Use of BrdU Labeling Assay to Study Skeletal Muscle Regeneration in Duchenne Muscular Dystrophy Mouse Model
Open AccessDuchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder caused by the absence of dystrophin, a protein that helps strengthen muscle cell membranes. DMD is characterized by progressive muscle wasting and weakness. As muscles fibers degenerate over time, the muscle strength also declines. However, we have no method for accurately measuring the amount of muscle fiber loss that occurs. Therefore, we tried to develop a method for measuring muscle loss in the Mdx52 knockout mouse model of DMD, which lacks the dystrophin protein and exhibits fragility of the outer membrane of the muscle cells.This results in severe necrosis of their muscle fibers, from 3 weeks of age, accompanied by compensatory repair from resident muscle precursors, but eventually leads to muscle atrophy and weakness.We labeled muscle fiber nuclei with Bromodeoxyuridine (BrdU), a synthetic thymidine analog that is incorporated into a cell's DNA when the cell is dividing. When BrdU is injected into newborn mice, it labels a cohort of myogenic cells that fuse into the muscle fibers and remain in the fibers until they die. As the mice grow, the BrdU-labeled myonuclei become diluted by fresh input from myogenic cells that are dividing in the absence of BrdU. In muscular dystrophic mice, this dilution is enhanced by the loss of muscle fibers containing labeled myonclei accompanied by repair with unlabelled nuclei. This excess drop in proportion of BrdU-labelled myonuclei in muscular dystrophic mice is an index of loss of muscle nuclei due to loss of muscle.We have used Mdx 52 dystrophin deficient mice and their C57BL/6 wildtype controls. Breeding pairs for both the strains were set up and pups were injected with BrdU up to 7 days. The pups were then sacrificed at 16, 36, and 50 days and frozen tissues were collected in liquid nitrogen chilled isopentane. The tissues were sectioned and immunohistochemistry was performed to evaluate BrdU labeled myonuclei. With the growth of the mice pups and subsequent cell divisions in the absence of BrdU, later-formed nuclei will not be labeled. In addition, in the mdx mice, labeled nuclei will be lost as muscle fibres die and are replaced by non-labelled nuclei formed during regeneration. The proportion of BrdU-labelled myonuclei diminished more rapidly in dystrophic than in control wild type mice.The final notion is to use the BrdU assay to assess the extent of degeneration and regeneration not only to understand myonuclei turnover but also to assess efficacy of drugs that protect skeletal muscle from degeneration or enhance its regeneration.
- All rights reserved
Notice to Authors
If you are the author of this work and you have any questions about the information on this page, please use the Contact form to get in touch with us.