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Tissue in Cases of Acute Rheumatism

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Tissue in Cases of Acute Rheumatism Ann Rheum Dis: first published as 10.1136/ard.16.3.307 on 1 September 1957. Downloaded from Ann. rheum. Dis. (1957), 16, 307. HISTOCHEMICAL INVESTIGATION OF CONNECTIVE TISSUE IN CASES OF ACUTE RHEUMATISM BY A. I. STRUKOV AND G. V. ORLOVSKAYA U.S.S.R. Academy of Medical Sciences, Moscow (RECEIVED FOR PUBLICATION JULY 5, 1957) The connective tissue is the chief place where the phases: soluble (procollagen) and insoluble (colla- pathological rheumatic process takes place. The stromine). Obtained by precipitation from a changes mainly affect its intercellular components. solution, procollagen has all the properties of On the basis of this localization of the pathological collagen fibrils (of collagen): it turns red when it is lesions, rheumatism has been included among the stained by Van Gieson stain, and its roentgenogram so-called collagen diseases (Klemperer, 1950). The shows the characteristic interplane intervals of mechanism of development of the pathological 2-9 A and 11-0 A. Under the electron microscope by copyright. processes in the intercellular substance of connective it shows the same transverse bands. tissue can be understood only when there is a clear In a roentgenogram, collastromine shows no idea of the nature, structure, and correlations of ring at 2 9 A which is characteristic for collagen; collagen and the amorphous cementing substance, it has no periodicity, does not turn red with Van both of which play a principal role in the develop- Gieson stain, and shows a distinct metachromasia; ment of the rheumatic process. staining with silver reveals argyrophilic fibres. The The investigations made in recent years with the results of fractioning show collagen as at least a aid of physical, chemical, and histochemical bi-phasic system; the known features of collagen methods have considerably advanced our concepts in toto are determined by its content of procollagen. about collagen as a protein-polysaccharide complex Both procollagen and collastromine consist of http://ard.bmj.com/ (Randall, 1953; Randall, Both, Burge, Fitton- many components. Collastromine contains poly- Jackson, and Kelly, 1955; Jackson, 1954, 1957; saccharides and argyrophilic and non-argyrophilic Partridge, 1948; Orekhovich, 1952; Schmitt, Gross, proteins. The last make up the main mass of and Highberger, 1955; Gross, Highberger, and collastromine, and its amino acid content is almost Schmitt, 1955; Banga, Balo, and Szabo, 1956), and identical with that of procollagen. have helped in elucidating the processes taking place Procollagen has been separated into its protein in the connective tissue in rheumatism. However, and polysaccharide components. The polysaccha- on September 30, 2021 by guest. Protected the concept that collagen was a monophasic system rides include hyaluronic-acid-type polysaccharides has hindered researchers from correctly analysing and chondroitin-sulphate. After the separation of the observed phenomena. the polysaccharides, procollagen loses its transverse A group of Soviet investigators have obtained new bands and is precipitated in the form of amorphous data on the structure of collagen; by biochemical, threads which, however, retain the collagen structure histochemical, roentgenological, and electron- in the roentgenogram. microscopic studies they have proved that collagen The reunion of individual polysaccharides with is a multi-phasic system consisting of many proteins and the subsequent dialysis of this com- components (Tustanovsky, Seidess, Orlovskaya, pound again leads to the formation of fibrils with and Michailov, 1954; Seidess, Tustanovsky, and characteristic bands. Thus, it may be considered Orlovskaya, 1955; Orlovskaya, Seidess, and that the presence of transverse bands is connected Tustanovsky, 1956). with the combination of polysaccharide with pro- Collagen has therefore been divided into two collagen. 307 Ann Rheum Dis: first published as 10.1136/ard.16.3.307 on 1 September 1957. Downloaded from 308 ANNALS OF THE RHEUMATIC DISEASES As a result of these investigations, a diagram of cardium, epicardium, and pericardium. The tissues collagen structure was worked out (Fig. 1). were fixed in cold acetone (+40 C.) and immersed The multi-phasic character of collagen is also in paraffin. The serial sections were stained by the proved by observation of the formation of collagen usual histological and histochemical methods and in embryo. Histochemical and roentgenological were also incubated with enzymes that specifically data show that, in the early stages of the embryonic destroy definite components of the tissue. The development of connective tissue, the fibrous following methods of staining were used: haemato- structures are preformed as collastromine threads. xylin and eosin; Mallory; Van Gieson; toluidin The formation of structures of the collagen type is blue; periodic acid+leucofuchsin, Shabadash- determined by the superimposition of procollagen Hotchkiss (PAS); silver, Bielschowsky-Foote; peri- on collastromine in a later stage of development. odic acid+silver, Bielschowsky-Foote (Tustanovsky Thus, polysaccharides take part in the formation and Orlovskaya, 1953); methyl-green-pyronine, of collagen up to the development of primary Brachet (1947); staining of fibrin, Weigert; staining collagen fibrils which are of a bi-phasic structure. of elastic tissue by fuchselyne. The tissues were This concept of collagen differs fundamentally also treated with testicular hyaluronidase (purified from the accepted idea that collagen is a protein- by the method of Hahn and Tint), collagenase (K- mucopolysaccharide complex with a wholly uniform toxin-Cl. perfringens), saliva, and crystalline (mono-phasic) structure. The latter view is pre- ribonuclease. sented by Banga and others (1956), who consider that procollagen and metacollagen form a mono- Results phasic structure. A detailed study of these cases of genuine rheu- In our study of the pathological alterations of the matism, based on the application of histochemical connective tissue, we have made a more accurate analysis, as well as our previous observations, showed analysis of the composition of the altered tissue that the rheumatic process cannot be reduced to a and have examined the part played in these altera- problem of granuloma. The alteration in the con- by copyright. tions not only by the entire collagen system but also nective tissue of the heart valves, the endocardium, by its separate components. and the chordae tendineae, leading to the final Material development of sclerosis, occurs whether there is a Our observations are based on the post-mortem study granulomatous process or not, and is the end-result of fifteen cases of genuine rheumatism. Four were of the so-called "primary" sclerosis which is specific children up to 10 years old, eight were aged from 15 to for rheumatism, as was pointed out by Talalayev 20 years, and three were between 30 and 40 years old. (1932). However, Talalayev considered it to be less The duration of the disease was: 1 to 2 years in three important in the development of rheumatic lesions cases, 2 to 10 years in five cases, and over 10 years in than the "secondary sclerosis", which arises as the the remaining seven cases. In all fifteen cases the pre- outcome of Our observa- http://ard.bmj.com/ dominant manifestation of the disease was cardiac specific granulomata. disorder; in five cases, joint affections had also been tions show that, in the connective tissue of the endo- observed at the onset of the disease. cardium and partially in the myocardium, sclerosing processes develop through a peculiar primary Method diffusive disintegration of the intercellular substance. The heart was the main object of our investiga- But the dystrophic lesions of the tissue with the tions. We studied the valves, endocardium, myo- corresponding cellular reaction may or may not on September 30, 2021 by guest. Protected Collagen Procollagen Complex Collastromine Procollagen Pcolysaccharide Polysaccharides Argyrop~iilic (protein) Component Protein Polysaccharides of the Polysaccharides of the Protein of the Hyaluronic Acid Type Chondroitin-sulphate Type Collagen Type Fig. 1.-Diagram of the collagen complex. Ann Rheum Dis: first published as 10.1136/ard.16.3.307 on 1 September 1957. Downloaded from HISTOCHEMICAL INVESTIGATION OF CONNECTIVE TISSUE 309 originate from such alterations of the connective polysaccharides appear. These alterations may be tissue. In the latter case, the process of disintegra- reversible. tion bears the same character. In the next stage of connective tissue alteration The composition of the altered connective tissue ("hyalinosis" or "fibrinoid" degeneration), the has been examined at different stages in the patho- tissue is not uniform. Two kinds of tissue area are logical process. The initial change is designated as observed, fibrin being deposited in one but not in the "mucoid swelling". At this stage the collagen other. In the latter, we observed an intense meta- bundles are swollen, yellowish, and light-refracting chromasia which disappeared after treatment with when stained with haematoxylin and eosin; toluidin hyaluronidase; PAS + +. Foote's stain gives an blue stain causes a diffuse metachromasia which is intense diffuse lilac colour, and Van Gieson's stain absent during preliminary treatment with hya- gives a bright diffused red. After treatment with luronidase. This metachromasia apparently de- collagenase, the red after Van Gieson staining and pends both on the mucopolysaccharides of the the blue-red after Foote
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