Evaluation of Alveolar
Cytokine Response to Aspiration of Gastric Contents*
Akella Chendrasekhar, MDa
Gregory A. Timberlake, MDa
Ganga Prabhakar, MDb
Leon S. Barringer, DVMc
aDepartment of Surgery Education, Iowa Methodist Medical
Center, Des Moines, IA
of Surgery, West Virginia University, Morgantown, WV
Agriculture, West Virginia University, Morgantown, WV
*This study was supported in part by an
unrestricted educational grant from Ballard Medical Products, Draper, Utah.
Background: Aspiration injury is
difficult to diagnosis clinically. Acute response to nonacidic gastric juice
within the lung parenchyma is unknown.
Design: We evaluated the acute response
in bronchoalveolar lavage (BAL) fluid with regard to albumin level, tumor
necrosis factor (TNF) level, interleukin-6 (IL-6) level, and myeloperoxidase
(MPO) level associated with pulmonary aspiration of gastric juice compared with
Materials and Methods: Pulmonary aspiration
injury was induced in 10 adult swine using 1 cc/kg of gastric juice (n = 5) and
saline (n = 5). BAL was performed immediately before and 1 hour after
aspiration injury in all animals. BAL fluid was analyzed for albumin level,
TNF-a, and IL-6 levels using commercially available ELISA kits as
well as MPO level.
Results: The levels of TNF-a
and IL-6 and MPO levels in BAL fluid with gastric juice aspiration showed a
significant increase over baseline levels compared with saline aspiration. BAL
albumin levels failed to show any increase from baseline to postaspiration.
Conclusions: Pulmonary aspiration with
gastric juice is associated with a strong local inflammatory response compared
Aspiration is a feared complication in
intensive care units (ICUs). The incidence of pulmonary aspiration of gastric
contents in ICU populations receiving enteral feedings varies widely, ranging
from 0.8% to 77%.1-4 Aspiration is associated with two main
detrimental sequelae, pneumonitis and pneumonia. The incidence of nosocomial
pneumonia in mechanically ventilated ICU patients has been found to range
between 21% to 38%.5 The high morbidity and mortality rates
(30% to 60%) associated with aspiration pneumonia and pneumonitis are a result
of the combined effects of a predisposing illness, a degree of acute airway
obstruction, and a direct chemical pulmonary injury.6 Aspiration may not be clinically
recognized unless it is accompanied by respiratory distress. Symptoms
associated with aspiration may occur hours after the episode,7
making correlation between the inciting event and the symptoms even more
difficult. Aspiration has also been identified as a strong general risk factor
in development of acute respiratory distress syndrome (ARDS).8 Huxley and colleagues have shown that
patients with depressed levels of consciousness are at increased risk of
aspiration compared with normal patients (70% versus 45%, respectively).9 Trauma and critical illness may further
increase the risk of aspiration injury by increasing the gastric secretion rate
and acidity level of gastric sections. As treatment of pulmonary aspiration is
largely supportive, early delineation of alveolar injury may allow more
aggressive treatment with possible avoidance of detrimental sequelae.
aspiration has been shown to increase alveolar protein content and pulmonary
macrophage accumulation with subsequent activation in various models.10-12
Tissue cytokine levels may be directly associated with further accumulation and
activation of pulmonary macrophages, thus leading to further lung injury.
Cytokines are low-molecular-weight proteins produced by activated immune cells
(eg, tumor necrosis factor [TNF], interleukins). These cytokines have been
shown to mediate the induction and amplification of the inflammatory response
to various types of injury, including hemorrhagic and endotoxic shock.13,14 However, alveolar (tissue level)
cytokines have not been studied as markers for detection of aspiration injury.
of alveolar and systemic cytokines (TNF-a, interleukin [IL]-6) have been shown to
change acutely (within 1 to 2 hours) in response to alveolar injury.11,12
Preliminary data from cytokine levels in bronchoalveolar lavage (BAL) fluid
have been shown to correlate with subsequent development and severity of ARDS
in high-risk patients.15,16
hypothesized that cytokine levels obtained from BAL fluid in pigs sustaining
gastric juice aspiration show significant increases compared with saline
aspiration in a porcine model of aspiration injury.
Cytokine levels correlate
with BAL myeloperoxidase (MPO) levels (a marker of tissue injury) in this
This protocol was approved by the University's
laboratory animal utilization committee. Animals were cared for in
accordance with the current guidelines of the National Institutes
of Health. Ten mixed-breed adult swine, weighing 75 to 85 kg, were
fasted overnight with free access to water. On the day of the experiment,
the animals were initially anesthetized with intramuscular telazol
(4 mg/kg). Intravenous (IV) access was obtained by cannulation of
an ear vein and anesthesia was continued with IV sodium pentobarbital
(3 to 5 mg/kg/hr). The animals were intubated endotracheally and mechanically
ventilated. The ventilator was adjusted to maintain eucarbia and a
Po2 of at
least 100 mm Hg.
lavage was performed with 50 mL normal saline using a non bronchoscopic lavage
catheter (BALCATH, Ballard Medical Products, Draper, Utah) placed through the
endotracheal tube. The BAL was performed immediately prior to and 1 hour after
instillation of the saline or gastric juice. The aspirated fluid was assayed
for albumin level, TNF, and IL-6 using commercially available ELISA kits
(Genzyme, Cambridge, Mass) and MPO level. The cytokine (TNF and Il-6) levels
(in pg/mL) are based on a standardization curve using human cytokines. All
assays were performed in duplicate. Statistical analysis was performed using
the averaged values for baseline and postaspiration values. Postaspiration
values were compared with baseline values using one way analysis of variance
with repeated measures. Statistical significance threshold was P < .05.
Two consecutive BALs before and after
aspiration were successfully carried out in all animals. The average recovery
volume from the BAL was 32 mL with recovery volumes ranging from 25 to 40
mL. No significant change in
oxygenation or ventilation was noted in the animals during the course of the
levels of TNF were statistically unchanged before and after aspiration of
saline (124.0 + 15.0 pg/mL versus 136.0 + 18.0 pg/mL, P = not significant). However, gastric
juice aspiration resulted in a significant change with the TNF level increasing
from 136.0 + 17.0 pg/mL to 915.0 + 26.0 pg/mL (P < .05; Table 1).
of IL-6 were elevated with both saline and gastric juice aspiration. With
saline the change from 128.0 + 18.0 pg/mL to 193.0 + 52.0 pg/mL
was not statistically significant. In contrast, gastric juice aspiration
produced a rise in the level of IL-6 from 115.0 + 19.0 pg/mL to 1217.0 +
25.0 pg/mL (P < .05; Table 1).
increases in cytokine levels were correlated with increases in BAL MPO activity
seen with gastric juice aspiration and not with saline aspiration (Table 1).
to our surprise, the BAL albumin level showed no significant change in either
the saline or gastric juice aspiration (Table 1).
gastric juice used for the pulmonary aspiration was checked for the presence of
TNF and IL-6, and MPO activity was found to have no detectable levels.
Aspiration is a feared complication in
ICUs. The incidence of pulmonary aspiration of gastric contents in ICU
populations receiving enteral feedings varies widely, ranging from 0.8% to 77%.1-4 The high morbidity and mortality rates
(30% to 60%) associated with aspiration are a result of the combined effects of
a predisposing illness, a degree of acute airway obstruction, and a direct
chemical pulmonary injury.6
Symptoms associated with aspiration may occur hours after the event,8 making the correlation between the
inciting event and the symptoms even more difficult. Trauma and critical
illness may further increase the risk of aspiration injury by increasing the
gastric secretion rate and acidity level of gastric sections.1,3 As aspiration treatment is largely
supportive, early delineation of alveolar injury may allow more aggressive
treatment with possible avoidance of detrimental sequelae.
lack of change in oxygenation and ventilation seen in our animals with gastric
aspiration was not surprising considering the insidious nature of aspiration
injury and the relative nonacidic pH of the aspirated fluids. The elevation of
alveolar cytokines may lead to delayed symptoms as described in the clinical
analysis has been vastly simplified by the recent introduction of commercially
available ELISA kits for mouse and human cytokines. Porcine cytokines have been
shown to be cross-reactive with human cytokines. Verification of the utility of
human cytokine ELISA with porcine models has been done with TNF-a
using a bioassay.17 Porcine IL-6 shares a significant homology with human IL-6
and does cross-react using the human ELISA kits; however, bioassay verification
has not been performed because of technical problems.17
of alveolar cytokines change acutely (within 1 hour) in response to high-
pressure and high-volume ventilation.11
Systemic levels of cytokines (TNF-a, IL-6) have been shown to change in
response to acid aspiration-related alveolar injury with a delayed time course.12 Cytokines have been found to be useful
markers of severity of injury in various clinical settings. Alveolar sampling
of albumin levels has been shown to be elevated in acid aspiration injury.10
Therefore, on re-evaluation with our level of nonacidic pH, the lack of
elevation of alveolar albumin level is not surprising.
origin of these cytokines is not entirely clear as the expected timeline for
accumulation of alveolar macrophages (if we assume that alveolar macrophages
are releasing these cytokines) does not seem to fit with the elevations noted
in this experiment. However, alveolar MPO activity was elevated and seemed to
correlate with the presence of cytokines. Another explanation for the etiology
of these cytokines might be the alveolar endothelial cells. Alveolar cytokine
levels may provide an effective window to look at subsequent increases in
pulmonary macrophage accumulation and activation. If the direct correlation of
aspiration with cytokine levels seen in our study is reproduced in patients,
the association of BAL cytokine levels with aspiration injury would allow for a
more aggressive directed therapeutic approach. Stated differently, if alveolar
cytokine levels are indeed elevated with significant aspiration injury and not
elevated with insignificant injury, an appropriate treatment algorithm can be
instituted. This might include more aggressive pulmonary care and antibiotics
in patients with significant injury. Conversely, in a majority of patients with
less injury, our cytokine analysis approach would allow for a more
cost-effective approach with resource conservation.
preliminary data looking at forced aspiration of gastric juice has shown
significant elevations in alveolar TNF-a and IL-6 levels compared with baseline
values. Acid aspiration injury has been shown to result in a rise in systemic
TNF levels12; however, our study is the first to look at alveolar
cytokine levels in this type of injury (ie, in which the contents of the
gastric juice [proteolytic enzymes, etc] may be causing the injury as opposed
to the acidity, as our pH was not very acidic). The association between BAL
cytokine levels and gastric juice aspiration is based on a relative change in
levels rather than exact numbers as our standardization curves were based on
human cytokines provided with the ELISA kits. We would have to assume 100%
cross reactivity to rely on exact numbers. Stated differently, the ELISA kits
rely on light absorption at a particular wavelength. The exact levels of
specific cytokines are based on extrapolation from a standardized curve plotted
using known levels of human cytokines. If we were checking for human cytokine
levels, the extrapolation along this standard curve would be exact; however, we
are sampling porcine cytokines using the human cytokine derived standard curve.
We assume a large degree of cross reactivity, although some degree of error may
be present. This error should be inherently negated as we used each animal as
its own control at baseline. Furthermore, the change in relative values
compared with baseline and saline aspiration is clear.
shortcoming of this data is that relative changes are clinically difficult to
use as most injury does not announce itself before arrival. This is a common
problem with using animal models to extrapolate the patient's condition.
However, this problem may be overcome in certain high-risk patients by
obtaining baseline levels at initiation of mechanical ventilation. If a patient
is subsequently suspected of having aspirated, a repeat BAL can be performed
and subsequent cytokine levels can be checked for elevation. There may be other
problems associated with using such sensitive markers as cytokine levels.
Alveolar cytokine levels have been shown to be elevated early (at 60 minutes)
in relation to injury by high-volume, high-pressure ventilation.11
Since alveolar cytokine changes may be associated with ventilator changes, this
may pose a problem in the mechanically ventilated patient in whom we are trying
to detect significant aspiration. Early elevations in alveolar cytokine levels
were seen in preliminary data studying various ventilator modalities by our
group as well,18 although the persistence of these changes over time
Such an early rise in BAL cytokine levels
has been seen in high-risk human populations and this has been shown by several
groups to be predictive of subsequent severe lung injury.15,16 The potential benefit of early detection
of significant aspiration injury is that intervention (resuscitation and
supportive therapy) can be more aggressive and focused. Further histologic
studies of injury classification and delineation studies of the relationship
between cytokine elevation and lung injury are needed.
Lavage Cytokine Levels with Acid Aspiration Injury
Necrosis Factor (pg/mL)
Preaspiration BAL (n = 5)
124 + 15
128 + 18
0.34 + 0.07
2.6 + 0.3
Postaspiration BAL (n = 5)
136 + 18*
193 + 52*
0.38 + 0.06*
2.8 + 0.5*
Juice Preaspiration BAL (n = 5)
136 + 17
115 + 19
0.34 + 0.08
2.7 + 0.3
Juice Postaspiration BAL (n = 5)
915 + 26+
1217 + 25+
1.30 + 0.11+
2.8 + 0.4*
*P = not significant.
+P < .05, mean + SEM
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