Quantifying Neurodegeneration and Neuroinflammation in MPTP Mice

Seven days after MPTP intoxication, mice were sacrificed and their brains fixed, embedded, and processed for tyrosine hydroxylase (TH) and thionin staining as described previously (Benner et al., 2004 (link); Ghosh et al., 2007 (link)). Total numbers of TH- and Nissl-stained neurons in SNpc were counted stereologically with stereo investigator software (MicroBrightfield, Williston, VT) by using an optical fractionator (Benner et al., 2004 (link); Ghosh et al., 2007 (link)). Quantitation of striatal TH immunostaining was performed as described (Benner et al., 2004 (link); Ghosh et al., 2007 (link)). Optical density measurements were obtained by digital image analysis (Scion, Frederick, MD). Striatal TH optical density reflected dopaminergic fiber innervation. For immunofluorescence staining on fresh frozen sections, rat anti-mouse CD11b (1:100), goat anti-mouse GFAP (1:100), rabbit anti NF-κB p65 (1:100), goat anti-NF-κB p65 (1:100), rabbit anti NF-κB p50 (1:100), and rabbit anti-mouse iNOS (1:250) were used. The samples were mounted and observed under a Bio-Rad MRC1024ES confocal laser scanning microscope.