Premature Rabbit Fetuses Research Articles

Treatment of Respiratory Distress Syndrome with Single Recombinant Polypeptides that Combine Features of SP-B and SP-C.

Treatment of respiratory distress syndrome (RDS) with surfactant replacement therapy in prematurely born infants was introduced more than 30 years ago; however, the surfactant preparations currently in clinical use are extracts from animal lungs. A synthetic surfactant that matches the currently used nature-derived surfactant preparations and can be produced in a cost-efficient manner would enable worldwide treatment of neonatal RDS and could also be tested against lung diseases in adults. The major challenge in developing fully functional synthetic surfactant preparations is to recapitulate the properties of the hydrophobic lung surfactant proteins B (SP-B) and SP-C. Here, we have designed single polypeptides that combine properties of SP-B and SP-C and produced them recombinantly using a novel solubility tag based on spider silk production. These Combo peptides mixed with phospholipids are as efficient as nature-derived surfactant preparations against neonatal RDS in premature rabbit fetuses.

Alterations of the C-terminal end do not affect in vitro or in vivo activity of surfactant protein C analogs

The secondary structure, orientation and hydrogen/deuterium exchange of SP-C33, a surfactant protein C analog, in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/egg phosphatidylglycerol (8:2, wt./wt.) bilayers, was studied by attenuated total reflection Fourier transform infrared spectroscopy. This showed a transmembrane α-helix, in which about 55% of the amide hydrogens do not exchange for up to 20h. Moreover, C-terminally modified SP-C33, either truncated after position 30, or having the methionine at position 31 exchanged for either lysine or isoleucine, showed the same secondary structure and orientation. The different peptides, suspended in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 68:31 (wt./wt.), were tested for surfactant activity in vitro in a captive bubble surfactometer and in vivo in an animal model of respiratory distress syndrome using premature rabbit fetuses. All preparations showed similar surface activity in the captive bubble surfactometer. Also, in the rabbit model, all preparations performed equally well and significantly better than non-treated controls, both regarding tidal volumes and lung gas volumes. Thus, truncation or residue replacements in the C-terminal part of SP-C33 do not seem to affect membrane association or surfactant activity.

Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome

Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.

Concentration Dependence of a Poly-Leucine Surfactant Protein C Analogue on in vitro and in vivo Surfactant Activity

Background: Modified natural surfactants currently used for treatment of respiratory distress syndrome contain about 0.5–1% (w/w phospholipids) of each of the surfactant proteins SP-B and SP-C. The supply of these preparations is limited and synthetic surfactant preparations containing lipids and peptides are under development. Objectives: To investigate the potential of different concentrations of the SP-C analogue SP-C33 in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (68:31, w/w). Methods: Surface activity was evaluated in pulsating and captive bubble surfactometers and in immature newborn rabbits. Results: Preparations containing ≧1% SP-C33 achieve minimum surface tension <5 mN/m indicating good biophysical activity, and increase tidal volumes in premature rabbit fetuses to the same level as a modified natural surfactant preparation does. Alveolar patency at end expiration, as evaluated by measurement of lung gas volumes, histological assessment of alveolar expansion and determination of alveolar volume density, was lower in the animals treated with synthetic surfactant than in those receiving modified natural surfactant. Conclusions: These data suggest that SP-C33 is similarly efficient as the native peptide in improving surface properties of phospholipids mixtures and in increasing lung compliance in surfactant-deficient states, but that other components are needed to maintain alveolar stability at low airway pressures.

Animal studies of surfactant replacement therapy.

Before using surfactant preparations in animal experiments or in clinical trials, it is necessary that they fulfil specific in vivo requirements. But conclusive criteria for the effectiveness of a surfactant have not yet been defined. Thus, some standard levels for improvement of lung function, or threshold levels, must be established in standardized animal models with surfactant deficiency. The respiratory distress syndrome model of premature rabbit fetuses is the most sensitive model, followed by the surfactant deficiency model by lung lavage, while the model of viral pneumonia is the least sensitive when testing exogenous surfactant. With a less-effective exogenous surfactat, a small improvement in lung function in the rabbit fetus model and the lung lavage model can be observed, while in the viral pneumonia model almost no improvement occurred. Only a functional effective surfactant fulfilled all our criteria for functional improvement in lung function.

Surfactant for Hyaline Membrane Disease

Since it has been demonstrated that hyaline membrane disease (HMD) is due to a relative deficiency of lung surfactant,1,2 one possible approach to the treatment or prevention of HMD in premature infants might be the introduction of surfactant into the lungs. Thus far, attempts at aerosolization of either synthetic surfactant (dipalmitoyl lecithin [DPL]) or natural surfactant into the lungs of patients or animals have failed to produce convincing benefits.3-5 On the other hand, direct instillation of a solution of natural surfactant into the trachea seems to produce striking results. Enhörning et al6 were the first to show that tracheal deposition of natural surfactant into premature rabbit fetuses before the first breath enhanced air intake and improved the pressure-volume relationships of the lungs; it also increased their survival time.7

Radiologic evaluation of the premature newborn rabbit after pharyngeal deposition of surfactant

Premature rabbit fetuses were delivered by hysterotomy and, prior to the first breath, a concentrated homologous surfactant suspension was deposited in the pharynx. With repeated radiograms, the effect of this treatment was studied during the first two hours of extrauterine life. The lung aeration was quantitated with densitometry, and the chest expansion was assessed by measuring the inferior angle formed by the fourth ribs. In comparison with control animals, the treated animals demonstrated a greater chest expansion, and their lungs were significantly better aerated. Clinical application has to await further studies.

Tracheal deposition of surfactant before the first breath.

Premature rabbit fetuses of gestational age 28 days underwent tracheostomy immediately after delivery, and thus had to take their first breath through a tracheal cannula. in 20 experimental animals, the tracheal tube contained 25 μliter of a concentrated surfactant suspension that had been prepared by centrifugation of alveolar wash from an adult rabbit for 1 hour at 1,000 g and 4° C. in 20 control fetuses, the tracheal tube contained an equal amount of saline or was empty. Thirteen of the fetuses treated with surfactant survived for 3 to 40 hours after the operation, whereas all but one of the control fetuses died within 45 minutes after tracheostomy (P < 0.001). the unopened thorax of each fetus was fixed in formalin, and the air expansion of the lungs was evaluated histologically. Alveolar air expansion, varying in degree from slight to prominent, was apparent in all fetuses treated with surfactant. Among control fetuses, slight alveolar air expansion was observed in 3 cases, whereas in 17, the lungs w...

Effect of tracheal deposition of surfactant on air expansion of lungs--study on premature rabbit fetuses.

Effect of tracheal deposition of surfactant on air expansion of lungs--study on premature rabbit fetuses.

Pharyngeal Deposition of Surfactant in the Premature Rabbit Fetus

A concentrated surfactant suspension was deposited in the pharynx of premature rabbit fetuses, before the onset of breathing. This treatment improved the air expansion of the lungs and prolonged fetal survival. Possibly, a similar technique might be employed as a prophylactic measure against respiratory distress in premature infants.

LUNG EXPANSION IN THE PREMATURE RABBIT FETUS AFTER TRACHEAL DEPOSITION OF SURFACTANT

Concentrated "surfactant" was obtained by centrifugation of alveolar wash from adult rabbits for 1 hour at 4°C, 1,000xG, and deposited in the trachea of premature rabbit fetuses, killed at the gestational age of 28 days, i.e., 1 day before appreciable amounts of surface active phospholipids are naturally present in the pulmonary fluid. Pressure-volume tracings revealed a mature pattern in the surfactant-treated animals, with wide hysteresis. Significantly less air entered the lungs of control fetuses from the same litters, and the pressure-volume tracings from these animals showed a premature pattern, with minimal hysteresis. The lungs of the fetuses were fixed in formalin under the endotracheal deflation pressure of 10 cm H20. Histological examination of the lungs revealed moderate or prominent air expansion of the parenchyma in surfactant-treated animals, whereas no or only slight air expansion characterized the controls. Tracheal deposition of surfactant prior to the first breath thus seems to enhance the air expansion of the premature lung, and might possibly be applied as a method for prophylaxis against the idiopathic respiratory distress syndrome, which is related to deficiency of surface active phospholipids in the pulmonary fluid.