The reporting of animal welfare details in liver research: A review of studies describing bile duct ligation in mice (2011–2013)
Authors: Judith Secklehner, Claire A. Richardson
Journal: Journal of Hepatology (2014)

Studies involving animals play a key role in basic liver research and are most commonly carried out in laboratory mice. For example, bile duct ligation (BDL) is frequently carried out to model a range of hepatic pathologies including fibrosis and cholestasis [[1]Georgiev P. Jochum W. Heinrich S. Jang J.H. Nocito A. Dahm F. et al.Characterization of time-related changes after experimental bile duct ligation.Br J Surg. 2008; 95: 646-656Crossref PubMed Scopus (171) Google Scholar]. When animals are used experimentally it is widely accepted that procedures should be refined to alleviate pain and distress [[2]Russell W.M.S. Burch R.L. The Principles of Humane Experimental Technique. Methuen, London1959Google Scholar]. Promoting laboratory animal welfare is not only of ethical value but of considerable scientific importance to minimize variability and improve validity of research involving animals [2Russell W.M.S. Burch R.L. The Principles of Humane Experimental Technique. Methuen, London1959Google Scholar, 3Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar, 4Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar].Given the clear importance of refining experimental procedures, we conducted a literature review to assess the reporting of details related to mouse welfare in studies that involved BDL in mice. Although the BDL procedure is widely used in rats and has been well characterized for many years [[5]Johnstone J.M. Lee E.G. A quantitative assessment of the structural changes in the rat's liver following the obstruction of the common bile duct.Br J Exp Pathol. 1976; 57: 85-94PubMed Google Scholar], it is increasingly carried out in mice because of the number of genetically altered mouse strains now available [[1]Georgiev P. Jochum W. Heinrich S. Jang J.H. Nocito A. Dahm F. et al.Characterization of time-related changes after experimental bile duct ligation.Br J Surg. 2008; 95: 646-656Crossref PubMed Scopus (171) Google Scholar]. The search was performed in July 2014, using the search engine SCOPUS and search terms "mouse OR mice" and "bile duct ligation OR BDL" within keyword, title and abstract to identify relevant studies, published in English between 2011 and 2013. A paper was eligible for inclusion if it described mice undergoing experimental BDL and was available in electronic format from Newcastle University. Conference papers, letters and reviews were excluded. Papers (including supplementary data) were then carefully screened to identify details relating to animal welfare.119 papers met our inclusion criteria (34 from 2011, 39 from 2012 and 46 from 2013); journals in which they were published are summarized in Table 1. Papers most commonly described mechanisms of cholestatic liver injury (66.4%) and therapeutic or toxic substances (17.6%). Transgenic mice were studied in 55.5% of the papers. The BDL procedure was carried out in North America (39%), Europe (27%) or Asia (24%). 10% of studies involved multiple institutions and the place where the animal work was carried out was not specified. 94% of papers made a statement related to ethical guidelines on the humane use of animals, with 86.6% of these studies referring to local ethical approval. The "Guide for the Care and Use of Laboratory Animals" [[6]NRC. Guide for the care and use of laboratory animals. 8th ed. National Academies Press; 2011:246.Google Scholar] was the most frequently cited guideline relating to animal care (22.7% of papers).Table 1Journals included in review.∗American Journal of Physiology, Behavioural Brain Research, BMC Gastroenterology, Clinical and Experimental Medicine, European Radiology, Food and Chemical Toxicology, Gut, Gut Microbes, Journal of Clinical Investigation, Journal of Gastrointestinal Surgery, Journal of Hepato-Biliary-Pancreatic Sciences, Journal of Immunology, Journal of Toxicological Sciences, Journal of Leukocyte Biology, Journal of Nuclear Medicine, Journal of Pharmacology, Journal of Surgical Research, Molecular Nutrition and Food Research, Molecular Biology Reports, Molecular and Cellular Biology, Pathophysiology, Peptides, Pharmaceutical Biology, PLoS Genetics, Proceedings of the National Academy of Sciences of the United States of America Plus, Psychopharmacology, Surgery, Toxicological Sciences, Toxicology, Toxicology and Applied Pharmacology. Open table in a new tab Surgical and anaesthetic details were described in 57% of the papers, whereas 43% of the papers only referred to methods described in previous studies. Of the papers citing previously published methodologies, 26.7% referred to studies carried out in rats, which is problematic given the species differences (e.g. the gallbladder is present in mice and absent in rats). When methods were not described, we recorded anaesthetic and analgesic details from papers whose methodologies had been cited. Overall, only 3.4% of studies describing BDL in mice specified administration of a systemic analgesic agent (non-steroidal anti-inflammatory or opioid agent); administration of buprenorphine (n = 3) and fentanyl (n = 1) was reported in 2 papers from North America and in 2 from Europe. Administration of a systemic analgesic agent to mice undergoing BDL is considerably lower than has been previously reported for mice and rats, undergoing a range of surgical procedures (typically 20%) [4Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar, 7Stokes E.L. Flecknell P.A. Richardson C.A. Reported analgesic and anaesthetic administration to rodents undergoing experimental surgical procedures.Lab Anim. 2009; 43: 149-154Crossref PubMed Scopus (143) Google Scholar]. This is surprising given that BDL is a painful procedure in rodents, although pain can be alleviated through systemic analgesic administration [[8]Liles J.H. Flecknell P.A. The influence of buprenorphine or bupivacaine on the post-operative effects of laparotomy and bile-duct ligation in rats.Lab Anim. 1993; 27: 374-380Crossref PubMed Scopus (52) Google Scholar]. Although it is possible that systemic analgesics were administered in these studies, but not reported (e.g. investigators may view analgesic administration as implicit in their statement relating to ethical guidelines), earlier work suggests that under-reporting of analgesic administration is relatively low [[4]Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar]. Furthermore, the reporting of details in animal research should be comprehensive to allow experiments to be repeated [[9]Kilkenny C. Browne W.J. Cuthill I.C. Emerson M. Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.PLoS Biol. 2010; 8: e1000412Crossref PubMed Scopus (4530) Google Scholar]. Space restraints for reporting methodological details in some journals, particularly those with a high impact factor, may also be perceived as an obstacle for some authors however, an increasing number of journals allow authors to include supplementary data. Alternatively, papers which report animal welfare information in detail could be cited with any modifications to the published protocol clearly described. Analgesics may have been withheld if investigators had concerns about potential effects of the analgesics, however, uncontrolled post-surgical pain is a source of considerable experimental variability [[4]Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar] and not a feature of cholestatic liver disease in most human patients.Thirty percent of studies had no information concerning the anaesthetic protocol used. When the anaesthetic regimen was specified, the most common agents were: ketamine/xylazine (30%), isoflurane (29%), pentobarbital (18%) and ether (12%). Only 27.7% of studies described the administration of an anaesthetic agents with analgesic properties (ketamine, alfentanyl, tribromoethanol, tiletamine). Three of the anaesthetic regimens used give rise to potential animal welfare concerns: irritancy on induction (ether), narrow safety margin (pentobarbital) and unpredictable adverse effects (tribromoethanol) [3Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar, 4Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar].As bile duct ligation induces progressive disease and is associated with considerable morbidity and mortality [[1]Georgiev P. Jochum W. Heinrich S. Jang J.H. Nocito A. Dahm F. et al.Characterization of time-related changes after experimental bile duct ligation.Br J Surg. 2008; 95: 646-656Crossref PubMed Scopus (171) Google Scholar], refinement of the BDL procedure should not just be limited to the peri-operative period and appropriate monitoring/supportive care is essential throughout the study [3Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar, 10Hunter J. Butterworth J. Perkins N. Bateson M. Richardson C. Using body temperature, food and water consumption as biomarkers of disease progression in mice with Eμ-myc lymphoma.Br J Cancer. 2014; 110: 928-934Crossref PubMed Scopus (15) Google Scholar]. Although 6.7% of studies specified saline injections and 5% described clinical monitoring, relatively few studies reported the provision of supportive care. A warm environment and soaked diet is generally beneficial to sick animals [[3]Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar], but only 16% of studies reported the room temperature, which was not increased after surgery (18–21 °C [2.5%], 21–23 °C [13.5%]) and provision of a soaked diet was not reported. Experimental and humane end points are also particularly important in progressive disease studies where pain and distress are more likely to occur as disease progresses [[11]Franco N.H. Correla-Neves M. Olsson I.A.S. How "humane" is your endpoint? – Refining the science-driven approach for termination of animal studies of chronic infection.PLoS Pathog. 2012; 8: e1002399Crossref PubMed Scopus (36) Google Scholar]. Although a small number of studies (less than 3%) continued for more than 6 weeks following BDL, when mice would have developed advanced disease, the majority (95%) specified clear experimental end points with 95% of end points at 4 weeks or earlier. Humane end points were referred to in one paper.Given the clear importance of the humane treatment of laboratory animals with respect to both animal welfare and the translation of in vivo studies to human patients with liver disease, we would encourage investigators, carrying out research involving animals, to implement refinements and to report the measures taken to minimise pain and distress according to the ARRIVE guidelines [[9]Kilkenny C. Browne W.J. Cuthill I.C. Emerson M. Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.PLoS Biol. 2010; 8: e1000412Crossref PubMed Scopus (4530) Google Scholar]. Specific recommendations for animals that have undergone surgery include provision and reporting of an appropriate systemic analgesic, or the rationale if analgesics had been withheld. Finally, both the anaesthetic regimen and refinement details relating to the post-operative period (e.g. monitoring, nursing care and humane end points) should be specified.Financial supportCAR is funded by a National Centre for the Replacement, Refinement and Reduction of Animals in Research David Sainsbury Fellowship (grant NC/K500513/1 ).Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Studies involving animals play a key role in basic liver research and are most commonly carried out in laboratory mice. For example, bile duct ligation (BDL) is frequently carried out to model a range of hepatic pathologies including fibrosis and cholestasis [[1]Georgiev P. Jochum W. Heinrich S. Jang J.H. Nocito A. Dahm F. et al.Characterization of time-related changes after experimental bile duct ligation.Br J Surg. 2008; 95: 646-656Crossref PubMed Scopus (171) Google Scholar]. When animals are used experimentally it is widely accepted that procedures should be refined to alleviate pain and distress [[2]Russell W.M.S. Burch R.L. The Principles of Humane Experimental Technique. Methuen, London1959Google Scholar]. Promoting laboratory animal welfare is not only of ethical value but of considerable scientific importance to minimize variability and improve validity of research involving animals [2Russell W.M.S. Burch R.L. The Principles of Humane Experimental Technique. Methuen, London1959Google Scholar, 3Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar, 4Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar]. Given the clear importance of refining experimental procedures, we conducted a literature review to assess the reporting of details related to mouse welfare in studies that involved BDL in mice. Although the BDL procedure is widely used in rats and has been well characterized for many years [[5]Johnstone J.M. Lee E.G. A quantitative assessment of the structural changes in the rat's liver following the obstruction of the common bile duct.Br J Exp Pathol. 1976; 57: 85-94PubMed Google Scholar], it is increasingly carried out in mice because of the number of genetically altered mouse strains now available [[1]Georgiev P. Jochum W. Heinrich S. Jang J.H. Nocito A. Dahm F. et al.Characterization of time-related changes after experimental bile duct ligation.Br J Surg. 2008; 95: 646-656Crossref PubMed Scopus (171) Google Scholar]. The search was performed in July 2014, using the search engine SCOPUS and search terms "mouse OR mice" and "bile duct ligation OR BDL" within keyword, title and abstract to identify relevant studies, published in English between 2011 and 2013. A paper was eligible for inclusion if it described mice undergoing experimental BDL and was available in electronic format from Newcastle University. Conference papers, letters and reviews were excluded. Papers (including supplementary data) were then carefully screened to identify details relating to animal welfare. 119 papers met our inclusion criteria (34 from 2011, 39 from 2012 and 46 from 2013); journals in which they were published are summarized in Table 1. Papers most commonly described mechanisms of cholestatic liver injury (66.4%) and therapeutic or toxic substances (17.6%). Transgenic mice were studied in 55.5% of the papers. The BDL procedure was carried out in North America (39%), Europe (27%) or Asia (24%). 10% of studies involved multiple institutions and the place where the animal work was carried out was not specified. 94% of papers made a statement related to ethical guidelines on the humane use of animals, with 86.6% of these studies referring to local ethical approval. The "Guide for the Care and Use of Laboratory Animals" [[6]NRC. Guide for the care and use of laboratory animals. 8th ed. National Academies Press; 2011:246.Google Scholar] was the most frequently cited guideline relating to animal care (22.7% of papers). ∗American Journal of Physiology, Behavioural Brain Research, BMC Gastroenterology, Clinical and Experimental Medicine, European Radiology, Food and Chemical Toxicology, Gut, Gut Microbes, Journal of Clinical Investigation, Journal of Gastrointestinal Surgery, Journal of Hepato-Biliary-Pancreatic Sciences, Journal of Immunology, Journal of Toxicological Sciences, Journal of Leukocyte Biology, Journal of Nuclear Medicine, Journal of Pharmacology, Journal of Surgical Research, Molecular Nutrition and Food Research, Molecular Biology Reports, Molecular and Cellular Biology, Pathophysiology, Peptides, Pharmaceutical Biology, PLoS Genetics, Proceedings of the National Academy of Sciences of the United States of America Plus, Psychopharmacology, Surgery, Toxicological Sciences, Toxicology, Toxicology and Applied Pharmacology. Surgical and anaesthetic details were described in 57% of the papers, whereas 43% of the papers only referred to methods described in previous studies. Of the papers citing previously published methodologies, 26.7% referred to studies carried out in rats, which is problematic given the species differences (e.g. the gallbladder is present in mice and absent in rats). When methods were not described, we recorded anaesthetic and analgesic details from papers whose methodologies had been cited. Overall, only 3.4% of studies describing BDL in mice specified administration of a systemic analgesic agent (non-steroidal anti-inflammatory or opioid agent); administration of buprenorphine (n = 3) and fentanyl (n = 1) was reported in 2 papers from North America and in 2 from Europe. Administration of a systemic analgesic agent to mice undergoing BDL is considerably lower than has been previously reported for mice and rats, undergoing a range of surgical procedures (typically 20%) [4Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar, 7Stokes E.L. Flecknell P.A. Richardson C.A. Reported analgesic and anaesthetic administration to rodents undergoing experimental surgical procedures.Lab Anim. 2009; 43: 149-154Crossref PubMed Scopus (143) Google Scholar]. This is surprising given that BDL is a painful procedure in rodents, although pain can be alleviated through systemic analgesic administration [[8]Liles J.H. Flecknell P.A. The influence of buprenorphine or bupivacaine on the post-operative effects of laparotomy and bile-duct ligation in rats.Lab Anim. 1993; 27: 374-380Crossref PubMed Scopus (52) Google Scholar]. Although it is possible that systemic analgesics were administered in these studies, but not reported (e.g. investigators may view analgesic administration as implicit in their statement relating to ethical guidelines), earlier work suggests that under-reporting of analgesic administration is relatively low [[4]Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar]. Furthermore, the reporting of details in animal research should be comprehensive to allow experiments to be repeated [[9]Kilkenny C. Browne W.J. Cuthill I.C. Emerson M. Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.PLoS Biol. 2010; 8: e1000412Crossref PubMed Scopus (4530) Google Scholar]. Space restraints for reporting methodological details in some journals, particularly those with a high impact factor, may also be perceived as an obstacle for some authors however, an increasing number of journals allow authors to include supplementary data. Alternatively, papers which report animal welfare information in detail could be cited with any modifications to the published protocol clearly described. Analgesics may have been withheld if investigators had concerns about potential effects of the analgesics, however, uncontrolled post-surgical pain is a source of considerable experimental variability [[4]Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar] and not a feature of cholestatic liver disease in most human patients. Thirty percent of studies had no information concerning the anaesthetic protocol used. When the anaesthetic regimen was specified, the most common agents were: ketamine/xylazine (30%), isoflurane (29%), pentobarbital (18%) and ether (12%). Only 27.7% of studies described the administration of an anaesthetic agents with analgesic properties (ketamine, alfentanyl, tribromoethanol, tiletamine). Three of the anaesthetic regimens used give rise to potential animal welfare concerns: irritancy on induction (ether), narrow safety margin (pentobarbital) and unpredictable adverse effects (tribromoethanol) [3Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar, 4Richardson C.A. Flecknell P.A. Anaesthesia and post-operative analgesia following experimental surgery in laboratory rodents: are we making progress?.Altern Lab Anim. 2005; 33: 119-127PubMed Google Scholar]. As bile duct ligation induces progressive disease and is associated with considerable morbidity and mortality [[1]Georgiev P. Jochum W. Heinrich S. Jang J.H. Nocito A. Dahm F. et al.Characterization of time-related changes after experimental bile duct ligation.Br J Surg. 2008; 95: 646-656Crossref PubMed Scopus (171) Google Scholar], refinement of the BDL procedure should not just be limited to the peri-operative period and appropriate monitoring/supportive care is essential throughout the study [3Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar, 10Hunter J. Butterworth J. Perkins N. Bateson M. Richardson C. Using body temperature, food and water consumption as biomarkers of disease progression in mice with Eμ-myc lymphoma.Br J Cancer. 2014; 110: 928-934Crossref PubMed Scopus (15) Google Scholar]. Although 6.7% of studies specified saline injections and 5% described clinical monitoring, relatively few studies reported the provision of supportive care. A warm environment and soaked diet is generally beneficial to sick animals [[3]Flecknell P.A. Laboratory animal anaesthesia.3rd ed. Elsevier, Amsterdam2009Google Scholar], but only 16% of studies reported the room temperature, which was not increased after surgery (18–21 °C [2.5%], 21–23 °C [13.5%]) and provision of a soaked diet was not reported. Experimental and humane end points are also particularly important in progressive disease studies where pain and distress are more likely to occur as disease progresses [[11]Franco N.H. Correla-Neves M. Olsson I.A.S. How "humane" is your endpoint? – Refining the science-driven approach for termination of animal studies of chronic infection.PLoS Pathog. 2012; 8: e1002399Crossref PubMed Scopus (36) Google Scholar]. Although a small number of studies (less than 3%) continued for more than 6 weeks following BDL, when mice would have developed advanced disease, the majority (95%) specified clear experimental end points with 95% of end points at 4 weeks or earlier. Humane end points were referred to in one paper. Given the clear importance of the humane treatment of laboratory animals with respect to both animal welfare and the translation of in vivo studies to human patients with liver disease, we would encourage investigators, carrying out research involving animals, to implement refinements and to report the measures taken to minimise pain and distress according to the ARRIVE guidelines [[9]Kilkenny C. Browne W.J. Cuthill I.C. Emerson M. Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.PLoS Biol. 2010; 8: e1000412Crossref PubMed Scopus (4530) Google Scholar]. Specific recommendations for animals that have undergone surgery include provision and reporting of an appropriate systemic analgesic, or the rationale if analgesics had been withheld. Finally, both the anaesthetic regimen and refinement details relating to the post-operative period (e.g. monitoring, nursing care and humane end points) should be specified. Financial supportCAR is funded by a National Centre for the Replacement, Refinement and Reduction of Animals in Research David Sainsbury Fellowship (grant NC/K500513/1 ). CAR is funded by a National Centre for the Replacement, Refinement and Reduction of Animals in Research David Sainsbury Fellowship (grant NC/K500513/1 ). Conflict of interestThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.