Minisymposium. Towards Evidence Based Translational Research. The Pros and Cons of Performing Systematic Reviews of Animal Studies

Transcription

1 Minisymposium Towards Evidence Based Translational Research The Pros and Cons of Performing Systematic Reviews of Animal Studies Radboud University Nijmegen Medical Centre Hippocrateszaal (route 77) Thursday, 30th August 2012

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3 About SYRCLE SYstematic Review Centre for Laboratory animal Experimentation SYRCLE is a unique and innovative research centre focusing on the continuous improvement of scientific quality and transparency of laboratory animal science and of animal welfare. Its current focus is on systematic reviews (SRs). These improve 1) the quality of scientific research, 2) the search for, and implementation of, the 3Rs (Refinement, Reduction, Replacement) and 3) patient safety. SYRCLE facilitates the development of guidelines for SRs, as well as the execution and education of SRs. Prof. Merel Ritskes-Hoitinga Founder and Project leader Dr. Marlies Leenaars Assistant Professor Dr. Carlijn Hooijmans Scientific Researcher Dr. Rob de Vries Scientific Researcher Judith van Luijk MSc PhD-student

4 Contact information 231 Central Animal Laboratory (CDL) SYRCLE Radboud University Nijmegen Medical Centre P.O. Box HB Nijmegen Visiting address Direction: UMC St Radboud Centraal Route: 231 Geert Grooteplein 29, Nijmegen Telephone:

5 Minisymposium Towards evidence-based translational research The pros and cons of performing Systematic Reviews of animal studies 30 August 2012, hr, Hippocrateszaal, route 77 Colleague researchers performing Systematic Reviews (SR) of animal studies will share their experiences with you. They will present highlights of the methodology, the usefulness/uselessness, (dis)advantages, hurdles, opportunities etc. And you can ask all your questions. Chair: Preliminary Program hr Prof. dr. Melvin Samsom: opening hr Dr. Henk Reinen, Ministry of Health: Systematic reviews in research using animals; a policy perspective hr Prof. dr. Maroeska Rovers: SRs of clinical versus animal studies: parallels and challenges hr Drs. Joris van Drongelen: Functional adaptation during pregnancy: a metaanalysis of animal studies hr Dr. Carlijn Hooijmans: What does it take to perform a SR of animal studies? hr Dr. Marlies Leenaars: How to facilitate SR hr break hr Drs. Marije Sloff: how does a PhD student experience doing an SR at the start of a PhD project? hr Drs. Linda Reus: Can animal models provide insight into neuromuscular functioning in Prader Willi syndrome? hr Drs. Kim Wever: A Systematic Review of ischemic preconditioning in animal models: the road to improved clinical trial design hr Dr. Erica van Oort: ZonMW call stimulating Synthesis of Evidence and publishing negative results. ZonMw programme More Knowledge with Fewer Animals 1

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7 Synthesis of Evidence (SE) and Systematic Reviews (SR) Also for preclinical studies to achieve evidence-based translation Prelude This minisymposium is aimed at presenting the experiences of performing Synthesis of Evidence (SE) and Systematic Reviews (SR) of animal studies. SE refers to the process of systematically locating, appraising and synthesizing evidence from scientific studies in order to obtain a reliable overview. SR is defined as a literature review focused on a single question which tries to identify, appraise, select and synthesize all high quality research evidence relevant to that question. SE is a broader term than SR. SR is a specific methodology for undertaking synthesis. The methodology of SR has been firmly established for clinical studies ( but not yet for animal studies. Janneke Horn MD was one of the first medical researcher in the world to perform a SR of animal studies 1. Her study demonstrated that when a SR of animal studies would have been performed, the actual side-effects seen in the clinical trial could have been predicted before the start of this trial. This would have prevented unnecessary adverse exposure of human patients, as was also demonstrated in the paper by Pound et al. 2 Within neurology, the CAMARADES collaboration ( has executed SR of animal studies on a routine basis since This need arose because of the low predictability of animal studies for the treatment of stroke in humans. These SRs have demonstrated that there is great potential for improving the quality and translational value of animal studies. Also SABRE ( was founded with the aim to promote SR of animal studies for a better health care of humans. Due to the lack of many details in publications, new guidelines for reporting of animal studies (GSPC; ARRIVE) have recently been published. The ARRIVE guidelines have been adopted by many journals and will hopefully lead to the publication of all necessary details of animal studies very soon 3-6. In August 2011 the 8 th World conference on Alternatives called for a change in culture and the Montreal Declaration was adopted accordingly ( Since 2009 SYRCLE ( is developing education, tools and guidelines in order to promote performing SR of animal studies. SYRCLE has chosen to focus on SR as one of the methodologies of SE, as this methodology is so well developed within the clinical field already. The first goal is to transfer this methodology to animal studies. 3

8 At this SYRCLE minisymposium, pioneer researchers in the field will share their experiences and discuss the advantages, disadvantages and pitfalls of doing SR of animal studies. Next to SR of clinical trials, SR of animal studies offer the important possibility to provide translational evidence on the possible value of animal models for humans. Providing better translational evidence is a need that recently has become expressed extensively both within and outside the scientific community. To be able to make translational calculations in meta-analyses, it will be necessary that individual data of animal studies become and remain stored in databases, next to human data. By nature, performing SR also requires interdisciplinary teamwork, and as such, this cooperation of diverse disciplines will also stimulate and integrate cooperation between preclinical and clinical scientists, again adding on to translation. Merel Ritskes-Hoitinga Professor in Laboratory Animal Science Centraal Dierenlaboratorium SYRCLE References 1) Horn, de Haan, Vermeulen, Luiten, Limburg. 2001; Nimodipine in animal model experiments of focal cerebral ischemia: a systematic review. Stroke;32(10): ) Pound, Ebrahim, Sandercock, Bracken, Roberts. 2004; Where is the evidence that animal research benefits humans? Bmj;328(7438): ) Hooijmans, Leenaars, Ritskes-Hoitinga. 2010; A gold standard publication checklist to improve the quality of animal studies, to fully integrate the Three Rs, and to make systematic reviews more feasible. Altern Lab Anim;38(2): ) Kilkenny, Browne, Cuthill, Emerson, Altman. 2010; Animal research: reporting in vivo experiments: the ARRIVE guidelines. J Gene Med;12(7): ) Hooijmans, de Vries, Leenaars, Curfs, Ritskes-Hoitinga M. 2011; Improving planning, design, reporting and scientific quality of animal experiments by using the Gold Standard Publication Checklist, in addition to the ARRIVE guidelines. Br J Pharmacol;162(6): ) Hooijmans, de Vries, Leenaars, Ritskes-Hoitinga M. 2011; The Gold Standard Publication Checklist (GSPC) for improved design, reporting and scientific quality of animal studies GSPC versus ARRIVE guidelines. Lab Anim;45(1):61. 4

9 Content: Abstracts presentations symposium... 7 Systematic reviews in research using animals; a policy perspective Systematic Reviews of clinical versus animal studies: parallels and challenges Functional adaptation during pregnancy: a meta-analysis of animal studies What does it take to perform a Systematic Review of animal studies How to facilitate implementation of Systematic Reviews of animal studies Writing a systematic review: a good start of a PhD-project Can animal models provide insight into neuromuscular functioning in Prader Willi syndrome? A Systematic Review of ischemic preconditioning in animal models: the road to improved clinical trial design ZonMw call stimulating Synthesis of Evidence and publishing negative results 17 ZonMw programme More Knowledge with Fewer Animals Addendum Basic science mechanisms of transcutaneous electrical nerve stimulation (TENS) in animal models of pain: results of a systematic literature search On the moral significance of the scientific quality of animal experiments. An ethical plea for systematic reviews

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13 Systematic reviews in research using animals; a policy perspective. Henk Reinen Nutrition, Health Protection and Prevention Department, Ministry of Health, Welfare and Sport, the Netherlands. ha.reinen@minvws.nl; Since the seventies of the previous century the number of experiments using animals in the Netherlands has seen an ongoing decrease. The last few years however, this number stays roughly the same (about annually). A stagnation so to say. It is evident that there is substantial progress. Otherwise these results could not have been achieved. However, now even stronger efforts seem necessary to realize a further reduction. Not an easy task, not a linear pathway either. Many developments of scientific and of societal nature influence the possibilities of positive changes. Some developments will tend to increase the number of animal experiments, such as the use of new materials, a reduced acceptation of health risks or new priorities in health care. Others can bring forth a decrease such as new innovative technologies, the use of human stem cells and by making a paradigm shift to develop research strategies focusing on 3R alternatives. Systematic Reviews belong to the latter group. It is very clear that the last decades public and political attention to the welfare of animals and especially for laboratory animals has increased. The upcoming of the Partij van de Dieren (member of Dutch Parliament since 2006) is a recent manifestation of this. There is a great need for better science, with less animals. There is also a great need for transparency and for openness, for monitoring actions, developments and results. Systematic Review in animal studies is a new step contributing to achieve the results needed and a step in line with public and political demands (enhancement of the quality of scientific research, a better use of existing animal experiments, more transparency and openness in the policy of the 3Rs ). A step the Dutch Parliament and the ministry will stimulate to become a method seen as normal and best practice in animal studies. 9

14 Systematic Reviews of clinical versus animal studies: parallels and challenges Maroeska Rovers a and Carlijn Hooijmans b a Departments of operating rooms and Health Evidence, Radboud University Nijmegen Medical Centre, M.Rovers@ok.umcn.nl b SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands Research has shown that the current methods used to assess and translate the medical value of animal research need to be improved. Systematic reviews are considered to be the most rigorous method of evaluating research. Important reasons to introduce systematic reviews in animal science are: 1) increasing the precision in estimating treatment effect estimates for trials in humans 2) reducing the risk of falsenegative results, 3) improving the applicability of preclinical animal research to clinical research, 4) assessing the strength of evidence to determine whether an effect exists in a particular direction 5) investigating heterogeneity among various studies, and 6) obtaining novel results and generating new hypotheses without the need to perform new or additional animal studies. Conducting such systematic reviews in laboratory animal science, however, involves several methodological challenges. Specific challenges comprise the often poor methodological quality of individual animal studies, the heterogeneity of species and strains used, the lack of generalisability and publication bias. In order to further implement systematic reviews in laboratory animal science, the above mentioned methodological and reporting issues need to be addressed and solved. Earlier clinical initiatives to further improve the conduct and reporting of systematic reviews could be followed. 10

15 Functional adaptation during pregnancy: a meta-analysis of animal studies. Joris van Drongelen a Carlijn Hooijmans b, Frederik Lotgering a, Paul Smits c, Marc Spaanderman ad a Department of Obstetrics and Gynecology, Radboud University Nijmegen Medical Centre, The Netherlands; j.vandrongelen@obgyn.umcn.nl b SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands c Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, The Netherlands d Department of Obstetrics and Gynecology, Research School GROW, Maastricht University Medical Centre, The Netherlands The vascular response to pregnancy has been frequently studied in mesenteric artery models, by investigating endothelial cell (EC) and smooth muscle cell (SMC) dependent responses to mechanical (flow-mediated vasodilation, myogenic reactivity, and vascular compliance) and pharmacological stimuli (G-protein coupled receptor responses: Gq EC, Gs SMC, Gq SMC ). It is unclear to what extent these pathways contribute to normal pregnancy-induced vasodilatation across species, strains, and/or gestational age, and at which receptor level pregnancy affects the pathways. We performed a meta-analysis on responses to mechanical and pharmacological stimuli associated with pregnancy-induced vasodilation of mesenteric arteries and included 55 (188 responses) out of 398 studies. Most included studies (84%) were performed in Wistar rats (WR) and Sprague Dawley (SDR), and compared late gestation versus non-pregnant controls (80%). Pregnancy promotes flow-mediated vasodilation in all investigated species. Only in SDR, pregnancy additionally stimulates both vasodilator Gq EC sensitivity ( EC 50 reduced by [-0.92, ] log[m]) and Gs SMC sensitivity (EC 50 reduced by [-0.82, ] log[m]), depresses vasopressor Gq SMC sensitivity (EC 50 increase in SDR by 0.23 [0.16, 0.31] log[m]) and enhances arterial compliance. We conclude that 1) Pregnancy facilitates flow-mediated vasodilation at term, amongst all investigated species; the contribution of additional vascular responses is species and strain specific, 2) Late pregnancy mediates vasodilation through changes at the receptor level, for the substances tested. The initial steps of vasodilation in early pregnancy remain to be elucidated. References 1) van Drongelen, Hooijmans, Lotgering, Smits, Spaanderman. 2012; Adaptive changes of mesenteric arteries in pregnancy: a meta-analysis. AJP-Heart and Circ (accepted) 11

16 What does it take to perform a Systematic Review of animal studies Carlijn Hooijmans a, Rob de Vries a, Marlies Leenaars a, Miranda Langendam c, Maroeska Rovers b, Merel Ritskes-Hoitinga a a SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands b Departments of operating rooms and Health Evidence, Radboud University Nijmegen Medical Centrethe Netherlands c Dutch Cochrane Centre, Academic Medical Center, Amsterdam, Netherlands. Conducting Systematic Reviews has been standard practice in the clinical field since decades. Professionals in evidence-based medicine regard these type of reviews as the highest level of medical evidence. In the field of animal experimentation these reviews are relatively new and not yet widely used nor undertaken, whereas a lot can be gained from this process. SYRCLE has been developing methodology for conducting SRs in Laboratory Animal Science. The methods used in the clinical field, especially those arising within the Cochrane Collaboration, form the inspiration for these developments in the field of animal studies. In a Systematic Review explicit methods are used to search comprehensively, critically appraise 1 and synthesize the available literature on a specific issue. In this presentation, Systematic Reviews will be compared to the more regular narrative reviews. The needs and steps involved in 1) the critical assessment of individual studies (Risk of bias analysis) and 2) the statistical combination of results of several independent studies (meta-analysis) will be presented. References: 1) Higgins, Altman, Gøtzsche, Jüni, Moher, Oxman, Savovic, Schulz, Weeks, Sterne. 2011; The Cochrane Collaboration's tool for assessing risk of bias in randomised trials. Cochrane Bias Methods Group; Cochrane Statistical Methods Group. BMJ. 18;343:d

17 How to facilitate implementation of Systematic Reviews of animal studies Marlies Leenaars a, Carlijn Hooijmans a, Alice Tillema b, Rob de Vries a, Merel Ritskes- Hoitinga a a SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands M.Leenaars@cdl.umcn.nl ; b Medical library, Radboud University Nijmegen Medical Centre Thorough analysis of already existing literature and data is a basic principle in science. Systematic reviews (SRs) are an effective method to carry out such a thorough analysis since all relevant studies are identified, appraised, selected and data are extracted in a transparent, thorough and systematic way. While within clinical research, SRs are common practice for already many years, this is not yet the case within animal research. SRs are a new topic within Laboratory Animal Science and limited experience on how to perform these reviews is available. To increase awareness of the advantages and benefits of performing SRs of animal studies, we develop education and teach on this topic in our mandatory FELASA category C Courses in Laboratory Animal Science in Nijmegen since This also includes a workshop on comprehensive search strategies. In the curriculum of Master students in Biomedical Sciences a one week module on SRs of animal studies has been incorporated. To ease the process of performing a SR, tools and guidelines are being developed. For example, the Gold Standard Publication Checklist 1, the step-by-step search guide 2 and search filters for PubMed and Embase 3,4 have been published. To improve the skills to perform a SR, hands-on training courses have been developed by our department. These courses are made available locally as well as (inter)nationally. References 1) Hooijmans, Leenaars, Ritskes-Hoitinga. 2010; A gold standard publication checklist to improve the quality of animal studies, to fully integrate the Three Rs, and to make systematic reviews more feasible. Alternatives to Laboratory Animals; 38(2): ) Leenaars, Hooijmans, van Veggel, ter Riet, Leeflang, Hooft, van der Wilt, Tillema, Ritskes- Hoitinga. 2012; Step-by-step guide to identify relevant animal studies. Laboratory Animals; 46(1), ) De Vries, Hooijmans, Tillema, Leenaars, Ritskes-Hoitinga. 2011; A search filter for increasing the retrieval of animal studies in Embase. Laboratory Animals; 45(4), ) Hooijmans, Tillema, Leenaars, Ritskes-Hoitinga ; Enhancing search efficiency by means of a search filter for finding all studies on animal experimentation in PubMed. Laboratory Animals; 44(3),

18 Writing a systematic review: a good start of a PhD-project Marije Sloff a, Rob de Vries b, Paul Geutjes a, Merel Ritskes-Hoitinga b, Egbert Oosterwijk a, Wout Feitz a a Department of Urology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen, The Netherlands. M.Sloff@uro.umcn.nl b Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands End-stage bladder disease is either caused by severe congenital malformations (e.g. spina bifida or bladder extrophy) or bladder cancer (~ deaths/year in Europe), necessitating urinary diversion in these patients. 1 The gold standard for treatment is the use of small intestine in a Bricker ileal conduit, even though this procedure requires invasive intestinal surgery with associated risks and complications. In 25-60% of the patients, complications occur as a consequence of intestinal surgery and/or unsuitability of the tissue for urinary diversion. 2 Regenerative medicine may provide an alternative solution through the construction of an artificial stoma from natural materials, which may prevent invasive surgery and associated complications. The Urology department (NCMLS, RUNMC) has shown that an artificial urostomy construct, combining the natural polymer collagen with a firming biodegradable polymer mesh, can be used for urinary derivation purposes in a preclinical pig model. 3 However, it is not clear whether this pig model is optimal for modeling urinary derivation in patients and provides the optimal basis for clinical translation. Accordingly, the aim of this project is to further develop an artificial stoma for urinary diversion in patients with extensive bladder disease. A systematic review on animal models for urinary diversion should yield the necessary information on selecting the optimal materials, methods and animal model for maximizing the relevant output of further experimentation. It teaches you to critically evaluate the quality and flaws of current in vivo studies. This systematic review on animal models for urinary diversion will provide an extensive and up-to-date overview of all relevant studies carried out so far and to generate a fundament for further research and discussion. References: 1) Nederlands Kankerregistratie, Cijferapplicatie ) Farnham, Cookson. 2004; Surgical complications of urinary diversion. World J Urol; 22: 157 3) Geutjes, Roelofs, Hoogenkamp, Walraven, Kortmann, de Gier, Farag, Tiemessen, Sloff, Oosterwijk, van Kuppevelt, Daamen, Feitz. 2012;Tissue engineered tubular construct for urinary diversion in a preclinical porcine model. J Urol; 188(2):

19 Can animal models provide insight into neuromuscular functioning in Prader Willi syndrome? Linda Reus a, Carlijn Hooijmans b, Janielle van Alfen-van der Velden c, Merel Ritskes- Hoitinga b,ria Nijhuis-van der Sanden ac a Department of Rehabilitation, Pediatric Physical Therapy, Radboud University Nijmegen Medical Centre, L.Reus@reval.umcn.nl b SYRCLE, Radboud University Nijmegen Medical Centre c Department of Pediatric Endocrinology, Radboud University Nijmegen Medical Centre d Scientific Institute for Quality of Healthcare, Radboud University Medical Centre At the department of pediatric physical therapy, Radboud University Nijmegen Medical Centre, motor development in infants with Prader Willy syndrome (PWS) is studied. PWS is a neurogenetic disorder with a prevalence of 1 in 10,000-30,000 births. PWS is characterized by a wide variety of physical, cognitive, and behavioral defects. The most significant characteristics are hypotonia, hypogonadism, hyperphagia, obesity, short stature, mild dysmorphic facial features, and cognitive/behavioral deficits. Especially during infancy, patients suffer from severe hypotonia and muscle weakness resulting into seriously delayed motor development. Motor performance is also decreased in childhood and adulthood is. It is presumed that the motor problems are related to decreased muscle mass combined with increased fat mass, however neuromuscular abnormalities may also contribute to the problem. The later is not studied much. This is however, a very interesting topic considering the fact that neuromuscular function is especially effected during infancy whereas body composition is only mildly effected during infancy. Studies with PWS animal models can provide more insight into neuromuscular functioning in PWS. Therefore we conducted a systematic literature search to provide an overview of the current insights. Based on this search we want to determine which animal model could be used to study the effect of training or medication on the neuromuscular system. In conclusion, our systematic search will enabled us to make an evidenced based decision about the most suitable animal model to study the effects of training or medication on de neuromuscular system in PWS. 1) Cassidy, Driscoll ; Prader-Willi syndrome. Eur J Hum Genet;17(1): ) Reus, van Vlimmeren, Bart, Otten, Nijhuis-van der Sanden; The effect of growth hormone treatment or physical training on motor performance in Prader-Willi syndrome: A systematic review. Neurosci Biobehav Rev. May 28. 3) Reus, Zwarts, van Vlimmeren, Willemsen, Otten, Nijhuis-van der Sanden; Motor problems in Prader-Willi syndrome: a systematic review on body composition and neuromuscular functioning. Neurosci Biobehav Rev;35(3):

20 A Systematic Review of ischemic preconditioning in animal models: the road to improved clinical trial design Kim Wever ab, Theo Menting b, Maroeska Rovers c, Adam van der Vliet b, Gerard Rongen ad, Rosalinde Masereeuw a, Merel Ritskes-Hoitinga e, Carlijn Hooijmans e, Michiel Warle b a Department of Pharmacology and Toxicology, Radboud University Nijmegen, the Netherlands. K.Wever@chir.umcn.nl b Department of Surgery, Radboud University Nijmegen, the Netherlands c Departments of Operating rooms and Epidemiology, Biostatistics & HTA, Radboud University Nijmegen, the Netherlands d Department General Internal Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. 2 SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands Ischemia-reperfusion injury (IRI) is the damage that occurs when an organ or tissue is temporarily occluded from the bloodstream. IRI occurs in many clinical conditions, such as infarction, transplantation, cardiac and aortic surgery, arterial stenosis and contrast administration. Current methods to reduce IRI are not sufficient to prevent complications and mortality in patients, and novel therapies are needed. Application of a brief ischemic pulse before the onset of IRI is a protective strategy known as ischemic preconditioning (IPC). IPC has been shown to have protective effects against IRI in many animal models of e.g myocardial infarction, renal IRI and liver transplantation. However, in spite of these promising results, this method has not yet been translated successfully into clinical practice. One of the reasons for this ambiguity may be the diversity in IPC protocols used: clinical trials all use similar IPC protocols, whereas the protocols used in animal studies vary greatly in terms of timing, duration and site of the IPC stimulus. At present, there is no consensus on the optimal design for the IPC protocol and it is unclear which factors influence the efficacy of the IPC stimulus. As a result, the IPC stimuli used in current clinical trials may be suboptimal or incorrectly applied. Can systematic review and meta-analysis of animal studies help to resolve this problem and design an optimal stimulus protocol for future clinical trials on IPC? We report the results of a systematic review and meta-analysis of animal studies, in which we identified factors modifying IPC efficacy in renal IRI (Wever et al. 2012), and present a work-in-progress of this strategy for animals studies on IPC of the heart. We propose that these results can be used to enhance the design of future clinical and animal studies, and identify a need for further standardization of animal experiments. References 1) Wever, Menting, Rovers van der Vliet, Rongen, Masereeuw, Ritskes-Hoitinga, Hooijmans, Warle; Ischemic preconditioning in the animal kidney, a systematic review and metaanalysis. PloS one 7; e

21 ZonMw call stimulating Synthesis of Evidence and publishing negative results ZonMw programme More Knowledge with Fewer Animals Erica van Oort a, Catherine Schuttevaer a, Rob Diemel a, Urmila Gangaram Panday a ZonMw, Team Wetenschap en Innovatie, Laan van Nieuw Oost Indië 334, 2593 CE Den Haag Introduction The social debate about animal testing is governed by opposite interests: the reduced acceptance of animal testing versus the demand for absolute risk exclusion that experimental animal research claims to achieve. However, the animal model also has limitations, notably in the translation to human beings. Methods to replace, reduce or refine the use of laboratory animals (the 3 Rs) are being developed across a broad spectrum. The More Knowledge with Fewer Animals (Meer Kennis met Minder Dieren / MKMD) programme aims to reduce, refine and replace the use of animal testing, without impairing the quality of the scientific research and the safety of developed products. The MKMD programme is subdivided into modules. Each (new) module is sponsored by a commissioning organisation, the commissioning organisation for amendment 21 module, is VWS. This module promotes and develops 3Rs knowledge infrastructure by training researchers in the use of synthesis of evidence (Systematic Reviews) and by providing open access to scientific sound unpublished negative or neutral data. The amendment 21 module comprises the entire breadth of fundamental experimental animal research. Information on other modules can be found via our website MKMD Amendment 21 Module Within the context of the MKMD programme, the amendment 21 module aims to: 1. Stimulate the publication of negative (or neutral) results involving the use of test animals to improve the quality of the data by requiring researchers to use the Gold Standard Publication Checklist (C.R Hooijmans et al. and/or the ARRIVE guidelines (C. Kilkenny et al Stimulate and embed a synthesis of evidence in experimental animal research This is a derivative of the Systematic Reviews which are commonplace within human studies (Cochrane Collaboration). Stimulate the publication of negative results involving the use of test animals. 17

22 The publication of negative results is not a priority for many researchers: the highimpact journals often reject these data and most researchers prefer not to publish in low impact journals as the invested time and costs outweigh the benefits. In addition, not all researchers are aware of the existence of journals that focus on negative results (if based on sound research). Due to the failure to publish negative results, this knowledge never reaches other researchers. This is unfortunate, because negative results are highly informative and should be publicly available (e.g. through publications). Of course, this applies to research in general. Negative results research may, for instance, contain information about the suitability of an animal model for a specific research question, information about control groups or the required dosage of chemicals or medication to produce a visible effect. Stimulate and embed synthesis of evidence in experimental animal research These are extremely extensive literature studies. A synthesis of evidence (SE) provides a general survey of the literature in which information from the original research is searched, selected, extracted, assessed and combined in a transparent and reproducible manner. In research involving human subjects, synthesis of evidence (or Systematic Reviews as these studies are called in relation to research involving human subjects) is already frequently applied because of the advantages it offers. Synthesis of evidence within experimental animal research can help to avoid repetition and can also lead to a more informed selection of the animal model. 18

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25 Basic science mechanisms of transcutaneous electrical nerve stimulation (TENS) in animal models of pain: results of a systematic literature search Jan Oosterhof a, Veerle Boomer b, Gert Jan van der Wilt c, Marlies Leenaars b a Department of Physiotherapy, Radboud University Nijmegen Medical Centre, the Netherlands J.Oosterhof@orthop.umcn.nl b SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands c Department of Epidemiology, Biostatistics and Health Technology Assessment, Radboud University Nijmegen Medical Centre, the Netherlands TENS is a widely used non-invasive treatment for chronic pain. However, the results for TENS in chronic pain are inconclusive 1, which may be due to differences in the TENS application and differences in pain conditions. TENS is applied clinically at varying frequencies, intensities and pulse durations of stimulation, High-frequency TENS is generally applied at sensory level and is referred to as conventional TENS, whereas low-frequency TENS and burst TENS are more commonly applied at motor level and are both referred to as acupuncture-like TENS. Several distinct types of pain exist: nociceptive, inflammatory, and neuropathic 2. Animal models have been developed, that mimic these clinical conditions, to measure effectiveness of medical treatment 3. Animal models minimize the placebo effect, allow for control of the extent of the injury, make application of TENS fully controllable, and allow one to assess the neurobiological mechanisms by which TENS produces a reduction of pain behaviours. We performed a systematic search for journal articles from the National Library of Medicine database and EMBASE 4, using the animal search filters 5,6. A total of 72 papers, all written in English, were reviewed and 44 papers were included. From the excluded papers two appeared human studies. Results showed consistent effects for nociceptive and inflammatory pain models for both high- and low-frequency TENS. However, contrary to low-frequency TENS, for the neuropathic pain model high-frequency TENS was not effective. This latter finding is in accordance with the results of our study of high-frequency TENS in chronic pain 7. Limitations: firstly, almost all animal studies used male animals (rats); however, chronic pain is more common in female patients compared to males. Secondly, for animals TENS treatment will be experienced as a stressor, in contrary, patients with chronic pain may expect pain relief during treatment, both include different adaptive processes; the consequences of these differences need further investigation. References 1) Carroll, Moore, McQuay, Fairman, Tramer, Leijon. 2004; Transcutaneous electrical nerve stimulation (TENS) for chronic pain (Cochrane Review). In: The Cochrane Library ;Issue 3:. Chichester, UK: John Wiley & Sons, Ltd. 21

26 2) Woolf. 2004; Pain: moving from symptom control toward mechanism-specific pharmacologic management. Annals of internal medicine;140(6): ) Dubner, Ren. Assessing transient and persistent pain in animals. In: Textbook of Pain Fourth Edition 1999; ) Oosterhof. Transcutaneous Electrical Nerve Stimulation (TENS) in the treatment of patients with chronic pain. 5) De Vries, Hooijmans, Tillema, Leenaars, Ritskes-Hoitinga. 2011; A search filter for increasing the retrieval of animal studies in Embase. Lab Anim; 45(4): ) Hooijmans CR, Tillema A, Leenaars M, Ritskes-Hoitinga M. 2010; Enhancing search efficiency by means of a search filter for finding all studies on animal experimentation in PubMed. Lab Anim; 44(3): ) Oosterhof, Samwel, de-boo, Wilder-Smith, Oostendorp, Crul. 2008; Predicting outcome of TENS in chronic pain; A prospective, randomized, placebo controlled trial. Pain;136(1-2):

27 On the moral significance of the scientific quality of animal experiments. An ethical plea for systematic reviews Rob de Vries a, Evert van Leeuwen b, Marlies Leenaars a, Gerben ter Riet c, Gert Jan van der Wilt d, Merel Ritskes-Hoitinga a a SYRCLE, Radboud University Nijmegen Medical Centre, the Netherlands; r.devries@cdl.umcn.nl b IQ healthcare, Section Ethics, Philosophy and History of Medicine, Radboud University Nijmegen Medical Centre, the Netherlands c Department of General Practice, Academic Medical Center, University of Amsterdam, the Netherlands d Department of Epidemiology, Biostatistics and Health Technology Assessment, Radboud University Nijmegen Medical Centre, the Netherlands Animal experiments in biomedical research keep raising moral concerns. Society wants as few animal experiments as possible, if any. The most obvious way to decrease the number of laboratory animals is by using and developing Replacement alternatives. However, even though the potential for such alternatives is not yet fully realised, not all animal experiments can be replaced by, for example, in vitro alternatives. What the biomedical research world can do to meet society s concerns is to carry out as few experiments as possible and to derive the most and most reliable information from the experiments that still have to be carried out. In order to attain this objective, an important step is a thorough analysis of all experiments that have been carried out on a particular topic, not only individually but also in relation to one another. The method that seems most suitable to perform such an analysis is a Systematic Review (SR), including, if feasible, a meta-analysis. SRs are scientifically sound summaries of all available evidence regarding a specific research question. SRs are already common practice in the field of clinical research. However, despite the fact that clinical research is often based on preceding animal experiments, SRs of animal experiments are still rather scarce. There are at least three types of ethical arguments why more SRs of animals experiments should be conducted: arguments from (a) research ethics, (b) animal ethics and (c) medical ethics (de Vries et al. 2010). (a) By revealing shortcomings in the execution and/or reporting of animal experiments (for instance, a lack of randomisation and blinding; cf. Kilkenny et al. 2009), SRs may lead to an improvement of the methodological quality of new animal experiments. Researchers who fail to properly design, execute and analyse their experiments and journals that do not demand such standards in effect show a lack of scientific virtue. (b) Because of the comprehensiveness of the search strategies they employ and the cumulative meta-analyses they may contain (e.g. Sena et al. 2010), SRs may prevent the unnecessary duplication of animal experiments and thereby lead to a reduction of the number of animals used. Moreover, they are likely to improve the ethical 23

28 evaluation of animal experiments by enabling members of Animal Ethics Committees to make more evidence-based decisions on whether new experiments are really necessary and whether the most suitable animal model has been selected (cf. de Vries et al. 2012). (c) SRs may increase patient safety by improving the translation of data from animals to humans. Particularly if SRs include a meta-analysis, they may produce information about the safety and efficacy of treatments that is not directly visible in the individual animal studies. In this way, they may prevent human patients from being exposed to the risks of severe side effects or pharmacologically ineffective treatments. In short, we owe it to science, laboratory animals and human patients that SRs become the standard in the field of animal experimentation as well. It is the joint responsibility of researchers, journal editors, funding agencies and ethical committees to realise that goal. References: 1) de Vries, Leenaars, van der Wilt, Ritskes-Hoitinga Systematische reviews van dierproeven. Over het ethisch belang voor dier én mens van het verhogen van de wetenschappelijke kwaliteit van dierexperimenteel onderzoek Tijdschrift voor Gezondheidszorg en Ethiek; 20(3): ) Kilkenny, Parsons, Kadyszewski, Festing, Cuthill et al Survey of the quality of experimental design, statistical analysis and reporting of research using animals PLoS ONE; 4(11): e ) Sena, Briscoe, Howells, Donnan, Sandercock, Macleod Factors affecting the apparent efficacy and safety of tissue plasminogen activator in thrombotic occlusion models of stroke: systematic review and meta-analysis J Cereb Blood Flow Metab; 30(12): ) de Vries, Buma, Leenaars, Ritskes-Hoitinga, Gordijn Reducing the number of laboratory animals used in tissue engineering research by restricting the variety of animal models. Articular cartilage tissue engineering as a case study Accepted by Tissue Engineering Part B. 24