Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α₁-antiprotease complex concentration or P_aO₂/F_iO₂ ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

High tidal volume ventilation induces NOS2 and impairs cAMP- dependent air space fluid clearance

Tidal volume reduction during mechanical ventilation reduces mortality in patients with acute lung injury and the acute respiratory distress syndrome. To determine the mechanisms underlying the protective effect of low tidal volume ventilation, we studied the time course and reversibility of ventilator-induced changes in permeability and distal air space edema fluid clearance in a rat model of ventilator-induced lung injury. Anesthetized rats were ventilated with a high tidal volume (30 ml/kg) or with a high tidal volume followed by ventilation with a low tidal volume of 6 ml/kg. Endothelial and epithelial protein permeability were significantly increased after high tidal volume ventilation but returned to baseline levels when tidal volume was reduced. The basal distal air space fluid clearance (AFC) rate decreased by 43% (P < 0.05) after 1 h of high tidal volume but returned to the preventilation rate 2 h after tidal volume was reduced. Not all of the effects of high tidal volume ventilation were reversible. The cAMP-dependent AFC rate after 1 h of 30 ml/kg ventilation was significantly reduced and was not restored when tidal volume was reduced. High tidal volume ventilation also increased lung inducible nitric oxide synthase (NOS2) expression and air space total nitrite at 3 h. Inhibition of NOS2 activity preserved cAMP-dependent AFC. Because air space edema fluid inactivates surfactant and reduces ventilated lung volume, the reduction of cAMP-dependent AFC by reactive nitrogen species may be an important mechanism of clinical ventilator-associated lung injury.     

呼吸机相关肺损伤模型建构的最佳潮气量

目的:应用大潮气量机械通气探讨制作兔的呼吸机相关肺损伤模型的最佳潮气量。方法:根据黄金分割法原理,采用三种不同潮气量68mL/kg、60mL/kg和45mL/kg各持续通气1分钟造成兔的急性肺损伤,取0h,12h,24h,48h四个时间点进行观察,观察兔存活情况,计算各时间点肺湿/干重比观察肺水肿严重程度及变化,组织病理学切片观察各时间点肺组织形态学改变。结果:1、潮气量68mL/kg组、60mL/kg组和45mL/kg组兔48h存活率分别为58.33%(7/12)、91.67%(11/12)和100%(12/12);与正常对照组相比,三组肺湿/干重比在0小时无明显变化,12小时明显增高,在24小时时达峰值,48小时后降低。3、镜下观察机械通气后不同时间肺组织均有不同程度形态学改变,68mL/kg组肺组织形态学改变非常明显,60mL/kg组肺组织形态学改变明显,45mL/kg组肺组织形态学改变不明显。结论:大潮气量通气成功建立兔的呼吸机相关肺损伤实验动物模型,潮气量指标为60mL/kg。     

Very low tidal volume ventilation with associated hypercapnia--effects on lung injury in a model for acute respiratory distress syndrome

Background

Ventilation using low tidal volumes with permission of hypercapnia is recommended to protect the lung in acute respiratory distress syndrome. However, the most lung protective tidal volume in association with hypercapnia is unknown. The aim of this study was to assess the effects of different tidal volumes with associated hypercapnia on lung injury and gas exchange in a model for acute respiratory distress syndrome.

Methodology/Principal Findings

In this randomized controlled experiment sixty-four surfactant-depleted rabbits were exposed to 6 hours of mechanical ventilation with the following targets: Group 1: tidal volume = 8–10 ml/kg/PaCO₂ = 40 mm Hg; Group 2: tidal volume = 4–5 ml/kg/PaCO₂ = 80 mm Hg; Group 3: tidal volume = 3–4 ml/kg/PaCO₂ = 120 mm Hg; Group 4: tidal volume = 2–3 ml/kg/PaCO₂ = 160 mm Hg. Decreased wet-dry weight ratios of the lungs, lower histological lung injury scores and higher PaO₂ were found in all low tidal volume/hypercapnia groups (group 2, 3, 4) as compared to the group with conventional tidal volume/normocapnia (group 1). The reduction of the tidal volume below 4–5 ml/kg did not enhance lung protection. However, oxygenation and lung protection were maintained at extremely low tidal volumes in association with very severe hypercapnia and no adverse hemodynamic effects were observed with this strategy.

Conclusion

Ventilation with low tidal volumes and associated hypercapnia was lung protective. A tidal volume below 4–5 ml/kg/PaCO₂ 80 mm Hg with concomitant more severe hypercapnic acidosis did not increase lung protection in this surfactant deficiency model. However, even at extremely low tidal volumes in association with severe hypercapnia lung protection and oxygenation were maintained.     

Urinary desmosine excretion is inversely correlated with the extent of emphysema in patients with chronic obstructive pulmonary disease

An enhanced proteolysis of lung interstitium is key event in the pathogenesis of emphysema, a major constituent of chronic obstructive pulmonary disease. To assess whether urinary desmosine and/or hydroxyproline may be used as a marker of lung destruction we studied urinary excretions of these products in 20 patients with chronic obstructive pulmonary disease and in 19 appropriate controls in 24h urine collection samples. For desmosine measurements, we developed a new indirect competitive enzyme-linked immunosorbent assay. The extent of emphysema was measured in high resolution computed tomography (CT) scans, by considering lung area with CT numbers <-950 Hounsfield units (HU).Urinary desmosine excretion was significantly higher in patients with chronic obstructive pulmonary disease than in controls (294+/-121 microg versus 183+/-93 microg, P=0.003), and was unrelated with both age and smoking habits. In patients with no evidence or only mild emphysema, desmosine excretion values were significantly higher (P=0.006) than those of patients with moderate to severe emphysema. In patients with chronic obstructive pulmonary disease, urinary hydroxyproline excretion was positively correlated with urinary desmosine excretion but on the average, it was not different from that of controls.These data indicate that urinary desmosine is a sensitive biological marker of lung elastin catabolism. The relatively low levels of urinary desmosine observed in patients with severe emphysema may be accounted for a decrease in elastin catabolism due to reduced lung elastin mass. Urinary desmosine may be used to identify subjects at risk of developing emphysema and to assess the efficacy of therapeutic interventions.     

大潮气量致急性肺损伤犬呼吸机相关性肺损伤模型的构建

目的建立大潮气量致急性肺损伤（ALI）犬呼吸机相关性肺损伤（VILI）模型。方法健康雄性杂种犬12只用油酸静脉注射法制备犬ALI模型,造模成功后进行支持通气15min过渡,然后随机分为VILI组及对照组行机械通气6 h,每组6只。VILI组潮气量（Vt）=20 mL/kg,对照组Vt=6 mL/kg,两组呼气末正压（PEEP）均为10 cmH2O。动态观察各组血气交换指标变化。通气6 h后取支气管肺泡灌洗液（BALF）作白蛋白浓度检查,取肺组织作病理切片肺损伤评分。结果各组在油酸静脉注射后（2.50±0.80）h达到ALI标准。VILI组在犬机械通气6 h后PaO2、SaO2及氧合指数（OI）较对照组略下降（P〈0.05）,而PaCO2波动不大,且心率、血压波动也较对照组小（P〈0.05）。VILI组BALF中蛋白浓度和肺组织损伤评分均较对照组显著升高（分别P〈0.05,P〈0.01）。结论本实验成功建立了大潮气量致ALI犬VILI模型。     

Mechanical ventilation of isolated rat lungs changes the structure and biophysical properties of surfactant.

Mechanical ventilation is an essential but potentially harmful therapeutic intervention for patients with acute lung injury. The objective of this study was to investigate the effects of mechanical ventilation on large-aggregate surfactant (LA) structure and function. Isolated rat lungs were randomized to either a nonventilated control group, a relatively noninjuriously ventilated group [1 h, 10 ml/kg tidal volume, 3 cmH(2)O positive end-expiratory pressure (PEEP)], or an injuriously ventilated group (1 h, 20 ml/kg tidal volume, 0 cmH(2)O PEEP). Injurious ventilation resulted in significantly decreased lung compliance compared with the other two groups. LA structure, as determined by electron microscopy, revealed that LA from the injurious group had significantly lower amounts of organized lipid-protein structures compared with LA obtained from the other groups. Analysis of the biophysical properties by using a captive bubble surfactometer demonstrated that adsorption and surface tension reduction were significantly impaired with LA from the injuriously ventilated lungs. We conclude that the injurious mechanical ventilation impairs LA function and that this impairment is associated with significant morphological alterations.     

Influence of tidal volume on histamine-induced bronchoconstriction in guinea pigs

The effects of tidal volume amplitude on bronchopulmonary reactivity were investigated in three groups of 14 anesthetized paralyzed mechanically ventilated guinea pigs. Animals of group 1 served as control; in animals of group 2, both the sympathetic and parasympathetic nervous systems were blocked; in animals of group 3, only the parasympathetic system was blocked. In each group, the animals were randomly divided into two subgroups characterized by their ventilatory pattern: rate of 60/min with a 6-ml/kg tidal volume or rate of 40/min with a 9-ml/kg tidal volume. Bronchopulmonary reactivity to infused histamine was assessed by the respiratory compliance and conductance values measured during bronchoconstriction and expressed as a percentage of the corresponding basal values. In group 1 the animals ventilated with a 9-ml/kg tidal volume were found significantly less reactive than those ventilated with a 6-ml/kg tidal volume. This difference was abolished in groups 2 and 3. These results demonstrate that the effects of increased tidal volume on bronchopulmonary reactivity are vagally mediated and suggest that the decrease observed in histamine-induced bronchoconstriction is mainly due to reflex effects evoked by stretch receptor stimulation.     

Interactive effects of mechanical ventilation and kidney health on lung function in an in vivo mouse model

We hypothesized that the influence of acute kidney injury (AKI) on the sensitivity of the lung to an injurious process varies with the severity of the injurious process. Thus, we thought that AKI would exacerbate lung injury from low degrees of lung trauma but attenuate lung injury from higher degrees of lung trauma. C57BL/6 mice underwent AKI (30-min kidney ischemia) or sham surgery, followed at 24 h by 4 h of spontaneous breathing (SB), mechanical ventilation with low tidal volume (7 ml/kg, LTV), or mechanical ventilation with high tidal volume (30 ml/kg, HTV). Compared with LTV, median bronchoalveolar lavage (BAL) protein leak was significantly lower with SB and greater with HTV in both sham and AKI mice. Compared with LTV, median Evans blue dye-labeled albumin extravasation in lungs (L-EBD) was also significantly lower with SB and greater with HTV. L-EBD showed a significant interaction between ventilatory mode and kidney health, such that AKI attenuated the L-EBD rise seen in HTV vs. LTV sham mice. An interaction between ventilatory mode and kidney health could also be seen in BAL neutrophil number (PMN). Thus, AKI attenuated the BAL PMN rise seen in HTV vs. LTV sham mice. These data support the presence of a complex interaction between mechanical ventilation and AKI in which the sensitivity of the lung to trauma varies with the magnitude of the trauma and may involve a modification of pulmonary neutrophil activity by AKI.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.     

High tidal volume ventilation induces lung injury after hepatic ischemia-reperfusion

Ischemia-reperfusion not only damages the affected organ but also leads to remote organ injuries. Hepatic inflow interruption usually occurs during hepatic surgery. To investigate the influence of liver ischemia-reperfusion on lung injury and to determine the contribution of tidal volume settings on liver ischemia-reperfusion-induced lung injury, we studied anesthetized and mechanically ventilated rats in which the hepatic inflow was transiently interrupted twice for 15 min. Two tidal volumes, 6 ml/kg as a low tidal volume (IR-LT) and 24 ml/kg as a high tidal volume (IR-HT), were assessed after liver ischemia-reperfusion, as well as after a sham operation, 6 ml/kg (NC-LT) and 24 ml/kg (NC-HT). Both the IR-HT and IR-LT groups had a gradual decline in the systemic blood pressure and a significant increase in plasma TNF-alpha concentrations. Of the four groups, only the IR-HT group developed lung injury, as assessed by an increase in the lung wet-to-dry weight ratio, the presence of significant histopathological changes, such as perivascular edema and intravascular leukocyte aggregation, and an increase in the bronchoalveolar lavage fluid TNF-alpha concentration. Furthermore, only in the IR-HT group was airway pressure increased significantly during the 6-h reperfusion period. These findings suggest that liver ischemia-reperfusion caused systemic inflammation and that lung injury is triggered when high tidal volume ventilation follows liver ischemia-reperfusion.     

Effects of volume history and vagotomy on pulmonary and chest wall mechanics in cats

Using the technique of rapid airway occlusion during constant-flow inflation, we studied the effects of inflation volume, different baseline tidal volumes (10, 20, and 30 ml/kg), and vagotomy on the resistive and elastic properties of the lungs and chest wall in six anesthetized tracheotomized paralyzed mechanically ventilated cats. Before vagotomy, airway resistance decreased significantly with increasing inflation volume at all baseline tidal volumes. At any given inflation volume, airway resistance decreased with increasing baseline tidal volume. After vagotomy, airway resistance decreased markedly and was no longer affected by baseline tidal volume. Prevagotomy, pulmonary tissue resistance increased progressively with increasing lung volume and was not affected by baseline tidal volume. Pulmonary tissue resistance decreased postvagotomy. Chest wall tissue resistance increased during lung inflation but was not affected by either baseline tidal volume or vagotomy. The static volume-pressure relationships of the lungs and chest wall were not affected by either baseline tidal volume or vagotomy. The data were interpreted in terms of a linear viscoelastic model of the respiratory system (J. Appl. Physiol. 67: 2276-2285, 1989).     

Benzamil, a blocker of epithelial Na(+) channel-induced upregulation of artery oxygen pressure level in acute lung injury rabbit ventilated with high frequency oscillation

The epithelial Na(+) transport via an epithelial Na(+) channel (ENaC) expressed in the lung epithelium would play a key role in recovery from lung edema at acute lung injury by removing the fluid in lung luminal space. The lung edema causes dysfunction of gas exchange, decreasing oxygen pressure level of artery [P(aO(2))]. To study if ENaC plays a key role in recovering P(aO(2)) from a decreased level to a normal one in acute lung injury, we applied benzamil (20microM, a specific blocker of ENaC) to the lung luminal space in acute lung injury treated with high frequency oscillation ventilation (HFOV) that is a lung-protective ventilation with a lower tidal volume and a smaller pressure swing than conventional mechanical ventilation (CMV). Benzamil facilitated the recovery of P(aO(2)) in acutely injured lung with HFOV but not CMV. The observation suggests that in acutely injured lung treated with HFOV an ENaC blocker, benzamil, can be applied as a therapeutic drug for acute lung injury combing with HFOV.     

High tidal volume upregulates intrapulmonary cytokines in an in vivo mouse model of ventilator-induced lung injury.

Mechanical ventilation has been demonstrated to exacerbate lung injury, and a sufficiently high tidal volume can induce injury in otherwise healthy lungs. However, it remains controversial whether injurious ventilation per se, without preceding lung injury, can initiate cytokine-mediated pulmonary inflammation. To address this, we developed an in vivo mouse model of acute lung injury produced by high tidal volume (Vt) ventilation. Anesthetized C57BL6 mice were ventilated at high Vt (34.5 +/- 2.9 ml/kg, mean +/- SD) for a duration of 156 +/- 17 min until mean blood pressure fell below 45 mmHg (series 1); high Vt for 120 min (series 2); or low Vt (8.8 +/- 0.5 ml/kg) for 120 or 180 min (series 3). High Vt produced progressive lung injury with a decrease in respiratory system compliance, increase in protein concentration in lung lavage fluid, and lung pathology showing hyaline membrane formation. High-Vt ventilation was associated with increased TNF-alpha in lung lavage fluid at the early stage of injury (series 2) but not the later stage (series 1). In contrast, lavage fluid macrophage inflammatory protein-2 (MIP-2) was increased in all high-Vt animals. Lavage fluid from high-Vt animals contained bioactive TNF-alpha by WEHI bioassay. Low-Vt ventilation induced minimal changes in physiology and pathology with negligible TNF-alpha and MIP-2 proteins and TNF-alpha bioactivity. These results demonstrate that high-Vt ventilation in the absence of underlying injury induces intrapulmonary TNF-alpha and MIP-2 expression in mice. The apparently transient nature of TNF-alpha upregulation may help explain previous controversy regarding the involvement of cytokines in ventilator-induced lung injury.     

The Effects of Lung Protective Ventilation or Hypercapnic Acidosis on Gas Exchange and Lung Injury in Surfactant Deficient Rabbits

Background

Permissive hypercapnia has been shown to reduce lung injury in subjects with surfactant deficiency. Experimental studies suggest that hypercapnic acidosis by itself rather than decreased tidal volume may be a key protective factor.

Objectives

To study the differential effects of a lung protective ventilatory strategy or hypercapnic acidosis on gas exchange, hemodynamics and lung injury in an animal model of surfactant deficiency.

Methods

30 anesthetized, surfactant-depleted rabbits were mechanically ventilated (FiO₂ = 0.8, PEEP = 7cmH₂O) and randomized into three groups: Normoventilation-Normocapnia (NN)-group: tidal volume (Vt) = 7.5 ml/kg, target PaCO₂ = 40 mmHg; Normoventilation-Hypercapnia (NH)-group: Vt = 7.5 ml/kg, target PaCO₂ = 80 mmHg by increasing FiCO₂; and a Hypoventilation-Hypercapnia (HH)-group: Vt = 4.5 ml/kg, target PaCO₂ = 80 mmHg. Plasma lactate and interleukin (IL)-8 were measured every 2 h. Animals were sacrificed after 6 h to perform bronchoalveolar lavage (BAL), to measure lung wet-to-dry weight, lung tissue IL-8, and to obtain lung histology.

Results

PaO₂ was significantly higher in the HH-group compared to the NN-group (p<0.05), with values of the NH-group between the HH- and NN-groups. Other markers of lung injury (wet-dry-weight, BAL-Protein, histology-score, plasma-IL-8 and lung tissue IL-8) resulted in significantly lower values for the HH-group compared to the NN-group and trends for the NH-group towards lower values compared to the NN-group. Lactate was significantly lower in both hypercapnia groups compared to the NN-group.

Conclusion

Whereas hypercapnic acidosis may have some beneficial effects, a significant effect on lung injury and systemic inflammatory response is dependent upon a lower tidal volume rather than resultant arterial CO₂ tensions and pH alone.     

肺保护性通气策略对老年患者腹腔镜结直肠癌手术的氧合功能及血浆炎症介质水平的影响

目的:探讨肺保护通气策略对老年患者行腹腔镜结直肠癌根治术肺部氧合功能及血清炎症介质水平的影响。方法:选择50例行择期腹腔镜结直肠癌根治术老年患者,ASA分级(美国麻醉医师协会体格情况评估分级)Ⅰ~Ⅱ级、年龄≥60岁,采用随机数字表法将其分为两组:VCV组和PCV组。在围术期行全麻机械通气中,VCV组采用容量通气模式,潮气量为8 m L/kg,PCV组采用肺保护通气,潮气量为6 m L/kg及5 cm H2O呼气末正压通气(positive end expiration pressure,PEEP),同时气腹后每30 min给予一次手法肺复张。记录患者气腹前5 min(T0)、气腹后5 min(T1)、气腹后30 min(T2)、气腹后60 min(T3)、气腹后120 min(T4)、气腹停止10 min后(T5)的呼吸力学指标、血流动力学指标于T0、T4、离开苏醒室时抽取血气,计算氧合指数(OI)值,于术前一天、T4、术后一天抽取静脉血,检测血浆CRP、IL-6的值。结果:与VCV组比较,PCV组在T4、T5时刻气道压降低,T4、T5肺顺应性增高(P<0.05)。两组患者血流动力学指标无明显差异。PCV组在离开苏醒室时氧合指数较高(P<0.05);PCV组在术后一天时刻IL-6和CRP值较低(P<0.05)。结论:肺保护性通气策略可以提高老年患者肺部氧合功能,减少炎症介质释放,减轻肺损伤。     

Respiratory adaptation to chronic hypercapnia in newborn rats

We asked 1) whether newborn rats respond to chronic hypercapnia with a persistent increase in ventilation and 2) whether changes in lung mass were accompanying the respiratory adaptation to chronic hypercapnia, as previously observed during neonatal chronic hypoxia. Five litters of rats were kept in 7% CO2 (with 21% O2) from day 1 to 7 after birth (CO2exp) and compared with six litters of control rats growing in normocapnia-normoxia (C). Body weight was similar between the two groups. Ventilation, measured by flow plethysmography, increased in CO2exp from day 2 and remained steadily elevated, and at day 7 it almost doubled (174%) the C value because of the large increase in tidal volume and mean inspiratory flow (192 and 189%, respectively) with no changes in respiratory frequency. Two days after return to normocapnia, ventilation was still 33% higher than in C; at this time, acute exposure to hypercapnia increased ventilation relatively less in the CO2exp than in C because of a lower increase in tidal volume. Neither the lung weight-to-body weight nor the heart weight-to-body weight ratios increased in CO2exp. We conclude that 1) chronic hypercapnia in newborn rats induces a steady increase in ventilation, which persists at least 2 days after return to normocapnia with a reduction in the acute response to CO2, and 2) hyperventilation per se is not the cause of the increased lung mass observed during chronic neonatal hypoxia.