Experimental calcification

Abstract

In 1905 1 -6 2 one of us was interested in the process of pathological calcification and indicated that in the human the condition was directly related to an antecedent fatty change in the tissues. From the studies then made it was demonstrated that the main factor leading to calcification of diseased organs was the accumulation of fatty materials in necrotic areas. The fat in these areas was liberated by the death of cells or was attracted to the areas of necrosis by the products of disintegration. The latter has been further demonstrated in the accumulation of fat by hyaline and amyloid substances.

Further proof of the relationship of fatty materials to the process of calcification is offered in a series of experiments where fats have been inoculated intravenously into rabbits. Two rabbits have been inoculated with pure olive oil and three with a mixture of cholesterin and olive oil (1-15) by the ear vein. Doses of 0.5 to 1 c.c. were given over a period of from one to eight weeks. The animals withstood the inoculations very well and showed no loss in weight. Temporary respiratory difficulty was sometimes observed for the first hour. The cholesterin mixture was usually warmed before giving.

The best results were obtained with the cholesterin-olive oil mixture after a period of four weeks. The major quantity of the oily injection was filtered out by the lung capillaries and only relatively small amounts reached the distant organs in its original condition. Reactions were recognized in the lung tissue at the end of two weeks when active proliferation of the endothelial lining of the capillaries frequently occluded their lumina or distorted the channels of the arterioles. The proliferating endothelium phagocyted the oil globules and frequently split the larger masses into small fragments which then were scattered through the protoplasm of these cells.

By staining with Sudan all the oil masses were equally colored. Sections stained with Nile blue sulphate showed the presence of neutral fats, fatty acids, and intermediate mixtures of these. With the polarizing microscope some anisotropic globules could be seen, while free cholesterin plates were also demonstrated. The majority of the fatty acid globules were intracellular. These were usually smaller than the globules of neutral fat. The fatty acid racidal was also demonstrated by the Fischler method.

Associated with the intra- and extra-cellular fatty acid globules, was found the deposition of calcium salts. The calcium became precipitated in the borders of these fatty acids and gradually irregular calcium precipitates encroached upon the center of the acid globule. This deposit took place in the protoplasm of the large phagocytic cells as well as in the fatty deposits which were lying free in the tissue or in the lymph channels. At the end of eight weeks we found the lung substance filled with these minute calcareous masses lying in areas of cell proliferation looking not unlike small tubercles. The calcareous process was also recognized in the walls of the blood vessels. In the latter, the process occurred in the deep intima and media where fatty deposits were also demonstrated. These calcium salts were recognized by staining with hematoxylin and their phosphatic radicals with silver nitrate. They could be removed by treating the tissue with hydrochloric acid.