Endotoxemia in pregnant cows: Comparisons of maternal and fetal effects utilizing the chronically catheterized fetus

Theriogenology

39:739-762,

ENDOTOXEMIA IN PREGNANT FETAL EFFECTS UTILIZING G.L.Foley,la

1993

COMPARISONS OF MATERNAL AND COWS: THE CHRONICALLY CATHETERIZED FETUS

D.H.Schlafer,l

T.H.Elsasser*

and M.Mitchel13

1Department of Patho ogy, Cornell University, Ithaca, NY 'USDA, Beltsville,MD & 13University of Utah, Salt Lake City,UT Received

for publication: MUZJ10, 1991 Accepted: January 11, 1993 ABSTRACT

We utilized the chronically catheterized bovine fetus to compare maternal and fetal responses to maternal lipoOur hypothesis was that LPSpolysaccharide (LPS) infusion. induced abortion was primarily a maternal luteolytic event with minimal transplacental fetal exposure. Fetal tibia1 arteries, amniotic, allantoic cavities and maternal carotid arteries were catheterized. Three cows had patent catheters with viable fetuses (190 to 200 days of gestation) 1 week after operation and were included in the study. Following a 2-day maternal and fetal baseline, 0.5 pg Salmonella tvohimurium LPS/kg was infused into a maternal jugular vein over a 2-hour period. Maternal and fetal responses were monitored clinically, biochemically and hormonally. The maternal response consisted of marked increases in plasma ,;.r;.aglandin F2a metabolite (PGFM), tumor necrosis factor, I ACTH and cortisol with a dramatic maternal leucopenia Progesterone concentrations decreased within 2 hours. within 7 hours (P
cow, lipopolysaccharide, reproduction

fetus, abortion,

Acknowledgements This work was partially supported by the Wetterburg Foundation and from NIH HD 21350. This work represents a portion of a thesis submitted by G.L. Foley to the Graduate School of Cornell University. The authors thank Dr. P.W. Nathanielsz and Dr. D. Sadowsky for their assistance. aPresent address: Department of Pathobiology, Veterinary Medicine, Urbana, Illinois 61801.

Copyright

0 1993 Butterworth-Heinemann

College

of

Theriogenology

740 INTRODUCTION

Lipopolysaccharides (LPS) have long been recognized as Zahl and Bjerknes(1) first reported LPSabortifacients. induced abortions with hemorrhages and colloidal dye localization in the placentas of mice and rabbits. Most of the articles examining the abortifacient effects of LPS in small laboratory animals have found placental hemorrhages and fibrin thrombi (l-3). Ovarian function (peripheral plasma progesterone) does not appear to be adversely affected by endotoxemia in rodents (4). Endotoxin-induced abortions in (cows (51, goats (6) and horses(7) have been associated primarily with luteolysis. The degree of LPS exposure of the embryo and fetus has been a subject of debate. Some researchers have found evidence of transplacental passage of LPS in rabbits (8) or of LPS-induced teratogenesis in a variety of laboratory animals (9,101. Lipopolysaccharides failed to cross human chorioamniotic membranes in an in vitro study (11). There are no reports of transplacental effects of LPS in domestic animals. Work on intravenous or subcutaneous exposure of 4 bovine fetuses to 500 Jig of LPS demonstrated fetal disseminated intravascular coagulation (12). When chronically catheterized fetal sheep were infused with LPS, the fetuses were able to tolerate much higher doses than adult pregnant sheep (13). The chronically catheterized fetus offers unique advantages in studying the pathogenesis of abortion and normal physiology of pregnancy. Catheterization of the placental compartments and fetal vasculature allow for concurrent maternal and fetal sampling over time. Additional information can be obtained by monitoring uterine electromyography (EMG) and pressure transducers placed on the vascular catheters. The purpose of this study was to utilize the chronically catheterized fetal calf to monitor and compare maternal and fetal responses to maternal endotoxemia. The hypothesis was that LPS-induced abortion in cows is a maternally-mediated event with a minimal fetal role. MATERIALS AND METHODS Animals Healthy first-calf Holstein heifers of known gestational stage were purchased from a university-owned herd. A total of 9 cows was initially used in the study. only animals with patent fetal and maternal catheters and viable, nonstressed fetuses, as indicated by fetal blood gas values, at 6 days post surgery were included in the study. Three

741

Theriogenology cows fulfilled these criteria. The cows were housed stanchions with free access to haylage and water.

in

Surgery Following a 3- to 5- day period of acclimatization to the facility, the cows and fetuses were catheterized. Surgery was done between Days 190 to 200 of gestation. Food was withdrawn for 48 hours and water for 24 hours prior to General anesthesia was induced by intravenous surgery. The animals were intubated thiamylala and guiaphenesin. and were maintained on halothane and oxygen for the duration The gravid uterus was exposed using a left of the surgery. paramedian incision. The uterine wall was opened and a hind leg of the fetus was exteriorized, as described previously (14). Instrumentation The fetal tibia1 artery was catheterized to the level An amniotic catheter was attached to of the caudal aorta. the skin of the fetal leg. The allantoic catheter was placed in the allantoic cavity either by a second uterine incision or by identification and insertion of the catheter into the allantoic cavity at the primary uterine incision site. A uterine vein within the uterine wall was catheterized in Cow 3. All fetal catheters were exteriorized through a separate paralumbar fossa site, dorsal to the The maternal carotid artery was also cathsurgical site. eterized. For LPS infusion, a jugular vein catheter was aseptThis ically placed 1 to 2 hours prior to LPS infusion. allowed separate sites for the infusion of LPS and for the All catheters were loaded with sampling of maternal plasma. sterile heparinized saline at surgery and were attached to Sterile, heparinized an infusion pump within 6 hours. saline (200 U heparin/ml) was continuously infused at 0.5 to Amniotic and 1.0 ml/hour,for the duration of the study. allantoic catheters were loaded with sterile heparinized saline and were flushed daily. Sampling

Protocol

Five to I days of post-operative stabilization allowed for the assessment of fetal preparation and surgical recovery. Baseline data were collected for 2 days prior to the LPS infusion. On the day of LPS infusion, 2 cows (Cows 1 and 2) were at Day 205 of gestation and the third cow (Cow 3) was at Day 203. A total of 0.5 )lg of Salmonella tvphimurium LPS/kg maternal body weight was infused over a 2-hour a Surital,

Park Davis, Morris Plains, NJ

Theriogenolog

742

y

Fetal plasma and fetal fluid samples were collected period. at -1, +2, +5, +7 and tll hours after maternal LPS infusion. Maternal plasma was collected at -1, +.25, f.5, +.75, +2, On the day following LPS infusion +3, +5, t7 and tll hours. On (Day 1 post infusion), plasma samples were taken twice. subsequent days, samples were collected once daily until Day 5. Plasma samples were collected into chilled heparinized plastic syringes and were immediately transferred to chilled tubes with either EDTA or heparin. Amniotic and allantoic samples were collected into chilled plastic tubes with no All samples were immediately centrifuged for preservatives. Plasma 15 minutes at 4OC and the plasma was harvested. samples for prostaglandin analyses were collected, mixed with EDTA and aspirin, and snap-frozen in liquid nitrogen. All samples were stored at -20°C until assay. Clinical

Pathology

White blood cell counts of maternal and fetal samples were determined by the Unopette microcollection system (Becton-Dickenson, Rutherford, NJ). Whole blood from EDTA tubes was diluted within 10 minutes of collection and white blood cells were counted on a hemacytometer within 2 hours. Blood gas analyses were done on paired maternal and Blood was collected into fetal arterial blood samples. chilled, plastic, heparinized l-ml syringes, then capped and transported in ice to a Radiometer ABL2 blood gas analyzer (Radiometer, Copenhagen, Denmark). All samples were A hemoximeter analyzed within 15 minutes of collection. (Radiometer) was used to measure the percentage of oxygen saturation of the sample. Biochemical analyses were performed on paired maternal and fetal heparinized plasma samples by the Clinical Pathology Laboratory of the New York State College of Veterinary Medicine. Tests performed on plasma samples included those of chloride, total carbon dioxide, total protein, albumin, globulin, glucose, calcium, phosphorous, aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), creatinine, blood urea nitrogen, sodium and potassium concentrations. The anion gap, albumin:globulin ratio and sodium:potassium ratio were also calculated. Assays Progesterone concentrations of maternal plasma were assayed by radioimmunoassay, as described by Beal et al. (15) - Briefly, petroleum ether was used to extract the heparinized plasma samples for the determination of progesterone concentrations. The aqueous phase was frozen using dry ice in an alcohol bath, then dried under nitrogen. The radioimmunoassay was done in duplicate on diluted samples and appropriate controls with standards. Following

Theriogenology

743

an overnight incubation at 4'C, free steroid was extracted Supernatant was decanted and by charcoal-dextran. All samples were done scintillation fluid was added. concurrently in the same assay. Maternal and fetal plasma ACTH was measured using the INC Star ACTH kit (Stillwater, MN) as described by McDonald The plasma was thawed on ice and 50 ~1 of et a1.(16). plasma was incubated with radioiodinated ACTH(l-24) antiserum for 24 hours at 4"C, and then with radioiodinated ACTH(l-24) for another 24 hours at 4'C. The bound fraction was precipitated with a goat anti-rabbit second antibody. The intra-assay coefficient of variation was 8.2%, for a All pool having a mean concentration of 1.6 pg/tube. samples were assayed together, thus making inter-assay The assay coefficients of variation unnecessary. sensitivity (90% of the bound to free ratio (BfBO) was approximately 11 pg/ml. Fetal and maternal plasma cortisol was measured using the Autopak Cortisol Kit (Micromedic Systems, Inc., Horsham, PA) in an assay validated for maternal and fetal plasma described previously in detail (17). Following dichloromethane extraction of 20 pl of plasma, assays were done in The antibody was raised against antibody-coated tubes. The intra-assay coefficient iodinated histamine-3-cortisol. of variation was 11%. All samples were assayed together thus making inter-assay coefficients of variation Assay sensitivity (95% of the bound to free unnecessary. ratio (B/BO) was 10 pg/tube. Tumor necrosis factor levels were determined by double radioimmunoassay, as previously described by Kenison et al. (18) in maternal and fetal plasma and in amniotic and Recombinant bovine TNFO( (Ciba Geigy, allantoic fluid. Basel, Switzerland) was used as a standard. Antibody was used in the assay at a final tube dilution of 1:96,000, which bound 34% of the tracer counts in the total binding Tracer for the assay was prepared by introducing U!es. I onto the recombinant monomeric TNF by the iodogen method (10 pg rBoTNF/l.S jig iodogen in 0.5M phosphate pH7.5 for 11 minutes: Similarly, the 99+% TCA precipitable). rBoTNF was used to construct the standard curve. Curves depicting displacement of tracer counts by increasing mass of standard or increasing volumes of plasma were parallel. Recovery of rBoTNF to plasma averaged 93% across doses of rBoTHF ranging between 125 and 1000 pg added/tube. Minimal sensitivity was 100 pg/ml in plasma. All samples were assayed in one lot using an equilibrium technique (24-hour incubation); the intra-assay coefficient of variation was 10s. Assays for prostaglandin F metabolite (PGFM) were done on maternal samples as previously described(lg), while

Theriogenolog

744

prostaglandin E2 (PGE2!, the primary eicosanoid in the near term fetus, was determlned in fetal plasma as well as in allantoic and amniotic samples (20,211. All samples were The solvent was extracted with acidified diethyl ether. evaporated under nitrogen, and the residue was redissolved in buffer or solvents for the further purification of plasma samples by silicic acid chromatography (22). The crossreactivities of the PGFM antiserum were 13-14-dihydro-PGF2cL (2.8%); 15(7%); 15-keto-PGF2 (3%); 13,14-dihydro-PGF1 keto-PGFlgl (2%); a!?1 others were (1% or less P. The cross reactivities of the PGE2 antiserum were: PGEl (50%); PGA2 (1.3%); all others were (1% or (6%); PGA (3%); 6-keto PGE T+I e intra-assay toe 6 ficients of variation were 6.4 less). and 7.5% for PGFM and PGE2, respectively. All samples for each animal were assayed together, thus making interassay The sensitivity (2 coefficients of variation unnecessary. SD of zero-point blanking) of the PGFM assay was 0.89 pg/tube, and the PGE2 assay was 0.52 pg/tube. The Limulus Amebocyte Lysate (LAL) assay (E-Toxate Assay, Sigma Chemical, St.Louis, MO) was used to detect endotoxin in the plasma and in fetal fluid samples. Plasma samples were diluted 1:lO with pyrogen-free water, followed by the heating of the diluted sample to 75°C for 5 minutes. Amniotic and allantoic fluids were assayed without treatment. Statistical

Analysis

Differences in plasma concentrations were determined within cows and within fetuses by paired t-tests. Statistical significance was set at P
y

745

Theriogenology

0

1

2

3

DAYOFSTUDY Figure

1. Average WBC counts of 3 pregnant cows (*-•) and their fetuses (r-r) following maternal intravenous infusion of O.Sj.lg LPS/kg in 2 hours (shaded area). * = Significantly different from Error bars = 1 SD. baseline.

3 a

1

0

1

2 DAY

Figure

3

OF STUDY

2. Average maternal plasma progesterone (P4) of Cows 1, 2 and 3 following maternal intravenous LPS infusion (shaded area). Error bars = 1 SD. Point with * indicates first significant decrease of plasma P4 from baseline.

i.

7.462~0.030

7.500+0.036

7.470+0.041

7.465kO.021

7.475+0.01a

7.450+0.008

7.437kO.029

-1

2

5

7

23

47

54

96

ND = Not done.

7.415kO.029

7.449+0.017

-24

7.412+0.040

Maternal

Fetal

7.341+0.008

ND

7.329kO.037

7.344kO.036

7.343kO.015

7.307kO.087

7.321kO.051

7.292kO.046

7.27320.092

7.318+0.024

pH level

Maternal and fetal arterial lipopolysaccharide infusion

-48

Hours Post Infusion

Table

96.1213.5

83.8k3.8

93.9k6.4

92.6k3.0

97.2k17.7

89.6219.2

79.927.5

93.9A10.7

90.8T12.7

96.226.5

Maternal

PO2

23.1k2.5

19.121.2

24.652.4

21.422.2

16.7t2.1

17.020.4

16.5~~0.1

23.721.8

23.124.4

23.622.1

Fetal

blood gas chemistries

maternal

97.823.0

96.9k5.2

98.424.0

98.8k4.3

100.5k3.7

99.4k3.4

98.6k4.1

98.522.5

98.9k3.8

98.423.0

Maternal

55.1k13.9

44.624.5

56.1k19.3

53.8216.9

37.227.8

35.8k6.0

30.3k6.4

60.824.8

56.1ilO.l

57.1~3.7

Fetal

$02 saturation

following

Theriogenology

747

Maternal arterial pH changes were also divergent from the fetal response. By 5 hours post infusion, maternal pH had increased by 0.02 to 0.05 units. Conversely, fetal pH initially decreased by 0.1 units at 5 hours and then increased above baseline by 7 hours. While maternal arterial pO2 values decreased markedly immediately following infusion, the percentage of oxygen saturation did not change in any of the cows. The fetuses had decreases in both the percentage of saturation and pO2 concurrent with the pH decreases. Of the biochemical parameters assessed (Table 21, there were maternal decreases in calcium, phosphorus, total protein and glucose, with concurrent increases in urea nitrogen, creatinine, GGT and AST. The fetal serum samples had no change in calcium, total protein, creatinine, GGT or AST. Fetal serum increases of phosphorus and urea occurred during decreased maternal phosphorous and concurrent with elevations in maternal urea. Fetal glucose was the only parameter that followed the same pattern of decrease seen in maternal plasma. Maternal progesterone (P4) briefly increased by 1.5 to 2 ng/ml in Cows 1 and 3 at 15 and 30 minutes, respectively. Thereafter, all cows had similar rapid decreases in peripheral piasma progesterone concentrations which were significantly different from baseline by 5 hours post infusion (Figure 2). By 23 hours post LPS infusion, P4 values for all 3 cows were less than 1.6 ng/ml, and by 48 hours, the P4 values were equal to or less than 1 ng/ml. Maternal arterial prostaglandin F metabolite (PGFM) concentrations started to increase by 15 minutes and peaked at 30 to 45 minutes post infusion (Figure 3A). Uterine vein samples from Cow 3 showed a similar pattern of PGFM increases but with higher serum concentrations. Fetal plasma PGE2 concentrations had a more gradual increase by the 23-hour sample (PcO.05; Figure 3B). Two fetuses ( Fetuses 1 and 3) had initial increases of plasma PGE2 by 11 and 23 hours, respectively, with decreases by the end of Day 1. A small increase of plasma PGE2 was seen in Fetus 3, approximately 8 hours prior to delivery. Fetus 1 plasma PGE2 levels continued to decline. Fetus 2 had a different pattern of PGE response. Levels of PGE2 did not increase as quickly as t z e other 2 fetuses, and the increased levels were sustained until the morning of Day 4. Prior to being aborted on the evening of Day 4, Fetus 2 had a slight increase in plasma PGE Amniotic and allantoic samples (Table 3) increased by 3' 4 to 48 hours, with progressive increases in Cows 2 and 3 up until just prior to aborticn when PGE? levels tended to decrease.

7.4+0.2a

8.820.5

11

23

120

14 25.2

14 211.3

23

120

7.522.0

7.5to.4

7.620.4

8.221.5

7.320.8

7.620.6

1.1 LO.3

1.4 +0.2a

1.4 kO.1

1.5 +0.2a

1.2 TO.1

1.2 TO.1

Maternal

Maternal

different

2.5 kO.9

2.5 20.3

2.5 kO.4

2.7 ~0.3

2.6 to.5

(PKO.05).

23 t10

35 216

67 ? 9

45 +20a

24 513

27 518

6 21

7 23

7 ?r5

7 23

7 +6

7 21

Fetal

34k20.5

41~23.2

31k5.7

44+15

40214.7

4923.6

Fetal

GGT (U/L) Maternal

70219.8

73220.1

68~2.1~

62~5.6~

8Ok2.9

84k2.8

levels

10 Tl

18 +9

13 +o

12 +3

11 +1

14 +3

from baseline

452 + 54

614 2691

600 +593

381 +404

319 +338

300 2234

Fetal

3.3kO.4

3.5kO.3

3.520.4

3.520.3

3.5kO.5

3.42.25

Maternal

Glucose (mT/dL)

lipopolysaccharide

Fetal

AST (U/L) Maternal

7.9+0.5a

6.920.6

6.1k0.4

6.3?0.4a

6.9AO.4

6.920.3

2.7 20.3

Fetal

maternal

Total Protein (g/dL)

following

Fetal

Creatinine (mg/dL)

8.8kO.5

7.420.2

7.2i-0.4a

7.8kO.2

9.6kO.3

9.320.2

are significantly

15 k12.0

14 +4.4a

16 ?3.5a

11 +5.6a

9 k5.0

8 ~5.2

Fetal

with superscripts

11 26.2

16 t5.7

5

8 25.5

11

8 23.4

-1

Maternal

BASELINE

a Values

12.420.9

12.621.0

12.5~1.4

12.8tO.6

12.620.7

12.620.3

Fetal

Urea (mg/dL)

7.2+0.4a

5

Hours Post Infusion

9.620.3

7.8~0.2~

-1

9.320.2

Maternal

Phosphorus (mg/dL)

chemistries

Maternal

and fetal plasma

Calcium (mg/dL)

Maternal infusion

BASELINE

Hours Post Infusion

Table ?.

2

8 s 0‘ 9

5! s.

W

749

Theriogenology

A

3000 2500 2000

1500 1000 500 0

1

0

2

3

4

5

DAYOFSTUDY

350 --

0

:... i:

0-0

2: p

*-A

FETUS 1 FETUS2 m--= FETUS 3

i$;

:>$.

1

2

3

4

5

DAYOFSTUDY Figure

3. Maternal carotid plasma prostaglandin F metabolite (A) and Fetal plasma PGE, (B) following maternal intravenous LPS infusion (shaded area). I = abortion of Fetus 2, 11 = abortion of Fetus3

Theriogenology

Table

3. Allantoic and amniotic lipopolysaccharide Fetus

Hours Post Infusion

Allantoic (pg/ml)

666.1

431.2

Amnion (pg/ml)

following Fetus

2

Allantoic (pg/ml)

ND

3

Amnion (w/ml)

Allantoic (pg/ml)

330.7

1033.6

-_ 224.4

-1

823.7

349.2

ND

361.2

894.1

2

876.5

416.2

ND

329.2

747.2

331.6

5

787.5

384.9

ND

224.3

815.5

336.4

11

797.0

473.1

ND

292.9

796.3

802.9

23

869.2

688.3

ND

654.4

955.3

ND

30

890.1

1141.8

ND

963.5

1307.2

ND

1291.4

ND

582.0

1380.6

ND

1637.4

41 54

ND

ND

ND

ND

1138.8

1193.4

59

ND

ND

ND

ND

977.4

1157.1

12

1018.7

411.6

ND

ND

ND = Not

Table

Fetus

1

Amnion (pg/ml)

-24

concentrations

PGE2

Abortion

done.

4. Limulus cows

Hours Post Infusion

Mata

Baseline

_

amebocyte cow Fetb

lysate

assay

of lipopolysaccharide-infused cow 2

1

cow

AmnC

Alld

Mat

Fet

All

Mat

+

+

+

-

-

-

0.5

+

NAe

NA

NA

+

NA

NA

+

0.75

+

NA

NA

NA

+

NA

NA

+

2

+

+

_

-

-

5

+

-

-

-

11

+

-

-

23

-

-

-

- = No gelation of sample and therefore no endotoxin + = Gelation of sample; indicating endotoxin ( >0.06 units/ml). E Mat = Maternal carotid plasma sample. Fet = Fetal tibia1 artery plasma sample. s Amn = Amniotic fluid sample. All = Allantoic fluid sample. ' IiA = Net Available.

3

Fet

Amn

All

NA

NA

NA

NA

NA

NA

-

detectable. endotoxin

Theriogenolog

y

751

Maternal plasma ACTH values began to increase significantly by 30 minutes after the start of infusion, with peak levels occurring at 2 hours (Figure 4). Cow 3 had the most marked ACTH increases, with a peak ACTH value of 594 pg/ml. Fetal plasma ACTH levels were increased in 2 of the 3 The third fetus had fetuses by 2 hours after infusion. minor elevations in plasma ACTH levels but these generally remained at baseline. Maternal and fetal plasma ACTH levels had returned to baseline levels by the 23-hour samples. Plasma ACTH did not increase in Cows 2 and 3 prior to abortion. Plasma cortisol concentrations began to increase by 15 minutes in Cows 1 and 3 and by 30 minutes in Cow 2 (Figure 5). Maternal plasma cortisol levels were significantly higher than baseline levels by 30 minutes post LPS infusion. Peak plasma levels occurred by 2 hours and persisted until 5 hours post infusion. Fetal plasma cortisol concentrations followed a similar though attenuated pattern, peaking at 2 Fetal plasma cortisol increases to 5 hours post infusion. were only significant at the 2 hour sample. Both maternal and fetal plasma cortisol levels gradually returned to baseline levels within 2 to 3 days. Fetus 2 had mild increases in plasma cortisol; however, corresponding maternal samples had no such increases in cortisol. Tumor necrosis factor (TNF) concentrations were measured in the plasma of both maternal and fetal samples as well as in the amniotic and allantoic fluids. Maternal plasma TNF concentrations were increased twofold over baseline by 30 minutes post LPS infusion, with a marked increase by 45 minutes. Significant increases in TNF occurred, with peak levels at 2 hours (Figure 6). No detectable significant increases in TNF occurred in any of the time period samples assayed in fetal plasma, amniotic fluid or allantoic fluid. Based on the limulus amoebocyte lysate (LAL) test, maternal plasma quickly cleared the infused endotoxins, with undetectable leveld even at the Z-hour time period samples (Table 4). Fetal plasma samples were negative at all collection times. In 2 of the 3 fetuses, no trace of LPS was found in the amniotic or allantoic samples. In the fetus of Cow 1, endotoxin was detected in amniotic and allantoic fluids during a baseline sample and in an immediate post infusion sample; the corresponding plasma samples from the fetus were all negative for LPS.

Theriogenology

752

A 600

--

500

--

400

--

300

--

200

--

.-. -A-b

GC _I#cc

100 --

DAYOFSTUDY

B

100 a-. FETUS 1 r-&FETUS2 n-mFzTUs3

90 60

\

70 60 50 40 30 20 10 0! 0

2

1

3

DAYOFSTUDY Figure

4. Maternal carotid (A) and fetal arterial (B) ACTH concentrations followingmaternalintravenous LPS infusion (shaded area). 5. = abortion of Fetus 3.

Theriogenology

40

753

I

A-A

cow 2

m-m

COW3

30

0

3

2

1

DAYOFSTUDY

B 0-0 4-A

m--m

FETUS 1 FEmJs 2 FETUS 3

8 5

0

1

2

3

DAYOFSTUDY Figure

5. Maternal carotid (A) and fetal arterial (B) cortis01 concentrations following a slow intravenous infusion of LPS (shaded area). Arrow indicates abortion of Fetus 3.

Theriogenology

754

150 125

100 75 50 25 0 DAY

Figure

OF STUDY

6. Average maternal carotid plasma tumor necrosis factor for Cows 1, 2 and 3 following intravenous Error bars = 1 SD. LPS infusion (shaded area). DISCUSSION

All 3 cows aborted following maternal endotoxin infusions. The cows did not become moribund following the Fetal death was not the cause slow infusion of endotoxin. of the abortion since clinical, biochemical, and hormonal data from all the cows and their fetuses were monitored up until abortion. The time of abortion was similar to that of another report on endotoxin infusion in pregnant cows (5). Fetal leucocytosis in the face of maternal endotoxemia was unexpected. The leucopenia has been demonstrated in the adults in a previous report (23). The lack of similar fetal leucopenia but rather the occurrence of fetal leucocytosis The fetuses may could be due to a variety of mechanisms. not have been exposed to the infused endotoxin, or the fetuses may not have had the ability to respond to endotoxins like adult cows. In previous studies of the bovine fetus, fetal white cell counts were similar to those of our baseline levels (24,25). Concurrent fetal infections with microbial agents were unlikely since the microbial examinations of the fetuses were negative. Moreover the LAL assays were negative for endotoxin in all the fetal samples examined. The maternal blood gas data reflects the pulmonary effects of endotoxins, including pulmonary endothelial damage and pulmonary edema (26). Clinically these changes were concurrent with the observed hyperventilation and coughing following infusion. The lack of change in the

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percentage of saturation in the maternal samples is most likely due to a shift in the oxygen dissociation curve due to changes in blood pH of the cows. The decreases seen in fetal pO2 and the percentage of oxygen saturation indicate a transient period of fetal hypoxia. The mechanisms responsible for the hypoxia include decreased uteroplacental blood flow, decreased maternal pO2 and the pH-induced shift of the oxygen dissociation curve. Decreases in uteroplacental perfusion has been demonstrated in sheep following endotoxin infusions (27). Additionally, maternal hypoxia has been shown to cause fetal acidosis Prostaglandin (28), similar to that in our findings. infusion has also been associated with decreases in the p02 of pigs(29) as well as with fetal hypoxia with increased uterine activity in sheep (30). The biochemical changes were most remarkable in that there were minimal fetal changes, despite the maternal Fetal calcium has previously been changes reported. reported to be independent of maternal levels (31). Glucose levels and blood urea nitrogen changes did parallel the maternal changes and reflected their ease of passage across Urea usually is transported in a fetal to the placenta. maternal gradient(32) while glucose is supplied to the fetus. The rapidity of plasma progesterone (P4) decrease supports the luteolytic cause of abortions in endotoxininfused cows. Two of the cows had a brief increase in P4 immediately after the start of infusion, and similar Experiincreases have been observed by others (7,33,34). mentally, both prostaglandins(35) and corticotrophin(36) are able to cause brief increases in P4 release. The timing of the increases in P4 corresponded to the observed PGFM and ACTH increases in all 3 cows. Luteolysis is typically defined as a decrease in P4 below 1 ng/ml. Luteolysis had occurred by 48 hours post infusion in all 3 cows. The dramatic increase in maternal PGFM seen at the 15and 30-minute samples demonstrates the rapidity of the maternal response to endotoxins. The rapid rise in PGFM following endotoxin infusion has been reported by other workers in the pregnant cow (5), the nongravid cow (34,37), the mare (33) and the goat (6). Conversely, fetal PGE2 concentrations showed a delayed increase at 23 hours and then a return to the baseline concentration. Amniotic and allantoic PGE2 levels gradually increased after the 23-hour sample. Because of the variability of the fetal PGE2 responses, the amniotic and allantoic increases were not significant (P>O.O5). The fetuses that were aborted during the study period did have gradual increases in PGE2 concentrations until the time of abortion.

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Cortisol and ACTH levels had similar transient increases in both the maternal and fetal plasma samples. The increases paralleled both the rapid increases in PGS and The rapid rise in the maternal cortisol is similar in TNF. to that observed in a previous report (23). The stress of The endotoxemia is responsible for the observed increases. fetal changes are similar to responses reported in fetal lambs during maternal stress (28). Plasma samples collected from Cow 3 during her abortion are conspicuous in the lack of change in fetal or maternal ACTH or in cortisol levels. During fetal-induced parturition, cortisol levels rise over the last week of gestation; yet if parturition is induced by maternally administered dexamethasone, fetal cortisol levels do not increase (38). The increases seen in TNF concentrations are similar to those of previous reports on LPS-induced changes in rats (39), dogs (40), mice (41) and humans (42). Previous work (G-L. Foley, unpublished observations) in our laboratory showed similar TNF changes following LPS infusion in cows. The lack of a fetal TNF increase supports our hypothesis that maternal endotoxemia does not expose the fetus to LPS. Murine fetuses are able to secrete TNF in response to LPS stimulation (43). Baseline levels of fetal plasma TNF were similar to or slightly higher than adult baseline concentrations. The lack of change in bovine amniotic or allantoic TNF following maternal endotoxemia is in contrast to increased TNF associated with endotoxin abortions in humans (44). Tumor necrosis factor within amniotic fluid has also been reported in normal human pregnancies, although in a biologically inactive form (45). The radioimmunoassay we used is specific for bovine TNF and does not require biologic activity for detection. However, if the conceptus is not exposed to LPS from maternal endotoxemia, no changes in amniotic or allantoic TNF would be expected. The ubiquity of low levels of endotoxins in our environment and in many reagents require careful handling of samples to avoid spurious results. The detection of endotoxins is primarily based on the gelation of the blood cell of the horseshoe crab. Detection of endotoxins in plasma samples has been a problem because of protein inhibitors in plasma that hinder gelation (46). Of the 3 most prevalent ways to destroy plasma inhibitors, we used the dilution-heating method since it is the most sensitive (46). Human amniotic fluid has also been assayed for endotoxin and rather than finding inhibitors of gelation, as in plasma, amniotic fluid has promoters of gelation (47). In our study, endotoxin in maternal plasma was cleared very rapidly from the systemic circulation to levels below the sensitivity of the assay (>0.06 endotoxin units/ml). In

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studies using bolus LPS injections, most of the LPS was cleared from the blood by 30 minutes and was followed by a change in buoyant density of the remaining LPS (48). The major organs involved in the clearance of endotoxin include the liver, spleen and lungs (49). All of these sites have macrophages which are able to quickly clear LPS from the systemic circulation. One cow (Cow 2) had positive LAL assays during the baseline period and at 11 hours post infusion. Fetus 1 also had positive amniotic and allantoic samples during the baseline measurement and at the 2-hour, post-infusion sampling. Positive baseline results may reflect a small, lingering post-surgical contamination or LPS contamination of samples during collection or handling. In the current experiment, the chronically catheterized bovine fetus allowed for the collection of a large database of hematologic, biochemical and endocrinologic data during maternal endotoxemia and subsequent abortion. While the sample was small, each of the 3 fetuses experienced a similar transient hypoxia and a brief acidosis, followed by a more prolonged alkalosis. In spite of the maternal leucopenia following exposure to LPS, the fetal response was one of leucocytosis. The maternal response included a rapid prostaglandin surge as well as increases in ACTH and TNF, followed by a rapid decline in peripheral progesterone. These data support the proposed hypothesis that endotoxininduced abortions are primarily a maternally mediated event (due to luteolysis) with a minimal fetal role in the induction of abortion. REFERENCES 1. Zahl, P.A. and Bjerknes, C. Induction of deciduaplacental hemorrhage in mice by the endotoxins of certain gram-negative bacteria. Proc. Sot. Exp. Biol. Med. 2:329-332 (1943). 2. Hall, G.A. An investigation into the mechanism of placental damage in rats inoculated with Salmonella dublin. Am. J. Pathol. =:299-312 (1974). 3. Reider, R.F. and Thomas, L. Studies on the mechanisms involved in the production of abortion by endotoxin. J. Immunol. a:189-193 (1960). 4. Baines, M.G. and Gendron, R.L. Are both endogenous exogenous factors involved in spontaneous foetal abortion. Res. Immunol. =:154-158 (1990).

and

Theriogenology 5. Giri, S.N., Emau, P., Cullor, J.S., Stabenfeldt, G.H., Bruss, M.L., BonDurant, R.H. and Osburn, B.I. Effects of endotoxin infusion on circulating levels of eicoprogesterone, cortisol, glucose and lactic sanoids, acid and abortion in pregnant cows. Vet. Microbial. =:211-231 (1990). 6. Edqvist, L.-E., Fredriksson, G. and Kindahl, H. Some aspects of endotoxins and corpus luteum function in Panel Proceedings of Nuclear Techniques ruminants. In: in Tropical Animal Disease. International Atomic Energy Agency, Vienna, 1984, ~~-57-68. 7. Daels, P.F., Starr, M., Kindahl, H., Fredriksson, G., Hughes, J.P. and Stabenfeldt, G.H. Effect of Salmonella typhimurium endotoxin on PGF-2Ci release and fetal death in the mare. J. Reprod. Fertil. 35 (Suppl.):485-492 (1987). 8.

Dzvonyar, J., Ruz&::,"d,1G,.;cDoqSfii~,dGdt~~ldC~~bt41eB. Distribution of P tissues of pregnant rabbits and of their fetuses. Am. J. Obstet. Gynecol. m:721-725 (1970).

9.

Haesaert, B. and Ornoy, A. Transplacental effects of endotoxemia on fetal mouse brain, bone and placental tissue. Ped. Pathol. 5:167-181 (1986).

10. Hellmann, W. Endotoxin, fever and anomalies of development in rabbits. Arzneim. Forsch. 2:1062-1064 (1979). 11. Romero, R., Lafreniere, D., Duff, G.W., Kadar, N., Durum, S. and Hobbins, J.C. Failure of endotoxin to cross the chorioamniotic membranes in vitro. Am. J. Perinatol. 4:360-362 (1987). 12. Culbertson, M.R. Bovine Hemostatic and Complement Systems: I. Ontogenetic Studies. II. Fetal Response to Bacterial Endotoxin. Doctorate thesis, University of Calif., Davis, 1978. 13.

Beth-Jansen, P., Brinkman, C.R., Johnson, G.H. and Assali, N.S. Circulatory shock in pregnant sheep. II. Effects of endotoxin on fetal and neonatal circulation. Am. J. Obstet.Gynecol. 113:37-43 (1972).

14. Schlafer, D.H. Applications for use of chronically instrumented bovine fetuses in abortion pathogenesis studies. Bov. Proc. a:168-169 (1988). 15. Beal, W.E., Milvae, R.A. and Hansel, W. Oestrous cycle length and plasma progesterone concentrations following administration of prostaglandin F2a early in the bovine oestrous cycle. J. Reprod. Fertil. =:393-396 (1980).

Theriogenolog

y

759

16. McDonald, T., Reimers, T-J., Figueroa, J.P. and Nathanielsz, P.W. The effect of continuous intracerebroventricular administration of arginine vasopressin (AVP) to the fetal lamb at 125 to 134 days of gestation on fetal plasma ACTH and cortisol concentrations. J. Dev. Physiol. =:35-40 (1989). 17. Binienda, Z., Massmann, A., Wimsatt, J., Honnebier, M.B.O.M., Figueroa, J.P., Reimers, T.J. and Nathanielsz, P.W. Endocrine changes during 48 hours of food withdrawal in the pregnant Rhesus monkey in the last third of gestation. J. Clin. Endocrinol. Metab. a:1184-1187 (1989). 18. Kenison, D.C., Elsasser, T.H. and Fayer, R. Radioimmunoassay for bovine tumor necrosis factor: concentrations and circulating molecular forms in bovine plasma. J. Immunoassay u:177--198 (1990). 19. Strickland, D.M., Brennecke, S.P. and Mitchell, M.D. Measurement of 13,14-dihydro-15-keto-prostaglandin F20: and 6-keto-prostaglandin Fla in plasma by radioimmunoassay without prior extraction or chromatography. Prostaglandins Leukotrienes Med. 2:491-493 (1982). 20. Maqness, R.R., Mitchell, M.D. and Rosenfeld. C.R. Uteroplacental production of eicosanoids in'ovine (1990). pregnancy. Prostaglandins 2:75-88 21

Binienda, Z., Rosen, E.D., Kelleman, A., Sadowsky, D.W., Nathanielsz, P.W. and Mitchell, M.D. Maintaining fetal normoglycemia prevents the increase in myometrial activity and uterine 13,14-dihydro-15-keto-prostaglandin F201 production during food withdrawal in late pregnancy in the ewe. Endocrinology 127:3047-3051 (1990).

22

Mitchell, M.D., Flint, A.P.F., Bibby, J., Brunt, J., Arnold, J.M., Anderson, A.B.M. and Turnbull, A.C. Plasma concentrations of prostaglandins during late human pregnancy: influence of normal and preterm labor. J. Clin. Endocrinol. Metab. j&:947-951 (1978).

23

Griel, L.C., Jr., Zarkower, A. and Eberhart, R.J. Clinical and clinico-pathological effects of Escherichia coli endotoxin in mature cattle. Can. J. Comp. Med. 39:1-6 (1975).

24

Hubbert, W.T. and Hollen, E.J. Cellular blood elements in the developing bovine fetus. Am. J. Vet. Res. =:1213-1219 (1971).

25

Reeves, J.T., Daoud, F.S. and Gentry, M. Growth of the fetal calf and its arterial pressure, blood gases and hematologic data. J. Appl. Physiol. x:240-244 (1972).

760

Theriogenology

26. Meyrick, B., Johnson, J.E. and Brigham, K.L. Endotoxininduced pulmonary endothelial injury. Prog. Clin. Biol. Res. =:91-99 (1989). 27. Beth-Jansen, P., Brinkman, C.R., Johnson, G.H. and Circulatory shock in pregnant sheep. I. Assali, N.S. Effects of endotoxin on uteroplacental and fetal umbilical circulation. Am. J. Obstet. Gynecol. =:1084-1094 (1972). 28. Challis, J.R.G., Richardson, B.S., Rurak, D., Wlodek, M.E. and Patrick, J.E. Plasma adrenocorticotropic hormone and cortisol and adrenal blood flow during sustained hypoxemia in fetal sheep. Am. J. Obstet. (1986). Gynecol. =:1332-1336 Induction of parturition in pigs: 29. Randall, G.C.B. short-term effects of prostaglandin F2a on chronically catheterised fetuses at term. Vet. Rec. m:61-63 (1990). 30

Jansen, C.A.M., Krane, E.J., Thomas, A.L., Beck, N.F.G., Lowe, K.C., Joyce, P., Parr, M. and Nathanielsz, P.W. Continuous variability of fetal PO2 in the chronically catheterized fetal sheep. Am. J. Obstet. Gynecol. m:776-783 (1979).

31

Chalon, S. and Garel, J.M. Plasma calcium control in the rat fetus. III. Influence of alterations in maternal plasma calcium on fetal plasma calcium level. Biol. Neonate 48:329-335 (1985).

32

Comline, R.S. and Silver, M. Some aspects of foetal and uteroplacental metabolism in cows with indwelling umbilical and uterine vascular catheters. J. Physiol. =:571-586 (1976).

33. Fredriksson, G., Kindahl, H. and Stabenfeldt, G. Endotoxin-induced and prostaglandin-mediated effects on the corpus luteum function in the mare. Theriogenology =:309-316 (1986). 34. Gilbert, R.O., Bosu, W.T.K. and Peter, A.T. The effect of Escherichia coli endotoxin on luteal function in Holstein heifers. Theriogenology 2:645-651 (1990). 35. Hixon, J.E., Pijanowski, P.G., Weston, P.G., Shanks, R.D. and Wagner, W.C. Evidence for an oscillator other than luteinizing hormone controlling the secretion of progesterone in cattle. Biol. Reprod. =:1155-1162 (1983).

Theriogenology

761

36.

Scholten, J.A. and Liptrap, R.M. A role for the adrenal cortex in the onset of cystic ovarian follicles in the sow. Can. J. Comp. Med. 42:525-533 (1978).

37.

Peter, A.T., BOSU, W.T.K. and Gilbert, R.O. Absorption of Escherichia coli endotoxin (lipopolysaccharide) from the uteri of postpartum dairy cows. Theriogenology 33:1011-1014 (1990).

38.

Comline, R.S., Hall, L-W., Lavelle, R.B., Nathanielsz, P.W. and Silver, M. Parturition in the cow: endocrine changes in animals with chronically implanted catheters in the foetal and maternal circulations. J. Endocrinol. Q:451-472 (1974).

39.

Waage, A. Production and clearance of tumor necrosis factor in rats exposed to endotoxin and dexamethasone. Clin. Immunol. Immunopathol. 45:348-355 (1987).

40.

LeMay, D.R., LeMay, L.G., Kluger, M.J. and D'Alecy, L.G. Plasma profiles of IL-6 and TNF with fever-inducing doses of lipopolysaccharide in dogs. Am. J. Physiol. Regul. Integr. Comp. Physiol. a:R126-R132 (1990).

41.

Mannel, D.N., Northoff, H., Bauss, F. and Falk, W. a cytokine involved in toxic Tumor necrosis factor: effects of endotoxin. Rev. Infect. Dis. (9 Suopl.) &:S602-5606 (1987).

42.

Ziegler, E.J. Tumor necrosis England J. Med. 318:1533-1535

43.

Yamasu, K., Onoe, H., Soma, G.-I., Oshima, H. and Mizuno, D.-I. Secretion of tumor necrosis factor during fetal and neonatal development of the mouse: ontogenic inflammation. J. Biol. Response Mod. _&:644-655 (1989).

44.

Casey, M.L., Cox, S.M., Beutler, B., Milewich, L. and MacDonald, P.C. Cachectin/tumor necrosis factor-a Potential role of cytokines formation in human decidua. in infection-induced preterm labor. J. Clin. Invest. =:430-436 (1989).

45.

Jaattela, M., Kuusela, P. and Saksela, E. Demonstration of tumor necrosis factor in human amniotic fluid and supernatants of placental and decidual tissues. Lab. Invest. %:48-53 (1988).

46.

Roth, R.I., Levin, F.C. and Levin, J. Optimization of detection of bacterial endotoxin in plasma with the Limulus test. J. Lab. Clin. Med. m:153-161 (1990).

factor in humans. New (1988).

762

Theriogenology

47. Romero, R., Kadar, N., Lafreniere, D., Durum, S., Hobbins, J.C. and Duff, G.W. Do blood and meconium affect the detection of endotoxin in amniotic fluid with the limulus amebocyte gel clot assay? Am. J. Perinatol. 4:356-359 (1987). 48. Munford, R.S., Hall, C.L., Lipton, J.M. and Dietschy, J.M. Biological activity, lipoprotein-binding behavior, and in vivo disoosition of extracted and native forms of Salmonella tvphimurium lipopolysaccharide. Clin. Invest. x:877-888 (1982). 49. Mathison, J.C. and Ulevitch, R.J. The clearance, tissue distribution and cellular localization of intravenously injected lipopolvsaccharide in rabbits. J. Immunol. u:2133-2143(1979).