Surfactant replacement attenuates the increase in alveolar permeability in hyperoxia

Rabbits exposed to hyperoxia develop surfactant deficiency, abnormal lung mechanics, and increased permeability to solute. We investigated whether replenishment of depleted alveolar surfactant by the intratracheal instillation of calf lung surfactant extract (CLSE) would mitigate the increase in alveolar permeability to solute. Twenty-eight rabbits were exposed to 100% O2 for 72 h and received intratracheal instillations of 125 mg CLSE (approximately 170 mumol dipalmitoyl phosphatidylcholine) at 24 and 48 h. The interlobar and intralobar distribution of CLSE was quantified by adding [14C]dipalmitoyl phosphatidylcholine liposes into the instillate and measuring the levels of activity in lung tissue. CLSE was nonuniformly distributed in the different lung lobes, the right lower lobe receiving more CLSE than the rest. Alveolar epithelial permeability to solute was assessed by instilling 10 ml isotonic saline, which contained a trace amount of [57Co]cyanocobalamin, in the right lower lobe and measuring the disappearance of the tracer from the alveolar saline and its appearance in the arterial blood during a 1-h period. CLSE treatment was associated with significantly increased 72-h survival in hyperoxia compared with saline-treated controls (number of survivors: 16/17 vs. 5/11, P less than 0.01). CLSE treatment significantly reduced the rate constant for the movement of cyanocobalamin out of the alveolar space (24 +/- 5 vs. 42 +/- 6 min-1 x 10(-3), P less than 0.01) and tracer appearance in the blood at the end of the study (7 +/- 1 vs. 34 +/- 13%, P less than 0.01) when compared with values in saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of elastase and cigarette smoke on alveolar epithelial permeability

To determine whether instilled porcine pancreatic elastase (PPE) increases alveolar epithelial permeability, we measured alveolar epithelium permeability X surface area (PS) for [14C]sucrose and 125I-bovine serum albumin (125I-BSA) in isolated perfused lungs from hamsters previously exposed to PPE and/or cigarette smoke. Saline (0.5 ml) with 0, 5, or 20 units PPE was instilled intratracheally in anesthetized hamsters. Those exposed to smoke for 4-6 wk received 0 or 5 units; PS was measured 3 h later. Nonsmokers received 0, 5, or 20 units; PS was measured 3 h, 24 h, or 5 days later. Control PS values were (cm3/s X 10(-4), +/- SE) 0.84 +/- 0.11 for sucrose and 0.030 +/- 0.006 for BSA. Three and 24 h following 20 units PPE, (PS)sucrose was twice the control valve. (PS)BSA was four times control at 3 h but not significantly increased at 24 h. Five days after PPE both were back to control levels. Five units PPE or smoke exposure alone caused no PS changes. Smoke exposure and 5 units PPE caused (PS)sucrose to increase markedly (1.85 +/- 0.32); (PS)BSA was not significantly increased (0.076 +/- 0.026). Thus instilled PPE causes reversible increases in alveolar epithelial PS; cigarette smoking potentiates this effect.     

Effects of hyperoxia on transdifferentiation of primary cultured typeII alveolar epithelial cells from premature rats

Hyperoxia exposure is a significant risk factor for the impaired alveolarization characteristic of bronchopulmonary dysplasia. Type II alveolar epithelial cells (AECIIs) may serve as "alveolar stem cells" to transdifferentiate into type I alveolar epithelial cells (AECIs). Here, we show that hyperoxia is capable of inducing transdifferentiation of AECIIs in premature rats in vitro. Hyperoxia-induced transdifferentiation was characterized by typical morphological changes, inhibition of cellular proliferation, decline in expression rate of Ki67, accumulation of cells in the G(1) phase of the cell cycle, increased expression of AECI-specific protein aquaporin 5, and decreased expression of AECII-associated protein surfactant protein C. These results suggest that hyperoxia may induce transdifferentiation of AECIIs into AECIs and the transdifferentiation may be responsible for repairing early lung injury.     

Effects of indomethacin on uterine capillary permeability and blastocyst development in rabbits

Increased capillary permeability at implantation sites was demonstrated in rabbits by extravasation of intravascular blue dye on day 7 of pregnancy. Subcutaneous administration of indomethacin (Id, 8 mg/kg twice daily) on days 4–6 of pregnancy inhibited this uterine blueing response and appeared to reduce the size of implantation swellings. To test the latter observation blastocyst diameter and development of the embryonic disk were assessed at 144 hr . In females receiving indomethacin at the dose level which inhibited uterine blueing, blastocysts were significantly smaller than those from control females. Developmental staging of embryonic disks revealed only slight differences between the smaller (Id-treated) blastocysts and control blastocysts. No effect of Id was seen on ovarian function as judged by luteal weights and plasma progesterone and estradiol levels. Since the major biological effects of indomethacin are due to its inhibition of prostaglandin synthesis, it appears that prostaglandins may play a role in the uterine response to blastocyst stimulation and in the expansion of blastocysts in the rabbit.     

Modification of alveolar hyperoxia induced pulmonary vasodilatation by indomethacin

Decrease in pulmonary vascular resistance was observed in neonatal minature pigs breathing 100% O₂ or 95% O₂:5% CO₂. The pulmonary vasodilator response to hyperoxia ventilation was reduced by indomethacin in the intact animal and in the isolated perfused lung preparation. In the isolated perfused lung preparation, it was also shown that lung alveolar pO₂ rather than pulmonary arterial pO₂ was responsible for the pulmonary vasodilation. The study suggests that alveolar hyperoxia induced decrease in pulmonary vascular resistance may be mediated in part by release of prostaglandins. The relevance of this study with oxygen therapy in newborn infants is also discussed.     

Effects of hyperoxia on postnatal intestinal development

Fetuses develop in a marked hypoxic environment in utero. Premature infants often require high concentrations of oxygen to survive and develop in an environment that would be considered an oxygen stress for the fetus. Postnatal hyperoxia alters organ development, but there is minimal research regarding the role of hyperoxia in intestinal development. We attempted to determine whether postnatal hyperoxia exposure alters intestinal growth and function by using a reliable, objective and sensitive set of methods to study region-specific postnatal intestinal maturation. Rat pups born naturally were placed in continual exposure to room air (normoxia) or 85% oxygen (hyperoxia) immediately after birth. Pups were sacrificed at 1 and 2 weeks of age. Intestines were removed and fixed in formalin. Average mucosal, submucosal, and muscularis thicknesses were measured on hematoxylin and eosin stained sections. Immunohistochemistry was performed using antibodies against NOS II. The staining intensity was determined and quantified for site-specific regions of intestinal sections. No differences in mucosal thickness, submucosal thickness, or muscularis thickness were measured in the duodenum, jejunum or colon at any age. At two weeks of age, the thickness of the ileal mucosa was significantly greater in the group reared in 85% oxygen, and the group exposed to room air demonstrated significantly greater NOS II protein concentration than the hyperoxia group within the distal villus, proximal villus/crypts, submucosa, and muscularis in the distal small intestine.     

Oxygen-induced lung microvascular injury in neutropenic rabbits and lambs

We did two studies to see if severe neutropenia might reduce the severity or delay development of O2-induced lung microvascular injury. First, we treated 11 rabbits with nitrogen mustard until their circulating neurophil count decreased to less than 50/microliters of blood, after which the rabbits breathed pure O2 until death; nine other rabbits received no nitrogen mustard and had normal numbers of circulating neutrophils during O2 breathing. All rabbits died of respiratory failure with pulmonary edema, and although chemotherapy decreased the number of neutrophils in the lungs by greater than 90%, it did not influence survival time or extravascular lung water content. To see if severe neutropenia might slow the development of O2-induced lung microvascular injury, we assessed the effects of sustained hyperoxia on lung fluid balance in unanesthetized lambs treated with hydroxyurea, so that their absolute neutrophil count was less than 50/microliters of blood. We measured pulmonary arterial and left atrial pressures, cardiac output, lung lymph flow, and concentrations of protein in lymph and plasma during a 2- to 4-h control period and then daily for 2 to 4 h as the lambs continuously breathed pure O2. After 3 days of hyperoxia, lymph flow doubled and the concentration of protein in lymph increased from 3.3 +/- 0.5 to 4.2 +/- 0.3 g/dl. Tracer studies with 125I-albumin before and 3 days after the start of O2 breathing confirmed the development of increased lung vascular permeability to protein. All lambs died of respiratory failure with pulmonary edema after 3-5 days in O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation between alveolar transmigration of neutrophils and lung injury in hyperoxia

The objective of this study was to quantitatively assess changes in cell adhesion molecule (CAM) expression on the pulmonary endothelial surface during hyperoxia and to assess the functional significance of those changes on cellular trafficking and development of oxygen-induced lung injury. Mice were placed in >95% O(2) for 0-72 h, and pulmonary injury and neutrophil (PMN) sequestration were assessed. Specific pulmonary CAM expression was quantified with a dual-radiolabeled MAb technique. To test the role of CAMs in PMN trafficking during hyperoxia, blocking MAbs to murine P-selectin, ICAM-1, or platelet-endothelial cell adhesion molecule-1 (PECAM-1) were injected in wild-type mice. Mice genetically deficient in these CAMs and PMN-depleted mice were also evaluated. PMN sequestration occurred within 8 h of hyperoxia, although alveolar emigration occurred later (between 48 and 72 h), coincident with rapid escalation of the lung injury. Hyperoxia significantly increased pulmonary uptake of radiolabeled antibodies to P-selectin, ICAM-1, and PECAM-1, reflecting an increase in their level on pulmonary endothelium and possibly sequestered blood cells. Although both anti-PECAM-1 and anti-ICAM-1 antibodies suppressed PMN alveolar influx in wild-type mice, only mice genetically deficient in PECAM-1 showed PMN influx suppression. Neither CAM blockade, nor genetic deficiency, nor PMN depletion attenuated lung injury. We conclude that early pulmonary PMN retention during hyperoxia is not temporally associated with an increase in endothelial CAMs; however, subsequent PMN emigration into the alveolar space may be supported by PECAM-1 and ICAM-1. Blocking PMN recruitment did not prevent lung injury, supporting dissociation between PMN infiltration and lung injury during hyperoxia in mice.     

Effects of hyperoxia on microvascular cells in vitro

Summary Microvascular cells are most vulnerable to direct oxygen damage. Using an in vitro model system we have investigated the effect of elevated oxygen on the proliferation, morphology, and integrity of microvascular endothelial cells (EC) and pericytes. Cultivation of these cells at oxygen concentrations of 40% for 1 wk resulted in the inhibition of EC proliferation but had no effect on the growth of the pericytes. Similarly, hyperoxia induced a dramatic change in the shape of the EC, increasing their spread area by close to six-fold. Under the same conditions, the spread area of the pericytes was unaffected. To understand the effect of the hyperoxic treatment on the cells, the integrity of various membrane systems was assessed.⁵¹Chromium release was used to monitor plasma membrane integrity. There was no difference in chromium release by EC and pericytes over the 7 d of growth under normoxic and hyperoxic conditions. Mitochondrial integrity was examined by staining the cells with Rhodamine 123, which is selectively accumulated by the mitochondria. The staining pattern of the mitochondria of both EC and pericytes was altered by growth in the elevated oxygen. Finally, the lysosomes were visualized using acridine orange. The acridine orange staining pattern revealed enlarged and perinuclear lysosomes in the EC but no change in the pericyte lysosomal staining pattern. Thus, the cells of the microvasculature seem to be differentially affected by hyperoxia, a fact that may be significant in the etiology of reperfusion injury, ischemic disease, and pathologies associated with prematurity. This research was supported by grant E4-05318 from the National Institutes of Health, Bethesda, MD.     

Polarity of alveolar epithelial cell acid-base permeability

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO-free Na(+) buffer (140 mM Na(+), 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked > or =80% by 100 microM dimethylamiloride, an inhibitor of Na(+)/H(+) exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na(+)/H(+) exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.     

Cytokines and oxygen radicals after hyperoxia in preterm and term alveolar macrophages

To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1beta and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2',7'-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.     

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.     

Effects of pre-exposure to hyperbaric hyperoxia on high-intensity exercise performance

This study comprised 2 main experiments: one was to determine the oxidative DNA damage under hyperbaric hyperoxia (HBO), and the other was to evaluate the effects of pre-exposure to HBO on high-intensity exercise performance. Healthy subjects (n = 8) inspired 100% O2 in an experimental chamber at a pressure of 1.3 atmospheres absolute (ATA) for 50 minutes once per week for 2 weeks. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured as a marker of DNA oxidative damage on day 0 and on days 1, 3, and 5 after each HBO exposure. To investigate the effects of pre-exposure to HBO on high-intensity exercise performance, subjects (n = 6) performed maximal isometric knee extensor exercise (30 repetitions x 2 sets) with and without HBO pre-exposure (100% O2 at 1.3 ATA for 50 minutes). Urinary 8-OHdG did not show any significant change after HBO exposure. Isometric knee extensor torque was significantly lower during the first half of the first set of exercises after HBO pre-exposure compared with the normobaric normoxia (NBO) trial. The decreased torque was associated with the lower integrated electromyography with respect to time. Changes in the degree of ischemia-reperfusion in the vastus lateralis muscle during exercise were larger in the HBO pre-exposure trial than in the NBO trial. Muscle fatigue index, serum lactate concentration, heart rate, and systolic blood pressure showed no differences between the 2 trials. These results indicated that HBO exposure was harmless to DNA, and HBO pre-exposure did not enhance high-intensity exercise performance. As a practical application, athletes who require maximal muscle strength should not inspire high-concentration of O2 just before their competitions because it might, as the case may be, impair their performance.