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.

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.

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.

Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0.

Improving the reproducibility of biomedical research is a major challenge. Transparent and accurate reporting is vital to this process; it allows readers to assess the reliability of the findings and repeat or build upon the work of other researchers. The ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) were developed in 2010 to help authors and journals identify the minimum information necessary to report in publications describing in vivo experiments. Despite widespread endorsement by the scientific community, the impact of ARRIVE on the transparency of reporting in animal research publications has been limited. We have revised the ARRIVE guidelines to update them and facilitate their use in practice. The revised guidelines are published alongside this paper. This explanation and elaboration document was developed as part of the revision. It provides further information about each of the 21 items in ARRIVE 2.0, including the rationale and supporting evidence for their inclusion in the guidelines, elaboration of details to report, and examples of good reporting from the published literature. This document also covers advice and best practice in the design and conduct of animal studies to support researchers in improving standards from the start of the experimental design process through to publication.

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.

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.

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.

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.

Applications of Nanotechnology in the Diagnosis and Therapy of Stroke.

Stroke is a leading cause of death and disability worldwide. The classification of stroke subtypes is difficult but critical for the prediction of clinical course and patient management, and limited treatment options are available. There is an urgent need for improvements in both diagnosis and therapy. Strokes have rapidly evolving phases of damage involving unique compartments of the brain, which imposes severe limitations for current diagnostic and treatment procedures. The rapid development of nanotechnology in other areas of modern medicine has ignited a widespread interest in its potential for the field of stroke. An important feature of nanoparticles is the relative ease in which their structures and surface chemistries can be modified for specific and potentially multiple, simultaneous purposes. Nanoparticles can be synthesized to carry and deliver therapeutics to specific cellular or subcellular compartments; they can be engineered to provide enhanced contrast for imaging based on the detection of changes in the blood flow; or possess ligand-specific chemistries which can facilitate diagnosis and monitor the treatment response. More specifically for a stroke, nanoparticles can be engineered to release their payload in response to the distinct extracellular processes occurring around the clot and in the ischemic penumbra, as well as aid in the detection of pathological hallmarks present at various stages of stroke progression. These capacities allow targeted release of disease-modifying agents in the affected brain tissue, increasing treatment efficacy, and limiting unwanted side effects. While nanospheres, liposomes, and mesoporous nanostructures all emerge as future prospects for stroke treatment and diagnosis, much of this potential is yet to be clinically realized. This review outlines aspects of nanotechnology identified as having potential to revolutionize the field of 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 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.

Olfactory Ensheathing Cell Transplantation in Experimental Spinal Cord Injury: Effect size and Reporting Bias of 62 Experimental Treatments: A Systematic Review and Meta-Analysis.

Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies (62 experiments, 1,164 animals) in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8-29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3-5). In conclusion, OEC transplantation exerts considerable beneficial effects on neurobehavioral recovery after traumatic experimental SCI. Publication bias was minimal and affirms the translational potential of efficacy, but safety cannot be adequately assessed. The data justify OECs as a cellular substrate to develop and optimize minimally invasive and safe cellular transplantation paradigms for the lesioned spinal cord embedded into state-of-the-art Phase I/II clinical trial design studies for human SCI.

Correction: Risk of Bias in Reports of In Vivo Research: A Focus for Improvement.

[This corrects the article DOI: 10.1371/journal.pbio.1002273.].

Risk of Bias in Reports of In Vivo Research: A Focus for Improvement.

The reliability of experimental findings depends on the rigour of experimental design. Here we show limited reporting of measures to reduce the risk of bias in a random sample of life sciences publications, significantly lower reporting of randomisation in work published in journals of high impact, and very limited reporting of measures to reduce the risk of bias in publications from leading United Kingdom institutions. Ascertainment of differences between institutions might serve both as a measure of research quality and as a tool for institutional efforts to improve research quality.

A call for transparent reporting to optimize the predictive value of preclinical research.

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

NXY-059, a Failed Stroke Neuroprotectant, Offers No Protection to Stem Cell-Derived Human Neurons.

BACKGROUND: Developing new medicines is a complex process where understanding the reasons for both failure and success takes us forward. One gap in our understanding of most candidate stroke drugs before clinical trial is whether they have a protective effect on human tissues. NXY-059 is a spin-trap reagent hypothesized to have activity against the damaging oxidative biology which accompanies ischemic stroke. Re-examination of the preclinical in vivo dataset for this agent in the wake of the failed SAINT-II RCT highlighted the presence of a range of biases leading to overestimation of the magnitude of NXY-059's effects in laboratory animals. Therefore, NXY-059 seemed an ideal candidate to evaluate in human neural tissues to determine whether human tissue testing might improve screening efficiency. MATERIALS AND METHODS: The aim of this randomized and blinded study was to assess the effects of NXY-059 on human stem cell-derived neurons in the presence of ischemia-like injury induced by oxygen glucose deprivation or oxidative stress induced by hydrogen peroxide or sodium nitroprusside. RESULTS: In MTT assays of cell survival, lactate dehydrogenase assays of total cell death and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic-like cell death, NXY-059 at concentrations ranging from 1 µm to 1 mm was completely without activity. Conversely an antioxidant cocktail comprising 100 µm each of ascorbate, reduced glutathione, and dithiothreitol used as a positive control provided marked neuronal protection in these assays. CONCLUSION: These findings support our hypothesis that stroke drug screening in human neural tissues will be of value and provides an explanation for the failure of NXY-059 as a human stroke drug.

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.

Identification and characterization of outcome measures reported in animal models of epilepsy: Protocol for a systematic review of the literature-A TASK2 report of the AES/ILAE Translational Task Force of the ILAE.

Current antiseizure therapy is ineffective in approximately one third of people with epilepsy and is often associated with substantial side effects. In addition, most current therapeutic paradigms offer treatment, but not cure, and no therapies are able to modify the underlying disease, that is, can prevent or halt the process of epileptogenesis or alleviate the cognitive and psychiatric comorbidities. Preclinical research in the field of epilepsy has been extensive, but unfortunately, not all the animal models being used have been validated for their predictive value. The overall goal of TASK2 of the AES/ILAE Translational Task Force is to organize and coordinate systematic reviews on selected topics regarding animal research in epilepsy. Herein we describe our strategy. In the first part of the paper we provide an overview of the usefulness of systematic reviews and meta-analysis for preclinical research and explain the essentials for their conduct. Then we describe in detail the protocol for a first systematic review, which will focus on the identification and characterization of outcome measures reported in animal models of epilepsy. The specific goals of this study are to define systematically the phenotypic characteristics of the most commonly used animal models, and to effectively compare these with the manifestations of human epilepsy. This will provide epilepsy researchers with detailed information on the strengths and weaknesses of epilepsy models, facilitating their refinement and future research. Ultimately, this could lead to a refined use of relevant models for understanding the mechanism(s) of the epilepsies and developing novel therapies.

Conventional protein kinase Cß-mediated phosphorylation inhibits collapsin response-mediated protein 2 proteolysis and alleviates ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

We previously reported that conventional protein kinase C (cPKC)ß participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCß interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCß, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCß-mediated CRMP2 phosphorylation via the cPKCß-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCß activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCß modulates CRMP2 phosphorylation and proteolysis, and cPKCß activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (cPKC)ß-mediated phosphorylation could inhibit CRMP2 proteolysis and alleviate ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

Evaluation of excess significance bias in animal studies of neurological diseases.

Animal studies generate valuable hypotheses that lead to the conduct of preventive or therapeutic clinical trials. We assessed whether there is evidence for excess statistical significance in results of animal studies on neurological disorders, suggesting biases. We used data from meta-analyses of interventions deposited in Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES). The number of observed studies with statistically significant results (O) was compared with the expected number (E), based on the statistical power of each study under different assumptions for the plausible effect size. We assessed 4,445 datasets synthesized in 160 meta-analyses on Alzheimer disease (n = 2), experimental autoimmune encephalomyelitis (n = 34), focal ischemia (n = 16), intracerebral hemorrhage (n = 61), Parkinson disease (n = 45), and spinal cord injury (n = 2). 112 meta-analyses (70%) found nominally (p≤0.05) statistically significant summary fixed effects. Assuming the effect size in the most precise study to be a plausible effect, 919 out of 4,445 nominally significant results were expected versus 1,719 observed (p<10⁻9). Excess significance was present across all neurological disorders, in all subgroups defined by methodological characteristics, and also according to alternative plausible effects. Asymmetry tests also showed evidence of small-study effects in 74 (46%) meta-analyses. Significantly effective interventions with more than 500 animals, and no hints of bias were seen in eight (5%) meta-analyses. Overall, there are too many animal studies with statistically significant results in the literature of neurological disorders. This observation suggests strong biases, with selective analysis and outcome reporting biases being plausible explanations, and provides novel evidence on how these biases might influence the whole research domain of neurological animal literature.