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* From the Département de Pneumologie (Drs. Marquette, Wermert, and Tonnel), Service de Bactériologie et Hygiène (Dr. Wallet), and Service d'Anatomopathologie (Dr. Copin), Hôpital A. Calmette, CHRU de Lille, France; Département Hospitalo-Universitaire de Recherche Expérimentale (Drs. Marquette and Wermert), Faculté de Médecine, Lille, France; and INSERM U416 (Drs. Marquette, Wermert, and Tonnel), Institut Pasteur, Lille.
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Abstract |
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Key Words: animal model • pneumonia
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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From studies by Johanson and coworkers9 ,10 ,11 originally aimed at evaluating lung repair processes in diffuse alveolar damage (induced by IV oleic acid), we knew that mechanically ventilated baboons can develop endogenous pulmonary infections (ie, VAP). From previous experiments,12 ,13 we knew that piglets rapidly develop endogenous pneumonia as a result of mechanical impairment of mucociliary clearance (ie, postobstructive pneumonia).
These data prompted us to investigate whether healthy piglets could develop endogenously acquired pulmonary infection as a result of prolonged mechanical ventilation (MV). We also investigated the capacity of these piglets to overcome massive intrabronchial challenge with bacterial pathogens in the absence of MV. In this article, we describe the standardization of this model of VAP and the similarities of the model with human VAP.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary Study
Several pharmacologic and technical issues first had to be
resolved to provide prolonged ventilatory support under general
anesthesia in our animals. Indeed, to our knowledge, no data were
available in the literature regarding prolonged anesthesia (
12 h)
in the pig. Attempts made with conventional drugs used for pigs'
anesthesia (pentobarbital, ketamine) failed because of the
cardiovascular toxicity of these drugs as soon as they were
administered for prolonged periods ( 24 h). We eventually succeeded
in developing a well-tolerated drug regimen for obtaining anesthesia,
analgesia, and muscle paralysis, combining midazolam, fentanyl, and
pancuronium bromide. Although it was clear from the medical literature
that large-volume fluid resuscitation was necessary to maintain stable
hemodynamic status in short-term (±12 h) experiments in the pig, we
had to make several attempts do define the "ideal" fluid and
electrolytes supply regimen to obtain a stable hemodynamic status and a
satisfactory electrolyte balance. Finally, we had to resolve the
trivial problem of urinary drainage since ureteral catheterization is
difficult and hazardous in male as well as in female piglets.
Percutaneous suprapubic catheterization may also be hazardous, due the
proximity of the pelvic arteries when the bladder is not overdistended.
We resolved this problem by inserting surgically a suprapubic vesical
catheter after surgical exposure of the bladder though a midline
minipelvitomy. Sixteen animals were used over a 1-year period for this
preliminary study (data not shown).
Animal Preparation
Random bred domestic Largewhite-Landrace piglets
(22 ± 2 kg) were used for these studies. Animals were
preanesthetized with IM ketamine (Ketalar, Parke-Davis, 250 mg) and
midazolam (Hypnovel, Roche, 5 mg). General anesthesia, analgesia, and
paralysis were produced by continuous IV infusion of midazolam (0.3
mg/kg/h), fentanyl (Fentanyl, Janssen-Cilag, 5 µg/kg/h), and
pancuronium bromide (Pavulon, Organon-Teknika, 0.32 mg/kg/h).
After orotracheal intubation with an 8-mm endotracheal low-pressure cuff tube (Portex; Hythe; Kent, England), the animals were mechanically ventilated by means of a volume-controlled respirator (Monal D; Taema, Antony, France). Humidification of inspired gases was obtained by means of a heat and moisture exchange filter (HMEF Clear Thermal 1941; Intersurgical Inc; Wokingham, UK) connected to the ventilatory circuit.
With the pig in the supine position, intravascular catheters were inserted into the jugular vein and femoral artery under surgical conditions. A 7.5F Swan-Ganz catheter (Baxter Healthcare Corporation; Santa Ana, CA) was placed into the pulmonary artery via the external jugular vein through a right cervical cutdown and a 3F polyethylene catheter (Plastimed; St. Leu la Forêt, France) was percutaneously inserted into the right or left femoral artery. These catheters were used for monitoring of hemodynamic and oxygenation parameters and for blood sampling. Finally, urinary drainage was obtained by vesical insertion of a 8F suprapubic catheter (Vesicoset; Angiomed; Karlsruhe, Germany) through surgical midline minipelvitomy. After this initial preparation, the animals were turned to the prone position with the snout positioned approximately 30° downwards from the neck axis to allow continuous drainage of oropharyngeal secretions onto an absorbent pad. Prone position was used since in pigs, as in sheep or cows, MV in the supine position results in lung atelectasis with severe ventilation/perfusion mismatch after a few hours.
Experimental Design
Three series of experiments were conducted. In the first series,
we sought to discover whether pigs undergoing prolonged MV would
develop VAP. Accordingly, once prepared as described above, a first
group of 23 animals (control group) was subjected to MV under general
anesthesia for a duration of 4 days. Ventilation parameters were
adjusted to maintain arterial PaCO2 between 35 and 45 mm Hg
and arterial oxygen saturation 90% throughout the study period.
Endotracheal suctioning was performed every 4 h for removal of
secretions in excess. Parenteral feeding, fluids, and electrolytes were
provided through continuous infusion of Ringer's lactate (125 mL/h)
and 10% glucose (40 mL/h). Additional vascular volume was administered
as needed with a fluid gelatin (Plasmion; Rhone Poulenc Rohrer; Antony,
France) to maintain cardiac output at 60 to 80% of the baseline level.
Since the occurrence of VAP in the control group was a nearly constant
feature, in a second series of experiments, we sought to discover
whether antibiotics administered prophylactically could prevent the
occurrence of pneumonia in this model. Accordingly, a second group of
nine animals (ATB group) were studied with the same protocol except
that ceftriaxone (Rocephin; Roche Laboratories), 1 g (IV), was
administered 15 min before intubation and then 1 g twice daily
until day 4. Ceftriaxone was chosen since we knew from the previous
experiments that this antibiotic was effective on most of the organisms
causing pneumonia in ventilated pigletts.
We then conducted a series of experiments in a third group of animals to investigate the consequences of intrabronchial inoculation of bacterial pathogens in the absence of MV (inoculated group). Clinical isolates of Pasteurella multocida and Klebsiella oxytoca were subcultured from positive blood cultures recovered from pigs that developed VAP in the control group. These organisms were considered as pathogens in the pigs since they were recovered both in lung and blood cultures in ventilated animals with pneumonia.
Organisms were grown overnight in 100 mL of brain heart infusion broth (Becton Dickinson Microbiology Systems; Cockeysville, MD) at 37°C. Centrifuged sediments of these actively growing organisms were resuspended in 50 mL of sterile saline solution. Quantification of the inocula was estimated by optical densitometry and precisely measured thereafter by quantitative serial 10-fold dilution cultures. The animals (inoculated group) were intubated under general anesthesia, mechanically ventilated, and turned into the prone position. A fiberoptic bronchoscope was passed through the endotracheal tube and wedged into the right middle lobe bronchus under direct vision. Bacterial inocula of P multocida or K oxytoca, ranging from 106 to 107 cfu/mL in 50 mL saline solution, were then gently injected through the working channel of the bronchoscope. The depth of anesthesia was maintained to prevent coughing and reflux of bacteria into other lung fields for approximately 30 min. The animals were then awaked, extubated, returned to the pigsty, and allowed free access to food and water. Animals in this group were killed 5 h, 3 days, or 2 weeks after intrabronchial inoculation, and lung specimens for bacteriologic and histologic studies were obtained as described below.
Measurements
In both control and ATB groups, the systemic arterial, central
venous, pulmonary arterial, and intermittent pulmonary wedge pressure
were measured with custom pressure transducers (Medex Medical;
Rossendale; England) and an amplifier (Kontron Instruments, type 128A;
Watford, England). Cardiac output was measured by thermodilution with a
cardiac output computer (Edwards model 9520A; Baxter Healthcare
Corporation; Santa Ana, CA). Core body temperature was measured with
the thermistor on the Swan-Ganz catheter. Arterial and mixed venous
blood were drawn twice daily for blood gas analysis. Venous blood was
drawn once daily for hematology (blood cell count) and clinical
chemistry (creatinine, urea, serum albumin, total protein levels and
electrolytes, and lactate concentrations) measurements.
Bacteriologic Samplings
On day 1, in both control and ATB groups, throat swabs were
obtained for cultures and fiberoptic bronchoscopy-guided BAL was
performed in the lingular bronchus (served as control). At completion
of the study (day 4), blood was sampled for culture. An endotracheal
aspirate was obtained by careful endotracheal suctioning using a sputum
suction trap and processed for microscopic examination and bacterial
cultures. Protected brush specimens and BAL were collected from the
right middle lobe bronchus and the apical bronchus of the right lower
lobe.
Collection of Lung Tissue Specimens
While general anesthesia and MV were maintained, heart and lungs
were exposed aseptically through a cervicothoracic midline incision.
Euthanasia was performed by means of massive exsangination though
direct cardiac puncture with a 8F polyethylene catheter. After careful
examination, six superficial tissue specimens (approximately 1
cm3 each) were excised from the pig lungs' most dependent
segments (lingula, middle lobe, anterior segments of the left and right
lower lobes) and from the most "nondependent" segments (apical
segments of the left and right lower lobes). Sampling was always
performed in areas showing gross abnormalities, when present. Each
specimen was cut in two parts in "vis-à-vis" (one for
quantitative cultures and one for histologic study) in order to compare
histologic and bacteriologic findings. Finally, right and left lungs
were weighed.
Bacteriologic Processing of Specimens
Bacteriologic processing of the endotracheal aspirate,
protected brush specimens, BAL, and lung tissue specimens was performed
as previously described13
,14
according to recommended
laboratory methods.15
,16
For lung tissue specimens, counts
of each identified bacterial species were expressed in colony forming
units per gram of tissue. In addition, for each specimen, the total
number of bacteria was calculated by adding the absolute number of
bacteria cultured from the specimen and the result was expressed in
colony forming units per gram of tissue.
Pathologic Study
Specimens were processed according to standard methods.
Evaluations were made by two observers, independently, without
knowledge of bacteriologic data. The lesions were graded as previously
described13
,14
,17
into six categories: no
pneumonia, purulent mucous plugging, bronchiolitis, pneumonia,
confluent pneumonia, and abscessed pneumonia. Classification of each
specimen was based on the worst category observed. The diagnosis of
pneumonia included only the pneumonia, confluent pneumonia, and
abscessed pneumonia categories. For further analysis, each specimen was
assigned a "histologic score" by grading no pneumonia, purulent
mucous plugging, bronchiolitis, pneumonia, confluent pneumonia, and
abscessed pneumonia as 0, 1, 2, 3, 4, and 5, respectively.
Data Analysis
Data are presented as mean ± SD, except otherwise specified.
The Fisher's Exact Test was used to compare categorical variables. For
continuous variables, the Mann-Whitney test for unpaired series was
used. Comparisons of measured parameters within each group were
assessed by two-way repeated measures analysis of variance or by the
Wilkoxon test for paired series depending on the size of the sample.
Correlation was assessed using the Spearman rank test. A p value of
< 0.05 was considered to indicate statistical significance.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histologic and Cytologic Findings
Mechanically Ventilated Animals (Control and ATB
Groups): In the control group, all animals but one (17/18)
developed histologically proven pneumonia. The incidence of pneumonia
(4/9) was significantly lower in ceftriaxone-treated animals.
Macroscopic examination revealed the following: (1) in all animals but one with histologically proven pneumonia, the signs of bronchopneumonia were obvious on gross examination; (2) in the majority of cases, < 30% of the lung was affected by the lesions of pneumonia; and (3) pneumonia was most often bilateral and predominated in the dependent lung segments. These latter two findings were confirmed by microscopic examination that showed that pneumonia was present in 54% of the dependent studied segments and in only 25% of the nondependent studied segments (p < 0.05) and that in only 5 out of 21 animals with pneumonia, the pneumonia was unilateral (right sided, n = 1; left sided, n = 4).
Histologic scores were higher in control animals than in ceftriaxone-treated animals (2.04 ± 1.62 vs 0.83 ± 1.24, p < 0.001). A significant difference was also observed between dependent and nondependent lung segments, at least in control animals (Fig 1 ). Minimal pleural empyema was present in only one animal with pneumonia.
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On day 1 (baseline), BAL cellularity, neutrophil, lymphocyte, and macrophage absolute counts (and percentages) were, respectively, 655 ± 370, 21 ± 22 (3 ± 2%), 85 ± 77 (13 ± 7%), and 549 ± 304 (84 ± 7%) cells per cubic millimeter. Segments with pneumonia displayed a dramatic increase in cellularity and neutrophil counts as compared with baseline values and as compared with values in segments without pneumonia (Fig 3 ). Segments without pneumonia in pigs with pneumonia (documented in another segment) also showed a significant increase in absolute neutrophil counts.
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104 cfu/g
tissue). As a whole, all (17/17) of the control animals with
histologically proven pneumonia yielded at least one biopsy specimen
growing P multocida 104 cfu/g and 10 of them
at least one biopsy specimen growing S suis
104 cfu/g. In ceftriaxone-treated animals, Gram-negative and Gram-positive species accounted for respectively, 37% and 63% of the isolates. Five isolates were resistant to ceftriaxone, the others were susceptible but were cultured at low concentrations (< 104 cfu/g tissue).
Blood cultures were positive in five cases (control animals). In four of them, the isolate was related to one of the organisms causative of the pneumonia. Finally, 79% of the organisms cultured at high concentration in pulmonary biopsy specimens and 57% of the organisms cultured at low concentration were also present in the upper airways as documented by throat swab cultures (data not shown).
Nonventilated Animals (Inoculated Group): BAL cultures failed to recover the inoculated microorganism in 25 of the 29 animals in this group (Table 1 ). Bordetella bronchiseptica, a microorganism commonly colonizing the pigs' respiratory tract, was occasionally cultured in BAL. Cultures of middle lobe biopsy specimens obtained 5 h after intrabronchial bacterial challenge recovered the inoculated microorganism only in the two animals challenged with 107 cfu K oxytoca. Cultures of middle lobe biopsy specimens obtained 3 days after intrabronchial bacterial challenge recovered the inoculated microorganism in five of six animals challenged with 107 or 108 cfu K oxytoca and in none of the animals challenged with the 106 cfu inocula. Cultures of middle lobe biopsy specimens obtained 2 weeks after intrabronchial bacterial challenge with K oxytoca or P multocida recovered the inoculated microorganism in only 3 of 15 animals. As for BAL, B bronchiseptica was occasionally cultured from lung biopsy specimens at 3 days or 2 weeks.
Hemodynamic Status and Electrolyte Balance
In the six animals that did not develop pneumonia, the main
hemodynamic parameters remained essentially stable throughout the study
period (Fig 5
).
In the 21 animals that developed pneumonia, the mean arterial pressure
showed a progressive and significant decrease from day 1 to day 4 and
the pulmonary vascular resistance index significantly increased.
However, despite these significant changes, the concomitant changes of
cardiac index and systemic vascular resistance index were not
consistent with the development of frank shock. Electrolyte balance as
judged by daily measurements of usual clinical chemistry (creatinine,
urea, serum albumin, total protein levels and electrolytes, and lactate
concentrations) remained essentially stable over the study period (data
not shown).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histologic, bacteriologic, and pathogenic aspects of pneumonia in this model resemble early-onset VAP in humans. Regarding standardization of the model, we must admit that the severity of pneumonia by day 4, as assessed by macroscopic examination, lung weight, and alteration in gas exchange was highly variable from one animal to another. However, in most cases, < 30% of the lungs were involved with the pneumonic process. As far as natural history is concerned, since the animals were not killed before day 4, we cannot precisely ascertain when pneumonia started during the time course of MV. In most of the cases, pneumonia became clinically suspected (purulent tracheal aspirates and need for higher FIO2) after 3 days of MV. Once gas exchange began to deteriorate, there was no trend toward spontaneous improvement. We can thus reasonably hypothesize that pneumonia would have extended if MV were continued after day 4. These findings are in accordance with those of Johanson et al9 ,10 ,11 in ventilated baboons.
The reasons why, without any exogenous bacterial challenge, such a high rate of pneumonia was observed in ventilated piglets remain unclear. Johanson et al9 already reported a 100% incidence of pneumonia in injured (oleic acid) and uninjured ventilated baboons. Primary deficiency in piglets' pulmonary antibacterial defense is unlikely since in ventilated animals with pneumonia, the ceftriaxone acted by inhibiting growth pathogens aspirated from the oropharynx at time of intubation or during the first days of ventilation. Another explanation for the high rate of pneumonia in the ventilated animals may be the influence of the route of nutrition. Indeed, as shown in mice by Kudsk and coworkers,21 parenteral feeding as compared with enteral feeding can impair upper respiratory tract immunity. For this author, this mechanism may explain the higher pneumonia rate in critically injured patients fed parenterally. Further studies in our model will be necessary to clarify this issue.
Through bronchoscopic-directed BAL, this model of endogenously acquired pneumonia resembling human VAP provides easy access to the alveolar compartment in affected and nonaffected lung areas at various times during the natural time course of pneumonia. Thereby, one may consider further investigations in this model regarding pulmonary inflammatory disorders related to MV and parenchymal infection.22
With respect to pharmacologic studies, the fact that bacteriology is not controlled limits the utility of the model in studying the effectiveness of antibiotics. In the opposite, immunotherapeutic compounds alone or in conjunction with antibiotics could be studied in this model. Granulocyte colony stimulating factor, for instance, has been shown to increase survival rates in nonneutropenic animal models of infection when administered either before or at the time of bacterial challenge.23 ,24 Its preventive effect, which has been suggested in VAP,25 could be tested in this model of endogenously acquired pneumonia. Likewise, it would be of interest to test whether new ventilation devices such as the endotracheal tube developed by Trawöger and coworkers,26 which has been shown to respect mucociliary clearance in ventilated sheep, would decrease the rate of ventilator-acquired infection in our model.
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Footnotes |
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Correspondence to: Dr. Charles-Hugo Marquette, Département de Pneumologie, Hôpital A. Calmette, CHU de Lille, 59 037 Lille cedex, France; e-mail: cmarquette@nordnet.fr
Abbreviations: ATB = antibioprophylaxis (group); FIO2 = fraction of inspired oxygen; MV = mechanical ventilation; VAP = ventilator-acquired pneumonia
Received for publication November 21, 1997. Accepted for publication May 6, 1998.
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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