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Tribromoethanol-medetomidine combination provides a safe and reversible anesthetic effect in Sprague-Dawley rats.

Tribromoethanol typically is used alone as a general anesthetic agent for rodent surgeries. In the present study, the alpha2-adrenergic agonists xylazine and medetomidine were combined with tribromoethanol to examine their use as alternate and safe anesthetic regimes in rats. We also tested the effect of atipamezole, an alpha2-adrenergic antagonist, in reversing the anesthetic effect of the tribromoethanol-medetomidine combination. Male Sprague-Dawley rats were used to evaluate the effects of tribromoethanol (400 mg/kg intraperitoneally [i.p.]) or tribromoethanol (150 mg/kg) and medetomidine (0.5 mg/kg i.p.). Tribromoethanol (400 mg/kg)-treated rats were anesthetized for an average of 10 min, whereas rats that received tribromoethanol (150 mg/kg) and medetomidine (0.5 mg/kg) remained anesthetized for an average of 55 min. Recovery time was approximately 6 min for the tribromoethanol (400 mg/kg) group compared with 21 min for the animals that received tribromoethanol and medetomidine. In a second study, three groups of rats were given tribromoethanol (150 mg/kg) and medetomidine (0.5 mg/kg). Group 1 received atipamezole (an alpha2-antagonist; 2.5 mg/kg i.p.) 10 min after anesthetic induction, and group 2 received the same dosage at 20 min post-induction. Group 3 was allowed to recover without atipamezole treatment. The anesthetic effects in animals from groups 1 and 2 were reversed after administration of atipamezole, whereas group 3 remained anesthetized. This study demonstrates the safe and effective use of tribromoethanol-medetomidine as an anesthetic in the rat.

Adrenergic alpha-Antagonists↗

Adverse effects of tribromoethanol as used in the production of transgenic mice.

Tribromoethanol is widely used as an anaesthetic agent for embryo-transfer surgery for the generation of transgenic mice. Potential side effects such as local irritation, fibrous adhesions in the abdominal cavity, and mortalities of unknown cause have been reported. Mice of three different strains (CD-1, OF-1, NMRI) received intraperitoneal injections of pentobarbiturate (60 mg/kg, 0.4%), tribromoethanol (240 mg/kg, 1.2%), tribromoethanol (450 mg/kg, 2.5%), ketamine/xylazine (120 mg/kg, 1.2%/16 mg/kg, 0.16%) or saline (NaCl, 0.9%). After 24 h the animals were sacrificed and blinded histopathological examination of abdominal organs was performed by light microscopy. Tribromoethanol caused focal to diffuse necrosis primarily of subperitoneal muscle fibres of the abdominal wall, and, occasionally, necrotic changes on the surface of abdominal organs. These changes were associated with acute peritoneal inflammation and fibrinous serositis of the abdominal organs. The severity of the findings increased with the concentration of tribromoethanol. The use of ketamine/xylazine yielded a comparable success rate in embryo transfer without undesirable side effects. Further use of tribromoethanol is not recommended.

Anesthetics↗

Effects of tribromoethanol anesthesia on echocardiographic assessment of left ventricular function in mice.

BACKGROUND AND PURPOSE: Pentobarbital and ketamine-xylazine anesthesia in mice result in markedly decreased left ventricular fractional shortening and cardiac output. However, to the authors' knowledge, the effect of short-acting, alcohol-based anesthesia on these parameters is unknown. METHODS: Fifteen mice (FVB/N, C57Bl/6J, A/J, n = 5 each) underwent high-resolution (15 MHz) 2-dimensional-directed M-mode echocardiography before and after undergoing 2.5% tribromoethanol anesthesia (0.01 ml/g of body weight). RESULTS: Tribromoethanol anesthesia resulted in significant heart rate slowing (29%) and left ventricular enlargement (20%), and a more modest (12%) reduction in left ventricular fractional shortening. Cardiac output was unchanged. The differences in left ventricular function between conscious and tribromoethanol studies were similar for each of the three strains of mice. CONCLUSIONS: Tribromoethanol anesthesia induced only modest effects on M-mode estimates of basal cardiac function and did not influence cardiac output. The effects to tribromoethanol anesthesia were similar among three commonly used mice strains.

Anesthesia↗

Sleep-time variation for ethanol and the hypnotic drugs tribromoethanol, urethane, pentobarbital, and propofol within outbred ICR mice.

To evaluate the phenotypic variation within a commercial outbred mouse stock, we examined sleep-time (or duration of loss of righting reflex) of outbred ICR mice after i.p. injection of ethanol (4.0 g/kg of body weight), urethane (1.3 g), tribromoethanol (250 mg), and pentobarbital (60 mg), and after i.v. injection of propofol (30 mg). We observed high-grade individual differences in sleep-time that ranged from 0 to 179 min, 83.1 +/- 4.3 (mean and SEM of 100 mice) for ethanol; 0 to 169 min, 64.5 +/- 3.1 for pentobarbital; 0 to 160 min, 36.6 +/- 3.6 for urethane; 0 to 120 min, 21.5 +/- 2.2 for tribromoethanol; and 3 to 20.5 min, 7.1 +/- 0.3 for propofol. This extensive phenotypic variance within the outbred stock was as great as the variation reported among inbred strains or selected lines, and the varied susceptibility within the colony was inherited by Jcl:ICR-derived inbred strains IAI, ICT, IPI, and IQI. The range of sleep-time variance for ethanol, pentobarbital, urethane, tribromoethanol, and propofol within four-way cross hybrid Jcl:MCH(ICR) mice was 86.6%, 63.3%, 124%, 61.0%, and 53.1% that of outbred Jcl:ICR mice, respectively. The present study indicates that phenotypic variance within an outbred Jcl:ICR stock was at high risk for susceptibility to the drugs that depress the central nervous system and that Jcl:ICR-derived inbreds may be an excellent source of animal models for studying the anesthesia gene.

Animals↗

A review of tribromoethanol anesthesia for production of genetically engineered mice and rats.

Tribromoethanol (TBE) is easy and inexpensive to make in the laboratory from readily available reagents, requires no special equipment for its administration, and is not subject to federal or state drug enforcement agency regulations. Intraperitoneal (i.p.) injection of TBE results in the simple and rapid induction of short-term surgical anesthesia; however, recent adverse reports about the efficacy and safety of TBE make its continued routine use as a rodent anesthetic controversial. The authors review the history and use of TBE as an animal anesthetic and conclude that TBE should be relegated to acute terminal studies when administered i.p.

Anesthesia, General↗

An evaluation of tribromoethanol (TBE) as an anaesthetic agent in the Mongolian gerbil (Meriones unguiculatus).

Mongolian gerbils injected i.p. with a 1.25% solution of tribromoethanol (TBE) quickly lost the righting reflex and showed good surgical anaesthesia, the duration of which was positively related to the dose administered. All levels (225-450 mg/kg bodyweight) of TBE injected s.c. resulted in loss of the righting reflex, though surgical anaesthesia was not attained. Following recovery from anaesthesia induced with 2.25% TBE i.p., or high doses of 1.25% TBE i.p., visceral adhesions and deaths occurred. To avoid these TBE-induced fatalities such extreme care must be taken during the preparation, storage and administration of the agent that it is not recommended as a suitable anaesthetic for general use in the gerbil.

Anesthesia, General↗

Fentanyl-fluanisone-midazolam combination results in more stable hemodynamics than does urethane alpha-chloralose and 2,2,2-tribromoethanol in mice.

Near-physiologic hemodynamic conditions for several hours were needed to study cardiovascular physiology in a murine model. We compared two commonly used anesthetic treatments, urethane alpha-chloralose (U-alphaCh; 968 mg U and 65 mg alphaCh/kg) and 2,2,2-tribromoethanol (TBE; 435 mg/kg) and fentanyl fluanisone midazolam (FFM; 3.313 mg fentanyl, 104.8 mg fluanisone, and 52.42 mg midazolam/kg) with respect to mean arterial blood pressure (MAP) and heart rate (HR) for 100 min at similar levels of surgical anesthesia. Assessed every 10 to 15 min, the U-alphaCh+TBE group maintained a significantly (P < 0.001) lower mean MAP (49 4 mmHg) than did the FFM group (78 5 mmHg). Mean HR in the U-alphaCh+TBE group significantly (P < 0.001) increased from 308 34 bpm at the beginning to 477 43 bpm at the end of the experiment. In comparison, the FFM group showed a stable HR of 431 37 bpm. The MAP and HR of the U-alphaCh+TBE group were extremely unstable, with sudden and unpredictable changes in MAP when examined at 1-min intervals. The results of our study show that U-alphaCh+TBE anesthesia should not be used in murine models in which stable, near-physiologic hemodynamics are needed for cardiovascular studies.

Anesthesia↗

An evaluation of preparation methods and storage conditions of tribromoethanol.

This reports the in vitro portion of a study designed to establish guidelines for the preparation, storage, and use of tribromoethanol (TBE). We evaluated: 1) the purity of TBE powder from three suppliers; 2) nine methods of preparation of a 25-mg/ml (working) solution for formation of particulates and breakdown products; 3) formation of particulates and breakdown products and pH change in 1-g/ml (stock) solutions and working solutions stored under four conditions (25 degrees C and 5 degrees C in light and in dark); and 4) stock and working solutions of TBE that caused lethal effects in mice. These objectives were met by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectroscopy, particle-size and turbidity analyses, and pH strips. TBE powder from three suppliers varied in purity. No significant differences in breakdown product formation, particle size, or turbidity were noted between the nine preparation methods evaluated. Stock solutions and the working solution stored at 5 degrees C in the dark maintained a pH of 6.5 to 7.0, whereas the pH dropped for all other working solutions. A low level of dibromoacetaldehyde (DBA), a potential breakdown product reported to cause toxic effects, was detectable in all newly prepared solutions. Regardless of the storage condition or pH, DBA concentration did not increase measurably in any of the solutions after 8 weeks. The stock and working solutions that demonstrated lethal effects in mice had a pH of 6.5 and did not differ notably from newly prepared, non-lethal solutions, when evaluated for DBA. A decrease in pH could not be correlated to an increase in DBA or potential lethality, as suggested in the literature. The toxicity associated with the lethal TBE in this study appears to be a result of a chemical reaction or breakdown product that has not yet been reported.

Acetaldehyde↗

Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice.

This study, performed in conjunction with an in vitro evaluation of tribromoethanol (TBE), consisted of three trials with three objectives. The first objective was to compare anesthetic efficacy and short-term pathologic findings of TBE, ketamine-xylazine (K-X), and sodium pentobarbital (NaP). The second objective was to evaluate how changes of TBE that occur during the perceived most favorable and least favorable storage conditions (8 weeks at 5 degrees C in the dark [5D] and 25 degrees C with exposure to light [25L], respectively) affect anesthetic efficacy and short-term pathology when compared to newly prepared TBE. The third objective was to perform a 6-week clinical assessment of animals that received newly prepared TBE. All animals that received TBE (400 mg/kg) and 14 of 15 that received K-X (K, 120 mg/kg; X, 16 mg/kg) were anesthetized, as defined by loss of pedal reflex. In comparison, only 8 of 15 animals administered NaP (60 mg/kg) were anesthetized. Anesthetic duration for animals that received K-X was 31.7 min, which was significantly (P = 0.0085) longer than animals that received TBE (18.5 min). Recovery times for TBE and K-X were not significantly different (26.5 and 27.5 min, respectively). Pathologic lesions associated with TBE administration were significantly (P = 0.001) greater than those associated with K-X. NaP was not associated with any pathologic lesions. The pH of newly prepared and 5D TBE was 6.5 to 7.0, whereas that for 25L TBE was 3.0. Anesthetic induction, duration, recovery times, and pathologic lesions were not significantly different, regardless of the pH or storage condition of the solution. It was noted, however, that the average anesthetic duration for animals administered newly prepared TBE in the second trial was longer (37.7 min) than the first trial that used newly prepared TBE. For the third trial (long-term clinical assessment), the average anesthetic duration for TBE was 46.5 min, significantly (P < 0.025) longer when compared to the first trial that used newly prepared TBE. During the third trial, 10 animals were found dead or moribund. All animals that were found moribund were necropsied and found to exhibit a marked ileus. Because of the variability in anesthetic effectiveness, pathology, and morbidity and mortality associated with the use of TBE, we do not recommend the use of this anesthetic agent in ICR mice.

Abdominal Wall↗

Comparing isoflurane with tribromoethanol anesthesia for echocardiographic phenotyping of transgenic mice.

Cardiac phenotyping of transgenic mice typically requires anesthesia. Chemical-grade tribromoethanol (TBE) is commonly used for this purpose due to its relatively short duration of action, modest cardiodepressive effects, and its noncontrolled status. In the present study, we used both genders of C57BL/6;C3H-Tg(Slc8a1)hKdp transgenic (TG) mice and C57BL/6;C3H wild-type (WT) mice to evaluate isoflurane (ISF) as a pharmaceutical-grade alternative to TBE for echocardiography and electrocardiography. Baseline target physiologic heart rates (beats per minute) were established by use of telemetry as 544 +/- 10 in WT mice and 580 +/- 21 in TG mice. TG and WT animals were anesthetized with either 0.8% to 1% inhalational ISF or 250 mg/kg intraperitoneal TBE. The following parameters were measured or calculated according to the previously defined physiologic heart rates: end diastolic and systolic dimensions; posterior wall and ventricular septal thicknesses; left ventricular mass, aortic ejection times; left ventricular fractional shortening; velocity of circumferential fiber shortening; and left ventricular ejection fraction. No significant difference between anesthetics was found for any measured cardiac parameters. However, the time required for data acquisition was significantly shorter for ISF (10 min) than for TBE (14 min). This study demonstrates that comparable echocardiographic results can be obtained at higher throughput by use of pharmaceuticalgrade ISF than with chemical-grade TBE.

Anesthetics, General↗

Comparison of tribromoethanol, ketamine/acetylpromazine, Telazol/xylazine, pentobarbital, and methoxyflurane anesthesia in HSD:ICR mice.

Variation in the duration of surgical anesthesia in mice prompted an evaluation of various commonly used anesthetics. Using biotelemetric technology, we evaluated the effects of six anesthetic regimens (tribromoethanol, ketamine and acetylpromazine in combination, Telazol and xylazine in two combinations, pentobarbital, and methoxyflurane) on temperature and activity. Six groups of four male HSD:ICR mice received one of the anesthetic regimens or an equivalent volume of saline. Induction time (time from anesthetic administration until righting reflex loss) and duration of anesthesia (loss of response to interdigital toe pinch) were evaluated. Methoxyflurane and both doses of Telazol combinations resulted in the shortest and most repeatable induction times. None of the mice in the ketamine/acetylpromazine- and pentobarbital-treated groups lost the interdigital toe pinch reflex. Duration of anesthesia was superior in the two Telazol/xylazine-treated groups. A direct correlation existed between duration of anesthesia and magnitude and duration of temperature reduction. Duration of anesthesia can be used to predict extent of hypothermia.

Acepromazine↗

Efficacy of tribromoethanol anesthesia in mice.

We undertook a retrospective study to evaluate the efficacy, safety, and suitability of tribromoethanol (0.2 ml/10 g body weight of a 1.2% solution) as a surgical anesthetic in mice. We compiled records of embryo transfer during a 2.5-year period (1989-1991) and examined mice subjected to several other procedures requiring anesthesia. We documented a low rate of mortality and morbidity (< 1%) and the absence of any significant abdominal adhesions or inflammatory response. The rapid induction and recovery, adequate surgical plane of anesthesia, and lack of complications make this anesthetic effective and simple to use. Precautions necessary to prevent decomposition of the anesthetic, storage in the dark at 4 degrees C, were minimal.

Anesthesia↗

Early effects of tribromoethanol, ketamine/xylazine, pentobarbitol, and isoflurane anesthesia on hepatic and lymphoid tissue in ICR mice.

We investigated the effects of various anesthetic agents on hepatic and splenic injury in mice. Three and six hours after intraperitoneal injection of TBE, intramuscular injection of ketamine/xylazine combination (K/X), intraperitoneal injection of pentobarbital (PB), and inhalation of isoflurane (IF), or intraperitoneal and intramuscular injection of control saline, mice were exsanguinated and serum was obtained for measurement of hepatic aspartate transaminase (AST), alanine transaminase (ALT) and gamma-glutamyltransferase (GGT). The spleen and liver also were obtained, and sections were examined by use of routine light microscopy for pathologic changes and for apoptosis, as determined by use of the in situ terminal deoxynucleotidyl transferase-mediated dUPT nick-end-labeling (TUNEL) histochemical analysis. Three hours after TBE or K/X administration, AST activity increased three- to fourfold above that in untreated and saline-injected control animals, and remained high at six hours. Administration of PB did not effect AST activity at three hours, but there was a significant increase at six hours. Activity of ALT was non-significantly increased three hours after TBE and K/X, but not PB administration. Administration of IF had no effect on hepatic enzyme activities, and GGT was not increased after administration of any of the agents. Markedly increased apoptosis was observed in splenic follicles and in hepatic Kupffer and endothelial cells at three hours after TBE and K/X administration, but apoptosis decreased to control levels by six hours. Increased apoptosis was not observed after IF administration. Administration of TBE and K/X causes injury to lymphocytes and to hepatic Kupffer and endothelial cells within three hours, and PB administration induces changes within six hours. Thus, use of these anesthetic agents should be avoided when experiments are being designed to test short-term effects of an experimental intervention on the spleen and possibly on all lymphoid tissues. In addition, they also should be avoided in experiments testing effects on hepatic tissue.

Alanine Transaminase↗