Involution of keloid implants in athymic mice treated with pirfenidone or with triamcinolone.

The experimental drug pirfenidone (PFD) has been evaluated as an inhibitor of keloid proliferation and compared with triamcinolone (TAC) injections by studying the involution of active human keloid implants in athymic nude mice (nu-nu). PFD was fed to mice with keloid implants at a level of 2.75 mg/g of feed. At this level PFD had no adverse effect on the body weights of the mice. Implant weights in both PFD-fed and control mice decreased with time. The weights of the implants from the PFD group were significantly lower than those of the control implants at 60 and 90 days after implantation. Consequently PFD may cause an increased degradation and absorption of keloid tissue. The implants from the PFD mice were not significantly different histologically from the implants of the mice with corresponding implants. The chondroitin-4-sulfate (C4S) levels of the implants from PFD-fed mice were not significantly different from those of the implants from control mice. Therefore the mechanism of action of PFD apparently is not mediated by an effect on C4S metabolism. In contrast, the injections of TAC at a level that caused temporary body weight loss in the mice resulted in significant decreases in both hyaluronic acid (HA) and C4S in the keloid implants. Histologically, fibroblasts disappeared from the implants treated with TAC by 20 days after injection. At 30 days after TAC injection, HA and C4S were not detected by electrophoresis in keloid implants; only dermatan sulfate appeared to be present.

Implants of keloid and hypertrophic scars into the athymic nude mouse: changes in the glycosaminoglycans of the implants.

Studies have been made of the glycosaminoglycan (GAG) composition of implants of keloid and hypertrophic scars in athymic nude mice in order to evaluate these implants as a model for studies of causation and therapy of these abnormal human scars. Changes in weight of implanted tissue were also recorded. Pieces of keloid, hypertrophic scar or normal human skin were placed in subcutaneous pockets of athymic nude mice and left for various times up to 246 days. The uronic acid content of the scar implants did not change significantly until after 80 days when the level decreased; the uronic acid level of normal skin increased slightly during the 110 days studied. The initially high percentage of chondroitin-4-sulfate of keloid and hypertrophic scar tissue decreased in the implants (averaging a 50% decrease at 164 days for keloids and at 176 days for hypertrophic scars). The average weight of the scar implants increased slightly after implantation and then decreased when expressed either as wet or dry weight. The regression lines of weight on time indicated an average loss of 50% dry weight at 66 days for keloid implants and 68 days for hypertrophic scars. Normal human skin increased in net weight until 20 days and dry weight until 40 days and then decreased, losing about 20% of weight (either wet or dry) at 110 days. On the basis of the glycosaminoglycan changes, the model should be useful for short term studies of therapy and causation.

Implants of hypertrophic scars and keloids into the nude (athymic) mouse: viability and morphology.

Pieces of hypertrophic scars and keloids were implanted into subcutaneous pockets of nude (athymic) mice and carried for varying times up to 246 days. No rejection phenomena were observed. Microvascular anastomosis occurred between host and implant within the first several days. Remodeling of the edges of the implant occurred very early. Multiple regression analysis of volume measurements suggests there was a size reduction of the implants with time. Yet virtually all implants retained their original histotypic character regardless of the length of implantation. The nodular character typical of all hypertrophic scars and keloids was retained in every case. Histologic analysis, confirmed by transmission electron microscopy, demonstrated a sustained cellular character. The degree and magnitude of microvascular occlusion was also sustained. Use of the nude mouse for implants of hypertrophic scar or keloid sustains true viability and morphology of the lesions. This procedure shows clear potential as an experimental model for the study of these lesions.

Characterization of implants from Dupuytren's contracture tissue into the nude (athymic) mouse.

Dupuytren's contracture tissues were obtained from six patients as excess surgical material. Pieces of these tissues (a total of 38 implants) were placed into subcutaneous pockets in the suprascapular area of nude (athymic) mice. The objective was to determine whether the implant tissues would be maintained in the mouse with the characteristics of Dupuytren's tissue. The implants were removed for study at 14-179 days after implantation. Microvascular anastomosis between implant and host skin was established within the first 14 days. Histologic character and electron microscopic structure of the implants did not change during the course of the study. The implants became reduced in size with time. However, neither the spatial pattern of collagen nor the appearance of fibroblast cells changed. The original high levels of chondroitin-4-sulfate were significantly decreased in the 66- to 179-day postimplantation group, but were not significantly different from the values for normal fascial bands. The hyaluronic acid of the implants increased significantly with time of implantation, but never reached the level found in the normal fascial bands. The use of implants into nude mice may be useful for further experimental studies of Dupuytren's contracture.

Zinc and ethanol: dietary interrelationships in pregnant rats.

Pregnant rats were fed three different liquid diets with ethanol providing 33.3, 35.6 and 50.8% of the total calories. The 35.6 and 50.8% ethanol diets, containing egg white as a source of protein, were zinc deficient and were fed to two groups of rats without zinc supplementation. At each level of ethanol, one additional group was supplemented with a moderate level of zinc and another group with a high level of zinc. The 33.3% ethanol diet contained casein as a source of protein and had a high level of zinc. Each rat in the ethanol group was yoked with another rat fed a diet in which the ethanol was replaced by an isocaloric amount of carbohydrate. Records were kept of food consumed, weight gained or lost, number of pups delivered, total weight of litter, and weights of each pup. Maternal and neonatal tissues were taken for zinc and copper analyses. The rats fed ethanol diets were found to ingest the same amount of ethanol-derived calories per day regardless of diet or concentration of ethanol. On the higher level of ethanol (50.8%), the rats, therefore, ingested fewer total calories and lost weight. No pups were delivered from this group of dams. Their respective pair-fed groups, although restricted in weight gain, delivered live pups at all levels of zinc. The pups from dams fed zinc-deficient diets had lower total body zinc levels and lower liver zinc levels. Sufficient dietary zinc improved the condition of the pregnant rats and their progeny, but there were no indications that higher levels of dietary zinc resulted in further improvement.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of athymic nude mice for the study of human keloids.

Keloid tissue has been implanted in the athymic nude mouse in order to develop an experimental animal model for the study of human keloids and hypertrophic scars. Untreated keloid tissues maintained essentially the same morphological patterns and glycosaminoglycan distributions for at least 60 days after implantation in the athymic mice. Normal human skin implanted in the same way was maintained without change in glycosaminoglycan distribution or morphologic characteristics. We suggest that this model may be useful for basic research of keloids and hypertrophic scars in that it will allow studies of morphologic, biochemical and therapeutic interrelationships under controlled conditions.

Brain glycoconjugates in alcohol and narcotic research.

The glycoconjugates may be divided into three major groups: glycolipids, glycosaminoglycans and glycoproteins. The brain contains a considerable amount of glycolipid and smaller but appreciable amounts of glycoproteins and glycosaminoglycans. The alterations of the brain glucoconjugates found in conditions which affect brain function have not been extensively studied. Alcohol intoxication has been reported to increase the ganglioside content of rat brain. On the other hand lowered gangliosides and glycoproteins have been noted in brain tissue of a human alcoholic. The sialic acid levels associated with gangliosides and with glycoproteins both decreased in morphine dependent rats after withdrawal of the drug. Chronic administration of lithium increased the amount of hyaluronic acid in the cerebellum of rats, but did not affect the chondroitin sulfate level. Further investigation of the glycoconjugates as affected by alcohol and other drug abuse may provide explanations for some of the changes in brain function in these conditions.

Immunoglobulins in hypertrophic scars and keloids.

Immunoglobulins A, G, and M were localized in normal skin, hypertrophic scars, keloids, and mature scars by the direct immunofluorescent antibody method. All three immunoglobulins appeared increased in the lesions above levels observed in normal skin. Extractions of the immunoglobulins from the same type of tissues also suggested an increase above levels from normal skin. The data suggest attritional leakage of several plasma proteins from the microvasculature in the lesions. No one immunoglobulin appears significantly increased in the lesions compared with others.

Hypertrophic scars and keloids: a review and new concept concerning their origin.

Hypertrophic scars and keloids are often the sequelae of deep injury to the skin of man. These lesions are characterized by excessive collage, in the form of discrete nodules, and an excess of microvessels, most of which are partially or totally occluded due to an excess of endothelial cells. The occlusion contributes to a measurable hypoxia. Hypertrophic scars and keloids contain elevated levels of fibronectin, immunoglobulins, other plasma proteins, histamine, type III collagen and chondroitin-4-sulfate. PO2 levels are lower than in normal skin and PCO2 levels are higher. Granulation tissue from deep injury contains predisposed patterns for nodule formation, excessive numbers of fibroblasts, high levels of fibronectin, demonstrates excessive synthesis of fibroblast products and microvascular occlusion. The likely candidate for stimulating excessive numbers of fibroblasts is fibrin polymer which persists in all granulation wounds. The stimulator for excessive synthesis activity is postulated to be related to a state of hypoxia. We propose that resolution of the lesions would be effected when microvascular patency is restored and PO2 levels returned to normal. Degradation of excess collagen would take place when collagenase and/or lysosomal hydrolases would be unmasked, activated or released. The etiology of the hypertrophic scar and keloid (and thus their resolution) is believed to be directly related to the quality of the microvessels and the leakage and deposition of blood products into the wound and lesion.

Microvasculature in hypertrophic scars and the effects of pressure.

The fine structure of the microvasculature was compared among eight samples of normal skin, 79 granulation tissues, 48 hypertrophic scars, 11 hypertrophic scars treated with mechanical pressure, and 13 mature scars. Increased synthesis activity is suggested in endothelial cells from granulation tissues, is less in hypertrophic scars, and low in mature scars. In hypertrophic scars most of the microvessels appear partially or completely occluded. Endothelial cell nuclei are crenated, many villous projections from the endothelial cell membranes exist on the blood side, and endothelial cell junctions are often complex, although no large gaps are observed. In all the granulation tissues studied fibrin polymer is present, occurring intraluminally and interstitially, which may be related to endothelial cell proliferation. Therapeutic mechanical pressure over 1 to 3 months effects striking changes in endothelial and perivascular satellite cells. Rented areas appear in endothelial cell cytoplasm. A few such areas were found in cases of nontreated hypertrophic scars but in no other group. Pressure-treated scars also demonstrate degenerating perivascular satellite cells, which also are observed in a few cases of mature scars but in no other group. A previously published theory that hypoxia is related to generation of the hypertrophic scar, and that pressure probably increases hypoxia, resulting in long-term focal degeneration of selective cells, appears further supported by the present findings.

Healing of myocardial infarction in animal models.

The experimental models used by investigators to study myocardial infarction have been considered as to their possible application for use in studies of the healing of myocardial infarction. The information concerning healing has also been surveyed. In general, the healing of the myocardial infarct in the dog and in the rat is by connective tissue replacement of the injured tissue resulting in a scar similar to skin scars. In the healing process, there is an early increase of glycoproteins, possibly from serum, and of hyaluronic acid in the injured tissue. Much of this is part of the general reaction to injury and may not be part of the healing process. Somewhat later (about 2-3 days in the dog) the chondroitin-4-sulfate fraction begins to rise. Collagen biosynthesis increases at the same time although this relationship is not well established. Much later (after 30 days in dog) the chondroitin-4-sulfate content of the injured tissue begins to decrease. At this time the scar is well formed. Much later (as late as 171 days) the scar in the myocardium still contains elevated amounts of chondroitin-4-sulfate. The dermatan sulfate is increased and the hyaluronic acid slightly decreased as compared to undamaged myocardium. These changes are typical in maturing scars of skin.

Electron microscopic studies of connective tissue repair after myocardial injury.

Myocardial infarcts were artificially induced in a series of dogs by ligation of the circumflex coronary artery. Infarcted areas were studied by transmission electron microscopy at varying times between 3 1/2 days to 126 days post-ligation. A granulation phase was demonstrated up to 13 days post-ligation, but fibrin and inflammatory cells were not usually seen after that. The fibroblasts showed increased rough endoplasmic reticulum and Golgi vesicles up to 30 days. Beyond that they reverted to a more quiescent morphology. Myofibroblasts were abundant at 13 days but were not found beyond 30 days. Many microvessels were occluded by excessive numbers of endothelial cells from 22 to 30 days. Beyond this period they, again, became patent. Many similarities exist between the healing stages of myocardial infarctions of dogs and deep injury to the body surface in man. It is suggested that animals form the equivalent of hypertrophic scar or keloid, but resolution of hypertrophy, or the self-limiting process, is much more rapid and efficient in animals than in man.

Urinary glycopeptides and glycosaminoglycans in children and adults as affected by age.

The glycopeptide and glycosaminoglycan excretion was studied in the urine of normal human subjects from newborns to 68 years of age. The glycopeptide and glycosaminoglycan preparations were made by digesting the urine with peoteolytic enzymes followed by removal of enzyme and undigested protein and finally freeze-drying of the supernatant. The contents of hexose, sialic acid and uronic acid were determined on these preparations. Because of the striking differences in body weight all values were expressed in terms of milligrams per 24 hours per kilogram of body weight. When corrected for body weight, all of the carbohydrate levels were significantly higher in infants and young children under 5 years of age than in older children and adults. No other significant changes with age were found after the age of 7 years. There were no significant differences between the sexes at any age.

Alteration of hypertrophic scars induced by mechanical pressure.

Hypertrophic scars and contractures may be rapidly resolved through application of pressure and forced extension. Examination of pressure-treated scars by scanning and transmission electron microscopy demonstrates a reduction in intercollagen cohesiveness and increasing numbers of vesicular fibroblasts. Assays of chrondroitin sulfate A show a decrease from the excessive levels found in untreated hypertrophic scars. It is suggested that the application of pressure increases an already present condition of hypoxia, which results in degeneration of many fibroblasts. The ratio of collagen synthesis to degradation would, then, be altered in favor of the latter, resulting in resolution of the scar.