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We studied 1) neutrophil mobilization in sheep given endotoxin (10 ng.kg-1.min-1, n = 5) for 4 h, 2) surviving (n = 17) and nonsurviving sheep (n = 8) during a 12-h infusion of endotoxin, and 3) adult sheep (n = 8) or lambs (n = 8) infused with endotoxin for 12 h. Bone marrow cells of sheep declined from a baseline value of 10,533 +/- 1,784 to 5,966 +/- 1,980 cells/microliter (P < 0.05) 4 h after endotoxin. After 12 h of endotoxin infusion, circulating neutrophils remained reduced from baseline values of 2,000-4,000 cells/microliter to 343 +/- 70 in lambs and 484 +/- 236 in nonsurviving sheep, while beginning to recover in surviving sheep to 1,838 +/- 467 cells/microliter. In lambs and nonsurviving sheep, a 12-h infusion of endotoxin increased lung lymph protein clearance tenfold compared with a fivefold increase in surviving sheep. Neutrophils cultured from sheep bone marrow exposed to lamb postendotoxin plasma failed to increase in cell number (609 +/- 229 to 610 +/- 182 cells/microliter), whereas similar cells exposed to adult sheep postendotoxic plasma showed a significant increase in cell number (1,069 +/- 101 to 2,293 +/- 448 cells/microliter, P < 0.05). Our results are consistent with the hypothesis that the ability to recruit neutrophils to the circulation during periods of inflammation is important in limiting the severity of acute lung injury.

We studied changes in cardiovascular and pulmonary function during prolonged endotoxemia in conscious sheep. Sheep with chronic lung lymph fistulas received a 12-h infusion of Escherichia coli endotoxin (10 ng x kg-1 x min-1) and allowed to recover for 12 h. Supportive therapies were withheld. Prolonged endotoxemia without volume support caused systemic hypotension associated with reduced cardiac output and increased systemic vascular resistance, pulmonary hypertension, and acute lung injury with progressive respiratory failure. Plasma tumor necrosis factor-alpha (TNF-alpha) concentrations increased transiently. Sustained pulmonary hypertension and increased pulmonary and systemic vascular resistances contributed to a poor outcome in 9 of 34 sheep (26%). Plasma TNF-alpha concentrations were significantly greater in survivors with sustained pulmonary hypertension and in nonsurviving sheep than in surviving sheep without pulmonary hypertension. Endotoxin (1 ng/ml) increased neutrophil expression of TNF-alpha in vitro. Addition of interleukin-6 prevented this response. Synthesis and release of TNF-alpha may be an important proximal event influencing the development of sustained pulmonary hypertension and progressive respiratory failure with endotoxemia. Interleukin-6 may contribute to the phasic nature of the TNF-alpha response.

Endotoxin stimulates synthesis of endothelin which can cause pulmonary and systemic vasoconstriction and bronchoconstriction. Prolonged endotoxemia in sheep results in dramatic increases in pulmonary and systemic vascular resistances in nonsurvivors compared with survivors. Experiments were conducted in 12 conscious sheep (seven survivors, five nonsurvivors) to determine if synthesis of endothelin might contribute to the pathophysiology in nonsurvivors. Endotoxin was infused at 10 ng/min/kg for 12 h followed by a 4 h recovery in survivors. Lung lymph endothelin concentration peaked at 38.7 +/- 5.8 pg/mL during the endotoxin infusion in survivors compared with a peak of 128.7 +/- 33.0 pg/mL (p < .05) in nonsurvivors. Cardiac output was lower, and systemic and pulmonary vascular resistances and the Aa gradient in PO2 were significantly greater in nonsurvivors compared with survivors. These variables are those likely to be affected by increased circulating endothelin concentrations which suggests that endothelin contributes to early mortality during prolonged endotoxemia.

We examined the hemodynamic response of awake sheep to prolonged endotoxin infusion (10 ng.kg-1 x min-1 for 12 h) and the in vitro endothelium-dependent relaxation of pulmonary arterial vessels excised 12 h after the end of endotoxin infusion to determine whether the development of pulmonary hypertension after endotoxin is associated with loss of endothelium-dependent relaxation. In seven of nine sheep, there was a maintained increase (4-68% of baseline) in pulmonary arterial pressure 24 h after the beginning of endotoxin infusion. The greater the increase in pulmonary arterial pressure in vivo, the greater was the in vitro deficit in endothelium-dependent relaxation of the pulmonary vessels. The maximum in vitro vessel dilation was 59% for pulmonary artery rings isolated from sheep without a sustained increase in pulmonary arterial pressure 24 h after endotoxin. Prolonged endotoxin infusion did not alter the in vitro response of pulmonary arterial vessels to KCl or 10(-5) M norepinephrine. Force development, response to 10(-5) M norepinephrine, and vasodilation in response to acetylcholine were also not altered in pulmonary vessels taken from control sheep and exposed in vitro to tumor necrosis factor-alpha (400 U/ml). Our results suggest that loss of endothelium-dependent relaxation in pulmonary vessels supports the sustained pulmonary hypertension that develops after prolonged exposure to endotoxin.

We used a continuous 12-h infusion of Escherichia coli endotoxin (10 ng.min-1.kg-1) in 10 awake sheep equipped with a lung lymph fistula and vascular catheters to determine the time course of increased plasma tumor necrosis factor-alpha (TNF-alpha) during the infusion and a 12-h postinfusion period. Lung lymph flow increased progressively during the infusion to a peak value averaging 8.6 +/- 2.0 times the baseline flow of 6.3 +/- 1.3 g/h. During the postinfusion period, lung lymph flow remained elevated at three to four times baseline. The lymph-to-plasma protein concentration ratio was unchanged from baseline over 24 h, indicating a dramatic increase in net protein flux across pulmonary microvessels. The TNF-alpha concentration peaked early in the infusion and then declined, despite the continuing presence of endotoxin. Plasma TNF-alpha concentration increased 10-fold (0.33 +/- 0.05 ng/ml at baseline to 3.89 +/- 0.78 ng/ml peak) 2 h into the endotoxin infusion. At the end of the endotoxin infusion, plasma TNF-alpha had decreased to 1.16 +/- 0.19 ng/ml. The circulating TNF-alpha concentration did not correlate with pathophysiology or outcome in these sheep.

We investigated the mechanism for the pressor effect of intravenous administration of naloxone in pentobarbital-anesthetized cats. Comparisons were made between groups of hemorrhaged animals that received either naloxone or an equivalent volume of saline after 1 h of hemorrhage. Two other groups of hemorrhaged animals were depleted of serotonin by pretreatment with para-chlorophenylalanine 40-48 h before the experiment. One group of serotonin-depleted animals received naloxone after 60 min of hemorrhage and the other group received saline. Animals with normal brain serotonin content showed a significant pressor effect following naloxone when compared with animals given saline. Animals with reduced brain serotonin content also had a pressor response following naloxone administration. Serotonin-depleted animals showed an increase in maximum left ventricular dP/dt following naloxone administration when compared to serotonin-depleted animals given saline. Our data are consistent with the hypothesis that naloxone can exert a pressor effect in hemorrhaged cats by actions at central and at peripheral sites. In cats with normal serotonin values, the peripheral action of naloxone is predominant. In serotonin-depleted animals given naloxone, central and peripheral sites contribute to the pressor effect.

We tested the effects of alpha-naphthylthiourea (ANTU) on lung fluid balance and prostanoid concentrations in anesthetized sheep acutely prepared for collection of pulmonary lymph. Sheep were given 20, 50, 75, or 100 mg/kg ANTU or the vehicle dimethylsulfoxide (DMSO) intravenously. The first phase of the response consisted of transient increases in pulmonary artery pressure and plasma and lymph thromboxane B2 concentrations. Lymph flow increased with no change in the lymph-to-plasma protein concentration ratio (L/P). These changes occurred in sheep given DMSO alone or DMSO with ANTU; they were not dependent on the dose of ANTU given. Two to 4 h after drug administration, pulmonary artery pressure and thromboxane concentrations were normal or near normal. Lymph flow rate reached steady-state levels averaging 1.5 (DMSO alone) to 5.3 (100 mg/kg ANTU) times base line with L/P ratios unchanged from base line. ANTU/DMSO produces transient, severe pulmonary hypertension that may be prostaglandin mediated. The sustained response consists of increased flow rate of protein-rich lymph.

Anesthetized cats were subjected to hemorrhage to a mean arterial blood pressure of 40 +/- 1 mmHg. After 60 min of oligemia pressure regulation was stopped, and naloxone hydrochloride or an equivalent volume of 0.9% saline was given intravenously as a bolus (2 mg x kg-1) followed by a continuous infusion (2 mg x kg-1 x hr-1). In other cats supraspinal systems were ablated by functional decapitation (FD) or serotonergic systems were ablated by IP injection of parachlorophenylalanine (pCPA, 300 mg/kg, 48 h prior to hemorrhage). Cats subjected to ablation of supraspinal or serotonergic neurons showed a distinct pressor response after naloxone. Although cats with intact neuronal systems failed to show a distinct pressor response to naloxone, the arterial pressures of these cats were significantly greater than hemorrhaged cats given saline (P less than 0.05, unpaired t). Our results suggest that a supraspinal system and a serotonergic system can interact to limit the pressor response to naloxone in hemorrhagic shock.

An investigation was made of the effect on cardiovascular function of electrical stimulation within the midline medullary region of the anesthetized cat. Stimuli consisted of low-intensity trains of pulses. Stimulation sites were defined histologically and in some experiments, detailed stimulus maps were obtained for the effect of electrical stimulation on mean arterial blood pressure (MABP). In other experiments, measurements were made of changes produced by electrical stimulation on heart rate, cardiac output, cardiac contractility, and total peripheral resistance. Comparisons were made between the effect of stimulation at sites on the midline and sites to 1.5 mm more lateral. At midline sites, electrical stimulation most often produced a depression of the MABP. Effective depressor sites were located preferentially in two regions along the anteroposterior axis of the brain stem. At more lateral sites, electrical stimulation elevated the MABP. At most sites, changes in MABP occurred concomitantly with changes in total peripheral resistance while cardiac output was unchanged. Sites where electrical stimulation directly altered heart rate and cardiac contractility had a different distribution within the medulla compared with sites where stimulation changed the MABP.

We measured pulmonary arterial (Ppa) and left atrial (Pla) pressures, lung lymph flow rate (QL), and protein clearance in 23 anesthetized dogs with ligation (MI) dogs or sham ligation (sham-MI dogs) of a coronary artery combined with either large transient increases in Pla or moderate steady-state increases in Pla. In steady-state experiments we also collected cardiac lymph. Plasma and lymph concentrations of several prostanoids including prostacyclin (PGI2) and thromboxane B2 (TXB2) were measured. Lung lymph flow rates and protein clearances were increased in MI dogs compared with those in sham-MI dogs in experiments with transient increases in Pla. Similarly in steady-state experiments lung QL and protein clearance were increased after MI. Cardiac QL increased after MI with no change in lymph protein clearance. Lung and cardiac lymph PGI2 and plasma TXB2 concentrations increased after MI. We suggest that acute MI results in increased lung vascular permeability and that activation of formed elements in blood may be involved in mediating MI-induced vascular injury.

Regional myocardial blood flow or myocardial creatine kinase activity and ultrastructure was studied in anesthetized cats subjected to sham operation, 5 h of circumflex-artery ligation, or 2 h of coronary-artery ligation with 3 h of reperfusion. Sham-operated cats were given dexamethasone sodium phosphate (8 mg/kg iv) at the beginning of the experiment. Cats subjected to coronary-artery ligation were given steroid or vehicle before ligation. In sham-operated cats the average blood flow in tissue of the anterior wall was 2.11 +/- 0.37 (SER) ml.g-1.min-1. The corresponding flow for tissue of the posterior wall was 2.22 +/- 0.42 ml.g-1.min-1. Ligation of the circumflex artery produced a range of average blood flow in the posterior basal myocardium that was 52-92% less than that of sham-operated cats. Within the tissue of the posterior left ventricular wall, steroid pretreatment did not prevent loss of creatine kinase activity after coronary-artery ligation. Moreover, steroid pretreatment was ineffective in maintaining myocardial structure in reperfused myocardial tissue. These results indicate that 1) dexamethasone does not effectively increase blood flow within ischemic or reperfused myocardial tissue, and 2) dexamethasone may stabilize ischemic myocardial tissue. However, upon reperfusion, dexamethasone is unable to maintain myocardial ultrastructure in moderately to severely ischemic tissue.

Hemorrhagic shock was produced in dogs by bleeding to a systemic blood pressure of 45 mm Hg for 3 hours, followed by reinfusion of the shed blood. A rapid decrease in pancreatic blood flow occurred and pancreatic perfusion remained at 15-25% of control over the entire 3-hour oligemic period. As a consequence of this marked degree of pancreatic hypoperfusion, autolytic changes occurred in pancreatic acinar cell ultrastructure, particularly in the enlarging of lysosomes which developed many vacuoles. Plasma proteolytic indices (e.g., cathepsin D activity and amino nitrogen concentration) markedly increased during shock as well as the activity of a myocardial depressant factor (MDF). MDF was also produced in incubated pancreatic homogenates obtained from nonshocked dogs and in non-incubated homogenates from shocked dogs. MDF activity in the homogenates was closely correlated with amino nitrogen concentration. These data suggest that pancreatic hypoperfusion plays a key role in MDF formation and ultimately in the pathogenesis of circulatory shock. Moreover, MDF activity was found not to be associated either with pentobarbital concentration or the salt content of active fractions of plasma and pancreatic tissue. Ashing of active fractions was very effective in destroying MDF activity. These data are consistent with the earlier findings that indicate MDF to be a peptide having a molecular weight of 500-1,000.

Cats of either sex anesthetized with sodium pentobarbital (30 mg/kg, IV) were subjected to sham pericardial tamponade or pericardial tamponade for five hours. Five cats underwent sham pericardial tamponade and six cats were subjected to pericardial tamponade which reduced the mean arterial blood pressure to approximately 40 mm Hg. Pericardial tamponade produced a 44-51% and a 28-38% decrease in the myocardial activities of cathepsin D and creatine phosphokinase (CPK), respectively. Observation of cellular and subcellular structure of left ventricular tissue of two sham operated cats and two cats after tamponade revealed myocardial injury in cats subjected to tamponade. Tissue stained with hematoxylin-basic fuchsin-picric acid stain showed extensive crimson-red basic fuchsin staining in cats subjected to pericardial tamponade and the absence of staining in cardiac tissue of sham-operated cats. Electron micrographs of cardiac tissue of cats subjected to acute pericardial tamponade revealed extensive vacuolization and extreme contracture. These results indicate ischemic injury with loss of cellular and subcellular enzymes in hearts of cats after five hours of pericardial tamponade sufficient to reduce the mean arterial blood pressure to about 40 mm Hg.

We evaluated the efficacy of intracoronary administration of verapamil hydrochloride in reducing myocardial injury during acute ischemia and reperfusion. Ischemia was induced by 30% and 45% reductions of circumflex arterial blood flow for successive 2-hour periods. A reperfusion period (1 hour and 45 minutes) followed ischemia upon deflation of a pneumatic occluder. Verapamil (30 micrograms/kg) was slowly injected into the circumflex artery as a bolus 15 minutes after each blood flow reduction step. To prevent verapamil-induced decreases in heart rate, ventricular pacing was established at 170 beats/min before a baseline period and maintained throughout the protocol. Creatine kinase activities (international units per milligram protein) measured in samples obtained from posterior papillary muscles were 15 +/- 1 (mean +/- SEM) and 10 +/- 2 for animals receiving verapamil or its saline vehicle, respectively (p less than 0.05). Quantitative morphometry was performed on left ventricular myocardium after staining with p-nitro blue tetrazolium. Intracoronary administration of verapamil reduced the extent of left ventricular infarction, as disclosed by positive tetrazolium staining of the tissue, from 34 +/- 4% of the left ventricle in vehicle-treated animals to 21 +/- 4% of the left ventricle in verapamil-treated animals (p less than 0.05). We conclude that intracoronary administration of verapamil reduced the extent of myocardial infarction acutely, independent of increases in blood flow through the circumflex coronary artery or decreases in heart rate. Administration of verapamil was not associated with decreases in ventricular afterload, the pressure-rate index, cardiac output, or the maximum rate of pressure development in the left ventricle. Verapamil treatment of animals subjected to ischemia was not associated with sustained elevations of left atrial pressure to values above those measured in animals receiving the vehicle.

We measured the extravascular water content of hearts and lungs of anesthetized dogs subjected to one of the following protocols: a)sham operation, b) circumflex artery ligation, c) increased left atrial pressure (Pla), or d) increased Pla and circumflex artery ligation. After 4 h, extravascular water of the heart and lungs increased significantly in the three experimental groups when compared with values from sham-operated dogs. After circumflex artery ligation, extravascular heart water increased 29% and lung water 8%, although Pla and calculated pulmonary microvascular pressure (Pmv) did not change. Extravascular heart water also rose 30% after increasing Pla from 23 to 37 cm H2O by inflating a left atrial baloon. In these dogs, extravascular lung water increased as a hyperbolic function of Pmv. Increasing Pla to 20 cm H2O in dogs with coronary artery ligation resulted in a 16% increase in heart water. Also at each Pmv, extravascular lung water was greater in dogs with coronary artery ligation than in dogs without. These data indicate that the increased extravascular lung water after coronary artery ligation cannot be explained solely by hemodynamic mechansims. We suggest that acute myocardial ischemia contributes to an increase in vascular permeability in the heart and lungs.

In cats the mean arterial blood pressure (MABP) was reduced to 40 or 30 mm Hg by controlled hemorrhage. In 3 animals the cochlea action potentials (CAP) are not changed between 30 and 380 Min by 40 mm Hg MABP. In 4 animals we saw a latent or permanent decrease of CAP between 4 and 115 Min by 30 mm Hg MABP. A singular experiment shows, that a CAP-decrease is caused by injection of an acetylcholin blockade substance. We did not find a correlation to the length of time of the ischemia and a CAP decrease. We found an exact correlation to the hypovolemic sympathetic palsy and a CAP decrease. In reduced blood perfusion the cochlea of cats is more resistant than the brain.