Signs in Pregnant Sheep Given Endotoxin. J.M. Naylor and D.S. Kronfeld*. ABSTRACT. Groups of four pregnant ewes were allocated to the following feeding ...
Relationships Between Metabolic Changes and Clinical Signs in Pregnant Sheep Given Endotoxin J.M. Naylor and D.S. Kronfeld*
ABSTRACT
Groups of four pregnant ewes were allocated to the following feeding and intravenous endotoxin treatments: 1) fed, Escherichia coli endotoxin (50 ,ug/kg.75), 2) fed, saline, 3) fasted, E. coli endotoxin (50 ,ug/kg-75) and 4) fasted, saline. Endotoxin administration resulted in depression, fever, hypoglycemia, hypocalcemia and a reduction in nonesterifled fatty acid and ketone body concentrations. Depression correlated best with body temperature (r = 0.76), fasted sheep showed smaller increases in body temperature and were less depressed following endotoxin. Three of eight endotoxin treated sheep died, mortality was not related to rectal temperature but was associated with lactic acidosis. Hypoglycemia was not associated with either death or depression. Fed sheep that were unable to stand had lower serum calcium concentrations than standing sheep.
Key words: Endotoxin shock, Escherichia coli, depression, survival, glucose, lactic acidosis. RESUME Cette experience portait sur quatre de brebis gravides, dont les deux premiers requrent une ration ordinaire, tandis que les deux autres furent soumis au je'une. Les brebis des premier et troisieme groupes requrent une injection intraveineuse de 50 ,ug/ kg75 d'endotoxine d'Escherichia coli, tandis que celles des deuxieme et quatrieme groupes ne requrent que 20 mL de saline, egalement par la voie groupes
intraveineuse. Llinjection de l'endotoxine provoqua de la depression, de la fievre, de l'hypoglycemie, de l'hypocalcemie et une baisse de la concentration sanguine en acides gras non esterifies et en corps cetoniques. La depression afficha une relation tres etroite avec la temperature corporelle (r = 0,76); cette derniere s'eleva cependant moins, chez les brebis soumises au jeiune. Trois des huit brebis qui avaient requ l'endotoxine precitee, succombierent; leur mort se revela reliee a l'acidose lactique, mais non a la temperature rectale. Lhypoglycemie n'afficha aucune relation avec la depression ou les mortalites. Les brebis alimentees, incapables de se tenir debout, affichaient une calcemie plus faible que celle des brebis qui pouvaient le faire. Mots cles : endotoxicose, Escherichia coli, depression, survie, glucose, acidose lactique.
INTRODUCTION Endotoxin produces a variety of metabolic changes. These may be important in the pathogenesis of endotoxic shock in sheep because a previous study suggested that disturbances in cardiovascular function were not responsible for death (1). It is of interest to determine which of these are of major clinical significance so that our understanding of the pathogenesis and treatment of endotoxemia can be improved. In other species, hypoglycemia, hypocalcemia and acidosis are important metabolic changes. Hypoglycemia has been incriminated as a cause of convul-
sions, cardiac failure and death in dogs given endotoxin (2-4). However, sheep are more resistant to nervous derangements induced by hypoglycemia than dogs, so this is less likely to be a cause of death (5-10). Hypocalcemia may be partly responsible for recumbency in cattle given endotoxin (11). Acidosis can depress central nervous function (12), increase peripheral resistance and diminish cardiac output (13-16) and has been observed in some shock models. The present study investigates the potential relevance of these changes to depression and death in ewes.
We were also concerned that pregnancy toxemia of sheep might involve a terminal endotoxemic component. Pregnancy toxemia is a disease of undernourished ewes in late pregnancy characterized by hypo-
glycemia, ketosis, elevated plasma nonesterified fatty acid concentrations and fatty liver (17). The liver constantly clears gut absorbed endotoxin from the portal circulation, and people with severe liver disease have periods of endotoxemia (18). We postulated that fatty infiltration of the liver might allow increased amounts of endotoxin to reach the general circulation, and that this might contribute to the metabolic changes in pregnancy toxemia. The potential of endotoxemia to exacerbate the metabolic changes of pregnancy toxemia is investigated in the present study.
MATERIALS AND METHODS ANIMALS AND CARE
Pregnant ewes were obtained from a university flock and acclimated to a
*Department of Veterinary Internal Medicine, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO (Naylor) and Department of Clinical Studies, University of Pennsylvania, New Bolton Center, Pennsylvania 19348 (Kronfeld). Submitted March 26, 1985.
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Can J Vet Res 1986; 50: 402-409.
hay and grain diet for at least five days. Hay was fed ad libitum and grain at the rate of 1.25 kg/day. The ewes were radiographed and the number of fetuses counted. EXPERIMENTAL DESIGN
The ewes were assigned to one of four treatment groups using a randomized block design. The treatments were:
1. Fed sheep, endotoxin 50 ,ug/kg75 in 20 mL 0.9% saline. 2. Fed sheep, 20 mL saline. 3. Fasted sheep, endotoxin 50 ,ug/ kg-75 in 20 mL saline. 4. Fasted sheep, 20 mL saline. There were four sheep in each treatment group, three were twin pregnant, the fourth had a single fetus. Fed sheep were offered food throughout. Fasted sheep were deprived of food but not water for 73 hours, before receving saline or endotoxin treatment at 0 hours. Endotoxin was from Escherichia coli strain 055:b5 (Difco Laboratories, Michigan) and was injected intravenously. Blood was collected from a catheter (Abbott Laboratories, Illinois) placed in the jugular vein, at -2 hours. Specimens were transported on ice and plasma was separated in a centrifuge at 4°C within 30 minutes of collection. Blood was collected at hourly intervals from -I to +11 hours and again at 24 hours, for glucose, calcium, ketone and nonesterified fatty acid estimation. Lactate and venous blood gas estimation were performed at -1, 0, 2, 4, 6, 8, 10 and 24 hours. Samples were collected for glucose analysis in tubes containing a mixture of sodium fluoride and oxalate and in test tubes containing lithium heparin (Vacutainer, Becton-Dickinson) for calcium and nonesterified fatty acid analysis. Lactate and ketone body analysis was performed on blood collected into test tubes containing ice cold perchloric acid. Samples for blood gas analysis were collected in a heparinized syringe, stored in an ice chest and analyzed within two hours of collection. BLOOD ANALYSIS
Lactate was measured with a Llactate dehydrogenase assay (Boehringer) and glucose with a glucose oxidase assay (19). Nonesterified fatty
acids were measured by the method of Dole (20), acetoacetate by Walker's method (21) and f3-hydroxybutyrate fluorimetically using a D-13 hydroxybutyric dehydrogenase linked assay (22). Blood gases were measured with an automated analyzer (ABLI, Radiometer, Copenhagen). Calcium was measured with an 0-cresolpthalein complexone method (Sigma Chemical Company, St. Louis, Missouri). CLINICAL EVALUATION
The ewes rectal temperature and degree of depression was assessed at hourly intervals. Depression was assessed on the basis of menace, palpebral, tactile and auditory responses:
between dying and surviving sheep made after calculating the mean value for the three surviving fed sheep. The Student's t-test (24) was then used to compare values for the two fasted and one fed dying sheep with the two fasted and the mean of the three fed sheep. Differences in calcium values in fed sheep were compared using the Student's t-test. Only differences that are significant at p < 0.05 are discussed in the text. were
RESULTS There were no significant treatment affects on blood pH, base excess or
Response
Method of Assessment
Menace
Hand towards eye, blink response
Palpebral
Finger on medial canthus of eyelids, blink response
Tactile
Pinch coronary band, withdraw foot
Auditory
Hand clap, orients to sound
The scores were summarized to give overall score for depression of cerebral nervous syustem mediated responsiveness to external stimuli. Normal sheep had a depression score of 0, severely affected sheep a maximum score of 8. The sheeps' ability to stand was also recorded at hourly intervals. an
STATISTICAL METHODS
All sheep survived for seven hours from treatment and values were compared using one way analysis of variance (23) at each time interval, means were compared by the method of Tukey (23). Linear regression and multiple linear regression (24) was used to compare score for depression with chemistry values. In sheep given endotoxin two of four fasted sheep and one of four fed sheep died. Comparisons of values
Interpretation 0 - Strong 1 - Weak 2 - Absent 0 - Strong 1 - Weak 2 - Absent 0 - Strong 1 - Weak 2 - Absent 0 - Strong I - Weak 2 - Absent
lactate concentrations. There were significant treatment affects on glucose concentration, and fed sheep given endotoxin went through a phase of hyperglycemia followed by hypoglycemia (Fig. 1). Fasted sheep had lower plasma glucose concentrations and a reduced hyperglycemic response; by five hours there was no significant difference between the hypoglycemic response of fed and fasted sheep to endotoxin. Endotoxin treatment decreased plasma nonesterified fatty acid (NEFA) concentrations in fasted sheep, this affect was not noticeable in fed sheep, which had low NEFA concentrations throughout the experiment (Fig. 2). Acetoacetate and ,Bhydroxybutyrate concentrations were also lower in those fasted sheep given endotoxin rather than saline, but interpretation is difficult because of pretreatment differences between the
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7
A FED ENDOTCXIN * FED SALINE * FAST ENDCTOXIN
6
*
FAST
SALINE
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4
3
2
1' -2
0
2
6
4
TIME.
8
HOURS
Fig. 1. Plasma glucose values in sheep receiving different treatments. Fasted sheep were deprived of food for 72 hours prior to experiment, endotoxin or saline was administered at 0 hours. Each point is the mean and 1 standard error of values from 4 sheep. At a given time period means with different letters are significantly different, p < 0.05.
groups. When the values were replotted as changes from pretreatment values, endotoxin lowered acetoacetate and /3-hydroxybutyrate concentrations in fasted sheep (Figs. 3 and 4).
Fasting significantly lowered baseline plasma calcium concentrations. Values (means ± 1 SE) for the pooled -1 and 0 hour samplng periods were 2.31 ± 0.4, 2.01 ± 0.25, 1.76 ± 0.07
A FED ENDOTOXIN * FED SALINE * FAST ENDOTOXIN * FAST SALINE
3
-j
0
E 0
-LJ
2
C-):
L0 CLi
CLA li
CI) CLi
z
0 z
0 -2
0
2
4
6
8
TIME. HOURS
Fig. 2. Plasma nonesterified fatty acid concentrations in sheep receiving different treatments. Fasted sheep were deprived of food for 72 hours, endotoxin or saline was administered at 0 hours. Each point is the mean and 1 standard error of values for 4 sheep. At a given time period means with different letters are significantly different, p < 0.05.
404
and 1.72 ± 0.12 mmol/L for the fedendotoxin, fed-saline, fastedendotoxin and fasted-saline groups, respectively. When the values following treatment with endotoxin or saline were plotted as changes from the pooled -I and 0 hour values it can be seen that endotoxin depresses plasma calcium by similar extents in both groups (Fig. 5). Plasma calcium did not have a significant correlation with pH in either fed or fasted sheep given endotoxin. Two of the fed endotoxin treated sheep were unable to rise on a total of four occasions. Fasted endotoxin treated sheep were able to stand throughout the experiment despite significantly lower serum calcium values than fed sheep. Plasma calcium values for standing and recumbent sheep were compared within the fed-endotoxin group. Recumbent sheep in the fed groups had slightly lower calcium values, 1.69 ± 0.07 mM (Mean ± SD) than were found in samples collected at the same time periods from standing sheep, 1.91 ± 0.23. Rectal temperature rose following endotoxin administration, but a higher peak temperature and longer duration of fever occurred in fed sheep (Fig. 6). Fed sheep given endotoxin became depressed, depression scores were significantly higher than for fasted endotoxin treated sheep (Fig. 7). The correlation coefficients are shown in Table I, rectal temperature had the best association with depression; overall 59% of the variation in depression was associated with rectal temperature. Blood pH, base excess and lactate concentrations also had significant correlation coefficients, but the association was much weaker than for temperature. A multiple linear regression using both body temperature and lactate as independent variables had a correlation coefficient, r, of 0.79. This is similar to the correlation coefficient for temperature alone, r = 0.77. Serum chemistry values for dying and surviving sheep were compared in two ways. The first method compared the blood sample drawn closest to death with samples drawn at the same time period from surviving sheep. Blood pH, base excess and lactate concentrations were significantly
t-- r2 1I0
A FED ENDOTOXIN
* FED SALINE * FAST ENDOTOXIN
>
120
*
teperature were compared, lactate was higher in dying sheep but temperature was similar in the two groups.
FAST SALINE
100
DISCUSSION
-80
40
20 C 1LU
-2
CD
z
