- Email: [email protected]
Heparin treatment of live Escherichia coli bacteremia in rats
JOURNAL
OF
SURGICIIL
RESEARCH,
HEPARIN
DAVID
L. HORWITZ,
M.D.,
3,
120-125
(1972)
TREATMENT OF LIVE BACTEREMIA IN PH.D.,*
THOMAS AND
V. N.
CLIFFORD
M.
SEPTICEMIA
IN MAN [4] and subhuman primates [6], and endotoxin shock in dogs [5] and rabbits [l], has been shown to be accompanied by disseminated intravascular coagulation (D.I.C.). For this reason, heparin has been advocated as an adjunctive treatment for shock accompanying septicemia. Both clinical and animal studies have produced conflicting data, however, on the efficacy of heparin treatment.
120 @ 1972 by Academic Preen, of reproduoiion in any form
Inc. reserved.
COLI
BALLANTINE, HERMAN,
M.D.,
t
ARNOLD
G.
CORAN,
M.D.,
M.D.
Hardaway and Johnson have shown that preheparinization of dogs subjected to Escherichia coli endotoxin will prevent the fall in fibrinogen and aid in maintaining blood pressure [5]. They gave a heparin dose of 600 units/kg intra-aortically just before the endotoxin, and 100 units/kg every 2 hr thereafter. Filkins and DiLuzio, studying SpragueDawley rats, found that heparin was protective against Salmonella enteriticlis endotoxin when a dose of 1000 units (about 4000 units/ kg) was given simultaneously with, or 16, 30, 60, or 120 min after endotoxin, but not at 180 or 240 min after endotoxin [3]. Margaretten, McKay, and Phillips also found increased survival in endotoxin shock after pretreating Columbia-Sherman rats with 2000 units intravenously [8]. On the other hand, Lillehei et al. have reported heparin (30 mg/kg) to be without effect on endotoxin shock in dogs [7]. Corrigan and Jordan have reported that in clinical cases heparin may reverse the consumption coagulopathy, but has no effect on survival [2]. Many of these conflicting data may result from speciesdifferences, as well as from differencesin dosage schedule and timing of heparin treatment. Furthermore, endotoxin shock has never been shown to be completely equivalent to septicemia caused by living bacteria. In many studies, the results have been ambiguous because of other treatments given simultaneously or because the volume of solution in which the heparin was administered has not been taken into account. We have therefore undertaken to study the effect of varying treatment schedules of heparin on septicemia pro-
5 The authors gratefully acknowledge the assistance of Navy Hospital Corpsman HM2 W. Hawker, and Mr. Frank Chang who performed a portion of this work while in the Summer Research Participation Program for High School Students sponsored by American University. Technical assistance was also provided by Navy Hospital Corpsmen W. Spenner, J. Ralston, S. Volker, S. Lucas, J. Fuller, F. Lane, and R. West. * Present address and address for reprints and correspondence : Department of Medicine, University of Chicago Hospital, Chicago, Illinois 60637. f Present address: Department of Surgery, Boston City Hospital, Boston, Massachusetts. From the Bureau of Medicine and Surgery, Navy Department, Research Task No. MR041.20.0287A2HB. Division of Experimental Surgery, Clinical Medical Sciences Department, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland 20014. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval service at large. The cxperimerits reported herein were conducted according to the principles set forth in Guide for Laboratory Animal Facilities and Care prepared by the Committee on the Guide for Laboratory Animal Resources, National Academy of SciencesNational Research Council. Submitted for publication December 6, 1971.
Copyright All rights
ESCHERICHIA
RATS5
HORWITZ
ET
AL.:
HEPARlN
duced by living E. coli. So that sufficient numbers of animals could be included, the study was conducted on rats.
TREATMENT
OF
Table
121
BACTEREMIA
1. Number
of Rats
Saline controls (no E. coli) Heparin (units/kg/hr)
Methods Male Sprague-Dawley rats (251), weighing 300-400 g each, were used in these studies. They were maintained on a normal laboratory diet with free access to water until the time of t,he study. At the onset of the study, each rat was anesthetized with pentobarbital sodium (Nembutal, Abbott), 25 mg/kg intraperitoneally. A left paramedian abdominal incision was made and the left kidney and renal vessels exposed transperitoneally. A no. 14 Jelco needle: was inserted into the left renal vein, and a catheter placed through it into the inferior vena cava. The catheter was secured in place with a silk ligature. A left nephrectomy was then performed, and the abdomen closed. While the method of infusion is admittedly crude, it was found to be the most reliable way to maintain the volume of infusion required for a sufficiently long period of time. In general, ten rats were studied at a time. Each group of ten was given the same heparin schedule, and sufficient groups of ten were studied for each heparin infusion schedule to attain at least ten animals surviving anesthesia and surgery, except for t’he control groups. Actual numbers in each group are shown in Table 1. Totals for each heparin schedule do not always equal a multiple of 10 because of complications of the cannulation procedure itself. Control groups were intermingled between experimental groups, Each rat was intermittently connected to a Harvard infusion pump and every 15 min infused for the number of seconds needed to give it one-fourth of its hourly heparin dose. The desired heparin doses were achieved by varying the concentration, but not the volume, of heparin solution infused. Sodium heparin U.S.P., 1000 units/cc, was diluted in normal saline so that rats could be infused with heparin solution at a rate of 0, 10, 30, 60, or 120 units/kg/hr while getting a constant volume of 2 cc/kg/hr regardless of heparin concentration. Septicemia was induced by intravenous in: Jelco
Laboratories.
10 30 60 120 Heparin 5 min before E. coli 0 10 30 60 120 Heparin 30 Fin after E. coli 10 30 60 120 120 Unit.s heparin/kg/hr after loading dose; minutes loading dose given after E. coli 15 30 60 120 5 min before B. coli
in Each
Group
No. rats surviving to E. coli 6 9 5 8
Anesthesia deaths
-
14 17 12 13 10
6 G 4 1 8
12 15 17 14
4 2 9 3
12 14 12 13 10
8 3 4 7 8
f&ion of live E. COG.For all studies, an enteropathic Dunwald strain of known serotype (kindly furnished by Dr. Lerner Hinshaw of the University of Oklahoma Medical Center) was grown in trypticase soy broth, washed free of unbound endotoxin (i.e., endotoxin not part of intact bacteria) with normal saline, and diluted in saline to a concentration of 2 x lO*O organisms per cubic centimeters. Each rat (except for controls) was infused with 7.5 cc/kg of the bacterial suspension. At either 5 min before or 15, 30, 60, or 120 min after E. coli infusion, each rat was given an intravenous loading dose of 200 units heparin/kg, after which the intermittent infusion described previously was begun. Rats were observed continuously for 24 hr after E. coli injection and their time of death, as evidenced by cessation of respiration, recorded to the nearest minute. No further anesthesia was administered and surviving rats were restrained with tape as they began to wake up. Statistical comparisons were by chi-square
122
JOURNAL
OF
SURGICAL
RESEARCH,
test for differences in survival frequencies, and by Friedman’s nonparametric test of ranked variates for comparisons between groups [lo]. The test used has been indicated in Results. Results In general, it took about 1 hr to prepare a group of ten rats for the study. During this interval, 23% of the rats died prior to E. coli administration. This figure, assumed to represent combined anesthetic and surgical deaths, did not vary significantly between the various groups studied (see Table 1). Therefore, while some deaths after E. coli may be due to late effects of surgery or anesthesia, we assume that this effect was present in all groups equally. Control groups, given a loading dose of 200 units/kg heparin followed by infusion at a rate of 10, 30, 60, or 120 units/kg/hr, showed no significantly excessive mortality above this (30% mortality in 24 hr) except for the group infused at 120 units/kg/hr where all of the rats expired within 24 hr (average survival was 9.2 hr). By chi-square test, this group differed significantly (P < .05) from all other control groups. Except for the 120 units/kg/hr group, all E. coli-infused rats had significantly higher mortality than their respective control groups. Figures l-3 show mortality curves (time after E. coli infusion plotted against percentage of animals still surviving at that time) for
Fig. 1. Survival of rats given a loading on the dose of heparin indicated.
dose of 200 units/kg
VOL.
13,
NO.
3,
SEPTEMBER
1972
various infusion schedules. In Fig. 1, which represents pretreatment with heparin 5 min before E. coli infusion, heparin has no significant effect in producing 24-hr survivors. Heparin infused at 10 or 30 units/kg/hr produced no significant change from saline-infused animals, while the group infused at 60 units/kg/ hr showed better survival for the first 6 hr but no greater long-term (24-hr) survival. As was the case in the control groups, heparin infused at 120 units/kg/hr had an initial detrimental effect. Animals surviving later into the septicemia period (over 400 min after E. coli infusion) did better with the higher doses of heparin (60 and 120 units/kg/hr, chi-square = 8.99, P < .Ol). Figure 2 shows the effects of beginning heparin infusion 30 min after E. coli infusion. After the initial 30 min after E. coli infusion (during which no heparin was given), all groups show a significantly better survival than the saline control of Fig. 1 (by Friedman’s test, chisquare = 6.125, P < 0.2). As before, increasing the heparin dose from 60 to 120 units/kg/hr seems detrimental early in the septic period, but slightly beneficial at later times. To investigate the effect of the time of the loading dose on survival, separate groups of rats were given the 200 units/kg loading dose at varying times in relation to E. coli infusion. All groups were then maintained on heparin at a rate of 120 units/kg/hr as this, from pre-
heparin
5 min
prior
to E. coli
infusion,
and maintained
HORWITZ
ET
AL.:
HEPARIN
TREATMENT
I’iy. 9. Survival of rats given the loading on the dose of heparin indicated.
dose of 200 units/kg
b’iy. 3. Survival heparin infusion
loading
of rats given a heparin of 120 units/kg/hr.
OF
heparin
30 min after
dose of 200 units/kg
at, the time
vious studies, seemed to give the best longterm (over 24-hr) survival. These results are shown in Fig. 3. As seen previously, rats given this larger dose showed better survival then it was begun late in the shock period (60 or 120 min after E. coli infusion). For rats surviving beyond 400 min, however, there was no significant difference in long-term survival. Random autopsy examination of the rats treated with 120 units/kg/hr showed no evidence of gross hemorrhage into the brain, although microscopic examination was not done. D&m&on Any study of septic shock or septicemia is complicated by the large number of variables
123
BACTEREMIA
E. coli infusion,
indicated,
and maintained
and maintained
on :t
involved. Clinical studies of necessity include patients with shock of differing etiologies and with varying and underlying diseases, and being treated in a large variety of ways. Animal studies differ in the dosage and origin of the bacteria or bacterial endotoxin used to induce shock or septicemia. We have attempted to eliminate all variables but a single therapeutic agent, heparin. Although the animals also received a form of t,herapy in fluid infusion, this was maintained constant for all groups. It is recognized that the rat is not an ideal animal for the study of shock. However, the type of data required can be obtained with statistical reliability only by using a suffi-
124
JOURNAL
OF
SURGICAL
RESEARCH,
ciently large number of animals. The data obtained should be useful in future studies in primate species. While our results cannot be strictly compared with those obtained on different species or using other means of inducing shock or septicemia, some comparisons are instructive. Basically, we confirm the results of Hardaway and Johnson [5], Filkins and DiLuzio [3], and Margaretten et al. [8] that very large doses (infused in repeated doses in the former study and as one dose in the latter two) cause an increased survival. Likewise, along with Lillehei [7], we have shown that smaller doses, particularly when given as a pretreatment dose, do not affect survival. Thus some of the conflicting data in previous studies may be attributed to differences in treatment schedules. Priano et al. showed no significant effect of heparin on endotoxin shock in dogs pretreated with 300 units/kg of heparin and maintained on a dosage of 300 units/kg/hr (9). While their average dose (75 units/kg/ hr) was comparable with ours, their study differed in species, in shock being induced by endotoxin rather than live bacteria, in pretreatment, and in heparin being given intermittently rather than continuously. On a units/kg/hr basis, the infusion rates of 10, 30, 60, and 120 units/kg/hr used by us would correspond to doses of 2800; 8400; 16,800; and 33,600 units given every 4 hrs to a 70-kg man. As we showed that only the higher doses are effective in improving survival, this may explain why Corrigan’s patients [2] who received a dose averaging 25 units/kg/hr did not show improved survival. It is quite interesting that his patients did, however, have their coagulation defects improved with heparin therapy. This suggests that the beneficial effect of heparin observed by us may be due to an undetermined property unrelated to its effects on the coagulation system. In interpreting data, it is important to note that although the groups treated with 60 or 120 units/kg/hr showed significantly longer survival, this refers only to short-term survival. Differences in 24-hr survivals were noted, but were not statistically significant. This suggests that 24-hr survival may not be the best criterion to use in evaluating any pos-
VOL.
13,
NO.
:j,
SEPTEMBER
1972
sible effects of heparin. While it may be argued that an effect is clinically significant only if it promotes long-term survival, we feel that in the treatment of septicemic conditions an important goal is interim support of the patient until effective antibiotic therapy is established. It is noteworthy that the highest dosage level of heparin studied, 120 units/kg/hr, had a detrimental effect if used prior to or in the early stages of septicemia, while it had a beneficial effect if used in the later stages of established septicemia. The reasons for this are not clear. Although the animals were studied immediately after a laparotomy for placement of the intravenous catheter, the rats receiving large heparin doses did not show any gross evidence of increased postoperative bleeding. While increased microscopic bleeding may have been responsible for the increased early mortality of these animals, there nevertheless seemed to be an advantage in the high doses of heparin when begun well after the onset of septicemia. In conclusion, our study indicates that very large doses of heparin result in improved survival when administered after septicemia has become well established. These effective doses of heparin are considerably larger than would be needed to affect the coagulation system. This suggests a need for further investigation into the possible therapeutic effects of heparin unrelated to its anticoagulant activity. The effect of heparin treatment on survival in 251 from septicemia was studied Sprague-Dawley rats. Both onset of treatment and dose of heparin were varied, while the dose of live E. coli used to induce septicemia and the volume in which the heparin was infused were kept constant. The data suggest that very high doses of heparin (60 or 120 units/kg/hr) are most effective, and that they are more effective when given 1 or 2 hr after onset of septicemia, rather than when given before or shortly after the infusion of live E. coli.
REFERENCES 1. Beller, nated
F. T., Graeff, intravascular
H., and Gorstein, coagulation during
T. Dissemithe contin-
HOR\Y
2.
3.
4.
5.
6.
ITZ
ET
.4L.
: HEI’AIZIN
uous infusion of cwlolosin in r:rl)l)ils. Arrlc!r. J. Ohtet. Gytaecol. 103:544. 1969. Corrigan. J. J. and Jordan, C. M. Hcpwin tllerttl)> in scptiwmia with disseminated intri1V:XXUl:U co:ymlation. N. Ejtgl. J. Med. 283:778. 1970. Fill&s. J. P. and DiT,uzio, 9. R. Hrparin protection in rndotosin ~!10&. Amer. *I. Physiol. 214 : 1074, 1968. Goldenfarh. I’. IS., %u~l
TliEATRlENT
7.
8.
9.
10.
OF
BACTEREXIIA
I’?-t .-)
Cuugul:ition cl~;u~pc~s of acptic shock in that b:r1,oon. ret/. Z’roc. 29:381A. 1970. I,illelwi. 1~. C.. I,ongrrheam, J. Ii.. Bloch, J. H.. rind M;tnax. W. 0. Hcmodynamic Anngrs in cntlotosin shock. In I,. C. Mills. and J. H. Moyel (Eds.). Shock rrntl Hypotension, 1,. 442. Ncn To&: Grune & Stratton, 1965. Margaretten, M’.. McKay, D. G., and Phillips. I,. I,. The effect. of hq)wrin on rndotoxin shock in t,hcx rat. Amer. J. Pnthol. 51:61. 1967. Priano, T,. I,., Wilson, R. D.. and Traher. D. L. T,:lcl< of significnnt protection afforded hy hcpnrin during cndotoxic shock. Amer. J. Physiol. 220:901. 1971. So!&. R. It. and Rohlf, I?. J. Biometry, p, 296. Snn Francisco: W. H. Freeman nnd Co.. 1969.
OF
SURGICIIL
RESEARCH,
HEPARIN
DAVID
L. HORWITZ,
M.D.,
3,
120-125
(1972)
TREATMENT OF LIVE BACTEREMIA IN PH.D.,*
THOMAS AND
V. N.
CLIFFORD
M.
SEPTICEMIA
IN MAN [4] and subhuman primates [6], and endotoxin shock in dogs [5] and rabbits [l], has been shown to be accompanied by disseminated intravascular coagulation (D.I.C.). For this reason, heparin has been advocated as an adjunctive treatment for shock accompanying septicemia. Both clinical and animal studies have produced conflicting data, however, on the efficacy of heparin treatment.
120 @ 1972 by Academic Preen, of reproduoiion in any form
Inc. reserved.
COLI
BALLANTINE, HERMAN,
M.D.,
t
ARNOLD
G.
CORAN,
M.D.,
M.D.
Hardaway and Johnson have shown that preheparinization of dogs subjected to Escherichia coli endotoxin will prevent the fall in fibrinogen and aid in maintaining blood pressure [5]. They gave a heparin dose of 600 units/kg intra-aortically just before the endotoxin, and 100 units/kg every 2 hr thereafter. Filkins and DiLuzio, studying SpragueDawley rats, found that heparin was protective against Salmonella enteriticlis endotoxin when a dose of 1000 units (about 4000 units/ kg) was given simultaneously with, or 16, 30, 60, or 120 min after endotoxin, but not at 180 or 240 min after endotoxin [3]. Margaretten, McKay, and Phillips also found increased survival in endotoxin shock after pretreating Columbia-Sherman rats with 2000 units intravenously [8]. On the other hand, Lillehei et al. have reported heparin (30 mg/kg) to be without effect on endotoxin shock in dogs [7]. Corrigan and Jordan have reported that in clinical cases heparin may reverse the consumption coagulopathy, but has no effect on survival [2]. Many of these conflicting data may result from speciesdifferences, as well as from differencesin dosage schedule and timing of heparin treatment. Furthermore, endotoxin shock has never been shown to be completely equivalent to septicemia caused by living bacteria. In many studies, the results have been ambiguous because of other treatments given simultaneously or because the volume of solution in which the heparin was administered has not been taken into account. We have therefore undertaken to study the effect of varying treatment schedules of heparin on septicemia pro-
5 The authors gratefully acknowledge the assistance of Navy Hospital Corpsman HM2 W. Hawker, and Mr. Frank Chang who performed a portion of this work while in the Summer Research Participation Program for High School Students sponsored by American University. Technical assistance was also provided by Navy Hospital Corpsmen W. Spenner, J. Ralston, S. Volker, S. Lucas, J. Fuller, F. Lane, and R. West. * Present address and address for reprints and correspondence : Department of Medicine, University of Chicago Hospital, Chicago, Illinois 60637. f Present address: Department of Surgery, Boston City Hospital, Boston, Massachusetts. From the Bureau of Medicine and Surgery, Navy Department, Research Task No. MR041.20.0287A2HB. Division of Experimental Surgery, Clinical Medical Sciences Department, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland 20014. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval service at large. The cxperimerits reported herein were conducted according to the principles set forth in Guide for Laboratory Animal Facilities and Care prepared by the Committee on the Guide for Laboratory Animal Resources, National Academy of SciencesNational Research Council. Submitted for publication December 6, 1971.
Copyright All rights
ESCHERICHIA
RATS5
HORWITZ
ET
AL.:
HEPARlN
duced by living E. coli. So that sufficient numbers of animals could be included, the study was conducted on rats.
TREATMENT
OF
Table
121
BACTEREMIA
1. Number
of Rats
Saline controls (no E. coli) Heparin (units/kg/hr)
Methods Male Sprague-Dawley rats (251), weighing 300-400 g each, were used in these studies. They were maintained on a normal laboratory diet with free access to water until the time of t,he study. At the onset of the study, each rat was anesthetized with pentobarbital sodium (Nembutal, Abbott), 25 mg/kg intraperitoneally. A left paramedian abdominal incision was made and the left kidney and renal vessels exposed transperitoneally. A no. 14 Jelco needle: was inserted into the left renal vein, and a catheter placed through it into the inferior vena cava. The catheter was secured in place with a silk ligature. A left nephrectomy was then performed, and the abdomen closed. While the method of infusion is admittedly crude, it was found to be the most reliable way to maintain the volume of infusion required for a sufficiently long period of time. In general, ten rats were studied at a time. Each group of ten was given the same heparin schedule, and sufficient groups of ten were studied for each heparin infusion schedule to attain at least ten animals surviving anesthesia and surgery, except for t’he control groups. Actual numbers in each group are shown in Table 1. Totals for each heparin schedule do not always equal a multiple of 10 because of complications of the cannulation procedure itself. Control groups were intermingled between experimental groups, Each rat was intermittently connected to a Harvard infusion pump and every 15 min infused for the number of seconds needed to give it one-fourth of its hourly heparin dose. The desired heparin doses were achieved by varying the concentration, but not the volume, of heparin solution infused. Sodium heparin U.S.P., 1000 units/cc, was diluted in normal saline so that rats could be infused with heparin solution at a rate of 0, 10, 30, 60, or 120 units/kg/hr while getting a constant volume of 2 cc/kg/hr regardless of heparin concentration. Septicemia was induced by intravenous in: Jelco
Laboratories.
10 30 60 120 Heparin 5 min before E. coli 0 10 30 60 120 Heparin 30 Fin after E. coli 10 30 60 120 120 Unit.s heparin/kg/hr after loading dose; minutes loading dose given after E. coli 15 30 60 120 5 min before B. coli
in Each
Group
No. rats surviving to E. coli 6 9 5 8
Anesthesia deaths
-
14 17 12 13 10
6 G 4 1 8
12 15 17 14
4 2 9 3
12 14 12 13 10
8 3 4 7 8
f&ion of live E. COG.For all studies, an enteropathic Dunwald strain of known serotype (kindly furnished by Dr. Lerner Hinshaw of the University of Oklahoma Medical Center) was grown in trypticase soy broth, washed free of unbound endotoxin (i.e., endotoxin not part of intact bacteria) with normal saline, and diluted in saline to a concentration of 2 x lO*O organisms per cubic centimeters. Each rat (except for controls) was infused with 7.5 cc/kg of the bacterial suspension. At either 5 min before or 15, 30, 60, or 120 min after E. coli infusion, each rat was given an intravenous loading dose of 200 units heparin/kg, after which the intermittent infusion described previously was begun. Rats were observed continuously for 24 hr after E. coli injection and their time of death, as evidenced by cessation of respiration, recorded to the nearest minute. No further anesthesia was administered and surviving rats were restrained with tape as they began to wake up. Statistical comparisons were by chi-square
122
JOURNAL
OF
SURGICAL
RESEARCH,
test for differences in survival frequencies, and by Friedman’s nonparametric test of ranked variates for comparisons between groups [lo]. The test used has been indicated in Results. Results In general, it took about 1 hr to prepare a group of ten rats for the study. During this interval, 23% of the rats died prior to E. coli administration. This figure, assumed to represent combined anesthetic and surgical deaths, did not vary significantly between the various groups studied (see Table 1). Therefore, while some deaths after E. coli may be due to late effects of surgery or anesthesia, we assume that this effect was present in all groups equally. Control groups, given a loading dose of 200 units/kg heparin followed by infusion at a rate of 10, 30, 60, or 120 units/kg/hr, showed no significantly excessive mortality above this (30% mortality in 24 hr) except for the group infused at 120 units/kg/hr where all of the rats expired within 24 hr (average survival was 9.2 hr). By chi-square test, this group differed significantly (P < .05) from all other control groups. Except for the 120 units/kg/hr group, all E. coli-infused rats had significantly higher mortality than their respective control groups. Figures l-3 show mortality curves (time after E. coli infusion plotted against percentage of animals still surviving at that time) for
Fig. 1. Survival of rats given a loading on the dose of heparin indicated.
dose of 200 units/kg
VOL.
13,
NO.
3,
SEPTEMBER
1972
various infusion schedules. In Fig. 1, which represents pretreatment with heparin 5 min before E. coli infusion, heparin has no significant effect in producing 24-hr survivors. Heparin infused at 10 or 30 units/kg/hr produced no significant change from saline-infused animals, while the group infused at 60 units/kg/ hr showed better survival for the first 6 hr but no greater long-term (24-hr) survival. As was the case in the control groups, heparin infused at 120 units/kg/hr had an initial detrimental effect. Animals surviving later into the septicemia period (over 400 min after E. coli infusion) did better with the higher doses of heparin (60 and 120 units/kg/hr, chi-square = 8.99, P < .Ol). Figure 2 shows the effects of beginning heparin infusion 30 min after E. coli infusion. After the initial 30 min after E. coli infusion (during which no heparin was given), all groups show a significantly better survival than the saline control of Fig. 1 (by Friedman’s test, chisquare = 6.125, P < 0.2). As before, increasing the heparin dose from 60 to 120 units/kg/hr seems detrimental early in the septic period, but slightly beneficial at later times. To investigate the effect of the time of the loading dose on survival, separate groups of rats were given the 200 units/kg loading dose at varying times in relation to E. coli infusion. All groups were then maintained on heparin at a rate of 120 units/kg/hr as this, from pre-
heparin
5 min
prior
to E. coli
infusion,
and maintained
HORWITZ
ET
AL.:
HEPARIN
TREATMENT
I’iy. 9. Survival of rats given the loading on the dose of heparin indicated.
dose of 200 units/kg
b’iy. 3. Survival heparin infusion
loading
of rats given a heparin of 120 units/kg/hr.
OF
heparin
30 min after
dose of 200 units/kg
at, the time
vious studies, seemed to give the best longterm (over 24-hr) survival. These results are shown in Fig. 3. As seen previously, rats given this larger dose showed better survival then it was begun late in the shock period (60 or 120 min after E. coli infusion). For rats surviving beyond 400 min, however, there was no significant difference in long-term survival. Random autopsy examination of the rats treated with 120 units/kg/hr showed no evidence of gross hemorrhage into the brain, although microscopic examination was not done. D&m&on Any study of septic shock or septicemia is complicated by the large number of variables
123
BACTEREMIA
E. coli infusion,
indicated,
and maintained
and maintained
on :t
involved. Clinical studies of necessity include patients with shock of differing etiologies and with varying and underlying diseases, and being treated in a large variety of ways. Animal studies differ in the dosage and origin of the bacteria or bacterial endotoxin used to induce shock or septicemia. We have attempted to eliminate all variables but a single therapeutic agent, heparin. Although the animals also received a form of t,herapy in fluid infusion, this was maintained constant for all groups. It is recognized that the rat is not an ideal animal for the study of shock. However, the type of data required can be obtained with statistical reliability only by using a suffi-
124
JOURNAL
OF
SURGICAL
RESEARCH,
ciently large number of animals. The data obtained should be useful in future studies in primate species. While our results cannot be strictly compared with those obtained on different species or using other means of inducing shock or septicemia, some comparisons are instructive. Basically, we confirm the results of Hardaway and Johnson [5], Filkins and DiLuzio [3], and Margaretten et al. [8] that very large doses (infused in repeated doses in the former study and as one dose in the latter two) cause an increased survival. Likewise, along with Lillehei [7], we have shown that smaller doses, particularly when given as a pretreatment dose, do not affect survival. Thus some of the conflicting data in previous studies may be attributed to differences in treatment schedules. Priano et al. showed no significant effect of heparin on endotoxin shock in dogs pretreated with 300 units/kg of heparin and maintained on a dosage of 300 units/kg/hr (9). While their average dose (75 units/kg/ hr) was comparable with ours, their study differed in species, in shock being induced by endotoxin rather than live bacteria, in pretreatment, and in heparin being given intermittently rather than continuously. On a units/kg/hr basis, the infusion rates of 10, 30, 60, and 120 units/kg/hr used by us would correspond to doses of 2800; 8400; 16,800; and 33,600 units given every 4 hrs to a 70-kg man. As we showed that only the higher doses are effective in improving survival, this may explain why Corrigan’s patients [2] who received a dose averaging 25 units/kg/hr did not show improved survival. It is quite interesting that his patients did, however, have their coagulation defects improved with heparin therapy. This suggests that the beneficial effect of heparin observed by us may be due to an undetermined property unrelated to its effects on the coagulation system. In interpreting data, it is important to note that although the groups treated with 60 or 120 units/kg/hr showed significantly longer survival, this refers only to short-term survival. Differences in 24-hr survivals were noted, but were not statistically significant. This suggests that 24-hr survival may not be the best criterion to use in evaluating any pos-
VOL.
13,
NO.
:j,
SEPTEMBER
1972
sible effects of heparin. While it may be argued that an effect is clinically significant only if it promotes long-term survival, we feel that in the treatment of septicemic conditions an important goal is interim support of the patient until effective antibiotic therapy is established. It is noteworthy that the highest dosage level of heparin studied, 120 units/kg/hr, had a detrimental effect if used prior to or in the early stages of septicemia, while it had a beneficial effect if used in the later stages of established septicemia. The reasons for this are not clear. Although the animals were studied immediately after a laparotomy for placement of the intravenous catheter, the rats receiving large heparin doses did not show any gross evidence of increased postoperative bleeding. While increased microscopic bleeding may have been responsible for the increased early mortality of these animals, there nevertheless seemed to be an advantage in the high doses of heparin when begun well after the onset of septicemia. In conclusion, our study indicates that very large doses of heparin result in improved survival when administered after septicemia has become well established. These effective doses of heparin are considerably larger than would be needed to affect the coagulation system. This suggests a need for further investigation into the possible therapeutic effects of heparin unrelated to its anticoagulant activity. The effect of heparin treatment on survival in 251 from septicemia was studied Sprague-Dawley rats. Both onset of treatment and dose of heparin were varied, while the dose of live E. coli used to induce septicemia and the volume in which the heparin was infused were kept constant. The data suggest that very high doses of heparin (60 or 120 units/kg/hr) are most effective, and that they are more effective when given 1 or 2 hr after onset of septicemia, rather than when given before or shortly after the infusion of live E. coli.
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