Fiber type composition of the human quadratus plantae muscle: a comparison of the lateral and medial heads

Eight female and three male human cadaveric foot specimens were obtained from the Department of Anatomy and Cell Biology, University of Saskatchewan. Each specimen was from a different individual, with four left and seven right feet included in this study. In all cases, embalming in 3.3% formalin occurred less than 24 hours post-mortem following which an interval between 1.5 and 2.5 years elapsed before the quadratus plantae was dissected. Mean age of individuals was 84 ± 9.0 (range 64-96) years. According to available medical information none had any history of neuromuscular disease, and specimens with major foot deformities or apparent joint disease were excluded from our study. Two additional individuals presented with only one muscle head, and were also excluded from this study. Ethics approval was obtained from the Biomedical Research Ethics Board, University of Saskatchewan (permit number 08-197).

Samples approximately 0.5 × 0.5 × 2-3 cm were excised by blunt and sharp dissection from medial and lateral heads of the quadratus plantae of each specimen (Figure 1). The long axis of each sample ran parallel to the direction of the muscle fascicles. Samples were then coated with Tissue Tek OCT Compound (Sakura Finetek, Torrance, USA), rapidly frozen in 2-methylbutane cooled by liquid nitrogen [15] and stored at -20°C.

Serial cross-sections of each specimen were cut to a thickness of 12 microns in the chamber of a Minitome PLUS Cryostat (Triangle Biomedical Sciences, Durham, USA) at -23°C. Pairs of successive serial sections were picked up on chilled ProbeOn Plus charged microscope slides (Fisher Scientific, Nepean, Canada) and quickly thawed. Consecutive slides were numbered and allowed to air dry for approximately 30 minutes at room temperature followed by storage at -20°C.

Immunohistochemical techniques follow our earlier protocols [21],[22], and were used to label consecutive slides prepared from lateral and medial quadratus plantae samples. Briefly, slides were removed from the freezer and air dried for 15 minutes. Blocking solution comprised of 2% bovine serum albumin and 5 mM ethylenediaminetetraacetic acid in phosphate buffered saline (0.02 M sodium phosphate buffer, 0.15 M sodium chloride, pH 7.2) was then applied to the slides for 30 minutes. Subsequently, primary monoclonal antibodies were used to label myosin heavy chains of Type I fibers (antibody A4.951) or Type II fibers (antibody A4.74). Primary antibodies utilized in this study were raised in mouse and obtained as hybridoma supernatant from the Developmental Studies Hybridoma Bank (DSHB; University of Iowa, Iowa City, USA). Primary antibody solutions consisted of antibody diluted in blocking solution, A4.951 at a 1:20 dilution and A4.74 at 1:50, and were applied to tissue sections overnight at 4°C. A very low frequency of fiber-like structures not labelled by primary antibody A4.74 or A4.951 were detected, and an antibody directed against the myosin heavy chains of all fiber types (antibody A4.1025, DHSB; used at a 1:20 dilution) was used to confirm these unlabelled structures as muscle fibers. As the ability of antibody A4.74 to recognize the Type IIX myosin isoform has not been definitively resolved [23],[24], these unlabelled fibers may represent Type II fibers containing purely IIX myosin.

The secondary antibody process utilized the Avidin Biotin Complex (ABC) method, and two commercially available kits; the ABC kit (Vector Laboratories, Burlington, Canada; PK-6200) and the DAB kit (Vector Laboratories; SK-4100). As per manufacturer instructions, the included universal secondary biotinylated IgG antibody was used to detect bound primary antibody. A 3% hydrogen peroxide solution in methanol was applied to prevent background staining. An avidin and biotinylated horseradish peroxidase macromolecular complex reagent, also included in the ABC kit, was pipetted onto tissue sections and incubated with tissue sections for 60 minutes in the dark at room temperature. Finally, using the DAB kit, a peroxidase substrate containing 3,3′diaminobenzidine was added to effect a specific colour reaction visible by light microscopy. Slides were then rinsed, mounted using Citifluor (Canemco and Marivac, Canton de Gore, Canada), and stored at 4°C. An example of immunohistochemical labelling is demonstrated in Figure 2.

Images of labelled tissue sections were captured with a Sony Cybershot DSC V3 digital still camera (Sony, Tokyo, Japan) attached to a Zeiss Axioskop 20 microscope (Carl Zeiss, Oberkochen, Germany). Images were then uploaded onto an iMac computer (Apple Computer, Cupertino, USA) for classification and calculation of fiber types.

The antibody labelling evident in images of serial sections was used to independently classify fibers as Type I (labelled by A4.951 only) or Type II (labelled by A4.74 only). These classifications were then compared to determine hybrid (labelled by both A4.951 and A4.74) and unlabelled (labelled by neither A4.951 nor A4.74) fibers. Classification was performed for at least one thousand fibers in total from each sample, and fiber classification for all eleven specimens was performed by the same investigator (KS). Data were entered into a Microsoft Excel spreadsheet, where the percentages of Type I, Type II, hybrid and unlabelled fibers in total classified fiber counts were determined for each specimen.