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Effect of hemorrhagic shock on wound healing in rats and guinea pigs
EFFECT
CERHARD
OF HEALING H.
HEMORRHAGIC IN RATS SCHMIDT,
M.D., M.D.,
WOUND disruption in patients who have undergone emergency operations for control of bleeding is not uncommon [ 111, A number of factors have been implicated: age, anemia, hurried surgical techniques, depletion of substances known to be required for tissue repair, metabolic derangements caused by shock, and persistent unrecognized hypovolemia. Experimental work with various animals has demonstrated no alteration in wound healing when only one of the factors is altered, such as age ( mice ) [ 151; acute anemia without hypovolemia (dogs and rabbits) [ 1, 281; acute hemolytic or chronic iron deficiency anemia (rats) [ 141. It has been suggested that shock may delay wound healing because it “accentuates the injury and appears to accentuate the duration and magnitude of the body’s response mechanisms . . .” [26]. However, the effect of hypovolemia sufficient to produce severe metabolic alterations on wound healing has not been studied in detail. Accordingly, a study was undertaken to determine whether a period of h emorrhagic hypovolemia suffiFrom the Department of Surgery, Western Reserve University School of Medicine, and the University Hospitals of Cleveland, Cleveland, Ohio. * Professor of Surgery, University of Toronto, and Surgeon-in-Chief, Toronto General Hospital, Toronto, Canada. Supported in part by U.S. Public Health Service Grant A-1253-10. Submitted for publication Dec. 5, 1966.
AND AND
SHOCK GUINEA WILLIAM
ON
WOUND PIGS R.
DRUCKER,
F.A.C.S.*
cient to alter intermediary metabolism would have a detrimental effect on the healing of experimental wounds in rats and guinea pigs. METHODS The initial studies were conducted in rats; guinea pigs were subsequently employed because these animals, similar to man, cannot synthesize ascorbic acid [ 18, 241. Male albino Sprague-Dawley rats weighing 300 to 400 gm. were anesthetized with intraperitoneal pentobarbital (40 mg. per kilogram). The hair over the abdomen and left groin was clipped and the skin was prepared with isopropyl alcohol. A 21-gauge scalp vein needle (Abbott) was inserted into the left femoral artery and the plastic tubing was attached via a three-way stopcock to a mercury manometer and a syringe which served as a reservoir for the withdrawn blood. Two hundred units of heparin were given to each animal through the femoral cannula. The rats were then bled to a mean arterial blood pressure (MABP) of 50 mm. Hg during a 15 minute period. This pressure was maintained by raising or lowering as necessary the syringe reservoir containing the shed blood. One hour after hemorrhagic shock was established, an abdominal laparotomy utilizing a 4 to 5 cm. midline incision was performed. Directly after it 513
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was produced, the wound was closed with interrupted 4-O black silk for the musculofascial layer, and 5-O silk for the skin. Hypovolemia was maintained for an additional hour after closure of the laparotomy wound. Following the two-hour period of hemorrhagic shock at a MABP of 50 mm. Hg, the hypovolemia was terminated by transfusion. The cannula was removed from the femoral artery and the wound in the groin was closed with a Michelle clip. All animals that survived this acute phase of the study were returned to their cages where they were kept in pairs and allowed water and rat chips ad libitum. Body temperature was monitored throughout the period of hypovolemia by a rectal probe. Heat was supplied as necessary by an external lamp to maintain the rectal temperature between 35°C. to 37.5”C. A series of control animals were subjected to a similar procedure except that hypovolemia was not produced. An incision was made in the left groin for ligation of the femoral artery. The abdominal laparotomy was performed in all respects similar to the ones performed in the hypovolemic animals. Since the femoral artery was not cannulated, it was unnecessary to give heparin. A clean set of heat-sterilized instruments was used each day. Between animals, instruments were wiped with isopropyl alcohol. The plastic tubing and stopcocks were rinsed with water and 70% alcohol, and air dried. All rats that survived the period of hypovolemia were sacrificed at intervals from three hours to seven days after wounding. Histological study of the wounds was performed on 20 of the rats sacrificed at varying time intervals (3 hours, 6 hours, 24 hours, 48 hours, 4 days, and 7 days), 10 from the control series and 10 from the experimental group. Cross sections of the abdominal wall, which included the wound, were fixed in 10% formalin. Sections from these tissues were stained with hematoxoline-eosine for morphological study, silver for reticulin, and azan-carmine for collagen. Fifty additional rats, including 25 in the experimental group and 25 in the control group were sacrificed seven days after the laparotomy for study of wound tensile 514
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strength. Directly after sacrifice, which was performed by decapitation without anesthesia, the abdominal wound was excised and sectioned into 8 mm. strips with a cutter made of parallel razor blades. The skin was sharply dissected off the musculofascial layer of each strip. All sutures were cut but not removed in both skin and musculofascial layers. Following these procedures, the skin strips were remeasured and corrected to a width of 5 mm. when the data were calculated. The elasticity of the skin, without fixation to prevent distortion, was found to result in strips which were not as uniform as the musculofascial strips. After this preparation in measurement, the strips were tested for tensile strength of the wounds. The apparatus for testing tensile strength was constructed with standard laboratory equipment. A clamp was placed on each end of the strip of tissue. The upper clamp was placed on a stationary platform. The tissue and lower clamp were allowed to hang down. A plastic bottle hooked to the lower clamp was filled with water at a rate of 250 gm. per minute until the wound was pulled apart. The weight of the bottle, water, and the lower clamp, expressed in grams, was assumed to represent the tensile strength of a strip of uniform length [2]. The study utilizing guinea pigs was generally similar to that performed with rats. Male albino guinea pigs weighing 400 to 500 gm. were anesthetized with intraperitoneal pentobarbital (30 mg. per kilogram). The right carotid artery was cannulated in the experimental group and ligated in the control group. Heparin, 300 units, was given to prevent clotting in the cannulated vessel and tubing. In contrast to the study in rats, the guinea pigs were bled to a predetermined volume rather than to a pressure because of the wide variability found in the control blood pressure of the guinea pigs. A degree of hypovolemia to be produced in the guinea pigs was determined by calculation of the data obtained from the study in 44 rats. The mean of the blood volume loss by rats was determined at three different periods: when the MABP was stabilized at 50 mm. Hg, at the
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time of laparotomy, and at the time of the maximal bleed out. Also, the mean time required for maximum bleed out was determined. A graph was then constructed to relate the weight of the guinea pig ( gm. ) to the volume of blood loss (ml. ) at each of the three periods. The prehemorrhage or normal blood volume for each guinea pig was calculated on the basis of body weight (ml./kg. ) [S]. Utilizing this data, it was possible to calculate the volume of blood to be withdrawn at each time period as a percent of initial blood volume (Table 2). Thus, at the first time period when the blood pressure was stabilized at 50 mm. Hg, all guinea pigs were bled 15.470 of their initial blood volume. The next time period when the laparotomy wound was performed, the volume loss was 31.77, of initial blood volume and the maximum bleed out volume was 42.7%. The mean time for maximum bleed out for rats was found to be at 100 minutes. The laparotomy incision was made one hour after onset of hypovolemia, to correspond with the time interval in the study with rats. After two hours of hypovolemia the guinea pigs were transfused with all shed blood and returned to their cages. Although the MABP was recorded at frequent intervals throughout the study, no attempt was made to adjust the pressure since the animals were bled to predetermined volumes. But the rapidity of bleeding was modified whenever the arterial pressure fell rapidly. Both control and experimental guinea pigs were maintained in clean cages and allowed water and rat food ad libitum without supplementary ascorbic acid. The guinea pigs were sacrificed with intraperitoneal pentobarbital. The abdominal wall was excised and sectioned in a manner similar to that utilized for rats. In the guinea pigs, only the musculofascial strips were tested for tensile strength.
RESULTS
IN
RATS
A total of 85 rats were subjected to shock and laparotomy wound. Of these, 9 animals died during the period of hypovolemia; 28
SHOCK
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HEALING
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RATS
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rats died within 96 hours of transfusion and were therefore unavailable for further study. The combined mortality in the total group of rats was 43.5%. Several of the rats that survived the period of shock were not included in the study of tensile strength because of timing errors in four rats, technical problems in four rats, and wound complications in four rats. The wound complications consisted of partial dehiscence in two rats and hematoma in two rats. Histological studies were performed in 10 rats for any changes in the timing and the extent of the inflammatory and reparative response to the laparotomy wound. By comparing the appearance of reticulin fibers and collagen fibers at each time period, as well as changes in inflammatory cells, no distinct difference was found between the histological appearance and staining characteristics of the wound of shock and control animals. Wound strength was tested seven days after injury on musculofascial strips 8 mm. in width. The results of these determinations, expressed in grams of weight applied at a constant rate required to rupture the wound, revealed that musculofascial strips from control animals were separated by a mean weight of 1,045 -+ 150 grams. The weight required to separate the wounds of experimental animals was 941 * 184 grams. The range of weight required to disrupt the skin wound in this series of animals was wider than that required for the musculofascial wounds. A mean of 197 _+ 87 grams was required to rupture the skin wound of control animals, and 153 I+ 45 grams was required for the experimental animals. Although the mean strength of the wounds obtained from shocked animals was slightly less than that of the conTable 1. Wound Strength in Rats Expressed as Weight Required To Cause Wound Separation Seven Days Following Hemorrhagic Shock
Group Control Shock
No. of Animals
Skina
25 25
197 2 87 153 e 45
Musculofasciata 1045 2 150 941 -r- 184
~1Mean ? standard deviation (grams). 515
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trol, the differences between the two groups were not found to be statistically significant (Table 1).
RESULTS
IN
GUINEA
PIGS
The volume of blood withdrawn from guinea pigs during the three stages of shock procedure is recorded in Table 2. The percent of initial blood volume removed at each of the three stages was calculated from the mean data of 44 rats so that it would be of comparable severity in the two species of animals. Of the I.2 guinea pigs subjected to hypovolemia and laparotomy, three animals died during the period of shock or immediately thereafter. The wound strength of the musculofascial layer alone was tested in nine of the surviving experimental animals and in nine nonbled control animals. An 8 mm. section of wound was found to require 669 * 97 gm. in the control animals and 709 + IO2 gm. in the experimental animals to produce a separation. This difference was not found to be statistically significant. Table 2. Withdrawn
Percent During
Calculated Two Hours Shock
of
Duration of MABP at 50 mm. Hg
Blood Volume of Hemorrhagic
Animals Ratsa Guinea pig+
0 15.7 -c 4.6 15.4 * 0.6 Initial hemorrhage 60 min. 33.0 * 9.8 31.7 1 2.7 Laparotomy Maximum 100 min. 46.3 -t 10.2 42.7 -C2.3 bleed out :I Mean 11 Mean
L- standard L- standard
deviation deviation
(in (in
44 rats). 12 guinea
pigs).
Table 3. Wound Strength in Guinea Pigs Expressed as Weight Required To Cause Wound Separation 7 Days Following Hemorrhagic Shock No. of Animals
Group Control Shock a Mean
516
9 9 -+
standard
deviation
Musculofascial” 669 t 97 709 c 102 (grams).
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DISCUSSION After injury or destruction of tissue cells, the early biological response is one of inflammation. A complex set of concurrent and consecutive events is initiated by vasodilation and an increase in capillary permeability, Exudation of fluid occurs, polymorphonuclear leukocytes in increasing numbers are seen within 2 to 3 hours after injury, and collagen fibers undergo swelling and hyalinization. Twenty-four to 48 hours after injury the polymorphonuclear cells, histiocytes, and monocytes increase in number. Before 24 hours have elapsed from the time of injury, fibroblasts along the wound margin assumea more embryonal appearance and mitoses appear. The concentration of fibroblasts is greatest at 72 hours after injury. The fibrin clot in the wound is gradually colonized by fibroblasts. As soon as the fibroblasts meet from the two sides of the wound, an ingrowth of capillary buds begins. Epithelialization starts only when the defect is filled with granulation tissue. This initial period has been termed the substrate phase. A collagen phase follows, starting about the fifth day after wounding with rapid subsequent gain in wound strength. Collagen deposition can be observed histologically as well as tested histochemically and indirectly by tensile strength studies. After this period, scar maturation occurs at variable rates so that some histological activity may be seen as late as one year following wounding [4, 281. Sample times for histological study, and tensile strength testing, were chosen to fall at suitable periods according to the outline of wound healing presented. It is reasonable to postulate that hypovolemia may cause inhibition of the healing process in many different mechanisms. With the degree of hypovolemia sufficient to reduce the MABP to 50 mm. Hg, circulation to both skin and muscle is severely curtailed as a homeostatic device to protect the central circulation, At this pressure, muscle in this study was pale and did not bleed. This reduction in peripheral blood flow may be responsible, at least in part, for the infrequency of hematoma observed in the present study despite the use
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of heparin. Further, the use of heparin may have reduced the incidence of microthrombi which have been observed following prolonged hemorrhagic shock [ 12, 271. The reduction of thrombi and hematoma formation would serve to promote wound healing, while the impeded circulation due to the reduction in blood volume might slow the inflammatory response characteristic of early wound healing. A decreased rate of delivery of leukocytes and substrate to the injured tissues as well as reduced rate of removal of breakdown products from cellular metabolism could result in interference with the normal inflammatory response. Further, proteolytic enzymes released either locally or systemically following prolonged hypovolemia, could cause additional damage to tissues already partially damaged by the wound and contribute to the quantity of debris which must be removed prior to healing [13, 231. Profound metabolic changes occur with prolonged hypovolemia. Significant changes are noted to appear within the first hour after hemorrhage sufficient to reduce the MABP to 50 mm. Hg [3]. With continued shock, the glycogen stores become depleted and the quantity of glucose available for energy rapidly declines, with a terminal hypoglycemia. The quantities of pyruvic and lactic acids increase progressively in association with a concomitant decline in blood pH [6]. Thus with prolonged shock, the decline in energy substrate and progressive acidosis may very likely contribute to inhibition of optimal enzyme activity and inflammatory response to tissue injury. Whether endotoxemia develops as a consequence of prolonged hemorrhagic shock is not conclusively determined [8, 201. But it is reasonably well established that numerous vasoactive polypeptides and such humoral agents as epinephrine and cortisol are released in increased quantities to the circulation following a significant degree of hypovolemia [ 191. In addition to the effect of these agents on energy metabolism, they may all have a direct effect on peripheral blood flow, vascular permeability and enzyme function involved in healing of wounds.
SHOCK
ON
WOUXD
HEALING
IN
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PIGS
The building blocks or substrates required for wound repair may be reduced significantly by a period of hemorrhagic shock [ 151. Thus, protein catabolism is increased with unrecognized and prolonged hypovolemia and may very well be responsible for the wound disruption in patients following persistent unrecognized hypovolemia [ 91. The blood levels of ascorbic acid, riboflavin, thiamine, and nicotinic acid have been found depleted following shock [ 161. A lower blood level of prothrombine has been observed following hypovolemic shock, and, if persistent, could be responsible for a tendency to hematoma formation in the wound [30]. \Vound contamination and infection can be a significant factor in healing. Decreased resistance by the host’s bacterial defense in man and dogs has been observed following hypovolemic shock [22]. However, rats are known to be resistant to the development of infection in cutaneous wounds. Therefore, no conclusion can be drawn regarding this factor in the current study. In view of the similarity in wound tensile strength found in the present study between the rats subjected to hemorrhage and the control rats, it might be questioned whether the magnitude of the hypovolemic insult was significant. Unquestionably, the metabolic alterations produced by hypovolemia become increasingly severe with time, but the rate of alteration may be accelerated by the severity of hypovolemia. In the present study, the degree of hypovolemia was sufficient to produce a metabolic acidosis, but the studies were not performed at a time when hepatic glycogen was depleted as evidenced by a fall in circulating blood glucose. Perhaps the most indicative measure of the severity of the shock procedure was the finding that 43% of the rats and 25% of the guinea pigs died consequent to the production of hypovolemia and prior to the time for tensile strength studies on the tissues. Most of the deaths were attributed to “irreversible shock” as defined by Wiggers [31]. It is conceivable that by seven days following the period of hypovolemia, the time at which the wound strength was tested, sufficient recovery of both metabolic and 517
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other alterations had occurred to obviate detectable influence on wound healing by the methods used in the study. It is known that the metabolic alterations that occur even during profound shock subside promptly after blood volume is restored [6, 291. A considerable range of variation is usually found in the tensile strength-not only in wounded but in normal tissues-despite handling by very constant and reproducible methods [lo, 251. The testing of wound tensile strength is not very precise [21]. However, by utilizing certain techniques, quite uniform results can be obtained. A midline abdominal incision provides constant landmarks. Bleeding is almost nonexistent. Uniformity of technique is improved by having the same individual perform the procedure of wounding and suturing. Tissue segment samples must be constant in width. Clamps must be applied to the tissues parallel to the wound [2]. Uniformity in animals is also required. This is done by utilizing animals of a narrow range in age and weight, of the same sex and strain, purchased from the same vendor, and maintained on a uniform diet and in a uniform environment. Some alterations in the healing of wounds associated with hypovolemia could probably be demonstrated by the histochemical and chemical methods described by Dunphy [7], if these were applied at suitable times after wounding.
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Besser, lationship SurgeTy
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D. T., Bains, J. A standard model J. Surg. Res. 6:265,
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Curtis, J. M., Stevens, D. P., and Drucker, W. R. Glucose transport by isolated intestine from hypovolemic rats. Fed. PTOC. 23:698, 1964. DeVito, R. V. Healing of wounds. Surg. Clin. N. Amer. 45:441, 1965.
4.
AND
CONCLUSION
Hemorrhagic shock in rats and guinea pigs was produced. The severity of the insult resulted in mortality rates of 43.5% and 257a, respectively, in these animals, despite transfusion. While the animals were hypovolemic, an abdominal wound was made and closed. Seven days after hemorrhagic shock, there was no difference in wound strength between animals that had been subjected to shock and controls. An episode of hemorrhagic shock alone can therefore not be considered as causing wound disruption, if the shock is adequately treated. 518
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J. L. wound
The rehealing.
W., and Ketcham for tensiometric 1965.
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Dittmer, D. S., and Grebe, R. M. Handbook of CiTcuZution. Philadelphia: ders, 1959. P. 66.
6.
Drucker, W. R., Kaye, M., Kendrick, R., Hoffman, N., and Kingsbury, B. Metabolic aspects of hemorrhagic shock. Surg. Forum 9:49, 1958.
7.
Dunphy, J. E., and Udupa, K. N. Chemical and histochemical sequences in the normal healing of wounds. New Eng. J. Med. 253:847, 1955. Fine, J., Frank, E. D., Ravin, H. A., Rutenberg, S. H., and Schweinburg, F. B. The bacterial factor in traumatic shock. New Eng. J. Med. 260:214, 1959.
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15.
16.
(Eds.). Saun-
Flear, C. T. G., and Clarke, R. Influence of blood loss and blood transfusion upon changes in metabolism of water, electrolytes, and nitrogen following civilian trauma. Clin. Sci. 14:575, 1955. Greever, E. F., Stein, J. M., and Levenson, S. M. Variations in breaking strength in healing wounds in young guinea pigs. J. Trauma 5:624, 1965. Guiney, E. J., Morris, P. J., and Donaldson, G. A. Wound dehiscence. Arch. Surg. (Chicago) 92:47, 1966. Hardaway, R. M., Greever, E. F., Burns, J. W., and Mock, H. P. Studies on the role of intravascular coagulation in irreversible hemorrhagic shock. Ann. Surg. 155:241, 1962. Janoff, A., Weissman, G., Zweifach, B. W., and Thomas, L. Pathogenesis of experimental shock: IV. Studies on lysozomes in normal and tolerant animals subjected to lethal trauma and endotoxemia. J. Exp. Med. 116:451, 1962. Jurkiewicz, M. J., and Garrett, L. P. Studies on the influence of anemia on wound healing. Amer. Surg. 30:23, 1964. Levenson, S. M., Nutritional and Fed. PTOC. Suppl. Levenson, S. M., L., Robinson, P.,
Einheber, A., and Malm, metabolic aspects of 9:99, 1961. Green, R. W., Taylor, Page, R. C., Johnson,
0. J. shock. F. H. R. E.,
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20. 21. 22.
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and Lund, C. L. Ascorbic acid, riboflavin, thiamin, and nicotinic acid in relation to severe injury, hemorrhage, and infection in the human. Ann. Surg. 124:840, 1946. Levenson, S. M., Greever, E. F., Crowley, L. V., Oates, J. F., Berard, C. W., and Rosen, H. The healing of rat skin wounds. Ann. Surg. 161:293, 1965. Levenson, S. M., Upjohn, H. J., Preston, J. A., and Steer, A. Effect of thermal burns on wound healing. Ann. Surg. 146:357, 1957. Levy, M. N., and Blattberg, B. Blood factors in shock. In S. G. Hershey (Ed.), Shock. Boston: Little, Brown, 1964. Lillehei, R. C. The intestinal factor in hemorrhagic shock. Surgery 42:1043, 1957. Milch, R. A. Tensile strength of surgical wounds. .I. Surg. Res. 5:377, 1965. Ollodart, R., and Mansberger, A. R. The effect of hypovolemic shock on bacterial defense. Amer. J. Surg. 110:302, 1965. Pagano, V. P., and Mersheimer, W. L. Immunochemical changes observed with shock. Surg. Forum 16:45, 1965. Pasqualini, C. D. The effect of ascorbic acid
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25. 26.
27.
28.
29.
30.
31.
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on hemorrhagic shock in the guinea pig. Amer. J. Physiol. 147:596, 1946. Pierce, J. A. Tensile strength of human lungs. J. Lab. Clin. Med. 66:652, 1965. Rhoads, J. E., Howard, J, M., and Janetta, P. J. The Chemistry of Trauma. Springfield, Ill.: Thomas, 1963. P. 82. Robb, H. J. The role of microemboli in the production of irreversible shock. Ann. Surg. 158:685, 1963. Robbins, S. L. Textbook of Pathology with CZinicaZ Application, 2nd ed. Philadelphia: Saunders, 1962. Seligman, A. M., Frank, H. A., Alexander, B., and Fine, J. Traumatic shock: XV. Carbohydrate metabolism in hemorrhagic shock in the dog. J. Clin. Inuest. 26:536, 1947. Tagnon, H. J., Levenson, S. M., Davidson, C. S., and Taylor, F. H. L. The occurrence of fibrinolysis in shock, with observations on the prothrombin time, and the plasma fibrinogen during hemorrhagic shock. Amer. J. Med. Sci. 211:88, 1946. Wiggers, C. J. Physiology of Shock. New York: The Commonwealth Fund, 1950.
519
CERHARD
OF HEALING H.
HEMORRHAGIC IN RATS SCHMIDT,
M.D., M.D.,
WOUND disruption in patients who have undergone emergency operations for control of bleeding is not uncommon [ 111, A number of factors have been implicated: age, anemia, hurried surgical techniques, depletion of substances known to be required for tissue repair, metabolic derangements caused by shock, and persistent unrecognized hypovolemia. Experimental work with various animals has demonstrated no alteration in wound healing when only one of the factors is altered, such as age ( mice ) [ 151; acute anemia without hypovolemia (dogs and rabbits) [ 1, 281; acute hemolytic or chronic iron deficiency anemia (rats) [ 141. It has been suggested that shock may delay wound healing because it “accentuates the injury and appears to accentuate the duration and magnitude of the body’s response mechanisms . . .” [26]. However, the effect of hypovolemia sufficient to produce severe metabolic alterations on wound healing has not been studied in detail. Accordingly, a study was undertaken to determine whether a period of h emorrhagic hypovolemia suffiFrom the Department of Surgery, Western Reserve University School of Medicine, and the University Hospitals of Cleveland, Cleveland, Ohio. * Professor of Surgery, University of Toronto, and Surgeon-in-Chief, Toronto General Hospital, Toronto, Canada. Supported in part by U.S. Public Health Service Grant A-1253-10. Submitted for publication Dec. 5, 1966.
AND AND
SHOCK GUINEA WILLIAM
ON
WOUND PIGS R.
DRUCKER,
F.A.C.S.*
cient to alter intermediary metabolism would have a detrimental effect on the healing of experimental wounds in rats and guinea pigs. METHODS The initial studies were conducted in rats; guinea pigs were subsequently employed because these animals, similar to man, cannot synthesize ascorbic acid [ 18, 241. Male albino Sprague-Dawley rats weighing 300 to 400 gm. were anesthetized with intraperitoneal pentobarbital (40 mg. per kilogram). The hair over the abdomen and left groin was clipped and the skin was prepared with isopropyl alcohol. A 21-gauge scalp vein needle (Abbott) was inserted into the left femoral artery and the plastic tubing was attached via a three-way stopcock to a mercury manometer and a syringe which served as a reservoir for the withdrawn blood. Two hundred units of heparin were given to each animal through the femoral cannula. The rats were then bled to a mean arterial blood pressure (MABP) of 50 mm. Hg during a 15 minute period. This pressure was maintained by raising or lowering as necessary the syringe reservoir containing the shed blood. One hour after hemorrhagic shock was established, an abdominal laparotomy utilizing a 4 to 5 cm. midline incision was performed. Directly after it 513
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was produced, the wound was closed with interrupted 4-O black silk for the musculofascial layer, and 5-O silk for the skin. Hypovolemia was maintained for an additional hour after closure of the laparotomy wound. Following the two-hour period of hemorrhagic shock at a MABP of 50 mm. Hg, the hypovolemia was terminated by transfusion. The cannula was removed from the femoral artery and the wound in the groin was closed with a Michelle clip. All animals that survived this acute phase of the study were returned to their cages where they were kept in pairs and allowed water and rat chips ad libitum. Body temperature was monitored throughout the period of hypovolemia by a rectal probe. Heat was supplied as necessary by an external lamp to maintain the rectal temperature between 35°C. to 37.5”C. A series of control animals were subjected to a similar procedure except that hypovolemia was not produced. An incision was made in the left groin for ligation of the femoral artery. The abdominal laparotomy was performed in all respects similar to the ones performed in the hypovolemic animals. Since the femoral artery was not cannulated, it was unnecessary to give heparin. A clean set of heat-sterilized instruments was used each day. Between animals, instruments were wiped with isopropyl alcohol. The plastic tubing and stopcocks were rinsed with water and 70% alcohol, and air dried. All rats that survived the period of hypovolemia were sacrificed at intervals from three hours to seven days after wounding. Histological study of the wounds was performed on 20 of the rats sacrificed at varying time intervals (3 hours, 6 hours, 24 hours, 48 hours, 4 days, and 7 days), 10 from the control series and 10 from the experimental group. Cross sections of the abdominal wall, which included the wound, were fixed in 10% formalin. Sections from these tissues were stained with hematoxoline-eosine for morphological study, silver for reticulin, and azan-carmine for collagen. Fifty additional rats, including 25 in the experimental group and 25 in the control group were sacrificed seven days after the laparotomy for study of wound tensile 514
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strength. Directly after sacrifice, which was performed by decapitation without anesthesia, the abdominal wound was excised and sectioned into 8 mm. strips with a cutter made of parallel razor blades. The skin was sharply dissected off the musculofascial layer of each strip. All sutures were cut but not removed in both skin and musculofascial layers. Following these procedures, the skin strips were remeasured and corrected to a width of 5 mm. when the data were calculated. The elasticity of the skin, without fixation to prevent distortion, was found to result in strips which were not as uniform as the musculofascial strips. After this preparation in measurement, the strips were tested for tensile strength of the wounds. The apparatus for testing tensile strength was constructed with standard laboratory equipment. A clamp was placed on each end of the strip of tissue. The upper clamp was placed on a stationary platform. The tissue and lower clamp were allowed to hang down. A plastic bottle hooked to the lower clamp was filled with water at a rate of 250 gm. per minute until the wound was pulled apart. The weight of the bottle, water, and the lower clamp, expressed in grams, was assumed to represent the tensile strength of a strip of uniform length [2]. The study utilizing guinea pigs was generally similar to that performed with rats. Male albino guinea pigs weighing 400 to 500 gm. were anesthetized with intraperitoneal pentobarbital (30 mg. per kilogram). The right carotid artery was cannulated in the experimental group and ligated in the control group. Heparin, 300 units, was given to prevent clotting in the cannulated vessel and tubing. In contrast to the study in rats, the guinea pigs were bled to a predetermined volume rather than to a pressure because of the wide variability found in the control blood pressure of the guinea pigs. A degree of hypovolemia to be produced in the guinea pigs was determined by calculation of the data obtained from the study in 44 rats. The mean of the blood volume loss by rats was determined at three different periods: when the MABP was stabilized at 50 mm. Hg, at the
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time of laparotomy, and at the time of the maximal bleed out. Also, the mean time required for maximum bleed out was determined. A graph was then constructed to relate the weight of the guinea pig ( gm. ) to the volume of blood loss (ml. ) at each of the three periods. The prehemorrhage or normal blood volume for each guinea pig was calculated on the basis of body weight (ml./kg. ) [S]. Utilizing this data, it was possible to calculate the volume of blood to be withdrawn at each time period as a percent of initial blood volume (Table 2). Thus, at the first time period when the blood pressure was stabilized at 50 mm. Hg, all guinea pigs were bled 15.470 of their initial blood volume. The next time period when the laparotomy wound was performed, the volume loss was 31.77, of initial blood volume and the maximum bleed out volume was 42.7%. The mean time for maximum bleed out for rats was found to be at 100 minutes. The laparotomy incision was made one hour after onset of hypovolemia, to correspond with the time interval in the study with rats. After two hours of hypovolemia the guinea pigs were transfused with all shed blood and returned to their cages. Although the MABP was recorded at frequent intervals throughout the study, no attempt was made to adjust the pressure since the animals were bled to predetermined volumes. But the rapidity of bleeding was modified whenever the arterial pressure fell rapidly. Both control and experimental guinea pigs were maintained in clean cages and allowed water and rat food ad libitum without supplementary ascorbic acid. The guinea pigs were sacrificed with intraperitoneal pentobarbital. The abdominal wall was excised and sectioned in a manner similar to that utilized for rats. In the guinea pigs, only the musculofascial strips were tested for tensile strength.
RESULTS
IN
RATS
A total of 85 rats were subjected to shock and laparotomy wound. Of these, 9 animals died during the period of hypovolemia; 28
SHOCK
ON
WOUND
HEALING
IN
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AND
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PIGS
rats died within 96 hours of transfusion and were therefore unavailable for further study. The combined mortality in the total group of rats was 43.5%. Several of the rats that survived the period of shock were not included in the study of tensile strength because of timing errors in four rats, technical problems in four rats, and wound complications in four rats. The wound complications consisted of partial dehiscence in two rats and hematoma in two rats. Histological studies were performed in 10 rats for any changes in the timing and the extent of the inflammatory and reparative response to the laparotomy wound. By comparing the appearance of reticulin fibers and collagen fibers at each time period, as well as changes in inflammatory cells, no distinct difference was found between the histological appearance and staining characteristics of the wound of shock and control animals. Wound strength was tested seven days after injury on musculofascial strips 8 mm. in width. The results of these determinations, expressed in grams of weight applied at a constant rate required to rupture the wound, revealed that musculofascial strips from control animals were separated by a mean weight of 1,045 -+ 150 grams. The weight required to separate the wounds of experimental animals was 941 * 184 grams. The range of weight required to disrupt the skin wound in this series of animals was wider than that required for the musculofascial wounds. A mean of 197 _+ 87 grams was required to rupture the skin wound of control animals, and 153 I+ 45 grams was required for the experimental animals. Although the mean strength of the wounds obtained from shocked animals was slightly less than that of the conTable 1. Wound Strength in Rats Expressed as Weight Required To Cause Wound Separation Seven Days Following Hemorrhagic Shock
Group Control Shock
No. of Animals
Skina
25 25
197 2 87 153 e 45
Musculofasciata 1045 2 150 941 -r- 184
~1Mean ? standard deviation (grams). 515
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trol, the differences between the two groups were not found to be statistically significant (Table 1).
RESULTS
IN
GUINEA
PIGS
The volume of blood withdrawn from guinea pigs during the three stages of shock procedure is recorded in Table 2. The percent of initial blood volume removed at each of the three stages was calculated from the mean data of 44 rats so that it would be of comparable severity in the two species of animals. Of the I.2 guinea pigs subjected to hypovolemia and laparotomy, three animals died during the period of shock or immediately thereafter. The wound strength of the musculofascial layer alone was tested in nine of the surviving experimental animals and in nine nonbled control animals. An 8 mm. section of wound was found to require 669 * 97 gm. in the control animals and 709 + IO2 gm. in the experimental animals to produce a separation. This difference was not found to be statistically significant. Table 2. Withdrawn
Percent During
Calculated Two Hours Shock
of
Duration of MABP at 50 mm. Hg
Blood Volume of Hemorrhagic
Animals Ratsa Guinea pig+
0 15.7 -c 4.6 15.4 * 0.6 Initial hemorrhage 60 min. 33.0 * 9.8 31.7 1 2.7 Laparotomy Maximum 100 min. 46.3 -t 10.2 42.7 -C2.3 bleed out :I Mean 11 Mean
L- standard L- standard
deviation deviation
(in (in
44 rats). 12 guinea
pigs).
Table 3. Wound Strength in Guinea Pigs Expressed as Weight Required To Cause Wound Separation 7 Days Following Hemorrhagic Shock No. of Animals
Group Control Shock a Mean
516
9 9 -+
standard
deviation
Musculofascial” 669 t 97 709 c 102 (grams).
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1967
DISCUSSION After injury or destruction of tissue cells, the early biological response is one of inflammation. A complex set of concurrent and consecutive events is initiated by vasodilation and an increase in capillary permeability, Exudation of fluid occurs, polymorphonuclear leukocytes in increasing numbers are seen within 2 to 3 hours after injury, and collagen fibers undergo swelling and hyalinization. Twenty-four to 48 hours after injury the polymorphonuclear cells, histiocytes, and monocytes increase in number. Before 24 hours have elapsed from the time of injury, fibroblasts along the wound margin assumea more embryonal appearance and mitoses appear. The concentration of fibroblasts is greatest at 72 hours after injury. The fibrin clot in the wound is gradually colonized by fibroblasts. As soon as the fibroblasts meet from the two sides of the wound, an ingrowth of capillary buds begins. Epithelialization starts only when the defect is filled with granulation tissue. This initial period has been termed the substrate phase. A collagen phase follows, starting about the fifth day after wounding with rapid subsequent gain in wound strength. Collagen deposition can be observed histologically as well as tested histochemically and indirectly by tensile strength studies. After this period, scar maturation occurs at variable rates so that some histological activity may be seen as late as one year following wounding [4, 281. Sample times for histological study, and tensile strength testing, were chosen to fall at suitable periods according to the outline of wound healing presented. It is reasonable to postulate that hypovolemia may cause inhibition of the healing process in many different mechanisms. With the degree of hypovolemia sufficient to reduce the MABP to 50 mm. Hg, circulation to both skin and muscle is severely curtailed as a homeostatic device to protect the central circulation, At this pressure, muscle in this study was pale and did not bleed. This reduction in peripheral blood flow may be responsible, at least in part, for the infrequency of hematoma observed in the present study despite the use
SCHMIDT
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of heparin. Further, the use of heparin may have reduced the incidence of microthrombi which have been observed following prolonged hemorrhagic shock [ 12, 271. The reduction of thrombi and hematoma formation would serve to promote wound healing, while the impeded circulation due to the reduction in blood volume might slow the inflammatory response characteristic of early wound healing. A decreased rate of delivery of leukocytes and substrate to the injured tissues as well as reduced rate of removal of breakdown products from cellular metabolism could result in interference with the normal inflammatory response. Further, proteolytic enzymes released either locally or systemically following prolonged hypovolemia, could cause additional damage to tissues already partially damaged by the wound and contribute to the quantity of debris which must be removed prior to healing [13, 231. Profound metabolic changes occur with prolonged hypovolemia. Significant changes are noted to appear within the first hour after hemorrhage sufficient to reduce the MABP to 50 mm. Hg [3]. With continued shock, the glycogen stores become depleted and the quantity of glucose available for energy rapidly declines, with a terminal hypoglycemia. The quantities of pyruvic and lactic acids increase progressively in association with a concomitant decline in blood pH [6]. Thus with prolonged shock, the decline in energy substrate and progressive acidosis may very likely contribute to inhibition of optimal enzyme activity and inflammatory response to tissue injury. Whether endotoxemia develops as a consequence of prolonged hemorrhagic shock is not conclusively determined [8, 201. But it is reasonably well established that numerous vasoactive polypeptides and such humoral agents as epinephrine and cortisol are released in increased quantities to the circulation following a significant degree of hypovolemia [ 191. In addition to the effect of these agents on energy metabolism, they may all have a direct effect on peripheral blood flow, vascular permeability and enzyme function involved in healing of wounds.
SHOCK
ON
WOUXD
HEALING
IN
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AND
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PIGS
The building blocks or substrates required for wound repair may be reduced significantly by a period of hemorrhagic shock [ 151. Thus, protein catabolism is increased with unrecognized and prolonged hypovolemia and may very well be responsible for the wound disruption in patients following persistent unrecognized hypovolemia [ 91. The blood levels of ascorbic acid, riboflavin, thiamine, and nicotinic acid have been found depleted following shock [ 161. A lower blood level of prothrombine has been observed following hypovolemic shock, and, if persistent, could be responsible for a tendency to hematoma formation in the wound [30]. \Vound contamination and infection can be a significant factor in healing. Decreased resistance by the host’s bacterial defense in man and dogs has been observed following hypovolemic shock [22]. However, rats are known to be resistant to the development of infection in cutaneous wounds. Therefore, no conclusion can be drawn regarding this factor in the current study. In view of the similarity in wound tensile strength found in the present study between the rats subjected to hemorrhage and the control rats, it might be questioned whether the magnitude of the hypovolemic insult was significant. Unquestionably, the metabolic alterations produced by hypovolemia become increasingly severe with time, but the rate of alteration may be accelerated by the severity of hypovolemia. In the present study, the degree of hypovolemia was sufficient to produce a metabolic acidosis, but the studies were not performed at a time when hepatic glycogen was depleted as evidenced by a fall in circulating blood glucose. Perhaps the most indicative measure of the severity of the shock procedure was the finding that 43% of the rats and 25% of the guinea pigs died consequent to the production of hypovolemia and prior to the time for tensile strength studies on the tissues. Most of the deaths were attributed to “irreversible shock” as defined by Wiggers [31]. It is conceivable that by seven days following the period of hypovolemia, the time at which the wound strength was tested, sufficient recovery of both metabolic and 517
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other alterations had occurred to obviate detectable influence on wound healing by the methods used in the study. It is known that the metabolic alterations that occur even during profound shock subside promptly after blood volume is restored [6, 291. A considerable range of variation is usually found in the tensile strength-not only in wounded but in normal tissues-despite handling by very constant and reproducible methods [lo, 251. The testing of wound tensile strength is not very precise [21]. However, by utilizing certain techniques, quite uniform results can be obtained. A midline abdominal incision provides constant landmarks. Bleeding is almost nonexistent. Uniformity of technique is improved by having the same individual perform the procedure of wounding and suturing. Tissue segment samples must be constant in width. Clamps must be applied to the tissues parallel to the wound [2]. Uniformity in animals is also required. This is done by utilizing animals of a narrow range in age and weight, of the same sex and strain, purchased from the same vendor, and maintained on a uniform diet and in a uniform environment. Some alterations in the healing of wounds associated with hypovolemia could probably be demonstrated by the histochemical and chemical methods described by Dunphy [7], if these were applied at suitable times after wounding.
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Curtis, J. M., Stevens, D. P., and Drucker, W. R. Glucose transport by isolated intestine from hypovolemic rats. Fed. PTOC. 23:698, 1964. DeVito, R. V. Healing of wounds. Surg. Clin. N. Amer. 45:441, 1965.
4.
AND
CONCLUSION
Hemorrhagic shock in rats and guinea pigs was produced. The severity of the insult resulted in mortality rates of 43.5% and 257a, respectively, in these animals, despite transfusion. While the animals were hypovolemic, an abdominal wound was made and closed. Seven days after hemorrhagic shock, there was no difference in wound strength between animals that had been subjected to shock and controls. An episode of hemorrhagic shock alone can therefore not be considered as causing wound disruption, if the shock is adequately treated. 518
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Dunphy, J. E., and Udupa, K. N. Chemical and histochemical sequences in the normal healing of wounds. New Eng. J. Med. 253:847, 1955. Fine, J., Frank, E. D., Ravin, H. A., Rutenberg, S. H., and Schweinburg, F. B. The bacterial factor in traumatic shock. New Eng. J. Med. 260:214, 1959.
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and Lund, C. L. Ascorbic acid, riboflavin, thiamin, and nicotinic acid in relation to severe injury, hemorrhage, and infection in the human. Ann. Surg. 124:840, 1946. Levenson, S. M., Greever, E. F., Crowley, L. V., Oates, J. F., Berard, C. W., and Rosen, H. The healing of rat skin wounds. Ann. Surg. 161:293, 1965. Levenson, S. M., Upjohn, H. J., Preston, J. A., and Steer, A. Effect of thermal burns on wound healing. Ann. Surg. 146:357, 1957. Levy, M. N., and Blattberg, B. Blood factors in shock. In S. G. Hershey (Ed.), Shock. Boston: Little, Brown, 1964. Lillehei, R. C. The intestinal factor in hemorrhagic shock. Surgery 42:1043, 1957. Milch, R. A. Tensile strength of surgical wounds. .I. Surg. Res. 5:377, 1965. Ollodart, R., and Mansberger, A. R. The effect of hypovolemic shock on bacterial defense. Amer. J. Surg. 110:302, 1965. Pagano, V. P., and Mersheimer, W. L. Immunochemical changes observed with shock. Surg. Forum 16:45, 1965. Pasqualini, C. D. The effect of ascorbic acid
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