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Morphological and Chemical Investigation of Dermal Elastic, and Collagenic Tissue During Jipiaermai1@' .1 1I'@'i @arcinogenesis - CHUNG K. MAt (From the Department of Anatomy, Washington Univerrity School of Medicine, St. Louis 10, Missouri) Before cancer develops in mouse epidermis in three zones. The first is the zone of tumor growth fluenced by methylcholanthrene, generations of devoid of elastic tissue; the second is the zone of epidermal cells live for months in a strangely a! cellular infiltration with decreased elastic tissue; tered kind of existence. Their lives are in a sense while the third is the zone of pressure character askew for they carry on despite many marked de ized by increased elastic tissue. Turning to the ficiencies and some excesses in essential materials. skins of mice, tTlesco-Stroganowa (11) induced can The features of their manner of life have been re cer by applications of coal tar and found in the re cently summarized (1). gion of epithelial hyperplasia a marked increase in Investigation of these epidermal cells is now be elastic tissue and in mast cells while about the ing supplemented by study of accompanying cancer elastic tissue was absent. It is to be noted changes in the underlying dermis. Thus, a new first that in both these tissues of humans and mice method for staining nerve endings and fibers has the investigators reported decrease in elastic tissue been devised (2, 3) and will be used. Sebaceous next to the cancer and a more distant increase, glands andhair follicles (4, 5), lodged in the der second that the conditions described were those mis, undergo interesting modifications. The lipase after cancer development. There is apparently no activity of sebaceous glands and of dermal fatty account in the literature of the sequence of altera tissues, as revealed by the Gomori reaction, is tions in dermal elastic and collagenic tissue during lost (6). Alterations observed in the number and epidermal carcinogenesis except one which was re fluorescence of mast cells (7) suggested operation ported by Bierich (12) who made a study of mouse of the spreading factor (hyaluronidase) in the skin at 5 minute, 6 hour, 24 hour, and 5 day inter breaking down of resistance to invading cancer vals after x-ray irradiation. But some useful infor cells possibly offered by hyaluronic acid in the mation is available concerning elastic and colla dermal tissue fluid. Preliminary experiments in genic tissue on nonmalignant conditions of the volving injections of hyaluronidase in the vicinity skin (13 to 24). I am grateful to Dr. E. V. Cowdry of cancer transplants seem to indicate that metas for suggestions. tasis is thereby promoted (8). On the theory that dermal mast cells may play some part in the pro MATERIAL AND METHODS duction of the hyaluronic acid other experiments histological data.—Female Swiss mice 4 weeks old are being made (9). Thus, data are being gathered were employed: (1) Five served as normal controls. on dermal modifications with a view to building up (2) Others received applications of 0.6 per cent methyl a comprehensive picture of what happens. cholanthrene in benzene delivered to their backs by a In this paper morphological and chemical modi No. 4 brush three times a week in exactly the same fications in dermal, elastic, and collagenic tissue manner that is regularly done in work by others in this group. Of these 30 mice, 5 were killed after 10, @0,30, are reported in the hope that they will supply clues 40, 50, and 60 days. (3) Still others received similar ap to the changing physical and chemical properties plications of pure benzene. Of these 10 mice, 5 were of the dermal ground substance. The literature on killed at 10 and @0days. this subject is scanty. It has been reported (10) Excised samples of skin were placed on thick paper that human skin with carcinoma is divisible into and fixed in fresh Helly's fluid. Some tissues were em @ S Aided by grants from the National Cancer Institute, the bedded in tissuemat and sections, cut @2,5thick, were C. F. Kettering Foundation, and the Women's Advertising stained in alcoholic orcein or in Weigert's resorcin Club of St. Louis. fuchsin. Others were embedded in hard paraffin and f In part fulfillmentfor the degreeof Doctor of Philosophy, sections, cut 8 ,.t thick, were colored by Mallory's stain, Washington University. the quad stain, or by Foot's method. The direction of 481 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1949 American Association for Cancer Research. 482 Cancer Research sections was transverse to the long axis of the body in to the slope of the hair, that is longitudinally to the some instances and longitudinal in others. long axis of the body and likewise to the hair fol Attempts were made quantitatively to measure licles. Part of the erector muscle is located to the alterations observed in the elastic tissue (25). A disc, right of the hair follicle in the obtuse angle formed ruled in large and small squares, inserted in the ocular between it and the epidermis. Elastic fibers en was calibrated by reference to a micrometer slide. At the magnification employed one of the small squares circle the distal portions of the hair follicles, @ covers square microns of tissue. Twenty-five of these stretch between them and along the erector small squares were included in each square. muscles. Some fibers are concentrated just be Chemical data.—Female Swiss mice of the same age neath the epidermis, especially at the attachments (4 weeks)wereusedandthemethylcholanthrenewas of these muscles, and seem, as so-catted Herx applied in the same manner. For elastin, chemical heimer's fibers, to be attached to the epidermis. analyses were made of 90 mice killed in lots of 18 on 0, Individual fibers are approximately 1.2js in thick 10, @0,30, and 60 days; for collagen, analyses were ness. made of 108 mice killed in lots of 18 on 0, 10, @0,30, 40, But the actual orientation and arrangement of and @0daysfrom the initiation of application. Six deter elastic tissue in living skin is undoubtedly some minations were made at each stage and the averages are given in the tables. what different. When still living skin is freshly cx For normal mice, the skin was removed from almost cised, it can be seen to shrink noticeably so that the entire back, while from the treated mice it was only when flattened out on a piece of stiff paper for fixa taken from the area painted with carcinogen. Pannicu tion it occupies a smaller area than it did originally lus carnosus was carefully removed from both with a in the animal. The extent of this shrinkage has scalpel. Epidermis was separated from dermis by the been measured in biopsy specimens of antecubital heat method of Baumberger, Suntzeff, and Cowdry skin by Evans, Cowdry, and Nielson (29). Skins (@6). of young persons in their thirties lost 38 to .50 per The elastin content of dermis for normal mice and mice of 10, @0,30, and 60 days, and the collagen con cent of their original areas. In mice this shrinkage tent of dermis for normal mice and mice of 10, @0,30, is more difficult to determine accurately because of 40, and 50 days after the first application of carcinogen extreme hairiness. There is also a further shrinkage were determined exactly as described by Lowry, @il1i- of at least 10 per cent owing to fixation and other gan, and Katersky (@7). Since the water content in the steps in the preparation of sections. Consequently dermis was markedly affected by carcinogen, all dermis the elastic tissue of the living animal is more samples were frozen and dried for 48 hours before ex spread out, made up of thinner fibers, and under traction with alkali in order that the results could be greater tension than Figure 1 indicates. calculated on a uniform dry weight basis. It is also to be noted that if the sections repre The percentages thus obtained sufficed to show the relative amounts of elastin and collagen. To compare sented in Figures 1 to 4 were of the thickness the elastin and collagen content in various stages of usually employed in histopathological examina carcinogenesis, an absolute measurement of elastin and tions, say 10 ;s, instead of 25 @,the amount of collagen is necessary. Hence, further work was done by elastic tissue contained in them would be much taking unit areas of dermis and weighing both before less. The advantage of these thick sections is that and after being dried at a temperature of 110°C.(23). with a binocular microscope the arrangement of Unit area of dermis was taken by means of a quadri fibers in depth can be better studied. Individual lateral razor-blade device which covers an area of @275 fibers can be traced through longer distances and square mm. (1 1 mm. X@25 mm.). The skin sample was alterations in their girth can be detected. The obtained directly from the carcass of the shaved mouse photomicrographs give only an artificial, flattened by using this instrument in as uniform a manner as pos sible to minimize distortion. The dry weight of the out picture in one plane of the fibers. dermis of a unit area of dermis was thus determined, By counting the elastic fibers in many 25 square and by multiplying it by the percentage of either elastin micra areas a rough measure was obtained of the or collagen of the corresponding stage, the absolute number of elastic fibers. The dermis selected for weight of elastin or collagen of that special stage was such determinations waslocated between hair fol obtained.

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