Effects of Antibiotic Therapy on Pseudomonas aeruginosa-Induced Lung Injury in a Rat Model

Male Sprague-Dawley rats (300 to 350 g; Simonsen, Gilroy, Calif.), certified pathogen free, were anesthetized with 30 mg of pentobarbital given intraperitoneally; anesthesia was maintained with 12 mg of intraperitoneally administered pentobarbital every 2 h. All rats remained anesthetized, intubated, and ventilated throughout the entire experiment. The right carotid artery was cannulated with a polyethylene tube (PE50; Clay Adams, Parsippany, N.J.), for monitoring arterial blood pressure and for blood sampling. The right jugular vein was cannulated with a PE50 tube, and the drugs were administered intravenously. Pancuronium bromide (0.3 mg/kg of body weight) was given intravenously every 2 h for neuromuscular blockade. An endotracheal tube (PE240; Clay Adams) was inserted into the trachea via a tracheostomy. The rats were ventilated with a constant-volume animal respirator (Harvard Apparatus, South Natick, Mass.) with an O₂ fraction of 1.0 and 3-cm positive end-expiratory pressure. The respiratory rate was adjusted to maintain an arterial PCO₂ between 35 and 45 mm Hg. All animal experiments were conducted in compliance with the guidelines of the Animal Care Committee of the University of California at San Francisco.

P. aeruginosa PA103 was instilled to induce lung injury, as it has been shown to significantly increase the measurements of lung injury (20). The strain was stored as a bacterial stock at −70°C in a 10% sterile skim milk solution. The bacteria were streaked onto a Vogel-Bonner minimal medium plate for 36 h at 37°C, and then the bacteria were cultured in tryptic soy broth for 13 h at 32°C in a shaking incubator.

The instillate consisted of a 1-ml iso-osmolar albumin solution prepared from Ringer’s lactate, 2 mg of Evans blue dye, and 3 μCi of¹²⁵I-labeled albumin. P. aeruginosa at a concentration of 10⁶, 10⁷, or 10⁸CFU, determined spectrophotometrically, was added to the instillate just prior to airspace instillation. A sample of the instillate was saved for measurement of radioactivity, protein concentration, and quantitative bacterial cultures to assure accurate inoculations.

The MICs of the antibiotics were determined by the standard microdilution technique, according to the methods of the National Committee for Clinical Laboratory Standards (21). Aztreonam (Squibb Inc., Princeton, N.J.) at 60 mg/kg or imipenem-cilastatin (Merck, Inc., Rahway, N.J.) at 30 mg/kg was administered as an intravenous bolus dose 1 h after the airspace bacterial instillation. Control rats received an equal volume of normal saline (Baxter, Roundlake, Ill.) as a bolus. The investigator was blinded to whether a drug was administered. Due to the length of surgery, only two rats were studied in 1 day to maintain reproducibility of the model; a control rat was always one of the experimental animals.

Serum samples for measurements of endotoxin were obtained just prior to and 4 h after the bacterial instillation. The samples were allowed to clot for 20 min and then were centrifuged and immediately frozen at −70°C until they were analyzed. The serum samples were measured for free endotoxin with theLimulus amebocyte lysate assay (Chromogenic-Limulus Amebocyte; Wittaker M. A. Bioproducts, Walkersville, Md.).

In all experiments, after the surgical preparation, 3 μCi of ¹³¹I-albumin (Merck-Frosst, Kirkland, Quebec, Canada) was injected intravenously. The rats were then placed in a right lateral decubitus position. With the use of 1-ml syringes and PE50 tubes, the instillates were delivered to the right lungs (primarily the right lower lobes) over a 30-min interval. The rats were injected with 500 mg ofd-galactosamine/kg intraperitoneally during the bacterial instillation in order to sensitize them to the effects of endotoxin (2, 15, 16). Systemic arterial and airway pressures were measured at 1-h intervals. Blood samples were obtained for¹³¹I-albumin and ¹²⁵I-albumin measurement every hour after the instillation for 6 h.

After 6 h, the rats were deeply anesthetized, their abdomens were opened, and they were exsanguinated. Samples of blood, urine, right pleural fluid, and liver were obtained for bacterial cultures and radioactivity counts. The lungs were removed, and any remaining instillate was aspirated with a 3-ml syringe and a PE50 tube passed into the right lower lobes. The total protein and radioactivity of the alveolar samples were measured. The right and left lungs were homogenized separately for water-to-dry-weight ratio measurements and for culture. Measurements of the total protein concentrations of plasma, pleural fluid, and initial and final instillate samples were performed by the Biruet method.

Lung injury was quantified in four ways, as previously described (29, 30). The first method quantified the movement of the alveolar protein tracer,¹²⁵I-albumin, from the lung into the blood by measuring the amount of residual ¹²⁵I-albumin in the lung as well as the accumulation of the tracer in the plasma over the 6-h course of the experiment. Total ¹²⁵I-albumin instilled into the lung was determined by measuring duplicate samples of the instillate for total radioactivity (counts per minute per gram) and multiplying this amount by the total volume instilled into the lung. To calculate the residual¹²⁵I-albumin in the lung after 6 h, the average of two 0.5-g samples obtained from the lung homogenate was multiplied by the total volume of the lung homogenate. The radioactivities in the lung homogenates were added to the recovered radioactive counts in the aspirated fluid from the lungs. Circulating plasma¹²⁵I-albumin was measured from a sample of plasma obtained at the end of 6 h. The plasma fraction was accounted for by multiplying the counts per milliliter by the plasma volume [body weight × 0.07 (1 hematocrit)].

The second method required the measurement of ¹³¹I-albumin (the vascular protein tracer) in the airspace compartment of the lung at the end of the experiment. This was accomplished by measuring the¹³¹I-albumin counts in the final airspace sample. Plasma¹³¹I-albumin counts were averaged over the course of the experiment, and the ¹³¹I-albumin counts in the airspaces were expressed as a ratio to the plasma counts. This ratio provides a good index of equilibration of the vascular protein tracer into the alveolar compartment, as had been shown previously (30).

Third, extravascular lung water was determined for each separate lung by calculating the water-to-dry-weight ratio. The excess water in the experimental lung was calculated with the same equation described previously (30). Briefly, the volume of excess lung water (ELW; in ml) of the serum-instilled experimental lung was calculated as follows: ELW = [W_e/(D_e − P) − W_c/D_c](D_e − P ), where W and D are extravascular lung water and blood-free dry weights, respectively, of the experimental lung (e) and the control lung (c) andP is the blood-free dry weight of the initial alveolar fluid multiplied by the fraction of ¹²⁵I-albumin remaining in the lung.

Finally, the accumulation of the vascular protein tracer into the extravascular space of the lung was calculated by determining the total extravascular count of ¹³¹I-albumin in the lung divided by the average counts in the plasma over the 6-h study period and was expressed as total plasma equivalents in milliliters.

All data presented are means ± standard errors. The data were analyzed by one-way analysis of variance followed by Dunnet’s multiple-comparison test. A P value of <0.05 was accepted as statistically significant.

First, we quantified the acute lung injury in rats receiving the airspace instillation of P. aeruginosa. Rats receiving three different doses (10⁶, 10⁷, and 10⁸ CFU/rat) of P. aeruginosa were compared with the control rats that received vehicle without bacteria. The measurements of alveolar epithelial injury (the alveolar protein tracer in blood) and two measurements of lung edema (extravascular lung water and total plasma equivalents) were calculated in the 4-h experiments. The alveolar epithelial injury quantified by the movement of the radiolabeled alveolar protein tracer was increased linearly with increasing doses of P. aeruginosa or by increasing the duration of bacterial exposure in the lung from 2 to 4 h (Fig.1). Both parameters of lung edema, the extravascular lung water (Fig. 2) and the total plasma equivalents (Fig. 3), were also increased linearly with increasing doses of P. aeruginosa. Therefore, in this rat model, increasing quantities of acute lung injury can be consistently produced by modifying the dose of bacteria and/or the interval of bacterial exposure.

Based on the results described above, we evaluated the effects of two antibiotics, aztreonam and imipenem-cilastatin, on acute lung injury. A dose (10⁷ CFU) of P. aeruginosa was administered to each rat to produce a moderate quantity of lung injury that might be modified by the administration of antibiotics. We observed the animals for 6 h in this series to evaluate the effect of antibiotics. In addition to the parameters of acute lung injury, we measured the number of bacteria in the instilled lung 6 h after the tracheal instillation of P. aeruginosa.

The MIC of imipenem-cilastatin was 16 μg/ml, considered to be resistant by the National Committee for Clinical Laboratory Standards (20). In contrast, the MIC of aztreonam was 0.5 μg/ml, considered sensitive by the same standards. Nonetheless, among the rats that received imipenem-cilastatin after the instillation of bacteria there was a trend toward fewer bacteria remaining in the lungs (3.0 × 10⁵ ± 2.0 × 10⁵CFU/ml) than among the rats that had received aztreonam (3.2 × 10⁶ ± 1.7 × 10⁶ CFU/ml) or the rats that had not received antibiotics (6.0 × 10⁶ ± 4.0 × 10⁶ CFU/ml) (Fig.4). The blood culture results from all rats at the end of the experimental period were negative. The culture results from a liver sample taken at the end of the experiment were positive for all rats. Imipenem-cilastatin significantly improved all the parameters of lung injury measured compared to the control, untreated rats or to the aztreonam-treated rats. The administration of imipenem-cilastatin resulted in significantly less alveolar epithelial injury (Fig. 5), significantly less extravascular lung edema (Fig. 6), and significantly fewer total plasma equivalents (Fig.7). The baseline blood pressure measurements of the groups were not different (P > 0.05). Treatment with aztreonam resulted in greater reduction in systolic and diastolic blood pressure 5 h after instillation compared to treatment with imipenem-cilastatin and the control (Table1). At 6 h, systolic blood pressure was significantly reduced in the aztreonam-treated group (P = 0.026), and there was a trend toward statistical significance for reduction in diastolic blood pressure in the aztreonam-treated group (P = 0.052) at this time point. The reduction in lung injury and hypotension in the imipenem-cilastatin-treated rats was not explained by the serum endotoxin levels 4 h after the tracheal instillation of P. aeruginosa; the levels were not different in the antibiotic-treated rats and the control animals (Table2).