Procion orange tracer dye technique vs. identification of intrafibrillar fibronectin in the assessment of sarcolemmal damage.
BACKGROUND
The use of Procion orange dye (POD) is one of the most widely accepted techniques to assess sarcolemmal damage. This phenomenon has been related to functional adaptation in skeletal muscles. The POD method includes intravenous injection of this colorant in vivo, enabling its identification inside those fibres with membrane leaks (fluorescence). However, the safety of the use of POD has not been proven.


OBJECTIVE
This study was designed to compare POD with a safer alternative, involving the identification of intracellular fibronectin using specific antibodies.


METHODS
Eight Swiss mice were submitted to electrical stimulation of the lower limbs at different frequencies (10-80 Hz). Subsequently, the POD solution was infused, and samples from the vastus medialis muscle were obtained 24 h later. Samples were processed and serial sections were analysed using immunohistochemistry (monoclonal antibodies against fibronectin) and epifluorescence microscopy.


RESULTS
Ninety-eight per cent of the fibres were equally classified by both techniques, which in addition showed good correlation (percentages of damaged fibres, r = 0.998, P < 0.001) and concordance (R1 = 0.82) in quantitative terms.


CONCLUSIONS
Although the two techniques compared here are based on different principles, both are comparable in assessing sarcolemmal damage. This would facilitate comparisons between human and experimental studies. In addition, the fibronectin technique appears to be a suitable alternative for long-term studies including repeated biopsies.
Palacio
J
J
Department of Pneumology and Pathology, Hospital Del Mar - IMIM, Universitat Pompeu Fabra, Barcelona, Spain.
Gāldiz
J B
JB
Alvarez
F J
FJ
Orozco-Levi
M
M
Lloreta
J
J
Gea
J
J
D000818:Animals; D004558:Electric Stimulation; D005353:Fibronectins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D012508:Sarcolemma / Q000378:metabolism / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods*; D014227:Triazines / Q000378:metabolism*
D005353:Fibronectins; D005456:Fluorescent Dyes; D014227:Triazines; C041397:Procion Orange MX-2R