Bz x800

Bladders were isolated from the rats in all groups and fixed in 4% paraformaldehyde. The tissues were frozen and cut on a cryostat at 5-μm thickness. Sections were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. The slides were incubated with mouse anti-F4/80 (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-iNOS (1:100; Abcam plc, Cambridge, UK), or rabbit anti-CD206 (1:500; Abcam plc) overnight at 4°C and then with anti-mouse Alexa Fluor Plus 594 (1:1000; Abcam plc), anti-rabbit Alexa Fluor Plus 488 (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies, and 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for 1 h at room temperature. Immunofluorescence images were obtained using an all-in-one fluorescence microscope (BZ-X800; KEYENCE) and analyzed using a BZ-X800 analyzer. Two random areas per bladder selection from each group were observed and the macrophage in the lamina propria was estimated within the visual fields observed at object lens 10× magnification. The percentage of iNOS- or CD206-positively stained cells per F4/80-positively stained cells in the fields was determined.

Detection of intracellular Fe²⁺ was performed using FerroOrange (excitation, 561 nm; emission, 570–620 nm) (DOJINDO, Kumamoto, Japan). Cells were cultured in glass-bottomed dishes for 3 days and washed with HBSS (Thermo Fisher), after which 1 µM FerroOrange was added for 30 min at 37°C, followed by observation under a microscope (BZ-X800; KEYENCE). To detect Fe²⁺ in mitochondria, 5 µM Mito-FerroGreen working solution (excitation, 488 nm; emission, 500–550 nm) (DOJINDO, Kumamoto, Japan) was added to the cells for 30 min at 37°C, followed by observation under a microscope. Lipid radicals were detected using LipiRADICAL Green (Funakoshi, Tokyo, Japan) (excitation, 470 nm; emission, 520–600 nm) and Liperfluo (excitation, 524 nm; emission, 535 nm) (DOJINDO, Kumamoto, Japan). Cells and cardiomyocyte were cultured in glass-bottomed dishes for 3 days and washed with HBSS, after which 1 µM LipiRADICAL Green or 4 µM Liperfluo working solution was added for 10 min at 37°C, followed by observation under a microscope (BZ-X800; KEYENCE).

All tissue sections were statistically processed by randomly selecting at least two sections per mouse. In the images acquired with a microscope (Olympus BX53, Keyence BZ-X800), each DRG neuron was identified as a dot and the numbers were counted using the image processing software GIMP 2.10.30 (GIMP Development Team) and the image processing and analysis software ImageJ (National Institutes of Health, Bethesda, MD, USA). Similarly, lumbar spinal cord sections from RCS mice were randomly selected, and a 20,000 μm² field of observation was cut from the images acquired by fluorescence microscopy using GIMP. Next, ImageJ was used to quantify the numbers of microglia (cells that co-expressed Iba1 and 4′,6-diamidino-2-phenylindole; n = 5 or 6 mice per group). The area occupied by GFP-positive fibers in the spinal cord was also quantified in the same manner (n = 5 mice per group). ChAT-positive and GFP- and ATF3-positive neurons in the spinal cord were similarly quantified (n = 4 or 5 mice per group). Images were adjusted identically and appropriately in each group, and were examined using the editing software supplied with the microscope (TiE-A1R Nikon: NIS-Elements Analysis; Keyence BZ-X800: BZ-X800 Analyzer) or GIMP.