Inhibition of the Nogo-pathway in experimental spinal cord injury: a meta-analysis of 76 experimental treatments.

Recovery after spinal cord injury (SCI) may be propagated by plasticity-enhancing treatments. The myelin-associated nerve outgrowth inhibitor Nogo-A (Reticulon 4, RTN4) pathway has been shown to restrict neuroaxonal plasticity in experimental SCI models. Early randomized controlled trials are underway to investigate the effect of Nogo-A/Nogo-Receptor (NgR1) pathway blockers. This systematic review and meta-analysis of therapeutic approaches blocking the Nogo-A pathway interrogated the efficacy of functional locomotor recovery after experimental SCI according to a pre-registered study protocol. A total of 51 manuscripts reporting 76 experiments in 1572 animals were identified for meta-analysis. Overall, a neurobehavioral improvement by 18.9% (95% CI 14.5-23.2) was observed. Subgroup analysis (40 experiments, N = 890) revealed SCI-modelling factors associated with outcome variability. Lack of reported randomization and smaller group sizes were associated with larger effect sizes. Delayed treatment start was associated with lower effect sizes. Trim and Fill assessment as well as Egger regression suggested the presence of publication bias. Factoring in theoretically missing studies resulted in a reduced effect size [8.8% (95% CI 2.6-14.9)]. The available data indicates that inhibition of the Nogo-A/NgR1pathway alters functional recovery after SCI in animal studies although substantial differences appear for the applied injury mechanisms and other study details. Mirroring other SCI interventions assessed earlier we identify similar factors associated with outcome heterogeneity.

Brain pericytes in culture display diverse morphological and functional phenotypes.

Pericytes play several important functions in the neurovascular unit including contractile control of capillaries, maintenance of the BBB, regulation of angiogenesis, and neuroinflammation. There exists a continuum of pericyte subtypes along the vascular tree which exhibit both morphological and transcriptomic differences. While different functions have been associated with the pericyte subtypes in vivo, numerous recent publications have used a primary human brain vascular pericytes (HBVP) cell line where this pericyte heterogeneity has not been considered. Here, we used primary HBVP cultures, high-definition imaging, cell motility tracking, and immunocytochemistry to characterise morphology, protein expression, and contractile behaviour to determine whether heterogeneity of pericytes also exists in cultures. We identified five distinct morphological subtypes that were defined using both qualitative criteria and quantitative shape analysis. The proportion of each subtype present within the culture changed as passage number increased, but pericytes did not change morphological subtype over short time periods. The rate and extent of cellular and membrane motility differed across the subtypes. Immunocytochemistry revealed differential expression of alpha-smooth muscle actin (αSMA) across subtypes. αSMA is essential for cell contractility, and consequently, only subtypes with high αSMA expression contracted in response to physiological vasoconstrictors endothelin-1 (ET1) and noradrenaline (NA). We conclude that there are distinct morphological subtypes in HBVP culture, which display different behaviours. This has significance for the use of HBVP when modelling pericyte physiology in vitro where relevance to in vivo pericyte subtypes along the vascular tree must be considered.

Microglia directly associate with pericytes in the central nervous system.

Cerebral blood flow (CBF) is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimer's disease (AD). Microglia associations with capillaries suggest they may play a role in the regulation of CBF or the blood-brain-barrier (BBB). We explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the control of CBF and maintenance of the BBB, discovering a spatially distinct subset of microglia that closely associate with pericytes. We termed these pericyte-associated microglia (PEM). PEM are present throughout the brain and spinal cord in NG2DsRed × CX3 CR1+/GFP mice, and in the human frontal cortex. Using in vivo two-photon microscopy, we found microglia residing adjacent to pericytes at all levels of the capillary tree and found they can maintain their position for at least 28 days. PEM can associate with pericytes lacking astroglial endfeet coverage and capillary vessel width is increased beneath pericytes with or without an associated PEM, but capillary width decreases if a pericyte loses a PEM. Deletion of the microglia fractalkine receptor (CX3 CR1) did not disrupt the association between pericytes and PEM. Finally, we found the proportion of microglia that are PEM declines in the superior frontal gyrus in AD. In summary, we identify microglia that specifically associate with pericytes and find these are reduced in number in AD, which may be a novel mechanism contributing to vascular dysfunction in neurodegenerative diseases.

A Systematic Review and Meta-Analysis on the Therapeutic Efficacy of Heparin and Low Molecular Weight Heparins in Animal Studies of Traumatic Brain Injury.

The identification of effective pharmacotherapies for traumatic brain injury (TBI) remains a major challenge. Treatment with heparin and its derivatives is associated with neuroprotective effects after experimental TBI; however, the optimal dosage and method of administration, modes of action, and effects on hemorrhage remain unclear. Therefore, this review aimed to systematically evaluate, analyze, and summarize the available literature on the use of heparin and low molecular weight heparins (LMWHs) as treatment options for experimental TBI. We searched two online databases (PubMed and ISI Web of Science) to identify relevant studies. Data pertaining to TBI paradigm, animal subjects, drug administration, and all pathological and behavior outcomes were extracted. Eleven studies met our pre-specified inclusion criteria, and for outcomes with sufficient numbers, data from seven publications were analyzed in a weighted mean difference meta-analysis using a random-effects model. Study quality and risk of bias were also determined. Meta-analysis revealed that heparin and its derivatives decreased brain edema, leukocyte rolling, and vascular permeability, and improved neurological function. Further, treatment did not aggravate hemorrhage. These findings must be interpreted with caution, however, because they were determined from a limited number of studies with substantial heterogeneity. Also, overall study quality was low based on absences of data reporting, and potential publication bias was identified. Importantly, we found that there are insufficient data to evaluate the variables we had hoped to investigate. The beneficial effects of heparin and LMWHs, however, suggest that further pre-clinical studies are warranted.

Vascular perfusion differs in two distinct PDGFRß-positive zones within the ischemic core of male mice 2 weeks following photothrombotic stroke.

Stroke therapy has largely focused on preventing damage and encouraging repair outside the ischemic core, as the core is considered irreparable. Recently, several studies have suggested endogenous responses within the core are important for limiting the spread of damage and enhancing recovery, but the role of blood flow and capillary pericytes in this process is unknown. Using the Rose Bengal photothrombotic model of stroke, we illustrate blood vessels are present in the ischemic core and peri-lesional regions 2 weeks post stroke in male mice. A FITC-albumin gel cast of the vasculature revealed perfusion of these vessels, suggesting cerebral blood flow (CBF) may be partially present, without vascular leakage. The length of these vessels is significantly reduced compared to uninjured regions, but the average width is greater, suggesting they are either larger vessels that survived the initial injury, smaller vessels that have expanded in size (i.e., arteriogenesis), or that neovascularization begins with larger vessels. Concurrently, we observed an increase in platelet-derived growth factor receptor beta (PDGFRß, a marker of pericytes) expression within the ischemic core in two distinct patterns, one which resembles pericyte-derived fibrotic scarring at the edge of the core, and one which is vessel associated and may represent blood vessel recovery. We find little evidence for dividing cells on these intralesional blood vessels 2 weeks post stroke. Our study provides evidence flow is present in PDGFRß-positive vessels in the ischemic core 2 weeks post stroke. We hypothesize intralesional CBF is important for limiting injury and for encouraging endogenous repair following cerebral ischemia.

Automated Quantification of Multiple Cell Types in Fluorescently Labeled Whole Mouse Brain Sections Using QuPath.

The quantification of labeled cells in tissue sections is crucial to the advancement of biological knowledge. Traditionally, this was a tedious process, requiring hours of careful manual counting in small portions of a larger tissue section. To overcome this, many automated methods for cell analysis have been developed. Recent advances in whole slide scanning technologies have provided the means to image cells in entire tissue sections. However, common automated analysis tools do not have the capacity to deal with the large image files produced. Herein, we present a protocol for the quantification of two fluorescently labeled cell populations, namely pericytes and microglia, in whole brain tissue sections. This protocol uses custom-made scripts within the open source software QuPath to provide a framework for the careful optimization and validation of automated cell detection parameters. Images obtained from a whole-slide scanner are first loaded into a QuPath project. Manual counts are performed on small sample regions to optimize cell detection parameters prior to automated quantification of cells across entire brain regions. Even though we have quantified pericytes and microglia, any fluorescently labeled cell with clear labeling in and around the nucleus can be analyzed using these methods. This protocol provides a user-friendly and cost-effective framework for the automated analysis of whole tissue sections.

Fibrin clot characteristics and anticoagulant response in a SARS-CoV-2-infected endothelial model.

Coronavirus disease 2019 (COVID-19) patients have increased thrombosis risk. With increasing age, there is an increase in COVID-19 severity. Additionally, adults with a history of vasculopathy have the highest thrombotic risk in COVID-19. The mechanisms of these clinical differences in risk remain unclear. Human umbilical vein endothelial cells (HUVECs) were infected with SARS-CoV-2, influenza A/Singapore/6/86 (H1N1) or mock-infected prior to incubation with plasma from healthy children, healthy adults or vasculopathic adults. Fibrin on surface of cells was observed using scanning electron microscopy, and fibrin characteristics were quantified. This experiment was repeated in the presence of bivalirudin, defibrotide, low-molecular-weight-heparin (LMWH) and unfractionated heparin (UFH). Fibrin formed on SARS-CoV-2 infected HUVECs was densely packed and contained more fibrin compared to mock-infected cells. Fibrin generated from child plasma was the thicker than fibrin generated in vasculopathic adult plasma (p = 0.0165). Clot formation was inhibited by LMWH (0.5 U/ml) and UFH (0.1-0.7 U/ml). We show that in the context of the SARS-CoV-2 infection on an endothelial culture, plasma from vasculopathic adults produces fibrin clots with thinner fibrin, indicating that the plasma coagulation system may play a role in determining the thrombotic outcome of SARS-CoV-2 infection. Heparinoid anticoagulants were most effective at preventing clot formation.

What has preclinical systematic review ever done for us?

Systematic review and meta-analysis are a gift to the modern researcher, delivering a crystallised understanding of the existing research data in any given space. This can include whether candidate drugs are likely to work or not and which are better than others, whether our models of disease have predictive value and how this might be improved and also how these all interact with disease pathophysiology. Grappling with the literature needed for such analyses is becoming increasingly difficult as the number of publications grows. However, narrowing the focus of a review to reduce workload runs the risk of diminishing the generalisability of conclusions drawn from such increasingly specific analyses. Moreover, at the same time as we gain greater insight into our topic, we also discover more about the flaws that undermine much scientific research. Systematic review and meta-analysis have also shown that the quality of much preclinical research is inadequate. Systematic review has helped reveal the extent of selection bias, performance bias, detection bias, attrition bias and low statistical power, raising questions about the validity of many preclinical research studies. This is perhaps the greatest virtue of systematic review and meta-analysis, the knowledge generated ultimately helps shed light on the limitations of existing research practice, and in doing so, helps bring reform and rigour to research across the sciences. In this commentary, we explore the lessons that we have identified through the lens of preclinical systematic review and meta-analysis.

Pharmacological PDGFRß inhibitors imatinib and sunitinib cause human brain pericyte death in vitro.

Capillary pericytes have numerous functions important for tissue maintenance. Changes in pericyte function are implicated in diseases such as cancer, where pericyte-mediated angiogenesis contributes to the blood supply that tumors use to survive. Some anti-cancer agents, like imatinib, target platelet-derived growth factor receptor-beta (PDGFRß). Healthy pericytes rely on PDGFRß phosphorylation for their survival. Therefore, we hypothesised that pharmacological agents that block PDGFRß phosphorylation could be used to kill pericytes. We treated human brain vascular pericytes, which express PDGFRß, with three receptor tyrosine kinase inhibitors: imatinib, sunitinib and orantinib. Imatinib and sunitinib, but not orantinib, inhibited PDGFRß phosphorylation in pericytes. Imatinib and sunitinib also reduced viability, prevented proliferation, and induced death, while orantinib only blocked pericyte proliferation. Overall, we found that receptor tyrosine kinase inhibitors that block PDGFRß phosphorylation cause healthy pericytes to die in vitro. While useful in cancer to limit tumor growth, these agents could impair healthy brain pericyte survival and impact brain function.

An Automated Approach to Improve the Quantification of Pericytes and Microglia in Whole Mouse Brain Sections.

Whole slide scanning technology has enabled the generation of high-resolution images from complete tissue sections. However, commonly used analysis software is often unable to handle the large data files produced. Here, we present a method using the open-source software QuPath to detect, classify and quantify fluorescently-labeled cells (microglia and pericytes) in whole coronal brain tissue sections. Whole-brain sections from both male and female NG2DsRed x CX3CR1+/GFP mice were analyzed. Small regions of interest were selected and manual counts were compared with counts generated from an automated approach, across a range of detection parameters. The optimal parameters for detecting cells and classifying them as microglia or pericytes in each brain region were determined and applied to annotations corresponding to the entire somatosensory and motor cortices, hippocampus, thalamus, and hypothalamus in each section. 3.74% of all detected cells were classified as pericytes; however, this proportion was significantly higher in the thalamus (6.20%) than in other regions. In contrast, microglia (4.51% of total cells) were more abundant in the cortex (5.54%). No differences were detected between male and female mice. In conclusion, QuPath offers a user-friendly solution to whole-slide image analysis which could lead to important new discoveries in both health and disease.

Circadian Biology and Stroke.

Circadian biology modulates almost all aspects of mammalian physiology, disease, and response to therapies. Emerging data suggest that circadian biology may significantly affect the mechanisms of susceptibility, injury, recovery, and the response to therapy in stroke. In this review/perspective, we survey the accumulating literature and attempt to connect molecular, cellular, and physiological pathways in circadian biology to clinical consequences in stroke. Accounting for the complex and multifactorial effects of circadian rhythm may improve translational opportunities for stroke diagnostics and therapeutics.

Acute Stroke Biomarkers: Are We There Yet?

Background: Distinguishing between stroke subtypes and knowing the time of stroke onset are critical in clinical practice. Thrombolysis and thrombectomy are very effective treatments in selected patients with acute ischemic stroke. Neuroimaging helps decide who should be treated and how they should be treated but is expensive, not always available and can have contraindications. These limitations contribute to the under use of these reperfusion therapies. Aim: An alternative approach in acute stroke diagnosis is to identify blood biomarkers which reflect the body's response to the damage caused by the different types of stroke. Specific blood biomarkers capable of differentiating ischemic from hemorrhagic stroke and mimics, identifying large vessel occlusion and capable of predicting stroke onset time would expedite diagnosis and increase eligibility for reperfusion therapies. Summary of Review: To date, measurements of candidate biomarkers have usually occurred beyond the time window for thrombolysis. Nevertheless, some candidate markers of brain tissue damage, particularly the highly abundant glial structural proteins like GFAP and S100ß and the matrix protein MMP-9 offer promising results. Grouping of biomarkers in panels can offer additional specificity and sensitivity for ischemic stroke diagnosis. Unbiased "omics" approaches have great potential for biomarker identification because of greater gene, protein, and metabolite coverage but seem unlikely to be the detection methodology of choice because of their inherent cost. Conclusion: To date, despite the evolution of the techniques used in their evaluation, no individual candidate or multimarker panel has proven to have adequate performance for use in an acute clinical setting where decisions about an individual patient are being made. Timing of biomarker measurement, particularly early when decision making is most important, requires urgent and systematic study.

Differences in fatigue-like behavior in the lipopolysaccharide and poly I:C inflammatory animal models.

Central fatigue is a condition associated with impairment of the central nervous system often leading to the manifestation of a range of debilitating symptoms. Fatigue can be a consequence of systemic inflammation following an infection. Administration of lipopolysaccharide (LPS) and polyriboinosinic:polyribocytidlic (poly I:C) to animals can induce systemic inflammation by mimicking a bacterial or viral infection respectively and therefore have been used as models of fatigue. We evaluated a range of phenotypic behaviors exhibited in the LPS and poly I:C animal models to assess whether they adequately replicate fatigue symptomology in humans. In addition to standard observation- and intervention-based behavioral assessments, we used powerful in-cage monitoring technology to quantify rodent behavior without external interference. LPS and poly I:C treated Sprague Dawley rats displayed 'sickness behaviors' of elevated temperature, weight loss and reduced activity in the open field test and with in-cage monitoring within 24 h post-treatment, but only LPS-treated rats displayed these behaviors beyond these acute timepoints. Once sickness behavior diminished, LPS-treated rats exhibited an increase in reward-seeking and motivation behaviors. Overall, these results suggest that the LPS animal model produces an extensive and sustained fatigue-like phenotype, whereas the poly I:C model only produced acute effects. Our results suggest that the LPS animal model is a more suitable candidate for further studies on central fatigue-like behavior.

Combined meta-analysis of preclinical cell therapy studies shows overlapping effect modifiers for multiple diseases.

INTRODUCTION: Cell therapy has been studied in many different research domains. Cellular replacement of damaged solid tissues is at an early stage of development, with much still to be understood. Systematic reviews and meta-analyses are widely used to aggregate data and find important patterns of results within research domains.We set out to find common biological denominators affecting efficacy in preclinical cell therapy studies for renal, neurological and cardiac disease. METHODS: We used datasets of five previously published meta-analyses investigating cell therapy in preclinical models of chronic kidney disease, spinal cord injury, stroke and ischaemic heart disease. We transformed primary outcomes to ratios of means to permit direct comparison across disease areas. Prespecified variables of interest were species, immunosuppression, cell type, cell origin, dose, delivery and timing of the cell therapy. RESULTS: The five datasets from 506 publications yielded data from 13 638 animals. Animal size affects therapeutic efficacy in an inverse manner. Cell type influenced efficacy in multiple datasets differently, with no clear trend for specific cell types being superior. Immunosuppression showed a negative effect in spinal cord injury and a positive effect in cardiac ischaemic models. There was a dose-dependent relationship across the different models. Pretreatment seems to be superior compared with administration after the onset of disease. CONCLUSIONS: Preclinical cell therapy studies are affected by multiple variables, including species, immunosuppression, dose and treatment timing. These data are important when designing preclinical studies before commencing clinical trials.

Acute Routine Leukocyte and Neutrophil Counts Are Predictive of Poststroke Recovery at 3 and 12 Months Poststroke: An Exploratory Study.

Background and Aims. White blood cell (WBC) and neutrophil counts (NC) are common markers of inflammation and neurological stroke damage and could be expected to predict poststroke outcomes. Objective. The aim of this study was to explore the prognostic value of early poststroke WBC and NC to predict cognition, mood, and disability outcomes at 3 and 12 months poststroke. Methods. Routine clinical analyses WBC and NC were collected at 3 time points in the first 4 days of hospitalization from 156 acute stroke patients. Correlations using hierarchical or ordinal regressions were explored between acute WBC and NC and functional recovery, depression, and cognition at 3 and 12 months poststroke, after covarying for age and baseline stroke severity. Results. We found significant increases in NC between <12 hours and 24 to 48 hours time points (P = .05). Hierarchical regressions, covaried for age and baseline stroke severity, found that 24 to 48 hours WBC (P = .05) and NC (P = .04) significantly predicted 3-month cognition scores. Similarly, 24 to 48 hours WBC (P = .05) and NC (P = .02) predicted cognition scores at 12 months. Increases in WBC and NC were predictive of increased cognition scores at both 3 and 12 months (positive recovery) though there were no significant associations between WBC and NC and disability or depression scores. Conclusions. Routine acute stroke clinical laboratory tests such as WBC and NC taken between 24 and 48 hours poststroke are predictive of cognition poststroke. It is interpreted that higher rapid immunological activation in the acute phase is an indicator for the trajectory of positive stroke recovery.

Longitudinal Stroke Recovery Associated With Dysregulation of Complement System-A Proteomics Pathway Analysis.

Currently the longitudinal proteomic profile of post-ischemic stroke recovery is relatively unknown with few well-accepted biomarkers or understanding of the biological systems that underpin recovery. We aimed to characterize plasma derived biological pathways associated with recovery during the first year post event using a discovery proteomics workflow coupled with a topological pathway systems biology approach. Blood samples (n = 180, ethylenediaminetetraacetic acid plasma) were collected from a subgroup of 60 first episode stroke survivors from the Australian START study at 3 timepoints: 3-7 days (T1), 3-months (T2) and 12-months (T3) post-stroke. Samples were analyzed by liquid chromatography mass spectrometry using label-free quantification (data available at ProteomeXchange with identifier PXD015006). Differential expression analysis revealed that 29 proteins between T1 and T2, and 33 proteins between T1 and T3 were significantly different, with 18 proteins commonly differentially expressed across the two time periods. Pathway analysis was conducted using Gene Graph Enrichment Analysis on both the Kyoto Encyclopedia of Genes and Genomes and Reactome databases. Pathway analysis revealed that the significantly differentiated proteins between T1 and T2 were consistently found to belong to the complement pathway. Further correlational analyses utilized to examine the changes in regulatory effects of proteins over time identified significant inhibitory regulation of clusterin on complement component 9. Longitudinal post-stroke blood proteomics profiles suggest that the alternative pathway of complement activation remains in a state of higher activation from 3-7 days to 3 months post-stroke, while simultaneously being regulated by clusterin and vitronectin. These findings also suggest that post-stroke induced sterile inflammation and immunosuppression could inhibit recovery within the 3-month window post-stroke.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration (E&E) document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research.

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the "ARRIVE Essential 10," which constitutes the minimum requirement, and the "Recommended Set," which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration (E&E) document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.