Computational protocol: Reductions in behavioral deficits and neuropathology in the R6/2 mouse model of Huntington’s disease following transplantation of bone-marrow-derived mesenchymal stem cells is dependent on passage number

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Protocol publication

[…] Images of the fluorescent labels were captured using a Zeiss Axiovert 200 M inverted fluorescent microscope with optical dissectors at 20× magnification. CYO labeled tissue was scanned using Nikon ScanPro (Nikon, Melville, NY, USA). Images were captured from five levels, centered at 0.5 mm anterior to the bregma (the transplant site) with two additional sections anterior and posterior to the transplant site, approximately 200 μm apart.All images were analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The average intensity of the fluorescent label, the counts of positively labeled cells, as well as the percentage of colocalization between the transplanted MSCs and NeuN or GFAP were analyzed between all groups. The average intensity of the fluorescent label, the number of labeled cells, and the percentage of cells that were co-labeled for both MSCs and either NeuN or GFAP were analyzed from samples of all transplant groups. Methods for rare-event stereology were modified from previously published protocols [, ]. Briefly, cell counts were made from the dorsal striatum within each section. Cells were counted as positive cells if they showed: antibody immunoreactivity within the cell body; the nucleus of that cell was within the counting frame without touching the exclusion lines; and the nucleus of that cell was in focus. For cell counts, the average number of cells per section was calculated.Densitometric measures of CYO and GFAP were taken from the striatum and the average intensities were normalized to the corpus callosum of each section. For the total brain area, sections were traced using ImageJ and the total area was calculated. [...] All statistical analyses were performed using SPSS v16 (IBM SPSS Statistics, Armonk, NY, USA) with an alpha level equal to 0.05. Assays between low-passage and high-passage BM MSCs in vitro were analyzed using an independent-samples t test. All behavioral data were analyzed using a repeated-measures analysis of variance (ANOVA), to measure changes between genotypes and treatments across weeks. Histological data were analyzed using a one-way ANOVA. When appropriate, a Tukey’s honestly significant difference (HSD) test was performed for post hoc analyses. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Huntington Disease, Genetic Diseases, Inborn