Anesthetic effect of phenobarbital sodium on female BALB/c mice

GUO Kai-kai, FENG Tong, WANG Peng-hui, GAO Chang✉

1.School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou 571199, China

2.School of Pharmacy, Hainan Medical University, Haikou 571199, China

Keywords:

ABSTRACT Objective: Anesthetics are of great importance in avoiding severe pain and suffering in animals and ensuring experimental progress.This study was aimed at elucidating the anesthesia score of phenobarbital sodium as a general anesthetic at different concentrations and doses in BALB/c mice, and finding the suitable anesthesia strategies for experimental surgeries.Methods:Phenobarbital sodium was administrated intraperitoneally at the doses of 75, 100, 125, 150,and 200 mg/kg and randomly in different concentrations (2%, 5%, and 10%) to female BALB/c mice.The anesthesia score was evaluated based on the stimulus index including tail-pinch,front and hind limb withdrawal, and eyelid reflexes.The speed and duration of anesthesia in different groups were recorded per the occurrence and duration of the righting reflex.Results:The anesthetic effect of phenobarbital sodium on female BALB/c mice showed an obvious dose-dependency.Respiratory suppression caused by high-dose anesthesia may lead to mouse death.Based on the anesthesia score, when the phenobarbital sodium treatment was greater than or equal to five percent or 200 mg/kg, more than 80% mice meet the anesthesia depth that surgical operation needed.The rates of achieving surgical anesthesia depth (standard-reaching rate) in mice treated with 2% sodium phenobarbital were 0% in the 75 mg/kg group, 0% in the 100 mg/kg group, 50% in the 125 mg/kg group, 66.7% in the 150 mg/kg group, and 100%in the 200 mg/kg group.The standard-reaching rate of mice treated with 5% concentration of phenobarbital sodium were: 0% in the 75 mg/kg group, 0% in the 100 mg/kg group, 83.33% in the 125 mg/kg group, 100% in the 150 mg/kg group, and 100% in the 200 mg/kg group.The standard-reaching rate of mice treated with 10% concentration of phenobarbital sodium were:50% in the 75 mg/kg group, 66.7% in the 100 mg/kg group, 100% in the 125 mg/kg group,100% in the 150mg/kg group, and 100% in the 200 mg/kg group.Sedation and hypnosis were induced in the low-concentration dose group, and anesthesia was induced in the highconcentration dose group.In the 5% and 125 mg/kg phenobarbital sodium groups, the mortality rate of mice was 0, the anesthesia induction time was (35.5 ± 7.92) minutes, and the anesthesia duration was (106 ± 39.59) minutes.In the 5% and 150 mg/kg phenobarbital sodium groups,the mortality rate of mice was 0, the anesthesia induction time was (34.83 ± 5.27) minutes, and the anesthesia duration was (131.7 ± 36.75) minutes.Conclusion: Phenobarbital sodium alone can provide appropriate general anesthesia in female BALB/c mice.Both the concentration and dose of phenobarbital sodium can affect the anesthetic effect.On the basis of our findings, we recommend the 5% and 125 mg/kg and 5% and 150 mg/kg concentration-dose combinations of phenobarbital sodium for anesthetizing mice according to the surgical requirement.

1.Introduction

The mouse is one of the most widely used mammals in biological and biomedical research.It is widely used in the establishment of a variety of human disease models.According to the 3Rs(replacement, reduction, and refinement), the guiding principles for ethical animal use in scientific research, steps must be taken to minimize the potential stress, pain, and suffering in experimental animals and promote good animal welfare during scientific experiments[1, 2].The use of anesthesia is required for many experimental operations in mice, such as sampling, modeling, and surgical research.An effective anesthesia program can alleviate the animals' pain, provide adequate immobilization, and thus,ensure experimental progress.In particular, in surgical procedures conducted in animals, appropriate anesthesia is the key to completing the experiment successfully.

General or surgical anesthesia in mice can be achieved by inhalation or injection of the anesthetic agent.Isoflurane,sevoflurane, halothane, or enflurane inhalation is an effective approach for anesthetizing mice[3-5].However, precision equipment and high technical skills are required to achieve close animal monitoring in inhalation anesthesia, and waste anesthetic gases may pose occupational risks to experimenters[6].Comparatively,injection anesthesia requires little supporting equipment and is easy to perform; therefore, it is much more commonly used in experiments involving mouse models[7].Many injectable anesthetics are available, each with its own efficacy and safety characteristics,including barbiturates, ketamine, etomidate, xylazine, propofol,tiletamine, alfaxalone, tribromoethanol, tramadol, medetomidine,midazolam, and butorphanol[8-16].In most countries and regions,anesthetics are controlled products and their application is strictly restricted by legal norms[6, 17].

Phenobarbital sodium (C12H11N2NaO3), 2,4,6(1H,3H,5H)-Pyrimidinetrione,5-ethyl-5-phenyl-, monosodium salt, is a barbiturate derivative.It was first synthesized in 1904 and introduced for clinical epileptic therapy in 1912[18].It remained a commonly prescribed sedative and hypnotic, which is considered a highly costeffective treatment for status epilepticus[19].Phenobarbital sodium acts by binding to the γ-aminobutyric (GABA) receptor and increasing the amount of time for which GABA-mediated chloride channels are open.Open chloride ion channels hyperpolarize the cell membrane and increase the action potential threshold, thereby inhibiting central nervous system activity[20].Phenobarbital sodium has been used for anesthetizing experimental animals for decades,and the effect of anesthesia induction was mostly evaluated by righting reflex in the early studies[21-23].However, a single index of righting reflex cannot fully reflect the depth of anesthesia, because experimental animals may still be awake after losing righting reflex,which does not meet the requirements of experimental surgery.In practice, the experimental animal may also wake up from the operation due to severe pain, strong light stimulation, temperature change, bleeding or prolonged operation time.Although awake experimental animals lose the righting reflex, it is often difficult for laboratory technicians to effectively identify whether the animals have recovered the perception of pain, so the laboratory technician cannot complete the duty of care accurately.This poses a potential ethical risk: the animal may suffer, and the experimenter may bear a psychological burden[24].Therefore, more and more researchers use the stimulation index method to determine whether experimental animals were able to reached the depth of surgical need[25-27].It's still unknown whether the anesthesia effect induced by phenobarbital sodium with different concentration/dose can reach the standard of experimental surgical depth.In order to better achieve the standard of experimental surgery in mice, and to provide reference for operators to choose anesthesia strategies, we observed the anesthetic effect of phenobarbital sodium in different concentrations and doses on female BALB/c mice.The scores of anesthesia were observed by the stimulus index including tail-pinch, front and hind limb withdrawal,and eyelid reflexes, and the relevant anesthesia time was recorded.

2.Materials and methods

2.1.Animals

Female BALB/c mice (8 to 12 weeks old) were purchased from Vital River (Charles River Co, Beijing, China).They were kept in climate-controlled conditions (12-/12-h light/dark cycle).The animals were cared for and handled per the principles of Good Laboratory Practice.

2.2.Phenobarbital sodium preparation

Injectable phenobarbital sodium was obtained from Shanghai Xinya Pharmaceutical Co., Ltd.(Shanghai, China).The drug was maintained in powder form at room temperature (20-26 ℃) and diluted to 2%, 5%, and 10% concentrations by using normal saline(0.9% NaCl) before being used.

2.3.Groups and administration

The animals were randomly divided into 15 groups.Each group(n = 6) received one anesthetic protocol of different combinations of concentration (2%, 5%, and 10%) and dose (75, 100, 125, 150,and 200 mg/kg).The anesthetic was administered intraperitoneally.Each mouse was weighed before being injected with phenobarbital sodium.The appropriate injection volume for each concentrationdose combination is shown in Table 1.After the injection, the animals' body temperature was maintained at 38 ℃ by using a homeothermic system for controlling the body temperature of small animals (SS20-2, YISHU Science and Technology Co., Ltd., Tianjin,China).

2.4.Anesthesia scores

The tail-pinch, front and hind limb withdrawal, and eyelid reflexes were evaluated for each mouse to evaluate the depth of anesthesia as described by Tsukamoto et al.[25].Regarding the tail-pinch reflex(total score: 1), non-traumatic tweezers were used to pinch the tail of the mouse, and a score of 1 was assigned if the tail did not move.To evaluate the front and hind limb withdrawal reflex (total score:2), non-traumatic tweezers were used to pull the front and rear limbs gently to observe the presence or absence of muscle tension, and a score of 1 was given if no reflex reaction was observed in the frontor rear limbs.Regarding the eyelid reflex (total score: 1), a Pasteur pipette was used to stimulate the animal’s eyes by airflow, and a score of 1 was assigned if the mouse did not blink.The animal was stimulated at 5, 10 20, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300,330, 360, 390, 420, and 450 min after injection.A score of 3 or 4 was defined as surgical anesthesia level.During the evaluation, all unexpected observations were also recorded, including dyspnea and death.

Tab 1 Injection volume of phenobarbital sodium at different concentrations and doses (every 20 g body weight mice)

2.5.Assessment of anesthesia-related time

Each mouse was continuously observed for 450 min.The animal was overturned twice in 30 s and observed for whether there was overturning to confirm the loss of the righting reflex (LORR).Time from the injection of phenobarbital sodium to LORR was defined as the induction time.The period between LORR and recovery of the righting reflex was defined as the anesthesia duration.

2.6.Statistical analysis

All analyses were performed using GraphPad Prism software(GraphPad Software Inc., San Diego, CA).The result rate (%) was expressed by Chi-square test and Fisher exact probability method.Measurement data results are expressed as the mean ± standard deviation (±s).Repeated-measure analysis of variance (ANOVA)was used to analyze the speed and duration of anesthesia.When multiple comparisons were made during the analyses, a Bonferroni correction was calculated and applied.A p-value of ＜0.05 was considered statistically significant.

3.Results

3.1.Safety study

The dyspnea rates of mice in different groups were shown in Table 2.At phenobarbital sodium concentrations of 10%, the percentage of mice with dyspnea in the 200 mg/kg group was significantly higher than that in 75 mg/kg, 100 mg/kg, 125 mg/kg and 150 mg/kg groups(P＜0.05).There was no significant difference in the rate of dyspnea among the other concentrations groups.

the number of female BALB/c mice that died in the different concentration and dose groups was shown in Table 3.There was no significant difference in mortality among the same concentration groups.In addition, the median lethal dose (LD50) of phenobarbital sodium is 10%-150 mg/kg combination.

Tab 2 Dyspnea of female BALB/c micein different groups (n=6, %)

Tab 3 Death of micein different groups (n=6, %)

3.2.Anesthesia score

As shown in Figure 1, at phenobarbital sodium concentrations of 2%, 5%, and 10%, the anesthesia scores increased with an increase of in the administered dose.

The percentage of mice who reach the depth of surgical anesthesia(compliance rate) in each concentration and dose of phenobarbital sodium group were shown in Table 4.At a phenobarbital sodium concentration of 2%, the compliance rate in the 200 mg/kg group was significantly higher than that in the 75 mg/kg group and 100 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 5%, the compliance rate in the 150 mg/kg group was significantly higher than that in the 75 mg/kg group and 100 mg/kg group(P＜0.05); while the compliance rate in the 200 mg/kg group was also significantly higher than that in the 75 mg/kg group and 100 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 10%, there was no significant difference in compliance rate among all groups.

Tab 4 Mice reached surgical anesthesia in different groups (n=6, %)

3.3.LORR situation

The percentage of LORR in mice of phenobarbital sodium at each concentration and dose group was shown in Table 5.At a phenobarbital sodium concentration of 2%, the percentage of LORR in the 200 mg/kg group was significantly higher than that in the 75 mg/kg group and 100 mg/kg group (P＜0.05); while the percentage of LORR in the 200 mg/kg group was also significantly higher than that in the 75 mg/kg group and 100 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 5%, the percentage of LORR in the 125 mg/kg group, 150 mg/kg, and 200 mg/kg group was significantly higher than that in the 75 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 10%, there was no significant difference in percentage of LORR among all groups.

Tab 5 LORR of mice in different groups (n=6, %)

3.4.Anesthetic induction time

The anesthetic induction time in mice of phenobarbital sodium at each concentration and dose group was shown in Table 6.At a phenobarbital sodium concentration of 2%, the anesthetic induction time in the 200 mg/kg group was significantly faster than that in the 125 mg/kg group and 150 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 5%, the anesthetic induction time in the 200 mg/kg group was significantly faster than that in the 100 mg/kg group, 125 mg/kg, and 150 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 10%, the anesthetic induction time in the 125 mg/kg group, 150 mg/kg, and 200 mg/kg group was significantly faster than that in the 75mg/kg group and 100 mg/kg group(P＜0.05).

3.5.Anesthesia duration

The anesthetic duration in mice of phenobarbital sodium at each concentration and dose group was shown in Table 7.At a phenobarbital sodium concentration of 2%, The 75 and 100 mg/kg groups showed a sedative effect but not an anesthetic effect; while the anesthesia duration in the 200 mg/kg group was significantly longer than that in the 100, 125, and 150 mg/kg groups (P＜0.05).At a phenobarbital sodium concentration of 5%, the 75 mg/kg group only showed a sedative effect; while the anesthesia duration in the 200 mg/kg group was significantly longer than that in the 100 mg/kg group, 125 mg/kg, and 150 mg/kg group (P＜0.05).At a phenobarbital sodium concentration of 10%, the anesthetic duration in the 125 mg group was significantly longer than that in the 75 and 100 mg/kg groups (P＜0.05).

Fig 1 Anesthesia score of mice after administration of phenobarbital sodium.

Tab 6 Anesthetic induction time in different groups (min, n=6, ±s)

Tab 6 Anesthetic induction time in different groups (min, n=6, ±s)

a Compared with the same concentration of 75 mg/kg group, there is a significant difference (P＜0.05); b Compared with the same concentration of 100 mg/kg group, there is a significant difference (P＜0.05); a Compared with the same concentration of 125 mg/kg group, there is a significant difference (P＜0.05); d Compared with the same concentration of 150 mg/kg group, there is a significant difference (P＜0.05).

75mg/kg 100mg/kg 125mg/kg 150mg/kg 200mg/kg F 2% - - - - 43.00±15.06 39.17±8.86 14.00±4.24cd 15.14 5% - - 43.00±19.7 35.50±7.92 34.83±5.27 14.67±3.01bcd 9.95 10% 27.5±7.18 33.83±5.71 10.50±1.05ab 10.67±2.66ab 11.83±1.47ab 34.99

Tab 7 Anesthetic duration in different groups (min, n=6, ±s)

Tab 7 Anesthetic duration in different groups (min, n=6, ±s)

a Compared with the same concentration of 75 mg/kg group, there is a significant difference (P＜0.05); b Compared with the same concentration of 100 mg/kg group, there is a significant difference (P＜0.05); a Compared with the same concentration of 125mg/kg group, there is a significant difference (P＜0.05); d Compared with the same concentration of 150 mg/kg group, there is a significant difference (P＜0.05).

75 mg/kg 100 mg/kg 125 mg/kg 150 mg/kg 200 mg/kg F 2% 0 10.17±24.9 48.00±44.77 68.33±37.98 331.80±162.30abcd 18.55 5% 0 46.50±62.38 106.00±39.59a 131.70±36.75ab 447.20±6.94abcd 135.50 10% 159.7±109.1 129.50±94.42 401.00±120.00ab 340.00±121.0 311.80±155.60 5.63

4.Discussion

The author of the 3R theory, Russell, proposed that anesthesia is “the greatest advance in humane technology” in the academic classic The Principles of Humane Experimental Technique published in 1959[28].Appropriate anesthesia is a significant component in the implementation of surgical experiments in animals.Use of anesthesia can not only safeguard animal welfare, but also ensure the integrity and accuracy of scientific research[29].General anesthesia provides unconscious and immobile animals to perform invasive procedures.However, there is no unified anesthesia program for experimental animals.Injection anesthesia has been considered suitable and useful for experimental studies in mice because of its easy operation[7].Phenobarbital sodium is a barbiturate with a large therapeutic index[30, 31].However, whether different concentrations and doses of phenobarbital sodium induced anesthesia in mice could meet the surgical standard have not been reported.In this study, we observed the anesthetic safety, score, induction and maintenance time of phenobarbital sodium in female BALB/c mice with different concentration-dose combinations, and investigated the appropriative concentration-dose combinations of phenobarbital sodium for surgical procedures in female BALB/c mice.

Phenobarbital sodium is a sedative-hypnotic drug that is used worldwide.Because it is effective and well tolerated, phenobarbital sodium has long been used for convulsive epilepsy treatment in both adults and children, especially newborns[32].Despite this fact,overdose does result in adverse effects, including neurotoxic effects,respiratory depression, and hypotension[33].Our results showed that phenobarbital sodium caused death at high concentration and dose combinations (2%/5%/10% and 200 mg/kg, and 10% and 150 mg/kg).Furthermore, 150 mg/kg phenobarbital sodium at a 10% concentration was the LD50.Our findings suggested that both a high concentration and a high dose are key risk factors for anesthesia-related death.Simultaneously, dyspnea was observed in the groups with animal death in this study.It has been proven in many studies that phenobarbital binding to GABA receptors keeps chloride channels open, which leads to the hyperpolarization of the membrane potential of respiratory medullary neurons and therefore a decline in respiratory frequency[20, 34].We speculated that respiratory depression may be the reason for mouse death caused by a high dose of phenobarbital sodium.Besides, no marked side effects were observed in the other groups.On the basis of our findings, we suggest that 150 mg/kg phenobarbital sodium at a concentration of 5% and 125 mg/kg at a concentration of 10% could be the maximum safe concentration and dose combinations for anesthesia in female BALB/c mice.

Unconsciousness and analgesia are essential to the evaluation of anesthesia depth as well as the welfare of experimental animals[6].LORR is a common index of anesthesia evaluation, but it is not persuasive enough to evaluate whether the anesthetic animals have reached the depth to surgery.In this study, the tail-pinch,front and hind limb withdrawal, and eyelid reflexes were tested to evaluate the depth of anesthesia in the different groups in this study.We considered the total score for the different reflexes as a sign of anesthesia depth, which should reach 3 or 4 for surgical need.Our work shown the AD50 values of phenobarbital sodium were 2% and 125 mg/kg and 10% and 75 mg/kg combinations,meaning that both the concentration and dose could affect the anesthesia effect.Our work also shown that the depth of anesthesia caused by intraperitoneal injection of phenobarbital sodium was significantly dose-dependent.Surgical anesthesia depth (score＞3) was achieved when 200 mg/kg phenobarbital sodium was administered at a concentration of 2%.However, the duration of this anesthesia strategy was up to 5 h, much higher than the 1-2 h which experimental surgery needed.Therefore, the combination of 2% and 200 mg/kg phenobarbital sodium may not adequate for surgical operations.Besides, more than 80% mice were able to reach the surgical standard when 5% and 10% phenobarbital sodium was administered at doses of 125, 150, and 200 mg/kg.The success rate of anesthesia was high in the 5% and 200 mg/kg, 10% and 125 mg/kg, 10% and 150 mg/kg, and 10% and 200 mg/kg dose groups.We suggest to select the appropriate dose of phenobarbital sodium from the above groups according to the needs of anesthesia maintenance duration.

The induction and duration of anesthesia are the basic indicators for the evaluation of the anesthetic effect in animals.After phenobarbital sodium was injected into the abdominal cavity of mice, the mice became agitated and retained autonomous activity for a period of time.Then, they went into a state of accelerated breathing,unstable gait, and forward crawling until LORR occurred.Previous studies have shown that 100 to 135 mg/kg phenobarbital sodium is the dose that induces a hypnotic effect in mice[35, 36].Similarly,our study showed that a low concentration and a small dose of phenobarbital sodium ( 2% and 100 mg/kg or 5% and 75 mg/kg) would induce sedative and hypnotic effects in mice.With an increase in concentration and/or dose, phenobarbital sodium induced an anesthetic effect, including LORR induction, and the loss of tailpinch, front and hind limb withdrawal, and eyelid reflexes.Therefore,the effect of phenobarbital was significantly dose-dependent.With an increase in the concentration and/or dose of phenobarbital sodium,the anesthesia induction time gradually shortened, and the duration of anesthesia obviously prolonged.However, we found that in the 10% phenobarbital sodium concentration groups, the anesthesia duration in the 150 mg/kg and 200 mg/kg groups were not longer than that in the 125 mg/kg group.We speculate that the animal death caused is the reason for the unexpected reduction in anesthesia duration in the high-dose phenobarbital sodium groups.Therefore,we recommend phenobarbital sodium in 5% and 125 mg/kg and 5%and 150 mg/kg combinations for surgical anesthesia in mice.

It is important to note that the anesthesia duration of phenobarbital sodium was quite long when high-dose phenobarbital sodium was applied, and some deaths occurred between 8 and 24 h after administration.We found that some of the deaths occurred between 8 and 24 h after administration (data not shown).Although there was thermal support, the animals’ body temperature was not measured in this study.Therefore, it remains unclear whether hypothermia occurred in the dead animals and if it was one of the causes of death.There are several limitations of the present study.First, we only investigated the anesthetic effect of phenobarbital sodium in female BALB/c mice aged 8 to 12 weeks.However, the effect of anesthetics is affected by the mouse strain, sex, and age[25, 37, 38].These factors require full consideration to achieve an even better anesthetic effect of phenobarbital sodium in practice.Second, we did not evaluate the changes in the physiological indexes caused by phenobarbital sodium.Further studies will be necessary to investigate changes in vital signs such as body temperature, oxygen saturation, blood pressure, heart rate, and muscle relaxation during phenobarbital sodium anesthesia.Besides, our study showed phenobarbital sodium had a slightly long anesthesia induction time and duration.Since the combined use of different anesthetics has become a trend in both basic research and clinical application[6, 39, 40].The combined effects of phenobarbital sodium and other anesthetics remain to be explored.In summary, this study provides basic information on the safety, anesthesia score, induction and duration of anesthesia of phenobarbital sodium anesthesia in mice.We found that phenobarbital sodium used singly could provide appropriate depth and optional duration for surgical anesthesia in female BALB/c mice.The anesthetic protocol is critical to the success of surgery in laboratory animals, and operators can select the appropriate concentration-dose combinations of phenobarbital sodium according to the anesthesia time required for the procedure.Our work may help improve the welfare of experimental animals.

Conflicts of Interest: The authors declare no competing interests.

Acknowledgments

The authors thank Shanying Cai, Chun Liu, Xiaoqing Ye (Hainan Institute for Drug Control) , and Shimin Chen, Chuan Lin (Hainan Medical University) for their helpful discussions and technical support.