Histopathology, immunohistochemistry, inflammation, and demyelination

Histological evaluation was done on paraformaldehyde-fixed, paraffin-embedded sections of spinal cords (SCs). Sections were stained with hematoxylin and eosin (HE), Luxol fast blue (LFB), and neurofilament (NF) [3] staining to assess inflammation, demyelination, and axonal pathology, respectively, as described previously [18]. We examined 12–16 longitudinal sections per mouse. Quantitative histological evaluation for inflammation and demyelination and axonal loss was done and scored blindly by two independent observers. Histopathology analysis was done at day 17 after start of RAM-589.555 treatment. Mice (n = 3) from each group received ketamine/xylazine and were perfused transcardially with PBS followed by 4% paraformaldehyde (PFA) in PBS. Spinal cords were removed and stored in 4% PFA at 4 °C. Longitudinal spinal cord sections were performed, stained by hematoxylin & eosin (H&E), Luxol fast blue (LFB), and neurofilament staining (NF, mouse anti-human NF protein, Dako) in 6 μm paraffin-embedded adjacent serial sections. The morphometric quantification of inflammation, demyelination, and axonal damage were performed using Olympus IX-73 microscope coupled to imaging software (Cellsens Entery digital imaging software, Olympus). The following parameters were evaluated: (a) inflammation: average number of lymphocyte infiltrations per square millimeter counted in spinal cord sections using a grid overlay (H&E); (b) demyelination: mean percent of demyelination areas per square millimeter in spinal cord sections (LFB); and (c) axonal damage: mean percent of axonal loss assessed by neurofilament loss per square millimeter in spinal cord sections. Both the cervical, thoracic, and lumbar regions were assessed. Quantitative analysis of immunopositive or stained region was carried out using NIH Image (ImageJ 1.43u).