PRIASE 2021 guidelines for reporting animal studies in Endodontology: explanation and elaboration
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doi:10.1111/iej.13481
REVIEW
PRIASE 2021 guidelines for reporting animal
studies in Endodontology: explanation and
elaboration
V. Nagendrababu1 , A. Kishen2 , P. E. Murray3 , M. H. Nekoofar4 , J. A. P. de
Figueiredo5 , E. Priya6 , J. Jayaraman7 , S. J. Pulikkotil8 , A. Jakovljevic9 &
P. M. H. Dummer10
1
Department of Preventive and Restorative Dentistry, College of Dental Medicine, University of Sharjah, Sharjah, UAE; 2Faculty
of Dentistry, University of Toronto, Toronto, ON, Canada; 3Private Consultant, Fort Lauderdale, FL, USA; 4Department of
Endodontics, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran; 5Federal University, Rio Grande do Sul,
Porto Alegre, Brazil; 6Division of Children and Community Oral Health, School of Dentistry, International Medical University,
Kuala Lumpur, Malaysia; 7Department of Developmental Dentistry, University of Texas Health School of Dentistry, San
Antonio, TX, USA; 8Division of Clinical Dentistry, School of Dentistry, International Medical University, Kuala Lumpur,
Malaysia; 9Department of Pathophysiology, School of Dental Medicine, University of Belgrade, Belgrade, Serbia; and 10School of
Dentistry, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK
Abstract Implementation of the PRIASE 2021 guidelines will
reduce potential sources of bias and thus improve the
Nagendrababu V, Kishen A, Murray PE, Nekoofar MH,
quality, accuracy, reproducibility, completeness and
de Figueiredo JAP, Priya E, Jayaraman J, Pulikkotil SJ,
transparency of reports describing animal studies in
Jakovljevic A, Dummer PMH. PRIASE 2021 guidelines for
Endodontology. The PRIASE 2021 guidelines consist of
reporting animal studies in Endodontology: explanation and
a checklist with 11 domains and 43 individual items
elaboration. International Endodontic Journal, 54, 858–886,
and a flowchart. The aim of the current document is to
2021
provide an explanation for each item in the PRIASE
Laws and ethics require that before conducting human 2021 checklist and flowchart and is supplemented
clinical trials, a new material, device or drug may have with examples from the literature in order for readers
to undergo testing in animals in order to minimize to understand their significance and to provide usage
health risks to humans, unless suitable supporting guidance. A link to the PRIASE 2021 explanation and
grandfather data already exist. The Preferred Reporting elaboration document and PRIASE 2021 checklist and
Items for Animal Studies in Endodontology (PRIASE) flowchart is available on the Preferred Reporting Items
2021 guidelines were developed exclusively for the for study Designs in Endodontology (PRIDE) website
specialty of Endodontology by integrating and adapting (http://pride-endodonticguidelines.org/priase/).
the ARRIVE (Animals in Research: Reporting In Vivo
Keywords: animal, consensus, endodontics, health
Experiments) guidelines and the Clinical and
research reporting guidelines.
Laboratory Images in Publications (CLIP) principles
using a validated consensus-based methodology. Received 20 January 2021; accepted 21 January 2021
Correspondence: V. Nagendrababu, Department of Preventive and Restorative Dentistry, College of Dental Medicine, University
of Sharjah, Sharjah, UAE (e-mail: hivenkateshbabu@yahoo.com, vnagendrababu@sharjah.ac.ae).
858 International Endodontic Journal, 54, 858–886, 2021 © 2021 International Endodontic Journal. Published by John Wiley & Sons LtdNagendrababu et al. PRIASE 2021 explanation and elaboration
included the essential items required to be included in
Introduction
peer-reviewed manuscripts of animal studies within
the specialty of Endodontology. This initial draft
The need for the Preferred Reporting Items for
checklist and flowchart were developed by integrating
Animal Studies in Endodontology (PRIASE) 2021
and adapting the ARRIVE guidelines (Kilkenny et al.
guidelines
2010, Percie du Sert et al. 2020) and Clinical and
Animal testing is important for evaluating the preclin- Laboratory Images in Publications (CLIP) principles
ical safety and effectiveness of new dental materials, (Lang et al. 2012).
drugs or devices to help identify and eliminate poten- The steering committee formed a PRIASE Delphi
tial health risks to humans. However, the translation Group (PDG) consisting of 31 experts from around
of research observations from animal studies to the world with the aim of building consensus through
humans has always been challenging (Yoneda et al. the use of the Delphi methodology to revise the items
2017). Sometimes the most promising products devel- of the preliminary PRIASE guidelines. The revised
oped using animal research can fail when used in PRIASE checklist and flowchart were then discussed
human trials and never become incorporated in daily during an online meeting conducted via Zoom on 9
clinical practice (Hackam & Redelmeier 2006, Pound September 2020 with a PRIASE Online Meeting
& Bracken 2014). Furthermore, poorly designed and Group (POMG) made up of 28 individuals (19 aca-
executed animal studies can produce unreliable and demics/clinicians, two postgraduate students, seven
inaccurate preclinical results (Pound & Bracken steering committee members). The details of each item
2014, Singh et al. 2016), which can defeat the pur- were discussed, and collective feedback was obtained
pose of animal testing, rendering it useless. that allowed the steering committee to further refine
The ARRIVE (Animal Research: Reporting In Vivo the items in the checklist and flowchart. The revised
Experiments) guidelines (Kilkenny et al. 2010, Percie du guidelines were then tested by several volunteer
Sert et al. 2020) and the SYRCLE (Systematic Review authors who drafted hypothetical manuscripts
Centre for Laboratory animal Experimentation) risk of describing animal studies in Endodontology when fol-
bias tool (Hooijmans et al. 2014) were developed to lowing the revised PRIASE guidelines. The final ver-
guide researchers and ultimately improve the quality of sion of the PRIASE 2021 guidelines consists of a
animal studies. However, animal studies in Endodontol- checklist with 43 items under 11 sections and a flow-
ogy often need information related exclusively to the chart (Nagendrababu et al. 2021).
specialty. Hence, the PRIASE 2021 guidelines were
developed with the objective of improving the standard
PRIASE 2021 explanation and elaboration
of manuscripts submitted to journals describing animal
document
studies linked to the specialty of Endodontology. It is
anticipated these guidelines will be of value to research- This explanation and elaboration document provides
ers, editors and peer reviewers of scientific journals a comprehensive explanation for each of the items
(Nagendrababu et al. 2021). in the checklist and for the contents of the flow-
chart. In addition, it reproduces extracts from
reports of published animal studies to provide fur-
The use of experts to develop the PRIASE 2021
ther help for authors and enhance understanding.
guidelines
In some of the real examples, citations or website
The PRIASE guidelines were developed by building a addresses have been removed, and abbreviations
consensus within a group of experts in the field of entered in full.
Endodontology (Nagendrababu et al. 2021) and fol-
lowed the Guidance for Developers of Health Research
Item 1a: Title – The specific animal species and its
Reporting Guidelines (Moher et al. 2010). The project
health or disease status (sometimes called ‘animal
leaders (VN, PD) identified the need for reporting
model’) must be provided
guidelines for animal studies in Endodontology. A
steering committee was formed with nine members Explanation
(PD, VN, AK, PM, MN, JF, EP, JJ and SP), that The type of animal (rat, mouse etc.) must appear in
included the project leaders. The steering committee the title to help readers identify the animal model
drafted a preliminary checklist and flowchart which used (Examples 1a.1, 1a.2). This information facilitates
© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 54, 858–886, 2021 859PRIASE 2021 explanation and elaboration Nagendrababu et al.
indexing in databases and may improve search
Item 2a: Keywords – Keywords such as ‘animal
results, for example Knockout mice with periapical
model or ‘in vivo model’ and the specific area(s) of
lesions, or Wistar rats with exposed molars etc. Other
interest must be provided
details of the animal model need only be included in
the title if they are a focus of the study, for example Explanation
age, gender, root canal disinfection, healing of peri- The inclusion of between two and five relevant key-
apical lesions etc. words can help to identify peer-reviewed manuscripts
of specific interest to readers, facilitate the indexing in
Example 1a.1 databases and improve the results of electronic litera-
From Conti et al. (2020) – ‘Relationship between api- ture searches. One of the keywords must be ‘animal
cal periodontitis and atherosclerosis in rats: lipid pro- model’ or ‘in vivo model’. Other keywords should
file and histological study’. include terms from the medical subject headings
(MeSH) terminology of the National Library of Medi-
Example 1a.2 cine (NLM; Examples 2a.1, 2a.2).
From Cotti et al. (2017) – ‘The Influence of Adalimumab
on the Healing of Apical Periodontitis in Ferrets’. Example 2a.1
From Altaii et al. (2016) – For the animal study enti-
tled ‘Endodontic regeneration and tooth revitalization
Item 1b: Title – The specific test, field, subject,
in immature infected sheep teeth’, the key words used
treatment of interest within the animal model
were ‘animal model, dental pulp necrosis, immature
must be provided
teeth, regeneration treatment, revitalization treat-
Explanation ment’.
The title must specify the treatment or study inter-
vention using descriptive terms and words for read- Example 2a.2
ers to identify the focus and key elements of the From Chang et al. (2020) – For the animal study enti-
study (Examples 1b.1, 1b.2), for example biocompat- tled ‘Regeneration of Tooth with Allogenous, Auto-
ibility, regenerative endodontics, sealer microleakage, claved Treated Dentin Matrix with Dental Pulpal
pulp capping, tooth replantation resorption, apexifi- Stem Cells: An In Vivo Study’, the key words used
cation, apexogenesis, periapical healing, root canal were ‘autoclaved, dental pulp stem cells, in vivo study,
disinfection, irrigation, analgesic effectiveness, stem tissue engineering, treated dentin matrix’.
cell therapy, etc. An exception can apply when the
animal experiment is only a small part of a larger
Item 3a: Abstract – The Introduction of the
multiphase study with several other components, for
Abstract must explain the significance of the study
example animals were used to test the biocompati-
bility of a newly developed sealer along with several Explanation
other laboratory-based tests. In this scenario, it may The introduction of the abstract (if provided) must
not be essential to include the animal model and identify the gap in knowledge and mention the signifi-
specific test in the title because of title word count cance and relevance of the study (Examples 3a.1,
limitation. 3a.2). The significance is an explanation of how the
study fills a gap in current knowledge, and the rea-
Example 1b.1 sons why the use of an instrument, device, material
From Lin et al. (2019) – ‘Dental Pulp Stem Cell Trans- or treatment may be beneficial or controversial. The
plantation with 2,3,5,4’-Tetrahydroxystilbene-2-O-b- information should be succinct, not confusing and
D-glucoside Accelerates Alveolar Bone Regeneration in focus on the important details.
Rats’.
Example 3a.1
Example 1b.2 From Chang et al. (2020) – ‘Biomaterials designed for
From Xu et al. (2016) – ‘Systemically Transplanted tissue engineering should be nontoxic and nonim-
Bone Marrow-derived Cells Contribute to Dental Pulp munogenic and should achieve their intended func-
Regeneration in a Chimeric Mouse Model’. tions. Treated dentin matrix (TDM), a bioactive
860 International Endodontic Journal, 54, 858–886, 2021 © 2021 International Endodontic Journal. Published by John Wiley & Sons LtdNagendrababu et al. PRIASE 2021 explanation and elaboration
extracellular matrix, is promising for tooth regenera-
Item 3c: Abstract – The most important details of
tion. However, the effect of sterilization on the surface
the animal and the experimental model must be
properties of allogenous TDM in the animal model is
provided
unclear’.
Explanation
Example 3a.2 The abstract must describe the details of the species,
From Tohma et al. (2020) – ‘Pulp capping materials strain and health/disease status of the animals with
allow healing of injured pulp with a layer of repara- enough specificity for a reader to identify the animal
tive dentin. Glucose is needed to cure the injured model. The type of model employed must be men-
area. Glucose is transported by glucose transporter tioned (Examples 3c.1, 3c.2).
(Glut) 2 and Glut4, which are transmembrane pro-
teins that act as gatekeepers. We hypothesized that Example 3c.1
the transport of glucose via Glut2/Glut4 might con- From Saito et al. (2020) – ‘A groove-shaped cavity
tribute to the production of a dentin bridge during was prepared on the mesial surface of the upper first
wound healing. Therefore, we explored Glut2 and molars in wild-type and Opn knockout (KO) mice’.
Glut4 expression during reparative dentinogenesis
after mineral trioxide aggregate capping’. Example 3c.2
From Azevedo et al. (2019) – ‘Experimental periapical
lesions (C57Bl/6 wild-type mice) were evaluated
Item 3b: Abstract – The unambiguous aim(s) and
regarding endogenous vasoactive intestinal peptide
objective(s) of the study must be provided
(VIP) expression correlation with lesion development
Explanation and the effect of recombinant vasoactive intestinal
The aim and objectives must be clearly described using peptide (VIP) therapy in lesion outcome’.
terms that do not confuse readers (Examples 3b.1, 3b.2).
Terms that have more than one clear assumed meaning
Item 3d: Abstract – Key details of the methodology
must be defined by using a specific criterion, such as suc-
must be provided
cess or failure. For example, success can mean several
things: survival of teeth, no pain, radiographic healing Explanation
or something else entirely. Failure can mean several The Methodology section within the Abstract must
things: lack of healing, flare up, pain, missed canal, irre- briefly explain what materials, devices, instruments,
versible pulpitis, necrotic pulp, loss of teeth, or some- motors, solutions, drugs and treatments were investi-
thing else entirely. To help authors improve the clarity gated, including the criteria used to describe the out-
of their aims and objectives, the use of PICO(T) elements: comes (Examples 3d.1, 3d.2).
Problem/Patient/Population, Intervention/Indicator,
Comparison, Outcome, and (optional) Time element or Example 3d.1
Type of Study, is recommended. From Conti et al. (2020) – ‘Atherosclerosis was
induced using a high-lipid diet associated with a sur-
Example 3b.1 gical ligature in the carotid artery and a super dosage
From Zaccaro Scelza et al. (2010) – ‘The present of vitamin D3. Apical periodontitis was induced via
study aimed to evaluate the inflammatory response of pulp exposure to the oral environment. At 45 and
17% EDTA, 17% EDTA-T, and 10% citric acid in 75 days, serum levels of total cholesterol (TC), triglyc-
bony defect created in rat jaws’. erides (TG), high-density lipoprotein cholesterol (HDL-
C) and low-density lipoprotein cholesterol (LDL-C)
Example 3b.2 were measured. The maxillary and mandibular jaws
From Tawil et al. (2009) – ‘The purpose of this study and carotid artery were collected and processed for
was to assess the healing of periapical tissues using histological analysis’.
three different materials (IRM [L.D. Caulk Inc, Dents-
ply International Inc, Milford, DE], Geristore [Den- Example 3d.2
Mat, Santa Maria, CA], and MTA [ProRoot MTA; From Jara et al. (2018) – ‘A standard serial root canal
Dentsply Tulsa Dental Specialties, Tulsa, OK]) after preparation technique was performed in the molar of
endodontic microsurgery in an animal model’. one side, whilst the opposite side was the control
© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 54, 858–886, 2021 861PRIASE 2021 explanation and elaboration Nagendrababu et al.
group. Rats were randomly divided into three experi- both in vivo and in vitro, and DMP1 compensates for
mental groups (n = 8), according to the diameter of the lack of OPN in regulating odontoblast like cell dif-
apical enlargement during root canal preparation: K- ferentiation after tooth injury’.
files size 20 (EG1), size 25 (EG2) and size 30 (EG3).
Each animal was its own positive control, because the Example 3f.2
opposite arch remained untreated. Root canals were From Conti et al. (2020) – ‘Apical periodontitis influ-
filled with a standard technique. After 3 weeks, the enced triglyceride levels, increasing them even in the
animals were euthanized. The main outcome of apical absence of atherosclerosis, and influenced the increase
periodontitis healing was evaluated radiographically in the thickness of the carotid artery intima tunic in
(mm2) and histologically (ordinal scores of inflamma- the presence of atherosclerosis. Atherosclerosis inten-
tion) using a HE staining technique’. sified the inflammatory reaction and increased bone
resorption in periapical lesions’.
Item 3e: Abstract – The most relevant and important
results must be presented succinctly including Item 4a: Introduction – The relevant background
differences amongst the means, medians or modes of information must be provided using terminologies
the dependent variables (treatment outcome and test consistent with professional standards and previous
results) and any significant P-values publications
Explanation Explanation
The mean, median or mode outcome(s) of the treat- The Introduction must accurately describe the rele-
ments should be reported, along with the differences, vant background information, using terminologies
and P-value significance (Examples 3e.1, 3e.2). consistent with professional standards and previous
publications (Examples 4a.1, 4a.2). Professional termi-
Example 3e.1 nologies (tooth number, root canal morphology, treat-
From Alves et al. (2018) – ‘There was no significant differ- ment etc.) must be used to avoid causing reader
ence in the bacterial penetration among groups A, B, and confusion. New terminologies should not be invented,
C at 45 days (P = 0.903) and 120 days (P = 0.211). No or old terminologies defined incorrectly. Multiple pro-
statistically significant difference was found (P = 0.608) fessional terminologies to describe the same issue
between the exposure time intervals’. must not be used, terminologies must be consistently
used throughout the manuscript, that is avoid invent-
Example 3e.2 ing a new term, novel root-end maturogenesis, to
From Berlin-Broner et al. (2020) – ‘Both groups devel- describe apexogenesis; avoid confusing root canal
oped a similar degree of atherosclerosis (mean lesion treatment with other endodontic treatments (e.g. par-
area 7.46 0.44% in the Tx group compared with tial-pulp capping, Cvek pulp capping, apexification,
7.65 0.46%, in the Sham group, P = 0.77), and a apexogenesis and regenerative endodontics).
similar degree of inflammation’.
Example 4a.1
From Choi et al. (2019) – ‘Vital pulp therapy such as
Item 3f: Abstract – Succinct conclusions supported
direct pulp capping, indirect pulp capping, and partial
by the results must be provided
or full pulpotomy can be used to preserve the health
Explanation status of teeth (1) because healthy pulp tissue is very
The Conclusions of the Abstract must be based only important for tooth longevity (2, 3). Up-regulation of
upon the results (Examples 3f.1, 3f.2). The best abstracts odontogenic differentiation, dentin formation, and
have memorable ‘take-away’ messages and give advice angiogenesis of human dental pulp cells (hDPCs) are
on future practice and research; however, over-general- key factors in vital pulp therapy (4). Materials used in
izing the conclusions or speculation must be avoided. vital pulp therapy should have adequate biocompati-
bility and bioactivity to promote dental pulp stem cell
Example 3f.1 activity and pulp healing in permanent teeth (5).
From Saito et al. (2020) – ‘These results suggest that Although mineral trioxide aggregate (MTA) provides
the expression of dentin matrix protein 1 (DMP1) is a very good sealing (6), acceptable biocompatibility,
up-regulated in osteopontin (OPN) knockout mice and dentin bridge formation in animal teeth (7) and
862 International Endodontic Journal, 54, 858–886, 2021 © 2021 International Endodontic Journal. Published by John Wiley & Sons LtdNagendrababu et al. PRIASE 2021 explanation and elaboration
human teeth 8, it has some drawbacks such as discol- etc.) and pets (dogs, cats etc.), a statement describing
oration potential, the presence of heavy metal, difficult the appropriateness of the animal model must be pro-
handling characteristics, a long setting time (9), and vided (Examples 4b.1, 4b.2, 4b.3). Extremely painful
high material cost. Thus, several new brands of MTA and extensive traumatic testing on some types of ani-
products such as Biodentine (Septodont, Saint-Maur- mals that maybe distressing to readers is generally
des-Fosses, France) and EndocemZr (Maruchi, Wonju, not acceptable for publication. Painful animal tests
Korea) have been introduced into the market in an must include pain monitoring and appropriate pain
attempt to overcome these shortcomings. These MTA relief measures. The animal model must have appro-
products have shown a relatively fast setting time, good priate tissues, cells, lesions, infections, anatomy, phys-
biologic outcomes, and acceptable color stability’. iology and an immune system to accomplish the aim
and objective of the study. Ideally, animals must have
Example 4a.2 a fully functioning immune system to study healing
From Lin et al. (2019) – ‘Dental pulp stem cells responses and disinfection, for example root canals
(DPSCs), the first identified dental stem cell source, must be infected to study disinfection. There should
have inherent mesenchymal characteristics and osteo- be a clear justification for using a particular animal
genic potential. DPSCs are harvested from adult tooth test method. For example, subcutaneous implantation
pulp tissues after enzyme treatment (3). Although of dental materials to study biocompatibility in accor-
gene profiles of DPSCs are similar to those of bone dance with ISO 10993 and 7405 standards.
marrow mesenchymal stem cells, DPSCs showed
higher colony-forming units and proliferation rates Example 4b.1
(4). In addition to a higher proliferation rate, DPSCs From Altaii et al. (2016) – ‘The possibility of
also possess the ability of mutilineage differentiation, endodontic regeneration/revitalization treatment of
such as osteogenic (5), neurogenic, and adipogenic immature infected teeth is a recent development offer-
lineages (6). In the past few decades, DPSCs for bone ing considerable biological advantages; but a more
tissue regeneration have been widely reported in tis- complete understanding of the treatment requires
sue engineering and regenerative medicine (7, 8). To in vivo research in a suitable animal model. Primates
begin with, significantly increased alkaline phos- have been used in many endodontic regeneration
phatase (ALP) levels and up-regulation of osteogenic studies because of their anatomical similarity to
markers, such as Runx2, osteopontin, and osteocal- human, but these animals are expensive, not readily
cin, were observed in DPSCs when cultured in osteo- available and can be difficult to manage. Dogs are
genic medium, suggesting that DPSCs were seen as pets in many cultures, and have substantially
undergoing osteogenic differentiation on the one hand different tooth anatomy to humans. Rodent incisor
(9, 10) After 40 days of culture, human DPSCs were teeth are small with wide-open apices and have a
reported to form a structure similar to a woven continuous growth. Larger animal models such as
fibrous bone with physical qualities of in vitro and pigs offer an alternative, but they can grow to an
in vivo bone on the other hand (11). Furthermore, unmanageable size and can be very unpleasant and
osteogenic induction of DPSCs and their combinations uncooperative. Sheep, on the other hand have been
with various biomaterials such as collagen were also used in many medical and dental studies due to their
investigated and resulted in positive outcomes (12, teeth being similar to humans in many anatomical
13, 14). Although the therapeutic potential of DPSCs and histological aspects. Sheep are widely available,
has been studied for bone regeneration, the therapeu- easy to handle and are comparatively cheap to keep
tic efficiency needs further consideration and exami- and maintain as they can be released to fields’.
nations for clinical applications (15)’.
Example 4b.2
From Kim et al. (2019) – ‘To achieve pulp-dentin
Item 4b: Introduction – The appropriateness of the
complex regeneration with tissue engineering, appro-
selected animal model to address the aims and
priate candidate substances have been proposed and
objectives of the study must be explained
tested in animal models. Unlike an in vitro environ-
Explanation ment in which several factors can be easily con-
In the context of the general public opposition to ani- trolled, in vivo experiments with animal teeth require
mal testing on nonhuman primates (monkeys, apes particularly advanced skills and techniques. Because
© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 54, 858–886, 2021 863PRIASE 2021 explanation and elaboration Nagendrababu et al.
of these difficulties, in vivo studies on pulp-dentin All factual statements must be supported by relevant
complex regeneration to date have usually involved literature citations (Examples 4c.1, 4c.2). That is, it is
ectopic transplantation of the candidate substance acceptable to cite reviews, but it is preferable to cite
into the subcutaneous tissue or renal capsule rather facts from original scientific publications. Whenever
than orthotopic transplantation directly into the there are guidelines for a professional standardized
teeth. Only several studies have been performed the approach relevant to the study, these should be
orthotopic transplantation of a candidate substance in described and conformed with: Institutional Animal
large animals such as dogs, pigs, ferrets, and mon- Care and Use Committees (IACUC), Institutional
keys. However, before applying these candidate sub- Review Board (IRB), International Organization for
stances in clinical trials, their treatment efficacies and Standardization (ISO), American National Standards
safeties should be evaluated using in vivo orthotopic Institute (ANSI), American Dental Association (ADA),
transplantation in a sufficient number of animals. Food and Drug Administration (FDA), etc. Any igno-
Experiments using sufficient numbers of animals are rance of the applicable standards will reflect poorly on
restricted by breeding, costs and ethical issues the authors’ depth of knowledge of the article topic.
involved in securing a sufficient number of experi-
mental animals. In contrast, mice are relatively inex- Example 4c.1
pensive, reproduce quickly, and can be easily From Berlin-Broner et al. (2020) – ‘In spite of the
manipulated genetically. Despite these advantages of numerous epidemiological studies suggesting a link
mice, most pulp-dentin complex regeneration studies between apical periodontitis (AP) and cardiovascular
have used large animals because the mouse tooth, of disease (CVD), causality has yet to be demonstrated.
which the diameter is only 1.5–2 mm, has been con- Performing a longitudinal study in humans to demon-
sidered too small’. strate causality is challenging due to the complexity
of the systemic conditions influencing inflammatory
Example 4b.3 status and the difficulty in controlling all potential
From Simon et al. (2008) – ‘To date, several animal confounders along with AP. The absence of animal
models of reparative dentinogenesis, including the rat, studies may be attributed to the complexity of the
dog, monkey and ferret, have been used; however, to experimental setting, which requires microsurgical
our knowledge, a mouse model has yet to be reported. techniques and long-term follow-up. Thus, there is a
The mouse represents an interesting and well-charac- gap in knowledge regarding the causality of the rela-
terized laboratory model, specifically with regard to tionship between AP and atherosclerosis, and the
transgenics. These models are predicted to be extre- mechanism(s) by which they may be linked, and an
mely informative in studies on the molecular signal- animal model is essential to study the role of AP as a
ing involved in pulp healing. The small size of the separate risk factor. The overall goal of this study was
animal, however, complicates surgical procedures twofold: first, to determine the feasibility of using the
during pulp capping, as traditional instrumentation is low-density lipoprotein receptor knockout (LDLR KO)
not suitable for use on molar teeth whose diameter is mouse, a classic and recognized model in the field for
approximately 1.4mm. Miniaturization of these proce- reliably mirroring aspects of human atherosclerotic
dures is therefore necessary to exploit the mouse as a disease, to study AP’.
laboratory model for pulp-capping research’.
Example 4c.2
From Leite et al. (2010) – ‘It was previously shown that
Item 4c: Introduction – A justification of the
dental pulp from diabetic rats stimulated catalase activ-
reasons why the investigation was necessary using
ity, suggesting an increase in oxidative stress in the den-
an animal model must be provided
tal pulp tissue of diabetic rats. The oxidative stress could
Explanation cause damage to biomolecules such as DNA, proteins
The Introduction must justify the use of an animal and lipids, compromising the functions of dental pulp.
model and adequately describe the background for Astaxanthin can be an adjunct in the treatment of dia-
using each of the treatments, materials, instruments betes, because it might restore some important cellular
and devices to allow readers to understand the rea- functions or at least prevent oxidative damage caused
sons for performing the investigation and to under- by a ROS-overproduction. Considering the excessive
stand any controversies or knowledge gaps that exist. generation of ROS in diabetes mellitus, it has been
864 International Endodontic Journal, 54, 858–886, 2021 © 2021 International Endodontic Journal. Published by John Wiley & Sons LtdNagendrababu et al. PRIASE 2021 explanation and elaboration
proposed that the supplementation of diabetic rats with should be submitted as supplemental materials with
astaxanthin might antagonize, or at least improve, the the manuscript to ensure ethical compliance with reg-
defect in their antioxidative status’. ulatory standards. The animal care methods and
treatments described in the ethical approval must
match precisely the words within the manuscript.
Item 4d: Introduction – The unambiguous aim(s)
The manuscript should also describe the housing,
and objectives(s) of the animal study must be
handling, diet, veterinary care and experimentation
provided
using animals, and how these care standards were
Explanation regulated, for example Animal [Scientific Procedures]
The aims and objectives must consider all the PICO(T) Act, U.K. (1986) or similar. The authors should need
elements (such as the Problem/Patient/Population, not name the institution who granted approval to
Intervention/Indicator, Comparison, Outcome, and maintain the blind peer review, the institution details
(optional) Time element or Type of Study) (Examples can be added after the peer-review has been com-
4d.1, 4d.2). In the interests of continuity and avoid- pleted.
ing reader confusion, the aim(s) and objective(s) in
the Introduction must be identical to the wording of Example 5a.1
the text of the Abstract. That is, to avoid having two From Palma et al. (2017) – ‘The study protocol was
different aim(s) and objective(s) for the same manu- approved by the Animal Welfare Committee of the
script, in different sections. Direcß~
ao-Geral de Veterin
aria of Portugal (no. 0420/
2011) and complied with the International Guiding
Example 4d.1 Principles for Biomedical Research Involving Animals
From Tawil et al. (2009) – ‘The purpose of the pre- (Geneva, 1985)’.
sent study was to evaluate the postsurgical periapical
healing response of three retro filling materials after Example 5a.2
6 months using a modern endodontic surgical proto-
From Mena-Alvarez et al. (2019) – ‘The present
col in beagle dogs’. research project has been approved by the Ethics
Committee for Research of University Alfonso X el
Example 4d.2 Sabio, by the Ethics Committee of the Animal
From Zaccaro Scelza et al. (2010) – ‘The aim of this Research Service of the Hospital Militar G
omez Ulla of
study was to verify the inflammatory response of Madrid (Ref. ES280790000187) and also by the Envi-
three decalcifying substances (17% EDTA, 17% ronment, Local Administration and Territorial Organi-
EDTA-T, and 10% citric acid) using an animal model zation Office of Madrid Autonomy (order number
in which critically sized mandibular defects were cre- PROEX 201/15). All sections of this report adhere to
ated that communicated from the buccal to the lin- the ARRIVE guidelines for reporting animal research
gual surfaces in rats’. 18. A completed ARRIVE guidelines checklist is
included in Checklist S1’.
Item 5a: Materials and Methods – The reference
Example 5a.3
number of the approval granted by the ethics
From Silva et al. (2020) – ‘All procedures were car-
board, such as an Institutional Review Board or
ried out in accordance with conventional guidelines
Institutional Animal Care committee, must be
in the Guide for the Care and Use of Laboratory
provided along with a reference to the applicable
Animals (US National Institutes of Health 85-23,
institutional and/or national regulations that were
revised 1996). The local Institutional Animal Care
enforced. Any identifying details about the authors
and Use Committee (register no. 1041) approved all
institution should not be disclosed during the blind
experimental protocols. This study is reported
peer review
according to the ARRIVE guidelines (Animal
Explanation Research: Reporting of in vivo experiments) [28]
The reference number of the approval granted by the and PREPARE guidelines (Planning Research and
ethics board must be provided (Examples 5a.1, 5a.2, Experimental Procedures on Animals: Recommenda-
5a.3). Ethical review board or Institutional Animal tions for Excellence) [29] with regard to the rele-
Care and Use Committee (IACUC) approval memos vant items. All efforts were made to minimize
© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 54, 858–886, 2021 865PRIASE 2021 explanation and elaboration Nagendrababu et al.
animal suffering and to reduce the number of ani- that correlated the volume of extruded dentine with
mals used with adherence to the 3Rs principles (re- inflammatory tissue reaction, an average effect size of
placement, reduction, and refinement)’. 0.7 was chosen. Other parameters included were:
alpha-error = 0.05, beta-power = 0.8 and correlation
between the repeated values of 0.5. The result indi-
Item 5b: Materials and Methods – The sample size
cated a minimum of 5 samples per group and per
must be justified by citing prior similar studies
experimental time’.
and/or be estimated by using statistical power
calculations to ensure an adequate sample size is
used to detect any significant differences and Item 5c: Materials and Methods – Details of how
answer the research questions. This is to avoid animal pain and disability was monitored and how
making any type I and type II errors animal suffering was prevented during all aspects
of experimentation must be provided
Explanation
Large numbers of animals are not always required to Explanation
obtain reliable results whilst using only a few ani- It is entirely unacceptable to cause preventable suffer-
mals should be avoided. In other words, the sample ing to animals during experimentation. Researchers
size should ensure a high probability of detecting a must never ignore pain, distress, discomfort, suffering,
significant P-value difference, if one truly exists. A disability, death, mayhem, excessive bleeding, gan-
reference to a similar study may be relevant provided grene, necrosis, hunger, thirst, lack of hygiene and
that the experimental design, the outcome and the general lack of animal care during experimentation.
minimal relevant difference are the same (Examples At the time of occurrence of an adverse event, correc-
5b.1, 5b.2, 5b.3). tive measures, pain relief, anaesthesia or euthanasia
must be provided to prevent animal suffering (Exam-
Example 5b.1 ples 5c.1, 5c.2). Details of pain monitoring and the
From Berlin-Broner et al. (2020) – ‘The number of pain relief measures taken to prevent animal suffering
mice in each group was based on a power calculation and disability must be described. In addition to pro-
from a previous periodontal disease study. In that viding details of animal housing conditions, bedding,
study, to achieve a P value < 0.05 with 90% power, light, food and temperature settings, to assure readers
the sample size was 11 mice per group. Based on the that the animals were being adequately cared for
length of the study and the degree of dermatitis expe- (Examples 5c.3, 5c.4).
rienced in the current facility, the number of mice
was increased; 17 in the treatment group (Tx) and Example 5c.1
22 in the Sham group completed the 16-week regi- From Silva et al. (2020) – ‘The animals were anes-
men’. thetized intraperitoneally with 1 mL/100 g of a solu-
tion containing 10% ketamine (1 mL/kg; Virbac; S~ ao
Example 5b.2 Paulo, SP, Brazil), 2% xylazine (0.5 mL/kg; For-
From Conti et al. (2020) – ‘Sample size was estimated tDodge; Rio de Janeiro, RJ, Brazil), 5% midazolam
based on data from previous studies. Considering an (0.6 mL/kg; Roche; Rio de Janeiro, RJ, Brazil), tra-
alpha error of 0.05% and 95% power to recognize a madol (0.2 mL/kg; Sun; Goi^ ania, GO, Brazil), and
significant difference of 1 in the median scores, a min- 0.9% saline solution (8.5 mL). During the postopera-
imum of seven animals per group was necessary. tive period, the rats received analgesia with 5 mg/kg
Considering possible animal deaths, three more ani- of meloxicam (Eurofarma; S~ ao Paulo, SP, Brazil) sub-
mals were added in each group, resulting in ten rats cutaneously every 24 h, starting immediately after
per group’. the surgical procedure and for 2 additional days’.
Example 5b.3 Example 5c.2
From Pappen et al. (2019) – ‘The minimum number From Alves et al. (2018) – ‘The dogs were sedated with
of samples needed to identify differences between an intramuscular injection of xylazine (Abbott, S~ ao
groups was determined using the G * Power 3.1 pro- Paulo, SP, Brazil) associated with a 10% ketamine
gramme for Mac (one-way ANOVA test from the F hydrochloride solution (Aster, S~ao Paulo, SP, Brazil),
family of tests). Due to the absence of previous studies and were anesthetized with a 5% solution of
866 International Endodontic Journal, 54, 858–886, 2021 © 2021 International Endodontic Journal. Published by John Wiley & Sons LtdNagendrababu et al. PRIASE 2021 explanation and elaboration
thionembutal (Abbott), injected intravenously at a dose fasting of the animals, carried out in accordance with
of 0.1 mL/kg. During the operative procedures, the ani- the guidelines of the National Institutes of Health
mals received an infusion of saline solution and intra- Guide for the Care and Use of Laboratory Animals,
venous anesthetics, as required. The dogs were and with the current international legislation on ani-
monitored throughout the entire experiment to ensure mal use in experimental research’.
that no clinical signs of infection or pathology were pre-
sent’.
Item 5e: Materials and methods – Specific details of
the animals must be provided, including their
Example 5c.3
species, strain, immune system, breeding
From da Fonseca et al. (2019) – ‘Sixty male Holtzman
programme, age, weight, health status and any
rats (Rattus norvegicus albinus) weighing 220 g
special characteristics
were housed in polyethylene cages under 12-h light/
12-h dark cycle at controlled temperature (23 2 °C) Explanation
and humidity (55 10%), with water and food (Guabi The source or supplier of animals must be identified.
rat chow, Paulınia, SP, Brazil) provided ad libitum’. If the animals were sourced from a breeding pro-
gramme, it should be described. The manuscript
Example 5c.4 should describe the animals using the international
From Reyes-Carmona et al. (2011) – ‘The experiments strain nomenclature, genetic modification status such
were conducted using 55 male Swiss mice aged 5 to as ‘knock out’ or ‘immunodeficient’, because these
7 weeks old (35–40 g) housed in polycarbonate cages specific variables could influence the results. The
placed in a ventilated, temperature-controlled room. manuscript should describe the animal’s average
Animals were kept in a 12-hour light/dark cycle, weight, species, strain, sex, age and tooth develop-
with controlled humidity (60% 5%) and tempera- mental stage to help readers adequately comprehend
ture (25°C 1°C). The commercial pellet diet and and replicate the study (Examples 5e.1, 5e.2).
distilled water were available ad libitum. Experiments
were performed during the light phase of the cycle. Example 5e.1
The animals were acclimatized to this environment From De Rossi et al. (2008) – ‘Male C57BL/6 wild-
for 5 days before testing’. type mice (WT) and mice deficient in IFN-c, IL-4, IL-
10, ICAM-1, and CCR5, 6 to 8 weeks old in the
beginning of the experiments, were used. The mice
Item 5d: Materials and methods – The job titles
were bred and maintained in microisolator cages in
and qualifications of the animal caretakers must
the animal housing facility of the Department of
be provided
Pathology, Faculty of Medicine of Ribeir~ ao Preto,
Explanation University of S~
ao Paulo. Mice with targeted disruption
The qualifications and job titles of the animal caretak- of IFN-c, IL-4, IL-10, ICAM-1, and CCR5 were
ers (e.g. certified animal technicians) must be obtained from Jackson Laboratories (Bar Harbor, ME).
described to ensure all animal care personnel were All knockout mice were originally generated in a
adequately qualified (Example 5d.1). The amount of mixed 129-B6-DBA background and then backcrossed
supervising veterinary care should be described, for to the C57BL/6J background for more than 8 genera-
example the animal housing facility had 24-h animal tions’.
care with emergency veterinarian support; the health
and welfare of each animal was monitored every hour Example 5e.2
for 3 days following surgery/intervention, and there- From Garlet et al. (2010) – ‘In this study, C57BL/6
after every 8 h. As good practice, the animal monitor- wild-type (WT) and CCR2 knockout (CCR2-KO) mice,
ing roles of the animal caretakers can be provided in obtained from Jackson Laboratory (Bar Harbor, ME),
a supplementary document. were used. CCR2-KO animals are generally healthy
and do not express any significant phenotype. All
Example 5d.1 experiments were performed with 8-week-old mice,
From Silva et al. (2020) – ‘A senior veterinarian con- weighing around 22 g, with at least 5 animals in
ducted all the nutritional recommendations and was each experimental group. Mice were bred and main-
in charge of the care and pre- and postoperative tained in FMRP animal house facilities’.
© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 54, 858–886, 2021 867PRIASE 2021 explanation and elaboration Nagendrababu et al.
Example 5f.2
Item 5f: Materials and methods – The
From Cotti et al. (2017) – ‘Three animals provided
experimental design must include details of the
the positive controls (control group) for the histologic
numbers of animals, numbers of experimental
evaluation of AP and 9 ferrets were randomly divided
units (e.g. teeth) and timelines (e.g. 5, 30 and
into 3 groups: Systemic group = RCT with systemic
60 days) used
adalimumab (Humira): 3 ferrets (12 teeth) received
Explanation conventional RCT and systemic subcutaneous admin-
Precise details of the experiments must be provided istrations of 0.2 mL adalimumab (50 mg/mL) every
and include the following: the numbers of animals, other week for the remainder of the study; local
the numbers of experimental units (defined as the group = RCT with local adalimumab: 12 teeth ran-
smallest unit to which a level of the treatment factor domly distributed in 6 ferrets received RCTs, and
can be administered, e.g. root canals) and post-treat- before canal obturation, had 0.1 mL adalimumab
ment timelines for each treatment data collection and administered via the root canal to the periapical
for the sacrifice of animals (Example 5f.1). In some areas; conventional RCT only (CRCT) group = RCT
studies, the experimental unit is the animal, in other only: 12 teeth randomly distributed in 6 ferrets
it may be the tooth (e.g. a split-mouth study; Example received conventional RCT. . . Postoperative CBCT
5f.2) or perhaps the root canal. That is, ‘After the six scans of the ferrets were obtained every 4 weeks for
animals reached two months of age (60 days 3 months, following completion of RCTs to monitor
3 days), the root canals were accessed and the healing of AP (ie, 30 [T2], 60 [T3], and 90 [T4]
infected/disinfected, after 7 days (1 day) three ani- days post-RCT scans)’.
mals were sacrificed, after 30 days (4 days) the final
three animals were sacrificed’. Any animals removed
Item 5g: Materials and methods – The primary
from the results or which died during the study (if
outcome data measures or categories as well as
any) must also be reported with an explanation.
any other secondary outcome data measures or
categories that will be assessed must be provided
Example 5f.1
From Reyes-Carmona et al. (2011) – ‘The animals Explanation
were divided into seven groups, with n = 10 for the The Materials and Methods must mention the unit of
12 hours and 1, 3, and 7-day experimental periods analysis for the outcome, for example single animal,
and n = 5 for the 15-, 30-, and 60-day time points. group of animals, samples/teeth/root
Mice were anesthetized with 80 mg/kg of ketamine canals/wounds/surgical sites/lesions/infections within
hydrochloride (Dopalen; Division Vetbrands Animal animals. In connection with the outcomes, the out-
Health, Jacareı, SP, Brazil) and 10 mg/kg of xyla- come measure(s) should also be mentioned, for exam-
zine (Anasedan; Agribrands do BrasilLtda, Paulınia, ple measured as X - Y or %X - %Y. If the outcomes
SP, Brazil). Then, four separate 1-cm incisions were were categories of healing, pulpitis, vitality, regenera-
made in the backs of mice at 1-cm intervals. The tion, success or failure etc., the published standardized
skin was deflected to create four subcutaneous criteria used must be described and cited (Example
pockets by a blunt dissection on one side of each 5g.1). Creating or inventing new criteria must be
incision, two in the cranial portion and two in the avoided when suitable standards exist such as ISO
caudal portion. Each mouse received three dentin 7405 and ISO 10993. Adherence to standardized
tubes, two filled with each material and one empty, methods used by prior high-quality peer-reviewed
whereas no specimen was inserted in the fourth publications will help increase the reproducibility,
pocket (sham). After 12 hours and 1, 3, 7, 15, 30, comparability, validity and reliability of the data.
and 60 days after implantation, the animals were
euthanized, the tubes with surrounding tissues were Example 5g.1
removed, and the surrounding tissues were col- From Wang et al. (2020) – ‘The degree of tooth re-erup-
lected. Half the samples (n = 5) from the 12-hour tion, pulp calcification, ankylosis, replacement root
to the 7-day time points were fixed in 4% resorption, and marginal bone loss in 2-dimensional
paraformaldehyde, at 4°C, for histological and and 3-dimensional image constructions were observed.
immunohistochemical staining’. The definitions of the observed outcomes are as follows:
868 International Endodontic Journal, 54, 858–886, 2021 © 2021 International Endodontic Journal. Published by John Wiley & Sons LtdNagendrababu et al. PRIASE 2021 explanation and elaboration
1. Degree of tooth re-eruption: the degree of tooth evaluating pain must describe how the pain was
re-eruption was divided into 3 categories: com- monitored, minimized, relieved and ended (Example
plete re-eruption, partial re-eruption and no re- 5h.1).
eruption. A completely re-erupted tooth was
when the occlusal surface of the intrusive tooth Example 5h.1
reached the occlusal level of the first molar. A no From Wu et al. (2010) – ‘Sixteen female wistar rats
re-erupted tooth referred to those whose occlusal weighing approximately 100g were obtained from
surface was still at the cervical level of the first Experimental Animal Center of Guangxi Medical
molar, and partially re-erupted referred to a state University. This study was approved by the animal
between completely re-erupted and no re-erupted. care and use committee of Guangxi Medical Univer-
2. Pulp calcification: radiopaque in the pulp cavity sity. The animals were divided randomly into two
and root canal. groups: Group I, Controls, animals were given tap
3. Ankylosis: the loss of periodontium space and the water containing 0.16 mg F per L; Group II ani-
integration of acellular cementum and alveolar mals were given sodium fluoride (NaF) in their drink-
bone. ing water at a concentration of 100 mg F per L.
4. Replacement root resorption: the root resorbed Each group consisted of eight female rats. Rats were
and replaced with bonelike tissue. fed regular laboratory rodent diet and were allowed
5. Marginal bone loss: the marginal bone in the buc- water ad libitum. After 3 months, the characteristic
cal side of the intruded tooth healed but recessed; enamel striations were apparent, which are indica-
marginal bone loss was evaluated from 30 days tions of dental fluorosis and altered mineralization of
after injury’. dentine and enamel. Rats were killed humanely by
cervical dislocation, and a pair of mandibular central
incisors was dissected from each animal, and the
Item 5h: Materials and Methods – Details must be
unmineralized proximal portion of the incisor was
provided on (1) steps in the interventions and
removed. The incisors were split longitudinally into
treatments, (2) instruments, medicaments or
two halves, and the pulp tissues removed using a
device allocation, and (3) concealment and
spoon excavator. One pulp of each rat was used for
randomization prior to data collection
microarray analysis. Every four pulps of each group
Explanation were pooled into one tube. Then, each group samples
Sufficient details of each step of the interventions or was divided into two pools for microarray analysis.
treatments must be described, including sterilization, Control pulps were divided into two pools of 4(c1), 4
disinfection, aseptic handling, the type of injury, infec- (c2); fluoride treatment group into two pools of 4(f1),
tion, or disease created, followed by the interven- 4(f2). The remaining pulps of each group were pooled
tion/surgery. All test materials, supplies, assays or into one tube, 8(c3, control group), 8 (f3, fluoride
equipment handling should be used according to the treatment group), which were submitted to RNA iso-
manufacturer’s directions to counter potential criti- lation for validation of microarray experiment data’.
cism of a lack of conformity. If image analysis soft-
ware was used to collect data from histology,
Item 5i: Materials and methods – Details regarding
radiographs or micro-CT images, how the data collec-
postdisease and postoperative care of the animals
tion was calibrated and validated must be explained.
must be provided
The wavelengths of spectrophotometers, light curing
units, flow cytometers and measurements from Explanation
Instron machines and microscopes must be calibrated, It is not ethically acceptable to mistreat animals used
to ensure accurate data collection. If any subjective in experimentation, by ignoring any severe pain, suf-
results were collected, the steps taken to prevent bias fering or disability. To ensure adequate care and wel-
must be described. The manuscript should describe fare of animals, the manuscript should provide details
how the investigators were not aware of the treat- about the postinjury, postdisease and postoperative
ments, materials or assignments of specimens (histol- care and monitoring to ensure steps were taken to
ogy, photographs, micrographs, radiographs, assays guarantee that animals were not disabled, disfigured
etc.) by randomizing them and concealing them with and did not experience severe pain as a result of the
blind codes during data collection. Animal studies experimentation. Pain relief medications and
© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 54, 858–886, 2021 869PRIASE 2021 explanation and elaboration Nagendrababu et al.
antibiotics should always be used (Examples 5i.1, 5i.2, statistical information is essential, including details of
5i.3, 5i.4, 5i.5). Withholding care and medicaments the statistical tests used for analysis, the type of soft-
from animals is always unacceptable. ware used, and any steps taken to validate or control
the accuracy of the data. Preferably, the steps of the
Example 5i.1 statistical analysis should be described in the same
From Paras et al. (2019) – ‘During the 30 days of the order as used when the results are presented. A com-
experiment, the health status of the animals (behaviour, plete statistical analysis must include an analysis of
changes in the skin and hair, food and water consum- the significance of the differences between each of the
mation, urinating and defecation) was checked daily’. test/treatment group means, and if relevant confi-
dence intervals (Examples 5j.1, 5j2, 5j.3). Nowadays,
Example 5i.2 when used correctly, the commercially available sta-
From Verma et al. (2017) – ‘An analgesic (carprofen, tistical software are highly reliable, so using two dif-
3 mg/kg, subcutaneous, every 24 h) was given ferent software packages is not always necessary to
immediately prior to the procedure and continued validate statistical analyses. The main problem with
until the following day to manage postoperative pain’. the statistical analyses submitted to journals is not
the quality of the software package, but the statistical
Example 5i.3 ignorance of the user. To prevent potential evaluator
From Silva et al. (2020) – ‘During the postoperative bias and to validate the accuracy of data, automated
period, the rats received analgesia with 5 mg/kg of data collection by machine using values or image
meloxicam (Eurofarma; S~ ao Paulo, SP, Brazil) subcu- analysis should be considered, or by using two-inde-
taneously every 24 h, starting immediately after the pendent data collectors. The use of methods of analy-
surgical procedure and for 2 additional days’. sis that enable quantification and parametric
statistical methods, when possible, should be
Example 5i.4 described. It is good practice to have the statistical
From Pappen et al. (2019) – ‘After the experimental analysis performed by someone who did not collect
procedures, the animals were placed in cages until the data, such as a statistician or colleague. If the
their recovery. Two animals were kept per house, statistics is unfamiliar or complex, a statistician
with a cycle of 12 h day night 1, temperature should be consulted to validate the analysis to avoid
between 19–23 °C, relative air humidity between reporting errors and artefacts. Datasets should be
40%–70%. To aid recovery, paracetamol designed to include positive and negative control sam-
(0.06 mg g 1 day 1) was added to their drink- ples/groups to help validate the accuracy of the data
ing water for 72 h’. (e.g. antibody specificity) and to identify problems
such as a failure to completely sterilize biomaterials
Example 5i.5 prior to testing, or the use of contaminated cultures
From Altaii et al. (2017) – ‘Experimental animals were of E. faecalis. If using absorbance or light curing
subjected to a treatment protocol comprising four ses- methods for material setting, all light sources and
sions. After each session, animals were given analgesics machines must be calibrated prior to experimentation.
(50 mg mL 1 Rimadyl IM injection, Pfizer, West
Ryde, Australia). Postoperatively, animals were given Example 5j.1
analgesics (2.2 mg kg 1 Rimadyl tablet) and visually From Jara et al. (2018) – ‘The distributions of the
monitored to check for signs of distress’. radiographic and histological parameters were anal-
ysed, and descriptive statistics (mean and standard
deviation) were calculated. Normality was ascertained
Item 5j: Materials and methods – Details on the
by Kolmogorov–Smirnov test. The measurement of
statistical analysis, statistical tests, the type of
effect was obtained between the three experimental
software used, and the steps taken to control,
groups (EG1, EG2 and EG3) by carrying out general-
interpret success or failure, and to validate the
ized estimating equations, with Poisson regression
accuracy of the data must be provided
with robust variance, pairing each EG with its respec-
Explanation tive CG within animals, and adjusted for the mean
All too often a single P-value is all that is given from within animal differences (Δ = CG side - EG side),
a multiple-group statistical analysis. Additional with a = 5%. Data were analysed using SPSS
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