0021-972X/90/7004-0939$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 4 Printed in U.S.A.
Somatropin and Wound Healing after Injury* H. J. C. R. BELCHERt AND H. ELLIS* Surgical Unit, Westminster Hospital, London SWl, England
trol rats on both the 6th and 14th days. The peak forces applied to their musculo-fascial and skin wounds were significantly decreased on the 6th day only. The control somatropin group had significantly stronger musculo-fascial wounds than the control placebo group on the 6th day. Somatropin administration had no effect on burned rats. It is concluded that the limited anabolic activity of somatropin in rats with normal pituitary function is abolished by injury. (J Clin Endocrinol Metab 70: 939-943,1990)
ABSTRACT. The anabolic effect of biosynthetic human GH (somatropin) has been assessed in both unburned and burned rats. Eighty rats, which had undergone standardized laparotomies, were randomly allocated to four groups (control placebo, control somatropin, burn placebo, and burn somatropin). Healing of the laparotomy wounds was assessed tensiometrically on the 6th and 14th postoperative days. Burned rats lost weight significantly and had significantly lower hemoglobin and serum albumin concentrations than con-
I
NJURY is followed by increases in resting energy expenditure and urinary nitrogen excretion (1). Failure to provide adequate nutritional support results in the rapid onset of protein-energy malnutrition, which is associated with impaired wound healing (2) and an increased morbidity and mortality in surgical patients (3). The consequences of protein-energy malnutrition can be quickly reversed by improved nutrition, but nitrogen retention is difficult to achieve in severely injured patients, and therefore, methods to either modify the metabolic response to injury or increase the efficiency of nutritional regimens are being sought (1). Biosynthetic human GH administration has recently been shown to improve nitrogen balance in postoperative patients (4, 5). A number of animal studies have also shown that it improves wound healing (6-8). It has, therefore, been suggested that biosynthetic GH may be useful adjunct to nutrition in hypercatabolic patients. It is the purpose of the present study to investigate the anabolic effects of biosynthetic human GH (somatropin) after injury by burning with particular reference to wound healing.
regulations. After a 6-day acclimatization period, during which they were individually caged and provided with 41B rat and mouse maintenance feed (Pilsbury's Ltd., Birmingham, England) ad libitum, all were anesthetised and then shaved circumferentially. A 6-cm midline laparotomy incision was made, after which the abdominal wall was repaired with a continuous over and over suture technique using 0000 monofilament nylon (Ethilon, Edinburgh, Scotland), and the skin was closed with skin clips, which were removed on the sixth postoperative day. At completion of the laparotomy, while still anesthetised, the rats were allocated by random selection to four experimental groups: CP, control rats to receive placebo (n = 16); CS, control rats to receive somatropin (n = 16); BP, burned rats to receive placebo (n = 24); and BS, burned rats to receive somatropin (n = 24). A 13 x 11-cm oval was marked on the backs of those rats randomized to be burned, and the marked areas were immersed for 30 s in water heated to 90 C. These rats then received an ip injection of 3 mL/kg- % burn Hartmann's solution (compound sodium lactate). The total burn surface area was calculated from the burn area and the body surface area (BSA) of each rat [BSA (cm2) = 10.65 x V(weight (g)2] (9). Somatropin (recombinant DNA-derived human biosynthetic human GH, Humatrope) and a placebo preparation were supplied by Lilly Research (Windlesham, Surrey). The identity of the active preparation was not known until the experiment had been completed. Somatropin/placebo was administered by sc injection at the completion of the operative procedures and then on alternate days at a dose of 0.2 mg/kg (0.1 mg/kg-day; 0.27 IU/kg-day), which was adjusted continuously for changes in weight. All rats also received a single sc injection of 10 mg/ kg Pethidine at the end of the operative procedures and thereafter were provided with approximately 100 mg/kg-day aspirin dissolved in their drinking water. The rats were housed at 20 C and provided with 85 g/kg • day
Materials and Methods The study was performed on 80 male 11-week old SpragueDawley rats which were cared for according to Home Office Received September 7, 1989. Address all correspondence and requests for reprints to: Mr. H. J. C. R. Belcher, Department of Plastic Surgery, West Norwich Hospital, Bowthorpe Road, Norwich, Norfolk, NR2 3TU England. * This work was supported by Lilly Research Ltd. This study is part of work that has been approved by London University for submission for a Mastery of Surgery degree (H.J.C.R.B.). t Blond Mclndoe Research Fellow. $ Professor of Surgery.
939
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BELCHER AND ELLIS
940
41B feed (249 Cal/kgday; 18.6% protein-derived energy), adjusted on alternate days for changes in body weight. The rats were weighed and food consumption was measured on alternate days to the nearest gram. Wound healing was assessed after 6 days in half of the rats in each group and after 14 days in the remainder. Blood was obtained under general anesthetic immediately before death, and a sample was collected for hemoglobin estimation; the remainder was centrifuged and stored at —20 C within 1 h of collection. Serum was also collected and pooled from three additional rats of identical strain and age that had not been anesthetised and had continued to receive an ad libitum intake of 41B feed during the experimental period. The serum samples were assayed for albumin by radial immunodiffusion using rabbit antirat albumin antiserum, and the results were expressed as the percentage of the normal pooled serum albumin concentration. The anterior abdominal wall was excised immediately after death. The skin and muscle were separated by sharp dissection, the sutures were removed, and using a metal template, two 1cm wide, dumbell-shaped strips were prepared for tensiometry from both the skin and muscle layers. Wound healing was assessed using an Instron 4301 tensiometer (Instron Ltd., High Wycombe, England). The specimens were extended at a rate of 20 mm/min from an initial jaw separation distance of 15 mm, and the maximum force (N/cm) was applied to a wound before disruption measured. The mean peak force was calculated for each animal from two measurements in both the skin and muscle layers. All results are presented as the mean and SE. Statistical significance, unless otherwise stated, has been assessed by either the unpaired t test or the two-tailed Mann-Whitney test according to the equivalence of variances.
JCE & M • 1990 Vol 70 • No 4
Food consumption
100
80
a*
60
O)
40 H
20-
0-2
2-4
4-6
6-8
8-10 10-12 12-14
Days FIG. 1. Mean food consumption in the four groups during the study (CP, CS, BP, and BS). *, Days 0-2, CP vs. BP and CS us. BS, P < 0.001.
Weight 450-
Results The BP and BS rats had full-thickness burns, with total burn surface areas of 18.5 ± 0.3% and 18.3 ±0.3%, respectively. One rat of the 24 in each of these groups died within the first 3 days after injury (4%). The food intake of the burned rats was significantly less than that of controls in the first 2 days after injury, but the intakes of all four groups were comparable thereafter (Fig. 1). There was no significant difference in weight among the four groups at the beginning of the study (Fig. 2). All groups lost weight on the first 2 postoperative days {P < 0.001, by paired t test). After this, the control groups gained weight (CP, P < 0.05; CS, P < 0.001; by paired t test), whereas the burned rats continued to lose weight rapidly (P < 0.001, by paired t test) and by the end of the study had lost 25.2 ± 2.2% of their initial weight. There was no significant difference between rats receiving somatropin or placebo. The mean hemoglobin concentration was significantly lower in burned rats on both the 6th and 14th days of the study compared with that in their control counter-
275"
250 8
10
12
14
Day FIG. 2. Mean weight of the four groups. By repeated measures analysis of variance: control vs. burned rats, P < 0.0001; placebo vs. somatropin treatment, not significant.
parts (Fig. 3). There were no changes in concentration between the 6th and 14th days in any treatment group, and there was no difference between rats receiving placebo or somatropin. The mean serum albumin concentration was close to the normal pooled serum value in the control rats on both the 6th and 14th days (Fig. 4). The burned rats had significantly lower concentrations
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SOMATROPIN AND WOUND HEALING
941
Muscle peak force
Hemoglobin CP
B
m
cs
0
BP BS
m•
CP CS BP BS
Day FlG. 3. Hemoglobin concentration on the 6th and 14th days postoperatively (mean ± SE). Day 6: CP vs. BP, P < 0.001; CS vs. BS, P < 0.05. Day 14: CP vs. BP, P < 0.05; CS vs. BS, not significant.
FIG. 5. Muscle peak force on the 6th and 14th days postoperatively (mean ± SE). • , CP vs. CS, P = 0.014. Day 6: CP vs. BP, P < 0.01; CS vs. BS, P < 0.001.
Skin peak force
Albumin
I
n •
CP CS BP BS
•
CP CS BP BS
Hi
Day FlG. 4. Serum albumin concentration on the 6th and 14th days postoperatively (mean ± SE). Days 6 and 14, CP vs. BP and CS vs. BS, P < 0.001.
than the control rats on both days. There were no differences between placebo- or somatropin-treated rats. On the 6th postoperative day, the mean peak forces applied to both skin and musculo-fascial wounds were significantly lower in the burned rats than in control rats (Figs. 5 and 6). The musculo-fascial wounds were significantly stronger in the control rats receiving somatropin than in those receiving placebo, but this difference was not evident in their skin wounds. There was no difference in the strength of either musculo-fascial or skin wounds between BP and BS rats. On the 14th day, there was no difference in wound strength among the four experimental groups.
FIG. 6. Skin peak force on the 6th and 14th days postoperatively (mean ± SE). Day 6, CP vs. BP and CS vs. BS, P < 0.05.
Discussion In the present study a 20% full-thickness burn caused rapid weight loss, despite the burned rats having comparable dietary intakes to control rats on all but the first 2 days. The rate of weight loss was similar to that in another study in which burned rats were provided with a fixed dietary intake and is due to the onset of a negative nitrogen balance (10). There was no evidence that somatropin administration caused any increase in weight over the 14 days of the study. We have found previously that somatropin fails to cause weight gain over a 5-day period in unwounded rats with normal pituitary function (11). Jorgensen and
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942
BELCHER AND ELLIS
Andreassen (8) showed that somatropin administration to rats causes increased weight gain in a dose-related manner, but the doses used exceeded that used in the present study, although the researchers failed to state the activity of their preparation. More recently, Damm Jorgensen et at. (12) showed that the administration of 0.5 IU/kg-day somatropin to normal male rats over a 90day period causes no significant weight gain. The dose of somatropin used in the present study was chosen in order to assess the effects of a clinically usable dose, as the use of higher doses is limited by their effect on glucose metabolism, particularly in burned patients (13). The administration of comparable doses of human pituitary GH and somatropin to hypophysectomized rats has been shown to result in a significant increase in the rate of growth (14-17). The dose of somatropin used in the present study is, therefore, analogous to those used in human studies by us (13) and others (4, 5), and approximates doses used for correcting hypopituitarism. There is evidence that mature rats with normal pituitary function and growth rates are relatively insensitive to the anabolic effects of exogenous GH while remaining sensitive to its effects on glucose metabolism (18). The clinical relevance of animal studies using large doses is, therefore, questionable. The fall in serum albumin in burned rats was marked and was principally due to the increase in capillary permeability that follows injury (19). There was no change in the albumin concentration after the sixth day despite the transience of this change. This probably reflects its large pool size, which limits its ability to either respond to short term changes in nutritional status (20) or recover after injury by burning, even in aggressively fed patients (13). There was no evidence that somatropin had any effect on serum albumin levels in either control or burned rats. Anemia is characteristic after injury by burning, due initially to destruction or damage to red cells by heat and, subsequently, to a shortened red cell lifespan and the unresponsiveness of bone marrow to erythropoietin (21). Although GH potentiates erythropoiesis in vitro (22), no improvement in hemoglobin level was seen in the rats receiving somatropin, but this may reflect the relative brevity of the study. The strength of both the musculo-fascial and skin wounds was decreased on the sixth postoperative day in the burned rats compared with that in controls. This finding is attributable to the presence of a number of factors that have previously been associated with impaired wound healing, including physical stress (23, 24), weight loss (25), hypoalbuminemia (26), anemia (27), and the presence of a distant source of infection (28). Despite their plurality, there was no long term reduction in the wound strengths of the burned rats. This is,
JCE & M • 1990 Vol70«No4
perhaps, surprising, but previous studies in burned animals have also shown only a short-lived reduction in wound strength, not extending beyond the fifth postoperative day (23, 29). There is now evidence that the loss of wound strength associated with stress and malnutrition is best correlated with the dietary intake at the time of healing (30, 31). Although the burned rats were clinically malnourished, they had only a transient fall in dietary intake compared with controls. The administration of somatropin resulted in a transient increase in musculo-fascial wound strength in control rats, consistent with previous studies that have shown increased breaking strengths in incised wounds and sc implanted sponges in rats receiving biosynthetic GH preparations (6-8). This was not observed in the skin wounds, but there is some indication that these are less responsive to dietary manipulation than those in muscle (25). It is thought that the growth-promoting and anabolic actions of GH are mediated by the somatomedins, principally insulin-like growth factor-I, which when administered to hypophysectomized rats is indistinguishable in its effect from GH (32). Fibroblasts can synthesize IGF-I, and there is evidence that IGF-I is required for normal fibroblast replication (33). It has been postulated that GH stimulates fibroblasts via the paracrine and/or endocrine release of somatomedins. Somatropin administration caused no increase in wound strength at any time in the burned rats. Plasma somatomedin levels fail after injury by burning (34). Furthermore, somatomedin release in response to somatropin is impaired after burn injury (11, 13). It can, therefore, be postulated that the failure of somatropin to improve wound strength in burned rats is due to an impaired somatomedin response. The present study has shown that somatropin has limited anabolic activity in rats with normal pituitary function. We have shown that somatropin has no effect in burned rats and provide further evidence that somatropin is unlikely to be effective as an anabolic agent soon after injury.
Acknowledgments We thank Cathy Godfrey for her technical assistance and Robert Beetham at the Protein Reference Unit, Westminster Hospital, for the albumin assays.
References 1. Douglas RG, Shaw JHF. Metabolic response to sepsis and trauma. Br J Surg. 1989;76:115-22. 2. Haydock DA, Hill GL. Improved wound-healing response in surgical patients receiving intravenous nutrition. Br J Surg. 1987;74:320-3. 3. Mullen JL, Buzby GP, Matthews DC, Smale BF, Rosato EF. Reduction in operative morbidity and mortality by combined preoperative and postoperative nutritional support. Ann Surg. 1980;192:604-13. 4. Ward HC, Halliday D, Sim AJW. Protein and energy metabolism
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SOMATROPIN AND WOUND HEALING
5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16.
17.
18.
with biosynthetic human growth hormone after gastrointestinal surgery. Ann Surg. 1987;206:56-61. Ponting GA, Halliday D, Teale JD, Sim AJW. Postoperative positive nitrogen balance with intravenous hyponutrition and growth hormone. Lancet. 1988;l:438-40. Hollander DM, Devereux DF, Marafino BJ, Hoppe H. Increased wound breaking strength in rats following treatment with synthetic human growth hormone. Surg Forum. 1984;35:612-4. Pessa ME, Bland KI, Sitren HS, Miller GJ, Copeland EM. Improved wound healing in tumor-bearing rats treated with perioperative synthetic human growth hormone. Surg Forum. 1985;36:6-8. Jorgensen PH, Andreassen TT. A dose-response study of the effects of biosynthetic human growth hormone on formation and strength of granulation tissue. Endocrinology. 1987;121:1637-41. San Roman F, Sanchez-Valverde MA, Bonafonte JI, SanchezValverde B. Methode de determination de la surface corporelle du rat Wistar adulte. Sci Tech Anim Lab. 1985;10:181-4. Caldwell FT. Metabolic response to thermal trauma. II. Nutritional studies with rats at two environmental temperatures. Ann Surg. l962;155:119-26. Belcher HJCR, Ellis H. An investigation of the anabolic activity of somatropin in normal and burned rats. Burns. In Press. Damm Jorgensen K, Svendsen 0, Greenough RJ, et al. Biosynthetic human growth hormone: subchronic toxicity studies in rats and monkeys. Pharmacol Toxicol. 1988; 62:329-33. Belcher HJCR, Mercer D, Judkins KC, et al. Biosynthetic human growth hormone in burned patients: a pilot study. Burns. 1989;15:99-107. Jansson JO, Albertsson-Wikland K, Eden S, et al. Effect of frequency of growth hormone administration on longitudinal bone growth and body weight in hypophysectomized rats. Acta Physiol Scand. 1982;114:261-5. Clark RG, Jansson JO, Isaksson 0, Robinson ICAF. Intravenous growth hormone: growth reponses to patterned infusions in hypophysectomized rats. J Endocrinol. 1985;104:53-61. Schoenle E, Zapf J, Hauri C, Steiner T, Froesch ER. Comparison of in vivo effects of insulin-like growth factors I and II and of growth hormone in hypophysectomised rats. Acta Endocrinol (Copenh). 1985;108:167-74. Moore JA, Rudraan CG, MacLachlan NJ, Fuller GB, Burnett B, Frane JW. Equivalent potency and pharmacokinetics of recombinant human growth hormones with or without an N-terminal methionine. Endocrinology. 1988;122:2920-6. Stred SE, Benedict MR, Kuehnling E, Richman RA. The effect of
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33. 34.
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growth hormone on growth and glucose tolerance of normal rats. Am J Dis Child. 1987;141:502-5. Carvajal HF, Linares HA, Brouhard BH. Relationship of burn size to vascular permeability changes in rats. Surg Gynaecol Obstet. 1979;149:193-202. Shetty PS, Watrasiewicz KE, Jung RT, James WPT. Rapid-turnover transport proteins: an index of subclinical protein-energy malnutrition. Lancet. 1979;2:230-2. Baar S. Anaemia of burns. Burns. 1979;6:l-8. Golde DW, Bersch N, Li CH. Growth hormone: species-specific stimulation of erythropoiesis in vitro. Science. 1977;196:1112-3. Chassin JL, McDougall HA, MacKay M, Localio SA. Effect of stress upon the healing of wounds in rats. Proc Soc Exp Biol Med. 1953;83:798-8O1. Crowley LV, Seifter E, Kriss P, Rettura G, Nakao K, Levenson SM. Effects of environmental temperature and femoral fracture on wound healing in rats. J Trauma. 1977;17:436-45. Irvin TT. Effects of malnutrition and hyperalimentation on wound healing. Surg Gynecol Obstet. 1978;146:33-7. Daly JM, Vars HM, Dudrick SJ. Effects of protein depletion on strength of colonic anastomoses. Surg Gynecol Obstet. 1972;134:15-21. Bains JW, Crawford DT, Ketcham AS. Effect of chronic anaemia on wound tensile strength: correlation with blood volume, total red blood cell volume and proteins. Ann Surg. 1966;164:243-6. De Haan BB, Ellis H, Wilks M. The role of infection in wound healing. Surg Gynecol Obstet. 1974;138:693-700. Levenson SM, Pirani CL, Braash JW, Waterman DF. The effect of thermal burns on wound healing. Surg Gynecol Obstet. l954;99:74-82. Greenhalgh DG, Gemelli RL. Is impaired wound healing caused by infection or nutritional depletion? Surgery. 1987; 102:306-12. Windsor JA, Knight GS, Hill GL. Wound-healing response in surgical patients: recent food intake is more important than nutritional status. Br J Surg. 1988;75:135-7. Schoenle E, Zapf J, Hauri C, Steiner T, Froesch ER. Comparison of in vivo effects of insulin-like growth factors I and II and of growth hormone in hypophysectomized rats. Acta Endocrinol (Copenh). 1985;108:167-74. Van Wyk JJ. The somatomedins: biological actions and physiologic control mechanisms. In: Li CH, ed. Hormonal proteins and peptides. Orlando: Academic Press; 1984;12:95-125. Coates CL, Burwell RG, Carlin SA, et al. Somatomedin activity in plasma from burned patients with observations on plasma cortisol. Burns. 1981;7:425-33.
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Vol. 70, No. 4 Printed in U.S.A.
Somatropin and Wound Healing after Injury* H. J. C. R. BELCHERt AND H. ELLIS* Surgical Unit, Westminster Hospital, London SWl, England
trol rats on both the 6th and 14th days. The peak forces applied to their musculo-fascial and skin wounds were significantly decreased on the 6th day only. The control somatropin group had significantly stronger musculo-fascial wounds than the control placebo group on the 6th day. Somatropin administration had no effect on burned rats. It is concluded that the limited anabolic activity of somatropin in rats with normal pituitary function is abolished by injury. (J Clin Endocrinol Metab 70: 939-943,1990)
ABSTRACT. The anabolic effect of biosynthetic human GH (somatropin) has been assessed in both unburned and burned rats. Eighty rats, which had undergone standardized laparotomies, were randomly allocated to four groups (control placebo, control somatropin, burn placebo, and burn somatropin). Healing of the laparotomy wounds was assessed tensiometrically on the 6th and 14th postoperative days. Burned rats lost weight significantly and had significantly lower hemoglobin and serum albumin concentrations than con-
I
NJURY is followed by increases in resting energy expenditure and urinary nitrogen excretion (1). Failure to provide adequate nutritional support results in the rapid onset of protein-energy malnutrition, which is associated with impaired wound healing (2) and an increased morbidity and mortality in surgical patients (3). The consequences of protein-energy malnutrition can be quickly reversed by improved nutrition, but nitrogen retention is difficult to achieve in severely injured patients, and therefore, methods to either modify the metabolic response to injury or increase the efficiency of nutritional regimens are being sought (1). Biosynthetic human GH administration has recently been shown to improve nitrogen balance in postoperative patients (4, 5). A number of animal studies have also shown that it improves wound healing (6-8). It has, therefore, been suggested that biosynthetic GH may be useful adjunct to nutrition in hypercatabolic patients. It is the purpose of the present study to investigate the anabolic effects of biosynthetic human GH (somatropin) after injury by burning with particular reference to wound healing.
regulations. After a 6-day acclimatization period, during which they were individually caged and provided with 41B rat and mouse maintenance feed (Pilsbury's Ltd., Birmingham, England) ad libitum, all were anesthetised and then shaved circumferentially. A 6-cm midline laparotomy incision was made, after which the abdominal wall was repaired with a continuous over and over suture technique using 0000 monofilament nylon (Ethilon, Edinburgh, Scotland), and the skin was closed with skin clips, which were removed on the sixth postoperative day. At completion of the laparotomy, while still anesthetised, the rats were allocated by random selection to four experimental groups: CP, control rats to receive placebo (n = 16); CS, control rats to receive somatropin (n = 16); BP, burned rats to receive placebo (n = 24); and BS, burned rats to receive somatropin (n = 24). A 13 x 11-cm oval was marked on the backs of those rats randomized to be burned, and the marked areas were immersed for 30 s in water heated to 90 C. These rats then received an ip injection of 3 mL/kg- % burn Hartmann's solution (compound sodium lactate). The total burn surface area was calculated from the burn area and the body surface area (BSA) of each rat [BSA (cm2) = 10.65 x V(weight (g)2] (9). Somatropin (recombinant DNA-derived human biosynthetic human GH, Humatrope) and a placebo preparation were supplied by Lilly Research (Windlesham, Surrey). The identity of the active preparation was not known until the experiment had been completed. Somatropin/placebo was administered by sc injection at the completion of the operative procedures and then on alternate days at a dose of 0.2 mg/kg (0.1 mg/kg-day; 0.27 IU/kg-day), which was adjusted continuously for changes in weight. All rats also received a single sc injection of 10 mg/ kg Pethidine at the end of the operative procedures and thereafter were provided with approximately 100 mg/kg-day aspirin dissolved in their drinking water. The rats were housed at 20 C and provided with 85 g/kg • day
Materials and Methods The study was performed on 80 male 11-week old SpragueDawley rats which were cared for according to Home Office Received September 7, 1989. Address all correspondence and requests for reprints to: Mr. H. J. C. R. Belcher, Department of Plastic Surgery, West Norwich Hospital, Bowthorpe Road, Norwich, Norfolk, NR2 3TU England. * This work was supported by Lilly Research Ltd. This study is part of work that has been approved by London University for submission for a Mastery of Surgery degree (H.J.C.R.B.). t Blond Mclndoe Research Fellow. $ Professor of Surgery.
939
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BELCHER AND ELLIS
940
41B feed (249 Cal/kgday; 18.6% protein-derived energy), adjusted on alternate days for changes in body weight. The rats were weighed and food consumption was measured on alternate days to the nearest gram. Wound healing was assessed after 6 days in half of the rats in each group and after 14 days in the remainder. Blood was obtained under general anesthetic immediately before death, and a sample was collected for hemoglobin estimation; the remainder was centrifuged and stored at —20 C within 1 h of collection. Serum was also collected and pooled from three additional rats of identical strain and age that had not been anesthetised and had continued to receive an ad libitum intake of 41B feed during the experimental period. The serum samples were assayed for albumin by radial immunodiffusion using rabbit antirat albumin antiserum, and the results were expressed as the percentage of the normal pooled serum albumin concentration. The anterior abdominal wall was excised immediately after death. The skin and muscle were separated by sharp dissection, the sutures were removed, and using a metal template, two 1cm wide, dumbell-shaped strips were prepared for tensiometry from both the skin and muscle layers. Wound healing was assessed using an Instron 4301 tensiometer (Instron Ltd., High Wycombe, England). The specimens were extended at a rate of 20 mm/min from an initial jaw separation distance of 15 mm, and the maximum force (N/cm) was applied to a wound before disruption measured. The mean peak force was calculated for each animal from two measurements in both the skin and muscle layers. All results are presented as the mean and SE. Statistical significance, unless otherwise stated, has been assessed by either the unpaired t test or the two-tailed Mann-Whitney test according to the equivalence of variances.
JCE & M • 1990 Vol 70 • No 4
Food consumption
100
80
a*
60
O)
40 H
20-
0-2
2-4
4-6
6-8
8-10 10-12 12-14
Days FIG. 1. Mean food consumption in the four groups during the study (CP, CS, BP, and BS). *, Days 0-2, CP vs. BP and CS us. BS, P < 0.001.
Weight 450-
Results The BP and BS rats had full-thickness burns, with total burn surface areas of 18.5 ± 0.3% and 18.3 ±0.3%, respectively. One rat of the 24 in each of these groups died within the first 3 days after injury (4%). The food intake of the burned rats was significantly less than that of controls in the first 2 days after injury, but the intakes of all four groups were comparable thereafter (Fig. 1). There was no significant difference in weight among the four groups at the beginning of the study (Fig. 2). All groups lost weight on the first 2 postoperative days {P < 0.001, by paired t test). After this, the control groups gained weight (CP, P < 0.05; CS, P < 0.001; by paired t test), whereas the burned rats continued to lose weight rapidly (P < 0.001, by paired t test) and by the end of the study had lost 25.2 ± 2.2% of their initial weight. There was no significant difference between rats receiving somatropin or placebo. The mean hemoglobin concentration was significantly lower in burned rats on both the 6th and 14th days of the study compared with that in their control counter-
275"
250 8
10
12
14
Day FIG. 2. Mean weight of the four groups. By repeated measures analysis of variance: control vs. burned rats, P < 0.0001; placebo vs. somatropin treatment, not significant.
parts (Fig. 3). There were no changes in concentration between the 6th and 14th days in any treatment group, and there was no difference between rats receiving placebo or somatropin. The mean serum albumin concentration was close to the normal pooled serum value in the control rats on both the 6th and 14th days (Fig. 4). The burned rats had significantly lower concentrations
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SOMATROPIN AND WOUND HEALING
941
Muscle peak force
Hemoglobin CP
B
m
cs
0
BP BS
m•
CP CS BP BS
Day FlG. 3. Hemoglobin concentration on the 6th and 14th days postoperatively (mean ± SE). Day 6: CP vs. BP, P < 0.001; CS vs. BS, P < 0.05. Day 14: CP vs. BP, P < 0.05; CS vs. BS, not significant.
FIG. 5. Muscle peak force on the 6th and 14th days postoperatively (mean ± SE). • , CP vs. CS, P = 0.014. Day 6: CP vs. BP, P < 0.01; CS vs. BS, P < 0.001.
Skin peak force
Albumin
I
n •
CP CS BP BS
•
CP CS BP BS
Hi
Day FlG. 4. Serum albumin concentration on the 6th and 14th days postoperatively (mean ± SE). Days 6 and 14, CP vs. BP and CS vs. BS, P < 0.001.
than the control rats on both days. There were no differences between placebo- or somatropin-treated rats. On the 6th postoperative day, the mean peak forces applied to both skin and musculo-fascial wounds were significantly lower in the burned rats than in control rats (Figs. 5 and 6). The musculo-fascial wounds were significantly stronger in the control rats receiving somatropin than in those receiving placebo, but this difference was not evident in their skin wounds. There was no difference in the strength of either musculo-fascial or skin wounds between BP and BS rats. On the 14th day, there was no difference in wound strength among the four experimental groups.
FIG. 6. Skin peak force on the 6th and 14th days postoperatively (mean ± SE). Day 6, CP vs. BP and CS vs. BS, P < 0.05.
Discussion In the present study a 20% full-thickness burn caused rapid weight loss, despite the burned rats having comparable dietary intakes to control rats on all but the first 2 days. The rate of weight loss was similar to that in another study in which burned rats were provided with a fixed dietary intake and is due to the onset of a negative nitrogen balance (10). There was no evidence that somatropin administration caused any increase in weight over the 14 days of the study. We have found previously that somatropin fails to cause weight gain over a 5-day period in unwounded rats with normal pituitary function (11). Jorgensen and
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BELCHER AND ELLIS
Andreassen (8) showed that somatropin administration to rats causes increased weight gain in a dose-related manner, but the doses used exceeded that used in the present study, although the researchers failed to state the activity of their preparation. More recently, Damm Jorgensen et at. (12) showed that the administration of 0.5 IU/kg-day somatropin to normal male rats over a 90day period causes no significant weight gain. The dose of somatropin used in the present study was chosen in order to assess the effects of a clinically usable dose, as the use of higher doses is limited by their effect on glucose metabolism, particularly in burned patients (13). The administration of comparable doses of human pituitary GH and somatropin to hypophysectomized rats has been shown to result in a significant increase in the rate of growth (14-17). The dose of somatropin used in the present study is, therefore, analogous to those used in human studies by us (13) and others (4, 5), and approximates doses used for correcting hypopituitarism. There is evidence that mature rats with normal pituitary function and growth rates are relatively insensitive to the anabolic effects of exogenous GH while remaining sensitive to its effects on glucose metabolism (18). The clinical relevance of animal studies using large doses is, therefore, questionable. The fall in serum albumin in burned rats was marked and was principally due to the increase in capillary permeability that follows injury (19). There was no change in the albumin concentration after the sixth day despite the transience of this change. This probably reflects its large pool size, which limits its ability to either respond to short term changes in nutritional status (20) or recover after injury by burning, even in aggressively fed patients (13). There was no evidence that somatropin had any effect on serum albumin levels in either control or burned rats. Anemia is characteristic after injury by burning, due initially to destruction or damage to red cells by heat and, subsequently, to a shortened red cell lifespan and the unresponsiveness of bone marrow to erythropoietin (21). Although GH potentiates erythropoiesis in vitro (22), no improvement in hemoglobin level was seen in the rats receiving somatropin, but this may reflect the relative brevity of the study. The strength of both the musculo-fascial and skin wounds was decreased on the sixth postoperative day in the burned rats compared with that in controls. This finding is attributable to the presence of a number of factors that have previously been associated with impaired wound healing, including physical stress (23, 24), weight loss (25), hypoalbuminemia (26), anemia (27), and the presence of a distant source of infection (28). Despite their plurality, there was no long term reduction in the wound strengths of the burned rats. This is,
JCE & M • 1990 Vol70«No4
perhaps, surprising, but previous studies in burned animals have also shown only a short-lived reduction in wound strength, not extending beyond the fifth postoperative day (23, 29). There is now evidence that the loss of wound strength associated with stress and malnutrition is best correlated with the dietary intake at the time of healing (30, 31). Although the burned rats were clinically malnourished, they had only a transient fall in dietary intake compared with controls. The administration of somatropin resulted in a transient increase in musculo-fascial wound strength in control rats, consistent with previous studies that have shown increased breaking strengths in incised wounds and sc implanted sponges in rats receiving biosynthetic GH preparations (6-8). This was not observed in the skin wounds, but there is some indication that these are less responsive to dietary manipulation than those in muscle (25). It is thought that the growth-promoting and anabolic actions of GH are mediated by the somatomedins, principally insulin-like growth factor-I, which when administered to hypophysectomized rats is indistinguishable in its effect from GH (32). Fibroblasts can synthesize IGF-I, and there is evidence that IGF-I is required for normal fibroblast replication (33). It has been postulated that GH stimulates fibroblasts via the paracrine and/or endocrine release of somatomedins. Somatropin administration caused no increase in wound strength at any time in the burned rats. Plasma somatomedin levels fail after injury by burning (34). Furthermore, somatomedin release in response to somatropin is impaired after burn injury (11, 13). It can, therefore, be postulated that the failure of somatropin to improve wound strength in burned rats is due to an impaired somatomedin response. The present study has shown that somatropin has limited anabolic activity in rats with normal pituitary function. We have shown that somatropin has no effect in burned rats and provide further evidence that somatropin is unlikely to be effective as an anabolic agent soon after injury.
Acknowledgments We thank Cathy Godfrey for her technical assistance and Robert Beetham at the Protein Reference Unit, Westminster Hospital, for the albumin assays.
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