizes the association between weight and temperature by table type and time. There was a significant trend toward heavier animals maintaining higher temperatures at each time point relative to lighter animals (P<0.05 by linear regression). Moreover, the association between weight and body temperature became stronger as time progressed (P<0.05, by linear regression).
The association between age and temperature by table type and time is summarized in Table 4. Among MF animals, there was a statistically significant increase in body temperatures on the downdraft table at 20 minutes for the younger age group (P=0.0121) and a statistically significant reduction in body temperatures on the downdraft table at 10 minutes for the older age group (P=0.0441). Among MM animals, there was a statistically significant reduction in body temperatures on the downdraft table at 10 and 20 minutes for the younger age group (P=0.0149 and P=0.0358, respectively).
Discussion
Downdraft tables are a commonly used form of engineering control in biocontainment research facilities as part of a biosafety program and animal research use. Several studies have shown the consequences of inadvertent hypothermia during anesthesia to include prolongation of recovery, prolongation of duration of action of drugs, increased incidence of surgical infections, impaired antibody and cell-mediated immune defense mechanisms and an increase incidence of postoperative adverse myocardial events in people among others.21,31 In addition to the animal welfare considerations regarding the adverse physiological effects that thermal stress and hypothermia may have, hypothermia could also confound results by affecting survival and affecting the reproducibility of animal studies.23
In our study, we evaluated the thermoregulatory response of anesthetized rhesus and cynomolgus macaques maintained either on a standard table or a downdraft table for up to 30 minutes. The main objective of this study was to explore the effect that using a downdraft table for 30 minutes may have on the temperature of anesthetized nonhuman primates. As for the effect of species on body temperature, comparing MF to MM animals, MM animals appeared to maintain higher temperatures throughout the study, which could be due to interspecies variability or their circadian rhythm. Since MM animals started at a higher temperature not only in this study, but also by looking at historical baseline temperatures, these higher baseline temperatures are most likely related to a normal species variability in the MM rather than an artifact of the study. The fact that MM animals have usually more fur present than MF animals, could potentially help MM animals maintain temperature better by helping insulate the body. Among MM animals on the downdraft table, mean body temperatures were between 0.63 °F and 0.90 °F lower than the standard table at the same time points. In contrast, we found no association between table type and body temperatures in the MF animals. The body’s initial reaction to a low body temperature is to activate the thermoregulatory defense mechanisms by increasing cardiac output followed by peripheral vasoconstriction and shivering.27 An impaired ability in peripheral vasoconstriction appears to be the most likely explanation for the increased susceptibility to hypothermia demonstrated not only in aged humans but also in older laboratory animals.32 Since MF animals were younger, we think that they had better thermoregulatory defense mechanisms to cope with cold exposure, potentially masking the true effect of the downdraft table on temperature on these younger animals.
One of the adverse effects of hypothermia is increased vascular resistance due to autonomic vasoconstriction, which in turn can raise blood pressure followed by a compensatory decrease in the heart rate.21 For example, temperature control in anesthetized mice and rats is so critical (especially in mice) that without proper warming, the heart rate in anesthetized mice can decrease to almost a half of the normal rate.11 We expected that the heart rate would decrease as the temperature decreased, especially for animals on the downdraft table, due to the increased air flow across the work surface. Among MM animals, a significant decrease in heart rate on the downdraft table was observed at 25 and 30 minutes, which matched the significant reduction in body temperature on the downdraft table for MM animals at 25 minutes. In contrast, among MF animals, a significant increase in heart rate on the downdraft table was observed at 10, 15, 25 and 30 minutes. This increase in heart rate could be observed if the animals were getting slightly lighter on anesthesia half way through the study, which is possible. Another reason for this increase in heart rate could be individual variability on how each animal responded to the dose or the anesthetic agent.
Overall there was not a large difference between sex and the effect of the downdraft table by species, however, the difference between genders increased over time in the cynomolgus macaques. As previously stated, MM animals maintained a higher temperature throughout the study, which agrees with the historical baseline temperatures for these animals. Most of the MF animals were below 98 °F at 30 minutes, with many of them requiring thermal support during recovery. This could be explained by the fact that MF animals had already started at a lower temperature at the beginning of the study compared to MM animals, which were above 98 °F at 30 minutes. None of the MM animals required thermal support during recovery.
In terms of the association between weight and temperature, there was a significant trend toward animals weighing more being able to maintain higher temperatures throughout the study, suggesting that weight had a protective effect related to low temperature. A possible explanation is that heavier animals have a smaller surface area. to volume ratio. In a study of hypothermia during preparation for rodent surgery6, researchers found that heavier mice tend to undergo a faster recovery of body temperature, which could be attributed to the fact that heavier mice have a smaller surface area to volume ratio, which counteracts hypothermia during anesthesia.6 Another possible explanation for this finding, is that heavier animals are thought to be able to use brown adipose tissue (BAT), a fat tissue specialized in non-shivering thermogenesis, to assist them in retaining their heat after cold exposure relative to lighter animals.6 BAT produces heat in response to cold-exposure by burning fat.33 In rhesus macaques, BAT is primarily found in the cervical and the axillary regions, similar to adult humans, although histologically it resembles that of obese mice.34 In cynomolgus macaques, Kates et al identified BAT in the axillary, interscapular, subscapular, and cervical regions.35 Yoneshiro et al found that a higher BAT activity in human subjects correlated with the degree of cold-induced thermogenesis.33
When comparing the effect of age on body temperature, a younger age appears to have a protective effect on body temperature. We observed a significant increase in body temperatures on the downdraft table in MF animals at 20 minutes in the younger age group. This increase in body temperature on the downdraft table could be caused by the body’s autonomic defense mechanisms against cold exposure. Previous research studies have shown that elderly individuals have an impaired ability to maintain body temperature and normothermia when exposed to cold ambient temperatures and are, therefore, more susceptible to the effects of hypothermia, most likely due to an age-related decrease in thermoregulatory vasoconstriction.30 This impaired ability to efficiently thermoregulate during cold temperatures, not only has been established in elderly humans, but also in laboratory animals. An altered regulation of plasma IGF-1 concentration is among the existing hypotheses regarding this impaired ability in elderly individuals.32 Since MF animals were younger and we did not see an association between table type and body temperatures on these animals, it is possible that a younger age might have played some role in the increased resistance to the effects of the downdraft table.
One of the limitations of this study is the use of anesthetics and the effect on thermoregulation. It is widely known that anesthetic agents influence temperature regulation and different anesthetic agents may affect the thermoregulatory response differently. Many anesthetic agents have been shown to alter thermoregulation and to decrease thermoregulatory vasoconstriction thus interfering with the homeostatic mechanisms responsible for temperature regulation.30 This is most likely due to the inhibition of thermoregulatory centers in the central nervous system that occurs during anesthesia, which suppresses both shivering and vasoconstriction.30 For example, in a study on female northern sea lions, a Telazol dosage of >3.5 mg/kg resulted in a tendency toward hypothermia; however, when used at the recommended 1.8 to 2.5 mg/kg or <1.8 mg/kg dosages, there was no change in vital parameters.36 For example, laboratory mice and rats undergoing anesthesia typically require additional thermal support in order to maintain normothermia and to prevent hypothermia.6 In order to control for some of the effect of anesthetics, we used a single anesthetic agent (Telazol) at the same dose (5 mg/kg) for each animal during each anesthetic procedure.
Yang et al showed that that the use of a laminar airflow operating room in addition to advanced age, were significant risk factors for developing intraoperative hypothermia,37 In a laminar airflow system, the air in the operating room recirculates between 20 to 300 times per hour. These ceiling-to-floor systems are commonly used in modern operating rooms because they help reduce dust, maintain positive pressure to reduce the number of microorganisms in the air, and help to prevent intra- and post-operative wound infections. 37Our study, for the first time, explored the relationship between the use of a downdraft table and the potential for hypothermia in anesthetized rhesus and cynomolgus macaques. The presence of both males and females with a range of ages from two species commonly used in research adds tremendous value to this study. In summary, our study found that, historically, rhesus macaques tend to maintain a higher body temperature, the effect of using the downdraft relative to the standard table was larger among rhesus macaques, and rhesus macaques also showed slower heart rates when the temperatures were lower. We also found that there was a significant trend for heavier animals, regardless of species or sex, to maintain higher temperatures relative to lighter animals which became stronger as time progressed. Even though we did not see the same effect of using the downdraft table on the cynomolgus macaques’ body temperatures, our findings suggest that the use of a downdraft table for up to 30 minutes could further impact an animal’s temperature compounding the anesthetic effects on thermoregulation and potentially leading to hypothermia, if no thermal support is planned while using the downdraft table.
In conclusion, because an anesthetized nonhuman primate has an impaired ability to maintain body temperature and therefore, can rapidly develop hypothermia in the absence of thermal supportive measures26, the potential practice of providing thermal support to nonhuman primates during extended downdraft table manipulations or procedures would be a refinement in accordance with the Russell and Burch’s three Rs (refinement, reduction, and replacement) of animal research.38 The preemptive use of warming devices such as forced-air warming blankets and conductive-fabric warming devices during and after anesthetic procedures, has animal well-being benefits. As a refinement, providing thermal support to nonhuman primates during longer downdraft manipulations would not only lead to improving animal welfare in biocontainment studies but has the potential to improve research outcomes and reproducibility of animal studies.
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