Onset of Inflammation With Ischemia: Implications for Donor Lung Preservation and Transplant Survival.

Lungs stored ahead of transplant surgery experience ischemia. Pulmonary ischemia differs from ischemia in the systemic organs in that stop of blood flow in the lung leads to loss of shear alone because the lung parenchyma does not rely on blood flow for its cellular oxygen requirements. Our earlier studies on the ischemia-induced mechanosignaling cascade showed that the pulmonary endothelium responds to stop of flow by production of reactive oxygen species (ROS). We hypothesized that ROS produced in this way led to induction of proinflammatory mediators. In this study, we used lungs or cells subjected to various periods of storage and evaluated the induction of several proinflammatory mediators. Isolated murine, porcine and human lungs in situ showed increased expression of cellular adhesion molecules; the damage-associated molecular pattern protein high-mobility group box 1 and the corresponding pattern recognition receptor, called the receptor for advanced glycation end products; and induction stabilization and translocation of hypoxia-inducible factor 1α and its downstream effector VEGFA, all of which are participants in inflammation. We concluded that signaling with lung preservation drives expression of inflammatory mediators that potentially predispose the donor lung to an inflammatory response after transplant.

Tumor necrosis factor-alpha and acute-phase proteins in early pregnant ewes after challenge with peptidoglycan-polysaccharide.

Bacterial infection shortly after mating interferes with establishment of pregnancy. Injection of peptidoglycan-polysaccharide (PG-PS), a component of gram-positive bacteria, into sheep on day 5 after mating reduces pregnancy rate. Experiments were designed to evaluate the acute-phase response (APR) in ewes to injection of PG-PS on day 5 after mating (day 0). Catheters were inserted into the jugular and posterior vena cava on day 4. On day 5, ewes were challenged with saline or 30 microg/kg body weight (BW) PG-PS (Exp 1) or 60 microg/kg BW PG-PS (Exp 2). Blood samples were collected every 15 min for 6 h (Exp 1) and every 15 min for 2 h, hourly for 12 h, and at 24, 36, and 48 h (Exp 2). Body temperature and clinical signs of infection were monitored in Exp 2. Plasma was assayed for concentrations of a pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha); 2 APR proteins, serum amyloid A (SAA) and haptoglobin (Hp); and progesterone (P(4)). Ewes injected with 60 microg/kg BW PG-PS exhibited fever, vaginal discharge, loss of appetite, and lethargy. After challenge with either 30 microg/kg or 60 microg/kg BW PG-PS, TNF-alpha increased in the posterior vena cava. Concentrations of SAA and Hp in the jugular increased after challenge with 60 microg/kg BW PG-PS. Only half (5/10) of the ewes treated with 60 microg/kg BW PG-PS had ultrasonically visible embryos, and none of them had functional corpora lutea (CL) (<1 ng/mL of P(4)) on day 21. On the other hand, 8/9 (88.9%) control ewes had visible embryos and all had functional CL on day 21. Using logistic regression, pregnancy on day 21 was predicted to depend on concentrations of TNF-alpha and Hp on day 5 and concentration of P(4) on day 14. In summary, injection of PG-PS on day 5 after mating resulted in fever; increased concentrations of TNF-alpha, Hp, and SAA on the day of and the day after the PG-PS challenge; and decreased concentrations of P(4) on days 14 and 21. These factors were related to failure to establish pregnancy.

Invasive arterial BP monitoring in trauma and critical care: effect of variable transducer level, catheter access, and patient position.

OBJECTIVES: (1) To determine the validity of current recommendations for direct arterial BP measurement that suggest that the transducer (zeroed to atmosphere) be placed level with the catheter access regardless of subject positioning: and (2) to investigate the effect of transducer level, catheter access site, and subject positioning on direct arterial BP measurement. DESIGN: Prospective, controlled laboratory study. SETTING: Large animal laboratory. SUBJECTS: Five Yorkshire pigs. INTERVENTIONS: Anesthetized animals had 16F catheters placed at three access sites: aortic root, femoral artery, and distal hind limb. Animals were placed in supine, reverse Trendelenburg 35 degrees, and Trendelenburg 25 degrees positions with a transducer placed level to each access site while in every position. MEASUREMENTS AND MAIN RESULTS: For each transducer level, five systolic and diastolic pressures were measured and used to calculate five corresponding mean arterial pressures (MAPs) at each access site. When transducers were at the aortic root, MAP corresponding to aortic root pressure was obtained in all positions regardless of catheter access site. When transducers were moved to the level of catheter access, as current recommendations suggest, significant errors in aortic MAP occurred in the reverse Trendelenburg position. The same trend for error was noted in the Trendelenburg position but did not reach statistical significance. CONCLUSIONS: (1) Current recommendations that suggest placing the transducer at the level of catheter access regardless of patient position are invalid. Significant errors occur when subjects are in nonsupine positions. (2) Valid determination of direct arterial BP is dependent only on transducer placement at the level of the aortic root, and independent of catheter access site and patient position.

Metalloproteinase inhibition prevents acute respiratory distress syndrome.

BACKGROUND: The acute respiratory distress syndrome (ARDS) occurs in patients with clearly identifiable risk factors, and its treatment remains merely supportive. We postulated that patients at risk for ARDS can be protected against lung injury by a prophylactic treatment strategy that targets neutrophil-derived proteases. We hypothesized that a chemically modified tetracycline 3 (COL-3), a potent inhibitor of neutrophil matrix metalloproteinases (MMPs) and neutrophil elastase (NE) with minimal toxicity, would prevent ARDS in our porcine endotoxin-induced ARDS model. METHODS: Yorkshire pigs were anesthetized, intubated, surgically instrumented for hemodynamic monitoring, and randomized into three groups: (1) control (n = 4), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 4), infusion of Escherichia coli lipopolysaccharide at 100 microg/kg; and (3) COL-3 + LPS (n = 5), ingestion of COL-3 (100 mg/kg) 12 h before LPS infusion. All animals were monitored for 6 h following LPS or sham LPS infusion. Serial bronchoalveolar lavage (BAL) samples were analyzed for MMP concentration by gelatin zymography. Lung tissue was fixed for morphometric assessment at necropsy. RESULTS: LPS infusion was marked by significant (P < 0.05) physiological deterioration as compared with the control group, including increased plateau airway pressure (P(plat)) (control = 15.7 +/- 0.4 mm Hg, LPS = 23.0 +/- 1.5 mm Hg) and a decrement in arterial oxygen partial pressure (P(a)O(2)) (LPS = 66 +/- 15 mm Hg, Control = 263 +/- 25 mm Hg) 6 h following LPS or sham LPS infusion, respectively. Pretreatment with COL-3 reduced the above pathophysiological changes 6 h following LPS infusion (P(plat) = 18.5 +/- 1.7 mm Hg, P(a)O(2) = 199 +/- 35 mm Hg; P = NS vs control). MMP-9 and MMP-2 concentration in BAL fluid was significantly increased between 2 and 4 h post-LPS infusion; COL-3 reduced the increase in MMP-9 and MMP-2 concentration at all time periods. Morphometrically LPS caused a significant sequestration of neutrophils and monocytes into pulmonary tissue. Pretreatment with COL-3 ameliorated this response. The wet/dry lung weight ratio was significantly greater (P < 0.05) in the LPS group (10.1 +/- 1.0 ratio) than in either the control (6.4 +/- 0.5 ratio) or LPS+COL-3 (7.4 +/- 0.6 ratio) group. CONCLUSIONS: A single prophylactic treatment with COL-3 prevented lung injury in our model of endotoxin-induced ARDS. The proposed mechanism of COL-3 is a synergistic inhibition of the terminal neutrophil effectors MMPs and NE. Similar to the universal practice of prophylaxis against gastric stress ulceration and deep venous thromboses in trauma patients, chemically modified tetracyclines may likewise be administered to prevent acute lung injury in critically injured patients at risk of developing ARDS.

Visual validation of the mechanical stabilizing effects of positive end-expiratory pressure at the alveolar level.

BACKGROUND: Positive end-expiratory pressure (PEEP) reduces ventilator-induced lung injury (VILI), presumably by mechanically stabilizing alveoli and decreasing intrapulmonary shear. Although there is indirect support for this concept in the literature, direct evidence is lacking. In a surfactant depletion model of acute lung injury we observed unstable alveolar mechanics referred to as repeated alveolar collapse and expansion (RACE) as measured by changes in alveolar area from inspiration to expiration (I - E(Delta)). We tested the hypothesis that over a range of tidal volumes PEEP would prevent RACE by mechanically stabilizing alveoli. MATERIALS AND METHODS: Yorkshire pigs were randomized to three groups: control (n = 4), Tween (surfactant-deactivating detergent) (n = 4), and Tween + PEEP (7 cm H(2)O) (n = 4). Using in vivo video microscopy individual alveolar areas were measured with computer image analysis at end inspiration and expiration over consecutive increases in tidal volume (7, 10, 15, 20, and 30 cc/kg.) I - E(Delta) was calculated for each alveolus. RESULTS: Surfactant deactivation significantly increased I - E(Delta) at every tidal volume compared to controls (P < 0.05). PEEP prevented this change, returning I - E(Delta) to control levels over a spectrum of tidal volumes. CONCLUSIONS: RACE occurs in our surfactant deactivation model of acute lung injury. PEEP mechanically stabilizes alveoli and prevents RACE over a range of tidal volumes. This is the first study to visually document the existence of RACE and the mechanical stabilizing effects of PEEP at the alveolar level. The ability of PEEP to stabilize alveoli and reduce shear during mechanical ventilation has important implications for therapeutic strategies directed at VILI and acute respiratory distress syndrome.

Altered alveolar mechanics in the acutely injured lung.

OBJECTIVES: Alterations in alveolar mechanics (i.e., the dynamic change in alveolar size during tidal ventilation) are thought to play a critical role in acute lung injuries such as acute respiratory distress syndrome (ARDS). In this study, we describe and quantify the dynamic changes in alveolar mechanics of individual alveoli in a porcine ARDS model by direct visualization using in vivo microscopy. DESIGN: Prospective, observational, controlled study. SETTING: University research laboratory. SUBJECTS: Ten adult pigs. INTERVENTIONS: Pigs were anesthetized and placed on mechanical ventilation, underwent a left thoracotomy, and were separated into the following two groups post hoc: a control group of instrumented animals with no lung injury (n = 5), and a lung injury group in which lung injury was induced by tracheal Tween instillation, causing surfactant deactivation (n = 5). Pulmonary and systemic hemodynamics, blood gases, lung pressures, subpleural blood flow (laser Doppler), and alveolar mechanics (in vivo microscopy) were measured in both groups. Alveolar size was measured at peak inspiration (I) and end expiration (E) on individual subpleural alveoli by image analysis. Histologic sections of lung tissue were taken at necropsy from the injury group. MEASUREMENTS AND MAIN RESULTS: In the acutely injured lung, three distinct alveolar inflation-deflation patterns were observed and classified: type I alveoli (n = 37) changed size minimally (I - EDelta = 367 +/- 88 microm2) during tidal ventilation; type II alveoli (n = 37) changed size dramatically (I - EDelta = 9326 +/- 1010 microm2) with tidal ventilation but did not totally collapse at end expiration; and type III alveoli (n = 12) demonstrated an even greater size change than did type II alveoli (I - EDelta = 15,418 +/- 1995 microm2), and were distinguished from type II in that they totally collapsed at end expiration (atelectasis) and reinflated during inspiration. We have termed the abnormal alveolar inflation pattern of type II and III alveoli "repetitive alveolar collapse and expansion" (RACE). RACE describes all alveoli that visibly change volume with ventilation, regardless of whether these alveoli collapse totally (type III) at end expiration. Thus, the term "collapse" in RACE refers to a visibly obvious collapse of the alveolus during expiration, whether this collapse is total or partial. In the normal lung, all alveoli measured exhibited type I mechanics. Alveoli were significantly larger at peak inspiration in type II (18,266 +/- 1317 microm2, n = 37) and III (15,418 +/- 1995 microm2, n = 12) alveoli as compared with type I (8214 +/- 655 microm2, n = 37). Tween caused a heterogenous lung injury with areas of normal alveolar mechanics adjacent to areas of abnormal alveolar mechanics. Subsequent histologic sections from normal areas exhibited no pathology, whereas lung tissue from areas with RACE mechanics demonstrated alveolar collapse, atelectasis, and leukocyte infiltration. CONCLUSION: Alveolar mechanics are altered in the acutely injured lung as demonstrated by the development of alveolar instability (RACE) and the increase in alveolar size at peak inspiration. Alveolar instability varied from alveolus to alveolus in the same microscopic field and included alveoli that changed area greatly with tidal ventilation but remained patent at end expiration and those that totally collapsed and reexpanded with each breath. Thus, alterations in alveolar mechanics in the acutely injured lung are complex, and attempts to assess what may be occurring at the alveolar level from analysis of inflection points on the whole-lung pressure/volume curve are likely to be erroneous. We speculate that the mechanism of ventilator-induced lung injury may involve altered alveolar mechanics, specifically RACE and alveolar overdistension.

A role for the anti-inflammatory properties of tetracyclines in the prevention of acute lung injury.

The etiology of inflammatory disorders involves many cellular, plasmatic and humoral pathways of signaling culminating in the production of enzymatic and free radical mediated tissue damage. Multiple redundant pathways of initiation and elusive temporal expression of initiators pose formidable barriers to effective treatment. Nowhere is this more evident than in the adult respiratory distress syndrome (ARDS), a systemic inflammatory disorder leading to pulmonary failure where, despite significant advances in intensive care management, mortality has improved only 10% over the last decade. Tetracyclines, in addition to their anti-microbial properties, exhibit inhibitory activity toward several initiators of the inflammatory cascade and mediators of tissue damage. In this review we discuss how the broad spectrum anti-inflammatory properties of the tetracyclines make them attractive candidates for use in the prevention of acute lung injury.

Evidence of increased matrix metalloproteinase-9 concentration in patients following cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response, which can result in acute lung injury known as "postperfusion syndrome." Neutrophil activation with concomitant serine protease release has been implicated in the pathogenesis of "postperfusion syndrome." Increased plasma levels of neutrophil elastase (NE) have been demonstrated in patients undergoing CPB, and it is well documented that both NE and matrix metalloproteinase-9 (MMP-9) have a synergistic role in pulmonary injury. We, therefore, hypothesized that plasma levels of MMP-9 would be elevated in patients after CPB. Human plasma was obtained after informed consent from eight patients undergoing CPB. Plasma was collected at the start of CPB, 5 minutes after the initiation of CPB, and at the termination of CPB (156 +/- 17 min). All samples were analyzed by both standard enzyme-linked immunosorbent assay (ELISA) and gelatin zymography for MMP-9 (free and total enzyme) concentration. Data were expressed as means +/-SE and assessed by analysis of variance (ANOVA). Plasma MMP-9 concentration was significantly increased at the end of CPB (191 +/- 30.4 ng/mL; p <.05) as compared to both the start of CPB (28.3 +/- 13.2 ng/mL) and 5 minutes after the initiation of CPB (44.3 +/- 15.4 ng/mL). Patients undergoing CPB show an increase in serum MMP-9 levels. Prior studies utilizing an animal model of "postperfusion syndrome" have shown that inhibition of MMP-9 and NE prevented pulmonary injury following CPB. The results of the current study suggest that such an approach may also have merit in the clinical setting of cardiopulmonary bypass.

The mechanism of lung volume change during mechanical ventilation.

To understand ventilator-induced lung injury (VILI) during positive pressure ventilation, mechanisms of normal alveolar mechanics must first be established. Isotropic "balloonlike" alveolar volume (VA) change has been viewed as the prevailing mechanism of normal lung volume (VL) changes. We hypothesized that change in VL is predominantly caused by alveolar recruitment-derecruitment (R/D). Fifteen mongrel dogs were anesthetized and intubated with a tracheal divider. Through a thoracotomy incision, in vivo microscopy of subpleural alveoli was performed as the degassed lung was inflated to 80% TLC, and then deflated to residual volume (RV). Still photomicrographs were evaluated to determine if change in VL is due to change in VA or R/D of alveoli. We noted a steady, significant increase in alveolar recruitment as VL increased to 80% TLC (p < 0.05). However, VA increased significantly, but only to 20% TLC (p < 0.05). Once recruited, alveoli did not demonstrate any further volume change, whereas the lung as a whole maintained a normal pressure/volume relationship. In our model, changes in VL predominantly are caused by R/D.

Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass.

BACKGROUND: Acute lung injury (ALI) after cardiopulmonary bypass (CPB) results from sequential priming and activation of neutrophils. Activated neutrophils release neutral serine, elastase, and matrix metalloproteinases (MMPs) and oxygen radical species, which damage alveolar-capillary basement membranes and the extracellular matrix, resulting in an ALI clinically defined as adult respiratory distress syndrome (ARDS). We hypothesized that treatment with a potent MMP and elastase inhibitor, a chemically modified tetracycline (CMT-3), would prevent ALI in our sequential insult model of ALI after CPB. METHODS AND RESULTS: Anesthetized Yorkshire pigs were randomized to 1 of 5 groups: control (n=3); CPB (n=5), femoral-femoral hypothermic bypass for 1 hour; LPS (n=7), sham bypass followed by infusion of low-dose Escherichia coli lipopolysaccharide (LPS; 1 microgram/kg); CPB+LPS (n=6), both insults; and CPB+LPS+CMT-3 (n=5), both insults plus intravenous CMT-3 dosed to obtain a 25-micromol/L blood concentration. CPB+LPS caused severe lung injury, as demonstrated by a significant fall in PaO(2) and an increase in intrapulmonary shunt compared with all groups (P<0.05). These changes were associated with significant pulmonary infiltration of neutrophils and an increase in elastase and MMP-9 activity. CONCLUSIONS: All pathological changes typical of ALI after CPB were prevented by CMT-3. Prevention of lung dysfunction followed an attenuation of both elastase and MMP-2 activity. This study suggests that strategies to combat ARDS should target terminal neutrophil effectors.

Videomicroscopy provides accurate in vivo assessment of pulmonary microvascular reactivity in rabbits.

When defining the mechanism of hypoxic pulmonary vasoconstriction (HPV), investigators have employed ex vivo preparations because of the belief that accurate, quantitative assessment of pulmonary microvessels could not be obtained in vivo. We hypothesize that accurate, quantitative assessment of pulmonary microvascular reactivity can be performed using a simple, in vivo preparation. Our aim was to provide this quantitative assessment in a defined animal model, and to confirm that the chosen preparation could discriminate changes in microvascular reactivity as influenced by endogenous mediators. New Zealand rabbits were instrumented for in vivo microscopy and direct measurement of subpleural arterioles. Rabbits were first randomized to either control (n = 7) or endotoxin (n = 5), infusion of Escherichia coli lipopolysaccharide (200 Fg/kg). All rabbits were then exposed to a repeated protocol of normoxia (21% O2) for 20 min and then hypoxia (15% O2) for 10 min over 2 h. The changes in arteriole diameter were measured at the end of each interval. Normal pulmonary arterioles repeatedly constrict 15+/-3.5% during hypoxia. Altering endogenous vasoactive mediators, as with infusion of endotoxin, caused a loss of hypoxia-induced vasoconstriction. The results of our study validate this experimental preparation for the reliable quantification of pulmonary microvascular reactivity and investigation of hypoxic pulmonary vasoconstriction under both normal and pathologic conditions.

Multiple sequential insults cause post-pump syndrome.

BACKGROUND: We hypothesize that post-pump syndrome (PPS) following cardiopulmonary bypass (CPB) can be caused by multiple minor insults and that the mechanism of PPS is a priming and subsequent activation of polymorphonuclear (PMN) leukocytes. In this study extensive pathophysiologic and morphometric assessment was undertaken in a porcine model of sequential insult PPS. METHODS: Pigs were anesthetized, placed on a ventilator, instrumented for measurements of hemodynamic function, and separated into five groups: (1) Control (n = 4)--surgery only, (2) CPB (n = 4)--placed on femoral-femoral hypothermic (28 degrees C) bypass for 1 h, (3) LPS (n = 6)--underwent sham CPB followed by infusion of low dose endotoxin [E. coli lipopolysaccharide (LPS-1 microg/kg)], (4) Heparin + protamine + LPS (HP + LPS, n = 4)--were heparinized without CPB for 1 h, following which protamine and LPS were infused and (5) CPB + LPS (n = 8)--subjected to both CPB and LPS. RESULTS: Only CPB + LPS resulted in acute respiratory distress typical of PPS as indicated by a significant decrease in PaO2 and increase in intrapulmonary shunt fraction (p<0.05). CPB + LPS significantly increased tissue density and the number of sequestered monocytes and PMNs (p<0.05) above all other groups. Alveolar macrophages (AM) increased equally in all groups receiving LPS. CONCLUSIONS: CPB primes the inflammatory system causing pulmonary PMN sequestration without lung injury. Exposure to an otherwise benign dose of endotoxin results in activation of the sequestered PMNs causing PPS. This study confirms that PPS can be caused by multiple minor insults.

Soluble tumor necrosis factor receptor prevents post-pump syndrome.

Post-pump syndrome is an acute lung injury following cardiopulmonary bypass (CPB) which is indistinguishable from the adult respiratory distress syndrome (ARDS). Tumor necrosis factor (TNF) is central to the inflammatory process and is capable of triggering the entire pathophysiologic response leading to ARDS. We hypothesized that treatment with a soluble TNF receptor-binding protein (TNFbp) would reduce the increase in serum TNF and prevent acute lung injury in our sequential insult model of ARDS following CPB. Anesthetized pigs were randomized to one of three groups: Control (n = 3), surgical preparation only; CPB + LPS (n = 6), femoral-femoral hypothermic bypass for 1 h followed by infusion of low dose Escherichia coli lipopolysaccharide (LPS; 1 microg/kg); and TNFbp + CPB + LPS (n = 4), pretreatment with intravenous TNFbp (2 mg/kg) followed immediately by both insults. CPB + LPS caused severe lung injury demonstrated by a significant fall in PaO2 and an increase in both intrapulmonary shunt and peak airway pressure as compared to all groups (P < 0.05). These changes were associated with a significant increase in plasma TNF level and pulmonary neutrophil sequestration. TNFbp significantly reduced plasma levels of TNF and prevented the lung injury typically observed with this ARDS model, but did not reduce pulmonary neutrophil sequestration. Thus, elevated serum TNF is not responsible for neutrophil sequestration but does play a role in neutrophil activation which causes lung injury. Prophylactic use of TNFbp in CPB patients may prevent neutrophil activation and reduce the incidence of post-pump ARDS.

Systemic inflammation induced by cardiopulmonary bypass: a review of pathogenesis and treatment.

The acute respiratory distress syndrome (ARDS) is a severe alteration in lung structure and function that develops secondary to a traumatic stimulus. When ARDS develops following cardiopulmonary bypass (CPB) it is know as postpump syndrome (PPS). ARDS can be caused by a single massive insult ("hit"); however, sequential minor insults ("hits") are more common clinically. The concept of multiple sequential insults causing ARDS has been termed the "two-hit" model of ARDS. The purpose of this article is to summarize our studies testing the hypothesis that PPS is caused by multiple sequential insults. To confirm our hypothesis, we developed a porcine model of "two-hit" PPS. Our model was composed of sequential benign insults, with CPB as the "first hit" and low dose of endotoxin as the "second-hit." It is our hypothesis that the mechanism of PPS is CPB-induced priming of polymorphonuclear leukocytes (PMNs) ("first-hit") with subsequent PMN activation by a second insult ("second-hit") such as endotoxin. Our model confirms this clinically relevant postulate, and we provide strategies to disrupt the inflammatory cascade leading to PPS.

Matrix metalloproteinase inhibitor: differential effects on pulmonary neutrophil and monocyte sequestration following cardiopulmonary bypass.

UNLABELLED: Acute respiratory distress syndrome (ARDS) following cardiopulmonary bypass (CPB), also known as "post-pump" or "post-perfusion syndrome" (PPS), results from sequential priming and activation of neutrophils. We hypothesized that chemically modified tetracycline (CMT-3) an inhibitor of neutrophil matrix metalloproteinase (MMP) and elastase, would prevent PPS. We performed histometric analysis of lung tissue from our porcine PPS model to correlate cellular sequestration and histologic injury with CMT-3 treatment. METHODS: Yorkshire pigs were randomized into five groups: Control (n = 3); CPB (n = 5); femoral-femoral bypass 1 hour; LPS (n = 7), Escherichia coli lipopolysaccharide (1 microgram/kg); CPB + LPS (n = 6); and CPB + LPS + CMT (n = 5), sequential insults and CMT-3. Protocol histometric analysis defined cellular and tissue components of lung injury. RESULTS: CMT-3 decreased neutrophil sequestration in the CPB + LPS + CMT-3 group (p < 0.0001 vs. CPB + LPS). There were no differences in monocytes between CPB + LPS and CPB + LPS + CMT treatment groups. CONCLUSIONS: CMT-3 attenuates neutrophil sequestration but has no effect on mononuclear sequestration in our PPS model. This finding supports current research on leukocyte chemokines and has important implications regarding mechanisms of CMT-3. Despite lack of monocyte response to CMT-3, PPS was prevented by inhibiting neutrophils alone; confirming the primary role of neutrophils in PPS.

Aerosolized surfactant improves pulmonary function in endotoxin-induced lung injury.

Surfactant dysfunction is a primary pathophysiologic component in patients with adult respiratory distress syndrome (ARDS). In this study we tested the efficacy of aerosolized surfactant (Sf ) replacement in a severe lung injury model of endotoxin-induced ARDS. Twenty-one certified healthy pigs were anesthetized, surgically prepared for measurement of hemodynamic and lung function, then randomized into one of four groups: (1) control (n = 5), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 6), infused with Escherichia coli LPS (100 microgram/kg) without positive end- expiratory pressure (PEEP) and ventilated with a nonhumidified gas mixture of 50% N2O and 50% O2; (3) LPS + PEEP (n = 4), infused with LPS, placed on PEEP (7.5 cm H2O), and ventilated with a humidified gas mixture; and (4) LPS + PEEP + Sf (n = 6), infused with LPS, placed on PEEP, and ventilated with aerosolized Sf (Infasurf, ONY, Inc.). All animals were studied for 6 h. Arterial PO2 significantly decreased in both the LPS and LPS + PEEP groups (LPS + PEEP = 74 +/- 19 mm Hg; LPS = 74 +/- 19 mm Hg, p < 0.05) while venous admixture (Q S/Q T) increased in these groups (LPS + PEEP = 43.3 +/- 3.9%; LPS = 47.7 +/- 11%, p < 0.05) as compared with the control group. PEEP + Sf reduced the fall in PO2 (142 +/- 20 mm Hg) and rise in Q S/Q T (15.1 +/- 3.6%) caused by LPS. Delayed induction of PEEP (2 h following LPS) did not significantly improve any parameter over the LPS group without PEEP in this ARDS model. LPS without PEEP (3.4 +/- 0.2 cells/6,400 micrometer2) caused a marked increase in the total number of sequestered leukocytes in the pulmonary parenchyma as compared with the control group (1.3 +/- 0.1 cells/6,400 micrometer2). LPS + PEEP + Sf (2.3 +/- 0.2 cells/6,400 micrometer2) significantly decreased while LPS + PEEP significantly increased (4.0 +/- 0.2 cells/6,400 micrometer2) the total number of sequestered leukocytes as compared with the LPS without PEEP group. In summary, aerosolized surfactant replacement decreased leukocyte sequestration and improved oxygenation in our porcine model of endotoxin-induced lung injury.

Exogenous surfactant and positive end-expiratory pressure in the treatment of endotoxin-induced lung injury.

OBJECTIVE: To evaluate the efficacy of treating endotoxin-induced lung injury with single dose exogenous surfactant and positive end-expiratory pressure (PEEP). DESIGN: Prospective trial. SETTING: Laboratory at a university medical center. SUBJECTS: Nineteen certified healthy pigs, weighing 15 to 20 kg. INTERVENTIONS: Pigs were anesthetized and surgically prepared for hemodynamic and lung function measurements. Animals were randomized into four groups: a) Control pigs (n = 4) received an intravenous infusion of saline without Escherichia colilipopolysaccharide (LPS); b) the LPS group (n = 5) received an intravenous infusion of saline containing LPS (100 microg/kg); c) the PEEP plus saline group (n = 5) received an intravenous infusion of saline containing LPS. Two hours after LPS infusion, saline was instilled into the lung as a control for surfactant instillation, and the animals were placed on 7.5 cm H2O of PEEP; d) the PEEP plus surfactant group (n = 5) received an intravenous infusion of saline containing LPS. Two hours following LPS infusion, surfactant (50 mg/kg) was instilled into the lung and the animals were placed on 7.5 cm H2O of PEEP. PEEP was applied first and surfactant or saline was instilled into the lung while maintaining positive pressure ventilation. All groups were studied for 6 hrs after the start of LPS injection. At necropsy, bronchoalveolar lavage was performed and the right middle lung lobe was fixed for histologic analysis. MEASUREMENTS AND MAIN RESULTS: Compared with LPS without treatment, PEEP plus surfactant significantly increased PaO2 (PEEP plus surfactant = 156.6 +/- 18.6 [SEM] torr [20.8 +/- 2.5 kPa]; LPS = 79.2 +/- 21.9 torr [10.5 +/- 2.9 kPa]; p<.05), and decreased venous admixture (PEEP plus surfactant = 12.5 +/- 2.0%; LPS = 46.9 +/- 14.2%; p< .05) 5 hrs after LPS infusion. These changes were not significant 6 hrs after LPS infusion. PEEP plus surfactant did not alter ventilatory efficiency index (VEI = 3800/[peak airway pressure - PEEP] x respiratory rate x PacO2), or static compliance as compared with LPS without treatment at any time point. Cytologic analysis of bronchoalveolar lavage fluid showed that surfactant treatment significantly increased the percentage of alveolar neutrophils as compared with LPS without treatment (PEEP plus surfactant = 39.1 +/- 5.5%; LPS = 17.4 +/- 6.6%; p< .05). Histologic analysis showed that LPS caused edema accumulation around the airways and pulmonary vessels, and a significant increase in the number of sequestered leukocytes (LPS group = 3.4 +/- 0.2 cells/6400 micro2; control group = 1.3 +/- 0.1 cells/6400 micro2; p < .05). PEEP plus saline and PEEP plus surfactant significantly increased the total number of sequestered leukocytes in the pulmonary parenchyma (PEEP plus surfactant = 8.2 +/- 0.7 cells/6400 micro2; PEEP plus saline = 3.9 +/- 0.2 cells/6400 micro2; p <.05) compared with the control and LPS groups. CONCLUSIONS: We conclude that PEEP plus surfactant treatment of endotoxin-induced lung injury transiently improves oxygenation, but is unable to maintain this salutary effect indefinitely. Thus, repeat bolus dosing of surfactant or bolus treatment followed by continuous aerosol delivery may be necessary for a continuous beneficial effect.

Endotoxin-stimulated alveolar macrophage recruitment of neutrophils and modulation with exogenous surfactant.

OBJECTIVE: To determine whether endotoxin-stimulated alveolar macrophages would attract neutrophils and whether exogenous surfactant treatment would modulate this chemoattraction. DESIGN: Alveolar macrophages were harvested from bronchoalveolar lavage fluid and neutrophils from the blood of anesthetized guinea pigs. SUBJECTS: Hartley guinea pigs. INTERVENTIONS: Alveolar macrophages were suspended in RPMI 1640 and stimulated with 1 microg/mL of lipopolysaccharide (LPS), the supernatant removed and the alveolar macrophages were incubated in either RPMI or RPMI with surfactant at two different doses (292 microg/mL or 875 microg/mL) for 16 hrs. MEASUREMENTS AND MAIN RESULTS: The supernatant was extracted from the alveolar macrophages and placed in a chemotaxis plate and the migration of neutrophils was measured. Chemotaxis of all cell types to be tested was measured by a change of absorbance on a microplate reader set at 492 nm. Results were compared with alveolar macrophages not stimulated with LPS, RPMI alone, and N formyl-methionyl-leucyl-phenylalanine (FMLP). The supernatant of the stimulated alveolar macrophages increased neutrophil chemotaxis as compared with unstimulated alveolar macrophages, and RPMI (p < .05). Surfactant treatment with 292 microg/mL significantly decreased LPS-stimulated alveolar macrophages induced neutrophil chemotaxis. Treatment with 875 microg/mL of surfactant did not alter neutrophil chemotaxis. CONCLUSIONS: Alveolar macrophages stimulation with LPS increased the chemotaxis of neutrophils. Treatment with surfactant at a concentration of 875 microg/mL did not alter neutrophil migration; however, treatment with 292 microg/mL significantly decreased neutrophil chemotaxis suggesting that at low concentrations, surfactant inhibits chemokine release and may reduce pulmonary neutrophil sequestration in vivo.

Surfactant replacement in the treatment of sepsis-induced adult respiratory distress syndrome in pigs.

OBJECTIVE: To evaluate the efficacy of treating sepsis-induced adult respiratory distress syndrome (ARDS) by instillation of exogenous surfactant in a porcine endotoxin model. DESIGN: Prospective trial. SETTING: Laboratory at a university medical center. SUBJECTS: Fifteen hybrid pigs, weighing 15 to 20 kg. INTERVENTIONS: Pigs were anesthetized and surgically prepared for hemodynamic and lung function measurements. Animals were randomized into three groups: a control group (group I; n=4) that received sham Escherichia coli lipopolysaccharide (endotoxin); an endotoxin group (group II; n=6) that received endotoxin (25 micrograms/kg); and an endotoxin + surfactant (Infasurf, ONY, Amherst, NY) instillation group (group III; n=5) that received endotoxin (25 micrograms/kg) followed by surfactant (100 mg/kg) instillation; all groups were studied for 6 hrs after the start of endotoxin injection. At necropsy, lung water and surfactant function (Wilhelmy balance) were measured and the right middle lung lobe was fixed for histologic analysis. Surfactant function was expressed as the surface tension at the minimum trough area. MEASUREMENTS AND MAIN RESULTS: Surfactant treatment (group III) significantly (p<.05) decreased venous admixture (group III = 41.5 +/- 9.1%; group II = 61.6 +/- 4.7%), PaCO2 (group III = 46.6 +/- 1.3 torr [6.2 +/- 0.2 kPa]; group II = 54.4 +/- 2.6 torr [7.25 +/- 0.34 kPa], and surface tension minimum (group III = 8.8 +/- 1.8 dyne/cm; group II = 20.0 +/- dyne/cm), as compared with endotoxin without treatment (group II) 6 hrs after endotoxin infusion. However, surfactant instillation did not significantly improve PaO2 (group III = 62.8 +/- 6.8 torr [8.4 +/- 0.9 kPa2]; group II = 50.3 +/- 3.7 torr [6.7 +/- 0.49 kPa]) or reduce the amount of pulmonary edema (group III = 7.1 +/- 0.39 ratio; group II = 6.8 +/- 0.24 ratio) seen 6 hrs following endotoxin injection. Histologic analysis showed that endotoxin caused edema accumulation around airways and pulmonary vessels, and a large increase in the number of marginated leukocytes with or without surfactant treatment. Surfactant treatment significantly increased the total number of leukocytes in the pulmonary parenchyma. CONCLUSIONS: We conclude that endotoxin caused lung injury typical of ARDS as demonstrated by pulmonary edema, an increase in PaCO2, and a decrease in PaO2, a decrease in static lung compliance and inhibition of surfactant function. Exogenous surfactant treatment effected only moderate improvements in lung function (i.e., reduced venous admixture and restored surfactant function) in this sepsis-induced ARDS model.