Determination of fibrosis from cryostat sections using high performance liquid chromatography: Skeletal muscle

Analysis of hydroxyproline (collagen) and pyridinoline (collagen cross-link s) in biopsies prepared for routine histological evaluation with OCT compou nd was performed. Frozen sections (250 mu m-thick) were cut from cardiac mu scle, diaphragm, liver, and soleus muscle from the rat. After removal of OC T compound by rinsing, the samples were dried, weighed and hydrolyzed in 6 N HCl. A portion of the hydrolysate was analyzed for hydroxyproline using h igh performance liquid chromatography with collagen type I as the standard. Collagen concentrations ranged from 6.6 mu g/mg dry weight (liver) to 74.7 mu g/mg dry weight (diaphragm). From the remainder of the hydrolyzate, pyr idinoline cross-links of collagen were separated and analyzed similarly by high performance liquid chromatography. The concentration of pyridinoline r anged 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) ro utinely used in clinical pathology. The method proved useful in the quantit ation of focal fibrosis in a partially denervated rat soleus. Denervation w as confirmed using fast myosin immunohistochemistry which revealed large ar eas of small myofibres containing fast myosin. Collagen concentration incre ased by five-fold and collagen cross-links by more than 7-fold consistent w ith fibrotic changes known to occur with denervation.