Sarah K. McCann

Systematic reviews and meta-analysis of preclinical studies: why perform them and how to appraise them critically

The use of systematic review and meta-analysis of preclinical studies has become more common, including those of studies describing the modeling of cerebrovascular diseases. Empirical evidence suggests that too many preclinical experiments lack methodological rigor, and this leads to inflated treatment effects. The aim of this review is to describe the concepts of systematic review and meta-analysis and consider how these tools may be used to provide empirical evidence to spur the field to improve the rigor of the conduct and reporting of preclinical research akin to their use in improving the conduct and reporting of randomized controlled trials in clinical research. As with other research domains, systematic reviews are subject to bias. Therefore, we have also suggested guidance for their conduct, reporting, and critical appraisal.

Early increase of Nox4 NADPH oxidase and superoxide generation following endothelin-1-induced stroke in conscious rats.

Oxidative stress contributes to the progression of brain injury following ischemic stroke and reperfusion. NADPH oxidase is a well‐established source of superoxide in vascular disease, but its contribution to tissue injury following ischemic stroke has yet to be fully elucidated. Here we show the spatiotemporal profile of NADPH oxidase subunits Nox2 and Nox4 and concurrent superoxide generation following stroke induced by middle cerebral artery constriction in conscious rats. Nox2 mRNA was progressively up‐regulated in both the ipsilateral cortex and the striatum from 6 hr to 7 days poststroke and reperfusion. Nox4 mRNA was also up‐regulated transiently in the cortex at 6 hr poststroke but returned to control levels after this time. In situ detection of superoxide generation with dihydroethidium fluorescence revealed an increase in superoxide within the ischemic core at 6 hr poststroke that was mostly colocalized with the neuronal marker NeuN. By 24 hr, this increase in superoxide production had spread to the boundary zone of the infarct, whereas it disappeared in the ischemic core as neuronal numbers declined. Subsequently, superoxide within the ischemic core again increased at 7 days and was mostly colocalized with the activated microglia/macrophage marker OX‐42. Immunoreactivity to Nox2 followed the same spatiotemporal pattern as that of OX‐42 immunostaining poststroke. Clearly, NADPH oxidase is an important mediator of oxidative stress and contributes to the progression of brain damage beyond the infarct core, via the activation of two catalytic subunits, Nox2 and Nox4. Selectively blocking these subunits might be useful for intervening in the progression of stroke brain injury.

Efficacy of antidepressants in animal models of ischemic stroke: a systematic review and meta-analysis

Background and Purpose— Poststroke depression is a prevalent complication of stroke with unclear pathogenesis. The benefits of antidepressants in this context and their effects on stroke recovery other than effects on mood are not clearly defined, with some studies suggesting efficacy in improving functional outcome in both depressed and nondepressed stroke patients. We have analyzed the preclinical animal data on antidepressant treatment in focal cerebral ischemia, modeled±depression, to help inform clinical trial design. Methods— We performed a systematic review and meta-analysis of data from experiments testing the efficacy of antidepressants versus no treatment to reduce infarct volume or improve neurobehavioral or neurogenesis outcomes in animal models of stroke. We used random-effects metaregression to test the impact of study quality and design characteristics and used trim and fill to assess publication bias. Results— We identified 44 publications describing the effects of 22 antidepressant drugs. The median quality checklist score was 5 of a possible 10 (interquartile range, 4–7). Overall, antidepressants reduced infarct volume by 27.3% (95% confidence interval, 20.7%–33.8%) and improved neurobehavioral outcomes by 53.7% (46.4%–61.1%). There was little evidence for an effect of selective serotonin reuptake inhibitors on infarct volume. For neurobehavioral outcomes there was evidence of publication bias. Selective serotonin reuptake inhibitors were the most frequently studied antidepressant subtype and improved neurobehavioral outcome by 51.8% (38.6%–64.9%) and increased neurogenesis by 2.2 SD (1.3–3.0). Conclusions— In line with current clinical data and despite some limitations, antidepressant treatments seem to improve infarct volume and neurobehavioral outcome in animal models of ischemic stroke.

NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives.

Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation.

Nox2 Knockout Delays Infarct Progression and Increases Vascular Recovery through Angiogenesis in Mice following Ischaemic Stroke with Reperfusion

Evidence suggests the NADPH oxidases contribute to ischaemic stroke injury and Nox2 is the most widely studied subtype in the context of stroke. There is still conjecture however regarding the benefits of inhibiting Nox2 to improve stroke outcome. The current study aimed to examine the temporal effects of genetic Nox2 deletion on neuronal loss after ischaemic stroke using knockout (KO) mice with 6, 24 and 72 hour recovery. Transient cerebral ischaemia was induced via intraluminal filament occlusion and resulted in reduced infarct volumes in Nox2 KO mice at 24 h post-stroke compared to wild-type controls. No protection was evident at either 6 h or 72 h post-stroke, with both genotypes exhibiting similar volumes of damage. Reactive oxygen species were detected using dihydroethidium and were co-localised with neurons and microglia in both genotypes using immunofluorescent double-labelling. The effect of Nox2 deletion on vascular damage and recovery was also examined 24 h and 72 h post-stroke using an antibody against laminin. Blood vessel density was decreased in the ischaemic core of both genotypes 24 h post-stroke and returned to pre-stroke levels only in Nox2 KO mice by 72 h. Overall, these results are the first to show that genetic Nox2 deletion merely delays the progression of neuronal loss after stroke but does not prevent it. Additionally, we show for the first time that Nox2 deletion increases re-vascularisation of the damaged brain by 72 h, which may be important in promoting endogenous brain repair mechanisms that rely on re-vascularisation.

Anesthetic Neuroprotection in Experimental Stroke in Rodents: A Systematic Review and Meta-analysis

Background: Patients undergoing endovascular therapy for acute ischemic stroke may require general anesthesia to undergo the procedure. At present, there is little clinical evidence to guide the choice of anesthetic in this acute setting. The clinical implications of experimental studies demonstrating anesthetic neuroprotection are poorly understood. Here, the authors evaluated the impact of anesthetic treatment on neurologic outcome in experimental stroke. Methods: Controlled studies of anesthetics in stroke using the filament occlusion model were identified in electronic databases up to December 15, 2015. The primary outcome measures, infarct volume, and neurologic deficit score were used to calculate the normalized mean difference for each comparison. Meta-analysis of normalized mean difference values provided estimates of neuroprotection and contributions of predefined factors: study quality, the timing of treatment, and the duration of ischemia. Results: In 80 retrieved publications anesthetic treatment reduced neurologic injury by 28% (95% CI, 24 to 32%; P < 0.0001). Internal validity was high: publication bias enhanced the effect size by 4% or less, effect size increased with study quality (P = 0.0004), and approximately 70% of studies were adequately powered. Apart from study quality, no predefined factor influenced neuroprotection. Neuroprotection failed in animals with comorbidities. Neuroprotection by anesthetics was associated with prosurvival mechanisms. Conclusions: Anesthetic neuroprotection is a robust finding in studies using the filament occlusion model of ischemic stroke and should be assumed to influence outcomes in studies using this model. Neuroprotection failed in female animals and animals with comorbidities, suggesting that the results in young male animals may not reflect human stroke.

D-galactose-induced brain ageing model: A systematic review and meta-analysis on cognitive outcomes and oxidative stress indices

Animal models are commonly used in brain ageing research. Amongst these, models where rodents are exposed to d-galactose are held to recapitulate a number of features of ageing including neurobehavioral and neurochemical changes. However, results from animal studies are often inconsistent. To better understand the characteristics of the model and effects of d-galactose on neurobehavioral and neurochemical outcomes in rodents we performed a systematic review and meta-analysis. We applied random-effects meta-analysis to evaluate the effect of study features. Our results give an overview of the characteristics of the d-galactose rodent ageing model, including neurobehavioral and neurochemical outcomes. We found that few studies took measures to reduce risks of bias, and substantial heterogeneity in the reported effects of d-galactose in included studies. This highlights the need for improvements in the use of the d-galactose rodent ageing model if it is to provide useful in the development of drugs to treat human ageing.

Risk of bias reporting in the recent animal focal cerebral ischaemia literature

Background: Findings from in vivo research may be less reliable where studies do not report measures to reduce risks of bias. The experimental stroke community has been at the forefront of implementing changes to improve reporting, but it is not known whether these efforts are associated with continuous improvements. Our aims here were firstly to validate an automated tool to assess risks of bias in published works, and secondly to assess the reporting of measures taken to reduce the risk of bias within recent literature for two experimental models of stroke. Methods: We developed and used text analytic approaches to automatically ascertain reporting of measures to reduce risk of bias from full-text articles describing animal experiments inducing middle cerebral artery occlusion (MCAO) or modelling lacunar stroke. Results: Compared with previous assessments, there were improvements in the reporting of measures taken to reduce risks of bias in the MCAO literature but not in the lacunar stroke literature. Accuracy of automated annotation of risk of bias in the MCAO literature was 86% (randomization), 94% (blinding) and 100% (sample size calculation); and in the lacunar stroke literature accuracy was 67% (randomization), 91% (blinding) and 96% (sample size calculation). Discussion: There remains substantial opportunity for improvement in the reporting of animal research modelling stroke, particularly in the lacunar stroke literature. Further, automated tools perform sufficiently well to identify whether studies report blinded assessment of outcome, but improvements are required in the tools to ascertain whether randomization and a sample size calculation were reported.

The development of an online database for interventions tested in transgenic mouse models of Alzheimer's disease

Abstract Despite many efforts by the research community, Alzheimers disease (AD) is still an incurable neurodegenerative condition that affects an estimated 44 million individuals worldwide and this figure is expected to increase to 135 million by the year 2050. As the research community currently reflects on previous endeavours, it is essential that we maximize the use of existing knowledge to inform future trials in the field. This article describes the development of a systematically identified data set relating to over 300 interventions tested in over 10,000 animals. The data set includes cohort‐level information for six structural outcomes and six behavioural assessments. We encourage others to use this dataset to inform the design of future animal experiments modelling AD and to promote effective translation to human health.

Animal Models of Stroke for Preclinical Drug Development: A Comparative Study of Flavonols for Cytoprotection

Laboratory animals have been used extensively to mimic human ischemic stroke with the aim of discovering drugs that minimize damage and reduce loss of motor and cognitive functions. To date, this has not led to successful clinical translation. Here we consider the available animal models of stroke with discussion focusing on their strengths and limitations for assessing neuroprotective actions. For assessment of drugs outside of thrombolytic therapies, we propose the use of the endothelin-1 (ET-1) model of stroke in conscious animals as a better model than the more common procedure of inducing focal stroke in anesthetized rats with a vascular occluder. The ET-1 model of ischemic stroke allows for stratification of animals to drug trial based on predictive outcomes assessed during stroke induction, mimicking the scenario of stroke patients who are assessed for functional outcome and survival for clinical management and to correctly stratify treatment groups for clinical trials. We highlight the value of applying a predictive outcome score to animals prior to treatment stratification using the water-soluble derivative of the flavonol, 3′,4′-dihydroxyflavonol: an effective neuroprotective drug in rats with modest focal strokes but not in rats with severe strokes. Finally, we discuss the use of flavonols as potential neuroprotectants and propose future ideas as to how the positive outcomes observed with flavonols in preclinical stroke models might indeed translate to clinical success.