Cardiovascular and Metabolic Responses to Carbon Dioxide Euthanasia in Conscious and Anesthetized Rats

Abstract

Euthanasia is a necessary component in research and must be conducted humanely. Currently, regulated CO₂ exposure in conscious rats is acceptable, but data are divided on whether CO₂ alone is more distressing than anesthesia prior to CO₂. To evaluate distress in rats, we compared physiologic responses to CO₂ euthanasia with and without isoflurane preanesthesia. Male Sprague–Dawley rats were implanted with telemetry devices to measure mean arterial pressure (MAP), heart rate (HR), and blood glucose. Animals recovered for 2 wk and were then exposed to either 5% isoflurane (n = 6) or 100% CO₂ (n = 7; calculated 30% chamber volume/min displacement) in their home cages to induce loss of consciousness. Euthanasia was then completed with CO₂ in both groups. MAP and HR increased when the gas delivery lids were placed on the home cages of both groups. Both MAP and HR gradually decreased with isoflurane exposure. MAP increased and HR decreased with CO₂ exposure. Glucose levels remained stable throughout the procedure, except for a small drop in conscious animals initially exposed to 100% CO₂. These data suggest that both gases affect the measured parameters in a similar manner, and that environmental factors, such as gas delivery lid placement, also change these measurements.

Euthanasia is a necessary component of research studies, and the method of euthanasia must balance humane animal treatment with the required study objectives. Regulated carbon dioxide (CO₂) exposure is a commonly-used euthanasia technique in rodents. The use of a displacement rate of 10% to 30% of the chamber volume per minute was the recommended standard used during the performance of this study.^5,7,28 Recently, the recommended displacement rate was changed in the AVMA Guidelines for the Euthanasia of Animals to 30% to 70%.⁶ Numerous studies have examined variations in flow rate, concentration, and adjunct use of anesthetics on the perceived humaneness of this method,^10,14,45,50 and published data present somewhat polarized conclusions on whether the currently recommended methods are optimal.^16,52

CO₂ is a relatively safe, inexpensive, and accessible method for euthanasia that causes respiratory acidosis leading to a loss of consciousness.⁴³ Positive pressure ventilation of 100% CO₂ is comparable to decapitation and cervical dislocation in terms of rapid cessation of cortical activity.¹³ CO₂ has also been used as a short-acting anesthetic^30,42 with analgesic effects after recovery,^37,46 but CO₂ may be aversive and can cause irritation to mucous membranes above specific concentrations.^31,40 CO₂ inhalation is reported to be distressing in humans¹⁸ and studies are divided on whether it causes distress in animals.^16,52 To minimize this risk of distress, the volatile anesthetic isoflurane has been used to pre-anesthetize animals prior to CO₂ exposure. The exact mechanism of action of isoflurane is unclear, but it may act at several types of receptors, including γ amino-butyric acid receptors (GABA_AR),^20,25 to decrease signal transduction and induce loss of consciousness. Isoflurane has recently been called a “mild” source of distress after both single and repeated exposures.²⁹ Others have suggested that even though isoflurane stimulates avoidance behaviors in rats, it is less aversive than CO₂ and therefore preferable.³⁴

Investigators face challenges when studying euthanasia because the animals must remain in a closed chamber for the duration of the exposure. Often, conclusions have been drawn from observed behaviors^31,50 and some postmortem blood and tissue sampling.^27,45 Serial sampling during the procedure is logistically challenging and disrupts the process. Behavioral observations and postmortem sampling will yield useful information but may be accompanied by observer bias or confounding postmortem effects. Thus, capturing quantitative and real-time changes in physiology as animals pass through the stages of loss of consciousness (LOC) and euthanasia is important to critically assess stress and associated distress.

According to the National Research Council, stress is defined as a perturbation to an organism's physiologic homeostasis or psychologic wellbeing.³⁹ Acute stress is known to impact cardiovascular and metabolic parameters, and changes in these parameters may be detectable during the various stages of consciousness and euthanasia.^49,50,55 Stress is similar to, but distinct from distress, which is considered an aversive state in which adaptation mechanisms (such as sympathoadrenomedullary system activation) fail to restore homeostasis.³⁹

Continuous physiologic monitoring via telemetry has been used in some euthanasia studies,^10,14,34,49 but thus far, none have observed the combination of cardiovascular and metabolic changes, such as plasma glucose, as real-time indicators of stress during euthanasia. The goal of this study was to identify real-time changes that occur in heart rate, blood pressure, and plasma glucose concentration in conscious, freely moving rats during euthanasia with CO₂ alone or with isoflurane anesthesia before euthanasia with CO₂. We hypothesized that heart rate, blood pressure, and plasma glucose would transiently increase during loss of consciousness in response to CO₂ or isoflurane, demonstrating that anesthesia induces a physiologic stress response. We predicted that exposure to CO₂ alone would elicit a greater increase of these parameters than exposure to isoflurane before CO₂ euthanasia, indicating a higher risk of distress with direct exposure to CO₂.

Materials and Methods

Animal Care and Use Statement.

All procedures executed during this study adhere to recommendations set forth in the Guide for the Care and Use of Laboratory Animals and were approved by the Data Sciences International IACUC.

Animals.

Thirteen male Sprague–Dawley rats (250 to 275 g, Charles River Laboratories, Raleigh, NC) were received and allowed to acclimate for a minimum of 5 d before surgery. Rats were housed 2 or 3 per 19”x10.5”x7” static cage with various enrichment items provided. Food and water were provided ad libitum, and animals were kept on a 12h:12h light-dark cycle. Room temperature and humidity were monitored daily.

Surgery.

Rats were surgically implanted with telemetric devices that measure systemic blood pressure and electrocardiogram (ECG) (HD-S11 F2, Data Sciences Intl., St. Paul, MN) and plasma glucose (HD-XG, Data Sciences Intl., St Paul, MN). All animals were anesthetized with 5% isoflurane in 1 l per min (LPM) oxygen and maintained with 2% to 2.5% isoflurane in 0.5 LPM oxygen for the duration of the surgery. Animals received 1 mg/kg meloxicam (Eloxiject; Henry Schein Animal Health, Dublin, OH) for analgesia once before surgery and once per day for 3 d postoperatively. In brief, the HD-XG glucose sensor was placed directly into the abdominal aorta and the device body and reference electrode were fixed in the intraperitoneal cavity. The HD-S11 catheter was placed into the caudal-most portion of the abdominal aorta via the femoral artery, and the ECG leads were placed intramuscularly in a modified lead II position. The HD-S11 device body was placed in a subcutaneous pocket on the flank. Animals recovered from surgery in warmed clean cages.

Postoperative period and data collection.

Animals were monitored daily and for the first 7 d after surgery and assessed using an inhouse perioperative pain assessment guide. Animals were singly housed in 19”x10.5”x7” static cages to facilitate data collection and were provided with various enrichment items. Standard Teklad 2014 rodent chow (Envigo, Indianapolis, IN) and tap water were provided ad libitum and animals were kept on a 12h:12h light-dark cycle. Baseline data were monitored with Ponemah software (v 6.40, Data Sciences International, St Paul, MN) until blood pressure and heart rate returned to normal to ensure sufficient postoperative recovery. After at least 1 wk of healing, animals were briefly and lightly anesthetized with isoflurane to remove wound clips.

The HD-XG required calibration via an intraperitoneal glucose tolerance test (IPGTT) once during this study, and this occurred approximately one week after surgery. For the IPGTT, the animals were fasted for 4 h and the tips of the rats’ tails were punctured with a 25-gauge needle to collect a droplet of blood with a hand-held Nova StatStrip Xpress glucometer (Nova Biomedical, Waltham, MA). The rat then received an intraperitoneal injection of 2.25 g/kg of 50% dextrose solution and one additional blood sample was collected shortly after the blood glucose peak was observed via live telemetry signal. The values from the glucometer were entered in the Ponemah software to calibrate the telemetry sensors’ readings to glucose concentrations in mg/dL.

One day prior to euthanasia, baseline data were collected over a 4-h period and reported as a single point average for each parameter. This 4-h data collection was performed in the morning at the same time as the scheduled euthanasia event to reduce any confounding circadian effects.

Experimental groups and euthanasia.

Animals were randomly assigned to one of 2 groups using a random number generator: Group CO₂exp (n = 7) underwent euthanasia via a calculated chamber displacement rate of 30% of the chamber volume per minute using 100% CO₂. Group Iso+CO₂ (n = 6) was first anesthetized using 5% isoflurane in 1 LPM oxygen until recumbent and then immediately euthanized in a chamber prefilled with 100% CO₂. Prior to euthanasia, animals were transferred one at a time to a separate room in their home cage, and the cage was placed on a telemetry receiver. CO₂exp animals remained in their home cages, and a specialized lid was placed on the top of the cage to deliver 100% CO₂ from a compressed gas cylinder at a controlled flow to obtain the calculated 30% chamber volume/min displacement rate. Iso+CO₂ animals also remained in their home cages, and a specialized lid was placed on the top of the cage to deliver 5% isoflurane in 1 LPM of oxygen. The animal was put into an induction chamber prefilled with 100% CO₂ from a compressed gas cylinder and was considered unconscious once it lost its righting reflex.^12,44 Animals were allowed a 2-min acclimation period once the lid was placed on the home cage before either gas was initially introduced. Time measurement started when the animal was first exposed to gas. All animals in both groups were video-monitored (Axis M1145-L Camera Kit, Axis Communications, Lund, Sweden) and relevant observed events, such as loss of righting reflex, were marked in the Ponemah software. Telemetry signals were monitored until death was declared, 2 min after heartbeat and respirations ceased. Respiration rate was monitored and heart rate was monitored with telemetry. Immediately after death, a blood sample was collected from the tail for glucose measurement with a handheld StatStrip Xpress glucometer. Pneumothorax was induced in all animals as a secondary method of euthanasia.

Behavioral Analysis.

Video footage was reviewed by blind observation for the presence of aversive behaviors as animals lost consciousness. Behaviors that were considered aversive included digging or burrowing, raising the head, sniffing, and head jerking.

Data and Statistical Analysis.

Euthanasia telemetry data collection began just before the modified cage lid was placed until death was declared. Data were averaged in 15 s intervals. Euthanasia data were aligned based on when the CO₂ chamber was turned on for CO₂exp group and when the animals were transferred to the CO₂ chamber for the Iso+CO₂ group (T₀). In addition, 15 s averages were reported for the 8 min prior to T₀ to account for isoflurane administration in the Iso+CO₂ group.

Individual and group mean data with standard errors were reported using Microsoft Excel spreadsheets, and treatment group averages were imported into OriginPro (Origin 2019, Northampton, MA) for graphical presentation.

Experimental groups were analyzed for normal distribution using a Shapiro-Wilk test. Each parameter or timepoint was compared using a student's paired t test, and the presence or absence of aversive behaviors was analyzed using a Chi-Squared Test (JASP Version 0.9.2, University of Amsterdam, Netherlands). Differences were deemed significant when P ≤ 0.05.

Results

Parenthetical values signify averages ± SEM.

Blood Pressure.

Mean arterial pressure (MAP) was not different between the 2 groups at baseline. MAP increased from baseline when the modified cage lid was placed in both the isoflurane (99 ± 2 mm Hg compared with 117 ± 3 mm Hg, P = 0.004) and CO₂ (101 ± 2 mm Hg compared with 118 ± 3 mm Hg, P = 0.003) groups, but there were no differences between the 2 treatment groups. MAP was not changed when either gas was first administered. As the Iso+CO₂ group lost consciousness, MAP steadily decreased back down to baseline levels. A rapid and significant decrease in MAP occurred when isoflurane-anesthetized animals were initially exposed to CO₂. MAP stabilized for approximately 1 min before permanently declining. In the conscious animals, MAP immediately increased for approximately 1.5 min after CO₂ delivery began. A consistent dip in MAP occurred around 2 min after delivery in this group before climbing again until approximately 3.5 min, after which MAP fell dramatically (Figure 1).

Figure 1.

Mean arterial pressure increased from baseline (T_-1400) in both groups when the modified cage lid was placed (*P < 0.05). Mean arterial pressure decreased as LOC occurred with isoflurane but increased through LOC with CO₂. Key timepoints are indicated by letter (a: modified cage lid placed; b: isoflurane delivery began; c: CO₂ delivery began; d: average time of loss of righting reflex indicating LOC). Black indicates Isoflurane + CO₂, light gray indicates CO₂ only, and medium gray (C) indicates both Isoflurane + CO₂ and CO₂ only.

Heart Rate.

Heart rate (HR) did not differ between the 2 groups at baseline, but it did increase from baseline in response to the modified lid placement in both the isoflurane (358 ± 7 bpm compared with 438 ± 14 bpm, P = 0.002) and CO₂ (358 ± 12 bpm compared with 393 ± 14 bpm, P = 0.004) groups (Figure 2). HR decreased steadily as the isoflurane induced unconsciousness. Once isoflurane-anesthetized animals were exposed to CO₂, HR fell rapidly and dramatically. When conscious rats were exposed to CO₂, HR decreased by nearly 40% in the first 1.5 min of exposure. Shortly after CO₂ exposure of the CO₂exp group, HR showed a transient and significant increase from their nadirs (P=0.009). A significant change did not occur in the Iso+CO₂ group.

Figure 2.

Significant increases from baseline (T_-1400) were evident in heart rate when the modified cage lid was placed for both groups (* P < 0.05). Heart rate remained elevated in anesthetized animals until CO₂ exposure began but returned to baseline levels for conscious animals when CO₂ delivery began. Key timepoints are indicated by letter (a: modified cage lid placed; b: isoflurane delivery began; c: CO₂ delivery began; d: average time of loss of righting reflex). Black indicates Isoflurane + CO₂, light gray indicates CO₂ only, and medium gray (c) indicates both Isoflurane + CO₂ and CO₂ only.

Plasma Glucose.

Plasma glucose did not change between baseline and lid placement or from baseline to initial gas exposure for either group. Upon initial exposure to CO_2, no significant change occurred in the isoflurane-anesthetized animals, but a consistent and significant decrease occurred in plasma glucose in the conscious animals (102 ± 3 mg/dL baseline to 98 ± 3 mg/dL (-4 ± 1% change from baseline), P < 0.001); Figure 3) approximately 1.5 min after exposure and remained stable for several minutes. Exposure to CO₂ did not affect blood glucose until approximately 3.5 min after exposure in the Iso+CO₂ group, and approximately 6 min after exposure in the CO₂exp group. At these timepoints, the plasma glucose concentration decreased in a rapid and linear fashion until death was declared; however, this decrease is suspected to be due to hypoxia rather than a physiologic drop in glucose, as glucose values were confirmed immediately after death with a hand-held Nova StatStrip Xpress glucometer.

Figure 3.

Plasma glucose was stable throughout induction with anesthesia, and decreased in conscious animals after approximately 1.5 min of exposure to CO₂ (*P < 0.05). Key timepoints are indicated by letter (a: modified cage lid placed; b: isoflurane delivery began; c: CO₂ delivery began; d: average time of loss of righting reflex). Black indicates Isoflurane + CO₂, light gray indicates CO₂ only, and medium gray (c) indicates both Isoflurane + CO₂ and CO₂ only.

Loss of Righting Reflex and Time to Death.

Conscious animals lost their righting reflex faster under CO₂ than isoflurane (184 ± 42 s compared with 335 ± 33 s respectively, P = 0.013). The average total time to death was also faster in the CO₂exp group than the Iso+CO₂ group (654 ± 24 s compared with 777 ± 41 s respectively, P = 0.002).

Avoidance Behaviors.

Four of 6 rats (67%) demonstrated one or more aversive behaviors (digging/burrowing, sniffing) in response to isoflurane, whereas only 1 of 7 rats (14%) exhibited an aversive behavior (head jerking) in response to CO₂. The difference between the groups was not significant (P = 0.053).

Discussion

Isoflurane and CO₂ are common and effective methods to induce LOC, but our data indicate differences in cardiovascular, metabolic, and behavioral parameters during euthanasia. The euthanasia process begins a complex physiologic cascade,^50,51,56 and for the purposes of this discussion, we focus primarily on events leading to LOC. In addition, the distinction between stress and distress is important to consider when evaluating the relative humaneness of euthanasia methods. Stress occurs when an internal or external change creates a physiologic shift in a system, and the body must compensate to stabilize the disturbance.³⁹ Stress is unavoidable and expected in euthanasia. Distress occurs when these natural compensatory mechanisms are overwhelmed, and the disturbance cannot be managed.³⁹ The foremost goal of this study was to examine physiologic markers and behavioral indicators of stress to make recommendations toward refining the current standard practice.

Mean Arterial Pressure.

MAP was significantly higher than baseline in both groups when the modified cage lid was placed on top of the home cage (Figure 1). This elevation of MAP may reflect a mild stress response to novel external environmental factors, that is the recent lid change, transfer to a different room with brighter lighting, or presence of unfamiliar personnel, but may also have contributed to possible distress. Intense light has been associated with increased incidence of aversive behaviors during euthanasia in albino mice,⁴⁵ and under normal conditions, our procedure room measured 550 lx, approximately equivalent to the experimental level at which the mice demonstrated these behaviors in the previous study.⁴⁵ Gas delivery itself did not seem to cause an additional stress effect; the distinct strong odor of isoflurane, the reported carbonic acid formation on mucous membranes from CO₂,⁵ the velocity of gas into the cage, or a sound associated with delivery did not provoke a significant rise in MAP as gas was introduced.

As isoflurane took effect, we observed a steady decline in MAP, which was an expected physiologic response to the anesthetic.^17,19,25 The exact mechanism of action of isoflurane is not yet fully understood, but it likely acts upon several types of receptors, such as GABA_AR, resulting in decreased signal transduction and subsequent loss of consciousness.^18,20,25 GABA_AR are ubiquitous in the rostral ventrolateral medulla, the area of the brain which mediates blood pressure reflexes.^3,21,36 Activation of GABA_AR is known to decrease blood pressure by vasodilation and decreasing total peripheral resistance. Excessive isoflurane administration has been shown to cause severe cardiac depression.^17,25 The immediate and drastic fall in MAP when anesthetized animals were exposed to CO₂ was likely due to nearly instantaneous asphyxia and associated cardiovascular effects. Hypercapnia is known to increase blood pressure due to rapid release of endogenous vasopressin⁴⁶ and angiotensin II⁴⁷ and activation of the central chemoreceptors.^32,54 This appears to be reflected by the elevation in MAP at 0.75 to 1.5 min after CO₂ exposure, before MAP plummeted and death was declared. Apart from the initial increase in MAP before gas delivery began, the pressure responses to the gases appear physiologic and not necessarily indicative of distress.

Heart Rate.

HR increased when the modified lid was placed on cages in both treatment groups and returned to baseline levels (P = 0.39) in the CO₂exp group at T₀. HR remained elevated and relatively stable through LOC under isoflurane, whereas HR decreased quickly and drastically when conscious animals were exposed to CO₂. Studies^12,14,49 have documented bradycardia during CO₂ exposure in rats prior to LOC. In dogs, HR slows as they become hypercapnic; this response also occurred if tachycardia was first induced with pentobarbital prior to hypercapnia.³⁸ We hypothesized that HR would increase due to pain and distress associated with carbonic acid formation, but any potential marker of distress seemed to be outweighed or negated by a normal physiologic stress response to excessive CO₂.^38,51

One group³⁵ documented elevated MAP and unchanged HR in humans when exposed to CO₂, and another⁵⁰ observed these same cardiovascular trends in rats. They attributed these in part to catecholamine release due to handling and the novel environment of the induction chamber. However, our experimental design involved euthanizing the animal in its home cage without any handling; therefore, our data suggest that a physiologic response may be responsible for CO₂-induced cardiovascular changes.

Previous work has shown that rats and mice demonstrate greater aversion to a repeat exposure to isoflurane as compared with CO₂.^29,55 GABA_AR are also involved in awareness and memory,²⁰ so the elevations in HR and blood pressure with isoflurane may be due to conditioned fear from repeated exposure to isoflurane during the initial surgical episode, the brief anesthesia required to remove wound clips, and the final euthanasia event. Therefore, the previous anesthetic episodes and/or recoveries may have been associated with stress, and anesthesia reversal may contribute to fear in subsequent episodes. In other studies, recovery from CO₂ also appeared to be stressful.² High flow rates of CO₂ have been shown to act on GABA_AR, resulting in an anxiogenic effect,^15,22 but these animals were naïve to CO₂. If recovery from CO₂ is unpleasant,² providing test runs to habituate animals may not be reasonable due to this conditioned fear response. Test runs with room air or oxygen, or even just with the modified cage lid, could habituate animals to the environment but the process would be time-consuming and may not be logistically feasible with large groups.

Plasma Glucose.

Previous studies have measured various metabolic markers to assess pain and distress, including adrenocorticotropic hormone (ACTH) and corticosterone. Immediate release of corticosterone in response to a stressor results in a rapid increase of blood glucose levels.⁴⁸ One group compared the effects of sedation, anesthesia, and/or handling on stress hormones and glucose during euthanasia with CO₂, and found no change in serum glucose or ACTH levels between sedated/anesthetized and nontreated rats.²³ Serum corticosterone did increase in animals injected with anesthesia or saline, indicating the injection itself was stressful enough to cause an increase in this hormone, even though rats were handled daily and were provided positive reinforcement in the form of a reward.²³ Another group⁴⁴ observed an increase in blood glucose in rats euthanized by pentobarbital injection but not by CO₂ inhalation or decapitation. Corticosterone release occurs relatively slowly and given the continued elevation throughout anesthesia in this study,⁴⁴ the authors conclude that handling for injection was the stimulating event, rather than CO₂ exposure, and that handling should be avoided when possible for euthanasia. A third group also found elevated serum corticosterone levels after brief anesthesia with either isoflurane or CO₂ in rats, but corticosterone levels were significantly higher with CO₂ compared with isoflurane for up to 24 h after exposure.² This suggests that CO₂ triggered a more severe stress response; the authors noted that recovery from CO₂ appeared more difficult than recovery from isoflurane. Therefore, the elevated corticosterone levels may be indicative of stress associated with recovery rather than induction and would not be a relevant factor during a euthanasia procedure.

Conversely, one group¹⁰ found no changes in corticosterone or ACTH immediately after euthanasia in mice exposed to different CO₂ chamber replacement rates, further suggesting that CO₂ itself may not be the primary stressor during euthanasia. In our study, plasma glucose concentrations dropped slightly only in conscious animals exposed directly to CO₂ but remained stable during the anesthetic episode until death. This decrease in circulating glucose could be due to activation of the orexin system,¹ which has also been associated with increased blood pressure and sympathetic nerve activity in hypercapnia,³² but further studies would be needed to explore this possibility. Our data combined with those of previous studies suggest that perhaps the duration of conscious exposure to these gas concentrations may be too brief to cause measurable increases in blood glucose, or the stressor is relatively mild through LOC.

One limitation of the present study is that the implantable sensor reports glucose levels with respect to oxygen. The device assumes a higher concentration of oxygen than glucose in the blood, so when oxygen is gradually displaced from the bloodstream in a hypercapnic/hypoxic environment, the sensor begins to reflect oxygen concentrations rather than glucose. We verified blood glucose values using a hand-held glucometer immediately after death was declared and all animals were normoglycemic (113 ± 4 mg/dL). Isoflurane was delivered in oxygen but the CO₂ was not admixed, so this could explain the relative stability in glucose as animals lost consciousness under isoflurane compared with CO₂. Admixture of oxygen with CO₂ has not previously been shown to result in improvements to welfare; in fact, adding oxygen resulted in a prolonged time to unconsciousness and increased incidence of tissue hemorrhage^4,18,24 so this method was not included in this study due to welfare concerns.

Behavioral Observations.

Behavioral observations in rodents^31,40 and studies in humans¹⁸ have concluded that CO₂ is irritating to the mucosa due to carbonic acid formation and that this may cause distress. However, the low incidence of observable aversive behaviors that occurred in our study when CO₂ was introduced at a gradual displacement rate of 30% chamber volume/minute suggests that other factors may have greater impact on stress responses. More aversive behaviors occurred as rats lost consciousness under isoflurane anesthesia, and times to LOC and death were longer than with CO₂. Our observations parallel those of others ^45,53 in mice in that the rats exposed to isoflurane exhibited more distress behaviors than those directly exposed to CO₂. Several groups have even suggested a sedative or anesthetic effect of CO₂,^9,30 and one study showed that mice ventilated with 70% CO₂ had mild decreases in cortical brain activity indicative of anesthesia.¹³ These studies, combined with our behavioral observations and telemetry parameters, suggest that CO₂ may initiate an anesthetic effect before distress can be perceived.

Considerations

A vast amount of research has studied specific species, strains, genders, and ages. However, the present study only included adult male rats. Numerous publications highlight sex-dependent stress responses in a variety of applications,^8,26,33 so translation to females must be approached with caution. Further studies are also warranted in different species, strains, and ages as these factors impact stress responses.^11,41

Summary

We hypothesized that both isoflurane and direct CO₂ exposure would result in elevated MAP, HR, and plasma glucose prior to LOC. We predicted that these elevations would be greater in the direct CO₂ exposure group, suggesting a higher risk of distress with CO₂ alone. Our telemetric data suggest that neither isoflurane nor CO₂ clearly induce distress prior to LOC in adult male rats, and any deviations from baseline once gas flow begins reflects an appropriate physiologic response. Coupled with the longer time to LOC and increased incidence of aversive behaviors with isoflurane, these data indicate that pre-anesthesia of rats with isoflurane offers no benefit and the current recommendations of conscious animals being exposed to CO₂ directly are more humane. Considerations should be given to light intensity and a smooth transfer of rats to the euthanasia area, and care should be taken to allow sufficient time to acclimate to the new macroenvironment when possible.