Partial Liquid Ventilation with Perfluorocarbon in the Treatment of Rats with Lethal Pneumococcal Pneumonia 

All methods were approved by the University of Massachusetts Medical Center Animal Research Committee and are in accordance with the National Institutes of Health guidelines for animal use.

Streptococcus pneumoniae, serotype 3 (ATCC 6303), was propagated on 5% sheep blood agar incubated at 37 [degree sign] Celsius in 5% CO²for 24 h. Immediately before infection of rats, bacteria were harvested from culture plates and suspended at a concentration of ca 10⁹colony-forming units/ml in sterile 0.85% NaCl containing 5% sucrose. Seventy-five antigen-free male Wistar rats (Charles River Laboratories, Portage, WI) weighing 275–300 g were maintained with free access to food and water for 2 weeks before infection to ensure the health of the experimental animals. At the time of infection, the rats were anesthetized with 2–3% halothane in oxygen. A 20-gauge catheter was placed into the trachea via direct laryngoscopy, and 1 ml of bacterial suspension was instilled directly through the catheter. Animals were allowed to recover briefly in 100% oxygen, returned to their quarters, and allowed free access to food and water.

Animals were divided into five groups of 15 each, and all animals were inoculated with Streptococcus pneumoniae (Figure 1). All treatments were instituted 24 h after inoculation. Treatments were neither randomized nor blinded. Controls received no treatment. Gas-ventilated animals were anesthetized with 2.5% halothane in oxygen and intubated via direct laryngoscopy with a 20-gauge catheter. A rodent pump (Harvard Apparatus, Inc., South Natick, MA) was used for ventilation and was set at a respiratory frequency of 120 breaths per minute and tidal volume of 5 ml. Ventilation time was 10 min. Animals who got partial liquid ventilation were intubated in the same fashion as the gas-ventilated animals. Fifteen milliliters of preoxygenated FC-77 (3M, St. Paul, MN) were instilled directly into the lungs followed by mechanical ventilation as described for the gas-ventilated animals. Any excess FC-77 above the functional residual capacity was exhaled around the uncuffed, loosely fitting endotracheal tube. Penicillin-treated animals received a single intramuscular dose of 200,000 U of penicillin G benzathine (Wyeth Laboratories, Philadelphia, PA). The last group (combined treatment) received a single intramuscular dose of penicillin G benzathine and partial liquid ventilation as described previously. Anesthetized animals were recovered in 100% oxygen for 1 h after treatment. The rats were observed every 24 h for survival for a total of 10 days.

In preliminary experiments, five infected but untreated controls were killed via exsanguination at one of three times (i.e., 4, 12, and 24 h), and the heart and lungs were removed en bloc. The lungs were inflated and placed in a 10% buffered formalin solution for preservation. Vertical sections were taken, fixed in paraffin, cut on a microtome, and stained with hematoxylin and eosin. The following system, which incorporated seven pathologic (two gross and five microscopic) criteria for acute lung disease, was used to define the extent of disease. The two gross pathologic parameters were in-duration and discoloration. The five microscopic parameters were extent of nonhomogeneous expansion, thickness of interalveolar septae, acute inflammation, proteinaceous exudate, and luminal debris. Each parameter was scored as absent (0), mild (1), moderate (2), or severe (3). The scores for all of the criteria were summed. Thus, a healthy lung would have a score of 0, and the score for an acutely injured lung could range from 1–21. One of us (AF), familiar with the scoring system but blinded to the time when the animals were killed, scored the gross and histologic specimens.

Four, 12, and 24 h after infection, animals were selected randomly for blood culture analysis. The tail of the rat was prepared with 10% povidone-iodine solution and snipped 1 cm from the tip. Approximately 0.1 ml of blood was aseptically transferred to the surface of a 5% sheep blood agar plate. Plates were incubated at 37 [degree sign] Celsius in 5% CO²for 72 h. A blood culture result was considered positive when colonies revealed alpha hemolysis and a mucoid appearance and the gram stain revealed gram-positive diplococci.

Survival curves were generated using a commercially available statistical software program (Statistica; Stat-Soft, Tulsa, OK). Cox's F test was used to compare survival curves. Seven comparison were made: combined therapy versus all the other groups, and intramuscular penicillin therapy versus the control group, gas-ventilation group, and partial liquid ventilation group. Bonferroni correction was used for the multiple comparisons. The null hypothesis was rejected for a corrected probability value < 0.05.

At 4 h, the mean lung pathology score of five infected but untreated control rats was 7.2 (range, 6–9). At 12 and 24 h, respectively, the mean scores were 14 (range, 12–16) and 15.8 (range, 13–18). Bacteremia was observed in 25% of rats at 4 h and in 100% of rats at 12 and 24 h. Representative gross and microscopic lung sections are depicted in Figure 2.

Survival curves are depicted in Figure 3. Between days 4 and 7, mortality was 100% in the control, gas-ventilated, and partial liquid ventilation animals. In the penicillin G benzathine-treated animals, survival was 40% with no death occurring after day 3 (P < 0.05 compared with control and partial liquid ventilation groups). The survival rate for animals treated with partial liquid ventilation and penicillin G benzathine was 80%(P < 0.05 compared with all other groups).

This is the first published study describing the use of PAGE in combination with antibiotic administration for the treatment of pneumonia. The combination of partial liquid ventilation with FC-77 and a systemic antibiotic was superior to treatment with either modality alone.

The beneficial effect of partial liquid ventilation in combination with antibiotic agents on outcome may be due to (1) a direct effect of perfluorocarbon on bacterial growth in the lung;(2) the lavaging of bacteria out of the lung;(3) an antiinflammatory effect of the perfluorocarbon; or (4) improvements in total pulmonary surfactant. [15]We have previously shown, however, that FC-77 does not affect the growth of pneumococci in vivo or in vitro [16]nor does it appear to lavage bacteria from the lung [16]; therefore, the third and fourth explanations are most likely.

The effects of perfluorocarbons on the inflammatory process are not well understood. Smith et al. [14]have shown that production of reactive oxygen species is decreased by lipopolysaccharide-stimulated alveolar macrophages in vitro when exposed to perflubron. Nesti et al. showed that exposure to perflubron enhanced healing and reduced inflammation of acute full-thickness burns [17]and the gastrointestinal tract in an animal model of ulcerative colitis. [18]Both FC-77 and perflubron are eight-carbon perfluorocarbons; the major difference between the two is a terminal bromide on perflubron. Whether FC-77 also reduces inflammation remains to be shown. If FC-77 possesses antiinflammatory properties, the use of this perfluorocarbon may provide a wider therapeutic window for systemic antibiotic agents in the treatment of pneumonia.

Pulmonary surfactant is deficient and abnormal in both animal models of pneumonia and clinical studies of pulmonary infection. [19]Recent data suggest that exposure of type II alveolar cells to perflubron results in increased production of surfactant. [20]FC-77 has a surface tension of 15 dynes/cm²at 25 [degree sign] Celsius [21]and may assist as a pulmonary surfactant. This effect may have contributed to the improved outcome in the group receiving combination therapy.

In this study, animals continued to die up to 48 h after initiation of appropriate antibiotic therapy. Penetration of antibiotic agents to the site of infection can be a limitation in the treatment of pneumonia. The delivery of antibiotic agents directly to the lung using perfluorocarbons as a carrier may represent a better therapeutic strategy than either oral or parenteral antibiotic administration. Preliminary data have failed to support this concept, however. [22] 

The pneumonia model that was used in this study is characterized by rapid disease progression and a fatal outcome in untreated control rats. All control animals had positive blood cultures and died within 5 days. Histologic examination revealed a consistent, severe pneumonic process. Penicillin G benzathine was chosen as the antibiotic agent because it is highly active against the strain of S. pneumoniae used in this study (mean inhibitory concentration [MIC]= 0.02 micro gram/ml). A single intramuscular dose of 2,500 U/kg in humans causes a rapid rise in serum concentration above the MIC of susceptible strains, with the concentration sustained for at least 2 weeks. [23]The dose used in this study ([nearly =] 600,000 U/kg) likely resulted in serum levels above the MIC shortly after injection to the end of the 10-day study period (package insert). Previous work [8]using this model reveal that penicillin G was uniformly effective when therapy was initiated within 12 h of infection (mortality = 0%), 40% effective at 24 h, and nearly ineffective at 36 h (mortality = 90%); therefore, we chose a time point when antibiotic agents were only partially effective and probably more clinically relevant.

Previous work in our laboratory has shown that partial liquid ventilation alone reduces 5-day mortality rates in animals ventilated 4 h after infection with S. pneumoniae. [8]The failure of PAGE as solo therapy to reduce mortality significantly in the current study may reflect the delay in treatment (4 vs. 24 h), shorter period of ventilation (10 vs. 30 min), and the longer period of observation (5 vs. 10 days). Unlike in the previous study, [8]in which animals underwent a tracheotomy, a small-caliber uncuffed endotracheal tube was used in this study. As a result, adequate ventilation (gas or partial liquid) could not be sustained beyond 10 min. It was our intent to provide a period of ventilation sufficiently long to transport the perfluorocarbons into the alveolar space while minimizing upper airway trauma. Although the mechanical ventilation time was decreased, exposure of pulmonary parenchyma to FC-77 continued beyond the period of ventilation as the perfluorocarbons had not specifically drained from the lung. Instead, animals were placed in oxygen chambers and FC-77 dissipated by evaporation. The half-life for FC-77 evaporation in the rat has not been determined. Estimates of perflubron evaporative loss in piglets indicate that approximately 12 h is required for complete evaporation of a functional residual capacity dose. [24] 

In this rat model of lethal pneumococcal pneumonia, partial liquid ventilation with FC-77 in combination with penicillin G therapy improved 10-day mortality rates to a greater extent than antibiotic agents or PAGE alone. PAGE is potentially effective in the treatment of the infected lung. Further studies to confirm the safety and efficacy of partial liquid ventilation with perfluorocarbons are warranted.