PRODUCTION OF FATAL ENDOTOXIC SHOCK BY VASOACTIVE SUBSTANCES

GASTROENTEROLOGY 64: 285-291, 1973 Copyright 1973 by The Williams & Wilkins Co. Vol. 64, No.2 Printed in U.S.A. PRODUCTION OF FATAL ENDOTOXIC SHOCK BY VASOACTIVE SUBSTANCES PETER CUEVAS, M.D" F,A,C,S" AND JACOB FINE, M,D, 'Harvard Surgical Unit, Boston City Hospital, Boston, Massachusetts Three vasoactive amines were given intravenously by continuous infusion for 3 to 4 hr into normal rabbits and into rabbits pretreated with intraintestinal kanamycin or made resistant to endotoxin, The untreated rabbits developed a fatal endotoxemia of intestinal origin. The titers of endotoxin in peritoneal fluid and plasma indicate that vasoactive substances do this by allowing endotoxin to move transmurally into the peritoneal cavity. Those rabbits in which the endotoxemia was only transient or prevented altogether did not develop lasting functional or structural damage and survived. Those in which it persisted developed the typical lesions of endotoxic shock in the lung and intestine and died. The persistence of the endotoxemia after stopping the infusion of the vasoactive substances suggests that circulating endotoxin initiates the further release from tissue sources and so creates a self-sustaining process. These data suggest that the endotoxemia which has been identified in various types of shock-producing septic or nonseptic trauma can be initiated by the continuous release of vasoactive substances from the site of trauma. They also demonstrate that these substances do not produce lasting injury in the absence of endotoxin. The vasoactive compounds released under normal conditions serve a physiologic function of brief duration. Even when a large amount is injected in a single bolus intravenously the response is rapidly dissipated. If such short-acting substances are involved in sustaining such a relatively protracted state as traumatic shock, their continuous production and release would seem to be required. Except for norepinephrine, the data on their respective mechanisms of action in shock are not well defined. 1-6 This report presents data Received July 27, 1972, Accepted October 4, 1972. Address requests for reprints to: Dr. Jacob Fine, Harvard Medical School, Department of Surgery, Sears Surgical Laboratory, Boston City Hospital, 818 Harrison Avenue, Boston, Massachusetts 02118. This work was aided by a grant from the National Institutes of Health, Bethesda, Maryland, (5R01-HL- 13908), and by a contract with the office of the Surgeon General, United States Army (DA-49-193- MD-2926). 285 from the experimental animal on the effect of a continuous infusion for 3 to 4 hr of three of the common vasoactive agents. They demonstrate a common response: (1) the development of a lethal endotoxemia at different intervals after beginning the infusion; and (2) recovery without significant injury when the endotoxemia is prevented. Materials and Methods Adult male New Zealand rabbits (2 1 /2 to 3 kg of body weight) were anesthetized with pentobarbital and the groin vessels exposed. One femoral artery was cannulated for continuous monitoring of the systemic blood pressure; one femoral vein served for sampling of blood to monitor the presence and amount of endotoxin by the Limulus lysate technique 7 at intervals until death or recovery; and the other femoral vein was used for infusion of the vasoactive compound. The first of three series of rabbits (N = 10) received an intravenous infusion of 30 cc of

286 CUEVAS AND FINE Vol. 64, No.2 normal saline containing bradykinin (0.1 mg per kg of body weight) during a period of 3 hr. The procedure was the same in the second series (N = 10) and in the third series (N = 5) except that serotonin (0.1 mg per kg of body weight) and histamine (1.0 mg per kg of body weight), respectively, were substituted for bradykinin. In a second set of three experiments, those in the first set were repeated except that all the rabbits in the second set received intraintestinal kanamycin, injected under pentobarbital anesthesia via a laparotomy incision, under direct vision into the duodenum (1 g in 30 cc of saline) and into the cecum via the appendix (1 g in 50 cc). The antibiotic was injected twice, the day before and again 4 hr before the infusion. In a third set of three experiments, the first set was repeated except that all the rabbits in the third set had been made resistant to endotoxin, as demonstrated in each case 1 week before the experiment by a full and quick recovery after an otherwise lethal dose of endotoxin. Resistance was induced either by serial intramuscular injections every other day for 10 days with graded doses of endotoxin, or by a single intramuscular injection of 100 Jl,g of endotoxin in 1 ml of mineral oil. 8 In a fourth set of rabbits (N = 11) the same dose (0.1 mg per kg) of each of the three vasoactive substances, serotonin (N = 4), histamine (N = 2), bradykinin (N = 2), and of prostaglandin F 2 a (N = 3), was dissolved in 5 ml of saline and infused in less than 5 min. The responses were monitored as in the other series. All rabbits that died were examined for the presence of the lesions characteristic of endotoxic shock,9-1o i.e., solidification of the lung by extravasated plasma and red cells ("shock lung"), and a similar process in the stomach and intestine, i.e., edema and hemorrhagic ulceration of the mucosa. All survivors were killed at corresponding survival times and likewise examined for such lesions. Postmortem assays of endotoxin 1 were made in every case on the heart's blood and the peritoneal fluid in order to determine the route by which endotoxin enters the circulation in animals treated with vasoactive agents. (Such titers do not change from the time of death up to 4 hr after death. ") Results Figure 1 shows the response of the systemic blood pressure. Bradykinin produced an immediate and pronounced hypotension followed by a rapid return to normal and then by a hypertensive state that persisted until the infusion was stopped. Thereafter the pressure fell slowly and progressively to a shock level. Death from refractory shock occurred 10 to 12 hr later. In the group receiving serotonin (0.1 mg per kg) there was an immediate drop in blood pressure which was progressive until death some 8 to 10 hr after beginning the infusion. The hypotensive response to histamine was less marked than for the other two substances and was followed after some 2 hr by a slow return to near normal upon completion of the infusion. Shock developed, however, within 1 or 2 hr there- Mm. 120 110 100 90 80 70 60 of 50 Hg. 40 30 20 10 lime of Infusion... o 2 3 4 HOURS 5 6 7 8 = BRADYKININ - SEROTONIN HISTAMINE FIG. 1. Blood pressure response of normal rabbits to continuous infusion of vasoactive agents. Mm. of Hg. 120 110 100 90 80 70 60 50 40 30 20 10.. Time of Infusion "" o 2 3 4 HOURS 5 6 8 = BRADYKININ - SEROTOIlIN... HISTAMINE FIG. 2. Blood pressure response of rabbits resistant to endotoxin or pretreated with intraintestinal kanamycin to continuous infusion of vasoactive agents.

February 1973 ENDOTOXIC SHOCK BY VASOACTIVE SUBSTANCES 287 TABLE 1. Intravenous bradykinin, endotoxin in plasma Experiment 1 hr 2 hr 4 hr 6 hr Postmortem Survival" No treatment 1 0 I 0 I 2 0 0 3 0 0 4 0 0 : 5 0 0 6 0 0 7 0 0 I 8 0 i 0 9 0 0 10 0 0 Intraintestinal antibiotic 11 0.04 0.04 12 0.01 0.001 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0 20 0 0 Endotoxin-resistant 21 0 0 22 0 0.001 23 0 0 24 0 0 25 0 0 26 0 0 27 0 0 28 0 0 29 0 0 30 0 0 a D, died; S, survived. Mimi 0 0.01 0.01 D 0 0.01 0.02 D 0.01 2.0 0.1 D 0 0.01 0.03 D 0 0.01 0.06 D 0 0 l.0 D 0 0 0.03 D 0 0.01 l.0 D 0 0.01 0.01 S 0 0.01 0.01 S 0 0 0.1 D 0 0 0. 1 D 0.01 0 0 D 0 0 0 D 0.04 0.001 0 S 0.001 after and death occurred some 6 to 7 hr later. The hemodynamic response of the animals treated with intraintestinal antibiotic or made resistant to endotoxin were similar to those in the corresponding untreated series, except that after the infusion was stopped the blood pressure returned to normal and recovery followed (fig. 2). In the untreated animals receiving serotonin or histamine endotoxemia developed within 2 hr; in those receiving bradykinin endotoxemia developed between 4 and 6 hr (tables 1 to 3). Circulating endotoxin was not found or, when present, was in most cases in lower titer in those that had received intraintestinal antibiotic or that had been made resistant to endotoxin. The survival rate was 20% or less in the untreated rabbits, 50% or more in those treated with intraintestinal antibiotics, and 80% or more in those made resistant to endotoxin. The gross lesions characteristic of endotoxic shock were present in all rabbits which showed an endotoxemia that persisted until death. Only 2 of the 30 rabbits that had been made resistant showed such lesions. Of the 30 that received intraintestinal antibiotics 12 had these lesions, although in general they were less

288 CUEVAS AND FINE Vol. 64, No.2 TABLE 2. Intravenous serotonin, endotoxin in plasma Experiment 1 hr 2 hr 4 hr 5 hr Post mortem Survival" No treatment 1 0 0.06 2 0 0.005 3 0.005 0.06 4 0 0.01 5 0 0.005 6 0 0.01 7 0 0.03 8 0 0.01 9 0 0 10 0.01 0.03 Intraintestinal antibiotic 11 0 0 12 0 0 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0 20 0 0 Rendered resistant 21 0.002 0.03 22 0 0.01 23 0 0.005 24 0 0.01 25 0 0 26 0.005 0.01 27 0 0 28 0 0.01 29 0.005 0.01 a D, died; S, survived. Ilg/ml 0 0.25 D 0.01 0.25 0.25 D 0.06 0.25 D 0.03 0.25 0.25 D 0.06 0.5 D 0.01 0.1 0.1 D 0.06 0.1 D 0.06 0.25 0.25 D 0.06 0.25 0.125 D 0 0 0.004 D 0 0 0.03 D 0 0 0.01 D 0 0 0.01 D 0 0 0.004 D 0.002 0 S 0.005 0 S 0.01 0.01 0.01 D severe than in the untreated rabbits. All rabbits that died had an endotoxemia at death. All survivors were free of endotoxemia when killed. The postmortem titer of endotoxin in the peritoneal fluid and plasma in survivors was zero. In those that died the average titers were less than 0.1 J-Lg per ml of plasma as compared with 1 to 2 J-Lg per ml of peritoneal fluid (table 4). In all but 2 of the 11 rabbits which received the entire dose of the vasoactive drug within 5 min there was a marked but transient hypotension, an endotoxemia (0.5 J-Lg per ml) which appeared within the first 15 min, and disappeared within 60 min, followed by a full and rapid return to the normal state. Discussion The foregoing data demonstrate that in the normal rabbit a continuous intravenous infusion for several hours of anyone of three of the vasoactive agents allows endotoxin to enter the circulation from the intestine in an amount sufficient to become a self-sustaining endotoxemia on discontinuing the infusion. The same result has been obtained with an infusion of norepinephrine. 10 The continuous release of

February 1973 END 0 TOXIC SHOCK BY VASOACTIVE SUBSTANCES 289 TABLE 3. Intravenous histamine, endotoxin in plasma Experiment 1 hr 2 hr 3 hr 4 hr Post- Survival a mortem Ilg/ml 1 0.03 0.06 0.06 0.61 0.01 D 2 0 0 0 0.12 1.0 D 3 0 0.03 0.03 0.03 0.03 D 4 1.0 1.0 1.0 1.0 1.0 D 5 1.0 0.25 0.06 1.0 0.03 D Intraintestinal antibiotic 6 1.0 0 1. 7 0 0.03 8 0.12 0.25 0.5 0.5 0.12 D 9 0 1.0 1.0 0 1.0 D 10 0 0 I 11 0.03 0.06 0.06 0.06 0.03 D 12 0 0 13 I 0.5 0.25 0.25 0.25 0.25 D 14 0 0 15 0.01 0.01 0.01 Endotoxin -resistant 16 0 0.06 1.0 0.12 0 S 17 0.06 0.5 0.5 2.0 2.0 D 18 0 1.0 0.5 19 0 1.0 0 2. 20 0 0.06 2.0 0.25 0 S 21 0 0 0 1. 22 0 0.5 0 0.25 1.0 D 23 0 0.12 0.12 0.06 0 S 24 0.005 0.01 0.03 25 0 0.03 0.002 a D, died; S, survived. TABLE 4. Endotoxin titer (micrograms per milliliter) in peritoneal fluid and plasma after a continuous 3-hr infusion of various vasoactive agents Vasoactive Treatment Peritoneal fluid Plas- Peritoneal fluid agent (survivors)a rna (nonsurvivors)a Plasma Bradykinin None N ~ 2 0.01 (±0.001) 0 N ~ 7 2.5 (±3)b 0.28 (±0.02) Antibiotic C N ~ 6 0.01 0 N ~ 2 1.0 (±0.03) 0.05 Resistant d N ~ 7 0 0 N ~ 1 0.1 0 Serotonin None N ~ 1 0 0 N ~ 7 2.7 (±0.3) 0.25 (±0.02) Antibiotic N ~ 4 0.01 (±0.001) 0 N ~ 4 1.0 (±0.0l) 0.01 (±0.001) Resistant N ~ 9 0 0 N ~ 1 0.1 0.01 Histamine None N ~ 0 N ~ 5 4 (±0.02) 0.5 (±0.26) Antibiotic N ~ 5 0.02 (±.00l) 0 N ~ 3 1.6 (±0.01) 0.35 (±0.005) Resistant N ~ 7 0 0 N ~ 2 3.0 1.5 (±0.5) a Samples were obtamed after death m nonsurvlvors and the day after the mfuslon m survivors. b Figures in parentheses ~ SD. C Antibiotic ~ intraintestinal kanamycin. d Resistant ~ induced resistance to endotoxin.

290 CUEVAS AND FINE Vol. 64, No.2 norepinephrine in states of sustained stress is a well established phenomenon. The continuous release of other vasoactive substances from a site of injury has been demonstrated in a current study of 30% immersion burns. Such a substance is detectable in the venous blood draining the burned area soon after the burn is inflicted. It produces an endotoxemia which is of intestinal origin because endotoxin is present in the arterial blood from 30 to 60 min before it appears in the venous blood from the burned area. If this substance is not one of those released to serve a physiologic function, it may be a protease or a peptidase from ruptured lysosomes. The induction of endotoxemia by the vasoactive substances was the result of their effect on the permeability of the intestinal wall, for the titer of endotoxin in the peritoneal fluid was 8 to 10 times that in the plasma of normal rabbits. In those pretreated with an intra intestinal antibiotic, the intraperitoneal titer was also higher than in plasma, but distinctly lower than in the untreated group, indicating a smaller intraintestinal amount of endotoxin available for absorption. The very low intraperitoneal titer in the endotoxinresistant rabbits can be accounted for by their capacity to extract circulating endotoxin fast enough to retard or prevent the mobilization of endotoxin from the intestine by a circulating residue. 12 The capacity of a persisting endotoxemia to become self-sustaining after the infusion of the vasoactive substance is discontinued appears to be because circulating endotoxin induces a continuing release of these substances from their sites of production.2. 5. 13 The endotoxemia of intestinal origin which has been observed in various types of shock-producing trauma (peritonitis, 14 intestinal ischemia, 15 and hemorrhagic hypotension 16, may also develop as a result of a similar process. The protection provided by intraintestinal antibiotics or by inducing increased resistance to endotoxin demonstrates that in all of these disorders the vasoactive substances, in so far as they are involved, do not produce lasting harm, provided the endotoxemia they evoke can be prevented or eliminated. This view accords with that of Webster and Clark that kallidin or bradykinin do not inflict lasting injury without the participation of endotoxin. 17 Since sympathetic denervation of a tissue protects it from damage by endotoxin,18 it appears that without the local release of norepinephrine other vasoactive agents cannot participate in the production of tissue damage by endotoxin. REFERENCES 1. Attar MA, Tingey HB, McLaughlin JS, et al : Bradykinin in human shock. Surg Forum 18:46, 1967 2. Hinshaw LB: Release of vasoactive agents and vascular effects of endotoxin, Microbial Tox ins, vol 5: Bacterial Endotoxins. New York, Academic Press, 1971, p 209-275 3. Lewis GP: Bradykinin-Biochemistry pharmacology and its physiological role in controlling local blood flow, The Scientific Bases of Medicine. London, Athlone Press, 1962, p 241-257 4. Miles AA: Local and systemic factors in shock. Fed Proc 20 (2):141-157,1961 5. Shayer RW: Relationship of induced histidine decarboxylase activity and histamine synthesis to shock from stress and from endotoxin. Am J Physiol 198 (6) :1187-1192, 1960 6. Zetterstrom BEM, Palmerio C, Fine J : Changes in tissue content of catechol amines in traumatic shock. Acta Chir Scand 128:13-19, 1964 7. Reinhold RB, Fine J: A technique for quanti tative measurement of endotoxin in human plasma. Proc Soc Exp Bioi Med 137:334-340, 1971 8. Prigal SJ: The induction of prolonged resistance against a lethal staphylococcic infection in mice with a single injection of an emulsified lipopolysaccharide. J Allergy 44:176-188, 1969 9. Cuevas P, de la Maza LM, Gilbert J, et al: The lung lesion in four different types of shock in rabbits. Arch Surg 104:319-322, 1972 10. Tamakuma S, Rojas-Corona R, Cuevas P, et al: Demonstration of a lethal endotoxemia of intestinal origin in refractory non-septic shock. Ann Surg 173:219-224, 1971 11. Woodruff PWH, O'Carroll DI, Koizumi S, et al: The role of the intestinal flora in major trauma. J Infect Dis (in press) 12. Caridis DT, Ishiyama M, Woodruff PWH, et al:

Febnwry 1973 END 0 TOXIC SHOCK BY VASOACTIVE SUBSTANCES 291 Role of the intestinal flora in clearance and detoxification of circulating endotoxin (in press) 13. Kimball HR, Melmon KL, Wolff SM: Endotoxin induced kinin production in man. Proc Soc Exp BioI Med 139: 1078-1082, 1972 14. Cuevas P, Fine J: Role of intra intestinal endotoxin in the death from peritonitis. Surg Gynecol Obstet 134:953-957, 1972 15. Cuevas P, Fine J: Demonstration of a lethal endotoxemia in experimental occlusion of the superior mesenteric artery. Surg Gynecol Obstet 133:81-83, 1971 16. Fine J, Caridis DT, Cuevas P,et al: Therapeutic implications of new developments in the study of refractory non-septic shock, Shock in Low and High Flow States. Amsterdam, Excerpta Medica, 1972 17. Webster ME, Clark WR: Significance of the callicrein-callidinogen-callidin system in shock. Am J Physiol 197:406-412, 1959 18. Zetterstrom BEM, Palmerio C, Fine J: Protection of functional and vascular integrity of the spleen in traumatic shock by denervation. Proc Soc Exp BioI Med 117:373-376, 1964