Evaluation of Alveolar Cytokine Response to Aspiration of Gastric Contents*

Akella Chendrasekhar, MD^a

Gregory A. Timberlake, MD^a

Ganga Prabhakar, MD^b

Leon S. Barringer, DVM^c

 

^aDepartment of Surgery Education, Iowa Methodist Medical Center, Des Moines, IA

^bDepartment of Surgery, West Virginia University, Morgantown, WV

^cSchool of Agriculture, West Virginia University, Morgantown, WV

 

*This study was supported in part by an unrestricted educational grant from Ballard Medical Products, Draper, Utah.

Key Words: aspiration, cytokine, inflammation

 

ABSTRACT

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 saline.

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 with saline.

 

INTRODUCTION

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.

            Acid 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.

            Levels 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

We 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 model.

 

 

MATERIALS AND METHODS

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.

            Gastric juice (pH = 4.57) from one of the fasted pigs was withdrawn using an orogastric tube and this served as the study aspiration material (1 mL/kg) for pigs in the study group (n = 5). Normal saline (pH = 4.5, 1 mL/kg) was instilled in to the endotracheal tube of the control animals (n = 5).

            Bronchoalveolar 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.

 

RESULTS

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 study.

            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).

            Levels 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).

            These increases in cytokine levels were correlated with increases in BAL MPO activity seen with gastric juice aspiration and not with saline aspiration (Table 1).

            Much to our surprise, the BAL albumin level showed no significant change in either the saline or gastric juice aspiration (Table 1).

            The 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.

 

DISCUSSION

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.

            The 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 literature.^3,4

            Cytokine 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

            Levels 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.

            The 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.

            Our 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.

            Another 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 is unclear.

 

CONCLUSION

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.

 

Table 1

Bronchoalveolar Lavage Cytokine Levels with Acid Aspiration Injury

	Tumor Necrosis Factor (pg/mL)	Interleukin-6 (pg/mL)	Myeloperoxidase U/mL	Albumin (mg/dL)
Saline Preaspiration BAL (n = 5)	124 + 15	128 + 18	0.34 + 0.07	2.6 + 0.3
Saline Postaspiration BAL (n = 5)	136 + 18*	193 + 52*	0.38 + 0.06*	2.8 + 0.5*
Gastric Juice Preaspiration BAL (n = 5)	136 + 17	115 + 19	0.34 + 0.08	2.7 + 0.3
Gastric Juice Postaspiration BAL (n = 5)	915 + 26+	1217 + 25+	1.30 + 0.11+	2.8 + 0.4*

^*P = not significant.

⁺P < .05, mean + SEM

BAL = bronchoalveolar lavage.

 

 

REFERENCES

1. Cataldi-Betcher EL, Seltzer MH, et al: Complications during enteral nutritional support: A prospective study. JPEN 7:546, 1983.

2. Metheny NA, Eisenberg P, Spies M: Aspiration pneumonia in patients fed through nasoenteral tubes. Heart Lung 15:526, 1986.

3. Kingston GW, Phang PT, Leathley MJ: Increased incidence of nosocomial pneumonia in mechanically ventilated patients with subclinical aspiration.

Am J Surg 161:589, 1991.

4. Elpern EH, Jacobs ER, Bone RC: Incidence of aspiration in tracheally intubated adults. Heart Lung 16:257, 1987.

5. Craven DE, Kunches LM, Kilinsky V, et al: Risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation.

Am Rev Respir Dis 133:792, 1986.

6. Kirsch CM, Sanders A: Aspiration pneumonia, medical management. Otolaryngol Clin North Am 21:677, 1988.

7. Kinni ME, Stout MM: Aspiration pneumonitis: Predisposing conditions and prevention. J Oral Maxillofac Surg 44:378, 1986.

8. Pepe PE, Potkin RT, Reus DH, et al: Clinical predictors of the adult respiratory distress syndrome. Am J Surg 144:124, 1982.

9. Huxley EJ, Viroslav J, Gray WR, et al: Pharyngeal aspiration in normal adults and patients with depressed consciousness. Am J Med 64:564, 1978.

10. Kennedy TP, Johnson KJ, Kunkel RG, et al: Acid aspiration lung injury in the rat: Biphasic pathogenesis. Anesth Analg 69:87, 1989.

11. Valenza F, Ribeiro SP, Slutsky AS: Large volume high pressure mechanical ventilation upregulates the production of tumor necrosis factor-a in an ex-vivo rat septic lung model. Crit Care Med 23S:A159, 1995.

12. Rabinovici R, Neville LF, Abdullah F, et al: Aspiration induced lung injury: Role of complement. Crit Care Med 23:1405, 1995.

13. Danner RL, Natanson C, Suffredini AE, et al: Microbial toxins: Role in the pathogenesis of septic shock and multiple organ failure, in Bihari DI, Cerra FB (eds): Multiple Organ Failure. Fullerton, CA, Society of Critical Care Medicine, 1989, p 151.

14. Chaudry IH, Ayala A, Ertel W, et al: Hemorrhage and resuscitation: Immunological aspects. Am J Physiol 259:R663, 1990.

15. Meduri GU, Headley S, Kohler G, et al: Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Chest 107:1062, 1995.

16. Antonelli M, Raponi G, Lenti L, et al: Leukotrienes and alpha tumor necrosis factor levels in the bronchoalveolar lavage fluid of patient at risk for the adult respiratory distress syndrome. Minerva Anestesiologica 60:419, 1994.

17. Vandermeer TJ, Menconi MJ, O'Sullivan BP, et al: Bactericidal/permeability increasing protein ameliorates acute lung injury in porcine endotoxemia.

J Appl Physiol 76:2006, 1994

18. Chendrasekhar A, Prabhakar G, Timberlake GA: Alveolar cytokine responses to different modes of mechanical ventilation in acute lung injury. FASEB J 10:A108, 1996.