Histological collagen architecture

Picrosirius red staining was performed similarly to previous studies (Acuña et al. n.d.; Trensz et al. 2010; Ardite et al. 2012; Smith & Barton, 2014). Samples were blotted, weighed, fixed in 4% paraformaldehyde overnight, and then stored in PBS at 4°C until use. Prior to sectioning, muscles were placed in 4% agarose gel. Longitudinal sections of 200 μm thickness were cut using a Leica VT1000S. Sections were rinsed, air-dried for 1 h, and stained for 1 h in 0.1% (wt/vol) Direct Red 80 (Fisher) dissolved in saturated aqueous picric acid (Fisher). Sections were then washed in two 1 min rinses of 0.5% acetic acid, dehydrated in three 1 min changes of 100% ethanol, cleared with CitriSolv (Fisher Scientific) for 3 min, and mounted with Permount (Fisher Scientific).

A complete longitudinal section was captured by tiling images using a 20× objective with brightfield illumination on a Leica DMi8 microscope and DFC9000GTC camera. To evaluate collagen alignment, muscle sections were also viewed under linearly polarized light by inserting a rotating polarizer into the beam path before and after the section, respectively. A series of 10 tiling arrays were captured at angles from 0 to 90° in 10° increments.

A custom script was written in MATLAB (Mathworks) to evaluate the parameters of collagen architecture. Briefly, the birefringent light intensity reaches a maximum at 45° from the angle of collagen molecules (Fig. 1). The amplitude of intensity changes through the range of angles relates to the degree of structural alignment at a sub-pixel level. Thus, the mean of each pixel’s amplitude was used to quantify the microECM alignment. The variability across pixels in the angle of maximum intensity was quantified using the circular standard deviation and labelled macroECM deviation. Pixels that did not reach a threshold of average intensity were excluded from analysis.

A, each muscle was imaged with a polarizer at the given angle and an analyser 90° from the polarizer. Images were taken at every 10° between 0° and 90°. The red, blue, and green circles are highlighting the same three pixels across all angles and analyses. B, example pixel intensities across the range of angles are demonstrated. The amplitude is used to determine the mean pixel microECM alignment. The circular standard deviation of the angle of maximum intensity is used to determine macroECM deviation. C, the image is scaled based on the mean amplitude (microECM alignment) of each pixel. D, the same image is represented by the angle of maximum intensity, with the primary orientation of the ECM being 45° from that angle as represented by the colour. The circular standard deviation across the muscle is used to calculate the macroECM. Pixels below an average intensity threshold were not included in C and D or the analysis. Scale bar = 100 μm.