Computational protocol: PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease

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Protocol publication

[…] Evaluation of gross striatal volume. Standard Nissl staining was employed on coronal step serial sections from rostral neostriatum through the level of anterior commissure (interaural 4.66 mm/bregma 0.86 mm to interaural 3.34 mm/bregma -0.46 mm) from six animals per group. Gross striatal volume was measured using Neurolucida Stereo Investigator software (Zeiss, Cochester, VT, USA).Striatal neuronal cell count. Single label immunofluorescence was performed using an antibody against Calbindin D-28K (marker of the Medium Spiny Neurons) (CALB, Immunological Sciences) to evaluate the number of surviving projection neurons in the striatum. Cell count were carried out in each of 19 1.0-mm-square confocal microscope fields, rostrocaudally spaced on both hemispheres of 6 mice from each group. For cell counts, we used Java image processing and analysis program Image J, developed by Wayne Rasband, available in the public domain (http://imagej.nih.gov/ij/docs/index.html). We used a manual approach, measuring the number of objects by means of the point selection tool.Microglial morphology. Microglial morphology was studied by immunolabeling our tissue with an antibody for microglia (mouse anti-CD-11b, Serotec). The diaminobenzidine–immunoperoxidase technique was employed as previously described [].Evaluation of NIIs. Sections were processed for single label EM-48 mutant huntingtin protein antibody (Clone mEM48, Millipore Corporation) by means of immunofluorescence, and were counterstained with NeuroTrace. A sample of about 250 neurons per hemisphere for each of three sections in each of 6 mice per treatment group was analyzed to determine the number of NIIs of striatal neurons in both saline and INO-1001 treated R6/2 mice (wild type littermates did not show NIIs–like mutant huntingtin immunoreactivity- data not shown). [...] The data collected were analyzed to compare the effect of INO-1001 on weight, clasping, and on surviving cell number, NIIs percentage, BDNF expression and CREB activation in the striatal of differently treated groups. Statistical analysis was performed by STATISTICA 10 software and ANOVA available on the software GraphPad Prism version 6.0. P values of less than 0.05 were considered statistically significant. Survival data were analyzed by means of a product limit method of Kaplan and Meier, and P value was set at <0.0001 for significant results. […]

Pipeline specifications

Software tools Neurolucida, ImageJ, Statistica
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Huntington Disease
Chemicals Poly Adenosine Diphosphate Ribose