Frederick T. Zugibe

THE DEMONSTRATION OF THE INDIVIDUAL ACID MUCOPOLYSACCHARIDES IN HUMAN AORTAS, CORONARY ARTERIES AND CEREBRAL ARTERIES. I. THE METHODS

A histochemical technique is described for identifying the individual acid mucopolysaccharides in human aortas, coronary arteries and cerebral arteries. Identifications of the various acid mucopolysaccharides depends on treating serial sections with an extract from Flavobacterium heparinum adapted to chondroitin sulfate A, an extract from Flavobacterium adapted to heparitin sulfate, a combination of these two extracts, testicular hyaluronidase, and streptococcal hyaluronidase respectively. Following this treatment and subsequent staining with alcian blue, the identification of the individual acid mucopolysaccharides was made by comparing the treated with the untreated sections. The mechanism of these enzymes is discussed.

MUCOPOLYSACCHARIDES OF THE ARTERIAL WALL

Fixatiots: The primary objective of fixatiolu is to preserve the t(utal coltuuplemnent of mucopolysaccharides in si/ui prior to staining. Each step in prolcessitsg nuust be carefully analyzed with regard to the kituds of tnucopolysacchanides to be preserved. A variety of techniques have been Pr(uPose(l for mucopolysacchani(Ie fixations, but the majority of thuese are actually effective only its precipitating proteimss. The existence of free ansd weakly boutud muiucopolysacchanides emphasizes the muecessit-y (If usitug neagetsts directed at t-lue polyansions per se. An example of the need for fixation was report-ed by Partridge (14) who extracted a significant amusolunt of the acid polysacchsa rides frotus cartilage using water alone. Lead acetate is a very effective fixative btit the formati(lti oof lead carbonate crystals creates a probletn. The tise (If cert-aiti quaternary anumonium salts alonie or in conjunction with formalin renders the aci(l polysacchanides insoluble by complex formati()n atthe same tituse that the formalin fixes the proteus (25, 27). Although this technique is partictilarlv indicated for use with frozen-dried canhowax sect ions amid for unsfixed cryostat sections, atsv aqueous fixative redluures a component

THE DEMONSTRATION OF THE INDIVIDUAL ACID MUCOPOLYSACCHARIDES IN HUMAN AORTAS, CORONARY ARTERIES AND CEREBRAL ARTERIES. II. IDENTIFICATION AND SIGNIFICANCE WITH AGING

Observations on the type and distribution of the acid mucopolysaccharides in grossly normal aortas, coronary arteries and basilar arteries of 130 individuals ranging from fetuses to 70 years of age utilizing new histochemical and histological methods, are summarized in Table One. Hyaluronic acid is present in the intima of the aortas of individuals ranging from fetuses to 6 years of age, in a variable number of intimas of the coronary arteries without regard to age, and in an occasional intima in the aorta of adults. Hyaluronic acid is also present in medias of the aortas (decreasing with age) and in a variable number of medias of the coronary arteries without regard to age. Hyaluronic acid is absent, irrespective of age, in the area of the internal elastic membrane of all arteries studied and in all layers of the basilar artery. It has been suggested that hyaluronic acid may play a role in nutrient transport. The major component of the intimal acid mucopolysaccharides in fetuses, infants and young juveniles is chondroitin sulfate A and/or C. This acid mucopolysaccharide is chiefly associated with the fibrous elements. There is an increase in chondroitin sulfate B with a variable decrease in chondroitin sulfate A and/or C with aging, from older juveniles to adults. The acid mucopolysaccharides in the area of the internal elastic membrane in fetuses, infants and juveniles is essentially chondroitin sulfate A and/or C, and is either associated with fine collagen fibers or with the absence of collagen. The ratio of chondroitin sulfate B to chonodroitin sulfate A and/or C increased with age and with severity of fragmentation and reduplication of the internal elastic membrane. There is a concomitant increase in the ratio of coarse collagen to fine collagen. The close association of chondroitin sulfate B with coarse collagen and elastic changes suggested a reinforcement mechanism to strengthen the artery wall. The presence of collagen in these areas was verified by the use of a highly purified collagenase preparation. It is suggested that chondroitin sulphate B may act as a cross-linking agent to form coarse collagen. The intimate association between the acid mucopolysaccharides and collagen in the remainder of the media is similar to the relationship in the proximal media and area of the internal elastic membrane. Heparitin sulfate and/or heparin is present in variable amount in the intima of all arteries, regardless of age. The clearing activity of such acid mucopolysaccharides suggests that they may play a role in release of lipids from lipoproteins in their passage through the intima. After completion of this manuscript a study of the acid mucopolysaccharides of human aortas by chemical extraction was published by Kaplan and Meyer (38) who reported that there is an increase in chondroitin sulfate B with aging and increasing degree of atherosclerosis and the reverse for hyaluronate and chondroitin sulphate C. There was no correlation of total acid mucopolysaccharides with age. These findings seem to substantiate our histochemical findings that the ratio of chondroitin sulfate B to chondroitin sulfate A and/or C increases with age and severity of fragmentation and reduplication of the internal elastic membrane.

Histochemical Studies in Atherogenesis: Human Aortas

There have been many conflicting reports concerning the relationships of lipid, acid mucopolysaccharides, and elastic changes in the early pathology of atherosclerosis. Various new histochemical methods have been applied to grossly normal and early lesions of aortas of 50 individuals ranging from fetuses to 40 years of age with a view to illuminating these problems. The following paper presents our results of these studies.

A NEW ION ASSOCIATION TECHNIQUE FOR DEMONSTRATING POLYANIONS IN TISSUE SECTIONS

A new ion association technique was developed in our laboratory to demonstrate polyanions histochemically. The identification of the polyanions was effected in two steps: (1) the formation of a polyanion-quaternary ammonium complex following treatment with a quaternary salt and (2) subsequent formation of a red ion association complex with the quaternary moiety following treatment with ferric thiocyanate solution. Chemical studies indicated that the ion association complexes are cetyltrimethylammonium-hexathiocyanatoferrate (iii) and cetylpyridinium-hexathiocyanatoferrate (iii). These are red in color, very soluble in organic solvents such as chloroform, ethanol, methanol, acetone and xylene and insoluble in water.

A new carbowax method for routinely performing lipid, hematoxylin and eosin and elastic staining techniques on adjacent freeze-dried or formalin-fixed sections.

1. A freeze-drying method is presented for the simultaneous processing of a large number of tissue specimens. 2. An infiltration medium consisting of carbowax 1000 and a blocking mixture consisting of carbowax 1000 and carbowax 4000 were developed and resulted in ribbons which could be cut as fine as 2 µ. 3. Diffusion currents encountered during flotation were obviated by the use of a General Flotation Solution consisting of potassium dichromate, glycerin and gelatin for routine sections and a Special Flotation Solution consisting of carbowax and glycerin for certain fragile tissues. 4. The application of routine stains to adjacent carbowax sections included a hematoxylin and cosin technique with good cosin differentiation and sharp hematoxylin staining, an elastic-collagen technique demonstrating distinct elastic and collagen fibers and a lipid technique with minimal fat loss. 5. This carbowax method allows for correlative, multiple staining and ultramicrochemical analysis on adjacent tissue sections.

A NEW TECHNIQUE FOR THE SIMULTANEOUS DEMONSTRATION OF LIPID AND ACID POLYSACCHARIDES ON THE SAME TISSUE SECTION

A new combination staining technique which utilizes alcian blue for acid polysaccharides and oil red O in carbowax 400 for lipid is described. This technique, combined with a carbowax embedding procedre, provides a method which fulfills all of the requirements for the study of these substances in the same tissue section; i.e., (1) lipid and polysaccharide loss during the preparation of the tissue for staining is minimal, (2) the lipid stains sharply, (3) the polysaccharide stain is specific, (4) the stains contrast sharply, (5) the lipids and polysaccharides are identifiable in statu nascendi, and (6) thin adjacent sections can routinely be prepared.

A NEW ION ASSOCIATION-FRACTIONATION TECHNIQUE FOR IDENTIFYING INDIVIDUAL MUCOPOLYSACCHARIDES AND OTHER POLYANIONS IN TISSUE SECTIONS

A new histochemical ions association-fractionation technique has been developed for identifying individual mucopolysaccharides and other biological polyanions in tissue sections. This method is based on the differential precipitation of individual polyanions with quaternary ammonium salts in solutions of specific ionic concentration and the subsequent formation of red ion association complexes with the bound quaternary moieties. Hyaluronic acid, the chondroitin sulfates, heparitin sulfate, heparin and RNA were readily identified. Further differentiation was effected by incorporating acetone-dried powder extracts from Flarobacterium heparinum which has been adapted to chondroitin sulfate and heparin.

Histochemical Studies in Atherogenesis: Human Cerebral Arteries

Histochemieal studies of grossly normal and early lesions of cerebral arteries of 75 iudividuals ranging from fetuses to 70 years of age were made, utilizing newer histochemieal techniques. The following conclusions were made: There is no apparent relationship between acid mucopolysaccharides and lipids in respect to staining intensity and/or distribution in any of the age groups. In older juveniles and adults, lipid material was consistently observed intimately associated with the internal elastic membrane and other reduplicated elastic fibers, even in the absence of gross lesions. Fragmentation, fraying, and/or reduplication of the internal elastic membrane were absent in the fetuses, infants, and young juveniles. There was no apparent association between lipid and these elastic changes. In fetuses, infants, and young juveniles there was an accumulation of acid mucopolysaccharides hydrolyzable by testicular hyaluronidase, principally in the area of the proximal media, suggesting that the polysaccharide material was hyaluronic acid and/or chondroitin sulfate A and/or chondroitin sulfate C. In the adults, there was an accumulation in acid mucopolysaccharides resistant to hydrolysis by testicular hyaluronidase in the proximal media and in the intima. This accumulation in acid mucopolysaccharides corresponded to the areas of collagen increase. It was further suggested that these polysaccharides were chondroitin sulfate B and/or heparitin sulfate and/or hepa-rin. The significance of these findings was discussed.

Histochemical Studies of Human Coronary Atherogenesis: Comparison With Aortic And Cerebral Atherogenesis

A series of new histochemical techniques developed in our laboratory was employed in a study of human coronary arteries obtained from individuals ranging in age from fetal life to 70 years of age. Our conclusions are as follows: No apparent relationship exists between lipid and acid mucopolysaccharides with respect to staining intensity or distribution in any of the groups studied. The earliest morphological alteration in coronary arteries is a primary deposition of lipid. This lipid was observed within endo-thelial cells, as small subendothelial aggregates within macrophages and muscle fibers, or as extracellular deposits. The presence of lipid within the internal elastic membrane and fragmented elastic fibers was an infrequent finding. A comparison of this study with our previous histochemical studies of aorta and cerebral atherogenesis resulted in the following conclusions: At least two independent mechanisms may be responsible for the fatty changes occurring in these arteries; (a) infiltration of lipid via the endothelium and (b) degenerative changes within elastic elements. Lipid was never found in endothelial cells and muscle fibers in cerebral arteries, and was observed only occasionally in aortas. The most frequent site of lipid in cerebral arteries was within the internal elastic membrane while lipid was rarely found in the internal elastic membrane of coronary arteries and only occasionally in aortas. The ratio of chondroitin sulfate B to chon-droitin sulfate A or C increased with aging and severity of changes of elastic fibers in the area of the internal elastic membrane, with a concomitant increase in the ratio of coarse-to-fine collagen. It is suggested that this relationship indicates a reinforcement mechanism which strengthens the arterial wall at sites of fragmentation of the internal elastic membrane.