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Failure of mestranol alone or with norethynodrel to prepare for the generalized Shwartzman reaction in rats
Failure of mestranol alone or with norethynodrel to prepare for the generalized Shwartzman reaction in rats LOUISE FAYE
LANG J.
HENRY Washington,
PHILLIPS,
CALHOUN, B.
SOLOWAY,
PH.D.* M.S. M.D.
D. C.
Female rats were given mestranol alone or in combination with norethynodrel for one or 2 months prior to intravenous injection of endotoxin. None of the drug-treated animals showed histologic evidence of the generalized Shwartrman reaction as did pregnant rats. Serial platelet counts and profibrinolysin levels indicate that intravascular coagulation in resfonse to endotoxin is minimal in the treated groutis as compared with the pregnant animals. Therefore, it may be concluded that administration of mestranol alone or in combination with norethynodrel does not potentiate the generalized Shwartzman reaction in rats as does pregnancy.
T H E R E L A T I 0 N s H I P between Oral contraceptive therapy and thromboembolic phenomena has been under investigation for some years. Recent data from England indicate that there is a seven- to ninefold increase in deaths from pulmonary embolus and cerebral thrombosis among women using oral contraceptives1 and a ninefold increase in hospital admissions from thrombophlebitis and thromboembolism.2 Studies of coagulation factors in women using oral contraceptives have shown changes which are similar to many of those observed in pregnancy. 3-6 There is no evidence, however, that the presence of elevated coagulation factors causes thrombotic episodes. Therefore, some additional stimulus is probably involved in the complications found among users of the hormonal contraceptives. From the Bureau Drug Administration, of Surgery, Walter of
Research.
The generalized Shwartzman reaction (GSR) refers to the process of disseminated intravascular coagulation induced by the administration of endotoxin. Two doses of endotoxin are usually required for the production of GSR in normal animals.7 ‘Pregnancy, however, prepares an animal for the development of GSR so that only a single dose of endotoxin is required.*? g The mechanism whereby this occurs is not known, but should it be related to elevated levels of circulating clotting factors, then oral contraceptive therapy should prepare for the GSR as does pregnancy. This mechanism could explain the increased incidence of thrombosis in women on birth control pills. The following experiments were undertaken to test this hypothesis using rats given prolonged courses of Enovid 5 or mestranol administered orally.
Medicine, Food and HEW, and Division Reed Army Institute
of
‘Present address: The Roosevelt Hospital, 428 West 59 Street, New New York 10019.
Material
and
methods
Osborne-Mendel female rats weighing 150 to 200 grams were randomly divided into groups of 8 animals each. They were
York,
210
Volume Number
107 2
Generalized
Shwartzman
reaction
in rats
211
Table I
Group : 3 % 6 7 8 9 10 11 12 13
NO. Of rats 8 8 8 8 8 8 8 8 8 8 8
Drug Corn oil Corn oil EnovidEnovidEnovidEnovidCorn oil Corn oil Mestranol Mestranol Mestranol Mestranol Pregnantcation
Dose (mcg./Kg. /day) 700 700 7,000 7,000 10.5 10.5 105 105 -no medi-
Months therapy
of
1 2 1 2 1 2 1 2 1 2 1 2 20 days’ gestation
dosed daily by gavage according to the schedule shown in Table I. The Enovid 5 (norethynodrel .and mestranol in a 67: 1 ratio) and mestranol were dissolved in corn oil in concentrations so that the desired dose could be administered in 0.1 to 0.2 ml. of oil. This amounted to approximately 7 and 70 times the human dosage. The control groups were given corn oil without added drugs. The pregnant animals were not medicated. Groups of rats were killed after one or 2 months therapy as indicated in Table I. The day before they were killed they were injected via the tail vein with 400 mcg. of E. coli endotoxin.” Platelet countslO were performed before endotoxin and again 5 minutes, 4 hours, and 24 hours after endotoxin. Twenty-four hours after injection of endotoxin the animaIs were anesthetized with ether. Blood was drawn from the inferior vena cava for profibrinolysin assaysI and tissues were collected in 10 per cent buffered formalin for histologic studies. T:he principles of laboratory care as promulgated by the National Research Council were observed. Results Initial platelet counts in all groups were similar with the: exception of those animals ‘Elacto Laboratory,
lipopolysarxharide Detroit, Michigan.
B.
E.
coil
0127:B8,
Difco
Fig. 1. Changes in platelets, expressed as per cent of original count, after injection of endotoxin to Enovid-treated and pregnant rats. Results of animaIs dosed for one and 2 months are averaged.
receiving high doses of mestranol. Since the values obtained in groups treated for one month and those threated for 2 months were virtually the same, the results have been averaged for purposes of presentation. Changes in platelet counts after injection of endotoxin to Enovid-treated and pregnant rats are shown in Fig. 1. Within 5 minutes of endotoxin, the counts fell to 40 to 50 per cent of the original level and were approximately the same 4 hours after injection. Twenty-four hours after endotoxin the platelet counts had risen in both control and Enovid-treated animals to 60 or 70 per cent of the original level but remained low in the pregnant group. There is a statistically significant difference between the pregnant and control groups, borderline significance between the pregnant and Enovid-treated
212
Phillips,
Calhoun,
and
Soloway
Amer.
PROFIEIRINOLYSIN
LEVELS
24 HOURS
AFTER
May J. Obstet.
15, 1970 Gynec.
ENDOTOXIN
unlts
20
10
5
0 Normal
Control
Low dose
Fig. 2. Profibrinoiysin levels obtained in control, after endotoxin. Levels of untreated nonpregnant Values are reported as units with one standard
High dose
Enovid-treated, rats without error,
Pregnant
and pregnant endotoxin are
rats 24 hours shown on left.
Table II. Rats dosed with mestranol for one of 2 months Groujs 7 and
8
9 and
Control (corn oil) Dose Platelets Time
after
No.
(10.5 %
10
I1 and
Low-dose mcg./Kg./day)
No.
12
High-dose (I 05 mcg./Kg./day) 70
No.
%
endotoxin
0 Count/c.c. P”
x IO-3
I,01 1
100
955 -
100 -
823 0.001
100 -
X 1O-3
550
55.2
436 0.01
46.3 -
372 0.001
45.6 -
X 1O-3
788
78.1
671
71.2 -
514 0.001
63.2 0.05
81.6
661
81.2
5 minutes Count/c.c. P* 4 hours Count/cc. P*
0.01
24 hours Count/cc. P+
881
X lO-$
87.7
775
Profibrinolysin Units Standard l P expresses
the probability
8.25 + 1.09
8.38 2 1.58
error of
stati&calIy
significant
differences
from
contro1
values.
8.02 _+ 0.99
Volume Number
1107 2
Fig. 3. Fibrin of rndotoxin.
Generalized
thrombi
found
in
the
glomerular
animals, but no significant differences between control rats and those treated with Enovid. Profibrinolysin levels obtained in controls, Enovi.d-treated, and pregnant rats 24 hours after endotoxin are shown in Fig. 2. Levels in untreated nonpregnant rats without endotoxin are also included for comparison. The increases in the corn oil (control) and Enovid-treated animals are not statistically significant and rnay be related in part to pneumonia which was evidenced by leukocyte infiltration into the lungs of 80 to 90 per cent of the animals in all groups subjected to repeated gavage. The low levels of profibrinolysin found in the pregnant anim,als are significantly different from the other groups with a probability of less than 0.01. Data on platelet counts and profibrinolysin values of mestranol-treated rats and their concomitant controls are recorded in Table II. Platelet counts are significantly louver than those of controls at all times (including pre-injection levels) in the group receiving the high dose of mestranol and after endotoxin in the low-dose group. The difference in per cent of original count, however, is significant only for the high dose group at 4 hours following endotoxin (p = 0.05).
capillaries
Shwartzman
of a pregnant
reaction
rat
after
in rats
213
injection
Profibrinolysin levels were similar in the three groups. The characteristic lesions of the generalized Shwartzman reaction are fibrin thrombi within the glomerular capillaries. These lesions found in pregnancy following endotoxin are illustrated in Fig. 3. Twenty-five per cent of the pregnant animals showed evidence of the GSR and another 12.5 per cent died in less than 24 hours after endotoxin from thrombi in areas other than the kidney. Fibrin thrombi were not observed in the kidneys of any of 64 animals pretreated lvith Enovid or mestranol. One control rat, which died three hours after endotoxin had fibrin deposits similar to those found in pregnancy. Since this was found in a control animal (1 of 32) it must be considered as an unusual chance occurrence in a rat particularly sensitive to endotoxin. Three other rats, one each from control? low-dose, and high-dose Enovid groups died within 4 to 6 hours after endotoxin. Microscopic examination failed to disclose evidence of GSR in these animals. One of them, however, had a platelet thrombus containing fibrin strands within an hepatic vessel (Fig. 4) . This rat had received ‘100 mcg. Enovid per kilogram per day for one month. The significance of this observation is not apparent.
214
Phillips,
Calhoun,
May 15, 1970 Amer. J. Obstet. Gynec.
and Soloway
Fig. 4. Platelet thrombus found in an hepatic vessel after endotoxin administered fed 700 mcg. Einovid per kilogram of body weight per day for one month.
to a
rat
Fig. 5. Centrilobular necrosis of the liver found in many control lowing injection of endotoxin.
rats
fol-
The control trilobular appears toxin. animals related
lesion most frequently observed in and experimental animals was cennecrosis of the liver (Fig. 5). This to be a nonspecific reaction to endoIt has been observed in heparinized following endotoxin and thus is unto intravascular coagulation.12
Comment The generalized Shwartzman reaction was originally described and defined as the re-
and
drug-treated
sponse in rabbits to two doses of endotoxin administered intravenously 24 hours apart.’ The first injection was called the preparative dose and the second the provoking dose. The reaction is now defined as the result of any process causing deposition and retention of fibrin thrombi in the glomeruli and leading to renal cortical necrosis.13 Many stimuli can take the place of the preparative dose of endotoxin. In rats the principal conditions which may substitute are pregnancy,g inhi-
Volume 107 Number
Generalized
Shwartzman
reaction
in rats
215
2
bition Iof the fibrinolytic system with epsilon of aminoc.aproic acid,14 or administration cortisone for three days before injection of endotoxin? Cooper and McKaylG showed that estrogen, progesterone, or their combination in doses comparable to those of cortisone did not prepare for the GSR. Therefore, the effect of cortisone seems to be a response to car-ticosteroids rather than a nonspecific response to steroids in general. The possibility remained, however, that chronic administration of these agents, by enhancement of the factors of the coagulation system, might prepare for the reaction. More recently, Nolan and Vilayat Alil’ demonstrated that estrogen injected intraperitoneally 18 to 48 hours prior to endotoxin increased the toxicity of endotoxin tenfold. Fibrin thrombi were not found in the kidneys and heparin did not protect against this enhanced lethality. Therefore, it was, not a generalized Shwartzman reaction and the response was not mediated through an effect on the coagulation mechanism. The data reported in the present paper indicate that neither mestranol alone nor the combination of norethynodrel and mestranol in large doses, prepares for the generalized Shwartzman reaction in rats as does pregnancy. No fibrin thrombi were found in the renal glomeruli in any of treated groups despite the fact that this same dose of endotoxin elicited the GSR in pregnant animals. The platelet thrombus found in the hepatic vessel of o-ne animal on Enovid can be found in normal animals following the first injection of endotoxin and is not neces-
sarily related to the Shwartzman reaction. The more frequently observed hepatic necrosis is also a nonspecific reaction to endotoxin and can be found in heparinized rats.l” In a previous study it was shown that activation of the fibrinolytic system as a secondary response to intravascular coagulation results in low levels of profibrinolysin in rats 24 hours after the challenge.ls The normal or elevated profibrinolysin levels found in control and drug-treated groups as compared to low levels found in pregnant rats is further evidence that extensive intravascular coagulation was not triggered by injection of endotoxin in these treated animals. Platelet counts were severely reduced in all groups of rats after administration of endotoxin. The failure of the pregnant animals to restore the platelet levels within 24 hours suggests a more intense effect of the endotoxin in this group. The mechanism by which platelet counts are depressed before endotoxin in the animals on high doses of mestranol remains unexplained and perhaps deserves further investigation. It may be concluded that high doses of mestranol alone or in combination with norethynodrel (Enovid) , administered orally for up to 2 months, do not prepare rats for the generalized Shwartzman reaction as does pregnancy. It would appear that the mechanism by which pregnancy prepares for the Shwartzman reaction is not due exclusively to an increase in coagulation factors. The data also suggest that the increase in thromboembolic disorders reported in users of oral contraceptives is not mediated through the Shwartzman phenomenon.
REFERENCES
1. Illman,
5.
2.
6.
3.
4.
W. H. W., and Vessey, M. P.: Brit. Med. J. 2: 193, 1968. Vessey, M. P., and Doll, R.: Brit. Med. J. 2: 199, 1968. Rutherford, R. N., Hougie, C., Banks, A. L., and Coburn, W. A.: Obstet. Gynec. 24: 886, 1964. Miller, S. P., Lee, S. L., and Ritz, N.: Metabolism 14: 398, 1965. (Suppl.)
7.
Brakman, R., Albrechtsen, 0. K., and Astrup, T.: J. A. M. A. 199: 69, 1967. Phillips, L. L., Turksoy, R. N., and Southam, A. L.: AMER. J. OBSTET. GYNEC. 82: 1216, 1961. Shwartzman, G.: Phenomenon of Local Tissue Reactivity and Its Immunological, Pathological and Clinical Significance, New York, 1937, Paul B. Hoeber, Inc.
216
8. 9. 10. 11. 12. 13.
14.
Phillips,
Calhoun,
Apitz, K.: Virchow 1, 1934. Wring, T. C.: AMER.
and
Arch.
Soloway
Path.
.T. OBSTET.
Amer.
Anat. GYNEC.
293:
84:
Brecher, G., and Cronkite, E. P.: J. Appl. Phvsiol. 3: 365. 1950. Phillips, L. L:, and Skrodelis, V.: J. Clin. Invest. 37: 965, 1958. Margaretten, W., McKay, D. G., and Phillips, L. L.: Amer. J. Path. 51: 61, 1967. McKay, D. G.: Disseminated Intravascular Coagulation, New York, 1965, Hoeber Medical Division, Harper & Row. Margaretten, W., and McKay, D. G.: Fed. Proc. 22: 251, 1963.
15. 16. 17. 18.
May 15, 1970 J. Obstet. Gynec.
Thomas, L., and Good, R. A.: J. Exp. Med. 95: 409, 1952. Cooper, A. B., and McKay, D. G.: Fed. Proc. 19: 246, 1960. Nolan, J. P.; and Vilayat Ali, M.: J. Lab. Clin. Med. 71: 501. 1968. Phillips, L. L., Maigaretten, W., and McKay, D. G.: AMER. J. OBSTET. GYNEC. 100: 319, 1968.
428 West 59th Street New York, New York 10019
LANG J.
HENRY Washington,
PHILLIPS,
CALHOUN, B.
SOLOWAY,
PH.D.* M.S. M.D.
D. C.
Female rats were given mestranol alone or in combination with norethynodrel for one or 2 months prior to intravenous injection of endotoxin. None of the drug-treated animals showed histologic evidence of the generalized Shwartrman reaction as did pregnant rats. Serial platelet counts and profibrinolysin levels indicate that intravascular coagulation in resfonse to endotoxin is minimal in the treated groutis as compared with the pregnant animals. Therefore, it may be concluded that administration of mestranol alone or in combination with norethynodrel does not potentiate the generalized Shwartzman reaction in rats as does pregnancy.
T H E R E L A T I 0 N s H I P between Oral contraceptive therapy and thromboembolic phenomena has been under investigation for some years. Recent data from England indicate that there is a seven- to ninefold increase in deaths from pulmonary embolus and cerebral thrombosis among women using oral contraceptives1 and a ninefold increase in hospital admissions from thrombophlebitis and thromboembolism.2 Studies of coagulation factors in women using oral contraceptives have shown changes which are similar to many of those observed in pregnancy. 3-6 There is no evidence, however, that the presence of elevated coagulation factors causes thrombotic episodes. Therefore, some additional stimulus is probably involved in the complications found among users of the hormonal contraceptives. From the Bureau Drug Administration, of Surgery, Walter of
Research.
The generalized Shwartzman reaction (GSR) refers to the process of disseminated intravascular coagulation induced by the administration of endotoxin. Two doses of endotoxin are usually required for the production of GSR in normal animals.7 ‘Pregnancy, however, prepares an animal for the development of GSR so that only a single dose of endotoxin is required.*? g The mechanism whereby this occurs is not known, but should it be related to elevated levels of circulating clotting factors, then oral contraceptive therapy should prepare for the GSR as does pregnancy. This mechanism could explain the increased incidence of thrombosis in women on birth control pills. The following experiments were undertaken to test this hypothesis using rats given prolonged courses of Enovid 5 or mestranol administered orally.
Medicine, Food and HEW, and Division Reed Army Institute
of
‘Present address: The Roosevelt Hospital, 428 West 59 Street, New New York 10019.
Material
and
methods
Osborne-Mendel female rats weighing 150 to 200 grams were randomly divided into groups of 8 animals each. They were
York,
210
Volume Number
107 2
Generalized
Shwartzman
reaction
in rats
211
Table I
Group : 3 % 6 7 8 9 10 11 12 13
NO. Of rats 8 8 8 8 8 8 8 8 8 8 8
Drug Corn oil Corn oil EnovidEnovidEnovidEnovidCorn oil Corn oil Mestranol Mestranol Mestranol Mestranol Pregnantcation
Dose (mcg./Kg. /day) 700 700 7,000 7,000 10.5 10.5 105 105 -no medi-
Months therapy
of
1 2 1 2 1 2 1 2 1 2 1 2 20 days’ gestation
dosed daily by gavage according to the schedule shown in Table I. The Enovid 5 (norethynodrel .and mestranol in a 67: 1 ratio) and mestranol were dissolved in corn oil in concentrations so that the desired dose could be administered in 0.1 to 0.2 ml. of oil. This amounted to approximately 7 and 70 times the human dosage. The control groups were given corn oil without added drugs. The pregnant animals were not medicated. Groups of rats were killed after one or 2 months therapy as indicated in Table I. The day before they were killed they were injected via the tail vein with 400 mcg. of E. coli endotoxin.” Platelet countslO were performed before endotoxin and again 5 minutes, 4 hours, and 24 hours after endotoxin. Twenty-four hours after injection of endotoxin the animaIs were anesthetized with ether. Blood was drawn from the inferior vena cava for profibrinolysin assaysI and tissues were collected in 10 per cent buffered formalin for histologic studies. T:he principles of laboratory care as promulgated by the National Research Council were observed. Results Initial platelet counts in all groups were similar with the: exception of those animals ‘Elacto Laboratory,
lipopolysarxharide Detroit, Michigan.
B.
E.
coil
0127:B8,
Difco
Fig. 1. Changes in platelets, expressed as per cent of original count, after injection of endotoxin to Enovid-treated and pregnant rats. Results of animaIs dosed for one and 2 months are averaged.
receiving high doses of mestranol. Since the values obtained in groups treated for one month and those threated for 2 months were virtually the same, the results have been averaged for purposes of presentation. Changes in platelet counts after injection of endotoxin to Enovid-treated and pregnant rats are shown in Fig. 1. Within 5 minutes of endotoxin, the counts fell to 40 to 50 per cent of the original level and were approximately the same 4 hours after injection. Twenty-four hours after endotoxin the platelet counts had risen in both control and Enovid-treated animals to 60 or 70 per cent of the original level but remained low in the pregnant group. There is a statistically significant difference between the pregnant and control groups, borderline significance between the pregnant and Enovid-treated
212
Phillips,
Calhoun,
and
Soloway
Amer.
PROFIEIRINOLYSIN
LEVELS
24 HOURS
AFTER
May J. Obstet.
15, 1970 Gynec.
ENDOTOXIN
unlts
20
10
5
0 Normal
Control
Low dose
Fig. 2. Profibrinoiysin levels obtained in control, after endotoxin. Levels of untreated nonpregnant Values are reported as units with one standard
High dose
Enovid-treated, rats without error,
Pregnant
and pregnant endotoxin are
rats 24 hours shown on left.
Table II. Rats dosed with mestranol for one of 2 months Groujs 7 and
8
9 and
Control (corn oil) Dose Platelets Time
after
No.
(10.5 %
10
I1 and
Low-dose mcg./Kg./day)
No.
12
High-dose (I 05 mcg./Kg./day) 70
No.
%
endotoxin
0 Count/c.c. P”
x IO-3
I,01 1
100
955 -
100 -
823 0.001
100 -
X 1O-3
550
55.2
436 0.01
46.3 -
372 0.001
45.6 -
X 1O-3
788
78.1
671
71.2 -
514 0.001
63.2 0.05
81.6
661
81.2
5 minutes Count/c.c. P* 4 hours Count/cc. P*
0.01
24 hours Count/cc. P+
881
X lO-$
87.7
775
Profibrinolysin Units Standard l P expresses
the probability
8.25 + 1.09
8.38 2 1.58
error of
stati&calIy
significant
differences
from
contro1
values.
8.02 _+ 0.99
Volume Number
1107 2
Fig. 3. Fibrin of rndotoxin.
Generalized
thrombi
found
in
the
glomerular
animals, but no significant differences between control rats and those treated with Enovid. Profibrinolysin levels obtained in controls, Enovi.d-treated, and pregnant rats 24 hours after endotoxin are shown in Fig. 2. Levels in untreated nonpregnant rats without endotoxin are also included for comparison. The increases in the corn oil (control) and Enovid-treated animals are not statistically significant and rnay be related in part to pneumonia which was evidenced by leukocyte infiltration into the lungs of 80 to 90 per cent of the animals in all groups subjected to repeated gavage. The low levels of profibrinolysin found in the pregnant anim,als are significantly different from the other groups with a probability of less than 0.01. Data on platelet counts and profibrinolysin values of mestranol-treated rats and their concomitant controls are recorded in Table II. Platelet counts are significantly louver than those of controls at all times (including pre-injection levels) in the group receiving the high dose of mestranol and after endotoxin in the low-dose group. The difference in per cent of original count, however, is significant only for the high dose group at 4 hours following endotoxin (p = 0.05).
capillaries
Shwartzman
of a pregnant
reaction
rat
after
in rats
213
injection
Profibrinolysin levels were similar in the three groups. The characteristic lesions of the generalized Shwartzman reaction are fibrin thrombi within the glomerular capillaries. These lesions found in pregnancy following endotoxin are illustrated in Fig. 3. Twenty-five per cent of the pregnant animals showed evidence of the GSR and another 12.5 per cent died in less than 24 hours after endotoxin from thrombi in areas other than the kidney. Fibrin thrombi were not observed in the kidneys of any of 64 animals pretreated lvith Enovid or mestranol. One control rat, which died three hours after endotoxin had fibrin deposits similar to those found in pregnancy. Since this was found in a control animal (1 of 32) it must be considered as an unusual chance occurrence in a rat particularly sensitive to endotoxin. Three other rats, one each from control? low-dose, and high-dose Enovid groups died within 4 to 6 hours after endotoxin. Microscopic examination failed to disclose evidence of GSR in these animals. One of them, however, had a platelet thrombus containing fibrin strands within an hepatic vessel (Fig. 4) . This rat had received ‘100 mcg. Enovid per kilogram per day for one month. The significance of this observation is not apparent.
214
Phillips,
Calhoun,
May 15, 1970 Amer. J. Obstet. Gynec.
and Soloway
Fig. 4. Platelet thrombus found in an hepatic vessel after endotoxin administered fed 700 mcg. Einovid per kilogram of body weight per day for one month.
to a
rat
Fig. 5. Centrilobular necrosis of the liver found in many control lowing injection of endotoxin.
rats
fol-
The control trilobular appears toxin. animals related
lesion most frequently observed in and experimental animals was cennecrosis of the liver (Fig. 5). This to be a nonspecific reaction to endoIt has been observed in heparinized following endotoxin and thus is unto intravascular coagulation.12
Comment The generalized Shwartzman reaction was originally described and defined as the re-
and
drug-treated
sponse in rabbits to two doses of endotoxin administered intravenously 24 hours apart.’ The first injection was called the preparative dose and the second the provoking dose. The reaction is now defined as the result of any process causing deposition and retention of fibrin thrombi in the glomeruli and leading to renal cortical necrosis.13 Many stimuli can take the place of the preparative dose of endotoxin. In rats the principal conditions which may substitute are pregnancy,g inhi-
Volume 107 Number
Generalized
Shwartzman
reaction
in rats
215
2
bition Iof the fibrinolytic system with epsilon of aminoc.aproic acid,14 or administration cortisone for three days before injection of endotoxin? Cooper and McKaylG showed that estrogen, progesterone, or their combination in doses comparable to those of cortisone did not prepare for the GSR. Therefore, the effect of cortisone seems to be a response to car-ticosteroids rather than a nonspecific response to steroids in general. The possibility remained, however, that chronic administration of these agents, by enhancement of the factors of the coagulation system, might prepare for the reaction. More recently, Nolan and Vilayat Alil’ demonstrated that estrogen injected intraperitoneally 18 to 48 hours prior to endotoxin increased the toxicity of endotoxin tenfold. Fibrin thrombi were not found in the kidneys and heparin did not protect against this enhanced lethality. Therefore, it was, not a generalized Shwartzman reaction and the response was not mediated through an effect on the coagulation mechanism. The data reported in the present paper indicate that neither mestranol alone nor the combination of norethynodrel and mestranol in large doses, prepares for the generalized Shwartzman reaction in rats as does pregnancy. No fibrin thrombi were found in the renal glomeruli in any of treated groups despite the fact that this same dose of endotoxin elicited the GSR in pregnant animals. The platelet thrombus found in the hepatic vessel of o-ne animal on Enovid can be found in normal animals following the first injection of endotoxin and is not neces-
sarily related to the Shwartzman reaction. The more frequently observed hepatic necrosis is also a nonspecific reaction to endotoxin and can be found in heparinized rats.l” In a previous study it was shown that activation of the fibrinolytic system as a secondary response to intravascular coagulation results in low levels of profibrinolysin in rats 24 hours after the challenge.ls The normal or elevated profibrinolysin levels found in control and drug-treated groups as compared to low levels found in pregnant rats is further evidence that extensive intravascular coagulation was not triggered by injection of endotoxin in these treated animals. Platelet counts were severely reduced in all groups of rats after administration of endotoxin. The failure of the pregnant animals to restore the platelet levels within 24 hours suggests a more intense effect of the endotoxin in this group. The mechanism by which platelet counts are depressed before endotoxin in the animals on high doses of mestranol remains unexplained and perhaps deserves further investigation. It may be concluded that high doses of mestranol alone or in combination with norethynodrel (Enovid) , administered orally for up to 2 months, do not prepare rats for the generalized Shwartzman reaction as does pregnancy. It would appear that the mechanism by which pregnancy prepares for the Shwartzman reaction is not due exclusively to an increase in coagulation factors. The data also suggest that the increase in thromboembolic disorders reported in users of oral contraceptives is not mediated through the Shwartzman phenomenon.
REFERENCES
1. Illman,
5.
2.
6.
3.
4.
W. H. W., and Vessey, M. P.: Brit. Med. J. 2: 193, 1968. Vessey, M. P., and Doll, R.: Brit. Med. J. 2: 199, 1968. Rutherford, R. N., Hougie, C., Banks, A. L., and Coburn, W. A.: Obstet. Gynec. 24: 886, 1964. Miller, S. P., Lee, S. L., and Ritz, N.: Metabolism 14: 398, 1965. (Suppl.)
7.
Brakman, R., Albrechtsen, 0. K., and Astrup, T.: J. A. M. A. 199: 69, 1967. Phillips, L. L., Turksoy, R. N., and Southam, A. L.: AMER. J. OBSTET. GYNEC. 82: 1216, 1961. Shwartzman, G.: Phenomenon of Local Tissue Reactivity and Its Immunological, Pathological and Clinical Significance, New York, 1937, Paul B. Hoeber, Inc.
216
8. 9. 10. 11. 12. 13.
14.
Phillips,
Calhoun,
Apitz, K.: Virchow 1, 1934. Wring, T. C.: AMER.
and
Arch.
Soloway
Path.
.T. OBSTET.
Amer.
Anat. GYNEC.
293:
84:
Brecher, G., and Cronkite, E. P.: J. Appl. Phvsiol. 3: 365. 1950. Phillips, L. L:, and Skrodelis, V.: J. Clin. Invest. 37: 965, 1958. Margaretten, W., McKay, D. G., and Phillips, L. L.: Amer. J. Path. 51: 61, 1967. McKay, D. G.: Disseminated Intravascular Coagulation, New York, 1965, Hoeber Medical Division, Harper & Row. Margaretten, W., and McKay, D. G.: Fed. Proc. 22: 251, 1963.
15. 16. 17. 18.
May 15, 1970 J. Obstet. Gynec.
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