JOURNAL
OF SURGICAL
RESEARCH
49,37-44 (1990)
LY171883 Preserves Mesenteric Perfusion in Porcine Endotoxic Shock’ STEPHEN M. COHN, M.D., MITCHELL P. FINK, M.D., PATRICK C. LEE, M.D., HAJLONG WANG, M.D., HEIDIE R. ROTHSCHILD, B.S., YAMO F. DENIZ, B.S., AND TAD BAUM, B.S. University
of Massachusetts
Medical Center, 55 Lake Avenue North, Submitted
for publication
uptake (v?,) is normally independent delivery (DO,) [6,42]. In contrast, in sepsis and septic shock, systemic vO2 dependent, even when cardiac output
1 Presented at the American Washington, DC, May 1989.
Federation
of Clinical
Research
01655
April 3, 1989
is normal or supranormal[2,4,28]. When VO, is limited by flow, tissue oxygenation is inevitably inadequate to meet metabolic demands. Therefore, tissue hypoxia may be an important factor contributing to organ system failure in the sepsis syndrome. The mucosa of the gastrointestinal tract seems to be among the tissues at risk for hypoperfusion and hypoxia in sepsis and septic shock. This view is supported by studies showing that gut intramucosal acidosis occurs in both septic patients [13, 241 and resuscitated, normodynamic endotoxic pigs [17, 181. The presence of intramucosal acidosis (presumably due to anaerobic glycolysis leading to excessive lactic acid production) is strong, albeit indirect, evidence that VO, across the mesenteric bed is flow limited [23]. Numerous endogenous vasoactive compounds are potential mediators of the apparent redistribution of cardiac output away from the mesenteric bed in sepsis and septic shock. Among these mediators are the peptidoleukotrienes (LTs), a group of lipoxygenase-derived metabolites of arachidonic acid that are potent mesenteric vasoconstrictors [3,8, 11, 311. These compounds have been implicated as contributing to some of the pathophysiological derangements occurring in sepsis and trauma. Elevated LT concentrations are detectable in the bile of rats subjected to endotoxic shock [26], traumatic shock, or burn injury. Agents that block LT synthesis [27, 401 or LT receptors [9,10,25,43] improve visceral perfusion and survival and prevent hemoconcentration in rat models of endotoxicosis or trauma. In the present study, we postulated that LTs are among the mediators responsible for the derangements in intestinal perfusion which occur in the sepsis syndrome. To test this hypothesis, we utilized LY171883, a specific LTDI/EI receptor antagonist [19], that has been shown to inhibit LT-mediated mesenteric vasoconstriction [lo, 311. Using a clinically relevant porcine model of endotoxic shock [17, 181, we assessed the adequacy of mesenteric perfusion in animals pretreated with either LY171883 or its vehicle.
Superior mesenteric arterial perfusion (a) decreases and gut intramucosal hydrogen ion concentration, [H’], increases in resuscitated normodynamic endotoxic pigs. The present study tested the hypothesis that these adverse phenomena can be prevented by pretreatment with LY171883, a specific leukotriene (LT) DJE, receptor antagonist. Pentobarbital-anesthetized pigs (14-18 kg) were instrumented to permit measurement of Q (ultrasonic flow probe) and [H+] (tonometer). Mesenteric O2 delivery (a,) and consumption (VO,) were calculated from the Oa contents of arterial and superior mesenteric venous blood. At t = -20 min, groups (N = 6) of pigs were pretreated with LY171883 (10 mg/kg) or vehicle. At t = 0 min, the pigs were infused over 20 min with lipopolysaccharide (LPS; 150 pg/kg) and resuscitated for 2 hr with saline (1.2 ml/kg min). Irrespective of treatment group, mean arterial pressure and systemic vascular resistance index decreased significantly after infusion of LPS. In general, cardiac index (CI) was well preserved, although in controls at t = 20, 100, and 120 min, CI decreased significantly with respect to the t = 0 min value. Normal mesenteric 4 and do, were maintained in the LY 171883 group, whereas, in controls, these parameters decreased significantly. Mesenteric VO, increased transiently but significantly in controls; this phenomenon was abrograted by the LT receptor antagonist. In controls, intramucosal [H+] increased by almost threefold; this adverse effect was significantly ameliorated by LY 171883. These data suggest that decreased mesenteric 4 and increased intramucosal [H+] may be mediated by LT in this o ieso AC&tic PWS, IIN. porcine endotoxic shock model.
Systemic oxygen of systemic oxygen many patients with is apparently flow
Worcester, Massachusetts
in
37
0022-4804/90$1.50
Copyright 0 1990 hy Academic Press, Inc. All rights of reproduction in any form reserved.
38
JOURNAL
OF SURGICAL
RESEARCH:
TABLE Baseline
Hemodynamic Control
VOL.
49, NO. 1, JULY
1990
1
and Oxygen
Transport
Data LY171883 group
group
t = -20 min
t = 0 min
t = -20 min
t = 0 min
MAP (Torr) CI (ml/min kg) SVRI (Torr min kg/liter)
111 268 428
+ 3 f 20 + 41
110 269 431
+ 5 f 25 f 59
105 280 380
+ 4 f 21 + 22
106 280 385
f 4 + 15 f 30
MPAP (Torr) SMA @ (ml/min)
16 474
f 2 t 40
17 486
* 1 f 41
16 371
+ 1 f 25
17 385
+ 1 f 35
Systemic rj0, (ml/min) Systemic ri0, (ml/min) Mesenteric DO2 (ml/min) Mesenteric VO, (ml/min)
559 142 64 17
f 39 + 6 -c 5 f 2
558 136 66 15
+- 47 f 10 + 5 + 2*
584 140 48 18
+ 47 + 13 It 4# + 2
570 145 48 19
+ 31 f 10 f 4# f 3
Intramural
49.1 +
[H+] (ti)
2.3
44.9 +
1.8
45.2 +
2.3
43.0 +
1.8
* P < 0.05 vs t = -20 min within group. # P < 0.05 vs time-matched value in control group.
METHODS
Studies were performed in accordance with the guidelines of the institutional review board for animal experimentation of the University of Massachusetts. Domestic pigs without clinical evidence of gram-negative infections were used in our experiments. The animal model used for these studies was adapted from one described by Breslow et al. [5] and was previously described [ 17,181. Male pigs (13-16 kg) were anesthetized with intramuscular ketamine (7 mg/kg) and intravenous sodium pentobarbital (17 mg/kg). Additional doses of pentobarbital (10 mg) were administered as necessary to maintain light general anesthesia. After tracheostomy, the animals were ventilated using a Harvard ventilator with 100% Oz at a tidal volume of 10 ml/kg. Respiratory rate was adjusted to maintain the pC0, equal to 40 _+5 mm Hg. Baseline core body temperature was maintained at 39.0 f 0.5”C by using heating pads and blankets. Polyethylene catheters (PE 160) were placed in the inferior vena cava and abdominal aorta via bilateral femoral cutdowns. A 2.5-French thermistor-tipped catheter (Edwards Division, American Hospital Supply Corp., Irvine, CA) was advanced into the thoracic aorta via a femoral artery. A flow-directed balloon-tipped catheter (Edwards) was inserted into the pulmonary artery via a femoral vein. Observing the characteristic pulmonary arterial pressure tracing on the monitor confirmed the location of the catheter. A 3.0-French catheter for injecting the thermal indicator was introduced into the right atrium via the right external jugular vein. A midline celiotomy was performed and a 4- to 6-mm ultrasonic flow probe (Transonic Systems, Inc.) was positioned around the superior mesenteric artery (SMA) near its origin from the abdominal aorta. The probe was connected to a previously calibrated Transonic Systems
Model TlOl blood flowmeter. A PE 90 catheter was advanced into the superior mesenteric vein (SMV) via a distal tributary in the mesentery of the small intestine. A tonometric catheter (Tonometrics, Inc., Bethesda, MD) was inserted into the ileum via an antimesenteric enterotomy and secured in place. The animals were allowed to stabilize for 30-60 min after closing the laparotomy incision. Arterial pressure was measured using Transpac-II transducers (Sorenson, Abbott Laboratories, North Chicago, IL) driving a Honeywell amplifier-monitor with digital readout. Cardiac output (CO) was determined by thermodilution using an Edwards Model 9520 computer and 3.0-ml injections of room-temperature normal saline injected into the right atria1 catheter. Cardiac output determinations were performed in triplicate and the results averaged. Cardiac index (CI) was determined by dividing CO by body weight. Systemic vascular resistance index (SVRI) was calculated by dividing mean arterial pressure (MAP) by CI. Arterial, mixed venous (pulmonary arterial), and superior mesenteric venous oxygen contents (Co,) were calculated after obtaining blood samples from the appropriate catheters and analyzing them for p02, hemoglobin concentration (Hgb), and percentage saturation (So,). An Instrumentation Laboratories Model 1302 blood gas analyzer was used to measure pop and an Instrumentation Laboratories Model 282 co-oximeter was used to measure Hgb and So,. Co, was calculated according to the equation Co, = (p02 X 0.003) + (Hgb X So, X 1.36). The following additional equations were utilized: systemic DO2 = cardiac output X arterial Co,; systemic ri0, = cardiac output X (arterial Co, - mixed venous Co,); mesenteric ho, = SMA flow X arterial Co,; mesenteric VO, = SMA flow X (arterial Co, - SMV Co,).
COHN
ET AL.: LY171883
AND
We calculated intramucosal hydrogen ion concentration, [H+], using a tonometric method originally described and validated by Fiddian-Green and co-workers [ 121. Because it diffuses readily, gut intramural COz is in equilibrium with CO2 in intraluminal fluid. If arterial bicarbonate concentration is an accurate estimate of the intramural bicarbonate concentration, then intramucosal [H+] can be estimated using a modified form of the HendersonHasselbalch equation: [Hf] = 24 X ( [HC03]/pCOz), where [HCOB] is the concentration of bicarbonate in arterial blood and pCOz is the partial pressure of carbon dioxide in gut luminal fluid. Rather than sampling luminal fluid directly, we measured pC0, in fluid (normal saline) contained in an intraluminal balloon tonometer constructed of a material (silicone) that is freely permeable to COZ. After inserting the tonometer into the gut lumen (see above), saline in the balloon was allowed to equilibrate for 20 min and then aspirated to permit measurement of pC0,; care was taken to discard the deadspace volume of the catheter. The tonometer balloon was refilled with fresh fluid every 20 min. The measured p COP was corrected to account for incomplete equilibration prior to calculating intramural [H+] [ 141. Blood samples for prostanoid determination were obtained from the femoral arterial catheter. The samples were collected into iced glass tubes containing EDTA (10.5 mg) and indomethacin (0.1 mg). Within 15 min of being obtained, the blood samples were centrifuged (15OOg for 15 min at 4°C). The plasma was collected and stored in polypropylene tubes at -70°C until assayed. Details of the radioimmunoassay for 6-ketoprostaglandin (PG) F1, and thromboxane (TX) Bz were described previously [ 151. The lower limits of sensitivity for the assay were ‘78 and 156 pg/ml for 6-keto-PGF1, and TxBz, respectively. Experimental Protocol Two groups of pigs were studied. Beginning at t = 0 min, both groups were infused over 20 min with lipopolysaccharide (LPS; Escherichia coli Olll:B4; DIFCO, Detroit, MI; 150 pg/kg). At t = -20 min, the experimental group (N = 6) was pretreated with LY171883 (10 mg/kg in 10 ml of 0.5 M NaHC03). Controls (N = 6) were pretreated with 10 ml of the 0.5 M NaHC03 vehicle. Starting at the beginning of the LPS infusion (t = 0 min) and continuing for the duration of the experiment, all animals were continuously resuscitated with normal saline (1.2 ml/kg min). Measurements to assess hemodynamics, oxygen metabolism, and intramucosal [H+] were made at t = -20 min (i.e., prior to infusing drug or vehicle), t = 0 min (i.e., after administering drug or vehicle, but prior to infusing LPS), and every 20 min thereafter for 2 hr. Blood samples for prostanoid determination were obtained at t = -20, 20, and 80 min. Animals were sacrificed at 120 min by lethal injection. Statistical Analyses Data were analyzed using commercially available software (CSS, Statsoft, Tulsa, OK) on a Kaypro 286i mi-
MESENTERIC
39
PERFUSION MEAN SYSTEMIC ARTERIAL PRESSURE 1
130 120
401 0
20
40
60
80
100
120
*
*
100
120
TIME (MN)
CARDIAC INDEX
130 120
#
110
T
I
60 50 0
20
40
60
80
TIME (MIN) FIG. 1. Effect of LY171883 on MAP (top) and CI (bottom) in endotoxic pigs. Results are expressed as percentages of the t = 0 min value for each animal. Symbols represent means + SE for pigs pretreated with LY171883 (squares) or vehicle (circles). *Values that are significantly different from the t = 0 min value for the same group. #Values that are significantly different from the time-matched values in the control group.
crocomputer. The results in the figures are expressed as percentages of the baseline (t = 0 min) value for each animal; the symbols represent means f SE. Overall group, time, and group X time effects were assessed using twoway analysis of variance (ANOVA) for repeated measures. Differences with respect to baseline within a group and time-matched differences between groups were assessed by analyzing using Duncan’s new multiple-range test [32]. The null hypothesis was rejected when P < 0.05. RESULTS
Baseline (i.e., t = -20 and 0 min) hemodynamic and oxygen transport data are presented in Table 1. Mesenteric BO, was significantly lower in the LY171883 group. For all other measured variables, the two treatment groups were quite comparable. Infusing LY 171883 or its vehicle significantly increased arterial pH; these effects were consistent with the alkaline nature of these solutions.
40
JOURNAL
.- . 0
2-O
;o
i0 TIME
FIG. 2. Effect of LY171883 as in Fig. 1.
OF SURGICAL
RESEARCH:
i0
li0
loo
VOL. 49, NO. 1, JULY
0
20
40
(MN)
on SVRI in endotoxic
1990
60
80
100
120
TIME (MIN) pigs. Symbols
The effects of LPS on systemic and pulmonary hemodynamics are shown in Figs. l-3. Irrespective of group, MAP decreased substantially to -60% of the t = 0 min (reference) value (Fig. 1). Although generally well preserved in both groups, CI decreased significantly in controls at t = 20, 100, and 120 min. At t = 80 min, CI was significantly higher in the drug-treated group. After increasing transiently at t = 20 min, SVRI decreased significantly in both groups, reaching a nadir at t = 80 min (Fig. 2). In both groups, mean pulmonary arterial pressure (MPAP) increased approximately threefold at t = 20 min and then declined to a plateau that was approximately twice the reference value (Fig. 3). At t = 40-60 min, MPAP was slightly, but significantly, lower in the LY171883 group. Mesenteric Q was fairly stable after LPS infusion in animals pretreated with LY171883 (Fig. 4). In controls? however, there was a marked decrease in mesenteric Q that was significant both compared to the baseline value within group and to matched time points in the LY 171883 group. By repeated-measures ANOVA, the group effect for this variable was significant (P = 0.022). The effects of LPS on systemic oxygen transport are shown in Fig. 5. Systemic DO2 tended to decrease over time in the control group and was significantly lower than in the LY171883 group at t = 20 and 80 min. Systemic VO, increased significantly at t = 20-60 min in controls. This phenomenon was abrogated by prior treatment with LY171883. By repeated-measures ANOVA, the group X time interaction for systemic VOz was significant (P = 0.032). In the drug-treated group, mesenteric ri0, was approximately normal until t = loo-120 min when this variable decreased to 80-90% of the reference value (Fig. 6). In the control group, however, mesenteric 60, decreased significantly to 50-60% of baseline. There was a statistically significant difference between groups (P = 0.012). Mesenteric VO, increased significantly in the
FIG. 3. Effect of LY171883 on MPAP as in Fig. 1.
in endotoxic
pigs. Symbols
control group, with respect both to the baseline value within group and to time-matched values in the LY171883 group. The group effect for mesenteric VO, was significant (P = 0.034). Table 2 shows the effect of LPS on arterial blood gases in control and drug-treated pigs. In both groups, arterial pOz decreased significantly during the second hour of the protocol. Oxygenation was transiently better in the LY171883 group at t = 40 min. By design,pCOz was nearly constant in both groups. Metabolic acidosis occurred in both groups, but this phenomenon was ameliorated in the animals pretreated with the LT receptor antagonist. Gut intramucosal [H+] increased dramatically after infusing LPS in controls (Fig. 7). Although [H+] also increased significantly in LY171883-treated animals, the magnitude of the change was substantially smaller than that observed in the control group. By ANOVA, both group and group X time effects were highly significant (P < 0.00001).
40 1 30 4 0
*
20
40
60
TIME
80
*
*
100
120
(MN)
FIG. 4. Effect of LY171883 on superior mesenteric flow in endotoxic pigs. Symbols as in Fig. 1.
arterial
blood
COHN
ET AL.: LY171883
AND
SYSTEMIC OXYGEN DELIVERY
130,
T
#
*
60-
1 *
A *
50, 0
20
40
60
80
100
120
TIME (MIN)
SYSTEMIC OXYGEN UPTAKE
220 200
*
MESENTERIC
this is the so-called hyperdynamic state [l, 22, 371. Decreased systemic vasomotor tone is so characteristic of the sepsis syndrome that this finding has been used in clinical research studies as a criterion for establishing the diagnosis [34, 381. During episodes of hypotension (i.e., septic shock), CI tends to decrease into the normal range while SVRI remains abnormally low [ 11. The porcine endotoxicosis model used for the present study satisfactorily mimicked the systemic hemodynamic changes commonly seen during episodes of septic shock in resuscitated human patients; i.e., CI was well-preserved and SVRI was markedly decreased. Consistent with current clinical practice, aggressive intravascular volume loading was a key feature of the protocol. In other similar models, resuscitation was guided by measurements of central venous [5] or left atria1 pressure [33], the volume of fluid administered being adjusted to maintain these indices of cardiac preload within the normal range. Instead of this approach, we chose to infuse’the same (large) volume of saline to all animals. We adopted this straightforward protocol for several reasons. First, myocardial performance is clearly impaired in sepsis [35,37] and enMESENTERIC OXYGEN DELIVERY
110
100
0
20
40
60
60
100
41
PERFUSION
#
#
120
TIME (MIN) FIG. 6. Effect of LY171883 on systemic 80, (top) and QO, (bottom) in endotoxic pigs. Symbols as in Fig. 1.
40
*
*
*
SO
100
120
30 0
20
40
After infusion of LPS, plasma levels of both 6-ketoPGF1, (prostacyclin metabolite) and TxBz (TxA2 metabolite) increased significantly. These changes were similar in both treatment groups (Table 3).
TIME (MIN)
MESENTERIC OXYGEN UPTAKE
200 190 180
DISCUSSION
These were the principal findings in the present study: (1) LY 171883 had no appreciable effect on any measured variable in normal, pentobarbital-anesthetized pigs; (2) pretreatment with. LY171883 substantially improved mesenteric 0 and DO, in our porcine model of resuscitated, endotoxic shock; (3) mesenteric VO, increased substantially in endotoxic controls, but this phenomenon was abrogated by the prior infusion of LY171883; (4) gut intramucosal [H+] increased significantly after infusion of LPS in both drug- and vehicle-treated animals, but the magnitude of this effect was much smaller in animals infused with the LTDI/E& receptor antagonist. In adequately resuscitated humans with compensated sepsis, CI is usually elevated and SVRI is typically low;
60
*
51 O-----I 170 If," .__ I
*
I.dV
*
4c.nJ
I
140 130 120
1
E w w
110
I
7’0
‘0
I/ /o
100
-
T
d
I/
I
I I
(
90 60 70 60 t
I
-y
I ,6-
- - -g
_ _ %&
#
i #
_ - -&
- - q-
#
T_ 1
- -0
T 1 #
50 4 0
20
40
60
SO
100
120
TIME (MIN) FIG. 6. Effect of LY171883 on mesenteric do, (top) and VO, (bottom) in endotoxic pigs. Symbols as in Fig. 1.
42
JOURNAL
OF SURGICAL
RESEARCH:
dotoxicosis [21]. Therefore, preload may need to be elevated to supranormal levels to maintain CI. Second, sepsis may lead to alterations in ventricular diastolic compliance [ 36 1. Thus, ventricular filling pressures are probably poor indices of preload (i.e., end-diastolic volume) in the setting of sepsis or endotoxicosis. Finally, a fixed-volume regimen markedly simplifies the experimental design and avoids problems of investigator bias. Tissue acidosis is a well-documented consequence of tissue hypoxia [39]. In the heart, tissue pH is strongly correlated with tissue concentrations of ATP [30]. In the gut, the onset of intramucosal acidosis correlates with the inflection point between flow-independent and tlow-dependent VO, [23]. In the present study, we assessed gut intramucosal pH using a tonometric method originally described by Fiddian-Green et al. 1121. The fundamental assumption underlying this method is that the concentration of bicarbonate is similar in plasma and tissue. This assumption is evidently valid, since Fiddian-Green et al. showed that there was good correlation (r = 0.98) between intramucosal pH measured directly and estimated tonometrically. We previously showed that gut intramucosal [Hf] increases in our normodynamic porcine model of endotoxic shock [17, 181. This observation was reproduced (in a new group of animals) in the present study. These data support the view that intramucosal VO, is inadequate in endotoxic shock. Pretreatment with LY171883 substantially ameliorated post-LPS intramucosal acidosis, suggesting that the adequacy of mucosal oxygenation was improved by the LTDl/Eq receptor antagonist. Two factors seem to be involved in this beneficial effect of LY171883. First, the drug clearly improved mesenteric Q in endotoxic pigs. As a consequence, mesenteric fi0, was significantly greater in the LY171883 group as compared to vehicle-treated controls. The effect of LY171883 on mesenteric perfusion in the present study is consistent with other previous data indicating that LTs are potent mesenteric vasoconstrictors [ll, 31, 381. The improvement
VOL. 49, NO. 1, JULY
240 bz
of LPS on Arterial
Blood
Gases and Derived
220
k!
20
0
40
60
100
80
120
TIME (FAN)
FIG. 7. Effect of LY171883 on ileal intramucosal pigs. Symbols as in Fig. 1.
[H+] in endotoxic
in mesenteric perfusion observed in our study is also consistent with data obtained by Etemadi et al. showing improved gut perfusion in a low-cardiac-output model of endotoxic shock in the rat [lo]. The data presented here support the notion that LTs are important mediators of the relative redistribution of cardiac output away from the mesenteric bed in resuscitated endotoxic shock. The second factor evidently contributing to the salutary effect of LY171883 on intramucosal [H+] was that the LTD4/E4 antagonist seemed to blunt the hypermetabolism induced by LPS. Thus, the drug apparently improved the adequacy of tissue oxygenation by both increasing mesenteric DO2 while, simultaneously, decreasing the metabolic demand for oxygen across this bed. It is well established that sepsis and endotoxicosis increase metabolic rate in adequately resuscitated animals [29] and humans [ 11. In the present study, this phenomenon was particularly evident across the mesenteric bed (Fig. 6). The mechanisms responsible for hypermetabolism in sepsis and endotoxicosis are incompletely understood. Circulating catecholamines, known to be elevated in sepsis/en-
TABLE Effect
1990
2
Values
in Vehicle-
and LY 171883-Pretreated
Pigs
Time Variable
Pox Vorr)
Group
Control LY171883
PcOz (Ton) [HCO;](mM) BE W@)
control LY171883 Control LY171883 Control LY171883
-20 454 450
c!
+11 *19
37.9 + 39.3 + 25.3 k 23.7 + 1.6 + -0.5 f
.4
1.9 .6 .9 .8
1.0
451 446 36.9 40.2 25.8-c 25.8 2.5 1.7
* P < 0.05 vs t = -20 min within group. # P < 0.05 vs time-matched value in control
20
+ 9 +15 ck .4 + 1.7 .8 + .8 f .9 + .b
group.
400 433
40
f20 +52
232 376
60
?50* +40#
35.0 * 41.9 k
1.0 2.4x
39.8 + 42.9 +
1.2 1.2
22.0 f 22.9 + -1.6 k -2.2 f
1.1* .7* 1.2*
19.5 k 21.8 * -5.9 + -3.8 +
.9* .7"
1.0*
1.w .9'#
258 307 40.6
+50* *57* 3~ 2.1*
42.1 18.5 20.4 -7.4 -5.4
* + + k
.8 .5* .8* .6*
+
1.1’.#
339 287 40.0 41.2 18.1 20.0 -8.0 -5.7
* 29* k51* k .8 + k + k *
120
100
80
1.3 .6* 7*,# .6' 1.0*.x
309 254
f27* +52*
38.1 + 40.8 + 17.7 f
.8 18
309 230 38.6
If: 29* f65' k .8
39.5 +
.5*
17.2 +
19.4 zk
.7*
19.0 +
-&O-1-6.3 +_
.6* .9**#
-8.8 -7.1
c +
1.0 .6* .6*,X .7' .9'*#
COHN
ET AL.: LY171883
AND
MESENTERIC
TABLE Effect
of LPS on Plasma
Prostanoid Control
6-K&o-PGF,,
TX& (pdml)
(pg/ml)
43
PERFUSION
3
Concentrations
in Vehicle-
and LY171883-Pretreated
group
Pigs
LY171883 group
t = -20 min
t = 20 min
t = 80 min
t = -20 min
t = 20 min
t = 80 min
707 t 113 474+ 99
721+ 162 3003 2 84*
2007+367* 2961+ 154*
780 + 153 253+ 97
596 f161 3180 + 226*
1868 f 291* 3650 f 508*
dotoxicosis [41], may be important, however, since exogenous catecholamines increase VO, in both animals [ 71 and patients [20]. The present data offer no clues as to the mechanisms underlying the effect of LYl71883 on mesenteric VOz. Clearly, however, this interesting phenomenon warrants further investigation. Consistent with numerous other studies [ 151, we showed that infusion of LPS leads to markedly elevated circulating levels of TxBz (TxAz metabolite) and 6-keto-PGF,, (PGIZ metabolite). It is noteworthy that pretreatment with LY171883 did not affect the plasma prostanoid response to LPS (Table 3). In contrast, another LT receptor antagonist, FPL 57231, seems to be less specific and may function as a cyclooxygenase inhibitor. The apparent lack of effect of LY171883 on the cyclooxygenase pathway renders the results presented here more amenable to interpretation. The present study is open to certain criticisms. First, we did not attempt to measure LT levels in either plasma or bile. Obviously, documenting elevated concentrations of LTs (or their metabolites) after administering LPS would greatly strengthen the case, implicating these lipids as mediators of some the physiological derangements observed in our endotoxic shock model. Unfortunately, the assay systems for these compounds are quite difficult, and, as yet, we have not perfected them in our laboratory. Second, we utilized only one LT receptor antagonist, leaving open the possibility that the pharmacologic effects described herein were due to an action of LY171883 unrelated to its activity as an LTD4/E4 receptor antagonist. LY 171883 is a phophodiesterase inhibitor [ 191. Ongoing studies in our laboratory using other compounds are directed at this criticism. Third, the animals were infused with LY171883 prior to the onset of endotoxemia. We deliberately chose a pretreatment design to maximize the chances of observing unambiguous effects. We recognize, however, that pretreatment is unlikely in the clinical setting, and, therefore, extrapolating our results to the treatment of human septic shock is clearly unwarranted. Fourth, mesenteric fi0, was lower at baseline in our pretreatment group. We do not know what effect this may have had on the protection obtained in this group, but recognize that this could have impacted on these data. Finally, we showed that LY 171883 ameliorated gut intramucosal acidosis in porcine septic shock, but provided no evidence that this phenomenon was beneficial in terms
of mucosal function or histology. Further experiments being planned to address this issue.
are
ACKNOWLEDGMENTS This work was supported by Grant 1 R29 GM-37631-0103 from the National Institutes of Health. Ms. Sandy Hinson and Ms. Penny Lucier assisted in the preparation of the manuscript.
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Bayorh, M. A., Faden, A. I., and Feuerstein, G. Differential hemodynamic effects of leukotriene D1 in anesthetized rats: Evaluation by directional pulse Doppler technique. Prostaglandin Leukotriene Med. 17: 229.1985.
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Bihari, D., Smithies, M., Gimson, A., and Tinker, J. The effects of vasodilation with prostacyclin on oxygen delivery and uptake in critically ill patients. N. Engl. J. Med. 317: 397, 1987.
5.
Breslow, M. J., Miller, C. F., Parker, S. D., Walman, A. T., and Traystman, R. J. Effect of vasopressors on organ blood flow during endotoxin shock in pigs. Amer. J. Physiol. 262: H291, 1987.
6.
Cain, S. M. Oxygen delivery and uptake in dogs during anemic and hypoxic hypoxia. J. Appl. Physiol. 42: 228, 1977.
7.
Cain, S. M., and Chapler, C. K. Effects of norepinephrine and beta-blockers on Ox uptake and blood flow in dog hind limb. J. Appl. Physiol. 5: 1245, 1981.
8.
Chapnick, B. M. Divergent influences of leukotriene Cd, D, and E4 on mesenteric and renal blood flow. Amer. J. Physiol. 246: H518,1984.
9.
Cook, J. A., Wise, W. C., and Halushka, P. V. Protective effect of a selective leukotriene antagonist in endotoxemia in the rat. J. Pharmacol. Exp. Thu. 235: 470,1985.
10.
Etemadi, A. R., Temple, G. E., Farah, B. A., Wise, W. C., Halushka, P. V., and Cook, J. A. Beneficial effects of a leukotriene antagonist on endotoxin-induced acute hemodynamic alterations. Circ. Shock 22:55,1987.
11.
Feigen, L. P. Differential effects of leukotriene Cd, D, and El in the canine, renal and mesenteric vascular beds. J. Pharmacol. Erp. Ther. 225: 682,1983.
12.
Fiddian-Green, R. G., Pittenger, G., and Whitehouse, W. M., Jr. Back-diffusion of CO2 and its influence on the intramural pH in gastric mucosa. J. Sug. Res. 33: 39, 1982.
13.
Fiddian-Green, R. G., McGough, E., Pittenger, G. L., and Rothman, E. Predictive value of intramural pH and other risk factors for
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massive bleeding from stress ulceration. GostroenteroZogy 86: 613, 1983. Fiddian-Green, R. G., Amelin, P. M., Herrmann, J. B., Arous, E., Cutler, B. S., Schiedler, M., Wheeler, H. B., and Baker, S. Prediction of the development of sigmoid ischemia on the day of aortic operations: Indirect measurements of intramural pH in the colon. Arch. Surg. 121: 654, 1986. Fink, M. P. Role of prostaglandins and related compounds in the pathophysiology of endotoxic and septic shock. Semin. Resp. Med. 7: 17, 1985. Fink, M. P., Gardiner, W. M., Roethel, R., and Fletcher, J. R. Plasma levels of 6-keto-PGF,, but not TxB2 increase in rats with peritonitis due to cecal ligation. Circ. Shock 16: 297, 1985. Fink, M. P., Cohn, S. M., Lee, P. C., Rothschild, H. R., Deniz, Y. F., Wang, H., and Fiddian-Green, R. G. Effect of lipopolysaccharide on intestinal intramucosal pH in pigs: Evidence of gut ischemia despite normal cardiac output and mesenteric oxygen consumption. Surg. Forum 39: 80, 1988. Fink, M. P., Cohn, S. M., Lee, P. C., Rothschild, H. R., Deniz, Y. F., Wang, H., and Fiddian-Green, R. G. Effect of lipopolysaccharide on intestinal intramucosal hydrogen ion concentration in pigs: Evidence of gut ischemia in a normodynamic model of septic shock. Crit. Care Med., 17(7): 641-646, 1989. Fleisch, J. H., Rinkema, L. E., Haisch, K. D., Swanson-Bean, D., Goodson, T., Ho, P. P. K., and Marshall, W. S. LY171883, 1-[2hydroxy-3-propyl-4-[4-(1 H-tetrazol-5-yl)butoxy]phenyl]ethanone, an orally active leukotriene D, antagonist. J. Phurmacol. Exp. Ther. 233: 148,1985. Gilbert, E. M., Haupt, M. T., Mandanas, R. Y., Huaringa, A. J., and Carlson, R. W. The effect of fluid loading, blood transfusion, and catecholamine infusion on oxygen delivery and consumption in patients with sepsis. Amer. Reu. Resp. Dis. 134: 873, 1986. Goldfarb, R. D., Nightingale, L. M., Kish, P., Weber, P. B., and Loegering, D. J. Left ventricular function during lethal and sublethal endotoxemia in swine. Amer. J. Physiol. 261: H364, 1986. Groenveld, A. B. J., Bronsveld, W., and Thijs, L. G. Hemodynamic determinants of mortality in human septic shock. Surgery 99: 140, 1986. Grum, C. M., Fiddian-Green, R. G., Pittenger, G. L., Grant, B. J. B., Rothman, E. D., and Dantzker, D. R. Adequacy of tissue oxygenation in intact dog intestine. J. Appl. Physiol. 66: 1065, 1984. Gys, T., Hubens, A., Neels, H., et al. The prognostic value of gastric intramural pH in surgical intensive care patients. Crit. Care Med., 16: 1222-1224,1988. Hagmann, W., and Keppler, D. Leukotriene antagonists prevent endotoxin lethality. Nuturwissenschuften 69: 594, 1982. Hagmann, W., Denzlinger, C., and Keppler, D. Role of peptide leukotrienes and their hepatobiliary elimination in endotoxin action. Circ. Shock 14: 223, 1984. Hall-Angeras, M., Saljo, A., and Hasselgren, P-O. Effect of methylprednisolone, indomethacin, and diethylcarbamazine on survival rate following trauma and sepsis in rats. Circ. Shock 20: 231,1986.
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Lange, Khuri, thermic itoring
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Michelassi, F., Shahinian, H. K., and Ferguson, M. K. Effect of leukotrienes Bq, C,, and D1 on rat mesenteric microcirculation. J. Surg. Res. 42: 475,1987.
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Miller, R. J., Jr. Simultaneous McGraw-Hill, 1966.
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Modig, J. Comparison of effects of dextran-70 and Ringer’s acetate on pulmonary function, hemodynamics, and survival in experimental septic shock. Crit. Care Med. 16: 266, 1988.
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Montgomery, W. V., Stager, M. A., Carrico, C. J., and Hudson, L. D. Causes of mortality in patients with the adult respiratory distress syndrome. Amer. Rev. Resp. Dis. 132: 485, 1985.
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Natanson, C., Fink, M. P., Ballantyne, H. K., MacVittie, T. J., Conklin, J. J., and Parrillo, J. E. Gram-negative bacteremia produces both severe systolic and diastolic cardiac dysfunction in a canine model that simulates human septic shock. J. Clin. Znuest. 78: 259, 1986.
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Natanson, C., Danner, R. L., Fink, M. P., MacVittie, T. J., Walker, R. I., Conklin, J. J., and Parrillo, J. E.: Cardiovascular performance with E. coli challenges in a canine model of human sepsis. Amer. J. Physiol. 254: H558, 1988.
37.
Parker, M. M., Shelhammer, J. H., Bachrach, S. L., Green, M. V., Natanson, C., Frederick, T. M., Danske, B. A., and Parrillo, J. E. Profound but reversible myocardial depression in patients with septic shock. Ann. Intern. Med. 100: 483,1984.
38.
Pepe, P. E., Potkin, R. T., Reus, D. H., Hudson, L. D., and Carrico, C. J. Clinical predictors of the adult respiratory distress syndrome. Amer. J. Surg. 144: 124, 1982.
39.
Poole-Wilson, P. A. Measurement of myocardial intracellular in pathological states. J. Mol. Cell. Curdiol. 10: 510, 1978.
40.
Rogers, F., Dunn, R., Nolin, P., Phunangsab, A., and Barrett, Diethylcarbamazine, a leukotriene inhibitor, improves survival endotoxemia in the rat. Circ. Shock 16: 52,1985.
41.
Schaller, M. D., Waeber, B., Nussberger, J., Brunner, H. R. Angiotensin II, vasopressin, and sympathetic activity in conscious rats with endotoxemia. Amer. J. Physiol. 249: H1086, 1985.
42.
Shibutani, K., Komatsu, T., Kubal, K., Sanchala, V., Kumar, V., and Bizzarri, D. V. Critical level of oxygen delivery in anesthetized man. Crit. Care Med. 11: 640, 1983.
43.
Smith, E. F., Kinter, L. V., Jugus, M., Wasserman, M. A., Eckardt, R. D., and Newton, J. F. Beneficial effects of the peptidoleukotriene receptor antagonist, SK&F 104353, on the responses to experimental endotoxemia in the conscious rat. Circ. Shock 25: 21,1988.
R., Kloner, R. A., Zierler, M., Carlson, N., Seiler, M., and S. S. Time course of ischemic alterations during normoand hypothermic arrest and its reflection by on-line monof tissue pH. J. Thorac. Cardiouasc. Surg. 86: 418, 1983.
Statistical
Inference.
New York:
pH J. of
OF SURGICAL
RESEARCH
49,37-44 (1990)
LY171883 Preserves Mesenteric Perfusion in Porcine Endotoxic Shock’ STEPHEN M. COHN, M.D., MITCHELL P. FINK, M.D., PATRICK C. LEE, M.D., HAJLONG WANG, M.D., HEIDIE R. ROTHSCHILD, B.S., YAMO F. DENIZ, B.S., AND TAD BAUM, B.S. University
of Massachusetts
Medical Center, 55 Lake Avenue North, Submitted
for publication
uptake (v?,) is normally independent delivery (DO,) [6,42]. In contrast, in sepsis and septic shock, systemic vO2 dependent, even when cardiac output
1 Presented at the American Washington, DC, May 1989.
Federation
of Clinical
Research
01655
April 3, 1989
is normal or supranormal[2,4,28]. When VO, is limited by flow, tissue oxygenation is inevitably inadequate to meet metabolic demands. Therefore, tissue hypoxia may be an important factor contributing to organ system failure in the sepsis syndrome. The mucosa of the gastrointestinal tract seems to be among the tissues at risk for hypoperfusion and hypoxia in sepsis and septic shock. This view is supported by studies showing that gut intramucosal acidosis occurs in both septic patients [13, 241 and resuscitated, normodynamic endotoxic pigs [17, 181. The presence of intramucosal acidosis (presumably due to anaerobic glycolysis leading to excessive lactic acid production) is strong, albeit indirect, evidence that VO, across the mesenteric bed is flow limited [23]. Numerous endogenous vasoactive compounds are potential mediators of the apparent redistribution of cardiac output away from the mesenteric bed in sepsis and septic shock. Among these mediators are the peptidoleukotrienes (LTs), a group of lipoxygenase-derived metabolites of arachidonic acid that are potent mesenteric vasoconstrictors [3,8, 11, 311. These compounds have been implicated as contributing to some of the pathophysiological derangements occurring in sepsis and trauma. Elevated LT concentrations are detectable in the bile of rats subjected to endotoxic shock [26], traumatic shock, or burn injury. Agents that block LT synthesis [27, 401 or LT receptors [9,10,25,43] improve visceral perfusion and survival and prevent hemoconcentration in rat models of endotoxicosis or trauma. In the present study, we postulated that LTs are among the mediators responsible for the derangements in intestinal perfusion which occur in the sepsis syndrome. To test this hypothesis, we utilized LY171883, a specific LTDI/EI receptor antagonist [19], that has been shown to inhibit LT-mediated mesenteric vasoconstriction [lo, 311. Using a clinically relevant porcine model of endotoxic shock [17, 181, we assessed the adequacy of mesenteric perfusion in animals pretreated with either LY171883 or its vehicle.
Superior mesenteric arterial perfusion (a) decreases and gut intramucosal hydrogen ion concentration, [H’], increases in resuscitated normodynamic endotoxic pigs. The present study tested the hypothesis that these adverse phenomena can be prevented by pretreatment with LY171883, a specific leukotriene (LT) DJE, receptor antagonist. Pentobarbital-anesthetized pigs (14-18 kg) were instrumented to permit measurement of Q (ultrasonic flow probe) and [H+] (tonometer). Mesenteric O2 delivery (a,) and consumption (VO,) were calculated from the Oa contents of arterial and superior mesenteric venous blood. At t = -20 min, groups (N = 6) of pigs were pretreated with LY171883 (10 mg/kg) or vehicle. At t = 0 min, the pigs were infused over 20 min with lipopolysaccharide (LPS; 150 pg/kg) and resuscitated for 2 hr with saline (1.2 ml/kg min). Irrespective of treatment group, mean arterial pressure and systemic vascular resistance index decreased significantly after infusion of LPS. In general, cardiac index (CI) was well preserved, although in controls at t = 20, 100, and 120 min, CI decreased significantly with respect to the t = 0 min value. Normal mesenteric 4 and do, were maintained in the LY 171883 group, whereas, in controls, these parameters decreased significantly. Mesenteric VO, increased transiently but significantly in controls; this phenomenon was abrograted by the LT receptor antagonist. In controls, intramucosal [H+] increased by almost threefold; this adverse effect was significantly ameliorated by LY 171883. These data suggest that decreased mesenteric 4 and increased intramucosal [H+] may be mediated by LT in this o ieso AC&tic PWS, IIN. porcine endotoxic shock model.
Systemic oxygen of systemic oxygen many patients with is apparently flow
Worcester, Massachusetts
in
37
0022-4804/90$1.50
Copyright 0 1990 hy Academic Press, Inc. All rights of reproduction in any form reserved.
38
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TABLE Baseline
Hemodynamic Control
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49, NO. 1, JULY
1990
1
and Oxygen
Transport
Data LY171883 group
group
t = -20 min
t = 0 min
t = -20 min
t = 0 min
MAP (Torr) CI (ml/min kg) SVRI (Torr min kg/liter)
111 268 428
+ 3 f 20 + 41
110 269 431
+ 5 f 25 f 59
105 280 380
+ 4 f 21 + 22
106 280 385
f 4 + 15 f 30
MPAP (Torr) SMA @ (ml/min)
16 474
f 2 t 40
17 486
* 1 f 41
16 371
+ 1 f 25
17 385
+ 1 f 35
Systemic rj0, (ml/min) Systemic ri0, (ml/min) Mesenteric DO2 (ml/min) Mesenteric VO, (ml/min)
559 142 64 17
f 39 + 6 -c 5 f 2
558 136 66 15
+- 47 f 10 + 5 + 2*
584 140 48 18
+ 47 + 13 It 4# + 2
570 145 48 19
+ 31 f 10 f 4# f 3
Intramural
49.1 +
[H+] (ti)
2.3
44.9 +
1.8
45.2 +
2.3
43.0 +
1.8
* P < 0.05 vs t = -20 min within group. # P < 0.05 vs time-matched value in control group.
METHODS
Studies were performed in accordance with the guidelines of the institutional review board for animal experimentation of the University of Massachusetts. Domestic pigs without clinical evidence of gram-negative infections were used in our experiments. The animal model used for these studies was adapted from one described by Breslow et al. [5] and was previously described [ 17,181. Male pigs (13-16 kg) were anesthetized with intramuscular ketamine (7 mg/kg) and intravenous sodium pentobarbital (17 mg/kg). Additional doses of pentobarbital (10 mg) were administered as necessary to maintain light general anesthesia. After tracheostomy, the animals were ventilated using a Harvard ventilator with 100% Oz at a tidal volume of 10 ml/kg. Respiratory rate was adjusted to maintain the pC0, equal to 40 _+5 mm Hg. Baseline core body temperature was maintained at 39.0 f 0.5”C by using heating pads and blankets. Polyethylene catheters (PE 160) were placed in the inferior vena cava and abdominal aorta via bilateral femoral cutdowns. A 2.5-French thermistor-tipped catheter (Edwards Division, American Hospital Supply Corp., Irvine, CA) was advanced into the thoracic aorta via a femoral artery. A flow-directed balloon-tipped catheter (Edwards) was inserted into the pulmonary artery via a femoral vein. Observing the characteristic pulmonary arterial pressure tracing on the monitor confirmed the location of the catheter. A 3.0-French catheter for injecting the thermal indicator was introduced into the right atrium via the right external jugular vein. A midline celiotomy was performed and a 4- to 6-mm ultrasonic flow probe (Transonic Systems, Inc.) was positioned around the superior mesenteric artery (SMA) near its origin from the abdominal aorta. The probe was connected to a previously calibrated Transonic Systems
Model TlOl blood flowmeter. A PE 90 catheter was advanced into the superior mesenteric vein (SMV) via a distal tributary in the mesentery of the small intestine. A tonometric catheter (Tonometrics, Inc., Bethesda, MD) was inserted into the ileum via an antimesenteric enterotomy and secured in place. The animals were allowed to stabilize for 30-60 min after closing the laparotomy incision. Arterial pressure was measured using Transpac-II transducers (Sorenson, Abbott Laboratories, North Chicago, IL) driving a Honeywell amplifier-monitor with digital readout. Cardiac output (CO) was determined by thermodilution using an Edwards Model 9520 computer and 3.0-ml injections of room-temperature normal saline injected into the right atria1 catheter. Cardiac output determinations were performed in triplicate and the results averaged. Cardiac index (CI) was determined by dividing CO by body weight. Systemic vascular resistance index (SVRI) was calculated by dividing mean arterial pressure (MAP) by CI. Arterial, mixed venous (pulmonary arterial), and superior mesenteric venous oxygen contents (Co,) were calculated after obtaining blood samples from the appropriate catheters and analyzing them for p02, hemoglobin concentration (Hgb), and percentage saturation (So,). An Instrumentation Laboratories Model 1302 blood gas analyzer was used to measure pop and an Instrumentation Laboratories Model 282 co-oximeter was used to measure Hgb and So,. Co, was calculated according to the equation Co, = (p02 X 0.003) + (Hgb X So, X 1.36). The following additional equations were utilized: systemic DO2 = cardiac output X arterial Co,; systemic ri0, = cardiac output X (arterial Co, - mixed venous Co,); mesenteric ho, = SMA flow X arterial Co,; mesenteric VO, = SMA flow X (arterial Co, - SMV Co,).
COHN
ET AL.: LY171883
AND
We calculated intramucosal hydrogen ion concentration, [H+], using a tonometric method originally described and validated by Fiddian-Green and co-workers [ 121. Because it diffuses readily, gut intramural COz is in equilibrium with CO2 in intraluminal fluid. If arterial bicarbonate concentration is an accurate estimate of the intramural bicarbonate concentration, then intramucosal [H+] can be estimated using a modified form of the HendersonHasselbalch equation: [Hf] = 24 X ( [HC03]/pCOz), where [HCOB] is the concentration of bicarbonate in arterial blood and pCOz is the partial pressure of carbon dioxide in gut luminal fluid. Rather than sampling luminal fluid directly, we measured pC0, in fluid (normal saline) contained in an intraluminal balloon tonometer constructed of a material (silicone) that is freely permeable to COZ. After inserting the tonometer into the gut lumen (see above), saline in the balloon was allowed to equilibrate for 20 min and then aspirated to permit measurement of pC0,; care was taken to discard the deadspace volume of the catheter. The tonometer balloon was refilled with fresh fluid every 20 min. The measured p COP was corrected to account for incomplete equilibration prior to calculating intramural [H+] [ 141. Blood samples for prostanoid determination were obtained from the femoral arterial catheter. The samples were collected into iced glass tubes containing EDTA (10.5 mg) and indomethacin (0.1 mg). Within 15 min of being obtained, the blood samples were centrifuged (15OOg for 15 min at 4°C). The plasma was collected and stored in polypropylene tubes at -70°C until assayed. Details of the radioimmunoassay for 6-ketoprostaglandin (PG) F1, and thromboxane (TX) Bz were described previously [ 151. The lower limits of sensitivity for the assay were ‘78 and 156 pg/ml for 6-keto-PGF1, and TxBz, respectively. Experimental Protocol Two groups of pigs were studied. Beginning at t = 0 min, both groups were infused over 20 min with lipopolysaccharide (LPS; Escherichia coli Olll:B4; DIFCO, Detroit, MI; 150 pg/kg). At t = -20 min, the experimental group (N = 6) was pretreated with LY171883 (10 mg/kg in 10 ml of 0.5 M NaHC03). Controls (N = 6) were pretreated with 10 ml of the 0.5 M NaHC03 vehicle. Starting at the beginning of the LPS infusion (t = 0 min) and continuing for the duration of the experiment, all animals were continuously resuscitated with normal saline (1.2 ml/kg min). Measurements to assess hemodynamics, oxygen metabolism, and intramucosal [H+] were made at t = -20 min (i.e., prior to infusing drug or vehicle), t = 0 min (i.e., after administering drug or vehicle, but prior to infusing LPS), and every 20 min thereafter for 2 hr. Blood samples for prostanoid determination were obtained at t = -20, 20, and 80 min. Animals were sacrificed at 120 min by lethal injection. Statistical Analyses Data were analyzed using commercially available software (CSS, Statsoft, Tulsa, OK) on a Kaypro 286i mi-
MESENTERIC
39
PERFUSION MEAN SYSTEMIC ARTERIAL PRESSURE 1
130 120
401 0
20
40
60
80
100
120
*
*
100
120
TIME (MN)
CARDIAC INDEX
130 120
#
110
T
I
60 50 0
20
40
60
80
TIME (MIN) FIG. 1. Effect of LY171883 on MAP (top) and CI (bottom) in endotoxic pigs. Results are expressed as percentages of the t = 0 min value for each animal. Symbols represent means + SE for pigs pretreated with LY171883 (squares) or vehicle (circles). *Values that are significantly different from the t = 0 min value for the same group. #Values that are significantly different from the time-matched values in the control group.
crocomputer. The results in the figures are expressed as percentages of the baseline (t = 0 min) value for each animal; the symbols represent means f SE. Overall group, time, and group X time effects were assessed using twoway analysis of variance (ANOVA) for repeated measures. Differences with respect to baseline within a group and time-matched differences between groups were assessed by analyzing using Duncan’s new multiple-range test [32]. The null hypothesis was rejected when P < 0.05. RESULTS
Baseline (i.e., t = -20 and 0 min) hemodynamic and oxygen transport data are presented in Table 1. Mesenteric BO, was significantly lower in the LY171883 group. For all other measured variables, the two treatment groups were quite comparable. Infusing LY 171883 or its vehicle significantly increased arterial pH; these effects were consistent with the alkaline nature of these solutions.
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2-O
;o
i0 TIME
FIG. 2. Effect of LY171883 as in Fig. 1.
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i0
li0
loo
VOL. 49, NO. 1, JULY
0
20
40
(MN)
on SVRI in endotoxic
1990
60
80
100
120
TIME (MIN) pigs. Symbols
The effects of LPS on systemic and pulmonary hemodynamics are shown in Figs. l-3. Irrespective of group, MAP decreased substantially to -60% of the t = 0 min (reference) value (Fig. 1). Although generally well preserved in both groups, CI decreased significantly in controls at t = 20, 100, and 120 min. At t = 80 min, CI was significantly higher in the drug-treated group. After increasing transiently at t = 20 min, SVRI decreased significantly in both groups, reaching a nadir at t = 80 min (Fig. 2). In both groups, mean pulmonary arterial pressure (MPAP) increased approximately threefold at t = 20 min and then declined to a plateau that was approximately twice the reference value (Fig. 3). At t = 40-60 min, MPAP was slightly, but significantly, lower in the LY171883 group. Mesenteric Q was fairly stable after LPS infusion in animals pretreated with LY171883 (Fig. 4). In controls? however, there was a marked decrease in mesenteric Q that was significant both compared to the baseline value within group and to matched time points in the LY 171883 group. By repeated-measures ANOVA, the group effect for this variable was significant (P = 0.022). The effects of LPS on systemic oxygen transport are shown in Fig. 5. Systemic DO2 tended to decrease over time in the control group and was significantly lower than in the LY171883 group at t = 20 and 80 min. Systemic VO, increased significantly at t = 20-60 min in controls. This phenomenon was abrogated by prior treatment with LY171883. By repeated-measures ANOVA, the group X time interaction for systemic VOz was significant (P = 0.032). In the drug-treated group, mesenteric ri0, was approximately normal until t = loo-120 min when this variable decreased to 80-90% of the reference value (Fig. 6). In the control group, however, mesenteric 60, decreased significantly to 50-60% of baseline. There was a statistically significant difference between groups (P = 0.012). Mesenteric VO, increased significantly in the
FIG. 3. Effect of LY171883 on MPAP as in Fig. 1.
in endotoxic
pigs. Symbols
control group, with respect both to the baseline value within group and to time-matched values in the LY171883 group. The group effect for mesenteric VO, was significant (P = 0.034). Table 2 shows the effect of LPS on arterial blood gases in control and drug-treated pigs. In both groups, arterial pOz decreased significantly during the second hour of the protocol. Oxygenation was transiently better in the LY171883 group at t = 40 min. By design,pCOz was nearly constant in both groups. Metabolic acidosis occurred in both groups, but this phenomenon was ameliorated in the animals pretreated with the LT receptor antagonist. Gut intramucosal [H+] increased dramatically after infusing LPS in controls (Fig. 7). Although [H+] also increased significantly in LY171883-treated animals, the magnitude of the change was substantially smaller than that observed in the control group. By ANOVA, both group and group X time effects were highly significant (P < 0.00001).
40 1 30 4 0
*
20
40
60
TIME
80
*
*
100
120
(MN)
FIG. 4. Effect of LY171883 on superior mesenteric flow in endotoxic pigs. Symbols as in Fig. 1.
arterial
blood
COHN
ET AL.: LY171883
AND
SYSTEMIC OXYGEN DELIVERY
130,
T
#
*
60-
1 *
A *
50, 0
20
40
60
80
100
120
TIME (MIN)
SYSTEMIC OXYGEN UPTAKE
220 200
*
MESENTERIC
this is the so-called hyperdynamic state [l, 22, 371. Decreased systemic vasomotor tone is so characteristic of the sepsis syndrome that this finding has been used in clinical research studies as a criterion for establishing the diagnosis [34, 381. During episodes of hypotension (i.e., septic shock), CI tends to decrease into the normal range while SVRI remains abnormally low [ 11. The porcine endotoxicosis model used for the present study satisfactorily mimicked the systemic hemodynamic changes commonly seen during episodes of septic shock in resuscitated human patients; i.e., CI was well-preserved and SVRI was markedly decreased. Consistent with current clinical practice, aggressive intravascular volume loading was a key feature of the protocol. In other similar models, resuscitation was guided by measurements of central venous [5] or left atria1 pressure [33], the volume of fluid administered being adjusted to maintain these indices of cardiac preload within the normal range. Instead of this approach, we chose to infuse’the same (large) volume of saline to all animals. We adopted this straightforward protocol for several reasons. First, myocardial performance is clearly impaired in sepsis [35,37] and enMESENTERIC OXYGEN DELIVERY
110
100
0
20
40
60
60
100
41
PERFUSION
#
#
120
TIME (MIN) FIG. 6. Effect of LY171883 on systemic 80, (top) and QO, (bottom) in endotoxic pigs. Symbols as in Fig. 1.
40
*
*
*
SO
100
120
30 0
20
40
After infusion of LPS, plasma levels of both 6-ketoPGF1, (prostacyclin metabolite) and TxBz (TxA2 metabolite) increased significantly. These changes were similar in both treatment groups (Table 3).
TIME (MIN)
MESENTERIC OXYGEN UPTAKE
200 190 180
DISCUSSION
These were the principal findings in the present study: (1) LY 171883 had no appreciable effect on any measured variable in normal, pentobarbital-anesthetized pigs; (2) pretreatment with. LY171883 substantially improved mesenteric 0 and DO, in our porcine model of resuscitated, endotoxic shock; (3) mesenteric VO, increased substantially in endotoxic controls, but this phenomenon was abrogated by the prior infusion of LY171883; (4) gut intramucosal [H+] increased significantly after infusion of LPS in both drug- and vehicle-treated animals, but the magnitude of this effect was much smaller in animals infused with the LTDI/E& receptor antagonist. In adequately resuscitated humans with compensated sepsis, CI is usually elevated and SVRI is typically low;
60
*
51 O-----I 170 If," .__ I
*
I.dV
*
4c.nJ
I
140 130 120
1
E w w
110
I
7’0
‘0
I/ /o
100
-
T
d
I/
I
I I
(
90 60 70 60 t
I
-y
I ,6-
- - -g
_ _ %&
#
i #
_ - -&
- - q-
#
T_ 1
- -0
T 1 #
50 4 0
20
40
60
SO
100
120
TIME (MIN) FIG. 6. Effect of LY171883 on mesenteric do, (top) and VO, (bottom) in endotoxic pigs. Symbols as in Fig. 1.
42
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OF SURGICAL
RESEARCH:
dotoxicosis [21]. Therefore, preload may need to be elevated to supranormal levels to maintain CI. Second, sepsis may lead to alterations in ventricular diastolic compliance [ 36 1. Thus, ventricular filling pressures are probably poor indices of preload (i.e., end-diastolic volume) in the setting of sepsis or endotoxicosis. Finally, a fixed-volume regimen markedly simplifies the experimental design and avoids problems of investigator bias. Tissue acidosis is a well-documented consequence of tissue hypoxia [39]. In the heart, tissue pH is strongly correlated with tissue concentrations of ATP [30]. In the gut, the onset of intramucosal acidosis correlates with the inflection point between flow-independent and tlow-dependent VO, [23]. In the present study, we assessed gut intramucosal pH using a tonometric method originally described by Fiddian-Green et al. 1121. The fundamental assumption underlying this method is that the concentration of bicarbonate is similar in plasma and tissue. This assumption is evidently valid, since Fiddian-Green et al. showed that there was good correlation (r = 0.98) between intramucosal pH measured directly and estimated tonometrically. We previously showed that gut intramucosal [Hf] increases in our normodynamic porcine model of endotoxic shock [17, 181. This observation was reproduced (in a new group of animals) in the present study. These data support the view that intramucosal VO, is inadequate in endotoxic shock. Pretreatment with LY171883 substantially ameliorated post-LPS intramucosal acidosis, suggesting that the adequacy of mucosal oxygenation was improved by the LTDl/Eq receptor antagonist. Two factors seem to be involved in this beneficial effect of LY171883. First, the drug clearly improved mesenteric Q in endotoxic pigs. As a consequence, mesenteric fi0, was significantly greater in the LY171883 group as compared to vehicle-treated controls. The effect of LY171883 on mesenteric perfusion in the present study is consistent with other previous data indicating that LTs are potent mesenteric vasoconstrictors [ll, 31, 381. The improvement
VOL. 49, NO. 1, JULY
240 bz
of LPS on Arterial
Blood
Gases and Derived
220
k!
20
0
40
60
100
80
120
TIME (FAN)
FIG. 7. Effect of LY171883 on ileal intramucosal pigs. Symbols as in Fig. 1.
[H+] in endotoxic
in mesenteric perfusion observed in our study is also consistent with data obtained by Etemadi et al. showing improved gut perfusion in a low-cardiac-output model of endotoxic shock in the rat [lo]. The data presented here support the notion that LTs are important mediators of the relative redistribution of cardiac output away from the mesenteric bed in resuscitated endotoxic shock. The second factor evidently contributing to the salutary effect of LY171883 on intramucosal [H+] was that the LTD4/E4 antagonist seemed to blunt the hypermetabolism induced by LPS. Thus, the drug apparently improved the adequacy of tissue oxygenation by both increasing mesenteric DO2 while, simultaneously, decreasing the metabolic demand for oxygen across this bed. It is well established that sepsis and endotoxicosis increase metabolic rate in adequately resuscitated animals [29] and humans [ 11. In the present study, this phenomenon was particularly evident across the mesenteric bed (Fig. 6). The mechanisms responsible for hypermetabolism in sepsis and endotoxicosis are incompletely understood. Circulating catecholamines, known to be elevated in sepsis/en-
TABLE Effect
1990
2
Values
in Vehicle-
and LY 171883-Pretreated
Pigs
Time Variable
Pox Vorr)
Group
Control LY171883
PcOz (Ton) [HCO;](mM) BE W@)
control LY171883 Control LY171883 Control LY171883
-20 454 450
c!
+11 *19
37.9 + 39.3 + 25.3 k 23.7 + 1.6 + -0.5 f
.4
1.9 .6 .9 .8
1.0
451 446 36.9 40.2 25.8-c 25.8 2.5 1.7
* P < 0.05 vs t = -20 min within group. # P < 0.05 vs time-matched value in control
20
+ 9 +15 ck .4 + 1.7 .8 + .8 f .9 + .b
group.
400 433
40
f20 +52
232 376
60
?50* +40#
35.0 * 41.9 k
1.0 2.4x
39.8 + 42.9 +
1.2 1.2
22.0 f 22.9 + -1.6 k -2.2 f
1.1* .7* 1.2*
19.5 k 21.8 * -5.9 + -3.8 +
.9* .7"
1.0*
1.w .9'#
258 307 40.6
+50* *57* 3~ 2.1*
42.1 18.5 20.4 -7.4 -5.4
* + + k
.8 .5* .8* .6*
+
1.1’.#
339 287 40.0 41.2 18.1 20.0 -8.0 -5.7
* 29* k51* k .8 + k + k *
120
100
80
1.3 .6* 7*,# .6' 1.0*.x
309 254
f27* +52*
38.1 + 40.8 + 17.7 f
.8 18
309 230 38.6
If: 29* f65' k .8
39.5 +
.5*
17.2 +
19.4 zk
.7*
19.0 +
-&O-1-6.3 +_
.6* .9**#
-8.8 -7.1
c +
1.0 .6* .6*,X .7' .9'*#
COHN
ET AL.: LY171883
AND
MESENTERIC
TABLE Effect
of LPS on Plasma
Prostanoid Control
6-K&o-PGF,,
TX& (pdml)
(pg/ml)
43
PERFUSION
3
Concentrations
in Vehicle-
and LY171883-Pretreated
group
Pigs
LY171883 group
t = -20 min
t = 20 min
t = 80 min
t = -20 min
t = 20 min
t = 80 min
707 t 113 474+ 99
721+ 162 3003 2 84*
2007+367* 2961+ 154*
780 + 153 253+ 97
596 f161 3180 + 226*
1868 f 291* 3650 f 508*
dotoxicosis [41], may be important, however, since exogenous catecholamines increase VO, in both animals [ 71 and patients [20]. The present data offer no clues as to the mechanisms underlying the effect of LYl71883 on mesenteric VOz. Clearly, however, this interesting phenomenon warrants further investigation. Consistent with numerous other studies [ 151, we showed that infusion of LPS leads to markedly elevated circulating levels of TxBz (TxAz metabolite) and 6-keto-PGF,, (PGIZ metabolite). It is noteworthy that pretreatment with LY171883 did not affect the plasma prostanoid response to LPS (Table 3). In contrast, another LT receptor antagonist, FPL 57231, seems to be less specific and may function as a cyclooxygenase inhibitor. The apparent lack of effect of LY171883 on the cyclooxygenase pathway renders the results presented here more amenable to interpretation. The present study is open to certain criticisms. First, we did not attempt to measure LT levels in either plasma or bile. Obviously, documenting elevated concentrations of LTs (or their metabolites) after administering LPS would greatly strengthen the case, implicating these lipids as mediators of some the physiological derangements observed in our endotoxic shock model. Unfortunately, the assay systems for these compounds are quite difficult, and, as yet, we have not perfected them in our laboratory. Second, we utilized only one LT receptor antagonist, leaving open the possibility that the pharmacologic effects described herein were due to an action of LY171883 unrelated to its activity as an LTD4/E4 receptor antagonist. LY 171883 is a phophodiesterase inhibitor [ 191. Ongoing studies in our laboratory using other compounds are directed at this criticism. Third, the animals were infused with LY171883 prior to the onset of endotoxemia. We deliberately chose a pretreatment design to maximize the chances of observing unambiguous effects. We recognize, however, that pretreatment is unlikely in the clinical setting, and, therefore, extrapolating our results to the treatment of human septic shock is clearly unwarranted. Fourth, mesenteric fi0, was lower at baseline in our pretreatment group. We do not know what effect this may have had on the protection obtained in this group, but recognize that this could have impacted on these data. Finally, we showed that LY 171883 ameliorated gut intramucosal acidosis in porcine septic shock, but provided no evidence that this phenomenon was beneficial in terms
of mucosal function or histology. Further experiments being planned to address this issue.
are
ACKNOWLEDGMENTS This work was supported by Grant 1 R29 GM-37631-0103 from the National Institutes of Health. Ms. Sandy Hinson and Ms. Penny Lucier assisted in the preparation of the manuscript.
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Statistical
Inference.
New York:
pH J. of
