Praca oryginalna Original paper
Safe and reliable anesthesia of mice has long been a leading concern among biomedical researchers. Pro-tocols with rapid recovery times can decrease the mor-tality associated with rodent anesthesia by minimizing hypothermia and hypoxia (1, 13). A wide variety of different injectable anaesthetic techniques have been described for use in mice. In most instances, injectable anaesthetics are administered by the intraperitoneal, subcutaneous, intravenous or intramuscular routes. The intraperitoneal injection of anaesthetic agents in mice is a simple and convenient method (2).
Most injectable anesthetic protocols used in mice are based on the combination of dissociative drugs or opiates with α2 agonists and tranquilizers or sedatives (11). Tiletamine, a phencyclidine intravenous
anes-thetic which is always compounded with zolazepam, xylazine, medetomidine, acepromazine, and a tilet-amine/zolazepam mixture (Telazol®), is widely used in
veterinary surgical practice (4, 14), and one particular combination, tiletamine-zolazepam and xylazine (TZX), has been used in mice (2). With advances in alpha-2 agonist development, xylazine is now less commonly used in animals, and has gradually been replaced by medetomidine or dexmedetomidine (5). However, medetomidine or dexmedetomidine cannot be obtained in some countries in veterinary medicine because of technical, economic, and legal restrictions. Thus, TZX is an alternative injectable combination for mice. Tramadol is a synthetic, centrally acting analge-sic used for the treatment of moderate to severe pain (6, 16). Tramadol has been studied in mice (20), and it is reasonable to assume that TZX combined with tra-madol (TZXT) would result in an injectable anesthetic
Anesthesia with intraperitoneal
tiletamine/zolazepam, xylazine, and tramadol in mice*
)
LIANG CHEN, YU-PENG YIN*, CHEN-CHEN WU**, SHENG JIANG**, HONG-GANG FAN**, DE-ZHANG LU*
Heilongjiang Institute of Veterinary Science, Qiqihaer, Heilongjiang 161006, P. R. China *Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University,
Yangling, Shaanxi 712100, P. R. China
**Department of Veterinary Surgery, College of Veterinary Medicine; Northeast Agricultural University Harbin, Heilongjiang 150030, P. R. China
Received 28.07.2014 Accepted 06.10.2014
*) This study was supported by the Natural Science Foundation of Shaanxi
Province of China (Grant No. 2014JQ3086) and by the Foundation for Talent of Northwest A&F University (Grant No. Z109021110).
Chen L., Yin Y. P., Wu C. C., Jiang S., Fan H. G., Lu D. Z.
Anesthesia with intraperitoneal tiletamine/zolazepam, xylazine, and tramadol in mice
Summary
A safe and reliable method for anesthetizing mice is very important for biomedical researches. The goals of this research were to find a safe intraperitoneal injection anesthesia protocol coupled with quick recovery for mice surgery.
This study was carried out to compare the anesthesia effect of a combination of tiletamine/zolazepam-xylazine-tramadol (TZXT) and tiletamine/zolazepam-xylazine (TZX) combinations and of the TZXT protocol plus atipamezole, given 30 min later to reverse xylazine (TZXTA). Sixty mice were immobilized by TZX, TZXT, and TZXTA on three different occasions.
The immobilization and analgesia scores, as well as baseline physiological parameters of the mice (heart rate and respiratory rate) were determined. All 3 combinations effectively induced anesthesia, and the mice assumed lateral recumbency within 5 min. Changes in physiological parameters after the administration of the drug combinations remained within biologically acceptable limits.
Although both TZX and TZXT appeared to be effective intraperitoneal anesthetic combinations, TZXT provided significantly better analgesia with a longer duration than TZX. Atipamezole administration provided effective antagonism, and no adverse effects were observed in this study.
protocol with a rapid onset of anesthesia and effective analgesia in mice, but to the authors’ knowledge, there are no published data available regarding the use of tramadol in combination with TZX in mice.
However, all anaesthetic combinations in current use that are administered to mice by intraperitoneal injec-tion produce prolonged sedainjec-tion, and the full recovery of consciousness may take several hours. This prob-lem can be reduced if the anaesthetic regimen can be completely or partially reversed by specific antagonist drugs. Of the drugs currently available, atipamezole (ATI) is the most selective and potent antagonist at the central and peripheral alpha-2-adrenoceptor (18) and reverses the sedative, cardiovascular, gastrointestinal, temperature, and neuroendocrine effects of medetomi-dine in mice (3).
Reversible anaesthetic regimens have the advantage that animals may recover quickly, which reduces the incidence of postoperative complications, such as hypothermia, and also provide a means of treating inadvertent anaesthetic overdose. This, however, does not allow an adjustment of the anaesthetic dose to meet the requirements of individual animals, and the relatively high dose rates required, in comparison with those associated with intraperitoneal administration, result in prolonged recovery times.
In the current study, we hypothesized that the com-bination of tiletamine/zolazepam with xylazine and tramadol would reduce the required doses of all drugs and yield appropriate anesthesia in mice, with good muscle relaxation and analgesia and minimal respira-tory depression. In addition, the reversal of xylazine effects with atipamezole would provide a rapid recov-ery, leading to a safe and practical anesthetic technique.
Material and methods
Animals. This study was conducted under the guidelines
of the Animal Care and Use Committee of the Northwest A&F University. Sixty Kunming mice of both sexes were used, with body weight ranging from 18 to 22 g, aged 8 to 10 weeks (purchased from The Fourth Military Medical University, China). On arrival, the mice were housed in a pathogen-free facility under strict veterinary supervision. The animals were housed in plastic cages and maintained under controlled environmental conditions throughout the study: 21-23°C, with a relative humidity of 60 ± 10%, 12:12 hour light–dark cycles (lights on from 07.00 to 19.00 hours), and with commercial mice diet and water available ad libitum except on the day of the experiment. The animals were housed in stable groups of six in open-top plastic cages type 3 (425 × 266 × 150 mm, floor area 820 cm2) provided
with corn-cob bedding material and tissue nesting mate-rial, and a cardboard tube. The cages were changed once a week. On arrival, the mice were adapted for two weeks before being used in the experiments.
Study design and experimental protocol.
TZX: Tiletamine/zolazepam (80 mg kg–1, Zoletil 100,
Virbac Corporation, France), Xylazine (10 mg kg–1,
Rompun, Bayer, Germany).
TZXT: Tiletamine/zolazepam (80 mg kg–1), Xylazine (10
mg kg–1), and Tramadol (15 mg kg–1, Tramal 100;
Grunen-thal GmbH, Germany).
TZXTA: Tiletamine/zolazepam (80 mg kg–1), Xylazine
(10 mg kg–1), and Tramadol (15 mg kg–1) and atipamezole
(1 mg kg–1, Antisedan; Orion Corporation Animal Health,
Turku, Finland).
To exclude any effects of the circadian rhythm, the experiments were always started at the same time (5 p.m.). Intraperitoneal administration was performed laterally to the midline, next to the umbilicus. A 1-inch 25-gauge needle was inserted at a 45° angle to the abdominal wall in the lower left quadrant of the abdomen. Each mouse was restrained and anesthetized by the same investigator. After administration, the mice were observed in order to identify any clinical signs associated with potential pain or discomfort, including guarding, licking, or scratching the potentially painful site, and a hunched posture. A pulse oximeter (MouseOx® Plus pulse oximeter, Harvard
Appa-ratus, Massachusetts, USA) placed on the upper right hind leg was used for monitoring oxygen saturation (SPO2) and the pulse rhythm during the main study. Oxygen (100%; 2 L/min) was delivered to each mouse by a face mask con-nected to a coaxial circuit.
All manipulations done on conscious animals were car-ried out in the animal’s home room. After weighing the animal, the drugs were administered intraperitoneally. The animal was placed back in its home cage until it lost its right-ing reflex. After anesthesia wore off, the animal first righted itself (rolled over from dorsal to sternal recumbency), then began to walk. At that time, the mouse was returned to its home cage and observed there for at least one additional hour to register any abnormal behavior. Body temperature was maintained in the range of 36-38°C throughout the period of anaesthesia by means of a homeothermic heating blanket (TT40X43-3X, Chang Sheng Ltd, Anshan, China), and body temperature was recorded to ensure that this appa-ratus was functioning effectively.
Measurements. A series of observations, simple tests,
and reflex measurements were carried out on each individual mouse to evaluate the depth and duration of anesthesia. The indicators were adapted from published protocols (1, 2, 8). To reduce sources of variation in response to the stimuli, all reflex tests were carried out and assessed by the same operator.
The time to the onset of sedation, loss of the righting reflex, loss of response to tail pinch, and loss of the pedal withdrawal reflex were recorded. The induction time was measured from the administration of an anesthetic com-bination to the loss of the righting reflex. The time from the loss of the pedal withdrawal reflex to the return of the pedal withdrawal reflex was the duration of surgical anesthesia. Tail pinch and pedal withdrawal reflexes were assessed by clamping with Kocher’s forceps. The pressure from the instrument was immediately released if the animal responded. Both responses were scored on a scale of 1-5, with 1 indicating no response (a score sufficient for surgical procedures) and 5 a marked response. Mice were consid-ered to have reached a surgical plane of anesthesia when they achieved a score of 1 on this scale (complete loss of
the pedal withdrawal reflex). This reflex was tested at 5, 10, 15, 20, 30, 45, 60, 90, and 120 min by the same investiga-tor, alternating between the left and right limbs after induc-tion time until the return of this reflex. The period of time between the loss and return of the pedal withdrawal reflex was defined as the duration of surgical anesthesia. When the animals did not lose the righting reflex for a period of 10 min after intraperitoneal injection, they were returned to their home cage and observed for the next 2 h for any occurrence of abnormal behavior by a researcher blinded to the doses administered.
Animal breathing was clinically assessed by monitor-ing thoracic movement, includmonitor-ing the type of movement observed (thoracic or abdominal) during anesthesia. Respi-ratory depression was defined as the absence of thoracic muscle movement or respiratory movements that became shallower and more abdominal, leading sometimes to apnea. If the animal stopped breathing for 10 s or longer, it was considered apneic.
The animals received atipamezole or the same volume of physiological saline solution (0.9%, Harbin Pharma-ceutical Group Company Limited, China) 30 min after the injection of the TZXT combination, in a dose of one-tenth of the xylazine dose administered by the intraperitoneal route. After the return of the righting reflex, the mouse was returned to its home cage and observed continuously for another 2 h for abnormal behavior. The duration of the total anesthetic period was defined as the time between the loss and return of the righting reflex. The animals were also observed during the following week to detect any postan-esthetic complications.
Data analysis. SPSS 13.0 was used for all statistical
analyses. Induction and anesthesia, as well as standing and walking times were compared between the groups by Student’s t-test. Data for pulse and respiratory rates were analyzed by one-way analyses of variance (ANOVA) for repeated measures to examine the effect of time within each treatment group and the treatment effect at each time point. When ANOVA was significant, Tukey’s test was used for multiple comparisons of the means. Data for time to recov-ery from anaesthesia were analysed by one-way ANOVA to assess the treatment effect, and Tukey’s multiple compari-son test was used to identify differences between means. Comparisons of the score data between treatment groups were performed by the Kruskal-Wallis test. A probability of 5% (p < 0.05) was considered significant.
Results and discussion
After drug administration, the anesthesia protocols used in this study were sufficient for excellent anes-thesia. All 3 combinations (TZX, TZXT, and TZXTA) induced sedation-anesthesia and lateral recumbency within 5 min after administration (Tab. 1). No signs of abdominal pain or discomfort were observed after intraperitoneal administration, and none of the mice died. In all animals that received intraperitoneal injec-tions of either TZX or TZXT, the onset of sedation and lateral recumbency were observed within 3-4 min after injection (Tab. 1). The anesthesia time with the TZXT treatment is significantly longer than that for the TZX treatment, and was not evaluated in the TZXTA treatment. The times from injection to the return of the righting reflex were significantly different for the 3 combinations. The recovery time in the TZXT treatment was significantly longer than for the other treatments, and atipamezole can significantly shorten the recovery time (Tab. 1).
Data for the pulse rhythm (Fig. 1), respiratory rate (Fig. 2), and depth of anesthesia (Fig. 3) are shown. The pulse rhythm, respiratory rate, and depth of anesthesia did not differ among the three groups until atipamezole administration at 30 min. The pulse rhythm was increased 10 min after the administration of all com-binations, and gradually decreased to baseline values at 120 min in the TZX and TZXT treatments. A regular pulse rhythm was observed throughout anesthesia in all mice. The respiratory rate increased significantly during the first 20 min after the administration of each combination, then decreased from 20 to 60 min in the TZX treatment and from 20 to 90 min in the TZXT treatment, and then increased at 90 min in the TZX treatment and at 120 min in the TZXT treatment. Oxygen saturation exceeding 98% was maintained in all mice. The administration of the antagonist drug atipamezole caused a prompt reversal of anesthesia, the anesthetic-induced pulse and respiratory depression.
The total score of the tail pinch reflex and the pedal withdrawal reflex were significantly lower for TZXT and TZX than for the TZXTA treatment after atipa-mezole administration, and for the TZXT treatment than for the TZX treatment at 60-120 min after the administration of the combinations.
This study demonstrated that both TZX and TZXT are effective injectable induction and anesthe-sia combinations in mice. There was no significant difference among the 3 protocols in the onset of sedation or time from injec-tion to the loss of the right-ing reflex. The duration of anesthesia was longer and tolerance to pinprick higher
Tab. 1. Anesthesia variables after intraperitoneal administration of tiletamine-zolazepam/ xylazine (TZX), tiletamine-zolazepam/xylazine/tramadol (TZXT), and tiletamine-zolazepam/ xylazine/tramadol antagonized with atipamezole (TZXTA) in mice (n = 20)
Variable Treatment
TZX TZXT TZXTA
Induction time (min) 3.55 ± 0.59 3.58 ± 0.41 3.60 ± 0.45 Duration of surgical anesthesia (min) 72.44 ± 6.38a 87.33 ± 5.26b Not evaluated
Time from injection to return of righting reflex (min) 120.35 ± 10.48b 141.04 ± 15.42a 43.12 ± 5.37c Explanations: a row without superscripts indicates no significant difference between treatment groups; different superscripts within a row indicate a significant difference between treatment groups (p < 0.05).
with the TZXT treatment, possibly indicating the com-bined effect of the analgesic properties of tramadol. In a clinical situation, the longer duration of sedation
anesthesia with the TZXT treatment is adequate for longer surgical procedures.
When all protocols were compared, there were similar tail pinch reflex and pedal withdrawal reflex scores for the TZXT treatment and the TZXTA treat-ment at 5 min, but a little higher in the TZX treatment. The degrees of anesthesia were greater in the TZXTA and TZXT treatments until 30 min as compared with the TZX treatment. This can be attributed to the effect of tramadol, which extends sedation-anesthesia time when compared with TZX alone. The influence of trama-dol may also explain the longer times to the return of the righting reflex observed with the TZXT treatment. Atipamezole is known to be effective in reversing sedation and analgesia caused by xylazine in mice (10). The dose of atipamezole selected for this study was considered adequate in terms of antagonism of xylazine for the antidotal procedure shortening the recovery period in mice anesthesia with TZXT, and adverse effects, such as rigidity of limbs, muscle tremors, and excitement were not observed. The atipamezole-to-xylazine ratio of 1:10 has previously been successfully used in pigs (15), and it did not cause any unde-sirable effects or resedation in this study.
We hypothesized that the antagonism of xylazine by atipamezole in the TZXTA group would ensure a high quality of recovery with a quicker return of the right-ing reflex. We expected the atipamezole antagonism of xylazine to be so clearly demonstrated that a sham injection of saline in the TZXT group at 30 min was planned. It was not a surprise that the administration of atipamezole significantly shortened the time of the return of the right-ing reflex, and the treated mice showed more signs of arousal between 30 and 45 min, including lower tail pinch reflex and pedal withdrawal reflex scores, increased head lifting, and more frequent attempts to assume sternal recumbency. On the basis of our findings, we recommend the use of atipamezole for shortening the profound depression of the central nervous system if a short (< 30 min), minimally painful procedure is performed on a mouse anes-thetized with a TZXT combination.
The pulse rhythm changed after the administration of all drug combinations, and no anticholinergic agent was used, but there was no significant difference among the 3 treatment groups until atipamezole was injected, which indicates that
Fig. 3. Median depth of anesthesia after intraperitoneal anesthesia (0 min) in mice given tiletamine/zolazepam-xylazine (TZX,
●
), tiletamine/zolaze-pam-xylazine-tramadol (TZXT,■
), and tiletamine/zolazepam-xylazine--tramadol (TZXTA, ▲) (depth of anesthesia was assessed by tail pinching, and pedal withdrawal reflexes were assessed by clamping with Kocher’s forceps) (n = 20) Explanation: as in Fig. 1. Depth of Anesthesia 0 1 2 3 4 5 6 0 5 10 15 20 30 45 60 90 120 Time ( in)m TZX TZXT TZXTA * * * * * *Fig. 2. Mean respiratory rate (in movements per minute, mpm) after in-duction of anesthesia (0 min) in mice given tiletamine/zolazepam-xylazine (TZX), tiletamine/zolazepam-xylazine-tramadol (TZXT) and tiletamine/ zolazepam-xylazine-tramadol (TZXTA) (n = 20)
Explanation: as in Fig. 1.
Fig. 1. Mean pulse rhythm (bpm) after induction of anesthesia (0 min) in mice given xylazine (TZX), tiletamine/zolazepam--xylazine-tramadol (TZXT), and tiletamine/zolazepamtiletamine/zolazepam--xylazine-tramadol (TZXTA) (n = 20)
Explanation: * indicates significant difference between treatment groups
Pulse Rate (bpm) 0 100 200 300 400 500 600 700 800 900 0 5 10 15 20 30 45 60 90 120 Time (min) * * * TZX TZXT TZXTA Respiratory Rate (mpm) 0 50 100 150 200 250 0 5 10 15 20 30 45 60 90 120 Time (min) TZX TZXT TZXTA * * * *
tramadol did not cause a pronounced cardiovascular depression, and suggests that the positive chronotropic effects of tiletamine/zolazepam may partially coun-terbalance the bradycardic effect of xylazine (12). Atipamezole has been shown to antagonize cardiovas-cular depression induced by xylazine (19), and in the current study, the pulse rhythm in the TZXTA treat-ment group increased significantly after atipamezole injection. An increased pulse rhythm after atipamezole injection may be caused by a central stimulant effect, which can induce transient vasodilative hypoten-sion and a reflex increase in the pulse rhythm (17). Tachycardia after the administration of atipamezole in rodents can be an undesirable side effect (9), but in our study the increases in the pulse rhythm after atipamezole administration were not severe.
Similar respiratory rates were observed in all 3 protocols until 30 min after drug administration, and the 3 combinations had an insignificant effect on
SpO2 because oxygen was delivered to each mouse
during this study. This may be partially due to trama-dol, which, compared with other opioids, has a major advantage of causing no cardiopulmonary depressant effects (7). Atipamezole has been shown to antagonize the respiratory inhibition induced by xylazine and medetomidine (18), and in the present study, respira-tory rates increased after the injection of the antagonist.
Conclusion
A single intraperitoneal injection of TZX or TZXT induced good anesthesia in mice. The duration of anesthesia induced by these combinations was approxi-mately 70-90 min, with durations of adequate surgical analgesia of 70 min for TZX and 90 min for TZXT. Cardiorespiratory responses were characterized by well-maintained pulse rhythm and respiratory rates, and no adverse effects were observed. The antagonism of xylazine by atipamezole was effective in shortening the anesthesia time and allowed mice to become alert somewhat earlier.
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Corresponding author: De-Zhang Lu, PhD, Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, P.R.China; e-mail: dezhanglu@hotmail.com
