Computational protocol: Apocynin prevents mitochondrial burdens, microglial activation, and pro-apoptosis induced by a toxic dose of methamphetamine in the striatum of mice via inhibition of p47phox activation by ERK

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Protocol publication

[…] Immunocytochemistry was performed as described previously []. Mice were perfused transcardially with 50 mL of ice-cold PBS (10 mL/10 g body weight) followed by 4 % paraformaldehyde (20 mL/10 g body weight). Brains were removed and stored in 4 % paraformaldehyde overnight. Series of every sixth sections (35 μm thickness, 210 μm apart) from striatum were selected and subjected to immunocytochemistry. Sections were blocked with PBS containing 0.3 % hydrogen peroxide for 30 min and then incubated in PBS containing 0.4 % Triton X-100 and 1 % normal serum for 20 min. After a 48-h incubation with primary antibody against TH [1:500; Chemicon (EMD Millipore)] and Iba-1 (1:500, Wako Pure Chemical Industries, Chuo-ku, Osaka, Japan), sections were incubated with the biotinylated secondary antibody (1:1000; Vector Laboratories, Burlingame, CA, USA) for 1 h. The sections were then immersed in a solution containing avidin–biotin peroxidase complex (Vector Laboratories) for 1 h, and 3,3′-diaminobenzidine was utilized as the chromogen.To examine TH-immunoreactivity, digital images were acquired at ×4 objective magnification using an Olympus microscope (BX51; Olympus) and a digital microscope camera (DP72; Olympus). ImageJ version 1.47 software (National Institutes of Health, Bethesda, MD, USA) was employed to measure the TH-immunoreactivity as described previously []. Briefly, the entire striatal region from each section was selected as the region of interest (ROI). Threshold values for hue (0–100), saturation (0–255), and brightness (175–255) were set in the “Adjust Color Threshold” dialog box, and then the mean density was measured. [...] To analyze morphological changes in microglia, digital images of Iba-1-immunostained sections were obtained at ×40 objective magnification under an upright microscope (BX51; Olympus) using an attached digital microscope camera (DP72; Olympus). Each section was acquired in 11 planes of focusing, and then the images were stacked and integrated into one image for morphological analysis []. The resolution of the resulting images was 1360 pixels × 1024 pixels (350 μm × 263 μm).Skeleton analysis was performed as described previously [–]. For skeleton analysis, the resulting images were subjected to background subtraction, converted to 8 bit, and binarized using ImageJ version 1.47 software. The “Skeletonize3D” plugin (fiji.sc/Skeletonize3D) and “AnalyzeSkeleton” plugin (fiji.sc/AnalyzeSkeleton) were applied to skeletonize and analyze skeleton, as shown in Additional file : Fig. S8a. The number of branches, the number of junctions, the number of triple points (junctions with exactly three branches), the average branch length, and summed branch length were determined.The cell size and cell body size in the area were determined as described previously [, ], using ImageJ version 1.47 software. Background was subtracted from each image to correct uneven background. To measure the cell size, all pixels that were darker than the background were selected by the auto-threshold command. Cell bodies were selected by manual intensity selection, as shown in Additional file : Fig. S8c. The “Analyze Particles” command was employed to measure the cell size and cell body size. The number of cell bodies was counted to normalize the cell size and cell body size per cell. The cell body size to cell size ratio (%) was also determined. […]

Pipeline specifications

Software tools ImageJ, AnalyzeSkeleton
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases Neurotoxicity Syndromes
Chemicals Methamphetamine, NADP, Superoxides