Determination of fibrosis from cryostat sections using high performance liquid chromatography: skeletal muscle.
Miller-GR; Smith-CA; Stauber-WT
Histochem J 1999 Feb; 31(2):89-94
Analysis of hydroxyproline (collagen) and pyridinoline (collagen cross-links) in biopsies prepared for routine histological evaluation with OCT compound was performed. Frozen sections (250 microm-thick) were cut from cardiac muscle, diaphragm, liver, and soleus muscle from the rat. After removal of OCT compound by rinsing, the samples were dried, weighed and hydrolyzed in 6 N HCl. A portion of the hydrolysate was analyzed for hydroxyproline using high performance liquid chromatography with collagen type I as the standard. Collagen concentrations ranged from 6.6 microg/mg dry weight (liver) to 74.7 microg/mg dry weight (diaphragm). From the remainder of the hydrolyzate, pyridinoline cross-links of collagen were separated and analyzed similarly by high performance liquid chromatography. The concentration of pyridinoline ranged from 2.6 ng/mg dry weight (liver) to 35.6 ng/mg dry weight (diaphragm). These techniques were adequate to analyze both collagen and pyridinoline (i.e. collagen cross-links) in small biopsy samples (< 1 mg dry weight) routinely used in clinical pathology. The method proved useful in the quantitation of focal fibrosis in a partially denervated rat soleus. Denervation was confirmed using fast myosin immunohistochemistry which revealed large areas of small myofibres containing fast myosin. Collagen concentration increased by five-fold and collagen cross-links by more than 7-fold consistent with fibrotic changes known to occur with denervation.
Fibrosis; Chromatographic-analysis; Collagen-fibrils; Histology; Muscle-tissue; Muscle-cells; Musculoskeletal-system-disorders; Laboratory-animals; Animals; Animal-studies
Department of Physiology, West Virginia University, PO Box 9229, Morgantown, WV 26506, USA
Disease and Injury: Musculoskeletal Disorders of the Upper Extremities
The Histochemical Journal
West Virginia University, Morgantown, West Virginia