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Departments of Physiology, Medicine, and Pathology, Lawson Health Research Institute, St. Joseph's Health Centre, University of Western Ontario, London, Ontario, Canada N6A 4V2
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The effects of mechanical
ventilation (MV) on the surfactant system and cytokine secretion were
studied in isolated septic rat lungs. At 23 h after sham surgery
or induction of sepsis by cecal ligation and perforation (CLP), lungs
were excised and randomized to one of three groups: 1) a
nonventilated group, 2) a group subjected to 1 h of noninjurious
MV (tidal volume = 10 ml/kg, positive end-expiratory pressure = 3 cmH2O), or 3) a group subjected to 1 h
of injurious MV (tidal volume = 20 ml/kg, positive end-expiratory
pressure = 0 cmH2O). Nonventilated sham and CLP lungs
had similar compliance, normal lung morphology, surfactant, and
cytokine concentrations. Injurious ventilation decreased compliance,
altered surfactant, increased cytokines, and induced morphological
changes compared with nonventilation in sham and CLP lungs. In these
lungs, the surfactant system was similar in sham and CLP lungs;
however, tumor necrosis factor-
and interleukin-6 levels were
significantly higher in CLP lungs. We conclude that injurious
ventilation altered surfactant independent of sepsis and that the CLP
lungs were predisposed to the secretion of larger amounts of cytokines
because of ventilation.
cecal ligation and perforation; sepsis; pulmonary surfactant
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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SEPSIS IS THE MOST COMMON predisposing condition leading to the acute respiratory distress syndrome (ARDS) and is associated with the highest mortality among causes of this condition (2, 17). The pathophysiology of sepsis and ARDS is complex and involves a variety of cytokines and other inflammatory mediators. In addition, the lung dysfunction associated with these conditions appears to be, in part, a consequence of alterations of the pulmonary surfactant system (10, 16, 25).
Interestingly, many patients with sepsis do not develop ARDS. In these patients, the host's inflammatory response to the systemic focus of infection may be adequately protective and thus lead to recovery. On the other hand, in situations in which the inflammatory response becomes overwhelming, ARDS ensues. Such an overwhelming inflammatory response may be the result of an additional insult to the lungs of septic patients. Recent studies suggest that mechanical ventilation (MV) may represent such an additional insult and, thereby, contribute to the development of ARDS in septic patients (21).
MV is a common and necessary therapeutic intervention in many critically ill patients. Unfortunately, a number of studies have shown that, in addition to its beneficial effects, MV can cause significant lung injury (5). These investigations demonstrated that ventilation strategies that caused overdistension and/or repeated collapse and reopening of lung units were damaging. Interestingly, as described for ARDS and sepsis (10, 16, 25), the mechanisms by which MV causes this lung damage have also been reported to involve an increased release of inflammatory cytokines into the alveolar space as well as alterations of the pulmonary surfactant system (23, 26).
On the basis of this information, we hypothesized that lungs from septic animals would be more susceptible to the harmful effects of MV than lungs from sham animals. We further hypothesized that this increased susceptibility may be mediated through alterations of the surfactant system and an increased secretion of inflammatory cytokines into the alveolar space. To examine this hypothesis, we utilized the cecal ligation-and-perforation (CLP) model of sepsis, in which adult rats were rendered septic but had no physiological evidence of lung injury. To investigate the lung-specific effects of ventilation, we then isolated the lungs from the septic and sham animals and subjected these lungs to MV for 1 h. Subsequently, the lungs were examined for changes in lung compliance, lung morphology, pulmonary surfactant, and inflammatory cytokines.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal preparation. Male Sprague-Dawley rats weighing 300-450 g were acclimatized to the laboratory environment for 1 wk before surgery. Free access to food and water was available for this period. For surgery, rats were anesthetized with 4% halothane in oxygen in an anesthetizing box. Once anesthesia was induced, the rats were transferred to a surgical table with a nose cone set up to maintain anesthesia. Catheters were inserted in the right external jugular vein and right carotid artery using PE-50 tubing. These lines were routed subcutaneously to the back of the neck and attached to a three-fluid channel (22-gauge) swivel system. The incision under the neck was closed with 3-0 silk suture.
To induce sepsis, the CLP technique was performed as previously described (16). The CLP procedure consisted of a laparotomy followed by ligation of the proximal one-third of the cecum. The cecum was then punctured twice with a 16-gauge needle: once just above the ligature and again at the tip of the cecum. The cecum was then compressed to extrude bowel contents into the peritoneum. A sham-operated control group was used for comparison with the animals undergoing the CLP procedure. In sham animals, induction of anesthesia and catheter placement were identical to that in the CLP animals, but no laparotomy or CLP procedure was performed. After attachment of the harness to secure the swivel system, each rat was placed in a plastic cage to recover. The swivel device allowed rats unlimited movement within the cage and free access to rat chow and water. Postoperatively, all animals received a continuous infusion of saline (7.5 ml · kg
1 · h1
iv) containing fentanyl (2 µg/ml) for analgesia. The arterial line
was infused with heparinized saline (1 U/ml) at 1 ml/h to maintain
patency. Measurements of arterial PO2 were
performed using a blood-gas analyzer (model ABL 500, Radiometer,
Copenhagen, Denmark). Arterial lactate levels were measured on a
glucose/lactate analyzer (model 2300 STAT Plus, Yellow Springs
Instruments). Mean arterial blood pressure (MAP) and heart rate (HR)
were recorded by attaching a pressure transducer to the arterial line
and reading the resulting MAP and HR on a blood pressure monitor. The
respiratory rate (RR) was recorded by counting the number of breaths
during a 15-s period. All of these measurements were recorded 4-5
h after surgery and again at 22-23 h, which was immediately before
death. At 22-23 h, in five animals in the sham and CLP groups, 6 ml of blood were withdrawn; this blood sample was used for blood
culture analysis under aerobic and anaerobic culture conditions. All
animals were killed by deeply anesthetizing the animal with
pentobarbital sodium, incising the abdominal cavity, and transecting
the descending aorta. Immediately after death, a cannula (14-gauge) was
inserted into the trachea, and the lungs were excised via a midline
sternotomy. All animals received humane care, and the Animal Care and
Use Committee of the University of Western Ontario approved all procedures.
Experimental protocol. The excised lungs were immediately placed into a 37°C humidified chamber. The lungs were inflated twice to a transpulmonary pressure (Ptp) of 25 cmH2O, and then static pressure-volume curves were determined by stepwise inflation of the lungs in 2-cmH2O increments to a Ptp of 25 cmH2O and deflation in 2-cmH2O increments to a Ptp of 0 cmH2O. The isolated lungs from sham and CLP animals were randomized to one of three groups: 1) a nonventilated control group, 2) a group subjected to 1 h of "noninjurious" MV [tidal volume (VT) = 10 ml/kg, positive end-expiratory pressure (PEEP) = 3 cmH2O, RR = 60 breaths/min], or 3) a group subjected to 1 h of "injurious" MV (VT = 20 ml/kg, PEEP = 0 cmH2O, RR = 30 breaths/min). The two latter groups were ventilated with room air by means of a volume-cycled rodent ventilator (Harvard Instruments, Saint Laurent, PQ, Canada). PEEP was added to the ventilation system by submerging the expiratory tube in water to a depth of 3 cm. During the ventilation period, airway pressures were monitored (model 400 monitor, Sechrist Industries, Anaheim, CA) and recorded every 30 min. On completion of 1 h of ex vivo ventilation, static lung compliance curves were measured as described above.
Lung lavage procedure.
Lungs randomized to the nonventilated group underwent a whole lung
lavage procedure immediately after the initial pressure-volume curve
determination. Both ventilated groups were lavaged subsequent to the
second determination of static lung compliance. Lungs were filled with
10-11 ml of 0.15 NaCl at room temperature until they appeared
fully distended, then the saline was withdrawn, and the same bolus of
saline was infused two more times. A 1.5-ml aliquot from this
first lavage was taken for cytokine analyses and processed as described
below. The remaining lavage fluid was collected in a beaker and
combined with the total fluid recovered from four additional lung
lavages. The volume of the combined lavage fluid was recorded, and a
5-ml aliquot of the total lavage was removed and stored at 
20°C.
The remainder of the lavage was centrifuged at 150 g for 10 min to yield a pellet containing cellular debris. This pellet was
resuspended with saline and stored at 20°C. A 5-ml aliquot of the
150-g supernatant was removed and stored at 20°C for
analysis of total surfactant pool size and for measurements of the
concentrations of surfactant-associated proteins (SP)-A and SP-D. The
remaining 150-g supernatant was then spun at 40,000 g for 15 min, yielding a supernatant that was designated the
small-aggregate surfactant fraction (SA). The 40,000-g
pellet was resuspended in 2 ml of saline and called the large-aggregate
surfactant fraction (LA). This technique for separating alveolar
surfactant subtypes has been previously described (13,
16). Both subfractions of surfactant were also stored at
20°C.
Protein measurements. The total protein content of the recovered lavage was determined using the method of Lowry and colleagues (15), with bovine serum albumin (BSA) as a standard.
An ELISA was used to quantitate SP-A and SP-D in the 150-g supernatant obtained from the six experimental groups, with lavage material obtained from normal rat lungs as a standard (26). Rabbit anti-rat SP-A or rabbit anti-rat SP-D polyclonal antibodies were diluted in 0.1 M NaHCO3 buffer (pH 9.6) to 1:1,500 (SP-A) or 1:1,000 (SP-D) and coated overnight at 4°C on a polystyrene 96-well microtiter plate (Maxisorb, Nunc, Roskilde, Denmark). After they were coated, the microtiter plates were blocked with 2% BSA in washing solution (50 mM Tris · HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4, 200 µl/well) for 60 min at room temperature and washed six times. Samples and standard were diluted in 0.1% BSA, 50 mM Tris · HCl, and 0.05% Tween 20, pH 7.4. Diluted samples and standards were applied to the microtiter plate (50 µl/well) and incubated for 1 h at room temperature. Plates were washed, and biotin-conjugated anti-rat SP-A or SP-D polyclonal antibody (50 µl/well), diluted in washing solution containing 0.1% BSA to 1:2,500 (SP-A) or 1:500 (SP-D), was applied to the plate for 1 h. After they were washed, the plates were incubated with horseradish peroxidase-conjugated streptavidin (0.1 µg/ml; Sigma Chemical, St. Louis, MO) diluted in washing solution containing 0.1% BSA (50 µl/well) at room temperature for 1 h. Subsequently, the plates were washed, and concentrations of SP-A or SP-D were determined by measuring the bound horseradish peroxidase with the use of tetramethylbenzidine reagent (150 µl/well; 100 µg/ml in 1 mM H2O2-0.1 M citric acid buffer, pH 4.0). The reaction was stopped by addition of 50 µl of 2 M H2SO4, and absorption was measured at 450 nm. On the basis of the data of the diluted normal rat sample, a linear standard curve was obtained. When samples were too concentrated, they were diluted until they fit on the linear portion of the standard curve. The results are expressed as relative amount of the specific protein measured in these samples compared with the amount recovered from the nonventilated sham lungs. Tumor necrosis factor- (TNF-), interleukin (IL)-6, and IL-1
protein concentrations were measured on the 1.5-ml aliquot of the first
lavage obtained from each lung. This 1.5-ml aliquot of lavage fluid was
immediately centrifuged at 200 g for 10 min. Four 350-µl
aliquots of the supernatants were then frozen in liquid nitrogen and
stored at 70°C until further analysis. In addition, cytokine
measurements were also performed on serum samples from sham and septic
animals collected at the time of lung harvest. These samples were also
frozen in liquid nitrogen and stored at 70°C until further
analysis. The cytokine measurements were performed using commercially
available ELISA kits (Biosource International, Camarillo, CA) following
the instructions provided by the supplier. The sensitivity of these
kits for TNF-, IL-6, and IL-1 were 4, 8, and 3 pg/ml,
respectively, and all were specific for rat. For each cytokine,
triplicates of all the samples were analyzed simultaneously.
Surfactant activity measurements.
Surface tension measurements of resuspended samples of LA were
performed using a pulsating bubble surfactometer, as described by
Enhorning (8). Aliquots of the appropriate samples were diluted to a final concentration of 2 mg phospholipid/ml in 150 mM
NaCl-1.5 mM CaCl2. All samples were incubated for 
90 min
at 37°C and analyzed using a pulsating bubble surfactometer at 37°C at 20 pulsations/min. Surface tension values at minimum bubble size,
after 100 pulsations, were expressed.
Bacterial culture of blood sample. Samples of blood obtained immediately after euthanasia were used to inoculate Columbia broth blood bottles for aerobic and anaerobic culture (BBL Septichek, Becton Dickinson). Blood cultures were incubated at 37°C for 24-120 h, and bacteria were identified by characteristic colony morphology, gram stain, lactose fermentation, spot indole, and oxidase tests.
Lung morphology and RNA analysis. Separate groups of rats (n = 3/group) were used to obtain lung tissue for morphological assessment (right lungs) and to obtain total lung RNA (left lungs). After completion of the final pressure-volume curve, the lung was inflated to a Ptp of 15 cmH2O, and the right bronchus was secured. The inflated right lungs were submerged and fixed in 10% neutral buffered formalin. The fixed right lung from each animal was sectioned sagittally and embedded in toto for histological evaluation. Sections (5-µm thick) were cut and stained with hematoxylin-and-eosin stain and examined by light microscopy. The lungs were examined for several different morphological features of early lung injury, such as atelectasis, overdistension and congestion, and edema. The severity of each of these variables was scored as normal, mild, moderate, or severe. All assessments were performed by a pathologist blinded with respect to the experimental groups.
The left lungs were used for RNA extraction immediately after removal of the right lung for morphological assessment. Total cellular RNA was extracted from fresh lung tissue using the TRIzol method (GIBCO Life Technologies, Paisley, UK), which was carried out according to the protocol recommended by the manufacturer. Cytokine RNA levels in the lungs were measured using a commercially available Multi-Probe RNase protection assay kit (PhaMingen) utilizing a multiprobe template set (rCK-1, Becton Dickinson, San Diego, CA). The RNase protection assay was carried out according to the instructions provided, with [-35S]UTP (1,250 Ci/mmol) utilized for probe
synthesis (Multi-Probe Template Set, rCK-1, catalog no. 45027P,
Dupont/NEN, Boston, MA). After the gel electrophoresis step, the gel
was dried and exposed to X-ray film (Eastman Kodak, Rochester, NY) for
identification of the specific RNA. The relative amounts of specific
cytokine RNAs were quantified by densitometry and standardized to the
L32-RNA levels.
Statistics. Values are means ± SE. Statistical significance between the ventilated and nonventilated groups and between sham and CLP groups was determined using a two-way analysis of variance followed by the Tukey post hoc analysis for multiple comparison. Analysis of physiological parameters and compliance was performed using paired t-test. P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Physiology.
The physiological parameters measured 4-5 h after surgery were not
significantly different between the sham and CLP animals, indicating
similar recoveries from anesthetic and surgery for both groups (data
not shown). The physiological parameters for all sham and CLP animals
just before death (22 h after surgery) are shown in Table
1. There were no significant differences
between sham and CLP rats with respect to body weight or RR at this
time. MAP was lower (P < 0.02) and HR was higher
(P < 0.001) in the CLP than in the sham group.
Arterial lactate levels were significantly higher in the CLP than in
the sham group (P < 0.001). There were no significant
differences in arterial PO2 or the
alveolar-arterial oxygen gradient between sham and CLP animals. In
addition, five animals in the CLP group that were tested for bacterial
cultures in the blood had positive cultures for gram-negative enteric
bacteria, whereas all sham animals tested had negative blood cultures.
Together, these parameters confirmed the systemic septic response in
animals undergoing the CLP procedure.
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Pressure-volume curves.
The results of the pressure-volume curves measured for the
nonventilated and ventilated groups are shown in Fig.
1. Within the nonventilated control
groups (Fig. 1A), there were no significant differences
between the sham and CLP lungs. Pressure-volume curves obtained from
lungs ventilated with the noninjurious strategy (Fig. 1B)
were not significantly different from those obtained from nonventilated
lungs. There were no significant differences between sham and CLP lungs
ventilated with the noninjurious strategy. All lungs ventilated with
the injurious ventilation strategy (Fig. 1C) had a
significant decrease in compliance and hysteresis compared with the
nonventilated and the noninjuriously ventilated groups (P < 0.05). There was no significant difference
between the pressure-volume curve obtained from lungs from the sham and
CLP animals within this latter ventilation group (Fig. 1C).
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Lung morphology.
Figure 2 shows representative
photomicrographs obtained from the lungs of each of the six groups
fixed at an inflation pressure of 15 cmH2O. Nonventilated
lungs had no evidence of cellular infiltrates or any other
morphological appearance indicating lung injury in the sham (Fig.
2A) or CLP (Fig. 2B) groups, except for some
focal overdistension in CLP lungs. Similarly, lungs from the
noninjuriously ventilated sham and CLP groups appeared normal with no
evidence of injury (Fig. 2, C and D). In
contrast, all lungs ventilated with the injurious ventilation strategy
showed morphological changes, with an area of atelectasis,
overdistension, and congestion (Fig. 2, E and F).
However, there were no detectable differences between the CLP and sham
lungs within the injuriously ventilated group.
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Surfactant analysis.
The recovered lavage volume obtained from the lungs was not
significantly different among the six groups. Figure
3 shows, for each of the different
groups, the total amount of surfactant obtained from the lavage as well
as the amounts of SA and LA. In the nonventilated group, there were no
significant differences between sham and CLP lungs in the total
surfactant pool, the amount of SA, or the amount of LA. After
noninjurious ventilation, the total surfactant pool was significantly
increased compared with the nonventilated groups, and this increase was
associated with a significantly greater amount of SA than in the
nonventilated controls. These increases in total surfactant and SA were
observed in the sham and CLP groups, and there was no significant
difference between these two groups. The injuriously ventilated animals
also had higher amounts of total surfactant and SA than the
nonventilated group; however, this difference reached statistical
significance only for the SA values. Similar to the nonventilated and
noninjuriously ventilated lungs, there were no significant differences
between sham and CLP lungs within this ventilation group.
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Cytokine analysis.
The concentrations of TNF-, IL-6, and IL-1 measured in the
alveolar lavage fluid are shown in Fig.
5. In the nonventilated group, TNF-
concentrations were ~40 pg/ml and were not significantly different
between sham and CLP animals. The concentrations of TNF- were higher
in lungs that were noninjuriously ventilated (100-200 pg/ml) than
in the nonventilated lungs; however, this difference did not reach
statistical significance (Fig. 5A). Lavage concentrations of
TNF- were also higher in the groups subjected to injurious
ventilation than in the nonventilated lungs. This difference was
statistically significant for the injuriously ventilated CLP group
compared with the nonventilated CLP group. Comparison between the sham
and CLP group revealed significantly higher TNF- concentrations in
injuriously ventilated CLP lungs than in sham lungs ventilated with the
injurious strategy.
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. In the lavages obtained from the nonventilated groups, IL-6 concentrations were low and not
significantly different between sham and CLP lungs. In noninjuriously ventilated lungs, IL-6 values were higher, but this did not reach statistical significance. IL-6 values were also higher in injuriously ventilated than in nonventilated and noninjuriously ventilated lungs,
and this was statistically significant for the CLP groups. IL-6 values
were significantly higher in the injuriously ventilated CLP lungs than
in the injuriously ventilated sham lungs.
IL-1 concentrations were similar for nonventilated sham and CLP
lungs (Fig. 5C). Noninjurious ventilation did not affect the
concentrations of IL-1 compared with the nonventilated groups. Injurious ventilation increased the lavage concentrations of IL-1, and this was statistically significant for the injuriously ventilated CLP animals compared with noninjuriously ventilated CLP lungs. There
was no difference between sham and CLP animals within any of the
ventilation groups.
Cytokine determinations were also performed on a small number of serum
samples obtained from CLP and sham rats at 23 h before lung
isolation. TNF- and IL-1 were undetectable in serum from sham and
CLP animals. IL-6 was also not detectable in serum samples from sham
rats; however, in samples from CLP rats, the average serum
concentration of IL-6 was 778 ± 251 pg/ml.
Normalized TNF-, IL-6, and IL-1 RNA levels are summarized in Fig.
6. Despite the lower n values
(n = 3/group), the RNA levels revealed a pattern
similar to the lavage protein concentrations of these cytokines (Fig.
5). In the nonventilated control groups, TNF- and IL-6 RNA were very
low and not significantly different between sham and CLP animals (Fig.
6, A and B). The RNA levels of TNF- and IL-6
were higher in ventilated than in nonventilated lungs. Comparison
between sham and CLP lungs revealed that TNF- and IL-6 RNA levels
were higher in ventilated CLP lungs. This difference between sham and
CLP lungs reached statistical significance for the IL-6 levels in the
noninjuriously ventilated groups.
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RNA levels are shown in Fig. 6C. These levels were
similar for the two nonventilated groups. Noninjurious and injurious ventilation increased the RNA levels; however, this increase did not
reach statistical significance. There was no significant difference between sham and CLP lungs within any of the ventilation groups.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Because not all patients with sepsis develop ARDS, it has been
suggested that additional insults to the septic lung may be involved in
the development of this condition. In the present study, we
investigated one such proposed additional insult, namely, MV
(21). We hypothesized that septic lungs were more
susceptible to MV-induced lung injury and that this would be reflected
by marked changes in the alveolar surfactant system and increased concentrations of inflammatory cytokines in the lung lavage. Overall, our results showed that sham and septic lungs responded similarly to an
injurious ventilation strategy with regard to decreases in lung
compliance, alterations of endogenous surfactant, and morphological
changes. However, the injuriously ventilated septic lungs produced
significantly more of the inflammatory cytokines TNF- and IL-6 than
the injuriously ventilated sham lungs. We conclude from these data that
sepsis predisposed the lung to release inflammatory mediators in
response to an injurious mode of MV and that sepsis had no effect on
the pulmonary surfactant system when the lungs were subjected to MV.
In this experimental study, the clinically relevant adult rat model of systemic sepsis induced by CLP was used. This CLP procedure, as described by Wichterman and colleagues (28), induces a peripheral focus of infection and peritonitis leading to sepsis. Rats undergoing the CLP procedure in the present study were confirmed to have sepsis 22 h after surgery. However, as indicated by the normal oxygenation values, lung morphology, and static lung compliance, these animals had no physiological evidence of lung injury at this time. The lungs from these animals were then isolated and ventilated in a humidified box. The ventilation strategies utilized in this experimental study were not based on current clinical practice, but on the available information on the injurious effects of overdistension of lung units and repeated alveolar collapse and reopening (5). It was believed that using these relatively severe conditions would provide insight into the development of lung injury due to MV over a short period of time. These isolated, nonperfused lungs were ventilated with room air, which may have resulted in changes in pH in the lung. This change in pH may have contributed to the alterations of surfactant and/or the development of lung injury observed in this study.
The rationale for using the isolated, nonperfused lung model for studying the effects of MV was to observe only the "lung-specific" effects of this intervention on pulmonary surfactant and inflammatory cytokines. Moreover, this model has been utilized in previous studies to elucidate mechanisms responsible for ventilation-induced lung injury (18, 23, 26). A potentially limiting factor of the isolated lung model is the effects of ischemia on the lung. However, because we utilized a relatively short period of ventilation and because we compared two different ventilation strategies with the same ischemic time, it is unlikely that the effects of ischemia had a major influence on our findings.
Pulmonary surfactant is a mixture of lipids and proteins responsible for stabilizing alveoli during end expiration (19). The importance of this biophysical function of surfactant is supported by studies on premature infants with neonatal respiratory distress syndrome and investigation of the lavage material from patients with ARDS. In these two conditions, the absence, in the former, or inactivation, in the latter, of surfactant results in decreased lung compliance (7, 10). Over the last several years, we and others have investigated the effects of MV on the pulmonary surfactant system and postulated that alteration of surface activity is a mechanism by which ventilation may contribute to the development of conditions such as ARDS (12, 24, 26). For example, certain models of MV were shown to alter the alveolar metabolism and surface activity of pulmonary surfactant. Specifically, high-VT ventilation was postulated to reduce the relative amount of the surface-active LA subtype of surfactant and to reduce the biophysical activity of these LA. The present study confirmed these previous observations, since the biophysical activity of LA obtained from the high-VT ventilation groups was significantly impaired compared with the other groups. This inactivation of surfactant likely contributed to the decreased lung compliance observed in the groups subjected to injurious MV. The exact mechanisms that led to the decreased activity of the LA is unknown at this time. It is possible that ventilation-induced reactive oxygen-nitrogen species affected the surfactant system, thereby impairing the surfactant system. Alternatively, increased protease and/or phospholipase activity during ventilation may have affected the proteins and lipids of the LA, resulting in the reduced activity. These and other possible mechanisms for the decreased surfactant activity require further study.
Interestingly, our results also showed that preexisting sepsis had no significant influence on these deleterious effects of MV on pulmonary surfactant. It was hypothesized that lungs isolated from septic animals would be more susceptible to injurious MV, resulting in more significant impairments of surfactant than lungs from the sham group. However, there were no significant differences between sham and septic lungs with regard to the total amounts of surfactant, the levels of surfactant proteins, the relative amounts of the surfactant aggregates, and the surface activity of the LA. Thus it is concluded that 1 h of high-VT ventilation decreases the surface activity of surfactant in a manner that is not influenced by sepsis.
In addition to the effects of MV on pulmonary surfactant, we also
hypothesized that MV-induced release of inflammatory cytokines would be
affected by sepsis. Tremblay and colleagues (23) showed that isolated normal rat lungs could produce a variety of inflammatory mediators in response to high-VT, zero-PEEP ventilation.
Higher concentrations of cytokines have also been reported in
brochoalveolar lavage material obtained from ventilated patients after
the onset of ARDS (9, 22). Furthermore, it was recently
shown that the particular MV strategy utilized in patients can affect
cytokine concentrations in the blood and bronchoalveolar lavage fluid
(20). These measurements of inflammatory mediators,
combined with studies showing that some of these mediators appear to
correlate with mortality, suggest that they play an integral rule in
the development of ARDS. However, the specific role of inflammatory
mediators in this context is not known. It is likely that the release
of inflammatory mediators in response to a specific insult represents a
protective effect, but in situations when this inflammatory response
becomes overwhelming, damage to various tissues may occur. Such an
overwhelming inflammatory response may result from additional insults
subsequent to the injury-inducing event (14). This latter scenario is supported by the present study, in which two inflammatory cytokines, IL-6 and TNF-, were markedly increased in the injuriously ventilated septic lungs compared with the sham lungs. RNA analysis revealed that these cytokines were likely produced within the lung
tissue, since the RNA values demonstrated a pattern similar to that
observed for the lavage protein levels of these cytokines. These
cytokine results were observed in septic lungs, despite the fact that,
before ventilation, lung compliance, surfactant systems, and
concentrations of cytokines were similar in septic and sham lungs.
In contrast to the results observed for TNF- and IL-6, the third
inflammatory cytokine that we measured, IL-1, was increased in
response to injurious MV but, unlike the others, was not affected by
sepsis. The reasons for these differences are unknown. However, it is
known that IL-1 does not contain a signal sequence for secretion and
thus is secreted by a mechanism different from TNF- and IL-6
(4).
When the results of the present study are interpreted, it is important
to consider the duration of ventilation. Lungs were ventilated for only
1 h, and thus these results reflect only the initial response to
MV. The inactivation of surfactant occurred within 1 h of MV at
high VT and likely contributed to the decreased compliance
observed in these lungs. In contrast, the cytokine levels did not
correlate with lung injury in this model and, therefore, appear to have
no direct role in the decreased lung compliance. However, it is
possible that these cytokines could affect lung function indirectly,
which may only be observed after prolonged ventilation. For example,
studies with isolated, surfactant-producing alveolar type II cells have
shown that TNF- can inhibit surfactant lipid and protein synthesis,
but this only occurred over longer time periods than used in the
present study (1, 11). It is, therefore, possible that
TNF- and other cytokines could significantly impact lung compliance
via their effects on the synthesis of surfactant. Studies involving a
longer period of ventilation are required to examine this possibility.
In summary, we have determined the effects of MV on the surfactant
system and cytokine secretion in isolated septic rat lungs. Surfactant
measurements differed depending on the ventilation strategy utilized,
but there were no significant differences between sham and septic
lungs. In contrast, TNF- and IL-6 levels were significantly higher
in injuriously ventilated septic lungs than in the sham group. These
results indicate that injurious ventilation altered surfactant,
regardless of the presence of sepsis, and that the lungs from septic
animals were predisposed to secrete larger amounts of TNF- and IL-6
in response to injurious ventilation than lungs of sham animals. We
speculate that the increase in cytokines in septic lung may contribute
to the development of ARDS in septic patients.
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ACKNOWLEDGEMENTS |
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The authors thank Fred Possmayer for use of the pulsating bubble surfactometer, Dr. Henk Haagsman and Bianca van Rozendaal for providing the antibodies and protocols for the SP-A and SP-D ELISAs, and Dr. L. Stitt for helpful suggestions regarding the statistical analysis.
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FOOTNOTES |
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This study was supported by the Canadian Institutes of Health Research, the Ontario Thoracic Society, and the Department of Medicine of the University of Western Ontario. T. Nakamura is the recipient of the Japanese-North American Medical Exchange Foundation Fellowship.
Address for reprint requests and other correspondence: R. Veldhuizen, Lawson Health Research Institute, Rm. G454, 268 Grosvenor St., London, ON, Canada N6A 4V2 (E-mail: rveldhui{at}julian.uwo.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 October 2000; accepted in final form 13 March 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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