title: Experimental Design and Data Analysis Issues Contribute to Inconsistent Results of C-Bouton Changes in Amyotrophic Lateral Sclerosis.
abstract: The possible presence of pathological changes in cholinergic synaptic inputs [cholinergic boutons (C-boutons)] is a contentious topic within the ALS field. Conflicting data reported on this issue makes it difficult to assess the roles of these synaptic inputs in ALS. Our objective was to determine whether the reported changes are truly statistically and biologically significant and why replication is problematic. This is an urgent question, as C-boutons are an important regulator of spinal motoneuron excitability, and pathological changes in motoneuron excitability are present throughout disease progression. Using male mice of theSOD1-G93Ahigh-expresser transgenic (G93A) mouse model of ALS, we examined C-boutons on spinal motoneurons. We performed histological analysis at high statistical power, which showed no difference in C-bouton size inG93Aversus wild-type motoneurons throughout disease progression. In an attempt to examine the underlying reasons for our failure to replicate reported changes, we performed further histological analyses using several variations on experimental design and data analysis that were reported in the ALS literature. This analysis showed that factors related to experimental design, such as grouping unit, sampling strategy, and blinding status, potentially contribute to the discrepancy in published data on C-bouton size changes. Next, we systematically analyzed the impact of study design variability and potential bias on reported results from experimental and preclinical studies of ALS. Strikingly, we found that practices such as blinding and power analysis are not systematically reported in the ALS field. Protocols to standardize experimental design and minimize bias are thus critical to advancing the ALS field.
authors.affiliation: Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine and College of Science and Mathematics, Wright State University , Dayton, Ohio 45435.
authors.forename: S Shekar
authors.identifier: 0000-0001-7770-3853
authors.initials: SS
authors.lastname: Dukkipati
authors.affiliation: Department of Biomedical, Industrial, and Human Factors Engineering, College of Engineering and Computer Science, Wright State University , Dayton, Ohio 45435.
authors.forename: Aouatef
authors.initials: A
authors.lastname: Chihi
authors.affiliation: Department of Biomedical, Industrial, and Human Factors Engineering, College of Engineering and Computer Science, Wright State University , Dayton, Ohio 45435.
authors.forename: Yiwen
authors.identifier: 0000-0002-9382-7546
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Neuroscience, Cell Biology, and Physiology, Boonshoft School of Medicine and College of Science and Mathematics, Wright State University, Dayton, Ohio 45435; Department of Biomedical, Industrial, and Human Factors Engineering, College of Engineering and Computer Science, Wright State University, Dayton, Ohio 45435.
authors.forename: Sherif M
authors.identifier: 0000-0002-6227-3894
authors.initials: SM
authors.lastname: Elbasiouny
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D003627:Data Interpretation, Statistical; D004195:Disease Models, Animal; D006652:Histological Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008297:Male; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009046:Motor Neurons / Q000378:metabolism / Q000473:pathology*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000473:pathology*; D015203:Reproducibility of Results; D012107:Research Design; D016037:Single-Blind Method; D013116:Spinal Cord / Q000378:metabolism / Q000473:pathology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
chemical_list: C109415:SOD1 G93A protein; D013482:Superoxide Dismutase; D000109:Acetylcholine
keywords: ALS; C-bouton; SOD1-G93A; amyotrophic lateral sclerosis; cholinergic synapse; motoneuron