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Cho, Lee, Kim, Lee, Kim, and Han: Evaluation on Efficacy and Safety of Tribromoethanol and Tribromoethanol plus α2-Adrenergic Agonists in Different Mouse Strains
Original Article

Lab. Anim. Res. 2010;26(3):241-247.

Published online: 17 September 2010

DOI: https://doi.org/10.5625/lar.2010.26.3.241

Evaluation on Efficacy and Safety of Tribromoethanol and Tribromoethanol plus α2-Adrenergic Agonists in Different Mouse Strains

Yoon Ju Cho†, Jae Won Lee, Jong In Kim, Young Ah Lee, Tae Yeon Kim, Jin Soo Han∗

Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul, Korea

†Current address: Department of Pet Science and Veterinary Nursing, Seojeong College University, Yangju, Korea

∗Corresponding author: Jin Soo Han, Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea Tel: +82-2-2049-6114 Fax: +82-2-3437-6114 E-mail: labvet@konkuk.ac.kr

Received 13 July 2010       Revised 29 July 2010       Accepted 31 August 2010

Copyright © 2010 Korean Association for Laboratory Animal Science

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The present study was carried out to provide a guideline for injecting tribromoethanol (TBE) as the main anesthetic agent, while adjusting the doses of xylazine (X) and medetomidine (M) according to different strains of mice (male ICR, C57BL/6, and BALB/c). Seven intraperitoneal injection anesthesia protocols using TBE and mixtures of TBE and α2–adrenergic agonists (TBE/X and TBE/M) were compared in terms of their efficacy and safety (anesthetic duration, death rate, and the development of pathological lesions of abdominal organs). All animals that were injected with a low dose of TBE (200 mg/kg) displayed clear signs of light anesthesia with a strong pedal withdrawal reflex. Despite the good anesthetic effect, a high dose of TBE (400 mg/kg) was not a suitable anesthetic for major surgery in all mouse strains because of the risk of pathologic changes in the abdominal organs, such as retention of the digestive tract, peritonitis, and fibrinoid adhesion. TBE200/X10 and TBE200/M0.5 (TBE, 200 mg/kg; X, 10 mg/kg; M, 0.5 mg/kg) appeared to be safe and provided satisfactory anesthesia in ICR mice. Finally, there were clear differences in anesthetic efficacy among ICR, C57BL/6, and BALB/c strains. TBE/M and TBE/X did not anesthetize BALB/c mice, and it anesthetized C57BL/6 mice for a short time. When administered with TBE/X and TBE/M maintained the sedation of ICR mice. We were able to establish different regimes for each strain (TBE200/X20 for C57BL/6, TBE300/X10 and TBE200/M1 for BALB/c). Our results showed that TBE/X and TBE/M could be recommended as an anesthetic mixture, with the dose appropriately adjusted according to mouse strain.

Keywords: Anesthetic dose, strain difference, tribromoethanol, xylazine, medetomidine

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Figure 1.
Appearance of a mouse's abdominal cavity at 10 dpi. (A) Gross necropsy of ICR mice that received saline intraperitoneally. Normal appearance of the abdominal cavity is noted. (B) Gross necropsy of ICR mouse that received 400 mg/kg tribromoethanol (TBE) intraperitoneally. The stomach is grossly empty (thick, dark arrow). The intestine is distended with fluid and digested food, supporting a gross description of ileus (thin, dark arrow). (C) Thin abdominal wall and distension of the gastrointestinal tract found upon gross necropsy of C57BL/6 mouse that was administered 400 mg/kg TBE. (D) BALB/c mouse that received 400 mg/kg TBE showed focally extensive area of fibrous adhesion between the small intestine and cecum, which were filled with digested food (open arrow).
lar-26-241f1.tif
Figure 2.
Microscopic photographs of gastrointestinal serosa in mice at 10 dpi. (A) Normal histology of large intestine from ICR mouse that was injected with saline intraperitoneally (bar, 100 µm). (B) Histopathology of a stomach from ICR mouse that received 400 mg/kg tribromoethanol (TBE) intraperitoneally. Section demonstrates intermixed mononuclear cells with the fibrous tissue in the serosa (arrow). Hematoxylin and eosin stain; bar, 50 µm (C) Histopathology of the large intestine from a C57BL/6 mouse that received 400 mg/kg TBE intraperitoneally. The section demonstrates fibrosis and inflammation of the serosa (arrow). Hematoxylin and eosin stain; bar, 200 µm. (D) Histopathology of the large intestine from a BALB/c mouse that was administered 400 mg/kg TBE intraperitoneally. This large intestine section demonstrates mononuclear cells intermixed with the fibrous tissue (arrow). Hematoxylin and eosin stain; bar, 200 µm.
lar-26-241f2.tif
Table 1.
Guidelines for tribromoethanol anesthesia in several facilities or literatures in mice
Dose∗ (mg/kg) References
  • NIH Anesthesia/Analgesia formulary
  • Cornell Center for Animal Resources and Education, in Cornell University
  • Animal Care Facility in Laurentian University
125-250 • Unit for Laboratory Animal Medicine in University of Michigan Medical School
  • Laboratory Animal Research Center in Samsung Biomedical Research Institute, Korea
  • Laboratory animal research center in Chungbuk National university, Korea
125-300 • (Flecknell, 2005)
  • (Fish et al., 2008)
125-400 • Safety Services in University of California, Davis
225-240 • Research Animal Resources in University of Minnesota
  • Guidelines in Duke University and Medical Center
250 • Guidelines in University of Rochestar Medical Center
  • (Hedenqvist et al., 2003)
250-500 • IACUC in University of California
300-600 • Animal Resource Center in Case Western Reserve University
310 • IACUC in University of Tennessee
360-800 • Guidelines in Baylor College of Medicine
400-600 • Office of the Campus Veterinarian in Washington State university
400-750 • Veterinary Services in Florida Atlantic University

∗ intraperitoneal injection

Table 2.
Summary of experiments and number of animals used
Protocol Substance Dose (mg/kg) Number of animals
ICR C57BL/6 BALB/c
sham PBS 0.5 mL/animal 5 5 5
P1 1.25% TBE 200 10 10 10
P2 2.50% TBE 400 10 10 10
P3 1.25% TBE+X 200+10 10 10 10
P4 1.25% TBE+M 200+0.5 10 10 10
P5 1.25% TBE+X 200+20 ND 10 ND
P6 1.25% TBE+X 300+10 ND ND 10
P7 1.25% TBE+M 200+1 ND ND 10

PBS, phosphate-buffered saline; TBE, tribromoethanol; X, xylazine (Rompun, Bayer HealthCare, Seoul, Korea); M, medetomidine (Domitor, Pfizer Animal Health Korea Ltd., Seoul, Korea); ND, not done.

Table 3.
Efficacy and safety of anesthesia after administration of each different regimes
      P2a     P3b     P4c   P5d P6e P7f
    ICR C57BL/6 BALB/c ICR C57BL/6 BALB/c ICR C57BL/6 BALB/c C57BL/6 BALB/c BALB/c
    ICR C57BL/6 BALB/c ICR C57BL/6 BALB/c ICR C57BL/6 BALB/c C57BL/6 BALB/c BALB/c
ANES rate (n/n) 10/10 10/10 4/10 10/10 10/10 0/10 8/10 10/10 7/10 10/10 8/10 10/10
  INDg 01:21 01:39 03:51 02:53 03:18 NA 02:54 02:04 07:10 03:23 03:30 04:16
  ±00:07 ±00:05 ±00:26∗† ±00:32 ±00:36 ±00:46 ±00:16 ±00:41∗† † ±00:19 ±00:23 ±00:25
Mean ±SEM (mm:ss) ANESh 39:05 31:02 08:48 39:28 13:26 NA 65:38 73:59 17:39 27:03 59:15 24:43
±03:01 ±03:31 ±01:24∗† ±05:08 ±00:56∗ ±06:40 ±07:34∗ ±04:06∗† † ±04:28 ±06:37 ±02:53
  RECi 26:45 51:31 27:29 09:13 18:49 NAj 48:28 42:26 73:04 23:34 41:31 57:35
  ±04:41 ±02:54∗ ±04:47† ±03:00 ±02:12∗ ±08:51 ±05:21∗ ±05:31† ±02:38 ±06:23 ±05:58
Death rate 0/10 0/10 0/10 0/10 0/10 0/10 2/10 0/10 0/10 0/10 2/10 0/10
Gross lesion 1 dpik 2/5 5/5 5/5 0/5 0/5 0/5 0/4 0/5 0/5 0/5 0/4 0/5
10 dpi 4/5 4/5 4/5 0/5 0/5 0/5 0/4 0/5 0/5 0/5 0/4 0/5

Data are presented as mean values and standard errors of anesthetized animals.

a tribromoethanol (TBE) 400 mg/kg;

b TBE 200 mg/ kg+xylazine 10 mg/kg;

c TBE 200 mg/kg+medetomidine 0.5 mg/kg;

d TBE 200 mg/kg+xylazine 20 mg/kg;

e TBE 300 mg/kg+xylazine 10 mg/kg;

f TBE 200 mg/kg+medetomidine 1 mg/kg;

g induction time;

h anesthetic duration;

i recovery time;

j not applicable (strong pedal withdrawal reflex);

k days of post injection;

† P<0.05 versus values for C57BL/6 at the same protocol (one-way ANOVA, Tukey-Kramer multiple comparisons test).

∗ P<0.05 versus values for ICR group at the same protocol;

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