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Not All Mice Are the Same: Standardization of Animal Research Data Presentation
COMMENTARY Not All Mice Are the Same: Standardization of Animal Research Data Presentation
A
s the editors of several journals, we have joined forces to highlight the importance of providing essential details related to animal experiments, particularly for studies that include mouse work. This is a critical issue that partially underlies the problem of irreproducible results that is attracting international attention1 as well as the attention of funding agencies such as the National Institutes of Health.2 For mouse studies, this can be a daunting problem given that many manuscripts do not provide sufficient details regarding the number of animals used for a given experiment, the sex of the animals, their age, and in some cases identification of the background genetic strains. Other variables that can also play an important role in shaping experimental findings and conclusions are the microbiome,3 making co-housing of control and genetically altered animals essential, diet, and even the composition of animal bedding.4 Several journals have supported the ARRIVE (Animal Research: Reporting of In Vivo Experiments) Guidelines that were originally proposed in 2010,5 which include an extensive checklist of information related to animal experiments that is considered essential to provide. However, it seems that the reporting standards may not have improved
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very much since initial presentation and acceptance of the guidelines by multiple journals.6 Several reasons may account for the observed ‘noncompliance’ with the ARRIVE guidelines,6 including difficulty ensuring that reviewers and editors carefully assess whether the guidelines have been followed, and the possibility that some authors may indicate that the guidelines have been followed (based on author interpretation and not because of any malintent) when in reality not all components have been pursued. Reviewers and editors frequently ask authors to expand on some of these necessary details, but the reviewers often focus on separate important issues while the specifics that are related to the mouse work may be overlooked. Another barrier might be the extensive nature of the ARRIVE checklist, which may not apply fully to many submitted manuscripts. This is relevant, because scientists (and physicians) are now facing an increasing barrage of regulatory documentation paperwork that is limiting their time for scientific investigation (or for their patients). Notwithstanding this limitation, we are uniting from different journals to highlight the importance of documenting what we consider to be the minimum list of information to improve transparency and the quality of data reporting. Our purpose is not to legislate a “one size fits all” philosophy, but rather to maximize the possibility of other researchers reproducing study findings from the same wild-type or mutant mouse strains. Our respective journals will be highlighting the criteria listed below in our Instructions to
Figure 1. Key elements to consider and highlight for mouse related studies. The schematic shows several important criteria that need to be considered when planning mouse (and other animal) experiments, and when submitting work for publication. FVB and C57BL/6 represent, as examples, commonly used mouse strains. Of note, vendor sources can also be important (eg, FVB/NJ from The Jackson Laboratory vs FVB/NTac from Taconic). Other considerations not displayed in the schematic, such as environment conditions, are highlighted in the text. þ/þ, wild-type littermate mice; -/-, knockout littermate mice. Cellular and Molecular Gastroenterology and Hepatology 2016;-:-–EDI 5.4.0 DTD JCMGH125 proof 5 May 2016 1:07 pm ce
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Commentary
Cellular and Molecular Gastroenterology and Hepatology Vol.
Authors, and some of our journals will also introduce an author-friendly checklist that will need to accompany manuscripts that use mice and other in vivo experimental models. The criteria that will be expected of authors include the following information (Figure 1): Sex and age of mice (or other in vivo experimental models) for all the experiments; The genetic background(s) of the mice or other experimental in vivo models; For transgenic or genetic mouse models, whether the controls were sibling littermates or were purchased separately (if purchased separately, were the animals cohoused to minimize potential microbiome effects); Specifics of the animal diet composition; Whether mice were fasted (and for how long) or not before a challenge or assessment is carried out; Type of bedding, caging system, and enrichment used for housing the mice; and If interventions were done, were they done during the light or dark cycle. Power analyses can be useful to estimate appropriate sample sizes; however, the standard deviations for relevant dependent variables are often not known a priori. Therefore, it is critical that the number of animals for each experimental arm or condition is reported together with biological replication of statistically significant results derived from independent groups of animals. There are also other variables that may need to be considered that we have not included as primary expectations, particularly those related to behavior, stress, and growth conditions. These variables include acclimation to a new environment (eg, when animals are shipped by the vendor or moved from the animal facility to a procedure room), other environment effects (eg, temperature, humidity, noise), littermate size, and pheromone effects. These additional variables can be very important depending on the biologic readout. Moreover, environment effects, such as avoidance of large temperature changes unless approved as part of an animal protocol, are expected to be part of the normal procedures of humane treatment of animals. What can journals and research institutions do moving forward toward a path to implementation?7 Certainly all stakeholders need to be engaged and many funding agencies now expect that applicants pay closer attention to this important issue, as exemplified by the recent requirement from the National Institutes of Health to include a section titled Authentication of Key Biological and/or Chemical Resources in grant applications.8 For journals, setting and enforcing clear expectations to authors, reviewers, and editors will be essential, because a checklist alone will not be sufficient, while making the process as user-friendly as possible. For institutions, several approaches can be
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No.
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176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 Q1193 M. BISHR OMARY Gastroenterology 194 195 DAVID E. COHEN 196 Hepatology 197 198 EMAD M. EL-OMAR 199 Gut 200 201 RAJIV JALAN 202 Journal of Hepatology 203 204 MALCOLM J. LOW 205 Gastroenterology 206 207 MICHAEL H. NATHANSON 208 Hepatology 209 RICHARD M. PEEK JR. 210 Gastroenterology 211 212 JERROLD R. TURNER 213 Cellular and Molecular Gastroenterology and Hepatology 214 215 216 References 217 1. Reardon SA. mouse’s house may ruin studies. Nature 218 2016;530:264. 219 2. Collins FS, Tabak LA. NIH plans to enhance reproduc- 220 ibility. Nature 2014;505:612–613. 221 3. Laukens D, Brinkman BM, Raes J, et al. Heterogeneity of 222 the gut microbiome in mice: guidelines for optimizing 223 experimental design. FEMS Microbiol Rev 2016;40: 224 117–132. 225 4. Ambery AG, Tackett L, Penque BA, et al. Effect of 226 Corncob bedding on feed conversion efficiency in a high227 fat diet-induced prediabetic model in C57Bl/6J mice. 228 J Am Assoc Lab Anim Sci 2014;53:449–451. 229 5. Kilkenny C, Browne WJ, Cuthill IC, et al. The ARRIVE 230 Guidelines Checklist: animal research: reporting in vivo ex231 periments. Available. www.nc3rs.org.uk/sites/default/files/ 232 documents/Guidelines/NC3Rs%20 ARRIVE%20Guide 233 lines%20Check list%20(fillable).pdf. 234 considered and implemented, that are aimed at investigators and trainees. For example, the curricula for students who are enrolled in bioscience-related undergraduate and graduate programs should include training not only in the ethics in conducting research, but also in the basic tenets of conducting and designing animal experiments. It is important for this to start early and to be reinforced as training advances. Similarly, postdoctoral fellows in biomedical disciplines should be expected to enroll in similar workshops that would be offered by their home institutions. For such workshops, centralized (rather than department- or unit-specific) oversight and administration will more likely ensure uniformity and implementation. We look forward to working together on this important effort and to receiving feedback from our authors, reviewers and readers.
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6.
7.
8.
2016
Commentary
Baker D, Lidster K, Sottomayer A, et al. Two years later: journals are not yet enforcing the ARRIVE guidelines on reporting standards for pre-clinical animal studies. PLoS Biol 2014;12:e1001756. Landis SC, Amara SG, Asadullah K, et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature 2012;490: 187–191. National Institutes of Health (NIH), Agency for Healthcare Research and Quality (AHRQ). Implementing rigor and transparency in NIH & AHRQ research grant applications. October 9, 2015. Available: http://grants.nih.gov/grants/ guide/noticefiles/NOT-OD-16–011.html.
3
Acknowledgments The authors thank the managing editors of our journals for helping facilitate the joint publication of this commentary. All authors contributed equally to this effort. This commentary is being jointly published by Cellular and Molecular Gastroenterology and Hepatology, Gastroenterology, Gut, Hepatology, and Journal of Hepatology. Conflicts of interest The authors disclose no conflicts.
© 2016 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X http://dx.doi.org/10.1016/j.jcmgh.2016.04.001
EDI 5.4.0 DTD JCMGH125 proof 5 May 2016 1:07 pm ce
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A
s the editors of several journals, we have joined forces to highlight the importance of providing essential details related to animal experiments, particularly for studies that include mouse work. This is a critical issue that partially underlies the problem of irreproducible results that is attracting international attention1 as well as the attention of funding agencies such as the National Institutes of Health.2 For mouse studies, this can be a daunting problem given that many manuscripts do not provide sufficient details regarding the number of animals used for a given experiment, the sex of the animals, their age, and in some cases identification of the background genetic strains. Other variables that can also play an important role in shaping experimental findings and conclusions are the microbiome,3 making co-housing of control and genetically altered animals essential, diet, and even the composition of animal bedding.4 Several journals have supported the ARRIVE (Animal Research: Reporting of In Vivo Experiments) Guidelines that were originally proposed in 2010,5 which include an extensive checklist of information related to animal experiments that is considered essential to provide. However, it seems that the reporting standards may not have improved
web 4C=FPO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
very much since initial presentation and acceptance of the guidelines by multiple journals.6 Several reasons may account for the observed ‘noncompliance’ with the ARRIVE guidelines,6 including difficulty ensuring that reviewers and editors carefully assess whether the guidelines have been followed, and the possibility that some authors may indicate that the guidelines have been followed (based on author interpretation and not because of any malintent) when in reality not all components have been pursued. Reviewers and editors frequently ask authors to expand on some of these necessary details, but the reviewers often focus on separate important issues while the specifics that are related to the mouse work may be overlooked. Another barrier might be the extensive nature of the ARRIVE checklist, which may not apply fully to many submitted manuscripts. This is relevant, because scientists (and physicians) are now facing an increasing barrage of regulatory documentation paperwork that is limiting their time for scientific investigation (or for their patients). Notwithstanding this limitation, we are uniting from different journals to highlight the importance of documenting what we consider to be the minimum list of information to improve transparency and the quality of data reporting. Our purpose is not to legislate a “one size fits all” philosophy, but rather to maximize the possibility of other researchers reproducing study findings from the same wild-type or mutant mouse strains. Our respective journals will be highlighting the criteria listed below in our Instructions to
Figure 1. Key elements to consider and highlight for mouse related studies. The schematic shows several important criteria that need to be considered when planning mouse (and other animal) experiments, and when submitting work for publication. FVB and C57BL/6 represent, as examples, commonly used mouse strains. Of note, vendor sources can also be important (eg, FVB/NJ from The Jackson Laboratory vs FVB/NTac from Taconic). Other considerations not displayed in the schematic, such as environment conditions, are highlighted in the text. þ/þ, wild-type littermate mice; -/-, knockout littermate mice. Cellular and Molecular Gastroenterology and Hepatology 2016;-:-–EDI 5.4.0 DTD JCMGH125 proof 5 May 2016 1:07 pm ce
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116
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117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175
Commentary
Cellular and Molecular Gastroenterology and Hepatology Vol.
Authors, and some of our journals will also introduce an author-friendly checklist that will need to accompany manuscripts that use mice and other in vivo experimental models. The criteria that will be expected of authors include the following information (Figure 1): Sex and age of mice (or other in vivo experimental models) for all the experiments; The genetic background(s) of the mice or other experimental in vivo models; For transgenic or genetic mouse models, whether the controls were sibling littermates or were purchased separately (if purchased separately, were the animals cohoused to minimize potential microbiome effects); Specifics of the animal diet composition; Whether mice were fasted (and for how long) or not before a challenge or assessment is carried out; Type of bedding, caging system, and enrichment used for housing the mice; and If interventions were done, were they done during the light or dark cycle. Power analyses can be useful to estimate appropriate sample sizes; however, the standard deviations for relevant dependent variables are often not known a priori. Therefore, it is critical that the number of animals for each experimental arm or condition is reported together with biological replication of statistically significant results derived from independent groups of animals. There are also other variables that may need to be considered that we have not included as primary expectations, particularly those related to behavior, stress, and growth conditions. These variables include acclimation to a new environment (eg, when animals are shipped by the vendor or moved from the animal facility to a procedure room), other environment effects (eg, temperature, humidity, noise), littermate size, and pheromone effects. These additional variables can be very important depending on the biologic readout. Moreover, environment effects, such as avoidance of large temperature changes unless approved as part of an animal protocol, are expected to be part of the normal procedures of humane treatment of animals. What can journals and research institutions do moving forward toward a path to implementation?7 Certainly all stakeholders need to be engaged and many funding agencies now expect that applicants pay closer attention to this important issue, as exemplified by the recent requirement from the National Institutes of Health to include a section titled Authentication of Key Biological and/or Chemical Resources in grant applications.8 For journals, setting and enforcing clear expectations to authors, reviewers, and editors will be essential, because a checklist alone will not be sufficient, while making the process as user-friendly as possible. For institutions, several approaches can be
-,
No.
-
176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 Q1193 M. BISHR OMARY Gastroenterology 194 195 DAVID E. COHEN 196 Hepatology 197 198 EMAD M. EL-OMAR 199 Gut 200 201 RAJIV JALAN 202 Journal of Hepatology 203 204 MALCOLM J. LOW 205 Gastroenterology 206 207 MICHAEL H. NATHANSON 208 Hepatology 209 RICHARD M. PEEK JR. 210 Gastroenterology 211 212 JERROLD R. TURNER 213 Cellular and Molecular Gastroenterology and Hepatology 214 215 216 References 217 1. Reardon SA. mouse’s house may ruin studies. Nature 218 2016;530:264. 219 2. Collins FS, Tabak LA. NIH plans to enhance reproduc- 220 ibility. Nature 2014;505:612–613. 221 3. Laukens D, Brinkman BM, Raes J, et al. Heterogeneity of 222 the gut microbiome in mice: guidelines for optimizing 223 experimental design. FEMS Microbiol Rev 2016;40: 224 117–132. 225 4. Ambery AG, Tackett L, Penque BA, et al. Effect of 226 Corncob bedding on feed conversion efficiency in a high227 fat diet-induced prediabetic model in C57Bl/6J mice. 228 J Am Assoc Lab Anim Sci 2014;53:449–451. 229 5. Kilkenny C, Browne WJ, Cuthill IC, et al. The ARRIVE 230 Guidelines Checklist: animal research: reporting in vivo ex231 periments. Available. www.nc3rs.org.uk/sites/default/files/ 232 documents/Guidelines/NC3Rs%20 ARRIVE%20Guide 233 lines%20Check list%20(fillable).pdf. 234 considered and implemented, that are aimed at investigators and trainees. For example, the curricula for students who are enrolled in bioscience-related undergraduate and graduate programs should include training not only in the ethics in conducting research, but also in the basic tenets of conducting and designing animal experiments. It is important for this to start early and to be reinforced as training advances. Similarly, postdoctoral fellows in biomedical disciplines should be expected to enroll in similar workshops that would be offered by their home institutions. For such workshops, centralized (rather than department- or unit-specific) oversight and administration will more likely ensure uniformity and implementation. We look forward to working together on this important effort and to receiving feedback from our authors, reviewers and readers.
EDI 5.4.0 DTD JCMGH125 proof 5 May 2016 1:07 pm ce
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235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293
6.
7.
8.
2016
Commentary
Baker D, Lidster K, Sottomayer A, et al. Two years later: journals are not yet enforcing the ARRIVE guidelines on reporting standards for pre-clinical animal studies. PLoS Biol 2014;12:e1001756. Landis SC, Amara SG, Asadullah K, et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature 2012;490: 187–191. National Institutes of Health (NIH), Agency for Healthcare Research and Quality (AHRQ). Implementing rigor and transparency in NIH & AHRQ research grant applications. October 9, 2015. Available: http://grants.nih.gov/grants/ guide/noticefiles/NOT-OD-16–011.html.
3
Acknowledgments The authors thank the managing editors of our journals for helping facilitate the joint publication of this commentary. All authors contributed equally to this effort. This commentary is being jointly published by Cellular and Molecular Gastroenterology and Hepatology, Gastroenterology, Gut, Hepatology, and Journal of Hepatology. Conflicts of interest The authors disclose no conflicts.
© 2016 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X http://dx.doi.org/10.1016/j.jcmgh.2016.04.001
EDI 5.4.0 DTD JCMGH125 proof 5 May 2016 1:07 pm ce
294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352