The chemical morphology of extracellular matrix in experimental rat liver fibrosis resembles that of normal developing connective tissue

Abstract

The time course of development of extracellular matrix (ECM) in experimentally induced fibrosis (thioacetamide administration followed for 12 weeks or bile duct ligation for 8 weeks) in adult rats was examined by light and electron microscopy, using Alcian blue or Cupromeronic blue staining for sulphated proteoglycans (PGs) in critical electrolyte concentration techniques. Proteodermatan sulphate (PDS) was regularly observed at the gap zone of the collagen fibrils. Morphometry of uranyl acetate-stained collagen fibrils, polarity of their banding patterns (a-e), statistics of d/e band occupancies by PDS, and lengths and thicknesses of PG filaments were quantified. Biochemical analyses showed that the ECM components collagen, hyaluronan, chondroitin and dermatan sulphates increased by 5-10 fold, roughly in parallel, as did heparan sulphate and DNA. Water and lipid contents also increased sharply. Thioacetamide treatment was much slower than bile duct ligation in producing fibrotic changes of equal severity. Sulphation of anionic glycosaminoglycans (AGAGs) decreased with increasing severity of fibrosis. Biochemical and ultrastructural methods correlated well. The large increase in dermatan sulphate was quantitatively as expected, given that it is collagen fibril surface-associated, and there was an increase of collagen content together with a decrease in fibril thicknesses. The increase in DNA reflected the marked increase in cell numbers in fibrotic livers. The chemical morphology of the new connective tissue closely resembled that in e.g. developing young tendon, in that fibrils were thinner, and AGAG levels were higher. The collagen fibrils were often disarranged, rather than ordered and parallel as in normal ECM. No other indication of abnormality in the new ECM was obtained.