Keywords
stroke, preclinical, systematic review, network metaanalysis, network meta-analysis,
This article is included in the Preclinical Reproducibility and Robustness gateway.
This article is included in the Iowa State University collection.
stroke, preclinical, systematic review, network metaanalysis, network meta-analysis,
NMA, network meta-analysis; DIC, deviance information criteria; NMD, normalized mean difference
Globally, an estimated 15 million people suffer a stroke; stroke is the second leading cause of death, with six million people dying and an additional five million becoming permanently disabled each year1,2. The costs of stroke are high due to a combination of immediate high costs from acute care and long-term costs from resulting disability. Worldwide cost estimates range from $266 billion to $1.038 trillion per year3. Despite the enormous human and economic burden, only four acute interventions are currently used clinically: patient care in a dedicated stroke unit3, reperfusion (by pharmacological thrombolysis or endovascular mechanical thrombectomy4), oral aspirin, and surgical decompression.
In the search for novel therapies for acute stroke, more than 1,000 potential neuroprotective therapeutics (e.g. anticoagulants, calcium channel blockers, free radical scavengers, GABA mimetics, etc.) have been evaluated in preclinical models5. Of these, only reperfusion with tissue plasminogen activators6, such as alteplase3, has had a preclinical basis. Despite its efficacy, alteplase has inherent limitations such as the risk of hemorrhagic transformation, which warrants exploration of novel adjunctive therapies that can maximize therapeutic benefit. Combination therapies with alteplase might limit reperfusion injury and cell death that can sometimes occur with this drug. However, given the multitude of therapies tested preclinically (and multiple mechanisms of action) it is difficult to assess which therapies should proceed to clinical testing.
Preclinical systematic reviews have served as a robust form of knowledge synthesis to evaluate transparently experimental therapies for more than a decade7–9. Previous preclinical systematic reviews have compared treatments in isolation using pair-wise meta-analyses, which limits the ability to simultaneously evaluate comparative effectiveness in the presence of many treatments of interest. Use of network meta-analysis (NMA) in comparative effectiveness research to study the relative benefits and harms of multiple interventions in humans9,10 has risen dramatically during the past decade11. Such analyses allow the comparison of many interventions based on all ‘direct’ and ‘indirect’ information. In addition, this approach has the potential to establish a more rigorous framework for decisions to embark on clinical trials while reducing risks to human trial participants and the enormous costs of preclinical translation3,7,12. Comparison of preclinical stroke therapeutics represents an excellent case study for such work. Given the novelty of this approach, this systematic review will also serve as a case study to empirically explore the methodological nuances of applying NMA in a preclinical setting.
This protocol will be registered in the international prospective register of systematic reviews (PROSPERO, CRD) following peer review. Our review protocol is reported in accordance with the Preferred Reporting Items in Systematic reviews and Meta-Analysis-Protocol guidelines (a complete checklist is available as Supplementary File 1)13. Post-protocol adjustments will be included in the final report.
Primary objective. We will perform a systematic review and NMAs to address the following question: amongst in vivo models of focal ischemic stroke, what are the relative benefits of competing therapies tested in combination with the gold standard treatment alteplase14 in (i) reducing cerebral infarction size, and (ii) improving neurobehavioural outcomes?
Secondary Objective. We will also (i) assess the risk of bias of the included studies, and (ii) explore what novel considerations for statistical adjustments are necessary for NMA of preclinical studies (e.g. method of ischemic induction, timing of treatment, species, sex, and comorbidities). We will also evaluate the challenges of applying NMA to preclinical studies (e.g. consistency, heterogeneity, availability of key study covariates).
An information specialist (RS) will construct a search strategy based on a previous review of comparative stroke therapies, and limit them to include studies which compared therapies to alteplase (representative search strategy is provided in Supplementary File 2)15. Search strategies will be peer reviewed by a second information specialist using the peer review of electronic search strategy method16. Searches of Ovid MEDLINE and Embase will be carried out for articles on the effects of combination therapies with alteplase (Supplementary File 2 contains the search strategy). Of note, no language or date restrictions will be used. We will also search the CAMARADES database which contains data extracted from existing preclinical systematic reviews on stroke15,17–28. In addition to this rigorous search, we will assess bibliographies of any new studies and reviews identified. Articles in foreign languages will be translated.
Eligibility criteria to identify relevant studies for the current review were established in considering the Population-Intervention-Comparators-Outcomes-Study design (PICOS) framework29.
Population. Preclinical in vivo models of experimentally induced focal ischemia will be sought. All species/strains of animals will be eligible. Both female and male animals will be included. Neonatal animals will be excluded; however, all other ages will be considered. Studies in which focal ischemic stroke was established by transient occlusion of the middle cerebral artery or anterior cerebral artery via any method (chemical, embolic, mechanical, thermal) will be eligible. Animal models of haemorrhagic stroke, global or hemispheric brain ischemia, models of permanent occlusion without reperfusion (e.g. photothrombosis, cauterization), or delayed reperfusion such that it is considered permanent will be excluded30. Human studies and tissue culture studies will be excluded.
Intervention and comparator. Studies where the treatment in combination with alteplase (e.g. alteplase + hypothermia) is compared with alteplase alone in animals that have experimentally induced focal ischemia will be eligible. Studies that compare more than one active treatment such as alteplase + hypothermia versus alteplase +FK506 (i.e. head to head comparisons) will also be included. Studies must include alteplase as a ‘foundational’ therapeutic in experimental arms to be eligible. All delivery routes and doses will be considered. To increase potential clinical relevance (i.e. construct validity), only studies that deliver therapies within 6 hours of induction of focal ischemic stroke will be included.
- Primary outcome. Infarct size is a measure of injury reduction at the infarct site in the brain and can be measured via a variety of quantifiable techniques through non-invasive techniques (e.g. T2-weighted magnetic resonance imaging) or post-mortem analysis (e.g. staining of brain sections using hematoxylin and eosin). This is the most widely reported outcome in preclinical stroke studies. Infarct size outcomes will be extracted at the latest time point for each study. Separate time-point specific analyses will be conducted (e.g. an early time point <30 days vs later time points >30 days).
- Secondary outcome. Neurobehavioural measures represent a valuable means of assessing functional recovery after treatment. Neurobehavioural assessment are sensitive to detecting the array of impairments, including motor/sensory deficits (e.g. ladder rung walking—foot slip errors) as well as memory/learning deficits (e.g. Morris water maze)31,32. These outcomes, while labour-intensive, are typically reported with less frequency than infarct volume even though functional outcomes may have the greatest clinical relevance)33,34. Neurobehavioral outcomes will be extracted at all timepoints and separate time-point specific analyses will be conducted as described above.
Controlled comparison studies testing the efficacy of therapies + alteplase versus alteplase alone will be sought.
Two reviewers (A.D. and H.S.C.), will review abstracts (Stage 1 screen) and full text reports (Stage 2 screen) from search results independently and in duplicate against the eligibility criteria below using Distiller SR® software (Evidence Partners, Ottawa, ON) to identify relevant articles. Discrepancies will be resolved through discussion with a senior team member (M.L. and D.C.). Both stages of screening will begin with a calibration exercise to ensure consistent application of eligibility criteria. A PRISMA flow diagram35 will be presented to document the process of study selection.
Two independent reviewers (A.D. and H.S.C.) will review studies and extract data into standardized, piloted forms implemented in Microsoft Excel (Microsoft Corporation, Seattle, Washington, USA). Discrepancies will be resolved through discussion with a senior team member. We will collect data related to, but not limited to, animal characteristics (Table 1); stroke model (Table 1); intervention (Table 2a, b); and outcomes (Table 3), as well as study ID (authors, year), and study design characteristics. Measures of central tendency (e.g. mean) and dispersion (e.g. standard deviation) will be extracted as reported. Data in graphical format will be extracted using Engauge Digitizer36. When measures of central tendency and dispersion or sample sizes are missing (or cannot be measured digitally), authors will be contacted; if authors do not respond, the data will be excluded.
Two independent reviewers will assess the risk of bias of each included study (quality of the design, conduct and analysis for the experiment)37. We will assess the risk of bias using a modified version of the Cochrane Risk of Bias Tool for randomized trials (Table 4). Risk of bias will be summarized38 with descriptive statistics and presented graphically using standard methods and radar charts. The assessment of risk of bias will play an important role in exploring potential limitations of the evidence base and establishing the feasibility of incorporating relevant adjustments in NMA models. The construct validity of included studies (i.e. degree to which experimental model and design reflect the clinical entity of stroke and its treatment) will be assessed using elements from the CAMARADES checklist alongside criteria established by expert consensus (Table 5).
We will begin by exploring the pattern of treatment comparisons represented by the included set of studies using network diagrams (or using a tabular approach if necessary, should the number and pattern of comparisons be too broad to be summarized graphically). Effect estimates from all included studies will be summarized. We will summarize traits of included studies focusing on clinical (e.g. age, sex, species, stroke model, reperfusion vs. permanent model, comorbidities, severity of infarct pre-treatment, infarct location)39 and methodological (e.g. risk of bias, timing of outcome assessment) features27, and review these with our clinical and preclinical experts to establish the degree of homogeneity within the included studies. For NMA, given the possibility that a large proportion of the studied interventions may have been evaluated in only a single study (and many could potentially yield very large effect sizes, which may not have been substantiated by more animals in more studies), we will exclude these interventions from NMAs performed; each of these treatments removed from NMA will neither benefit from “borrowing strength” through NMA, nor end up with a summary estimate and confidence interval different from what was reported in a single study. The reported findings for the outcomes of interest from studies removed from the NMA according to these criteria will be summarized separately in descriptive tables to ensure all relevant data are summarized. This approach will also restrict the network to a more practical size and reduce the risk of computational challenges.
Where there is homogeneity of important effect modifiers, we will perform NMAs to compare interventions9,10,40, following procedures to assess the validity of the assumptions of homogeneity, similarity, and consistency41. Based upon the extracted study characteristics, we will work with our clinical and preclinical experts to establish any additional novel aspects of preclinical studies that may be important to consider in relation to judgements regarding study homogeneity beyond those anticipated in preparing this protocol. We have anticipated different species of animals (rats, mice, gerbils, dogs, sheep, non-human primates) across studies. We also anticipate that multiple reporting formats will have been used to assess both infarct volume (e.g. mm3, % of hemisphere or total brain, etc.) and neurobehavioral changes (e.g. seconds, % of baseline).
For meta-analysis of preclinical studies, the normalized mean difference (NMD) scale is useful in serving the purpose of synthesizing the complexity of data aforementioned42. Prior to performing NMAs, we will perform traditional pairwise meta-analyses on the NMD scale for each comparison in the treatment networks where two or more studies are available to explore heterogeneity based on the I2 statistic29. To perform network meta-analyses on the NMD scale, we will use an established model from the National Institute in Health and Care Excellence’s TSD series43, adapting its identity link to the log link in order to conduct the NMA on a log ratio of means (logRoM) scale. The log ratio of means of the kth treatment and the “stroke only” control, dk = logRoMC,Tk, can be estimated after model fitting, and the corresponding NMD estimate is:
1 – exp{logRoMC,Tk} = 1 – exp (dk)
The NMD of the kth treatment in comparison with alteplase (k=1) is 1 – exp (dk – d1).
Both fixed- and random-effect Bayesian NMAs will be performed using a common heterogeneity parameter according to established methods10,40,43. Model fit will be assessed by comparing the model’s posterior total residual deviance with the number of unconstrained data points43. Selection between models will be based on deviance information criteria (DIC), with a difference of five points suggesting an important difference43. All pairwise comparisons between interventions will be expressed with both summary point estimates and corresponding 95% credible intervals. Vague prior distributions will be assigned for all measures of treatment effect, as well as for the between-study variance parameter in random effects analyses. NMAs will be performed using OpenBUGS software version 3.2.344 and the R Package R2OpenBUGS45. Model convergence will be assessed using established methods including assessment of Rhat (the potential scale reduction factor) and the Gelman-Rubin convergence diagnostic to see if they are near 19. Surface under the cumulative ranking (SUCRA) values, and the mean rank of each intervention (with 2.5% and 97.5% quantiles) will also be estimated for each intervention46. Forest plots of treatment comparisons versus “stroke with no treatment” control as well as versus stroke + alteplase will be prepared for each outcome. Given the anticipated high number of interventions assessed in only a single study, a tabular approach to summarizing findings will be employed for them. We will also undertake forest plots of effects wherein interventions are ordered according to mean rank estimated from NMA.
To check the validity of the consistency assumption (i.e., transitivity of the effect size through common comparators), a consistency model as well as an unrelated mean effects model will be fit to the data47. We will compare their respective DIC values to check model fitting and their posterior mean deviance contribution per study to check the consistency assumption. We will also assess the magnitude of the estimated between-study SD measure from both models, as a reduction in this parameter in the inconsistency model also provides evidence of inconsistency.
The likelihood of important clinical and methodological heterogeneity between studies is anticipated by the research team to be high and may include several nuances which are unique to the pre-clinical setting. First, several vital aspects of preclinical studies from our risk of bias assessments (described earlier) may be important adjustment factors that could have an important impact on the findings from NMAs, including randomization and blinding24,48. In this work, we will use subgroup analyses or covariate-adjusted analyses to address and explore the impact that covariates have on findings and to establish the robustness of findings from primary syntheses49,50. We will assess the possibility to adjust for the following group level factors: animal species (e.g. mouse) and strain (e.g. C57Bl6 strain of mice), model of stroke, average animal age, percentage of female subjects, average time since stroke induction, combination therapies, cerebral blood flow, temperature, infarct location and severity, use of randomization and blinding of experimenters and outcome assessments. Alternatively, when combining data from different species, we could model animal species as an extra level in the hierarchical model for treatment effect, allowing for heterogeneity across species and assuming that treatment effects are similar across species around an overall mean effect. For the network structure, primary analyses will be performed at the treatment level. As dose may have an important effect on intervention benefits, we will also explore the range of doses associated with each intervention across studies to consider additional analyses. However, as dose response characteristics of different agents may also vary between animal species and an a priori source of information to establish appropriate dose categories is not available, any analyses pursued in relation to dose will be appropriately indicated as post-hoc. Findings from all analyses will be reported. Given the anticipated complexity of this novel application of NMA, we anticipate separate publications will be required for the primary and secondary outcomes.
Current approaches to evaluating the relative therapeutic benefit of preclinical treatments for stroke are limited. Although systematic reviews have been conducted comparing more than a thousand candidates, many have never been systematically assessed, nor have they been assessed relative to one another, or more importantly, to the best available clinical treatment (alteplase). Use of NMA to synthesize data on all relevant available therapies may help address this knowledge gap. Thinking more broadly, the proposed review, with the application and evaluation of NMA to preclinical therapeutics, will inform translational scientists’ knowledge of which preclinical stroke therapeutics have the most promise for either further preclinical research or translation to clinical trial.
In addition to addressing an important question for clinical research, we anticipate this study will inform empirical explorations of anticipated challenges of evidence synthesis that are unique to the pre-clinical setting. First, a debate among preclinical and clinical scientists is likely to exist regarding both the appropriateness and approach to synthesizing outcome data from different species as well as different models of stroke. Second, there exists an especially important need to consider a broad range of adjustments to account for between-study heterogeneity related to animal characteristics or other features; lack of availability of these key data may be sub-optimal. Our study will provide an empirical evaluation of the degree of missingness of features, such as those significant to experimental design (e.g. randomization). This will provide an indication of the changes to the available evidence when exploring adjustments of comparisons. More specifically, if the lack of reporting proves to be severe, this will provide further high-level evidence that educational efforts are needed to improve the completeness of reporting of preclinical research51.
Other challenges potentially requiring consideration will include identifying optimal strategies for presenting findings (including those with many comparators rendering analysis unfeasible), analysis of studies with small sample sizes, and strategies to select the most promising therapy to translate clinically. We anticipate that this systematic review will provide insight into these and other methodologic challenges and thereby serve as an exemplar for future NMA of preclinical data to build upon.
Findings from this review will be shared with several key knowledge users including (i) the Stroke Treatment Academic Industry Roundtable52 for development of future guidelines; (ii) the Heart & Stroke Foundation and the Canadian Partnership for Stroke Recovery to inform future potential trials51; (iii) the Cochrane Stroke Group to inform a future clinical systematic review and NMA; and (iv) stroke survivors, via sharing of findings with our knowledge users.
No data are associated with this article.
M.M.L. contributed to protocol design, with specific methodological input on systematic review methods (B.H., D.M., D.A.F.) and statistical analysis and synthesis (B.H., W.C., D.M., D.F., N.J.W.). R.S. designed the search strategy. D.C., D.D., S.K.M., provided substantive topic-specific input that informed the protocol's revision and refinement. M.M.L. and B.H. drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by Canadian Institutes of Health Research (Grant #365473). M.M.L. is supported by The Ottawa Hospital Anesthesia Alternate Funds Association and the Scholarship Protected Time Program, Department of Anesthesiology and Pain Medicine, uOttawa. D.M. is supported by a University Research Chair. N.J.W. was supported by the NIHR Biomedical Research Centre at the University Hospitals Bristol NHS Foundation Trust and the University of Bristol. The views expressed in this publication are those of the authors and not necessarily those of the funding bodies.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Supplementary File 1. PRISMA-Protocols checklist.
Click here to access the data.
Supplementary File 2. Representative search strategy.
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Is the rationale for, and objectives of, the study clearly described?
Yes
Is the study design appropriate for the research question?
Yes
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Systematic review and meta-analysis of pre-clinical studies
Is the rationale for, and objectives of, the study clearly described?
Yes
Is the study design appropriate for the research question?
Yes
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: preclinical meta-analysis, translational cardiology.
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | ||
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Version 1 03 Jan 19 |
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Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
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