Dosage selection and effect evaluation of sodium pentobarbital in tree shrew anesthesia

Abstract

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To achieve surgical anesthesia in animal experimentation, it is important to select the appropriate anesthetic dose. However, few studies have investigated the reasonable anesthetic dose in tree shrew (Tupaia belangeri). The aim of the study was to review the literature to determine the most commonly used anesthetic dose in tree shrew and to calculate the reasonable equivalent dose between tree shrew and rat based on the body surface area conversion. Two groups of 10 adult tree shrews each were anesthetized with 1% sodium pentobarbital through intraperitoneal injection separately at doses of 62 mg/kg (equivalent dose) and 40 mg/kg (reported dose). Anesthetic depth and times were assessed in addition to vital signs. The results showed that the dosage was quite different across studies, ranging from 15 mg/kg to 80 mg/kg, with 40 mg/kg being the most frequently reported dose. However, the group of tree shrews anesthetized with the commonly reported dose were unable to meet the requirements of surgery. In contrast, the equivalent dose (62 mg/kg, intraperitoneal injection with sodium pentobarbital) calculated by body surface area conversion could achieve an anesthetic time of 44.28 ± 3.95 min with no serious or fatal effects. During anesthetic monitoring, we found that sodium pentobarbital had an inhibitory effect on the blood pressure, pulse rate, respiratory rate and rectal temperature in tree shrews, especially on the respiratory rate. Thus, our study indicated that the use of the equivalent dose of sodium pentobarbital was effective in anesthetizing tree shrews.

Keywords

Anesthesia tree shrew equivalent dose body surface area animal welfare

Introduction

Tree shrew (Tupaia belangeri), a small squirrel-like mammal widely distributed in Southeast Asia, has been bred as an experimental animal in the laboratory because of its small size, fast reproduction and easy capture. In addition, some studies have confirmed that tree shrew belongs to the order Scandentia, which is more closely related to humans than to other non-primate laboratory animals;^1–4 therefore, it is a widely used animal model in the field of biomedical research.^5–10

Anesthesia is a common operation in the process of animal modeling, and the appropriate dose is the key to successful anesthesia.^11,12 Unfortunately, as far as we know, neither data on the evaluation of anesthesia effects on various body systems nor the comparison of the effects on the anesthetic depth and efficiency between different anesthetic protocols used in tree shrews are available so far. By reviewing the published literature that reported tree shrew experiments involving anesthesia in the past five years, we found that the intraperitoneal injection of sodium pentobarbital was the most common method used for anesthetizing tree shrews Figure 3(a); however, we found considerable variability with respect to dosages reported in these experimental studies, even for the same drugs and routes of administration Figure 3(b). This practice is unsafe as it often results in an excessive or insufficient amount of drug administered and may lead to tree shrew death, seriously harming the welfare of experimental animals and violating the ‘3Rs’ principle of animal experiments.^11,12

Figure 1.

Flowchart of studies enrolled in this systematic review. *Tree shrew. Forty-nine articles reported both anesthesia and euthanasia.

In our previous study, we calculated the equivalent dose between tree shrew and rat using the body surface area (BSA) conversion.¹³ Therefore, in the current study, we verified the validity of the equivalent dose of sodium pentobarbital for tree shrews. In addition, we compared the most frequently reported dose of sodium pentobarbital with the equivalent dose.

Materials and methods

Literature review

We searched the literature databases, including PubMed, China National Knowledge Infrastructure and Chinese Wanfang database, using the keywords ‘Tupaia belangeri’ ‘Tupaia glis’ and ‘tree shrew’ from January 2015 to February 2020. Subsequently, we screened the literature using the following conditions: 1) the animals used were adult; 2) the manuscript contained the name, method and dose of the anesthetic used. Consequently, 95 articles were included in the evaluation. The flowchart and literature screening results are shown in Figure 1 and Supplementary material Appendix 4 online.

Animals

The sample size was calculated using the PASS software, version 15.0 (NCSS, LLC). The minimum number of animals that would provide sufficient results was 10 (power > 0.9). Thus, a total of 20 tree shrews were divided into two groups of 10 animals, with each group containing five males and five females (F1 generation; production approval number: SCXK (Dian) 2020–0004; average age, 1 year ± 6 months; average body weight, 121.5 ± 9.9 g). All animal experiments were performed in a semi-barrier zone at the Guangxi Medical University Laboratory Animal Research Center and Health Monitoring, according to the institutional guidelines (DB53/T 328.1-2010). The tree shrews were housed alone in a single cage (35 cm length × 25 cm width × 30 cm height), with a rest room set up in each cage for sleeping (15 cm length × 12 cm width × 12 cm height). The tree shrews were housed under controlled conditions of temperature (23–25°C) and relative humidity (40–50%) and a light/dark cycle of 12 h. The equipment and sterile food used in tree shrew houses were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China. Water was provided ad libitum, and cages were changed every week.

The experimental animals were randomly divided into two groups, with 10 animals in each group, using randomization cards because of the probable presence of uncontrolled confounding factors. The two groups were anesthetized by an intraperitoneal injection of 1% sodium pentobarbital (Solarbio Science & Technology Company, Beijing, China). In group D-BSA (62 mg/kg), tree shrews were anesthetized with a dose of 62 mg/kg, which was converted from the anesthetic dose used in rats (40–50 mg/kg^14–17). Taking the median dose of 45 mg/kg and the conversion coefficient between tree shrews and rats as 1.368,¹³ the dosage calculated based on the BSA conversion was 45 mg/kg × 1.368 =61.56 mg/kg ≈ 62 mg/kg). In group D-Rev (40 mg/kg), tree shrews were anesthetized with the most frequently reported dose (40 mg/kg). The protocols used in this study were approved by the Animal Ethics Review Committee of Guangxi Medical University (approval number: 201911061). The experimental protocols strictly followed the Guiding Principles for the Use and Care of Experimental Animals issued by the Ministry of Science and Technology of China.

Assessment of depth and time of anesthesia

To assess the depth of anesthesia, the following four parameters were used: tail-pinch reflex, forelimb pull-back reflex, hindlimb pull-back reflex, and corneal reflex.^18–20 For the tail-pinch reflex, forceps were used non-invasively to gently pinch six positions near the tail in a non-venous walking area. For the pull-back reflex, hemostatic forceps were used non-invasively to squeeze the interfinger ribbon of both limbs and pull them back. The pull-back reflex was evaluated in both a forelimb and a hindlimb. For the corneal reflex, the cotton wool from a medical cotton swab was loosened and used to gently sweep the surface of the cornea. To reduce sources of variation in response to stimuli, all reflex tests were evaluated by the same operator, and stimuli were repeated from the loss of the righting reflex to the recovery of the righting reflex every 3 min. The score was 0 when a reflex response to a stimulus was observed, and the score was 1 when no reflex response was observed. The score 0 for the reflexes also included the presence of a decreased reflex, whereas the score 1 indicated the absolute absence of reflex. The person performing the anesthetic depth assessments and scoring was unaware of the treatments administered. Each parameter was scored, and the depth of anesthesia was expressed as the total score calculated for each tree shrew. The depth of surgical anesthesia was defined as a score ≥3.²¹ In addition, the time of loss/recovery of reflexes was recorded. The induction time, anesthetic time, emergence time, immobilization time and recovery time were evaluated according to reflexes in each animal. The induction time was defined as the time required for the anesthesia depth score to reach three points after administration. The anesthetic time was defined as the length of time for which the anesthetic score remained ≥3.^20,21 The emergence time referred to the time from the end of surgical anesthesia to the recovery of the righting reflex. The immobilization time referred to the interval between the loss and the recovery of the righting reflex. The recovery time was defined as the time from the recovery of the righting reflex to the recovery of locomotor activity (restored alertness when someone approached the cage). The distribution of each time period in the whole process of anesthesia is shown in Figure 2.

Figure 2.

Time-related parameters of anesthesia recorded in this study.

Vital sign monitoring

Vital signs, including the blood pressure (systolic, diastolic and mean arterial), pulse rate, rectal temperature and respiratory rate, were monitored before and after anesthesia. The blood pressure and pulse rate were measured using a small animal blood pressure monitor (BP-2010, Softron, Tokyo, Japan) by attaching a detector to the beginning of the tail. The rectal temperature was measured by inserting a lubricated mercury thermometer 2 cm into the rectum. The respiratory rate was defined as the number of thoracic movements per minute. Anesthetized tree shrews were positioned on a nylon pad to maintain a constant body temperature. The above vital signs were measured again prior to the anesthetic injection, and the results were regarded as ‘0 min’ values. Vital signs were measured every 5 min within 30 min after the anesthetic injection.

Subsequently, all tree shrews were returned to the cages and kept for future research.

Statistical analysis

Outliers were first detected and removed using the ‘ROUT’ method to clean the data. Clean data were assessed for normality using the Shapiro–Wilk normality test. The two groups were compared using the unpaired Student’s t-test when data were normally distributed and the Mann–Whitney U-test when data did not conform to normal distribution. Data were analyzed using Graph Pad Prism, version 8.0.2 (Graph Pad Software, San Diego, CA, USA). p < 0.05 was set as a statistically significant level.

Results

Anesthetic use reported in the literature

Of the 95 articles, 75 (78.95%) reported the use of a single drug for anesthesia, with sodium pentobarbital (28/95, 29.47%) being the most frequently used anesthetic, as shown in Figure 3(a). Further analysis revealed that 23/28 (82.14%) of articles reported intraperitoneal administration of anesthesia; however, the dosage reported in different articles was quite different, ranging from 15 mg/kg to 80 mg/kg, with 40 mg/kg being the most frequently reported dose, as shown in Figure 3(b) (see Supplementary material Appendix 1 for details).

Figure 3.

The anesthetics used in adult tree shrews reported in the articles. (a) The type and proportion of anesthetics used in adult tree shrews and (b) The dosage of pentobarbital sodium used in adult tree shrews by intraperitoneal injection. For further details about ‘combination’, see Supplementary material Appendix 1 online.

Verification of the anesthetic dose effect

All tree shrews survived after completion of the experiment, with no obvious complications. In group D-Rev (40 mg/kg), only three animals showed the disappearance of the corneal reflex, and none of them lost their tail-pinch reflex. The depth of anesthesia scores during the whole process of anesthesia were less than 3 (Figure 4). As no induction or anesthetic period met the required definition, this dose was obviously unable to meet the requirements of surgery. In group D-BSA (62 mg/kg), the anesthetic effect was stable, the effective period (depth of anesthesia score ≥ 3) lasted 44.28 ± 3.95 min, the immobilization time during the whole anesthetic process was 95.57 ± 4.34 min, and the tree shrews showed restored activity in the feeding cage after the recovery of the righting reflex (14.79 ± 1.86 min). Table 1 and Table 2 summarize the time points of these state changes during anesthesia and the durations of the induction, anesthetic, immobilization and recovery periods (see Supplementary Appendix 2 for details).

Figure 4.

The anesthetic scores for the two kinds of doses.

Table. 1.

Time points of major events during anesthesia.

Agent	Loss of righting reflex (min)	Commencement of surgical anesthesia (min)	End of surgical anesthesia (min)	Recovery of righting reflex (min)	Locomotor activity (min)
D-BSA (62 mg/kg)	2.26 ± 0.40	4.95 ± 0.81	49.23 ± 4.00	97.83 ± 4.34	112.60 ± 5.25
D-Rev (40 mg/kg)	3.83 ± 0.50	–	–	42.71 ± 5.73	52.53 ± 5.32

Time courses are expressed in minutes and presented as the means ± SD (n = 10). ‘–’means that animals cannot meet the anesthesia requirements of this time period. Commencement of surgical anesthesia: the beginning of anesthesia score ≥3; end of surgical anesthesia: the end of anesthesia score ≥3; locomotor activity: restored tree shrew alertness when someone approached the cage. D-BSA (62 mg/kg): the tree shrew dose converted based on the BSA; D-Rev (40 mg/kg): the most commonly used dose for tree shrews in the literature.

BSA: body surface area.

Table 2.

Duration of each stage during anesthesia.

Agent	Induction time (min)	Anesthetic time (min)	Emergence time (min)	Immobilization time (min)	Recovery time (min)
D-BSA (62 mg/kg)	4.95 ± 0.81	44.28 ± 3.95	48.60 ± 5.66	95.57 ± 4.34	14.79 ± 1.86
D-Rev (40 mg/kg)	–	–	–	38.88 ± 5.85	9.83 ± 1.83

Time courses are expressed in minutes and presented as the means ± SD (n = 10). ‘–’ means that animals cannot meet the anesthesia requirements of this time period. D-BSA (62 mg/kg): the tree shrew dose converted based on the BSA; D-Rev (40 mg/kg): the most commonly used dose for tree shrews in the literature.

BSA: body surface area.

Vital sign monitoring

To further study changes in the vital signs of tree shrews during anesthesia, we monitored changes in the blood pressure, pulse rate, respiratory rate and rectal temperature within 30 min after the injection of sodium pentobarbital (Figure 5). In group D-BSA (62 mg/kg), the blood pressure, pulse rate, respiratory rate and rectal temperature decreased rapidly within the first 10 min after the anesthetic injection, and all vital signs tended to be stable after 10 min. In group D-Rev (40 mg/kg), the blood pressure, pulse rate, respiratory rate and rectal temperature also decreased rapidly within the first 15 min after the anesthetic injection; however, there were no obvious stable periods for all vital signs, which returned to normal values 15 min after the administration of the anesthetic (see Supplementary Appendix 3 for details).

Figure 5.

Vital signs during anesthesia. (a) Blood pressure under each type of anesthesia. (b) Pulse rate under each type of anesthesia. (c) Respiratory rate under each type of anesthesia. (d) Rectal temperature under each type of anesthesia.

To evaluate the degree of inhibition of sodium pentobarbital on the blood pressure, pulse rate, respiratory rate and rectal temperature of the tree shrews, we compared the percentage of decrease for each parameter during anesthesia with respect to the physiological value before the anesthetic injection and found that the greatest inhibition was exerted on the respiratory rate (group D-BSA (62 mg/kg), 47.09%; group D-Rev (40 mg/kg), 40.52%; p = 0.018). Subsequently, we compared the lowest values of all four parameters in the process of anesthesia between the two doses. No significant difference was found in the pulse rate. The lowest blood pressure (p = 0.003), respiratory rate (p = 0.018) and rectal temperature (p = 0.032) in group D-BSA (62 mg/kg) were significantly lower than those in group D-Rev (40 mg/kg) (p < 0.05) (Table 3).

Table 3.

Minimum values of vital signs during the study period.

Vital signs	Mean blood pressure (mmHg)	Pulse rate (beats/min)	Respiratory rate (breaths/min)	Rectal temperature (°C)
Normal	116.4 ± 10.32	390.5 ± 21.49	120.2 ± 12.04	38.38 ± 0.46
D-BSA (62 mg/kg)	71.60 ± 3.24 (30 min)	313.00 ± 33.85 (20 min)	63.60 ± 6.38 (15 min)	36.30 ± 0.46 (30 min)
D-Rev (40 mg/kg)	86.70 ± 5.44 (15 min)	328.70 ± 27.47 (20 min)	71.50 ± 7.22 (15 min)	36.73 ± 0.36 (15 min)
R-BSA (62 mg/kg)	−38.49	−19.85	−47.09	−5.42
R-Rev (40 mg/kg)	−25.52	−15.83	−40.52	−4.3
p value	0.003	0.270	0.018	0.032

‘Normal’ represents the measurement result at 0 min. Values are presented as the means ± SD. The time at which the lowest value occurs. D-BSA and D-Rev were compared using independent samples t-tests. D-BSA (62 mg/kg): the tree shrew dose converted based on the BSA; D-Rev (40 mg/kg): the most commonly used dose for tree shrews in the literature; R-BSA (62 mg/kg): the relative change in the group D-BSA (62 mg/kg); R-Rev (40 mg/kg): the relative change in the group D-Rev (40 mg/kg); ‘−’: indicates a decrease, and the value is expressed as %.

BSA: body surface area.

Discussion

A suitable anesthetic dose can effectively reduce both stress response and mortality in experimental animals; moreover, it can avoid experimental errors caused by improper anesthesia and ensure the accuracy and repeatability of results.^11,12 As shown in Figure 2, the immobilization time comprises emergence time and anesthetic time. However, the emergence period does meet the needs of surgical anesthesia because of the recovery of reflexes, such as forelimb and hindlimb pull-back reflexes, in this period. Currently, tree shrews are receiving increasing attention because of their high suitability as experimental models in biomedical research; however, no suitable anesthetic protocol has been reported for use in tree shrews so far. In the published literature, the most common intraperitoneal dose of sodium pentobarbital used in tree shrews is 40 mg/kg, which is similar to the dose commonly used in rats,^16,17 probably because of the fact that the weight and shape of rats are closer to tree shrews’. However, this relatively empirical approach was found inaccurate, and our results showed that this dose could not meet the requirements of surgical anesthesia. In contrast, the equivalent dose calculated by BSA conversion (62 mg/kg) could achieve an anesthetic time of 44.28 ± 3.95 min, with no serious or fatal effects, indicating that BSA is an appropriate parameter for determining the drug dosage for different species.^22–25 We recognized that our study also had a number of limitations, one of which was that this protocol lacked the use of any analgesia, thus making it applicable only in non-painful procedures or minor procedures with the addition of an analgesic agent. The main purpose of the current study was to verify the validity of the equivalent dose of sodium pentobarbital for tree shrews, which would eliminate the need for additional examinations after recovery (e.g. body weight, grimace, response to stimuli, behavior assessment). Further exploration would be required to understand the effect of the drugs on long-term safety in tree shrews.

During anesthetic monitoring, sodium pentobarbital was found to have an inhibitory effect on the blood pressure, pulse rate, respiratory rate, and rectal temperature in tree shrews, with the degree of inhibition being dose-dependent for blood pressure, respiration rate and rectal temperature. Moreover, sodium pentobarbital exerted the highest inhibition on the respiratory rate. These effects of anesthesia with sodium pentobarbital in tree shrews were similar to those reported in mice and other animals.^26,27 Thus, the above findings suggest that sodium pentobarbital should be used carefully in respiratory disease models.

This study provides a practical suggestion for a suitable anesthetic dosage in tree shrews, but many gaps remain to be addressed (e.g. other administration routes, other anesthetic drugs and assessment of pain or discomfort in the post-anesthetic period). Moreover, the experimental operation may cause tissue irritation because of the inadequate analgesic effect of sodium pentobarbital. Another anesthetic drug commonly used in clinical practice, ketamine, functioned well in analgesia and anesthesia. However, animals do not reach surgical levels of anesthesia when ketamine is administered as a sole agent, and they typically receive a ketamine-containing cocktail with other agents, such as xylazine, to reach an adequate plane of anesthesia. Further experiments are needed to fully evaluate these anesthetic drugs. In the present study, we chose sodium pentobarbital as an anesthetic agent, because it was the most frequently used anesthetic in tree shrews. Gas anesthesia system (e.g. isoflurane) is preferred for animals when the equipment for gas anesthesia is available; however, no devices are available specifically for the tree shrew at present. This is worth exploring further in the near future to meet the needs of personalized anesthesia in different situations.

Conclusion

This study shows that the use of an anesthetic dose of sodium pentobarbital for tree shrews was quite different across studies and may not meet the requirements of surgical anesthesia. However, the equivalent dose (62 mg/kg of sodium pentobarbital, intraperitoneal injection) calculated through BSA conversion could achieve an anesthetic time of 44.28 ± 3.95 min, with no serious or fatal effects. Thus, our method of calculating the equivalent dose through BSA conversion may be extended to calculate the equivalent dose of other drugs in tree shrews.

Supplemental Material

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Supplemental material, sj-xlsx-1-lan-10.1177_00236772221146419 for Dosage selection and effect evaluation of sodium pentobarbital in tree shrew anesthesia by Wei Xia, Zong-jian Huang, Nan Shi, Yi-wei Feng and An-zhou Tang in Laboratory Animals

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Supplemental material, sj-xlsx-2-lan-10.1177_00236772221146419 for Dosage selection and effect evaluation of sodium pentobarbital in tree shrew anesthesia by Wei Xia, Zong-jian Huang, Nan Shi, Yi-wei Feng and An-zhou Tang in Laboratory Animals

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Supplemental material, sj-xlsx-3-lan-10.1177_00236772221146419 for Dosage selection and effect evaluation of sodium pentobarbital in tree shrew anesthesia by Wei Xia, Zong-jian Huang, Nan Shi, Yi-wei Feng and An-zhou Tang in Laboratory Animals

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Supplemental material, sj-xlsx-4-lan-10.1177_00236772221146419 for Dosage selection and effect evaluation of sodium pentobarbital in tree shrew anesthesia by Wei Xia, Zong-jian Huang, Nan Shi, Yi-wei Feng and An-zhou Tang in Laboratory Animals

Footnotes

Acknowledgments

We thank the Kunming zoology institute, Chinese Academy of Science for providing experiment animal sources and the Experimental Animal Center of Guangxi Medical University for providing technical support for animal feeding. We thank the members of our research groups for providing technical assistance and participating in discussions.

Data availability

The original data used for analyses presented are available in Supplementary Appendices 1–4 online.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethics statement

All Institutional and National Guidelines for the care and use of animals were followed.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (grant no. 81760189 and grant no. 81760188).

ORCID iD

Wei Xia

Supplemental material

Supplemental material for this article is available online.

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