In order to observe the effects of serum albumin and fibrinogen on biophysical surface properties and the morphology of pulmonary surfactant in vitro, we measured the surface adsorption rate, dynamic minimum and maximum surface tension (min-, max-ST) by Pulsating Bubble Surfactometer, and demonstrated ultrastructures on a series of mixtures with varying concentrations of albumin or fibrinogen and Surfactant-TA. The albumin and fibrinogen significantly inhibited the adsorption rate and ST-lowering properties of surfactant through increasing STs of adsorption rate, min-ST, and max-ST. The characteristic morphology of the Surfactant-TA changed from lamellar rod-like structure with open ends into spherical structures with loss of their open ends by mixing with albumin or fibrinogen. These inhibitory effects of albumin and fibrinogen on surface properties of surfactant were dependent upon the increasing concentration of albumin or fibrinogen. We concluded that albumin and fibrinogen significantly altered surfactant function and its ultrastructural morphology in vitro. These findings support the concept that albumin and fibrinogen-induced surfactant dysfunction may play an important role in the pathophysiology of adult respiratory distress syndrome, and this adverse effect of albumin and fibrinogen on surfactant might be overcome by administration of large doses of exogenous surfactant.
Adsorption ; Animal ; Cattle ; Fibrinogen/pharmacology* ; Human ; Pulmonary Surfactants/ultrastructure* ; Pulmonary Surfactants/drug effects ; Serum Albumin, Bovine/pharmacology* ; Surface Properties

OBJECTIVETo observe the ultrastructures of rat alveolar type II cells and change of composition of phospholipid(PL) and content of protein in pulmonary surfactant(PS), to investigate the relation between change in composition of PL and activity of alveolar type II cells.

METHODSThe rats lung injury models were made by intratracheally instilling bleomycin(BLM) (4 mg/ml, 5 mg/kg). 28 rats were divided into four groups: 3-day group, 7-day group, 14-day group and 28-day group. Preparations of each group were stained histochemically and examined by electron microscope, content of PL in BALF, composition of PL and content of protein of each group were determined respectively.

RESULTS(1) Rats lungs in experimental groups were found that PS lost continuously, appeared homogenous and chorionic, dropped in the pulmonary alveolies. 3-day group was more apparent. Ruthenium red attaching on pulmonary surfactant was thicker in 3-day group, and the colour deeper, no difference in 7-day group and 14-day group, thinner in 28-day group. Content of PL in PS of BALF was increasing. Content of phosphatidylglycerol(PG) increased in 3-day group, decreased in 7-day, 14-day and 28-day group. The change of content of phosphatidylinositol(PI) was reversed. (2) Alveolar type II cells degenerated, necrotized, even disintegrated in 3-day group and 7-day group. 3-day group was more apparent. Proliferations of alveolar type II cells were found in each group, 7-day group was more apparent. We found that type II cells transformed to type I cells in 14-day group, extended and attached on bare basement membrane. Content of protein in PS was the highest in 3-day group, almost equal to the content of the control group in 28-day group.

CONCLUSIONMorphologic change and alternation of quality and quantity of PS after bleomycin-induced pulmonary injures specifically reflect the activity of alveolar type II cells. Measuring content of PL in BALF is one of simple and feasible method judging activity of alveolar type II cells when lungs of the rats are injured early by bleomycin.

Animals ; Antibiotics, Antineoplastic ; toxicity ; Bleomycin ; toxicity ; Bronchoalveolar Lavage Fluid ; chemistry ; Lung ; drug effects ; pathology ; ultrastructure ; Pulmonary Alveoli ; chemistry ; Pulmonary Surfactants ; analysis ; Rats

OBJECTIVETo investigate the effects of methylprednisolone pretreatment on pulmonary lung permeability index and the content of the pulmonary surfactant dipalmitoylphosphatidylcholine (DPPC) in a rabbit model of reexpansion pulmonary edema.

METHODSTwenty-one male New Zealand white rabbits were randomly divided into control group, reexpansion, and reexpansion+methylprednisolone pretreatment groups. The rabbit model of reexpansion pulmonary edema was established using Sakaos method. A bolus dosage of methylprednisolone (3 mg/kg) in reexpansion+methylprednisolone group group or 2.0 ml/kg normal saline in the other two groups was administered intravenously 20 min before reexpansion pulmonary edema. Bronchoalveolar lavage fluid (BALF) and arterial blood samples were collected for measurement of the total protein (TP) and DPPC contents 4 h after reexpansion, and the pulmonary permeability index was calculated.

RESULTSThe pulmonary permeability index in methylprednisolone pretreatment group was significantly lower than that in the reexpansion group (0.007∓0.002 vs 0.177∓0.004, P<0.05). Methylprednisolone pretreatment significantly increased DPPC concentration in the BALF as compared with saline treatment in the reexpansion group (61.815∓28.307 vs 101.955∓24.544 µg/ml, P<0.05).

CONCLUSIONMethylprednisolone pretreatment can increase pulmonary surfactant content and improve pulmonary permeability in the rabbit model of reexpansion pulmonary edema.

1,2-Dipalmitoylphosphatidylcholine ; analysis ; Animals ; Bronchoalveolar Lavage Fluid ; Capillary Permeability ; drug effects ; Male ; Methylprednisolone ; pharmacology ; Permeability ; Pulmonary Edema ; metabolism ; physiopathology ; Pulmonary Surfactants ; metabolism ; Rabbits

OBJECTIVETo analyze the effect of hyperoxia on the proliferation and surfactant associated protein messenger RNA levels of type II alveolar epithelial cells (AECIIs) of premature rat, and to investigate the effect of amygdalin on the change resulted from hyperoxia in AECIIs isolated from premature rat lung in vitro.

METHODSThe lung tissue of 20-day fetal rat was digested by trypsin and collagenase. AECIIs and lung fibroblasts (LFs) were isolated and purified at different centrifugal force and different adherence, then cultured. The nature of the cultures was identified by cytokeratin staining, vimentin staining and transmission electron micrography. For establishing hyperoxia-exposed cell model, purified AECIIs were cultured for 24 hours after culture flasks were filled with 95% oxygen-5% CO2 at 3 L/min for 10 min, and then sealed. Oxygen concentrations were tested in CYS-1 digital oxygen monitor after 24 hours of exposure. A sample was discarded if its oxygen concentration was < 90%. Cell proliferating vitality was examined by MTT assay after treatment with amygdalin at various concentrations. DNA content, protein expression of proliferating cell nuclear antigen (PCNA) and mRNA levels of SPs of AECIIs were analyzed with flow cytometric assay, Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively after 24 hours of air or hyperoxia exposure in the presence or absence of 200 micromol/L amygdalin.

RESULTSExcellent yields of highly purified, culturable AECIIs could be obtained from 20-day fetal lungs. The expression of cytokeratin in AECIIs was positive and that of vimentin negative by immunocytochemistry. Those, however, in LFs were just opposite. Lamellar bodies in purified AECIIs were revealed by transmission electron micrography. The established hyperoxia-exposed cell model assured the oxygen concentrations of culture flasks more than 90%. Amygdalin at the concentration range from 50 micromol/L to 200 micromol/L stimulated the proliferation of AECIIs in a dose-dependent manner; however, at the concentration of 400 micromol/L inhibited the proliferation of AECII. Flow cytometric analysis showed that the apoptosis rate and G0/G1 phase percentage increased significantly (P < 0.01), S phase and G2/M phase percentage decreased significantly (P < 0.01), in hyperoxia group compared with that of air group. The apoptosis rate of air plus 200 micromol/L amygdalin group, compared with air group, was not significantly different (P > 0.05); however, G0/G1 phase percentage decreased markedly, S phase percentage increased significantly, G2/M phase percentage did not significantly change (P > 0.05). The apoptosis rate of hyperoxia plus 200 micromol/L amygdalin group was not significantly different (P > 0.05) from that of hyperoxia group, S phase and G2/M phase percentage increased significantly (P < 0.01), G0/G1 phase percentage decreased significantly (P < 0.01). Western blot analysis showed that the protein expression levels of PCNA in all group was significantly different, in turn, hyperoxia group < hyperoxia plus 200 micromol/L amygdalin < air group < air puls 200 micromol/L amygdalin (P < 0.01). SPs mRNA levels were significantly decreased in hyperoxia group, as compared with air group (P < 0.01). After amygdalin was added, SPs mRNA levels were elevated in air plus amygdalin group and hyperoxia plus amygdalin group, as compared with hyperoxia group (P < 0.01, P < 0.05, respectively), but compared with air group, SP mRNA levels were not significantly elevated (P > 0.05).

CONCLUSIONAECIIs of premature rats were isolated, purified and cultured successfully. Hyperoxia-exposed cell model was established in AECIIs of premature rat in this experiment. Amygdalin promotes the proliferation of premature rat AECII exposed to air or hyperoxia, the concentration of amygdalin with the best effect was 200 micromol/L. Hyperoxia inhibited the proliferation and decreased SPs mRNAs levels in AECIIs in vitro, which may contribute to hyperoxia-induced lung injury in premature rats. Amygdalin could inhibit the changes of SPs mRNAs levels and cell proliferation of AECIIs resulted from hyperoxia and may play partial protective role in hyperoxia-induced premature lung injury.

Amygdalin ; pharmacology ; Animals ; Animals, Newborn ; Cell Proliferation ; drug effects ; Cytoprotection ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Hyperoxia ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; analysis ; Pulmonary Alveoli ; drug effects ; metabolism ; pathology ; Pulmonary Surfactants ; analysis ; RNA, Messenger ; analysis ; Rats

OBJECTIVETo investigate the efficacy of nitric oxide (NO) inhalation on lung function and modulation of inflammation in ventilated premature piglets.

METHODSPremature piglets were obtained at gestation of 101-103 days (89% of full term) with a mean body weight of 870 g. All animals were subjected to mechanical ventilation (PEEP 4-6 cmH2O, Vt 6-8 mL/kg, RR 40-60/min) and randomized into 4 group (n=8 each): Ventilation control, NO inhalation (5-10 ppm), Surfactant (100 mg/kg) or NO plus surfactant. Before and during the ventilation blood gas and lung mechanics were monitored. At the end of the experiment, the lung samples were taken for measuring the NF-kappaB activity, wet/dry weight ratio and histopathology, and the results were compared with those from 10 non-ventilated premature piglets.

RESULTSThe oxygenation index was significantly lower in the NO+surfactant-treated group compared with that of the Control group (2.3 +/- 1.9 vs 9.5 +/- 7.5, P < 0.05). The alveolar aeration in the lungs was similar among the treatment groups. Both NO and NO+surfactant treatments significantly improved the ventilation index. The NO-treated and the Non-ventilated groups had a significantly lower NF-kappaB activity and wet/dry lung weight ratio compared with the Control group. Neither methemoglobin and NO2 levels nor inflammatory lung injury was significantly increased in the NO and combined with surfactant-treated groups.

CONCLUSIONSEarly treatment with NO alone or combined with surfactant can improve oxygenation and ventilation efficacy without obvious adverse effects on immature lungs of premature piglets. The beneficial effects of NO may be due to the suppression of NF-kappaB activity.

Administration, Inhalation ; Animals ; Lung ; drug effects ; pathology ; physiology ; NF-kappa B ; metabolism ; Nitric Oxide ; administration & dosage ; pharmacology ; Premature Birth ; physiopathology ; Pulmonary Surfactants ; pharmacology ; Respiration, Artificial ; Swine

OBJECTIVETo investigate the effect of L-Arginine (L-Arg) on pulmonary surfactant (PS) expression and alveolar macrophage (AM) in rats with pulmonary injury induced by lipopolysaccharide (LPS).

METHODSModel of acute lung injury (ALI) was made by injection (iv) with LPS 5 mg/kg in rats. Fourty-eight male SD rats were randomly divided into 3 groups(n = 16): control, model (LPS) and L-Arg groups. L-Arg (500 mg/kg ip ,L-Arg group) or saline (control and LPS group) was administrated at 3 h or 6 h after LPS injection respectively for 3 h. The expression of surfactant protein A (SP-A) mRNA in the lung tissue was detected by ISH. The total protein (TP) in the bronchoalveolar lavage fluid (BALF) was detected. Rat AM were isolated from the bronchial alveolar lavage fluid of SD rats and harvested by selective plating technique. LPS and L-Arg were added to the culture medium. The concentration of nitric oxide (NO),the activity of lactate dehydrogenase (LDH), the contents of tumor necrosis factor alpha (TNF-alpha) and interleukin- 6 (IL-6) in the culture supernatants were respectively measured.

RESULTSCompared with the control group, the expression of SP-A mRNA was significantly decreased, the TP concentration was significantly increased in LPS group. Compared with LPS group at the same time points, treatment with L-Arg at 3 h after LPS, the expression of SP-A mRNA in lung tissue was increased markedly, whereas TP concentration was decreased significantly. In cultured rat AM, LDH activity, NO, TNF-alpha and IL-6 contents in culture medium were significantly increased in LPS group to compared with those of control group. LDH activity, TNF-alpha and IL-6 contents were decreased in L-Arg group compared with those of LPS group.

CONCLUSIONL-Arg can protect the lung against LPS-induced pulmonary injury by up-regulating the expression of PS and inhibiting inflammatory transmitters from AM.

Acute Lung Injury ; chemically induced ; drug therapy ; metabolism ; Animals ; Arginine ; pharmacology ; therapeutic use ; Lipopolysaccharides ; adverse effects ; Macrophages, Alveolar ; metabolism ; Male ; Pulmonary Surfactants ; metabolism ; Rats ; Rats, Sprague-Dawley

To treat respiratory distress syndrome, surfactant is currently delivered via less invasive surfactant administration (LISA) or INtubation SURfactant Extubation (INSURE). The aim of this study was to compare the effect of the two delivery methods of surfactant on cerebral autoregulation. Near infrared spectroscopy monitoring was carried out to detect cerebral oxygen saturation (ScO), and the mean arterial blood pressure (MABP) was simultaneously recorded. Of 44 preterm infants included, the surfactant was administrated to 22 via LISA and 22 via INSURE. The clinical characteristics, treatments and outcomes of the infants showed no significant differences between the two groups. The correlation coefficient of ScOand MABP (r) 5 min before administration was similar in the two groups. During surfactant administration, rincreased in both groups (0.44±0.10 to 0.54±0.12 in LISA, 0.45±0.11 to 0.69±0.09 in INSURE). In the first and second 5 min after instillation, rwas not significantly different from baseline in the LISA group, but increased in the first 5 min after instillation (0.59±0.13, P=0.000 compared with the baseline in the same group) and recovered in the second 5 min after instillation (0.48±0.10, P=0.321) in the INSURE group. There were significant differences in the change rates of rbetween the two groups during and after surfactant administration. Our results suggest that cerebral autoregulation may be affected transiently by surfactant administration. The effect duration of LISA is shorter than that of INSURE (<5 min in LISA vs. 5-10 min in INSURE).
Administration, Intranasal ; adverse effects ; Brain ; metabolism ; Female ; Homeostasis ; Humans ; Infant, Newborn ; Infant, Premature ; Intubation ; adverse effects ; Male ; Oxygen Consumption ; Pulmonary Surfactants ; administration & dosage ; therapeutic use ; Respiratory Distress Syndrome, Newborn ; drug therapy ; therapy

Newfactan(R) is a domestically developed, bovine lung-derived, semi-synthetic surfactant. The aim of this study was to compare the clinical efficacy of Newfactan(R) with that of Surfacten(R) in the treatment of respiratory distress syndrome (RDS). Newfactan(R) or Surfacten(R) was randomly allocated to 492 newborn infants who were diagnosed as RDS and required surfactant instillation in four participating hospitals. The comparisons were made individually in two subsets of infants by birth weight (<1,500 g group [n=253] and >or=1,500 g group [n=239]). Short-term responses to surfactant and acute complications, such as the total doses of surfactant instilled, response type, extubation rate, ventilator settings, changes in respiratory parameters, air leak, patent ductus arteriosus, pulmonary hemorrhage, and intraventricular hemorrhage, and mortality during the 96 hr after surfactant instillation were measured. Long-term outcome and complications, such as total duration of intubation, bronchopulmonary dysplasia and periventricular leukomalacia, and ultimate mortality were measured. There were no significant differences in demographic and perinatal variables, shortterm responses to surfactant and acute complications, and long-term outcome and complications between Newfactan(R) and Surfacten(R) in both birth weight groups. We concluded that Newfactan(R) was comparable to Surfacten(R) in the clinical efficacy in the treatment of RDS in both birth weight groups.
Comparative Study ; Female ; Humans ; Infant, Newborn ; Lung/drug effects/pathology/physiopathology ; Male ; Pulmonary Surfactants/*therapeutic use ; Research Support, Non-U.S. Gov't ; Respiratory Distress Syndrome, Newborn/*drug therapy/mortality ; Survival Rate ; Time Factors ; Treatment Outcome

The effects of endothelin-1 (ET-1) at low concentration (1-100 pmol/L) on the reactive oxygen-induced inhibition of both pulmonary surfactant (PS) lipid synthesis and the activity of CTP: phosphorylcholine cytidylyltransferase (CCT), a rate-limiting enzyme in biosynthesis of phosphoatidylcholine (PC), were studied in cultured lung explants without serum. The xanthine-xanthine oxidase superoxide anion generating system decreased (3)H-choline incorporation into PC in a dose-dependent manner in cultured lung explants. ET-1 reduced both the reactive oxygen-induced decrease in (3)H-choline incorporation and the increase in malondialdehyde (MDA) content of lung tissues, but did not change the levels of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and the total antioxidant capability in the lung explants. ET-1 enhanced microsomal CCT activity of the lung tissues, while it decreased cytosolic CCT activity of lung tissues. ET-1 also prevented the inhibitive effect of reactive oxygen on microsomal CCT activity in the lung explants. These results suggest that ET-1 at low concentration can protect the microsomal CCT activity and reduce the inhibition of PS lipid synthesis induced by oxidant lung injury. The protective mechanism of ET-1 is not relative to the pulmonary endogenous antioxidant defense system.
Animals ; Choline-Phosphate Cytidylyltransferase ; metabolism ; Endothelin-1 ; administration & dosage ; pharmacology ; Female ; In Vitro Techniques ; Lung ; drug effects ; enzymology ; metabolism ; Male ; Phospholipids ; biosynthesis ; Pulmonary Surfactants ; chemistry ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; toxicity

OBJECTIVETo evaluate dose response of inhaled nitric oxide (iNO) for surfactant-treated rabbits with meconium aspiration-induced acute lung injury (ALI) and hypoxemic respiratory failure (HRF), and variation of measured iNO by continuous NO delivery in pressure support ventilation (PSV).

METHODSAdult rabbits (2.0 - 3.5 kg, n = 33) were randomized to receive intratracheal meconium instillation for 30 min and subjected to following treatment (n = 6 - 8). There were 4 groups: Control (C); NO, iNO at 1, 10, 20 and 40 x 10(-6) each for 60 min at 30 min interval of disconnection; Surf, intratracheal instillation of porcine lung surfactant phospholipids (100 mg/kg); SNO, both iNO and surfactant as in the NO and Surf groups; and a normal group (N), which did not undergo meconium aspiration but received sham deliveries of normal saline. All the animals were treated with PSV for 6 h. iNO levels at different input and sampling sites in the NO and SNO groups were detected by on-line chemiluminescent technique. The blood gas and lung mechanics were measured during the experiments every 2 h.

RESULTS(1) Meconium aspiration induced ALI and severe HRF (PaO(2)/FiO(2) < 200 mmHg) and depressed dynamic compliance of respiratory system (Cdyn) and airway resistance (Raw). In both Surf and NO groups modestly improved oxygenation was observed. In the SNO, values for PaO(2)/FiO(2) were improved from (185 +/- 39) mmHg at baseline to (301 +/- 123) mmHg at 6 h, while moderate or transient improvement was observed in both Surf and NO groups. Cdyn and Raw were only improved for short time in the Surf, NO and SNO groups. iNO had a mild response at 1 x 10(-6) to good response at 10 and 20 x 10(-6), but no further improvement occurred at 40 x 10(-6). The response of iNO in NO group was relatively transient compared to the SNO group. (2) When iNO was connected to the ventilator circuit, the connected site should be placed before humidifier to minimize fluctuation of iNO concentration, and sampling site for iNO monitoring should be placed adequately to eliminate artifact.

CONCLUSIONSiNO synergistically improved surfactant effects on oxygenation and lung mechanics. Continuous supply of iNO with non-continuous flow ventilator provided stable NO within accepted target range with least variation.

Administration, Inhalation ; Animals ; Drug Therapy, Combination ; Female ; Humans ; Infant, Newborn ; Lung ; drug effects ; pathology ; physiopathology ; Male ; Meconium ; Meconium Aspiration Syndrome ; complications ; Nitric Oxide ; administration & dosage ; therapeutic use ; Phospholipids ; therapeutic use ; Pulmonary Surfactants ; therapeutic use ; Pulmonary Ventilation ; Rabbits ; Random Allocation ; Respiratory Distress Syndrome, Adult ; etiology ; therapy ; Treatment Outcome