O2-induced Lung Injury Research Articles

Regulation of hyperoxia-induced neonatal lung injury via post-translational cysteine redox modifications

Preterm infants and patients with lung disease often have excess fluid in the lungs and are frequently treated with oxygen, however long-term exposure to hyperoxia results in irreversible lung injury. Although the adverse effects of hyperoxia are mediated by reactive oxygen species, the full extent of the impact of hyperoxia on redox-dependent regulation in the lung is unclear. In this study, neonatal mice overexpressing the beta-subunit of the epithelial sodium channel (β-ENaC) encoded by Scnn1b and their wild type (WT; C57Bl6) littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FiO2)-induced lung injury. Results showed that O2-induced lung injury in transgenic Scnn1b mice is attenuated following chronic O2 exposure. To test the hypothesis that reversible cysteine-redox-modifications of proteins play an important role in O2-induced lung injury, we performed proteome-wide profiling of protein S-glutathionylation (SSG) in both WT and Scnn1b overexpressing mice maintained at 21% O2 (normoxia) or FiO2 85% (hyperoxia) from birth to 11–15 days postnatal. Over 7700 unique Cys sites with SSG modifications were identified and quantified, covering more than 3000 proteins in the lung. In both mouse models, hyperoxia resulted in a significant alteration of the SSG levels of Cys sites belonging to a diverse range of proteins. In addition, substantial SSG changes were observed in the Scnn1b overexpressing mice exposed to hyperoxia, suggesting that ENaC plays a critically important role in cellular regulation. Hyperoxia-induced SSG changes were further supported by the results observed for thiol total oxidation, the overall level of reversible oxidation on protein cysteine residues. Differential analyses reveal that Scnn1b overexpression may protect against hyperoxia-induced lung injury via modulation of specific processes such as cell adhesion, blood coagulation, and proteolysis. This study provides a landscape view of protein oxidation in the lung and highlights the importance of redox regulation in O2-induced lung injury.

A Locus on Chromosome 15 Contributes to Acute Ozone-induced Lung Injury in Collaborative Cross Mice.

Ozone (O3)-induced respiratory toxicity varies considerably within the human population and across inbred mouse strains, indicative of gene-environment interactions (GxE). Though previous studies have identified several quantitative trait loci (QTL) and candidate genes underlying responses to O3 exposure, precise mechanisms of susceptibility remain incompletely described. We sought to update our understanding of the genetic architecture of O3 responsiveness using the Collaborative Cross (CC) recombinant inbred mouse panel. We evaluated hallmark O3-induced inflammation and injury phenotypes in 56 CC strains after exposure to filtered air or 2 ppm O3, and performed focused genetic analysis of variation in lung injury, as reflected by protein in lung lavage fluid. Strain-dependent responses to O3 were clear, and QTL mapping revealed two novel loci on Chr (Chromosomes) 10 (peak, 26.2 Mb; 80% confidence interval [CI], 24.6-43.6 Mb) and 15 (peak, 47.1 Mb; 80% CI, 40.2-54.9 Mb), the latter surpassing the 95% significance threshold. At the Chr 15 locus, C57BL/6J and CAST/EiJ founder haplotypes were associated with higher lung injury responses compared with all other CC founder haplotypes. With further statistical analysis and a weight of evidence approach, we delimited the Chr 15 QTL to an ∼2 Mb region containing 21 genes (10 protein coding) and nominated three candidate genes, namely Oxr1, Rspo2, and Angpt1. Gene and protein expression data further supported Oxr1 and Angpt1 as priority candidate genes. In summary, we have shown that O3-induced lung injury is modulated by genetic variation, identified two high priority candidate genes, and demonstrated the value of the CC for detecting GxE.

Effect Of Chemerin Deficiency On Ozone-Induced Lung Injury And Lung Inflammation

Chemerin is a non-chemokine chemoattractant for macrophages, natural killer (NK) cells, and plasmacytoid dendritic cells. Macrophages and NK cells promote lung injury and lung inflammation induced by exposure to ozone (O3), a criteria pollutant, an occupational irritant, and a non-atopic asthma stimulus. We hypothesized that genetic deficiency of chemerin in mice would lessen the severity of O3-induced lung injury and lung inflammation since macrophages and NK cells are unable to respond to a chemotactic signal from chemerin.

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

Asiaticoside attenuates hyperoxia-induced lung injury in vitro and in vivo.

Objective(s):Asiaticoside (AS) displays anti-inflammation, and anti-apoptosis effect, but the role of AS in hyperoxia-induced lung injury (HILI) treatment is undefined. Therefore, the aim of this study was to investigate the effects of AS on HILI on premature rats and alveolar type II (AEC II) cells.Materials and Methods:Sprague-Dawley premature rats (n=25/group) were exposed to 80% O2 with or without AS. Then, we detected 80% O2-induced lung injury and survival rate of premature rat. We tested the concentration of malondialdehyde (MDA), myeloperoxidase (MPO), total antioxidant capacity (TAOC), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β) in premature rats’ blood. Then, the AEC II cell apoptosis was observed by Hoechst 33258 staining and flow cytometry. Simultaneously, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway was measured by Western blot.Results:Our results found that AS-treated group rats had significantly higher survival rates than 80% O2 group at day 14 (P<0.05). AS protected HILI, decreased the MPO and MDA concentration, and reversed TAOC level (P<0.05). AS also downregulated the levels of TNF-α, IL-1β, and IL-6 in the premature rat’s blood (P<0.01). Moreover, AS markedly attenuated AEC II cell apoptosis and increased Nrf2 and Heme oxygenase 1 (HO-1) expression in the nucleus (P<0.05).Conclusion:AS showed protective effects on premature rats of HILI in vitro and in vivo. AS can potentially be developed as a novel agent for the treatment of HILI diseases.

Common Transcriptomic Changes Regulated by Innate Immune Receptors and Nuclear Factor NF-kappa-B in Mice Exposed to Ozone

Ozone (O3) is a predominant oxidant in air pollution and has been linked to diminished lung function, respiratory inflammation, and worsening of preexisting airway diseases. Tumor necrosis factor (TNF)-α and toll-like receptor 4 (TLR4) play critical roles in innate immune responses. Signaling through TNF receptor (TNFR) and TLR4 activates nuclear factor NF-kappa-B (NF-κB), leading to the transcriptional regulation of immunity genes. The injurious roles of TNF-α, TLR4, and NF-κB in O3-induced lung pathogenesis have been determined in mice. The current study was designed to examine lung transcriptome changes and downstream mechanisms regulated by these innate immune response factors. Mice lacking Tnfr (Tnfr-/-) or Nfkb (Nfkb-/-), Tlr4 mutant mice (C3H/HeJ), and their wild-types (Tnfr+/+, Nfkb+/+, C3H/HeOuJ) were exposed to air or 0.3 ppm O3. Lung RNAs were isolated for cDNA microarray analysis. Significantly varied genes between the wild-type and gene knockout mice after O3 exposure were determined (2-way ANOVA) to elucidate commonly changed genes between the TNFR-NF-κB, TLR4-NF-κB, and TNFR-TLR4 pathways (Venn Diagram analysis). Downstream mechanisms affected by the common differentially regulated genes and chemicals/drugs that may act on O3-responsive genes were predicted by bioinformatics analysis tools. qPCR, ELISA, Western blotting, and chromatin immunoprecipitation assay validated the results. Tnfr- and Nfkb-dependently changed genes by O3, including matrix metalloproteinase 9 (Mmp9) and endothelin 1 (Edn1), were involved in cell death, connective tissue injury, and inflammation. Interleukin 12 (IL-12) and transforming growth factor (TGF)-β1 were suggested as upstream regulators. Genes changed in common by Tlr4 and Nfkb during O3 exposure (e.g. D-box binding PAR BZIP transcription factor, Dbp; lipocalin 2, Lcn2; serum amyloid A 3, Saa3) were related to lipid derangement, cell morphology, and lymphoid tissue hyperplasia. Common gene transcripts regulated by TNFR and TLR4 such as fatty acid binding protein 1 (Fabp1) and tissue inhibitor of metalloproteinase 1 (Timp1) contributed to the chemotaxis and fatty acid metabolism. In conclusion, the TNFR-NF-κB and TLR4-NF-κB pathways modulated distinct transcriptomes during the development of O3-induced lung injury in mice. Results will provide significant insights into the pulmonary O3 pathogenesis and therapeutic interventions.

Recombinant human elafin promotes alveologenesis in newborn mice exposed to chronic hyperoxia.

Elastase inhibitors reverse elastin degradation and abnormal alveologenesis and attenuate the lung structural abnormalities induced by mechanical ventilation with O2-rich gas. The potential of these molecules to improve endothelial function and to ameliorate severe bronchopulmonary dysplasia (BPD) during lung development is not yet understood. We sought to determine whether the intratracheal treatment of newborn mice with the elastase inhibitor elafin would prevent hyperoxia-induced lung elastin degradation and the cascade of events that cause abnormal alveologenesis. Newborn mice were exposed to 85% O2 for 3, 7, 14 or 21days. Recombinant human elafin was administered by intratracheal instillation from the first day every two days for 20days. We next used morphometric analyses, quantitative RT-PCR, immunostaining, Western blotting, and ELISA methods to assess the key variables involved in elastogenesis disruption and the potential signaling pathways noted below in recombinant human elafin-treated mouse pups that had been exposed to 85% O2. We found that impaired alveolar development and aberrant elastin production were associated with elevations in whole lung elastase levels in 85% O2-exposed lungs. Elafin attenuated the structural disintegration that developed in the hyperoxia-damaged lungs. Furthermore, elafin prevented the elastin degradation, neutrophil influx, activation of TGF-β1 and apoptosis caused by 85% O2 exposure. Pulmonary elastase plays an important role in disrupting elastogenesis during O2-induced damage, which is the result of a pulmonary inflammatory response. Elafin prevents these changes by inhibiting elastase and the TGF-β1 signalling cascade and may be a new therapeutic target for preventing O2-induced lung injury in neonates.

Hypoxia-induced pulmonary arterial hypertension augments lung injury and airway reactivity caused by ozone exposure

Ozone (O3)-related cardiorespiratory effects are a growing public health concern. Ground level O3 can exacerbate pre-existing respiratory conditions; however, research regarding therapeutic interventions to reduce O3-induced lung injury is limited. In patients with chronic obstructive pulmonary disease, hypoxia-associated pulmonary hypertension (HPH) is a frequent comorbidity that is difficult to treat clinically, yet associated with increased mortality and frequency of exacerbations. In this study, we hypothesized that established HPH would confer vulnerability to acute O3 pulmonary toxicity. Additionally, we tested whether improvement of pulmonary endothelial barrier integrity via rho-kinase inhibition could mitigate pulmonary inflammation and injury. To determine if O3 exacerbated HPH, male C57BL/6 mice were subject to either 3 weeks continuous normoxia (20.9% O2) or hypoxia (10.0% O2), followed by a 4-h exposure to either 1ppm O3 or filtered air (FA). As an additional experimental intervention fasudil (20mg/kg) was administered intraperitoneally prior to and after O3 exposures. As expected, hypoxia significantly increased right ventricular pressure and hypertrophy. O3 exposure in normoxic mice caused lung inflammation but not injury, as indicated by increased cellularity and edema in the lung. However, in hypoxic mice, O3 exposure led to increased inflammation and edema, along with a profound increase in airway hyperresponsiveness to methacholine. Fasudil administration resulted in reduced O3-induced lung injury via the enhancement of pulmonary endothelial barrier integrity. These results indicate that increased pulmonary vascular pressure may enhance lung injury, inflammation and edema when exposed to pollutants, and that enhancement of pulmonary endothelial barrier integrity may alleviate such vulnerability.

Exogenous Hydrogen Sulfide (H2S) Protects Alveolar Growth in Experimental O2-Induced Neonatal Lung Injury

BackgroundBronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT). Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S), carbon monoxide and nitric oxide (NO), belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD.MethodsWe took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate) to study its lung protective potential in vitro and in vivo.Results In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation.ConclusionsH2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.

Ozone induces pulmonary activating transcription factor 3 (ATF3) expression

Ozone, a major component of air pollution, has been linked to detrimental health effects including inflammation, asthma, cardiovascular disease, and increased mortality. While recent evidence suggests that transcription factor ATF3 is a negative regulator of pulmonary inflammation, its role in O3-induced lung injury remains unknown. To address this question, we exposed 8-10 wk male C57BL/6 mice to 2 ppm O3 or filtered air (FA) for 3 hrs and allowed subsequent recovery in room air for up to 24 hrs. Compared to FA, ATF3 protein was up-regulated by 5-fold after exposure to O3 for 3 hrs (p<0.01) while the expression of ATF4 and ATF5 proteins remained unchanged. O3 increased the nuclear accumulation of ATF3, but not ATF4. Quantitative PCR revealed a 3-fold increase in ATF3 after O3 but no change in ATF4 or ATF5. Both ATF3 protein and mRNA levels remained elevated during early recovery (4 hrs post-exposure), but declined by 24 hrs. Induction of ATF3 expression involves multiple overlapping signal transduction pathways known to be sensitive to oxidative stress. We found that O3 increased the phosphorylation of ERK1/2, JNK, and p38 MAPK, but not the phosphorylation of eIF2α. In addition, p38 phosphorylation remained elevated during early recovery. Altogether, these results illustrate that O3 induces expression of ATF3 in the lung. The increase in ATF3 may play a role in the negative regulation of O3-induced inflammation.

Decreased Protein C Activation is Associated with Increased Lung Injury in Response to Prolonged Oxygen Exposure in Mice

We have shown significant decreases in the pulmonary protein expression and functional activity of the endothelial surface protein thrombomodulin (TM) by 24h of exposure to >95% oxygen (O2) in 8–10 wk C57/B6 mice. Given the key role of TM in binding thrombin and activating protein C (PC), abnormalities in the PC cascade could contribute to O2-induced lung injury. To investigate this hypothesis, we studied two transgenic mouse models with an impaired ability to activate PC. First, we found plasma APC levels generated after murine thrombin (80U/kg IV) in heterozygous PC deficient (PC+/−) mice (n=5) were 65±21% those in wildtype (WT) mice (n=7; p=0.05). Second, we studied mice deficient in platelet factor 4 (PF4−/−), a platelet α-granule-specific chemokine shown to accelerate thrombin·TM complex APC generation. Plasma APC generation by thrombin was 74 ± 6% in PF4−/− mice (n=7) vs. WT (n=15, p<0.1). Next, mice were exposed to >95% O2 for 72h. Lung injury, as assessed by bronchoalveolar lavage fluid (BAL) protein was significantly increased about 2 -fold in both the PC+/−mice vs. WT (2.46 + 0.55, n = 4 vs. 1.37 + 0.11mg/ml, n=6; mean + SEM; p<0.05) and in the PF4−/− mice vs. WT (11.11 + 2.48, n = 7 vs. 6.68 + 1.03 mg/ml, n = 9; mean+SEM; p<0.05). Survival rates were also lower in the PC+/− and PF4−/− mice. Total WBC counts in BAL were similar between groups, suggesting changes in pulmonary inflammation did not account for the increased lung injury. Given the added role of protein C in the coagulation and fibrinolytic cascades, increased intra-alveolar fibrin deposition could contribute to the increased lung injury in these mice. Supported by NIH K08HL67913-01

Expression of Urokinase-Type Plasminogen Activator in Platelets Decreases Oxygen-Induced Lung Injury in Mice.

We have previously shown that urokinase (uPA) can be ectopically expressed and stored in platelets in a transgenic mouse model and that such platelets can effectively prevent untoward thrombosis. One major advantage of this uPA delivery system is that there is no systemic fibrinolysis and the uPA is selectively released at sites of vascular injury. We have now tested whether such a delivery model may be of use in the care of patients with lung injury where inhibition of fibrinolytic activity and intra-alveolar fibrin deposition are of pathogenic importance. In this study, we evaluated (O2)-induced lung injury in transgenic mice expressing platelet uPA compared to wildtype (WT) littermate controls. Given that pulmonary sequestration of activated inflammatory cells, including neutrophils (PMN) and platelets, occurs relatively early in response to O2, and given the ability of the transgenic uPA platelets to enhance fibrin lysis, we hypothesized that hyperoxic-induced lung injury would be lessened in the transgenic animals. Lung injury was assessed by changes in bronchoalveolar lavage fluid (BAL) protein concentration. In this model, a significant increase in BAL protein concentration, indicative of injury to the alveolar capillary barrier, occurred in WT mice after 72 hr of >95% O2 exposure compared to controls [6.9 ± 0.9 vs. 0.14 ± 0.01 mg/ml (mean ± SEM)], respectively. BAL protein was significantly decreased in platelet uPA+ mice [1.5 ± 0.4 vs. 6.9 ± 0.9 mg/ml, n=6–8, p<0.05]. Immunohistochemical detection of fibrin in control and platelet uPA+ mice lungs using anti-murine fibrin(ogen) antibody showed that very little fibrin(ogen) was detected in control (0 hr) mice or WT mice exposed to >95% O2 for 24 hr. By 72 hr of O2 in WT mice, however, fibrin(ogen) immunoreactivity was seen within the alveolar space and diffusely throughout the interstitium. Fibrin clots within small blood vessels were also seen. In contrast, fibrin(ogen) immunoreactivity was greatly reduced in uPA+ mice. Moreover survival time of uPA+ mice was significantly longer than WT animals (127 ± 28 hr vs. 97 ± 14 hrs, respectively, n=12, p<0.003). Total white blood cell counts and % PMN in the BAL were significantly higher after 72 hr exposure to O2, suggesting that increased pulmonary inflammation is not necessarily associated with increased lung injury, a finding consistent with other studies showing that neutrophils do not play a significant role in the genesis of this lung injury. These data support the hypothesis that a decrease in lung fibrinolytic activity is a primary factor in the development of O2-induced lung injury and that platelet delivery of uPA may be an especially effective and safe way to prevent the development of such lung injury.

Airspace Surface Chemistry Mediates O3-Induced Lung Injury

Ozone (O3) produces diverse pulmonary pathophysiologies but with marked heterogeneities relative to species, age, anatomic site, disease, and exposure history. These pronounced susceptibility variations have remained largely undefined. We have postulated that interactions between inhaled O3 and the airspace surfaces appreciably govern the distribution and extent of lung injury. O3 displays unique absorption properties wherein chemical reaction with the epithelial lining fluid (ELF) maintains the net flux from the gas phase and couples O3 uptake with the generation of products that lead to cell injury. Diversities in respiratory tract geometry and interfacial physicochemical conditions leads to spatially heterogeneous rates of O3 flux into the ELF which combine with the local production of bioactive species to dictate the local dose. We have observed that both the uptake and distribution of acute epithelial injury is principally localized to the conducting airways. O3 preferentially reacts with ELF ascorbic (AH2) and uric acids (UA) although reaction with GSH and unsaturated fatty acids (UFA) occur to a lesser extent. UFA reactions may not generate sufficient bioactive materials to account for acute cell injury. Reactions with AH2 and GSH, but not UA or Trolox, form secondary oxidants that initiate oxidation of model membranes and in vitro cell damage. However, secondary oxidant production is antioxidant concentration-dependent with a hyperbolic-shaped dose/response curve. Acquisition of species-specific data characterizing the pharmacodynamics of ELF substrate turnover under both basal and exposure conditions are critical to further our understanding of how surface chemistry regulates the balance between quenching of inhaled O3 and conditions that promote production of bioactive/cytotoxic species and, therefore, biological outcomes.

Tolerance of SP-A-deficient mice to hyperoxia or exercise.

Mice carrying a null mutation of the surfactant-associated protein A (SP-A) gene have normal respiratory function, but their surfactant lacks tubular myelin, is sensitive to protein inactivation in vitro, and contains decreased pool sizes of the biophysically active large-aggregate surfactant. We hypothesized that SP-A-deficient mice would be more susceptible to exercise-induced stress and O(2)-induced lung injury. SP-A-(-/-) and SP-A-(+/+) mice tolerated 1 h of swimming or 45 min of running on a treadmill at 15 m/min equivalently, without alterations of the amount of alveolar saturated phosphatidylcholine. After 3 days of hyperoxia, SP-A-(-/-) mice had increased alveolar protein, but pressure-volume curves were not different between groups. Alveolar protein concentration was similarly increased in SP-A-(-/-) and SP-A-(+/+) mice after 4 days of exposure to hyperoxia. Survival rates were similar after 4 days of hyperoxia. SP-A-(-/-) mice were equally tolerant to exercise and 4 days of hyperoxia, indicating that the SP-A-dependent alterations in surfactant did not result in functional deficits.

Inhibition of macrophages with gadolinium chloride abrogates ozone-induced pulmonary injury and inflammatory mediator production.

Acute inhalation of toxic doses of ozone (O3) induces macrophage accumulation in the lung and the release of cytotoxic and proinflammatory mediators. To evaluate the role of macrophages and their mediators in the pathophysiologic response of the lung to O3, we examined the effects of the macrophage inhibitor, gadolinium chloride (GdCl3), on O3-induced inflammation, mediator production, and lavage fluid protein levels. Rats were pretreated with GdCl3 (7 mg/kg, intravenously) or control 24 h prior to exposure to air or O3 (2 parts per million, 3 h). Animals were killed 48 h after exposure. GdCl3 pretreatment of rats was found to abrogate O3-induced increases in the number of cells, as well as the amount of protein recovered in bronchoalveolar lavage fluid. Following GdCl3 pretreatment of rats, lung lavage cells consisting of > 90% macrophages were found to produce significantly less nitric oxide and express less inducible nitric oxide synthase (iNOS) when compared to cells from rats exposed to O3. O3-induced alterations in superoxide anion production by alveolar macrophages, both in vitro and in situ, were also attenuated by GdCl3 pretreatment of rats. In addition, increases in tumor necrosis factor alpha (TNF-alpha) and fibronectin in lung tissue induced by O3 were reduced. Taken together, these data provide support for the hypothesis that macrophages contribute to the pathogenesis of O3-induced lung injury.

Response of alveolar macrophage-depleted rats to hyperoxia.

Recently an alveolar macrophage (AM)-depleted rat model has been characterized and it has been demonstrated that AM are required for the endotoxin-induced tumor necrosis factor (TNF) release into the alveolar space (J Appl Physiol 1993;74:2812-2819). The current study investigated the response of AM-depleted rats to hyperoxia and evaluated the potential role of AM in the pathogenesis of pulmonary O2 toxicity. Rats were insufflated with Hanks' balanced salt solution (HBSS), liposome-encapsulated phosphate-buffered saline (PBS-liposomes), or liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP-liposomes) and 2 days later exposed to 100% O2. The effect of hyperoxia was assessed by parameters of O2-induced lung injury (e.g., hematocrit value, pleural effusion volume, effusion protein to plasma protein ratio, and alveolar lavage fluid protein content), TNF release into the alveolar space, and survival. Insufflation of Cl2MDP-liposomes, but not HBSS or PBS-liposomes, caused a sustained depletion of > 70% AM, which was associated with a slight but significant increase in the number of lavageable neutrophils. Twenty percent of AM-depleted rats survived longer than 74 h of O2 exposure, while all rats insufflated with HBSS or PBS-liposomes died within 74 h (p < .05). No significant differences were detected in alveolar TNF release or in the extent of O2-induced lung injury.

Effect of Inhaled Nitric Oxide on 100% O2-induced Lung Injury in Intact Rats

Effect of Inhaled Nitric Oxide on 100% O2-induced Lung Injury in Intact Rats

Pentoxifylline Attenuates Oxygen-Induced Lung Injury

Patients requiring mechanical ventilation can develop severe pulmonary injury. Although pentoxifylline (PTX) is known to attenuate endotoxin and tumor necrosis factor (TNF)-induced lung injury, as well as decrease interleukin-6 (IL-6) levels following hemorrhage and resuscitation, it remains unknown if this agent has any beneficial effects against O2-induced lung injury. The aim of this study, therefore, was to determine if PTX attenuates pulmonary oxygen toxicity. To investigate this, male Sprague-Dawley rats were injected subcutaneously with 1 ml saline or 1 ml PTX (50 mg/kg) and immediately exposed to either 21% O2 or ⩾95% O2 for 52 hr. The animals were then reweighed and euthanized. Pleural fluid was collected, blood samples were obtained, and lung lavage was performed. Lactate dehydrogenase (LDH) activity, protein content, and IL-6 concentrations were determined in the lavage fluid and serum. The supernatant LDH activity, protein content, pleural fluid accumulation, and IL-6 concentration were significantly decreased (P < 0.05) in those animals pretreated with PTX prior to exposure to hyperoxia compared to those animals exposed to hyperoxia and not treated. Furthermore, the hematocrit and serum IL-6 concentration were also decreased in the treated group and not significantly different from the controls. Thus, PTX appears to attenuate O2-induced lung injury and may play a role in protecting those patients at risk for developing pulmonary oxygen toxicity.

Failure of Premature Rabbits to Increase Antioxidant Enzymes During Hyperoxic Exposure: Increased Susceptibility to Pulmonary Oxygen Toxicity Compared with Term Rabbits

Although the prematurely born are known to have decreased baseline levels of protective antioxidant enzymes (Frank L, Sosenko IRS: J Pediatr 110:9 and 106, 1987), the ability to augment the baseline values during high O2 exposure is the key factor determining O2 tolerance versus O2 susceptibility. We have compared the pulmonary antioxidant enzyme responses of prematurely delivered rabbits (gestational d 29 of 32) and full-term rabbits to 48-72 h of hyperoxic exposure. We found that although full-term newborns exposed to greater than 90% O2 consistently showed elevated superoxide dismutase, catalase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase activities, the premature animals repeatedly failed to respond to hyperoxia with increased antioxidant enzyme activity levels. Consistent with the comparative antioxidant enzyme responses were the evidences of O2 toxicity in the two age groups. The prematurely born rabbits had significantly increased lung lavage protein content, lung conjugated diene levels, and more severe light microscopic lung pathology compared with the full-term animals during equal O2 exposure time. This first reported comparison of prematurely born versus full-term animal responses to hyperoxia might help to explain the clinical observation that the very prematurely born infant is excessively prone to the development of O2-induced lung injury and the progressive development of bronchopulmonary dysplasia.

In vivo exposure to hyperoxia increases airway responsiveness in rats. Demonstration in vivo and in vitro.

Studies regarding O2-induced lung injury have concentrated on damage to alveolar structures and pulmonary vasculature without consideration of alterations that may be occurring in airways. This study was undertaken to determine the effects of in vivo hyperoxic exposure on airway responses to excitatory stimuli in intact, anesthetized rats and in intrapulmonary bronchi isolated from hyperoxia-exposed rats. Using lung conductance (G1) as an index of bronchoconstriction, intravenously administered 5-hydroxytryptamine (5HT) elicited greater bronchoconstrictor responses in anesthetized, mechanically ventilated rats that had been exposed to 85% O2 for 7 days rather than to air. Further, airways of hyperoxia-exposed rats were more sensitive to the effects of intravenously administered 5HT as evidenced by the lower log dose of 5HT required to decrease G1 30%. Cylindrical segments of intrapulmonary bronchi isolated from hyperoxia-exposed rats were more responsive to the contractile effects of 5HT and electrical field stimulation. However, no differences in responsiveness to bethanechol or KCl were observed between the two groups. The log concentration of 5HT and the log frequency of electrical field stimulation that elicited half-maximal responses were smaller in bronchi isolated from hyperoxia-exposed animals, indicating an increase in sensitivity of the airways to these stimuli. These results suggest that prolonged exposure to greater than ambient levels of O2 can alter airway function; however, the mechanism responsible for these changes remains to be determined.