|Ceglia, Lisa -|
|Niramitmahapanya, Sathit -|
|Price, Lori Lyn -|
|Liu, Beiyun Caitlin -|
|Harris, Susan -|
|Dawson-Hughes, Bess -|
|Fielding, Roger -|
Submitted to: Biological Procedures
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 17, 2013
Publication Date: March 13, 2013
Citation: Ceglia, L., Niramitmahapanya, S., Price, L., Liu, B., Harris, S., Dawson-Hughes, B., Fielding, R. 2013. An evaluation of the reliability of muscle fiber cross-sectional area and fiber number measurements in rat skeletal muscle. Biological Procedures. 15(1):6. Interpretive Summary: The manual assessment of the size and number of fibers in the skeletal muscle of a rat is time-consuming and labor-intensive. Whether selecting only a subset of muscle fibers to accurately and reliably estimate average muscle fiber size or total fiber number has not been systematically evaluated. This study described the variability in estimates of mean fiber cross-sectional area (measure of size) as a function of the number of fibers measured, and the relationship between total fiber number and a subset of fibers from 1-4 fields in the muscle cross-section in older rats. A statistical resampling approach showed that the variability in the distribution of resamples of mean fiber cross-sectional area decreased and gradually stabilized as the number of fibers measured increased; the largest declines occurred at numbers below about 150-200 fibers. For the evaluation of fiber number, the fiber count of 3 to 4 fields in the section was more representative of the total fiber number than counts from 1 to 2 fields. Therefore, our data describe a pattern of improved accuracy and precision in estimating fiber size in older rats as sample size of fibers measured increases, particularly beyond 150-200 fibers. Fiber counts from 3 fields within a muscle section provide similar time-saving advantages when reporting total fiber number.
Technical Abstract: Background: The reliability of estimating muscle fiber cross-sectional area (measure of muscle fiber size) and fiber number from only a subset of fibers in rat hindlimb muscle cross-sections has not been systematically evaluated. This study examined the variability in mean estimates of fiber cross-sectional area as a function of the number of fibers measured, and tested whether counting a subset of fibers in a cross-section could predict total fiber number in middle-aged rats. Results: Soleus and extensor digitorum longus (EDL) muscle cross-sections from 23-month-old, male Fisher 344 x Brown Norway rats were stained for myofibrillar ATPase activity to identify muscle fiber type (either type I [slow-twitch] or II [fast-twitch]) and laminin to facilitate fiber cross-sectional measurements. We outlined the circumference of 1000 to 1600 single muscle fibers for measurement of fiber cross-sectional area within muscle sections. Mean type I fiber cross-sectional area was based on soleus muscle sections which were predominantly composed of type I muscle fibers. Mean type II fiber cross-sectional area was based on EDL muscle sections which were predominantly composed of type II muscle fibers. A bootstrapping resampling technique demonstrated that variability in sampling distribution of mean type I and II fiber cross-sectional areas decreased and gradually stabilized as the number of fibers measured increased with large declines in variability occurring at numbers below 150 fibers. Coefficients of variation for bootstrapped mean type I fiber cross-sectional areas were lower than for type II. In the same muscle sections, total fiber number was compared to fiber numbers within 1, 2, 3, and 4 fixed field areas (10x magnification; 1000 x 1500 pixels in size/field) on the cross-section. Fiber numbers from 3 to 4 fields (approximating 15 to 20% of the cross-section) provided a reasonably predictive value of total fiber number (r=0.57-0.59, P=0.003). Conclusions: These data describe a pattern of improved precision in estimating mean fiber cross-sectional area as sample size of fibers measured increases to at least 150 in this rat model. Counting 15-20% of the fibers in cross-sections provides a reasonably reliable estimate of the total fiber number.