Age-related changes in two- and three-dimensional morphology of type-identified endplates in the rat diaphragm

To examine the age-related morphological changes in the motor endplate of type-identified muscle fibers, 20 male Wister rats were divided into 2-month-, 10-month-, 24-month- and 30-month-old groups (n = 5 in each group). Three segments of mid-costal diaphragm muscle were removed, and then a fluorescent double-labeling technique was used to visualize the endplates on type-identified muscle fibers. Endplates were labeled with alpha-bungarotoxin-tetramethylrhodamine. Muscle segments were first incubated in antibodies to MHC isoforms (I and/or IIa) and then labeled by a second antibody with FITC. The endplates were imaged using 3D confocal microscopy with two lasers. In each age group, the planar area and volume of endplates on type-IIx/b muscle fibers were larger than those on type-I and -IIa muscle fibers, while the normalized planar areas of the endplate (endplate area/muscle fiber diameter) and the mean thickness of the endplate (volume/endplate area) were identical on all fiber types within the same age group. Decreased endplate density (endplate area/surrounding area) in the old diaphragm indicated fragmentation of the endplate, especially on type IIx/b fibers. These morphological changes may lead to functional deficiency and selective denervation of type-IIx/b muscle fiber with aging.