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Histochemical response to experimental skin injury in rats
Biochemical Pharmacology, Supplement, pp. 51-62. Pergamon Press. 1968. Printed in Great Britain
HISTOCHEMICAL RESPONSE TO EXPERIMENTAL SKIN INJURY IN RATS* B. R. BHUSSRY and SAROJA RAO Department of Anatomy, Georgetown University Schools of Medicine and Dentistry, Washington, D.C. Abstract--In this investigation, the sequence of histological and histochemical responses
of skin wounds was studied in rats treated with papain and with cortisone. The site of experimental injury, with adjacent uninjured skin, was excised and fixed in 10% alcoholic formalin; these specimens were processed by routine histological and histochemical procedures. Radioautographs were prepared from specimens from animals injected with proline-H 3 and S35. The microscopic observations revealed that the wounds of both the papain-treated and the cortisone-treated animals demonstrated a decrease in the concentration of acid polysaccharides. There was a delay in the differentiation and proliferation of fibroblasts and in the formation of reticular and collagen fibers, resulting in retardation of the wound healing process. In the cortisone-treated animals, a reduced uptake of proline-H a and Sa~ was noted, indicating a decreased metabolic turnover of these components in dermal wounds.
THE SEQUENCE o f changes that occur during the healing o f skin wounds depends on the regenerative capacity o f the injured tissues. The basic determinants o f this biological p h e n o m e n o n are related to the metabolism and response of the connective tissue after injury. Various agents 1 which are k n o w n to influence the connective tissue components m a y be utilized under experimental conditions for assessing the metabolism o f normal and wounded skin. Since papain 2, a and cortisonO, s alter the dermal connective tissue in experimental animals, the role o f these two agents in w o u n d healing was of particular interest in this investigation. T h o m a s o demonstrated that intravenous administration o f papain appeared to liberate the acid polysaccharides from the rabbit ear cartilage within 2 A 48 hr. Spicer and Bryant 7 confirmed these findings and showed a diffusion o f acid polysaccharides from cartilage into the surrounding tissues and blood vessels. Irving and R o n n i n g 8 reported severe alterations o f the epiphyseal cartilages in rats after i.p. injection o f papain. We z observed a decrease in the concentration o f acid polysaceharides in the intact skin o f papain-treated rats and a delayed formation and maturation o f collagen in dermal wounds. H o u c k and Patel a showed a significant loss o f insoluble collagen from the rat dermis after administration o f activated papain. These investigations suggest that papain m a y influence the fibrillar elements as well as the g r o u n d substance of dermal connective tissue. * Supported by research grant NONR(G)00029 from the Office of Naval Research and by contract no. DA49-193-MD(2414) from the Department of the Army. 51
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B . R . BHUSSRY and SAROJA RAO
The relation of cortisone to connective tissue morphology and metabolism has been extensively investigated (excellent reviews have been published by AsboeHansen 4 and by PinterS). Cortisone and hydrocortisone are generally considered to delay the formation of granulation tissue after injury. 9, 10 Howes et al. 1~ demonstrated a decrease in the mucopolysaccharide content and retardation of granulation tissue formation in wounds in cortisone-treated rats and rabbits. On the basis of radioautographs made after administration of SaS, Layton 12 suggested that cortisone inhibits the synthesis of the chondroitin sulfate necessary for the metabolism of connective tissue intercellular substances, resulting in delayed healing of wounds. Clarke and Umbreit la confirmed these findings but were doubtful whether this was primarily due to the effect of cortisone. Upton and Coon 14 concluded that adrenal hormones did not influence wound healing in either normal or scorbutic guinea pigs. Sobel et al. 15 reported that administration of cortisone to rats resulted in a loss of dermal hexosamine but had no effect on collagen content, but Sethi et al. 16 observed a decrease of both dermal hexosamine and hydroxyproline in rats treated with cortisone. Houck and Jacob 17 demonstrated a decrease in the dermal concentrations of hexosamine and nitrogen in young rats given a single dose of cortisol. Castor ~8 reported that, in ~,itro, fibroblasts synthesized less hyaluronic acid in the presence of cortisone. It is obvious that papain (a proteolytic enzyme) and cortisone (a hormone) are both capable of influencing the metabolism of connective tissue components, resulting eventually in retardation of the healing of wounds. Whether the series of events leading to delayed collagen formation and maturation are similar in both cases is not well understood. This investigation was therefore designed to compare the effects of papain and of cortisone on the sequence of histological and histochemical responses in experimental skin wounds in rats.
MATERIALS AND METHODS Protocol
Adult albino rats, weighing 150-200 g, were divided into three groups. The dorsal skin was cleaned and shaved along the midline, and a circular experimental wound, 1 cm in dia. was excised down to the subpannicular tissue. The boundaries of the skin wounds, approximately the same in all rats, were delineated by the cut surfaces of the epidermis and the dermis. The wound floor was formed by the deep layer of the fascial sheath of the panniculus carnosus. The cut edges of the muscle could always be observed microscopically at either end of the wound floor. After injury, the experimental animals were treated with either papain or hydrocortisone. Group 1. Papain-treated animals. Three days after creation of the wound 24 rats were given a single i.p. injection of activated crude papain in distilled water (20 mg/kg body wt. in 0"5 ml). Twelve untreated animals were used as controls. Groups of 6 rats (4 papain-treated and 2 controls) each were sacrificed at days 1, 2, 4, 6, 8, and 10 after administration of papain (i.e., 4, 5, 7, 9, 11, and 13 days after injury). Group 2. Cortisone-treated animals. Daily doses (3 mg/kg body wt.) of an aqueous suspension of hydrocortisone acetate (Cortril) were given s.c. to 56 rats for 7 days after injury. Twenty-eight animals, used as controls for this group, received an equivalent volume of isotonic saline. At days 4 and 7 after wounding, groups of 6 rats (4 cortisone-treated and 2 controls) each were sacrificed. On day 7, half of the
Models and methods used in the study of inflammation
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remaining cortisone-treated rats (24) and control animals (12) were given proline-H 3 (5 f~c/g body wt.) and the other half received Na2S3~O4 (5/zc/g body wt.), both i.p. Groups of 6 rats (4 cortisone-treated and 2 controls) were sacrificed at 1, 1, 6, and 24 hr and 4 and 7 days later. Group 3. Cortisone pretreatment. Twelve rats received daily doses of hydrocortisone (3 mg/kg body wt.) beginning 7 days prior to wounding. This was continued for 7 days after injury, at which time all the experimental animals and their six corresponding controls were sacrificed.
Histological and histoehemical procedures The area of experimental injury with the adjacent uninjured skin was excised from all animals at the predetermined sacrifice schedule. Specimens of normal skin were also taken from the anterior dorsal region, distant from the site of injury. All tissues were fixed in 1 0 ~ alcoholic formalin, processed by routine histological procedures, and embedded in paraffin. Serial sections were cut at 6-8 t~ thickness; some were stained initially with hematoxylin and eosin for routine observations. To evaluate specific changes in the connective tissue components, additional sections were stained as follows: van Gieson stain for collagen fibers, BielschowskyoFoot silver stain for reticular fibers, periodic acid-Schiff (PAS) stain for glycogen and glycoproteins, and Mowry's colloidal iron-PAS procedure for acid and neutral mucopolysaccharides. Enzymatic digestions with diastase, hyaluronidase, and neuroaminidase (sialidase) prior to staining were always performed on adjacent sections. This technique permitted an immediate comparison of enzyme-treated and untreated areas on the same slide and facilitated evaluation of a procedure. Since the interpretation of microscopic findings in this study were based upon the intensity of staining reactions in various parts of the wounds, the sections from experimental and control animals were stained simultaneously in a single batch of stain in the same staining rack and were inserted and removed from the stains at the same time.
Preparation of radioautographs The sections were deparaffinized in xylene and hydrated in descending alcohols. Under darkroom conditions, with a 15 W bulb and Wratten No. 2 filter at a distance of 4 ft, the slides were dipped in Kodak NTB-2 nuclear track emulsion warmed to 40 ° in a water bath. 19 The coated slides were dried, placed in microscope slide boxes containing a package of Drierite, and sealed with light-proof tape. They were then placed inside light-proof cardboard boxes similarly sealed and stored in a refrigerator at 4 °. Sections from animals receiving proline-H 3 were removed after 3 weeks of exposure while those given NazS3504 were removed after 5 weeks of exposure. The slides were warmed to room temperature and developed in a fresh solution of Kodak D-19 developer at 20 ° for 1 min, washed in running tap water for 2 rain, and fixed in Kodak fixer with hardener for 3 rain. They were then washed in running tap water for 15 rain. Some of these sections were stained with Harris's hematoxylin for the differentiation of cells and others were stained with the van Gieson stain for collagen fibers. OBSERVATIONS No significant histological or histochemical differences could be observed in th wounds of experimental and control animals during the first 24 hr. Within a few
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Fl(~. 2. Effect of papain. Sections of collagen. (a) Comrol. (Van Gieson; Same section as in (a): P Prussian Same section as
skin wounds 9 days after injury: F libroblast; N ne~ 400). (b) After papain treatment. (Van Gieson; 400). (c) blue-positive material. (Mowry's colloidal iron; 400). (dl in (b). (Mowry's colloidal iron; 4001.
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FiG. 3. Effect of cortisone. Sections of 7-day-old skin wounds. (a) Control. (Van Gieson: 400). (b) From cortisone-treated rat. (Van Gieson; 400). (c) Same section as in (a). (Mowry's colloidal iron; 400). (d) Same section as in (b). (Mowry's colloidal iron; 400).
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[:Jc;. 4. Effect of cortisone. Sections of 7-day-old skin wounds. Le/t. Control. (Silver stain, R~i~,ht. From cortisone-treated rat. (Silver stain : 400).
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FJ•. 5. Effect of coitisone. Sections of 14-day-old skin wounds, (a) Control. (Van Gieson; 400). (b) From cortisone-treated rat. (Van Gieson; 400). (c) Same section as in (a). (Mowry's colloidal iron; 4001. (d) Same section as in (b). (Mowry's colloidal iron; 400).
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Fie;. 6. Effect of cortisone. R a d i o g r a p h s of 7-day-old w o u n d s after labeling with proline-H:L (a) ( ' o n trol, 1 hr. ( H e m a t o x y l i n ; • 400). (b) F r o m cortisone-treated animal, I hr. ( H e m a t o x y l i n ; 400). (c) Control, 6 hr. (Vall Gieson ; • 400). (d) F r o m cortisone-treated animal, 6 hr. ( Hematoxylin ; 400).
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FIG. 7. Effect of cortisone. Radioaulographs of 7-day-old wounds after labeling with proline-H;L (a) Control, 24 hr. (Van Gieson; 400). (b) From cortisone-treated animal, 24 hr. (Hematoxylin; • 4001. (c) Control, 7 days. (Van Gieson: 400). (d) From cortisone-treated animal, 7 days. (Hemaioxylin ; 400).
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Fi¢;. 8. Effect of cortisone. R a d i o a u t o g r a p h s of 7-day-old w o u n d s after labeling with S :~.~.(a) Control, I hr. ( H e m a t o x y l i n : • 400). Ib) F r o m cortisone-treated animal, I hr. ( H e m a t o x y l i n : 400). ~c) ('ontrol, 6 hr. (Hen~atoxylin: • 400). (d) F r o m cortisone-treated animal. 6 hr. CHematoxylin; 400).
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t:1¢+i. 9. Effect of cortisone. RadJoautographs of ?-day-old wounds after labeling with S :~,': M mast cell. (a) Control, 24 hr. (Hematoxylin: 400). (b) From cortisone-treated animal, 24 hr. (Hematoxyfin: 400). (c) Control, 7 days. {Hematoxylin~ 400). (d) From corfisone-trealed animal, 7 days. (Hematoxylin : 400).
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t-t[;. I0. EI~'ccl of" cortistm¢ r)rctrcalmcnl. Sc¢lions o1 7-day-old skin wouind~,. (a) Van (~icscm ; (b) Mo~ry's colloidal iron: 400.
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FIG. I. Effect of papain. Sections of 4-day-old skin wounds: C clot; E epithelium; I inflammatory cells; U : uninjured skin; W wound. (a) Control. (Van Gieson; < 400). (b) After papain treatment (24 hours). (Van Gieson; 400.) (c) Same section as (a). (Mowry's colloidal iron; 400). (dl Same section as (b). (Mowry's colloidal iron; . 400).
Bio-Sltpp. J'ucitt<~ pak, e 54
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B.R. BHUSSRYand SAROJARAO
hours, all wounds contained an exudate consisting of fibrin, various types of leukocytes (particularly neutrophils), erythrocytes, and some macrophages. In some wounds one could see swollen masses of collagen undergoing necrosis. Beginning on the second day, there was a well-formed clot beneath which the dense band 2° of neutrophils (polyband) was evident. The underlying zone of fibrin network was well organized. Dispersed in the interstices of fibrin were the polymorphonuclear leukocytes, in various stages of disintegration, and an increased number of mononuclear cells and macrophages. The epithelial proliferation and migration was initiated at the edge of the wound under the polyband and proceeded towards the center of the injury. The base of the crater was formed by the subp~mnicular connective tissue and contained large numbers of dilated blood vessels, leukocytes, and many new fibroblasts. At this stage, the wounds of the control and experimental animals also appeared to be generally similar. Beginning at 3-4 days after injury, significant differences in the healing patterns were observed. Furthermore, it became obvious that different portions of the wound consistently demonstrated variations in histological and histochemical patterns. It was therefore considered erroneous to compare the events at the edge of the wound in one specimen with those at the base of the wound in another. Because the greatest activity was noted in the well-demarcated area formed by the lateral junction of the injured and uninjured skin, this paper will be restricted to a comparison of the healing process in this zone (the edge of the wound) in the experimental and control animals.
Papaht-treated anhlzals Beginning at 24 hr after papain treatment, there was a generalized decrease in the eosinophilic staining of the dermis of the uninjured skin. Although there was no significant difference in the PAS staining reaction of the uninjured skin from control and from papain-treated animals, there was a gradual but definite decrease in the concentration of colloidal-iron-binding material (acid polysaccharides) in the ground substance in the latter. There was also a considerable degranulation of the mast cells. At 8 days after papain treatment, the components of the uninjured skin appeared normal in the histological and histochemical staining reactions, suggesting that the initial decrease of the acid polysaccharide concentrations lasted for a short period after the papain treatment. The earliest histological and histochemical responses in the wounds of papaintreated animals were observed within 24 hr of papain administration (Figs. 1(a) and (b)). The connective tissue portion at the edge of the wound demonstrated large amounts of edema with many inflammatory cells, and the adjacent uninjured collagen appeared to have lost its discrete bundle formation. Patches of swollen collagen undergoing necrosis were observed. The exudate was poorly organized, with a considerable shrinkage of the crater, and no distinct fibroblasts were evident. The epithelial proliferation and migration was not affected. The control wounds showed a well-organized exudate covered with actively proliferating layers of epithelial cells and a few newly differentiated fibroblasts. Sections stained by Mowry's procedure (Figs. l(c) and (d)) demonstrated an intensely positive PAS reaction of the inflammatory exudate, with scattered islands of Prussian blue-positive material. The area of injury in the papain-treated animals showed a profound depletion of the colloidaliron-binding and the PAS-positive substances. Enzymatic digestions with diastase
Models and methods used in the study of inflammation
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did not remove the magenta-colored PAS-positive substances from the area of injury, while hyaluronidase digestion removed most of the Prussian-blue-stained material from experimental and control specimens. This suggests the possibility that the nature of the colloidal-iron-binding and PAS-positive substances in both specimens was similar but there was considerably less of it in the tissues of the papaintreated animals. At 48 hr after papain treatment, there was a significant increase in the inflammatory cells; the wounds of control animals demonstrated an increased fibroblastic activity in a well-organized granulation tissue. Occasional reticular fibers were also seen. The colloidal-iron-binding material was almost gone from the area of injury. Since some of this material could be seen in the tissues and blood vessels of the subcutaneous connective tissue, there may have been release of the acid polysaccharides into the circulation and tissues. Although there was no fibroblastic activity at the edge of the wound, an occasional fibroblast was apparent in the center of the wound but these cells were not as well differentiated as those in the controls. At 4-6 days after papain treatment (7-9 days after injury), the wounds in experimental animals showed a fairly well organized granulation tissue, a few newly differentiated fibroblasts, and large numbers of inflammatory cells scattered in the interstices of a fibrin network; the healing process in the control animals seemed well advanced. There was an increased fibroblastic activity, a fine network of reticular fibers, and an increased amount of collagen formation in the wound (Figs. 2(a) and (b)). Sections stained by Mowry's procedure (Figs. 2(c) and (d)) demonstrated a relative decrease in the amount of colloidal-iron-binding material in this area, as compared to the wounds of control animals. The colloidal-iron-binding material in both the experimental and the control wounds was removed by hyaluronidase digestion, These findings indicate a possible direct relationship between the concentration of acid polysaccharides in the wound and the rate of reticular and collagen fiber formation. The observations suggest a delayed histochemical response in the wounds of papain-treated animals, resulting in retardation of the fiber formation. In the papain-treated animals, this histological and histochemical pattern continued up to 14 days after injury, at which time the fibroblastic activity, concentration of acid polysaccharides, and formation of new reticular and collagen fibers were similar to those of 11-day-old wounds in control animals.
Cortisone-treated animals Histochemical study. At day 4 after injury, the edge of the wound in the cortisonetreated animals showed a poorly organized inflammatory exudate with relatively large numbers of inflammatory cells and negligible fibroblastic activity. Patches of swollen collagen undergoing necrosis were seen. Sections stained by Mowry's procedure demonstrated an intense PAS reaction with very little colloidal-iron-binding material, as compared to the control wounds in which a heavy concentration of Prussian-blue reaction was seen. At day 7 the size of the crater was unusually large, as compared to the control animals. The injury in the control animals showed an advanced stage of organization: the surface epithelium had proliferated for some distance toward the center of the wound and large numbers of active fibroblasts and stellate cells were present at the
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B. R. BHUSSRYand SAROJARAO
edge of the wound interspersed among the interstices of newly forming collagen and reticular fibers (Figs. 3(a) and (b)). There were only a few inflammatory cells present. Although the sequence of events in the cortisone-treated animals were similar to that in the controls, the various stages of organization and cell differentiation were retarded. An unusually large number of inflammatory cells and immature fibroblasts were evident in the granulation tissue. The fine network of collagen and reticular fibrils was poorly organized and appeared fragile (Fig. 4). Sections stained for acid polysaccharides (Figs. 3(c) and (d)) demonstrated a significant decrease in the Prussianblue reaction as compared to the controls, and the stain was diffusely distributed in the intercellular spaces. During days 9-11 after injury, there was a progressive increase in the fibroblastic activity and the formation of reticular and collagen fibers in both groups. A concomitant decrease in the inflammatory cells was evident. However, beginning at day 9, there was a sudden increase in the colloidal-iron-binding material in the wounds in cortisone-treated animals relative to the controls. During this period, fibroblastic activity and formation of collagen was most pronounced in the experimental wounds. This series of events appeared to coincide with the discontinuation of the cortisone treatment, indicating that the hormone may have suppressed the acid polysaccharide activity and thus caused delay in collagen formation. At day 14, the surface of the control wound was completely covered by keratinized epithelium; the scar tissue had been exfoliated. The edge of the wound contained large amounts of advanced collagen formation, with a tendency towards the organization of the collagen into bundles (Figs. 5(a) and (b)). Most of the cells in this area were fibroblasts; no unusual number of inflammatory cells was noted. Sections stained by Mowry's procedure (Figs. 5(c) and (d)) showed an increase in the colloidal-ironbinding material and a significant distribution of PAS-stained fibers. Although there was no remarkable difference in the amount of acid polysaccharide material in the wounds of cortisone-treated animals and controls at the same interval, there were fewer fibroblasts. The reticular and collagen fiber formation was not as well advanced. suggesting a retardation of this response in experimental wounds. Radioautographs: tritiated proline. Only 7-day-old skin wounds of untreated and cortisone-treated animals were studied. At 30 rain after administration of proline-H 3, a large number of fibroblasts in the control wounds demonstrated grains over their cytoplasm. A few grains were observed over the collagen and the extracellular spaces. By 1 hr, there was a significant increase in the intensity of the label, both in the cells and in the extracellular spaces at the edge of the wound. The organization of the granules along the newly forming collagen fibers was apparent. At 6 hr, the grain density was at a maximum over the cells and along the newly forming collagen fibers. The newly forming fibrils were labeled more heavily than the relatively mature collagen fibers in the wound. At 24 hr, the grain density was markedly decreased over the cells while over the collagen fibers it remained at the high level and progressively increased till the fourth day. By the seventh day, there was a negligible grain density over cells while the collagen fibers showed a significant label. In the wounds of cortisone-treated animals the pattern of distribution was similar to that in control animals but the grain density was relatively lower at 30 min, 1 hr, and 6 hr (Fig. 6). This suggests a decreased metabolic turnover of proline in the wounds of cortisone-treated animals. It was interesting to observe that at 24 hr the
Models and methods used in the study of inflammation
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grain counts (Figs. 7(a) and (b)) on cells and extracellular space in the wounds of cortisone-treated animals reached a maximum and were similar to those in 6-hr control wounds. Furthermore, at day 4, more grain density was evident over the new collagen fibers in these control wounds, indicating a retardation in uptake and release of proline by wounds in cortisone-treated animals. It is also possible that the amount or the process of entry of proline into the cells and passage from them to the new collagen fibrils differs from that in the control animals, thus delaying the release of this amino acid. By day 7 (Figs. 7(c) and (d)) and day 14, there was very little grain density, and no significant difference could be detected between the control and experimental animals. Radioautographs: S35. Again, only 7-day-old skin wounds of untreated and cortisone-treated animals were compared after administration of S35. In control sections, the most concentrated area of labelling with S~5 was at the edge of the wound. Although the pattern of distribution was similar in the wounds of experimental and of control animals, the grain density was relatively lower in cortisone-treated animals at 30 min, 1 hr, and 6 hr after S~5 administration (Fig. 8). The maximal intensity of the label was reached at 6 hr in control wounds and at 24 hr in wounds in cortisonetreated animals (Fig. 9). This suggested a reduced uptake and delayed release of S35 by wounds in the cortisone-treated animals. Grains could be observed along the newly forming collagen fibers beginning at 1 hr and continuing till 4 days after S3,~ administration.
Cortisone pretreatment The alterations in the wounds of animals treated with hydrocortisone for 7 days prior to injury and after injury for an additional 7 days were more pronounced than those in the wounds of animals receiving cortisone only after injury. The collagen of the uninjured skin had begun to lose the normal discrete bundle formation and appeared homogeneous. A breakdown of the fibrillar material into smaller units in some areas was apparent. In a few animals the wounds were severely shrunken and filled with colonies of inflammatory cells and exudate, while in others the organization of granulation tissue was advanced. The wounds in cortisone-pretreated animals (Fig. 10) demonstrated a few plump fibroblasts, increased capillary vascularity, and more inflammatory cells as compared with the cortisone-treated animals. A relative decrease in the colloidal-iron-binding material was observed, suggesting its role in further delaying the organization of granulation tissue, fibroblastic differentiation, and formation of fibrillar elements in the wound. DISCUSSION These observations suggest that the histological and histochemical patterns of response of skin wounds in rats are similar after i.p. administration of activated papain and of cortisone. Both agents influenced the dermal connective tissue components by decreasing the acid polysaccharide content of the ground substance and delaying differentiation of the fibroblasts and formation of collagen. This resulted in retardation of the healing process. These results suggest a direct relationship between concentration of acid polysaccharides and collagen formation
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B . R . BHUSSRY and SAROJA RAO
after injury. Since papain is a proteolytic enzyme and hydrocortisone is a hormone, it is possible that the mechanism of action of these two agents may be different.
Effect of papain Our results suggest that i.p. administration of papaiu to rats decreased the concentration of acid polysaccharides in the ground substance of normal skin and of skin wounds. This effect lasted only for a short period, 24-48 hr after injection. Although the influence of papain on skin is similar to that reported for cartilage, 6, v it appeared to act slower and for a longer period than in cartilage. It is possible that the rat is more resistant to papain action than is the rabbit. 21, 2") Furthermore, it is conceivable that the diffusion of this enzyme from the peritoneal cavity into the connective tissue of the skin may be slower than diffusion from the serum into the cartilage. The initial delay in the wound healing process in the papain-treated animals was manifested by the presence of fine, immature reticular tibers and retardation in formation of collagen fibers. This may be related to the slower rate of fibroblastic differentiation in these wounds. The effect of papain in depleting the connective tissue ground substance of acid polysaccharides, which are presumed to play an important role in the conversion of reticular fibers to collagen bundles, may partially explain this observation. Papain-induced depletion of the ground substance of its acid polysaccharide component was observed in this investigation as a decrease in the Prussian-blue reaction in experimental wounds within 24-48 hr following papain treatment. Based on the loss of S35 from rabbit cartilage following papain treatment, Tsaltas 23 and Engfeldt and Westerborn 24 concluded that chondroitin sulfate was liberated from cartilage. As the healing progressed, the intensity of the colloidal iron staining was at a maximum at 6 days after papain treatment, at which time a considerable decrease in the staining reaction of control wounds had begun. Concomitant with the increased accumulation of acid polysaccharides was the increased formation of new collagen. Sheldon and Robinson 2~ suggested a similar alteration in the amorphous component of cartilage matrix in papain-treated rabbits. There is still considerable disagreement over whether the action of papain is directly on the cellular elements or on the intercellular ground substance. It has also been postulated that papain (exogenous protease) influences the endogenous collagenase :~ which digests dermal collagen. I f this is so, then it is possible that the acid polysaccharides of the ground substance may be depolymerized and released into the circulation. 7 This may last for a short period and indirectly influence the fibroblastic activity and collagen formation, thus retarding the healing process in skin wounds.
Effect Of cortisone Although the sequence of events in the wounds of experimental and control animals were similar, there was a reduced concentration of ground substance acid polysaccharides in the wounds of cortisone-treated animals. There were fewer fibroblasts, and new collagen formation was retarded. Radioautographic observations indicated a decreased metabolic turnover and a delay in the release of tritiated proline in the cortisone-treated animals. A reduced uptake of S3~ and a possible delay in the release of this material in cortisone-treated animals were also noted. The histochemical patterns of mucopolysaccharides in healing skin wounds observed in this study are
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similar to those r e p o r t e d by D u n p h y and U d u p a . 2~ H o w e s e t aL 11 explained the r e t a r d a t i o n o f g r a n u l a t i o n tissue f o r m a t i o n in w o u n d s o f cortisone-treated animals as being due to a decrease in the m u c o p o l y s a c c h a r i d e c o n c e n t r a t i o n a n d suggested reduction o f c h o n d r o i t i n sulfate as the p r i m a r y c o m p o n e n t . F u r t h e r m o r e , the acid polysaccharides r e m a i n i n g in these w o u n d s were r e m o v e d by h y a l u r o n i d a s e a n d sialidase, suggesting the influence o f cortisone on h y a l u r o n i c acid a n d sialic acid c o m p o n e n t s o f the g r o u n d substance. These observations are in agreement with the findings o f Layton, 12 C l a r k and Umbreit, 13 K o d i c e k and Loewi 27 and Castor. is A l t h o u g h various investigators have studied the m e t a b o l i c t u r n o v e r rates o f tritiated proline in scorbutic animals, i n f o r m a t i o n r e g a r d i n g the m e t a b o l i c p a t t e r n o f this a m i n o acid in c o r t i s o n e - t r e a t e d animals is sparse. The present investigation d e m o n s t r a t e d a reduced u p t a k e o f p r o l i n e - H 3 by the fibroblasts a n d in the extracellular spaces in w o u n d s after cortisone a d m i n i s t r a t i o n . It was the a m o u n t a n d process o f entry o f p r o l i n e into cells and passage from t h e m to the new collagen fibrils which differed f r o m those in the c o n t r o l animals. It has been suggested ~4 that the sulfated m u c o p o l y s a c c h a r i d e s o f g r o u n d substance influence the fibroblastic p r o l i f e r a t i o n and activity in c u t a n e o u s lesions. The r e d u c e d uptake o f S 35 and decreased a m o u n t s o f nonsulfated m u c o p o l y s a c c h a r i d e in w o u n d s in cortisone-treated a n i m a l s r e p o r t e d in this p a p e r m a y influence the u p t a k e a n d m e t a b o l i s m o f p r o l i n e - H 3 by the fibroblasts, resulting in a r e t a r d a t i o n o f their activity - - t h a t is, f o r m a t i o n o f new collagen fibers d u r i n g the healing process. Acknowledgements--The authors are grateful to Mrs. Danuta Mikolajczyk and Miss Joanne Ruocco
for their assistance and to Mrs. Nancy Springer for her help in this study. REFERENCES 1. R. W. CHEN and R. W. POSTLETHWAIT,Surgical Sci. 1,215 (1964). 2. B. R. BHUSSRYand S. RAO, Proc. Soc. exp. Biol. Med. 115, 1010 0964). 3. J. C. HoucK and Y. M. PATEL, Am. J. Path. 45, 1045 (1964). 4. G. ASBOE-HANSEN,Am. J. Med. 26, 370 (1959). 5. K. PINTER, Am. J. reed. Sci. 240, 387 (1960). 6. L. THOMAS,J. exp. Med. 104, 245 (1956). 7. S. S. SPICERand J. H. BRYANT,Am. J. Path. 34, 61 (1958). 8. J. T. IRVINGand O. RONMNG, Archs oral Biol. 7, 357 (1962). 9. L. RAGAN,E. L. HOWLS,C. M. PLOTZ,K. MEYERand J. W. BLUNT,Proc. Soc. exp. Biol. Med. 72, 718 (1949). 10. O. M. SPAIN, N. MOLOMUTand A. HABER,Science 112, 335 (1950). 11. E. L. HOWLS,C. M. PLOTZ,J. W. BLUNTand C. RAGAN,Surgery 28, 177 (1950).
12. L. L. LAYTON,Proc. Soc. exp. Biol. Med. 76, 596 (1951). 13. I. CLARKand W. W. UMBREIT,Proc. Soc. exp. Biol. Med. 86, 558 (1954). 14. A. C. UPTON and W. W. COON, Proc. Soc. exp. Biol. Med. 77, 153 (1951). 15. H. SOBEL,S. GABAYand C. JOHNSON,Proc. Soc. exp. Biol. Med. 99, 296 (1959). 16. P. SETHI, E. R. RAMEYand J. C. HOUCK, Proc. Soc. exp. Biol. Med. 108, 74 (1961). 17. J. C. HOUCK and R. A. JAcoa, Proc. Soc. exp. Biol. Med. 112, 273 (1963). 18. C. W. CASTOR,Arthritis Rheum. 5, 105 (1962). 19. B. KOPRIWAand C. P. LEBLOND,J. Histochem. Cytochem. 10, 259 (1962). 20. G. HADFIELD,Br. J. SirrA. 50, 751 (1963).
21. 22. 23. 24.
A. HAULTHand O. WESTERBORN,J. Bone Jt Surg. B-41, 836 (1959). L. MERKOWand J. J. LALICH,J. Bone Jr Surg. A-43, 679 (1961). T. TSALTAS,J. exp. Med. 108, 507 (1958). B. ENGFELDTand O. WESTERBORN,Acta path. microbiol, scand. 49, 73 (1960).
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B . R . BHUSSRY and SAROJA RAO
25. H. SHELDON and R. A. ROmNSON, J. Biophys. Biochem. Cytol. 8, 151 (1960). 26. J. E. DUNPHV and K. N. UDUPA, New Engl. J. Med. 253, 857 (1955). 27. E. KODlCEK and G. LOEWI, Proc. R. Soc. Biol. 144, 100 (1955-56).
COMMENTS Dr. Bentley indicated that his chemical data, to be presented later in the meeting, also indicated an intimate relationship between collagen synthesis and mucopolysaccharide synthesis in the rat. This relationship described histochemically by Dr. Bhussry and chemically by Dr. Bentley was in conflict with previous ieports by Prockop et al. Dr. Houck pointed out that the latter workers studied chickens in the egg and not rats on the paw. Dr. Bentley also challenged the concept that acid mucopolysaccharides provided an essential matrix for collagen synthesis as had been described by Dunphy. Dr. Bhussry agreed that this probably was not proved. Drs. Bentley and Tsaltas then discussed with Dr. Bhussry the inadequacies of histochemical techniques coupled with enzyme digestion studies. Dr. Fainstat then indicated that these histochemical changes in skin wounds were essentially similar to those of the uterus during pregnancy which, he felt, was in essence an inflammatory response of the uterus to the growing fetus.* Dr. Bhussry concurred in these findings by Dr. Fainstat because he too had seen these pregnancyrelated alterations in the collagen and elastin components in the uterus. Dr. Rebuck reported that, in inflammation in patients with Hurler's disease, the mononuclear cells which have phagocytosed the acid mucopolysaccharides (AMPS) resemble typical Hurler's cells. He then asked if any information was available as to variations in AMPS degradation with various diseases or was it always stereotyped in pattern? Dr. Bhussry thought that most AMPS was not re-utilized in the formation of newly synthesized material. Dr. Houck added that Bollet as well as Davidson had found a lysosomal hyaluronidase which could degrade AMPS and that AMPS was insoluble only because of complexing with cationic proteins. During inflammation the various cellular proteases degraded the proteins to which the AMPS were bound and thus they usually drained away from the lesion and hence were lost to analysis.
Changes in uterine connective tissue during pregnancy (the uterine stromal CCC phenomenon) WE CAN consider, from the point of view of this conference, that one feature of the state of pregnancy is a relentless series of "injurious" stimuli to the connective tissue in the uterus. As a result, changes occu~ in uterine tissues that resemble the changes seen in the healing of dermal wounds, but the temporal relationships among the different phases of the response to injury are altered. Throughout gestation the uterine connective tissue manifests sustained responses to persisting stimuli; however, only the early changes seen in the healing of skin wounds are also seen in the rat uterus during pregnancy. 1 Thereafter, the connective tissue changes in the uterus rapidly progress beyond the earliest stages of healing, and are unmistakeably similar to those in healing dermal wounds. ~, a In a previous report 1 some of the easily observed changes in early pregnancy in the rat were termed the "uterine stromal CCC phenomenon" (Fig. 1). The letters represent Cell hypertrophy and hyperplasia, Collagen bundle splitting, fraying, disaggregation, degeneration, and disappearance (a readily apparent example of extracellular changes), and Capillary (and othe; blood vessel) proliferation and expansion. Attachment of blastocysts to the uterine epithelium and surgical trauma to a progestational endometrium in the rat are both partly responsible fol what we may call "induction of nidation media." In association with ovoimplantation, a pattern of changes in the uterine stroma unfolds. For brevity we may refer to these changes collectively as the "uterine stromal CCC phenomenon." Collagen bundle disappearance (Fig. 2a, b, c, d and Fig. 3) may be associated with lessened rigidity or increased plasticity of the tissue, thus permitting the growth of decidua (Fig. 4) and trophoblast in the implantation area while contributing to the supply of extracellular constituents used in nourishment of the growing cells. Failure of dense and rigid collagen bundles to give way to the rapid * Because of interest in these observations, Dr. Fainstat was asked to expand his remarks for inclusion at the end of this section.
HISTOCHEMICAL RESPONSE TO EXPERIMENTAL SKIN INJURY IN RATS* B. R. BHUSSRY and SAROJA RAO Department of Anatomy, Georgetown University Schools of Medicine and Dentistry, Washington, D.C. Abstract--In this investigation, the sequence of histological and histochemical responses
of skin wounds was studied in rats treated with papain and with cortisone. The site of experimental injury, with adjacent uninjured skin, was excised and fixed in 10% alcoholic formalin; these specimens were processed by routine histological and histochemical procedures. Radioautographs were prepared from specimens from animals injected with proline-H 3 and S35. The microscopic observations revealed that the wounds of both the papain-treated and the cortisone-treated animals demonstrated a decrease in the concentration of acid polysaccharides. There was a delay in the differentiation and proliferation of fibroblasts and in the formation of reticular and collagen fibers, resulting in retardation of the wound healing process. In the cortisone-treated animals, a reduced uptake of proline-H a and Sa~ was noted, indicating a decreased metabolic turnover of these components in dermal wounds.
THE SEQUENCE o f changes that occur during the healing o f skin wounds depends on the regenerative capacity o f the injured tissues. The basic determinants o f this biological p h e n o m e n o n are related to the metabolism and response of the connective tissue after injury. Various agents 1 which are k n o w n to influence the connective tissue components m a y be utilized under experimental conditions for assessing the metabolism o f normal and wounded skin. Since papain 2, a and cortisonO, s alter the dermal connective tissue in experimental animals, the role o f these two agents in w o u n d healing was of particular interest in this investigation. T h o m a s o demonstrated that intravenous administration o f papain appeared to liberate the acid polysaccharides from the rabbit ear cartilage within 2 A 48 hr. Spicer and Bryant 7 confirmed these findings and showed a diffusion o f acid polysaccharides from cartilage into the surrounding tissues and blood vessels. Irving and R o n n i n g 8 reported severe alterations o f the epiphyseal cartilages in rats after i.p. injection o f papain. We z observed a decrease in the concentration o f acid polysaceharides in the intact skin o f papain-treated rats and a delayed formation and maturation o f collagen in dermal wounds. H o u c k and Patel a showed a significant loss o f insoluble collagen from the rat dermis after administration o f activated papain. These investigations suggest that papain m a y influence the fibrillar elements as well as the g r o u n d substance of dermal connective tissue. * Supported by research grant NONR(G)00029 from the Office of Naval Research and by contract no. DA49-193-MD(2414) from the Department of the Army. 51
52
B . R . BHUSSRY and SAROJA RAO
The relation of cortisone to connective tissue morphology and metabolism has been extensively investigated (excellent reviews have been published by AsboeHansen 4 and by PinterS). Cortisone and hydrocortisone are generally considered to delay the formation of granulation tissue after injury. 9, 10 Howes et al. 1~ demonstrated a decrease in the mucopolysaccharide content and retardation of granulation tissue formation in wounds in cortisone-treated rats and rabbits. On the basis of radioautographs made after administration of SaS, Layton 12 suggested that cortisone inhibits the synthesis of the chondroitin sulfate necessary for the metabolism of connective tissue intercellular substances, resulting in delayed healing of wounds. Clarke and Umbreit la confirmed these findings but were doubtful whether this was primarily due to the effect of cortisone. Upton and Coon 14 concluded that adrenal hormones did not influence wound healing in either normal or scorbutic guinea pigs. Sobel et al. 15 reported that administration of cortisone to rats resulted in a loss of dermal hexosamine but had no effect on collagen content, but Sethi et al. 16 observed a decrease of both dermal hexosamine and hydroxyproline in rats treated with cortisone. Houck and Jacob 17 demonstrated a decrease in the dermal concentrations of hexosamine and nitrogen in young rats given a single dose of cortisol. Castor ~8 reported that, in ~,itro, fibroblasts synthesized less hyaluronic acid in the presence of cortisone. It is obvious that papain (a proteolytic enzyme) and cortisone (a hormone) are both capable of influencing the metabolism of connective tissue components, resulting eventually in retardation of the healing of wounds. Whether the series of events leading to delayed collagen formation and maturation are similar in both cases is not well understood. This investigation was therefore designed to compare the effects of papain and of cortisone on the sequence of histological and histochemical responses in experimental skin wounds in rats.
MATERIALS AND METHODS Protocol
Adult albino rats, weighing 150-200 g, were divided into three groups. The dorsal skin was cleaned and shaved along the midline, and a circular experimental wound, 1 cm in dia. was excised down to the subpannicular tissue. The boundaries of the skin wounds, approximately the same in all rats, were delineated by the cut surfaces of the epidermis and the dermis. The wound floor was formed by the deep layer of the fascial sheath of the panniculus carnosus. The cut edges of the muscle could always be observed microscopically at either end of the wound floor. After injury, the experimental animals were treated with either papain or hydrocortisone. Group 1. Papain-treated animals. Three days after creation of the wound 24 rats were given a single i.p. injection of activated crude papain in distilled water (20 mg/kg body wt. in 0"5 ml). Twelve untreated animals were used as controls. Groups of 6 rats (4 papain-treated and 2 controls) each were sacrificed at days 1, 2, 4, 6, 8, and 10 after administration of papain (i.e., 4, 5, 7, 9, 11, and 13 days after injury). Group 2. Cortisone-treated animals. Daily doses (3 mg/kg body wt.) of an aqueous suspension of hydrocortisone acetate (Cortril) were given s.c. to 56 rats for 7 days after injury. Twenty-eight animals, used as controls for this group, received an equivalent volume of isotonic saline. At days 4 and 7 after wounding, groups of 6 rats (4 cortisone-treated and 2 controls) each were sacrificed. On day 7, half of the
Models and methods used in the study of inflammation
53
remaining cortisone-treated rats (24) and control animals (12) were given proline-H 3 (5 f~c/g body wt.) and the other half received Na2S3~O4 (5/zc/g body wt.), both i.p. Groups of 6 rats (4 cortisone-treated and 2 controls) were sacrificed at 1, 1, 6, and 24 hr and 4 and 7 days later. Group 3. Cortisone pretreatment. Twelve rats received daily doses of hydrocortisone (3 mg/kg body wt.) beginning 7 days prior to wounding. This was continued for 7 days after injury, at which time all the experimental animals and their six corresponding controls were sacrificed.
Histological and histoehemical procedures The area of experimental injury with the adjacent uninjured skin was excised from all animals at the predetermined sacrifice schedule. Specimens of normal skin were also taken from the anterior dorsal region, distant from the site of injury. All tissues were fixed in 1 0 ~ alcoholic formalin, processed by routine histological procedures, and embedded in paraffin. Serial sections were cut at 6-8 t~ thickness; some were stained initially with hematoxylin and eosin for routine observations. To evaluate specific changes in the connective tissue components, additional sections were stained as follows: van Gieson stain for collagen fibers, BielschowskyoFoot silver stain for reticular fibers, periodic acid-Schiff (PAS) stain for glycogen and glycoproteins, and Mowry's colloidal iron-PAS procedure for acid and neutral mucopolysaccharides. Enzymatic digestions with diastase, hyaluronidase, and neuroaminidase (sialidase) prior to staining were always performed on adjacent sections. This technique permitted an immediate comparison of enzyme-treated and untreated areas on the same slide and facilitated evaluation of a procedure. Since the interpretation of microscopic findings in this study were based upon the intensity of staining reactions in various parts of the wounds, the sections from experimental and control animals were stained simultaneously in a single batch of stain in the same staining rack and were inserted and removed from the stains at the same time.
Preparation of radioautographs The sections were deparaffinized in xylene and hydrated in descending alcohols. Under darkroom conditions, with a 15 W bulb and Wratten No. 2 filter at a distance of 4 ft, the slides were dipped in Kodak NTB-2 nuclear track emulsion warmed to 40 ° in a water bath. 19 The coated slides were dried, placed in microscope slide boxes containing a package of Drierite, and sealed with light-proof tape. They were then placed inside light-proof cardboard boxes similarly sealed and stored in a refrigerator at 4 °. Sections from animals receiving proline-H 3 were removed after 3 weeks of exposure while those given NazS3504 were removed after 5 weeks of exposure. The slides were warmed to room temperature and developed in a fresh solution of Kodak D-19 developer at 20 ° for 1 min, washed in running tap water for 2 rain, and fixed in Kodak fixer with hardener for 3 rain. They were then washed in running tap water for 15 rain. Some of these sections were stained with Harris's hematoxylin for the differentiation of cells and others were stained with the van Gieson stain for collagen fibers. OBSERVATIONS No significant histological or histochemical differences could be observed in th wounds of experimental and control animals during the first 24 hr. Within a few
(a)
(b)
(c)
(d)
Fl(~. 2. Effect of papain. Sections of collagen. (a) Comrol. (Van Gieson; Same section as in (a): P Prussian Same section as
skin wounds 9 days after injury: F libroblast; N ne~ 400). (b) After papain treatment. (Van Gieson; 400). (c) blue-positive material. (Mowry's colloidal iron; 400). (dl in (b). (Mowry's colloidal iron; 4001.
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(b)
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FiG. 3. Effect of cortisone. Sections of 7-day-old skin wounds. (a) Control. (Van Gieson: 400). (b) From cortisone-treated rat. (Van Gieson; 400). (c) Same section as in (a). (Mowry's colloidal iron; 400). (d) Same section as in (b). (Mowry's colloidal iron; 400).
(a)
(b)
[:Jc;. 4. Effect of cortisone. Sections of 7-day-old skin wounds. Le/t. Control. (Silver stain, R~i~,ht. From cortisone-treated rat. (Silver stain : 400).
400).
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t> 5 .
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.
.
.
.
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FJ•. 5. Effect of coitisone. Sections of 14-day-old skin wounds, (a) Control. (Van Gieson; 400). (b) From cortisone-treated rat. (Van Gieson; 400). (c) Same section as in (a). (Mowry's colloidal iron; 4001. (d) Same section as in (b). (Mowry's colloidal iron; 400).
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Fie;. 6. Effect of cortisone. R a d i o g r a p h s of 7-day-old w o u n d s after labeling with proline-H:L (a) ( ' o n trol, 1 hr. ( H e m a t o x y l i n ; • 400). (b) F r o m cortisone-treated animal, I hr. ( H e m a t o x y l i n ; 400). (c) Control, 6 hr. (Vall Gieson ; • 400). (d) F r o m cortisone-treated animal, 6 hr. ( Hematoxylin ; 400).
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(b) N
\
(c)
(d)
FIG. 7. Effect of cortisone. Radioaulographs of 7-day-old wounds after labeling with proline-H;L (a) Control, 24 hr. (Van Gieson; 400). (b) From cortisone-treated animal, 24 hr. (Hematoxylin; • 4001. (c) Control, 7 days. (Van Gieson: 400). (d) From cortisone-treated animal, 7 days. (Hemaioxylin ; 400).
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(b)
(c)
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Fi¢;. 8. Effect of cortisone. R a d i o a u t o g r a p h s of 7-day-old w o u n d s after labeling with S :~.~.(a) Control, I hr. ( H e m a t o x y l i n : • 400). Ib) F r o m cortisone-treated animal, I hr. ( H e m a t o x y l i n : 400). ~c) ('ontrol, 6 hr. (Hen~atoxylin: • 400). (d) F r o m cortisone-treated animal. 6 hr. CHematoxylin; 400).
(a)
(b)
(c)
(d)
t:1¢+i. 9. Effect of cortisone. RadJoautographs of ?-day-old wounds after labeling with S :~,': M mast cell. (a) Control, 24 hr. (Hematoxylin: 400). (b) From cortisone-treated animal, 24 hr. (Hematoxyfin: 400). (c) Control, 7 days. {Hematoxylin~ 400). (d) From corfisone-trealed animal, 7 days. (Hematoxylin : 400).
(a)
(b)
t-t[;. I0. EI~'ccl of" cortistm¢ r)rctrcalmcnl. Sc¢lions o1 7-day-old skin wouind~,. (a) Van (~icscm ; (b) Mo~ry's colloidal iron: 400.
400.
(a)
(b)
(c)
(d)
FIG. I. Effect of papain. Sections of 4-day-old skin wounds: C clot; E epithelium; I inflammatory cells; U : uninjured skin; W wound. (a) Control. (Van Gieson; < 400). (b) After papain treatment (24 hours). (Van Gieson; 400.) (c) Same section as (a). (Mowry's colloidal iron; 400). (dl Same section as (b). (Mowry's colloidal iron; . 400).
Bio-Sltpp. J'ucitt<~ pak, e 54
54
B.R. BHUSSRYand SAROJARAO
hours, all wounds contained an exudate consisting of fibrin, various types of leukocytes (particularly neutrophils), erythrocytes, and some macrophages. In some wounds one could see swollen masses of collagen undergoing necrosis. Beginning on the second day, there was a well-formed clot beneath which the dense band 2° of neutrophils (polyband) was evident. The underlying zone of fibrin network was well organized. Dispersed in the interstices of fibrin were the polymorphonuclear leukocytes, in various stages of disintegration, and an increased number of mononuclear cells and macrophages. The epithelial proliferation and migration was initiated at the edge of the wound under the polyband and proceeded towards the center of the injury. The base of the crater was formed by the subp~mnicular connective tissue and contained large numbers of dilated blood vessels, leukocytes, and many new fibroblasts. At this stage, the wounds of the control and experimental animals also appeared to be generally similar. Beginning at 3-4 days after injury, significant differences in the healing patterns were observed. Furthermore, it became obvious that different portions of the wound consistently demonstrated variations in histological and histochemical patterns. It was therefore considered erroneous to compare the events at the edge of the wound in one specimen with those at the base of the wound in another. Because the greatest activity was noted in the well-demarcated area formed by the lateral junction of the injured and uninjured skin, this paper will be restricted to a comparison of the healing process in this zone (the edge of the wound) in the experimental and control animals.
Papaht-treated anhlzals Beginning at 24 hr after papain treatment, there was a generalized decrease in the eosinophilic staining of the dermis of the uninjured skin. Although there was no significant difference in the PAS staining reaction of the uninjured skin from control and from papain-treated animals, there was a gradual but definite decrease in the concentration of colloidal-iron-binding material (acid polysaccharides) in the ground substance in the latter. There was also a considerable degranulation of the mast cells. At 8 days after papain treatment, the components of the uninjured skin appeared normal in the histological and histochemical staining reactions, suggesting that the initial decrease of the acid polysaccharide concentrations lasted for a short period after the papain treatment. The earliest histological and histochemical responses in the wounds of papaintreated animals were observed within 24 hr of papain administration (Figs. 1(a) and (b)). The connective tissue portion at the edge of the wound demonstrated large amounts of edema with many inflammatory cells, and the adjacent uninjured collagen appeared to have lost its discrete bundle formation. Patches of swollen collagen undergoing necrosis were observed. The exudate was poorly organized, with a considerable shrinkage of the crater, and no distinct fibroblasts were evident. The epithelial proliferation and migration was not affected. The control wounds showed a well-organized exudate covered with actively proliferating layers of epithelial cells and a few newly differentiated fibroblasts. Sections stained by Mowry's procedure (Figs. l(c) and (d)) demonstrated an intensely positive PAS reaction of the inflammatory exudate, with scattered islands of Prussian blue-positive material. The area of injury in the papain-treated animals showed a profound depletion of the colloidaliron-binding and the PAS-positive substances. Enzymatic digestions with diastase
Models and methods used in the study of inflammation
55
did not remove the magenta-colored PAS-positive substances from the area of injury, while hyaluronidase digestion removed most of the Prussian-blue-stained material from experimental and control specimens. This suggests the possibility that the nature of the colloidal-iron-binding and PAS-positive substances in both specimens was similar but there was considerably less of it in the tissues of the papaintreated animals. At 48 hr after papain treatment, there was a significant increase in the inflammatory cells; the wounds of control animals demonstrated an increased fibroblastic activity in a well-organized granulation tissue. Occasional reticular fibers were also seen. The colloidal-iron-binding material was almost gone from the area of injury. Since some of this material could be seen in the tissues and blood vessels of the subcutaneous connective tissue, there may have been release of the acid polysaccharides into the circulation and tissues. Although there was no fibroblastic activity at the edge of the wound, an occasional fibroblast was apparent in the center of the wound but these cells were not as well differentiated as those in the controls. At 4-6 days after papain treatment (7-9 days after injury), the wounds in experimental animals showed a fairly well organized granulation tissue, a few newly differentiated fibroblasts, and large numbers of inflammatory cells scattered in the interstices of a fibrin network; the healing process in the control animals seemed well advanced. There was an increased fibroblastic activity, a fine network of reticular fibers, and an increased amount of collagen formation in the wound (Figs. 2(a) and (b)). Sections stained by Mowry's procedure (Figs. 2(c) and (d)) demonstrated a relative decrease in the amount of colloidal-iron-binding material in this area, as compared to the wounds of control animals. The colloidal-iron-binding material in both the experimental and the control wounds was removed by hyaluronidase digestion, These findings indicate a possible direct relationship between the concentration of acid polysaccharides in the wound and the rate of reticular and collagen fiber formation. The observations suggest a delayed histochemical response in the wounds of papain-treated animals, resulting in retardation of the fiber formation. In the papain-treated animals, this histological and histochemical pattern continued up to 14 days after injury, at which time the fibroblastic activity, concentration of acid polysaccharides, and formation of new reticular and collagen fibers were similar to those of 11-day-old wounds in control animals.
Cortisone-treated animals Histochemical study. At day 4 after injury, the edge of the wound in the cortisonetreated animals showed a poorly organized inflammatory exudate with relatively large numbers of inflammatory cells and negligible fibroblastic activity. Patches of swollen collagen undergoing necrosis were seen. Sections stained by Mowry's procedure demonstrated an intense PAS reaction with very little colloidal-iron-binding material, as compared to the control wounds in which a heavy concentration of Prussian-blue reaction was seen. At day 7 the size of the crater was unusually large, as compared to the control animals. The injury in the control animals showed an advanced stage of organization: the surface epithelium had proliferated for some distance toward the center of the wound and large numbers of active fibroblasts and stellate cells were present at the
56
B. R. BHUSSRYand SAROJARAO
edge of the wound interspersed among the interstices of newly forming collagen and reticular fibers (Figs. 3(a) and (b)). There were only a few inflammatory cells present. Although the sequence of events in the cortisone-treated animals were similar to that in the controls, the various stages of organization and cell differentiation were retarded. An unusually large number of inflammatory cells and immature fibroblasts were evident in the granulation tissue. The fine network of collagen and reticular fibrils was poorly organized and appeared fragile (Fig. 4). Sections stained for acid polysaccharides (Figs. 3(c) and (d)) demonstrated a significant decrease in the Prussianblue reaction as compared to the controls, and the stain was diffusely distributed in the intercellular spaces. During days 9-11 after injury, there was a progressive increase in the fibroblastic activity and the formation of reticular and collagen fibers in both groups. A concomitant decrease in the inflammatory cells was evident. However, beginning at day 9, there was a sudden increase in the colloidal-iron-binding material in the wounds in cortisone-treated animals relative to the controls. During this period, fibroblastic activity and formation of collagen was most pronounced in the experimental wounds. This series of events appeared to coincide with the discontinuation of the cortisone treatment, indicating that the hormone may have suppressed the acid polysaccharide activity and thus caused delay in collagen formation. At day 14, the surface of the control wound was completely covered by keratinized epithelium; the scar tissue had been exfoliated. The edge of the wound contained large amounts of advanced collagen formation, with a tendency towards the organization of the collagen into bundles (Figs. 5(a) and (b)). Most of the cells in this area were fibroblasts; no unusual number of inflammatory cells was noted. Sections stained by Mowry's procedure (Figs. 5(c) and (d)) showed an increase in the colloidal-ironbinding material and a significant distribution of PAS-stained fibers. Although there was no remarkable difference in the amount of acid polysaccharide material in the wounds of cortisone-treated animals and controls at the same interval, there were fewer fibroblasts. The reticular and collagen fiber formation was not as well advanced. suggesting a retardation of this response in experimental wounds. Radioautographs: tritiated proline. Only 7-day-old skin wounds of untreated and cortisone-treated animals were studied. At 30 rain after administration of proline-H 3, a large number of fibroblasts in the control wounds demonstrated grains over their cytoplasm. A few grains were observed over the collagen and the extracellular spaces. By 1 hr, there was a significant increase in the intensity of the label, both in the cells and in the extracellular spaces at the edge of the wound. The organization of the granules along the newly forming collagen fibers was apparent. At 6 hr, the grain density was at a maximum over the cells and along the newly forming collagen fibers. The newly forming fibrils were labeled more heavily than the relatively mature collagen fibers in the wound. At 24 hr, the grain density was markedly decreased over the cells while over the collagen fibers it remained at the high level and progressively increased till the fourth day. By the seventh day, there was a negligible grain density over cells while the collagen fibers showed a significant label. In the wounds of cortisone-treated animals the pattern of distribution was similar to that in control animals but the grain density was relatively lower at 30 min, 1 hr, and 6 hr (Fig. 6). This suggests a decreased metabolic turnover of proline in the wounds of cortisone-treated animals. It was interesting to observe that at 24 hr the
Models and methods used in the study of inflammation
57
grain counts (Figs. 7(a) and (b)) on cells and extracellular space in the wounds of cortisone-treated animals reached a maximum and were similar to those in 6-hr control wounds. Furthermore, at day 4, more grain density was evident over the new collagen fibers in these control wounds, indicating a retardation in uptake and release of proline by wounds in cortisone-treated animals. It is also possible that the amount or the process of entry of proline into the cells and passage from them to the new collagen fibrils differs from that in the control animals, thus delaying the release of this amino acid. By day 7 (Figs. 7(c) and (d)) and day 14, there was very little grain density, and no significant difference could be detected between the control and experimental animals. Radioautographs: S35. Again, only 7-day-old skin wounds of untreated and cortisone-treated animals were compared after administration of S35. In control sections, the most concentrated area of labelling with S~5 was at the edge of the wound. Although the pattern of distribution was similar in the wounds of experimental and of control animals, the grain density was relatively lower in cortisone-treated animals at 30 min, 1 hr, and 6 hr after S~5 administration (Fig. 8). The maximal intensity of the label was reached at 6 hr in control wounds and at 24 hr in wounds in cortisonetreated animals (Fig. 9). This suggested a reduced uptake and delayed release of S35 by wounds in the cortisone-treated animals. Grains could be observed along the newly forming collagen fibers beginning at 1 hr and continuing till 4 days after S3,~ administration.
Cortisone pretreatment The alterations in the wounds of animals treated with hydrocortisone for 7 days prior to injury and after injury for an additional 7 days were more pronounced than those in the wounds of animals receiving cortisone only after injury. The collagen of the uninjured skin had begun to lose the normal discrete bundle formation and appeared homogeneous. A breakdown of the fibrillar material into smaller units in some areas was apparent. In a few animals the wounds were severely shrunken and filled with colonies of inflammatory cells and exudate, while in others the organization of granulation tissue was advanced. The wounds in cortisone-pretreated animals (Fig. 10) demonstrated a few plump fibroblasts, increased capillary vascularity, and more inflammatory cells as compared with the cortisone-treated animals. A relative decrease in the colloidal-iron-binding material was observed, suggesting its role in further delaying the organization of granulation tissue, fibroblastic differentiation, and formation of fibrillar elements in the wound. DISCUSSION These observations suggest that the histological and histochemical patterns of response of skin wounds in rats are similar after i.p. administration of activated papain and of cortisone. Both agents influenced the dermal connective tissue components by decreasing the acid polysaccharide content of the ground substance and delaying differentiation of the fibroblasts and formation of collagen. This resulted in retardation of the healing process. These results suggest a direct relationship between concentration of acid polysaccharides and collagen formation
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B . R . BHUSSRY and SAROJA RAO
after injury. Since papain is a proteolytic enzyme and hydrocortisone is a hormone, it is possible that the mechanism of action of these two agents may be different.
Effect of papain Our results suggest that i.p. administration of papaiu to rats decreased the concentration of acid polysaccharides in the ground substance of normal skin and of skin wounds. This effect lasted only for a short period, 24-48 hr after injection. Although the influence of papain on skin is similar to that reported for cartilage, 6, v it appeared to act slower and for a longer period than in cartilage. It is possible that the rat is more resistant to papain action than is the rabbit. 21, 2") Furthermore, it is conceivable that the diffusion of this enzyme from the peritoneal cavity into the connective tissue of the skin may be slower than diffusion from the serum into the cartilage. The initial delay in the wound healing process in the papain-treated animals was manifested by the presence of fine, immature reticular tibers and retardation in formation of collagen fibers. This may be related to the slower rate of fibroblastic differentiation in these wounds. The effect of papain in depleting the connective tissue ground substance of acid polysaccharides, which are presumed to play an important role in the conversion of reticular fibers to collagen bundles, may partially explain this observation. Papain-induced depletion of the ground substance of its acid polysaccharide component was observed in this investigation as a decrease in the Prussian-blue reaction in experimental wounds within 24-48 hr following papain treatment. Based on the loss of S35 from rabbit cartilage following papain treatment, Tsaltas 23 and Engfeldt and Westerborn 24 concluded that chondroitin sulfate was liberated from cartilage. As the healing progressed, the intensity of the colloidal iron staining was at a maximum at 6 days after papain treatment, at which time a considerable decrease in the staining reaction of control wounds had begun. Concomitant with the increased accumulation of acid polysaccharides was the increased formation of new collagen. Sheldon and Robinson 2~ suggested a similar alteration in the amorphous component of cartilage matrix in papain-treated rabbits. There is still considerable disagreement over whether the action of papain is directly on the cellular elements or on the intercellular ground substance. It has also been postulated that papain (exogenous protease) influences the endogenous collagenase :~ which digests dermal collagen. I f this is so, then it is possible that the acid polysaccharides of the ground substance may be depolymerized and released into the circulation. 7 This may last for a short period and indirectly influence the fibroblastic activity and collagen formation, thus retarding the healing process in skin wounds.
Effect Of cortisone Although the sequence of events in the wounds of experimental and control animals were similar, there was a reduced concentration of ground substance acid polysaccharides in the wounds of cortisone-treated animals. There were fewer fibroblasts, and new collagen formation was retarded. Radioautographic observations indicated a decreased metabolic turnover and a delay in the release of tritiated proline in the cortisone-treated animals. A reduced uptake of S3~ and a possible delay in the release of this material in cortisone-treated animals were also noted. The histochemical patterns of mucopolysaccharides in healing skin wounds observed in this study are
Models and methods used in the study of inflammation
59
similar to those r e p o r t e d by D u n p h y and U d u p a . 2~ H o w e s e t aL 11 explained the r e t a r d a t i o n o f g r a n u l a t i o n tissue f o r m a t i o n in w o u n d s o f cortisone-treated animals as being due to a decrease in the m u c o p o l y s a c c h a r i d e c o n c e n t r a t i o n a n d suggested reduction o f c h o n d r o i t i n sulfate as the p r i m a r y c o m p o n e n t . F u r t h e r m o r e , the acid polysaccharides r e m a i n i n g in these w o u n d s were r e m o v e d by h y a l u r o n i d a s e a n d sialidase, suggesting the influence o f cortisone on h y a l u r o n i c acid a n d sialic acid c o m p o n e n t s o f the g r o u n d substance. These observations are in agreement with the findings o f Layton, 12 C l a r k and Umbreit, 13 K o d i c e k and Loewi 27 and Castor. is A l t h o u g h various investigators have studied the m e t a b o l i c t u r n o v e r rates o f tritiated proline in scorbutic animals, i n f o r m a t i o n r e g a r d i n g the m e t a b o l i c p a t t e r n o f this a m i n o acid in c o r t i s o n e - t r e a t e d animals is sparse. The present investigation d e m o n s t r a t e d a reduced u p t a k e o f p r o l i n e - H 3 by the fibroblasts a n d in the extracellular spaces in w o u n d s after cortisone a d m i n i s t r a t i o n . It was the a m o u n t a n d process o f entry o f p r o l i n e into cells and passage from t h e m to the new collagen fibrils which differed f r o m those in the c o n t r o l animals. It has been suggested ~4 that the sulfated m u c o p o l y s a c c h a r i d e s o f g r o u n d substance influence the fibroblastic p r o l i f e r a t i o n and activity in c u t a n e o u s lesions. The r e d u c e d uptake o f S 35 and decreased a m o u n t s o f nonsulfated m u c o p o l y s a c c h a r i d e in w o u n d s in cortisone-treated a n i m a l s r e p o r t e d in this p a p e r m a y influence the u p t a k e a n d m e t a b o l i s m o f p r o l i n e - H 3 by the fibroblasts, resulting in a r e t a r d a t i o n o f their activity - - t h a t is, f o r m a t i o n o f new collagen fibers d u r i n g the healing process. Acknowledgements--The authors are grateful to Mrs. Danuta Mikolajczyk and Miss Joanne Ruocco
for their assistance and to Mrs. Nancy Springer for her help in this study. REFERENCES 1. R. W. CHEN and R. W. POSTLETHWAIT,Surgical Sci. 1,215 (1964). 2. B. R. BHUSSRYand S. RAO, Proc. Soc. exp. Biol. Med. 115, 1010 0964). 3. J. C. HoucK and Y. M. PATEL, Am. J. Path. 45, 1045 (1964). 4. G. ASBOE-HANSEN,Am. J. Med. 26, 370 (1959). 5. K. PINTER, Am. J. reed. Sci. 240, 387 (1960). 6. L. THOMAS,J. exp. Med. 104, 245 (1956). 7. S. S. SPICERand J. H. BRYANT,Am. J. Path. 34, 61 (1958). 8. J. T. IRVINGand O. RONMNG, Archs oral Biol. 7, 357 (1962). 9. L. RAGAN,E. L. HOWLS,C. M. PLOTZ,K. MEYERand J. W. BLUNT,Proc. Soc. exp. Biol. Med. 72, 718 (1949). 10. O. M. SPAIN, N. MOLOMUTand A. HABER,Science 112, 335 (1950). 11. E. L. HOWLS,C. M. PLOTZ,J. W. BLUNTand C. RAGAN,Surgery 28, 177 (1950).
12. L. L. LAYTON,Proc. Soc. exp. Biol. Med. 76, 596 (1951). 13. I. CLARKand W. W. UMBREIT,Proc. Soc. exp. Biol. Med. 86, 558 (1954). 14. A. C. UPTON and W. W. COON, Proc. Soc. exp. Biol. Med. 77, 153 (1951). 15. H. SOBEL,S. GABAYand C. JOHNSON,Proc. Soc. exp. Biol. Med. 99, 296 (1959). 16. P. SETHI, E. R. RAMEYand J. C. HOUCK, Proc. Soc. exp. Biol. Med. 108, 74 (1961). 17. J. C. HOUCK and R. A. JAcoa, Proc. Soc. exp. Biol. Med. 112, 273 (1963). 18. C. W. CASTOR,Arthritis Rheum. 5, 105 (1962). 19. B. KOPRIWAand C. P. LEBLOND,J. Histochem. Cytochem. 10, 259 (1962). 20. G. HADFIELD,Br. J. SirrA. 50, 751 (1963).
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A. HAULTHand O. WESTERBORN,J. Bone Jt Surg. B-41, 836 (1959). L. MERKOWand J. J. LALICH,J. Bone Jr Surg. A-43, 679 (1961). T. TSALTAS,J. exp. Med. 108, 507 (1958). B. ENGFELDTand O. WESTERBORN,Acta path. microbiol, scand. 49, 73 (1960).
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25. H. SHELDON and R. A. ROmNSON, J. Biophys. Biochem. Cytol. 8, 151 (1960). 26. J. E. DUNPHV and K. N. UDUPA, New Engl. J. Med. 253, 857 (1955). 27. E. KODlCEK and G. LOEWI, Proc. R. Soc. Biol. 144, 100 (1955-56).
COMMENTS Dr. Bentley indicated that his chemical data, to be presented later in the meeting, also indicated an intimate relationship between collagen synthesis and mucopolysaccharide synthesis in the rat. This relationship described histochemically by Dr. Bhussry and chemically by Dr. Bentley was in conflict with previous ieports by Prockop et al. Dr. Houck pointed out that the latter workers studied chickens in the egg and not rats on the paw. Dr. Bentley also challenged the concept that acid mucopolysaccharides provided an essential matrix for collagen synthesis as had been described by Dunphy. Dr. Bhussry agreed that this probably was not proved. Drs. Bentley and Tsaltas then discussed with Dr. Bhussry the inadequacies of histochemical techniques coupled with enzyme digestion studies. Dr. Fainstat then indicated that these histochemical changes in skin wounds were essentially similar to those of the uterus during pregnancy which, he felt, was in essence an inflammatory response of the uterus to the growing fetus.* Dr. Bhussry concurred in these findings by Dr. Fainstat because he too had seen these pregnancyrelated alterations in the collagen and elastin components in the uterus. Dr. Rebuck reported that, in inflammation in patients with Hurler's disease, the mononuclear cells which have phagocytosed the acid mucopolysaccharides (AMPS) resemble typical Hurler's cells. He then asked if any information was available as to variations in AMPS degradation with various diseases or was it always stereotyped in pattern? Dr. Bhussry thought that most AMPS was not re-utilized in the formation of newly synthesized material. Dr. Houck added that Bollet as well as Davidson had found a lysosomal hyaluronidase which could degrade AMPS and that AMPS was insoluble only because of complexing with cationic proteins. During inflammation the various cellular proteases degraded the proteins to which the AMPS were bound and thus they usually drained away from the lesion and hence were lost to analysis.
Changes in uterine connective tissue during pregnancy (the uterine stromal CCC phenomenon) WE CAN consider, from the point of view of this conference, that one feature of the state of pregnancy is a relentless series of "injurious" stimuli to the connective tissue in the uterus. As a result, changes occu~ in uterine tissues that resemble the changes seen in the healing of dermal wounds, but the temporal relationships among the different phases of the response to injury are altered. Throughout gestation the uterine connective tissue manifests sustained responses to persisting stimuli; however, only the early changes seen in the healing of skin wounds are also seen in the rat uterus during pregnancy. 1 Thereafter, the connective tissue changes in the uterus rapidly progress beyond the earliest stages of healing, and are unmistakeably similar to those in healing dermal wounds. ~, a In a previous report 1 some of the easily observed changes in early pregnancy in the rat were termed the "uterine stromal CCC phenomenon" (Fig. 1). The letters represent Cell hypertrophy and hyperplasia, Collagen bundle splitting, fraying, disaggregation, degeneration, and disappearance (a readily apparent example of extracellular changes), and Capillary (and othe; blood vessel) proliferation and expansion. Attachment of blastocysts to the uterine epithelium and surgical trauma to a progestational endometrium in the rat are both partly responsible fol what we may call "induction of nidation media." In association with ovoimplantation, a pattern of changes in the uterine stroma unfolds. For brevity we may refer to these changes collectively as the "uterine stromal CCC phenomenon." Collagen bundle disappearance (Fig. 2a, b, c, d and Fig. 3) may be associated with lessened rigidity or increased plasticity of the tissue, thus permitting the growth of decidua (Fig. 4) and trophoblast in the implantation area while contributing to the supply of extracellular constituents used in nourishment of the growing cells. Failure of dense and rigid collagen bundles to give way to the rapid * Because of interest in these observations, Dr. Fainstat was asked to expand his remarks for inclusion at the end of this section.