Morphological Changes In Endplate On Type-identified Muscle Fiber In Aged Rat Diaphragm

Abstract

PURPOSE: Previous studies demonstrated a decrease in neuromuscular activity with aging and the extent of the decrease could influence neuromuscular junction morphology and function. In this study, we compared 3-dimensional (3D) morphological properties of motor endplate on type-identified muscle fibers in diaphragm muscle between young and old rats. METHODS: Young (4 months, n=5) and old (over 2 years, n=5) male Wistar rats were used. Three segments of mid-costal diaphragm muscle were removed and then a fluorescent double-labeling technique was applied to visualize the type-identified endplate. Endplates were labeled with a-bungarotoxin conjugated tetramethylrhodamine. Myosin heavy chain (MHC) isoforms were first incubated in antibodies to MHC isoforms (I and/or IIa) and then labeled by second antibody with FITC. The endplates on type-identified muscle fibers were imaged using 3D confocal microscopy (Nikon, E600 & C1) with 2 lasers (543nm for rhodamine and 488nm for FITC) and software (solution systems, 3D doctor). RESULTS: In young diaphragm, the planar area and volume of endplate on type-I and -IIa muscle fiber were smaller than those on type-IIb/x fiber, while the normalized planar areas of endplate to muscle fiber diameter were identical on all fiber types. The receptor densities (endplate area/outer surrounding area) on all fiber types were identical (70, 67 and 68 % on type I, IIa and IIb/x fibers). With aging, the planar area and volume of endplate increased on all fiber type, and there were no significant changes in the normalized endplate area to fiber diameter. However, the receptor densities on endplates of all fiber types were significantly lower in specimens from old rats (63, 56 and 45 % on type I, IIa and IIb/x fibers) compared to those in young diaphragm. Furthermore, the density on type IIb/x fiber was significant lower than those on type I and IIa fibers in old diaphragm. CONCLUSIONS: The identical values of normalized endplate area to fiber diameter between young and old diaphragm suggest the compensatory expansion of endplate with aging. Decreased receptor density in the old diaphragm indicates fragmentation of the endplate. These morphological changes might lead to functional deficiency and selective denervation on type-IIb/x muscle fiber with aging.