For visualization of nerve and neuromuscular junctions, slides were imaged with scanner laser confocal microscopy (model FV1000; Olympus, Tokyo, Japan) to sequentially capture images in the same focal plane with different filters. Neuromuscular junctions were imaged at 20× oil immersion magnification and nerves at 10× magnification. Z-stacks through the thickness of the sections were collected. Every 20th section was imaged for analysis. With a random starting point, every third field throughout the entire muscle specimen was taken.
Z-stacks were collapsed for each field, and the following measurements were taken using ImageJ software (National Institutes of Health, Bethesda, MD, USA): muscle area, mean diameter for myofibers positive or negative for slow MyHC, total number of neuromuscular junctions, and area of each neuromuscular junction endplate as determined by hand circling. For every fiber with a neuromuscular junction, it was also noted whether the fiber was positive or negative for slow myosin. For each field, the muscle layer (orbital, global) and region (proximal, endplate zone, distal) were noted. Neuromuscular junctions were categorized based on slow myosin expression, endplate area, and morphology to distinguish between en grappe and en plaque endplates. For en plaque neuromuscular junctions in the endplate zone, an additional measurement of neuromuscular junction length was performed. For assessment of neuromuscular junction size, only counts of complete neuromuscular junctions were included. For a subset of sections, every fifth field was imaged at 60× oil immersion magnification to visualize neuromuscular junction morphology fully. Additional staining among control and experimental muscles with double-labeling of SNAP-25 (1:1000 dilution; Covance; Dedham, MA, USA) or synaptophysin (1:300 dilution; Abcam; Cambridge, MA, USA), nerve (smi-31), and α-bungarotoxin revealed that all α-bungarotoxin-labeled neuromuscular junctions in the EOM specimens colocalized with presynaptic components. Based on these results, we are confident that most α-bungarotoxin-positive neuromuscular junctions in our samples were active endplates.
For calculation of average myofiber cross-sectional area, slides were visualized with a light microscope, and fields were randomly chosen throughout the length of the muscle in both the global and the orbital layers. A minimum of 200 myofibers per section were analyzed, and a minimum of three slides per muscle specimen was measured.
For calculation of embryonic MyHC and neonatal MyHC density and mean myofiber diameter, every 40th section along the treated and control medial rectus slides was visualized using light microscopy. For analysis, every third field throughout the slide was counted with a random starting point. The percentage of fibers in each field staining positive for each myosin was determined. The average fiber diameter for fibers positive and negative for each myosin was also determined for each field. Mean myofiber size and percent positive for both embryonic and neonatal MyHC isoform expression were assessed with imaging analysis software (BioQuant Nova Prime morphometry program; BioQuant, Nashville, TN, USA).
All data were analyzed statistically (Prism software; GraphPad, San Diego CA, USA). For all measurements, comparisons were made between the treated medial rectus and control medial rectus muscle, or between untreated lateral rectus muscle from experimental subjects and control lateral rectus muscle. All data were analyzed for statistical significance using the Student's t-test with correction for multiple comparisons using the Holm-Sidak method, except for analysis of cross-sectional area, which was analyzed with one-way ANOVA, followed by the Tukey test to correct for multiple comparisons. Data were considered statistically significant if P < 0.05. For all graphs, error bars signify standard deviation.