Ana Antonic

Meta-analysis of data from animal studies: A practical guide

Meta-analyses of data from human studies are invaluable resources in the life sciences and the methods to conduct these are well documented. Similarly there are a number of benefits in conducting meta-analyses on data from animal studies; they can be used to inform clinical trial design, or to try and explain discrepancies between preclinical and clinical trial results. However there are inherit differences between animal and human studies and so applying the same techniques for the meta-analysis of preclinical data is not straightforward. For example preclinical studies are frequently small and there is often substantial heterogeneity between studies. This may have an impact on both the method of calculating an effect size and the method of pooling data. Here we describe a practical guide for the meta-analysis of data from animal studies including methods used to explore sources of heterogeneity.

Stem cell‐based therapy for experimental stroke: A systematic review and meta‐analysis

Stem cell therapy holds great promise in medicine, but clinical development should be based on a sound understanding of potential weaknesses in supporting experimental data. The aim of this article was to provide a systematic overview of evidence relating to the efficacy of stem cell-based therapies in animal models of stroke to foster the clinical application of stem cell-based therapies and to inform the design of large-scale clinical trials. We conducted a systematic search for reports of experiments using stem cells in animal models of cerebral ischaemia, and performed DerSimmonian and Laird random effects meta-analysis. We assessed the impact of study characteristics, of publication bias and of measures to reduce bias. We identified 6059 publications, 117 met our prespecified inclusion criteria. One hundred eighty-seven experiments using 2332 animals described changes in structural outcome and 192 experiments using 2704 animals described changes in functional outcome. Median study quality score was 4 (interquartile range 3 to 6) and less than half of studies reported randomization or blinded outcome assessment; only three studies reported a sample size calculation. Nonrandomized studies gave significantly higher estimates of improvement in structural outcome, and there was evidence of a significant publication bias. For structural outcome autologous (i.e. self-derived) stem cells were more effective than allogeneic (donor-derived) cells, but for functional outcome, the reverse was true. A significant dose–response relationship was observed only for structural outcome. For structural outcome, there was an absolute reduction in efficacy of 1.5% (−2.4 to −0.6) for each days delay to treatment; functional outcome was independent of the time of administration. While stem cells appear to be of some benefit in animal models of stroke the internal and external validity of this literature is potentially confounded by poor study quality and by publication bias. The clinical development of stem cell-based therapies, in stroke and elsewhere, should acknowledge these potential weaknesses in the supporting animal data.

Stem cell transplantation in traumatic spinal cord injury: a systematic review and meta-analysis of animal studies.

A systematic analysis of the literature shows that stem cell implantation can improve function in animal models of spinal cord injury, depending on the methods used.

Systematic review and meta-analysis of therapeutic hypothermia in animal models of spinal cord injury

Background Therapeutic hypothermia is a clinically useful neuroprotective therapy for cardiac arrest and neonatal hypoxic ischemic encephalopathy and may potentially be useful for the treatment of other neurological conditions including traumatic spinal cord injury (SCI). The pre-clinical studies evaluating the effectiveness of hypothermia in acute SCI broadly utilise either systemic hypothermia or cooling regional to the site of injury. The literature has not been uniformly positive with conflicting studies of varying quality, some performed decades previously. Methods In this study, we systematically review and meta-analyse the literature to determine the efficacy of systemic and regional hypothermia in traumatic SCI, the experimental conditions influencing this efficacy, and the influence of study quality on outcome. Three databases were utilised; PubMed, ISI Web of Science and Embase. Our inclusion criteria consisted of the (i) reporting of efficacy of hypothermia on functional outcome (ii) number of animals and (iii) mean outcome and variance in each group. Results Systemic hypothermia improved behavioural outcomes by 24.5% (95% CI 10.2 to 38.8) and a similar magnitude of improvement was seen across a number of high quality studies. The overall behavioural improvement with regional hypothermia was 26.2%, but the variance was wide (95% CI −3.77 to 56.2). This result may reflect a preponderance of positive low quality data, although a preferential effect of hypothermia in ischaemic models of injury may explain some of the disparate data. Sufficient heterogeneity was present between studies of regional hypothermia to reveal a number of factors potentially influencing efficacy, including depth and duration of hypothermia, animal species, and neurobehavioural assessment. However, these factors could reflect the influence of earlier lower quality literature. Conclusion Systemic hypothermia appears to be a promising potential method of treating acute SCI on the basis of meta-analysis of the pre-clinical literature and the results of high quality animal studies.

Hypothermia protects human neurons

Background and Aims Hypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury. Methods Human embryonic stem cells were cultured and differentiated to provide a source of neurons expressing β III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H2O2-induced oxidative stress were used to induce relevant injury. Results Hypothermia to 33°C protected these human neurons against H2O2-induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% (P ≤ 0·0001; 95% confidence interval 34·8–71·04) and 42% (P ≤ 0·0001; 95% confidence interval 27·5–56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3–71·3 and 26%, P ≤ 0·001; 95% confidence interval 12·4–52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H2O2-induced oxidative stress even when hypothermia was initiated six-hours after onset of injury (P ≤ 0·05; 95% confidence interval 0·57–43·1). Conclusion We conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H2O2-induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances.

Human In Vitro Models of Ischaemic Stroke: a Test Bed for Translation

Our increased understanding of the ischaemic cascade hasdriven the rush to develop neuroprotective interventions.Over 500 have been reported to effectively improve out-come in experimental animal models of stroke. Although anumber of well-conducted clinical trials have been per-formed, we have failed to reproduce these effects in humans[1]. Despite these difficulties in translation, neuroprotectionremains an important potential therapy. If safe and easilyadministered, potentially in a pre-hospital setting, evensmall absolute benefits might have a significant impact.Translational failure has been hotly debated, and manyplausible explanations have been put forward. These includethe possibility that clinical trials may have failed to detectneuroprotection where it does in fact exist, that animalstudies may overstate the efficacy of neuroprotective inter-ventions they test, and that the experimental models that weuse may not replicate human stroke with sufficient fidelity.We do not believe that animal models are inherently faulty;indeed, the evidence suggests that the main themes of strokebiology hold true across all mammals studied so far. Clinicaltrial design and the strength and weaknesses of animalmodels and their employment are the subject of intensestudy [1–5], and in vivo testing will remain an importantpart of our overall armamentarium.However, we need to consider the possibility that rodentmolecular targets might not all be present in humans. More-over, without robust target identification and proof-of-principal demonstration that attacking these targets elicitsappropriate cellular responses, the foundations of our trans-lational pyramid are weak and the pyramid itself is subjectto collapse. Importantly, when the final target species isHomo sapiens, there is little logic to in vitro testing in cellsfrom other species if human cell cultures are available atsimilar cost.Why might humans and rodents not share the samemolecular targets? While both species share all the hall-marks of mammals, they are separated by 80 million yearsof evolution [6]. Humans have large gyrencephalic brainswith a high proportion of white matter, while most exper-imental animals have small smooth brains with relativelylittle white matter [7]. Within the cortex, the detailedarchitecture is different, with cross-species variations infunctional maps and synaptic density [8]. Although wemay share 90 % of our genome with rodents [9] and have93 % homology with the rhesus macaque (Macacamulatta)[10], a 10 % difference implies that up to 3,000genes may be different. Even those genes with homologymay still have evolved different biochemistry and function.Forexample,mutationsthatcauseornithine transcarbamylaseand phenylalanine hydroxylase deficiency in humans arepresent in the macaque genome, but are not associated withdisease [10].The recent disastrous clinical trial of TGN1412 that near-ly killed six healthy volunteers provides another example ofthe risks of assuming molecular targets are identical acrossspecies. TGN1412 is believed to act through the CD28receptor, of which the extracellular domain shares nearly100 % homology between humans and rhesus monkeys[11]. It is now believed that the binding characteristics ofthe two proteins differ and that this accounts for the severeinflammatory reactions seen in humans, but not in rhesusmonkeys [12]. Excitotoxicity, one of the critical early eventsin the ischaemic cascade, differs between species. In mice,these events, as measured in the extracellular fluid of the

A systematic review and meta-analysis of gene therapy in animal models of cerebral glioma: why did promise not translate to human therapy?

Abstract Background The development of therapeutics is often characterized by promising animal research that fails to translate into clinical efficacy; this holds for the development of gene therapy in glioma. We tested the hypothesis that this is because of limitations in the internal and external validity of studies reporting the use of gene therapy in experimental glioma. Method We systematically identified studies testing gene therapy in rodent glioma models by searching three online databases. The number of animals treated and median survival were extracted and studies graded using a quality checklist. We calculated median survival ratios and used random effects meta‐analysis to estimate efficacy. We explored effects of study design and quality and searched for evidence of publication bias. Results We identified 193 publications using gene therapy in experimental glioma, including 6,366 animals. Overall, gene therapy improved median survival by a factor of 1.60 (95% CI 1.53–1.67). Study quality was low and the type of gene therapy did not account for differences in outcome. Study design characteristics accounted for a significant proportion of between‐study heterogeneity. We observed similar findings in a data subset limited to the most common gene therapy. Conclusion As the dysregulation of key molecular pathways is characteristic of gliomas, gene therapy remains a promising treatment for glioma. Nevertheless, we have identified areas for improvement in conduct and reporting of studies, and we provide a basis for sample size calculations. Further work should focus on genes of interest in paradigms recapitulating human disease. This might improve the translation of such therapies into the clinic.

WDR62 Regulates Early Neural and Glial Progenitor Specification of Human Pluripotent Stem Cells

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker, TBR2, and also glial marker, S100β. In contrast, inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers, PAX6 and EAAT1, respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation.

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-059s 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.

Derivation of phenotypically diverse neural culture from hESC by combining adherent and dissociation methods

BACKGROUND Differentiation of human embryonic stem cells (hESCs) into distinct neural lineages has been widely studied. However, preparation of mixed yet neurochemically mature populations, for the study of neurological diseases involving mixed cell types has received less attention. NEW METHOD We combined two commonly used differentiation methods to provide robust and reproducible cultures in which a mixture of primarily GABAergic and Glutamatergic neurons was obtained. Detailed characterisation by immunocytochemistry (ICC) and quantitative real-time PCR (qPCR) assessed the neurochemical phenotype, and the maturation state of these neurons. RESULTS We found that once neurospheres (NSs) had attached to the culture plates, proliferation of neural stem cell was suppressed. Neuronal differentiation and synaptic development then occurred after 21 days in vitro (DIV). By 49DIV, there were large numbers of neurochemically and structurally mature neurons. The qPCR studies indicated that expression of GABAergic genes increased the most (93.3-fold increase), followed by glutamatergic (51-fold increase), along with smaller changes in expression of cholinergic (3-fold increase) and dopaminergic genes (6-fold increase), as well as a small change in glial cell marker expression (5-fold increase). COMPARISON WITH EXISTING METHOD (S) Existing methods isolate hESC-derived neural progenitors for onward differentiation to mature neurons using either migration or dissociative paradigms. These give poor survival or yield. By combining these approaches, we obtain high yields of morphologically and neurochemically mature neurons. These can be maintained in culture for extended periods. CONCLUSION Our method provides a novel, effective and robust neural culture system with structurally and neurochemically mature cell populations and neural networks, suitable for studying a range of neurological diseases from a human perspective.