INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Despite advances in the treatment of respiratory distress syndrome (RDS) in infants, neonatal chronic lung disease (CLD) remains a major complication among immature infants (1). A variety of insults may contribute to the pathogenesis of CLD including oxygen toxicity, volutrauma, and infection (5- 8). Animal and human studies of pulmonary effluent have demonstrated elevation and activation of neutrophils and macrophages, as well as increased levels of proinflammatory cytokines in association with lung injury (5).

Short-term studies show that both high-frequency oscillatory ventilation (HFOV) and surfactant replacement therapy reduce oxygen- and volutrauma-associated lung injury in animal models of hyaline membrane disease (HMD) (6, 14). Animals treated with HFOV have decreased recruitment and activation of neutrophils in the lungs and airways (17, 18). Other studies describe reduced concentrations of proinflammatory cytokines in the pulmonary effluent of HFOV-treated animals compared with animals supported with conventional mechanical ventilation (16, 19, 20).

Recently, long-term studies have for the first time reported chronic lung injury among immature animals with HMD managed with early surfactant replacement, minimal oxygen exposure, and prolonged mechanical ventilation (21, 22). In chronically ventilated preterm lambs, larger tidal volume (VT) ventilation (15 ml/kg versus 6 ml/kg) appeared to worsen the degree of chronic lung injury. More immature preterm baboons (67% versus 75% gestation) had significant elevations in tracheal leukocytes and persistent upregulation of proinflammatory cytokines when compared with more mature animals. The potential for HFOV to reduce inflammation and chronic lung injury in these models has not been reported.

Introduction of HFOV and surfactant replacement therapy to the care of premature humans with HMD has contributed to improved pulmonary outcome and survival (23). However, controversy exists regarding the benefit of combined HFOV/surfactant therapy versus low VT positive pressure ventilation (LV-PPV)/surfactant therapy in reducing lung inflammation and long-term lung injury (25).

The purpose of this study was to compare the effects of early, prolonged HFOV with LV-PPV after prophylactic surfactant replacement therapy in an immature primate model of neonatal CLD. Specifically, we hypothesized that early, sustained HFOV would improve gas exchange during the phase of acute lung injury, enhance pulmonary mechanics, reduce tracheal concentrations of proinflammatory cytokines, and improve long-term alveolization.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Delivery and Instrumentation

All animal studies were performed at the Southwest Foundation for Biomedical Research in San Antonio, TX. All animal husbandry, animal handling, and procedures were reviewed and approved to conform to American Association for Accreditation of Laboratory Animal Care guidelines. Timed gestations were determined by observing characteristic sex skin changes and confirmed by serial fetal ultrasound examinations. Pregnant baboon dams (Papio papio) were treated with 6 mg of intramuscular betamethasone 48 and 24 h before elective hysterotomy under general anesthesia. Study animals were delivered at 125 ± 3 d (67% of term gestation at 185 d). At birth, infants were weighed, treated with intramuscular ketamine hydrochloride (10 mg/kg) for anesthesia, and intubated with a 2.5-mm endotracheal tube. Before the first breath, tracheal lung liquid was collected, then infants were given a single 4 ml/kg bolus of exogenous surfactant (Survanta; donated by Ross Laboratories, Columbus, OH) through the endotracheal tube. Ventilation was initiated in all infants with a humidified, pressure-limited, time-cycled infant ventilator (InfantStar; donated by Infrasonics, San Diego, CA). The initial rate was set at 40 breaths/ min, peak inspiratory pressure (PIP) adequate to move the chest, positive end-expiratory pressure (PEEP) at 4 cm H₂O, and fraction of inspired oxygen (FI_O2) at 0.40. For the first 5 min of life, PIP was weaned to maintain minimal but not excessive chest wall motion. At 5 min age, based on prior antenatal assignment, the animals were converted to HFOV or maintained on LV-PPV as described subsequently. Animals were subsequently instrumented with an umbilical arterial catheter and percutaneous central venous catheter and nursed in a servo-controlled, infrared-warmed, body plethysmograph (VT1000; VitalTrends Technology, New York, NY) capable of continuous VT measurements and computer-regulated intermittent pulmonary function testing. Ventilator adjustments were made based on chest radiograph, clinical examination, arterial blood gas measurement, and VT measurement as described subsequently. Intermittent sedation was provided as needed with ketamine (5 mg/kg) or diazepam (0.1 mg/kg), or both.

Ventilator Management

The approach applied to conventionally ventilated infants (n = 12) was based on a strategy to maintain VT at 4 to 6 ml/kg as continuously measured by the VitalTrends system and associated with adequate chest motion by clinical exam. This technique is referred to as LV-PPV. Rate was adjusted as required to regulate Pa_CO2 between 45 and 55 mm Hg. HFOV (SensorMedics 3100A; SensorMedics, Anaheim, CA) was initiated in six animals at 5 min of life as follows. The rate was set at 10 Hz, mean airway pressure () was set at 2 cm H₂O above the value measured on the conventional system, and amplitude was adjusted to produce visible chest wall vibration.

Target goals for Pa_O2 were 55 to 70 mm Hg. During LV-PPV oxygenation was primarily manipulated through changes in PEEP and FI_O2. During HFOV oxygenation was primarily manipulated by changes in and FI_O2. In an effort to minimize exposure to high FI_O2, if Pa_O2 was above target goals, FI_O2 was initially weaned until < 0.40, then PEEP/ or FI_O2 were decreased as tolerated. If Pa_O2 was below target guidelines, a chest radiograph was obtained to evaluate lung inflation. Appropriate adjustments in PEEP/ were made in an effort to optimize inflation of the lung. If lung inflation was deemed adequate, FI_O2 was adjusted as indicated.

Target goals for Pa_CO2 were 45 to 55 mg Hg. During LV-PPV, ventilation was maintained by changes in rate and adjustment of pressures to maintain the desired VT measurements. During HFOV, Pa_CO2 was maintained primarily by modification of amplitude.

Pulmonary Function Testing (PFT)

PFT was performed using the VT1000 body plethysmograph (VT1000; VitalTrends Technology). This system is a flow-through whole body plethysmograph patterned after that reported by Hjalmarson and Olsson and similar to that described by Schulze and coworkers (29, 30). The system uses a differential piezoresistive pressure transducer interfaced with a single-screen pneumotachometer to detect airflow in and out of the sealed plethysmograph. Designed specifically for neonatal use, the VT range is from 1.0 to 50.0 ml (resolution 0.1 ml), frequency response is to 5 Hertz, and flow range is ± 175 ml/s. The system interfaces with two dedicated microcomputers capable of pattern recognition, data storage, data analysis, and real-time presentation of flow-volume and pressure-volume curves. VT was monitored continuously for the first 48 h of life. As an esophageal pressure catheter was not used, compliance and resistance measurements, obtained at 6, 12, 18, 24, 36, and 48 h and every 24 h thereafter while intubated, were of the respiratory system as a whole. Ten breaths (meeting five predefined breath selection criteria) were recorded at each time point and averaged for determination of VT, dynamic respiratory system compliance, and expiratory resistance. Variability between measurements was compared by periodic triplicate recording of PFT and was consistently < 5%. For data analysis VT was corrected for birth weight; dynamic compliance was corrected for birth weight and measured VT. Functional residual capacity was not determined. This system is not capable of PFT during HFOV. Infants on HFOV were changed to LV-PPV for 5 min before assessment, to ensure appropriate VT, and immediately switched back to HFOV after pulmonary function measurements were completed.

Nutritional Management

During the first 24 h of life all animals received heparinized normal saline via the umbilical artery catheter and a 5% dextrose/water infusion with supplemental calcium via the central venous catheter. Initial volume intakes for the first day of life were calculated to deliver 250 to 300 ml/kg/d but subsequently decreased over the first 3 to 4 d to approximately 175 to 200 ml/kg/d. These initial fluid requirements were necessary to maintain electrolyte homeostasis, to provide minimal urine output at 1 to 2 ml/kg/h, to maintain acceptable blood pressure, and to minimize metabolic acidosis. Parenteral nutrition was initiated at 24 h of life with amino acids at 1.25 g/kg/d (Trophamine; McGaw, Irvine, CA), electrolytes, vitamins (Pediatric MVI; Astra, Westborough, MA or Cernevit; Clintec, Deerfield, IL), and trace elements (MTE-5; Fujisawa USA, Deerfield, IL). Amino acid intake was increased to 2.5 g/kg/d at 48 h of life and L-cysteine (0.60 mmol/kg/d) was added at 72 h of life. A 20% lipid emulsion (Intralipid; Pharmacia & Upjohn, Clayton, NC) was initiated on day of life 7 and increased to 2.5 g/kg/d. If clinically stable, enteral nutrition was initiated on day of life 7. Donated human breast milk was given by intermittent gastric infusion at an initial volume of 10 ml/kg/d and advanced by 5 to 10 ml/kg/d, as tolerated. Once enteral intakes of 100 ml/kg/d were tolerated, enteral feeding was changed to Primilac (Bio-Serv, Frenchtown, NJ). Nutritional goals included a volume intake of 150 to 200 ml/kg/d, 80 to 120 calories/kg/d, and 3.0 g/kg/d of protein.

Patent Ductus Arteriosus

Animals were monitored by clinical examination and echocardiography for evidence of patent ductus arteriosus (PDA). Management of the PDA initially included attempted volume restriction and use of dopamine as required to maintain blood pressure and urine output. Operative ligation within 48 h of the onset of clinical signs of PDA was performed.

Other Care Plans

Arterial blood gases were measured hourly for the first 24 h, every 2 h between 24 and 48 h, every 4 h from 48 to 96 h, and then every 6 to 12 h as determined by clinical needs. Electrolytes and hematocrit were monitored every 12 to 24 h. Complete chemistries and blood counts were performed weekly. To maintain hematocrit between 30% and 45%, packed red blood cells were administered periodically using fresh heparinized blood obtained from adult baboons.

All animals were initially treated with antibiotics (ampicillin and gentamycin) for the first 7 to 10 d of life. Subsequent antibiotic use, as needed for clinically suspected infection, included the use of vancomycin and an anti-Pseudomonas regimen. Prophylactic fluconazole was initiated in all animals with 6.0 mg/kg/dose at 12, 96, and 168 h age. Doses were then given every other day until day of life 28. Prophylactic intravenous immunoglobulin (Sandoglobulin; Swiss Red Cross, Berne, Switzerland) was given at a dose of 400 mg/kg on day of life 5 and 21.

Significant hypotension was defined as a transduced mean blood pressure less than 25 mm Hg accompanied by either increasing base deficit or decreasing urine output. Hypotension was initially treated with additional volume supplementation (10 to 20 ml/kg at least twice over a 1-h period) and the use of dopamine (5 to 20 µg/kg min). If this approach failed to improve mean blood pressure within 2 to 4 h, then a stress dose of hydrocortisone (1.0 mg/kg) was administered at 6-h intervals until either mean blood pressure increased to greater than 25 mm Hg or a maximum of 4 doses of hydrocortisone were received.

Pathology: Light Microscopy and Immunocytochemistry

At the time of necropsy, the right lower lobe was removed, weighed, and intrabronchially fixed with phosphate-buffered 4% paraformaldehyde and 0.1% glutaraldehyde at 20 cm H₂O constant pressure for 24 h. After fixation, the volume of the right lower lobe was determined by volume displacement. The lobe was cut into three serial, equally spaced horizontal tissue sections. The entire cut surfaces of all three horizontal sections were processed for light microscopic study. These specimens were dehydrated in alcohol, embedded in paraffin, cut at 4 µm, and stained with hematoxylin-eosin. The presence or absence of secondary crests/alveoli, the extent of saccular/alveolar wall fibrosis, if present, and the presence or lack of airway involvement were assessed subjectively in all animals, and the presence of infection was evaluated in all specimens. Mild, focal bronchopneumonia was defined as the presence of scattered neutrophils within occasional bronchioles and the immediately subjacent alveoli/saccules. The presence of confluence or consolidation, and/or necrosis of the lung parenchyma by a fibrinopurulent exudate, was designated as severe or necrotizing bronchopneumonia. Mean linear intercept and total internal surface area (ISA) were determined by standard methods on 10 micrographs of resin-embedded sections, photographed at ×10 magnification. Using elastic stained preparations, color 8 × 10 photographs of peribronchiolar saccular/alveolar ductal septae and walls were photographed randomly, printed, and graded blindly for increased elastic tissue deposition by two of the authors.

Tracheal Aspirates (TA)

TA were collected at 24, 48, 72, and 96 h, and 6 to 8, 9 to 10, 13 to 15, and 16 to 44 d. Animals were disconnected from the ventilator, and 1 ml of sterile normal saline was instilled through the endotracheal tube. The animal was reconnected to the ventilator and allowed to breathe 4 to 5 breaths. The animal was disconnected again, and a sterile 5- or 6-French suction catheter was inserted through the endotracheal tube to 1 to 2 cm past the tip of the tube until it reached the carina. The suction catheter was connected to a sterile suction trap and suction applied until there was no longer a liquid return. The animal was then reconnected to the ventilator.

Each aspirate was separated as follows. Cell count and differential specimens were transferred to a Nunc tube, placed in wet ice, and transferred to the laboratory. Cytokine samples were centrifuged for 10 min at 2,500 rpm, the supernatant removed and allocated in 0.25-ml aliquots, then frozen at 70° C until assay.

Cytokine/Chemokine Assays

Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-) concentrations were determined in TA aliquots by specific and sensitive radioimmunoassays. TNF- was measured using a specific antiserum to human TNF- (Caltag Laboratories, San Francisco, CA) at a final dilution of 1/100,000, radiolabeled human TNF- from New England Nuclear (Boston, MA), and purified human TNF- for the standard (Collaborative Research, Bedford, MA). Assay sensitivity was 16 pg/tube and the intra-assay and interassay coefficients of variation were 5.5% and 6.9% respectively. IL-6 was measured using a specific antiserum to human IL-6 (Sigma Immunochemicals, St. Louis, MO) at a final dilution of 1/100,000, radiolabeled human IL-6 from New England Nuclear, and purified human IL-6 for the standard (Austral Biologicals, San Ramon, CA). Assay sensitivity was 0.6 pg/tube and the intra- assay and interassay coefficients of variation were 6.5% and 11.9%, respectively. IL-1 was measured by radioimmunoassay using a specific rabbit antiserum to human IL-1 purchased from Sigma Chemical (St. Louis, MO). Radioiodinated IL-1 was purchased from New England Nuclear and standard was IL-1 obtained from DPC (Bad Nauheim, Germany). Assay sensitivity was 9 pg/tube, having an intra-assay and interassay coefficient of variation of 8% and 14%, respectively. An enzyme immunoassay (PerSeptive Diagnostics, Ann Arbor, MI) was used to measure IL-8. Assay sensitivity was 100 pg/ml and the intra-assay and interassay coefficients of variation were 10% and 24%. IL-10 was measured by radioimmunoassay using a specific rabbit antiserum to human IL-10 purchased from Chemicon International (Temecula, CA). Antigen for radioiodination and standard was recombinant human IL-10 also purchased from Chemicon International. Assay sensitivity was 4 pg per tube, with an intra-assay and interassay coefficient of variation of 10% and 15%, respectively. Although there is no consensus as to the appropriate reference marker for small-volume bronchoalveolar lavage, protein determinations were performed using the Pierce BCA Protein Assay (Pierce Chemical Co., Rockford, IL).

Data Analyses

Data are presented as mean ± SEM, unless otherwise noted. Intergroup differences for continuous data were compared by unpaired t tests and Mann-Whitney U test. Categorical data were compared by Fisher exact test. All tests were two-tailed. Intergroup comparisons among various time points were made using one-way analysis of variance (ANOVA) for repeated measures. For histopathologic comparison a panel of five standards was derived from study animals that represented the spectrum of changes from most normal to most abnormal (Grade 1 to Grade 5, respectively). A Zeiss photomicroscope (Carl Zeiss, Inc., Thornwood, NY) fitted with a ×1 objective was used to photograph the entire surface area present on the same serial section level (level 3 from the three serial, equally spaced horizontal tissue sections obtained for microscopic study), of each animal in the study. The 35-mm negatives were photographically enlarged, using the same magnification, and printed to 8 × 10 black-and-white photographs. As has been previously described, each photograph was graded independently by three different observers using the derived panel of standards (31, 32). Agreement between observers was measured by Cronbach's  test. The mean of the raters' scores was calculated and compared between groups by unpaired t test and Mann-Whitney U test. A p value  0.05 was required for significance. Statistical results were generated using SPSS, version 9.0 (SPSS, Chicago, IL).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Group Characteristics

All animal studies were performed over the same 3-yr period. Of eight animals randomized to HFOV, six survived between 22 and 41 d before necropsy. The early HFOV deaths were due to cardiovascular failure from an aortic clot (n = 1, Day 5) and postoperative complication after PDA ligation (n = 1, Day 11). Of 20 LV-PPV animals delivered over the same time period, 12 survived 23 to 45 d before sacrifice. Several of these animals were included in our initial report of this model for neonatal CLD (21). All early deaths in the LV-PPV group occurred by 11 d age with the majority (5 of 8) due to cardiovascular compromise secondary to vascular thrombosis. Characteristics of the two study groups are shown in Table 1. All animals had clinical and radiographic evidence for HMD, moderated by the early administration of surfactant. Although gestational ages were similar, mean birth weight was significantly greater in the LV-PPV group. Median age and range for necropsy age were similar in both groups. As previously reported, hypotension was a common problem for these immature animals and a majority received volume and pressor support in the first 48 h of life (21). Clinically significant PDA was identified and treated in a majority of animals in each group between 7 and 10 d age. Cholestasis, when noted, manifested between 14 and 28 d of age. Late-onset nosocomial infection was common and, because of the critical importance of pathologic endpoints in study design, was the most frequent reason for elective necropsy. During the course of this study, several animals achieved low enough ventilator support to attempt extubation. Animals were extubated to nasal continuous positive airway pressure (specially manufactured nasal prongs were provided courtesy of Dr. Alex Stenzler, SensorMedics, Anaheim, CA). A significantly greater number of HFOV animals met criteria and had attempted extubation (67% versus 8%; 2-tailed Fisher exact test, p = 0.022). Due to problems maintaining a patent pharyngeal airway, no animal was able to sustain effective spontaneous ventilation for more than 1 week. All extubated animals were subsequently reintubated and ventilated as previously described.

Pulmonary Function and Mechanics

Throughout the study there were no significant differences in arterial pH or Pa_CO2 values at any time point compared between or within the two groups (Table 2). Pa_O2 was significantly higher in the HFOV group at 72 and 144 h. Although FI_O2 was similar in both groups over the initial 24 h of life, LV-PPV animals had significantly higher FI_O2 requirements between 48 and 96 h of life (Figure 1). As HMD resolved between Days 4 to 6, the FI_O2 requirements of the two groups became similar and remained so until necropsy.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Sequential changes in mean FIO2 by mode of ventilation. *p < 0.05 by Student's t test for HFOV versus LV-PPV.

Recorded mean airway pressures were consistently higher among HFOV animals in the initial 20 h of life, then decreased to levels consistently lower by 24 h of life through hour of life 120. At no time point analyzed were these differences significantly different. In the LV-PPV group, PEEP was consistently maintained between 3 and 5 cm H₂O. Because of inherent difficulties in comparing conventional ventilation to high-frequency ventilation, particularly differences in mean airway pressure measurement and rate, we chose the arterial/alveolar (a/A) ratio to assess the effectiveness of oxygen exchange, rather than oxygenation index or a ventilation efficiency index. HFOV animals had significantly higher a/A ratios during the initial 4 d of life and again at 10 d of age (Figure 2). By 14 d of age the LV-PPV group demonstrated improved a/A ratios which remained similar to that of the HFOV group for the duration of the study.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Sequential changes in a/A ratio by mode of ventilation. *p < 0.05 by Student's t test for HFOV versus LV-PPV.

Comparisons of respiratory system mechanics are shown in Figures 3A-3C. Corrected dynamic respiratory compliance was similar between the two groups at 12 h age (Figure 3A). Subsequently, animals maintained on HFOV had significantly greater compliance at all time points measured out to 28 d, except at 144 h, the point of optimal resolution of the initial HMD. Consistent with the differences measured in respiratory compliance, PIP (measured during the acquisition of pulmonary mechanics on conventional ventilator support) were significantly lower among HFOV infants at all time points compared except 12 h of age (Figure 3B). VT during PFT measurements were maintained within the defined range, and were not significantly different between groups at any time point (data not shown). Animals ventilated with HFOV had consistently lower airway resistance than LV-PPV managed animals at the majority of time points compared (Figure 3C).

View larger version (27K): [in this window] [in a new window]   Figure 3.   Measurement of pulmonary mechanics. (A) Sequential changes by mode of ventilation in respiratory system dynamic compliance (ml/cm H2O/kg/VT), *p < 0.05 by ANOVA for repeated measures LV-PPV 336-672 h versus  96 h. (B) Peak inspiratory pressure via conventional ventilation during PFT, p < 0.05 HFOV versus LV-PPV for all times except 12 h. (C ) Expiratory airway resistance (cm H2O/ml/s), p < 0.05 by Student's t test for HFV versus LV-PPV.

Cytokines and Cell Differential Counts

Median values and ranges for the cytokines assayed are shown in Table 3. On the whole, there was wide variability within each study group at each time point evaluated and across time points. No consistent differences or trends were noted between HFOV and LV-PPV animals for IL-1, IL-10, or TNF-. IL-6 was significantly greater in HFOV animals at 24 h age, but not thereafter. IL-8 concentrations were higher among LV-PPV animals, significantly so between 6 and 10 d of ventilator support. None of the cytokines measured correlated with FI_O2, VT, a/A ratio, or lung mechanic measurements.

Total cell counts were elevated, though not significantly, in the LV-PPV group at the 6-8 and 9-10 d study periods (Figure 4). These study times coincided with the elevated tracheal IL-8 concentrations in the LV-PPV group. Tracheal aspirates of the LV-PPV animals also had fourfold increases in macrophages/monocytes at the 6-8 and 9-10 d study times. These values were significantly greater than the HFOV animals (p  0.05). Increases in eosinophil and lymphocyte counts were measured in tracheal aspirates at several time points in LV-PPV animals, and were significantly elevated at 9 to 10 d when compared with HFOV animals (p  0.05).

View larger version (24K): [in this window] [in a new window]   Figure 4.   Total and differential cell counts of tracheal aspirates collected from HFOV versus LV-PPV animals are compared. LV-PPV animals had significantly greater numbers of macrophages/monocytes and eosinophils (p < 0.05) at 6 to 8 and 9 to 10 d of ventilation. Lymphocytes were also significantly greater (p < 0.05) in the LV-PPV animals at 9 to 10 d age. *Value greater than HFOV group, p < 0.05.

Pathology: Light Microscopy and Immunocytochemistry

At necropsy there were no obvious differences in the appearance of the lungs. Both groups manifested focal atelectasis and small areas of congestion. Light microscopy in both groups revealed few alveoli, and variable wall thickness within the saccular/alveolar compartment. In some lung specimens of the HFOV animals, an inflation difference of overinflated thin-walled air spaces abutting on zones with less but evenly inflated distal air spaces was seen (Figure 5). Mucous plugs were only occasionally identified in an adjoining airway. Focal bronchopneumonia was evident in 60% of the HFOV animals, and in these foci, alveolar walls were more cellular and thickened, and the alveolar spaces contained cellular exudate. Seventy-three percent of the LV-PPV group had evidence of pulmonary infection, but half of this group had more severe pneumonia involvement than animals in the HFOV group. Overall, the HFOV animals appeared to have better-inflated and thin-walled distal lung parenchyma. This was substantiated using the panel of standards analysis. Three of the panels (Grades 1, 3, and 5) are depicted in Figure 6 to show the range of microscopic changes found. The most normal grade (Grade 1, left panel ) reflects well-expanded and thin-walled saccules/alveoli, whereas the worst grade (Grade 5, right panel ) reflects findings of more thickened saccular/alveolar walls, focal atelectasis, and focal pneumonia. Using the panel of standards analysis, Table 4 reveals that HFOV animals had significantly better inflation patterns at necropsy than LV-PPV animals, with a high degree of interobserver agreement (Cronbach's  > 0.95). HFOV-ventilated animals also had lower mean linear intercepts and higher internal surface area measurements (Figure 7), suggesting a trend toward improved alveolization; however, these differences did not achieve statistical significance. Despite the overall improved histologic appearance of the lungs of HFOV-managed infants, given the median age at necropsy of 29 to 30 d, the degree of expected alveolization is clearly reduced in both animal groups (Figure 8). Subjectively, there were no differences in the airway epithelial appearance and wall thickness between the two groups. Sites of increased elastic fiber staining were evident only focally in both study groups.

View larger version (110K): [in this window] [in a new window]   Figure 5.   HFOV specimen, 41-d survivor. Note the overinflated air spaces (asterisk) that abut on airways and saccular/alveolar spaces with a more normal and even inflation pattern. The thickness of saccular/ alveolar walls is similar in both areas. Hematoxylin-eosin; original magnification: ×40.

View larger version (62K): [in this window] [in a new window]   Figure 6.   Grades 1, 3, 5; panel of standards depicting the gradation of the best to worst morphology in the lungs of both groups. Grade 1 (left panel ) reflects evenly inflated and thin-walled distal parenchyma. Grade 3 (middle panel ) shows thicker saccular walls and more uneven inflation to which sites of bronchopneumonia and/or hemorrhage are added to comprise Grade 5 findings (right panel ). Hematoxylin-eosin; original magnification: ×10.

View larger version (10K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 7.   Mean linear intercept and internal surface area determinations tend to support that HFOV animals had more alveolization and greater surface area when compared with LV-PPV group, but the values did not reach significance.

View larger version (53K): [in this window] [in a new window]   Figure 8.   Alveolization: normal term versus LV-PPV versus HFOV. In the term animal (a), the saccular/alveolar walls are very thin, and several alveolar structures are apparent in the alveolar duct area (arrows). A comparable alveolar duct site in a LV-PPV specimen is shown in (b). The saccular walls contain more cells and are thickened. Abortive secondary crests, seen as protuberances or elevations along the saccular wall, are evident (arrows), but formed alveoli are absent. There is a similar lack of formed alveoli in (c), but the saccular walls of the HFOV animal are thinner and less cellular than the LV-PPV specimen, though still more cellular than the term specimen in (a). Hematoxylin-eosin; original magnification: ×100.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this extremely immature animal model for neonatal CLD we demonstrated a beneficial effect of early, prolonged HFOV compared with LV-PPV on acute lung function. HFOV-treated animals had early and sustained improvement in pulmonary mechanics, and significantly more normal lung histology than LV-PPV-treated animals. The long-term benefits of HFOV on pulmonary mechanics and histology are notable in light of the fact that more typical clinical assessments of lung function such as FI_O2, a/A ratio, and chest radiographs did not suggest measurable differences by mode of ventilation beyond 2 wk of age. This study is the first to provide detailed microscopic analyses and sequential pulmonary function assessments related to the effect of prolonged HFOV in such immature infants. This critical information is lacking from the numerous studies involving premature human infants with CLD.

There is considerable controversy regarding possible beneficial effects of HFOV in reducing lung injury among premature infants with respiratory disease. Acutely, in both animal and human studies, HFOV has been shown to significantly improve gas exchange (14, 26, 31, 33, 34). Short-term studies in surfactant-deficient animals have consistently demonstrated decreased lung injury during HFOV compared with conventional positive pressure ventilation. This has been measured in a variety of ways, including reduced neutrophil migration, lower concentrations of proinflammatory cytokines in lung lavage, reduced protein leak, improved pulmonary compliance, and marked improvement in lung histopathology (14, 20). None of these studies, however, was performed in the presence of surfactant replacement. Concomitant use of surfactant replacement therapy with HFOV has yielded conflicting findings in other premature animal studies. Walther and coworkers, in premature lambs, and Jackson and coworkers, studying premature monkeys, found improved oxygenation and reduced lung injury with combined surfactant-HFOV treatment compared with surfactant plus conventional ventilation (35, 36). However, in both studies animals were mildly hyperventilated (Pa_CO2 < 40 mm Hg) and exposed to 100% oxygen. Either of these factors, alone or combined, could have contributed to the increased lung injury found with conventional ventilation.

Contrary findings were reported by Heldt and colleagues and Ikegami and colleagues (37, 38). Studying premature rabbit pups given delayed surfactant replacement therapy, Heldt and colleagues found similar lung compliance and morphometry between HFOV and conventionally ventilated animals killed at 1 h of age. More recently, Ikegami and colleagues reported on the effects of ventilation style in premature lambs treated at birth with sheep surfactant. They compared the effects of high VT (15 ml/kg) conventional ventilation, low VT (8 ml/kg) conventional ventilation, and HFOV on lung function and surfactant metabolism. Importantly, in an attempt to minimize lung injury, they adjusted ventilator rates to maintain Pa_CO2 levels between 45 and 50 mm Hg. After 24 h of ventilation they found no important differences between modes of ventilator support, suggesting that early surfactant replacement therapy combined with avoidance of hyperventilation may lead to minimal lung injury independent of ventilator style. These findings are intriguing but are limited by the relatively short time period of ventilator support and the more mature stage of lung development found in the premature lamb at the gestational age studied (approximately 80% term). Our study is the first to report on what effect, if any, the style of ventilation may have on lung function, pulmonary mechanics, tracheal aspirate cytokines, and lung morphometry in surfactant-treated, extremely immature animals managed with extended ventilator support typical of the human condition today.

The TA cell count data in this study suggest that, over time, a low VT strategy may induce significant lung injury. The initial site of injury may occur at several levels within the lung, including the airway (39). Significantly increased numbers of alveolar macrophages/monocytes, eosinophils, and lymphocytes after 9 to 10 d of low VT ventilation support the presence of an inflammatory response in the LV-PPV group. Both groups were treated with prophylactic exogenous surfactant replacement, had comparable oxygen exposure, and had similar nutritional and fluid support. As previously reported, tracheal colonization was a common finding in all animals evaluated (7). Infection (perinatal and/or postnatal) appears to play a very important role in the development of chronic lung injury in the premature human (9, 13, 40). Each group received comparable prophylactic antimicrobial therapy, and there were no clinical or radiographic indications of infection in either study group during the initial 14 d of support. These similarities suggest that the cellular and cytokine differences at the 6-8 and 9-10 d time points are secondary to the imposed differences in ventilator strategy. This overlap of cellular and molecular components of the inflammatory process during the early stages of RDS also suggests that this may be an important window for consideration of anti-inflammatory therapies.

The lack of tracheal cytokine correlation with physiologic and lung mechanic measurements is of interest. It is possible that acute elevations in these cytokines may not be reflected immediately in how the lung functions. A certain time delay between cause and effect should not be unexpected. TA may not be the ideal process to assess bronchopulmonary cytokines. Bronchoalveolar lavage may be a better method but early attempts at bronchoalveolar lavage in these animals resulted in severe cardiopulmonary decompensation. Given the relatively small number of animals studied, the wide range of variability in cytokine values may also hinder correlation analysis.

The participation of the alveolar macrophage in inducing lung injury is well documented (8, 13, 45). The significantly increased numbers of eosinophils and lymphocytes in the TA of the LV-PPV group were perplexing. A blinded reexamination of the airways revealed only rare eosinophils (one or two) in the submucosa in two of the 11 LV-PPV animals. It was these same two animals that accounted for the elevated eosinophil counts in the TA. A difference in number of airway lymphocytes was not apparent by light microscopic examination. The significance of the finding of increased TA eosinophils, if any, is unknown. Peripheral eosinophilia has been reported in human infants with persistent pulmonary disease during the initial 4 wk of life (46). Significantly elevated eosinophilic cationic protein levels have been described in the TA of infants with bronchopulmonary dysplasia (BPD), and correlated with neutrophil elastase levels in TA (46).

Controversy has arisen related to the possible long-term pulmonary benefits of HFOV in the premature human (25, 34, 47). These studies have suffered from a variety of limitations, including inadequate numbers of high-risk infants, delayed use of HFOV, lack of surfactant replacement therapy, inappropriate HFOV or conventional ventilation strategy, other confounding therapies (postnatal steroids), and the inability to adequately blind investigators. Recently, Thome and coworkers reported a randomized, multicenter trial comparing HFOV and high-rate ("low tidal volume") positive pressure ventilation in a large group of high-risk infants designed to correct for many of the deficiencies of previous trials (28). Specifically, infants were quite immature with a mean gestation of 27 wk, a large majority received prenatal steroids, and surfactant replacement therapy was frequently used. Additionally, HFOV was initiated with a high lung volume strategy early in the course of respiratory disease (mean time of 28 min from intubation) whereas PPV was applied with a low VT high-rate strategy. With a study population of 284 infants, they were unable to distinguish any significant differences between groups in survival (90%), CLD (25%), or survival without CLD (69%). Both groups of infants had low median FI_O2 (0.25) by 6 h after randomization, and postnatal dexamethasone treatment was used equally in both groups (40%). This study suggested that either mode of ventilation, when managed "optimally" and in association with other widely used current interventions (prenatal and postnatal steroids, surfactant replacement, nutritional support, etc.) resulted in similar clinical outcomes at discharge. A key remaining question from their study relates to any possible difference in future long-term pulmonary development and function (50). This may be particularly important given the histologic findings we report. Subtle improvements in lung architecture during the development of the premature lung may contribute to clinically important differences later in life.

It is of interest that Thome and coworkers have also reported on cytokine changes in TA of a subpopulation from the study described previously (53). They measured concentrations of albumin, IL-8, and leukotriene B₄ serially over the first 10 d of support. They were unable to identify any significant differences or trends between HFOV and LV-PPV infants in cytokine production or protein leak. Contrary to their findings, we did identify a significant increase in TA concentrations of IL-8 in LV-PPV-ventilated immature baboons. The increase in tracheal IL-8 concentrations was first noted late in the first week of LV-PPV ventilation, peaked around Day 10, and then remained consistently greater thereafter. Importantly, as previously noted, we found that the primary cell lines contributing to IL-8 concentrations, monocytes and macrophages, were also significantly increased in the TA of LV-PPV animals during this time.

There were important methodological differences between the study by Thome and coworkers and ours in this area. They corrected cytokine concentrations to the concentration of secretory IgA also present in the TA. We normalized our data for albumin. They excluded all infants with colonization of the airway, which effectively eliminated over 80% of their study patients by Day 10. We have previously demonstrated that nearly all intubated animals are colonized within the first week of life (7). It is possible that microbial colonization contributed to some of the cytokine values we obtained (44). We did not prospectively monitor tracheal colonization in relationship to cytokine levels. It is possible that the wide range of cytokine concentrations found in our study, as well as that of Thome and coworkers, may be a reflection of the proinflammatory role of bacteria or other microorganisms that colonize or infect the pulmonary system in these immature infants. Importantly, of the animals included in this study, all received broad-spectrum antibiotics for the first 10 d of life and none had clinical or radiographic evidence of pneumonia or infection during the initial 10 to 14 d of life. Despite this, all of the animals had tracheal colonization by 14 d of age, with the predominant species being gram-negative rods. A number of studies have suggested that infectious processes, prenatal and/or postnatal, may contribute significantly to the development of neonatal CLD (41- 43). We cannot rule out that late-onset infection could have contributed to the differences we found in lung inflation on histologic review. Perhaps most importantly, Thome and coworkers studied premature human infants in comparison to our premature baboons. Gestational ages were equivalent (68 to 70%), and lung development at this time in gestation is similar. It is possible that events leading to human premature birth, specifically the high incidence of chorioamniotic infection with fetal inflammatory response, could alter the subsequent pulmonary cytokine response of the human premature as compared with that of premature baboons delivered by elective hysterotomy (54).

The significant differences in pulmonary mechanics present throughout the course of this study suggest that the histologic differences between HFOV and LV-PPV animals could be related to differences in how these two techniques maintain lung inflation. Improved compliance would be expected if more uniform lung inflation could be sustained during HFOV. Likewise, static airway inflation during HFOV versus intermittent airway expansion during LV-PPV may minimize biochemical and pathologic factors associated with increased airway resistance. Short-term animal studies in surfactant-deficient and surfactant-treated models have demonstrated improved pulmonary compliance with the use of early HFOV applied with a strategy to promote lung recruitment and maintain lung volume (16, 36, 58). This is the first study to suggest that the improved compliance and lower airway resistance can be sustained over prolonged periods of HFOV.

In summary, we found that early and prolonged use of HFOV in an antenatal steroid-treated, surfactant-treated immature nonhuman primate model of neonatal chronic lung disease was associated with significant improvements in the histopathologic appearance of the lung. Reduced concentrations of inflammatory cells and IL-8 in TA during the post-HMD recovery phase, and sustained improvements in airway resistance and dynamic respiratory compliance accompanied this improvement. Given the multifactorial nature of chronic lung disease and the relatively small number of animals studied, these findings must be considered preliminary and in need of confirmation through additional studies.