Multiple sclerosis, neuromyelitis optica, Guillain–Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy are representative demyelinating diseases of the central and peripheral nervous system. Remyelination by myelin forming cells is important for functional recovery from the neurological deficits caused in the demyelinating diseases. Lysophosphatidylcholine-induced demyelination in mice is commonly used to identify and study the molecular pathways of demyelination and remyelination. However, detection of focally demyelinated lesions is difficult and usually requires sectioning of demyelinated lesions in tissues for microscopic analysis. In this review, we describe the development and application of a novel vital staining method for labeling demyelinated lesions using intraperitoneal injection of neutral red (NR) dye. NR labeling reduces the time and effort required to search for demyelinated lesions in tissues, and facilitates electron microscopic analysis of myelin structures. NR labeling also has the potential to contribute to the elucidation of pathologies in the central and peripheral nervous system and assist with identification of drug candidates that promote remyelination.

While the rapid decrease in estrogen is well known as the main cause of postmenopausal osteoporosis in women, the precise pathogenesis of senile osteoporosis in the elderly regardless of gender is largely unknown. The age-related epigenetic regulation of receptor activator NF-κB (RANK) gene expression was investigated with the use of a high-passaged mouse osteoclast progenitor cell line, RAW264.7, as an in vitro model of aging. In the RAW264.7 cells after repeated passages, receptor RANK expression was downregulated, resulting in decreased soluble RANK ligand (sRANKL)-induced osteoclastogenesis, expression of tartrate-resistant acid phosphatase-5b (TRAcP) and cathepsin K (CTSK). Methylation-specific PCR and bisulfite mapping revealed hypermethylation of CpG-loci located in the RANK gene promoter in multiple-passaged cells. ICON probe-mediated in situ assessment of methylated-cytosine at the CpG loci revealed an increase in the percentage of methylated RAW264.7 cells in the RANK gene in a passage-dependent manner. Conversely, upon treatment with demethylating agent 5-aza-2-deoxycytidine (5-aza-dC), high-passaged RAW264.7 cells displayed restored expression of the RANK gene, osteoclastogenesis, TRAcP and CTSK. Ex vivo cultures of splenic macrophages from young (10.5 W) and aged (12 M) mice also showed that CpG methylation was predominant in the aged animals, resulting in reduced RANK expression and osteoclastogenesis. Reduced RANK expression by age-related accumulation of DNA methylation, albeit in a limited population of osteoclast precursor cells, might be, at least in part, indicative of low-turnover bone characteristic of senile osteoporosis.