Paul-Georg Germann

Comparison of rSP-C surfactant with natural and synthetic surfactants after late treatment in a rat model of the acute respiratory distress syndrome

1 In a previous paper we showed that an SP‐C containing surfactant preparation has similar activity as bovine‐derived surfactants in a rat lung lavage model of the adult respiratory distress syndrome. In this study surfactant was given ten minutes after the last lavage (early treatment). In the present investigation we were interested how different surfactant preparations behave when they are administered 1 h after the last lavage (late treatment). 2 Four protein containing surfactants (rSP‐C surfactant, bLES, Infasurf and Survanta) were compared with three protein‐free surfactants (ALEC, Exosurf and the phospholipid (PL) mixture of the rSP‐C surfactant termed PL surfactant) with respect to their ability to improve gas exchange in this more stringent model when surfactant is given one hour after the last lavage. For better comparison of the surfactants the doses were related to phospholipids. The surfactants were given at doses of 25, 50 and 100 mg kg−1 body weight. The surfactants were compared to an untreated control group that was only ventilated for the whole experimental period. 3 Tracheotomized rats (8–12 per dose and surfactant) were pressure‐controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths min−1, inspiration expiration ratio of 1: 2, peak inspiratory pressure of 28 cmH2O at positive endexpiratory pressure (PEEP) of 8 cmH2O. Animals were ventilated for one hour after the last lavage and thereafter the surfactants were intratracheally instilled. During the whole experimental period the ventilation was not changed. 4 Partial arterial oxygen pressures (Pao2, mmHg) at 30 min and 120 min after treatment were used for statistical comparison. All protein containing surfactants caused a dose‐dependent increase of the reduced Pao2 values at 30 min after treatment. The protein‐free surfactants showed only weak dose‐dependent increase in Pao2 values at this time. This difference between the protein‐containing and the protein‐free surfactants was even more pronounced when comparing the Pao2 values at 120 min after treatment. Only rSP‐C surfactant, bLES and Infasurf showed a dose‐dependent increase in Pao2 at this time. 5 With this animal model of late treatment it is possible even to differentiate between bovine derived surfactants. The differences between protein‐containing and protein‐free surfactants become even more pronounced. From the comparison of rSP‐C surfactant with bovine‐derived surfactants and the PL surfactant without rSP‐C, it can be concluded that addition of rSP‐C is sufficient to achieve the same activity as that of natural surfactants.

A rat model of acute respiratory distress syndrome (ARDS): Part 1. Time dependency of histological and pathological changes.

The time course of histopathological changes in a rat lung lavage model of the acute respiratory distress syndrome (ARDS) was analyzed by sacrificing animals 10, 30, 60, 180, and 210 min after the last lung parenchyma lavage which was performed with physiological saline solution. This lavage depleted the lung from its natural surfactant resources leading into a pathophysiological cascade similar to that of the acute respiratory distress syndrome. Tracheotomized rats (12 animals per time point) were pressure-controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths/min, inspiration-expiration ratio of 1:2, peak inspiratory pressure of 28 cm H2O at positive end-expiratory pressure (PEEP) of 8 cm H2O. During the whole experimental period, the ventilation was not changed. Blood gases (partial arterial oxygen pressures [PaO2, mmHg] and partial arterial carbon dioxide pressures [PaCO2, mmHg]) were estimated before, directly after, and 10, 30, 60, 90, 120, 150, 180, and 210 min after the last lavage. For grading lung lavage-induced histopathological changes associated with the time-dependent development of ARDS, slides were coded and evaluated without any knowledge of the sacrifice time. A semiquantitative grading was performed with respect to the severity of the following parameters: hyaline membrane formation (HM), interstitial and intraalveolar edema edema (E), and margination and infiltration of polymorphonuclear neutrophil leukocytes (PMNL) into the lung alveoli. The severity of these parameters showed a time-dependent increase after the last lavage. This was accompanied by a time-dependent decrease in partial arterial oxygen pressure (PaO2) values during the early postlavage period (up to 30 min). Thereafter, PaO2 levels remained fairly stable. The severity of intraalveolar and/or perivascular hemorrhages within the lung was not time dependent. The rat lavage model shows similarities to the pathophysiological sequelae occuring during the acute phase of the acute respiratory distress syndrome in humans. Most of the characteristic pathognomic histological changes seen in humans can be observed in this lung lavage model. This ARDS model is brief and easy in its experimental design, showed a good and homogeneous reproducibility of pathophysiological and histopathological parameters, and is therefore a useful model to estimate the influence of therapeutic pharmacological treatments of ARDS.

A RAT MODEL OF ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS). PART 2, INFLUENCE OF LAVAGE VOLUME, LAVAGE REPETITION, AND THERAPEUTIC TREATMENT WITH RSP-C SURFACTANT

UNLABELLED The influence of lavage volume, and lavage repetition with physiological saline solution (groups 1-3: 3x4, 4x4, 5x4, groups 7-9: 3x8, 5x8, 7x8, mL per animal) was studied in a rat lung lavage model of the acute respiratory distress syndrome (ARDS). Anesthetized and tracheotomized rats (12 rats/group) were pressure-controlled ventilated with 100% oxygen at a respiratory rate of 30 breaths/min, inspiration: expiration ratio of 1:2, peak inspiratory pressure of 28 cm H2O at positive end-expiratory pressure of 8 cm H2O during the whole experimental period. To investigate the influence of therapeutic treatment, a recombinant surfactant protein C (rSP-C) containing surfactant was used. Therefore, rats which received a lavage of 4x4 mL per animal (groups 4 to 6) or 7x8 mL per animal (groups 10-12) were treated intratracheally with surfactant doses of 12.5, 25, or 100 mg phospholipids (PL) per kg body weight (bw). In all groups, partial arterial oxygen pressures (PaO2, mm Hg) and partial arterial carbon dioxide pressures (PaCO2, mm Hg) were determined 30 min before, directly after, and 5, 30, 60, 90, 120, 150, 180, and 210 min after the last lavage. Additionally, animals were euthanized 210 min after the last lavage for semiquantitative histopathological grading of coded lung slides. Grading was performed with respect to the severity of hyaline membrane formation (HM), margination and infiltration of polymorphonuclear neutrophil leukocytes (PMNL) into the lung alveoli and interstitial and intraalveolar edema (E). The intrapulmonary distribution of intratracheally applied rSP-C was estimated in selected lung slides stained with polyclonal anti-rSP-C antibody and was compared to unlavaged control rats and unlavaged rats which received 100 mg/kg bw rSP-C. The repetitive lavage depleted the lung from its natural surfactant resources leading to a pathophysiological cascade similar to that of the acute respiratory distress syndrome. PaO2 levels and HM formation showed a lavage-induced decrease. Both changes were significantly dependent on the repetition and volume of the lavage; however, the parameters PMNL and E did not show such a dependence. Treatment with rSP-C surfactant significantly improved oxygenation and reduced HM-formation in a dose-dependent manner independent from the lavage volume. All doses of rSP-C surfactant showed no clear influence on the parameters PMNL and E independently from the lavage volume. In lavaged rat lungs (ARDS-model), the exogenously applied rSP-C was distributed homogeneously along the alveolar lining. Unlavaged rats that received a similar dose of rSP-C showed a marked inhomogeneous extracellular distribution, mainly associated with larger bronchi, while the type II pneumocytes were stained positively in unlavaged control and unlavaged rSP-C treated rats. CONCLUSION This model mimics very closely the wide spectrum of the clinical situation of human acute lung injury (ALI) because the variation of lavage volume and repetition lead to reproducible different severity grades and states of ALI. The significant reduction of pathognomic changes due to treatment with rSP-C surfactant showed that this is a useful model to estimate the influence of therapeutic concepts in ALI and ARDS.

Development of neutralizing antibodies correlates with resolution of interstitial pneumonia after measles virus infection in cotton rats

Abstract Cotton rats (Sigmodon hispidus, inbred strain COTTON/NIco) have been shown to be a good animal model to investigate measles virus (MV) immune suppression and to assess alternative vaccine candidates against MV infection. Here we demonstrate that cotton rats develop a bronchusassociated and interstitial pneumonia with necrotic lesions after intranasal infection resembling findings from fatal cases of human MV pneumonia. In the absence of superinfection restitutio ad integrum is observed and overcoming of lung infection correlates with the development of MV specific neutralizing antibodies.

Cyclooxygenase-inhibition enhances the effects of rSP-C surfactant therapy in a rat lavage model of acute respiratory distress syndrome (ARDS).

UNLABELLED The effects of additional i.v. therapy with a cyclooxygenase-inhibitor Eltenac to a recombinant surfactant protein C (rSP-C) based surfactant were investigated in a rat lung lavage model of acute lung injury. Treatment was done at 60 min after the induction of acute lung injury by lavage. The influence of the different treatments were tested with regard to improving oxygenation, histopathological changes (hyaline membrane formation and alveolar influx of neutrophil leukocytes). These effects were further compared to a fixed combination of Eltenac with rSP-C surfactant which was administered intratracheally (i.tr.), 60 min after lavage. To prove that fibrinogen is involved in the formation of hyaline membranes in this animal model confocal microscopy was applied. Furthermore, for selected cases the influence of Eltenac or rSP-C surfactant on fibrinogen leakage was investigated by using confocal microscopy. Results of additional i.v. therapy exhibited an improved oxygenation with rSP-C surfactant, while a high dose of Eltenacalone did not influence oxygenation as compared to untreated controls. Addition of Eltenac lead to improved oxygenation using the low dose of rSP-C surfactant. The rSP-C surfactant prevented further hyaline membrane formation. Furthemore, addition of Eltenac to the low dose of rSP-C surfactant lead to improved hyaline membrane formation at a dose of 100 micromol/kg b.w. Results of combined i.tr. therapy confirmed the results of the additional therapy. Again, rSP-C surfactant improved oxygenation and further hyaline membrane formation, while even the high dose of i.tr. administered Eltenacalone only prevented further hyaline membrane formation. Using the low dose of rSP-C surfactant, combined treatment with Eltenac showed additional effects on oxygenation and inhibition of hyaline membrane formation. The maximum therapeutic effect of combined treatment was achieved at 0.3 mg Eltenac per kg b.w. which is equivalent to approximately 1 micromol. The inflammatory cell infiltration into the lung was not influenced by any of the therapeutic approaches. Confocal microscopy gave evidence that fibrinogen is involved in hyaline membrane formation in this animal model. Furthermore, as was shown by the explorative investigations with confocal microscopy, addition of the cyclooxygenase-inhibitor decreases the diffuse interstitial leakage of fibrinogen into the lung while surfactant monotherapy did not exhibit any influence on the fibrinogen influx into the alveoli. CONCLUSIONS Confocal microscopy may be an effective method to investigate the connection between fibrinogen leakage and hyaline membrane formation. Effects of additional or combined treatment were superior when compared to each treatment alone leading to the conclusion that a rSP-C surfactant containing a cyclooxygenase-inhibitor, acts synergistically in this animal model of acute lung injury. Lower doses of Eltenac could be used to reach similar effects on oxygenation and prevention of hyaline membrane after combined i.tr. treatment than after additional i.v. treatment together with surfactant. This leads to the conclusion that a fixed combination of rSP-C surfactant and a cyclooxygenase-inhibitor may be an effective treatment. Further testing may be warranted to prove whether this is a promising treatment for patients with acute lung injury.

Practical Aspects of Discovery Pathology

Pathologists are uniquely qualified to play a central role in driving drug discovery and development programs by: 1) establishing disease models to assess potential therapies, 2) characterizing modifications in the disease state in response to therapies, 3) characterizing toxicologic mechanisms and responses to drug candidates, and 4) facilitating multidisciplinary efforts to monitor for the clinical occurrence, progression, and reversibility of adverse events. Such nontraditional deployment of resources must, to be viable, produce benefits to the pharmaceutical industry comparable to those of more conventional activities such as delivery of data in nonclinical safety studies. Additionally, benefits must be tangible from standpoints such as timesavings or improved quality of research decisions, manifesting as either program acceleration or improved candidate survival.