ABSTRACT— By means of staining with Sirius Red F3BA in a saturated picric acid solution, the collagen contents of rat livers with varying degrees of fibrosis have been measured quantitatively in fixed and sectioned material, using both histophotometry in situ and extraction of bound dye with colorimetric analysis. These findings have been correlated with chemical assays of the hydroxyproline content in homogenates from the same livers. It appears that a highly significant correlation exists between both section‐based analysis methods and the hydroxyproline content, the Spearman‐Rank correlation coefficients being virtually identical. For analysis of collagen accumulation in rat liver, both section‐based methods seem to be useful and reliable, the extraction method giving the quickest results for large‐scale screening, and the histophotometric method being more appropriate to take readings in selected areas. With human liver material, indications have been obtained for the existence of large sampling errors due to inhomogeneous distribution of collagen deposits. Using the extraction method, no significant changes could be observed in the volume density of collagen during postnatal growth from 1 week to 21 months in rat liver: only on the third day after birth was a higher value of collagen/total protein obtained, possibly due to a higher water content of the hepatocytes. Partial hepatectomy was found to have no influence at all on the collagen content of rat liver during the period of restorative growth or after it.
The development of fibrosis in the liver of 16 rats treated for 1, 2, 3 or 4 weeks with CCl4, has been followed with chemical hydroxyproline determination and histophotometric analysis of histological sections stained with Sirius Red F3BA in saturated aqueous picric acid. The readings were taken with a scanning and integrating microphotometer and corrected for picric acid absorbance as a measure for mean protein mass per unit area of the section. It appears that the integrated absorbance readings of Sirius Red absorbing material in the section show a highly significant correlation with the hydroxyproline determinations. It is concluded that picrosirius photometry can be used to give a measure of the volume density of collagen in sections. An advantage of the photometric assay is that measurements are taken on the basis of the microscopic image, so that it is also possible to estimate collagen density in a selected area, e.g. a tumour formation amidst normal tissue, or to exclude necrotic areas.
Ultrastructural morphometric analysis was used to study time-dependent variations in macro- and microautophagy in rat hepatocytes. Except during periods of short-term starvation for up to 24 h, animals were kept under standardized conditions of food intake. In hepatocytes of meal-fed rats the volume fraction of macroautophagic vacuoles is significantly higher at 23:00 h, i.e., immediately before food intake, compared to 11:00 h, i.e., 12 h following feeding. During fasting, macroautophagy drops to a low level. Microautophagic vacuoles in hepatocytes of meal-fed rats, sacrificed at 11:00 or 23:00 h respectively, do not show any significant quantitative differences. However, during 12 h of starvation, the volume fraction of microautophagic vacuoles rises significantly, whereas the numerical density remains constant. Subsequently, during the second 12-h period of fasting, the volume fraction of microautophagic vacuoles remains unchanged, but the numerical density increases. Over a period of 24 h of starvation the volume fraction of the total lysosomal system does not change significantly, whereas the numerical density rises. The time-dependent changes of the macroautophagic vacuolar system correlate with the circadian, food-related variations in the protein content of individual hepatocytes from meal-fed animals. The increase in volume fraction and thereafter in number of microautophagic vacuoles, as observed during starvation, coincides with a large decrease in protein content of individual hepatocytes.
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