[Show abstract][Hide abstract] ABSTRACT: Inflammatory cell numbers are important endpoints in clinical studies relying on endobronchial biopsies. Assumption-based bidimensional (2D) counting methods are widely used, although theoretically design-based stereologic three-dimensional (3D) methods alone offer an unbiased quantitative tool. We assessed the method agreement between 2D and 3D counting designs in practice when applied to identical samples in parallel.
Biopsies from segmental bronchi were collected from healthy non-smokers (n = 7) and smokers (n = 7), embedded and sectioned exhaustively. Systematic uniform random samples were immunohistochemically stained for macrophages (CD68) and T-lymphocytes (CD3), respectively. In identical fields of view, cell numbers per volume unit (NV) were assessed using the physical disector (3D), and profiles per area unit (NA) were counted (2D). For CD68+ cells, profiles with and without nucleus were separately recorded. In order to enable a direct comparison of the two methods, the zero-dimensional CD68+/CD3+-ratio was calculated for each approach. Method agreement was tested by Bland-Altmann analysis.
In both groups, mean CD68+/CD3+ ratios for NV and NA were significantly different (non-smokers: 0.39 and 0.68, p<0.05; smokers: 0.49 and 1.68, p<0.05). When counting only nucleated CD68+ profiles, mean ratios obtained by 2D and 3D counting were similar, but the regression-based Bland-Altmann analysis indicated a bias of the 2D ratios proportional to their magnitude. This magnitude dependent deviation differed between the two groups.
2D counts of cell and nuclear profiles introduce a variable size-dependent bias throughout the measurement range. Because the deviation between the 3D and 2D data was different in the two groups, it precludes establishing a 'universal conversion formula'.
PLoS ONE 01/2014; 9(3):e92510. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The authors investigated the protective effects and dose dependency of perfluorohexane (PFH) vapor on leukocyte-mediated lung injury in isolated, perfused, and ventilated rabbit lungs. Lungs received either 18 vol.% (n = 7), 9 vol.% (n = 7), or 4.5 vol.% (n = 7) PFH. Fifteen minutes after beginning of PFH application, lung injury was induced with formyl-Met-Leu-Phe (fMLP). Control lungs (n = 7) received fMLP only. In addition 5 lungs (PFH-sham) remained uninjured receiving 18 vol.% PFH only. Pulmonary artery pressure (mPAP), peak inspiratory pressure (P(max)), and lung weight were monitored for 90 minutes. Perfusate samples were taken at regular intervals for analysis and representative lungs were fixed for histological analysis. In the control, fMLP application led to a significant increase of mPAP, P(max), lung weight, and lipid mediators. Pretreatment with PFH attenuated the rise in these parameters. This was accompanied by preservation of the structural integrity of the alveolar architecture and air-blood barrier. In uninjured lungs, mPAP, P(max), lung weight, and lipid mediator formation remained uneffected in the presence of PFH. The authors concluded that pretreatment with PFH vapor leads to an attenuation of leukocyte-mediated lung injury. Vaporization of perfluorocarbons (PFCs) offers new therapeutic options, making use of their protective and anti-inflammatory properties in prophylaxis or in early treatment of acute lung injury.
Experimental Lung Research 08/2010; 36(6):342-51. · 1.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regeneration of the gas exchange area by induction of neoalveolarisation would greatly improve therapeutic options in destructive pulmonary diseases. Unilateral pneumonectomy is an established model to remove defined portions of gas exchange area and study mechanisms of compensatory lung growth. The question of whether new alveoli are added to the residual lung after pneumonectomy in mice was addressed. Left-sided pneumonectomy was performed in 11 adult C57BL/6 mice. Alveolar numbers were analysed in lungs fixed at days 6 and 20 after pneumonectomy and in 10 age-matched controls using design-based stereology based on a physical fractionator. Post-fixation lung volume was determined by fluid displacement. Complete restoration of lung volume was observed 20 days after pneumonectomy. Alveolar numbers were significantly increased by 33% in residual right lungs at day 20 in comparison with control right lungs. In control left lungs, an average of 471+/-162 x 10(3) alveoli was estimated, 49% of which were regenerated by residual lungs at day 20. Of the newly formed alveoli seen at day 20, 74% were already present at day 6. The present data demonstrate that, in addition to growth in size of existing alveoli, neoalveolarisation contributes to restoration of the gas exchange area in adult mice and is induced early after pneumonectomy.
European Respiratory Journal 04/2008; 31(3):515-22. · 6.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prophylactic exogenous surfactant therapy is a promising way to attenuate the ischemia and reperfusion (I/R) injury associated with lung transplantation and thereby to decrease the clinical occurrence of acute lung injury and acute respiratory distress syndrome. However, there is little information on the mode by which exogenous surfactant attenuates I/R injury of the lung. We hypothesized that exogenous surfactant may act by limiting pulmonary edema formation and by enhancing alveolar type II cell and lamellar body preservation. Therefore, we investigated the effect of exogenous surfactant therapy on the formation of pulmonary edema in different lung compartments and on the ultrastructure of the surfactant producing alveolar epithelial type II cells.
Rats were randomly assigned to a control, Celsior (CE) or Celsior + surfactant (CE+S) group (n = 5 each). In both Celsior groups, the lungs were flush-perfused with Celsior and subsequently exposed to 4 h of extracorporeal ischemia at 4 degrees C and 50 min of reperfusion at 37 degrees C. The CE+S group received an intratracheal bolus of a modified natural bovine surfactant at a dosage of 50 mg/kg body weight before flush perfusion. After reperfusion (Celsior groups) or immediately after sacrifice (Control), the lungs were fixed by vascular perfusion and processed for light and electron microscopy. Stereology was used to quantify edematous changes as well as alterations of the alveolar epithelial type II cells.
Surfactant treatment decreased the intraalveolar edema formation (mean (coefficient of variation): CE: 160 mm3 (0.61) vs. CE+S: 4 mm3 (0.75); p < 0.05) and the development of atelectases (CE: 342 mm3 (0.90) vs. CE+S: 0 mm3; p < 0.05) but led to a higher degree of peribronchovascular edema (CE: 89 mm3 (0.39) vs. CE+S: 268 mm3 (0.43); p < 0.05). Alveolar type II cells were similarly swollen in CE (423 microm3(0.10)) and CE+S (481 microm3(0.10)) compared with controls (323 microm3(0.07); p < 0.05 vs. CE and CE+S). The number of lamellar bodies was increased and the mean lamellar body volume was decreased in both CE groups compared with the control group (p < 0.05).
Intratracheal surfactant application before I/R significantly reduces the intraalveolar edema formation and development of atelectases but leads to an increased development of peribronchovascular edema. Morphological changes of alveolar type II cells due to I/R are not affected by surfactant treatment. The beneficial effects of exogenous surfactant therapy are related to the intraalveolar activity of the exogenous surfactant.
Respiratory research 02/2008; 9:5. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary graft dysfunction, characterised by intra-alveolar oedema, is a major obstacle in pulmonary transplantation. The present study evaluates the potential of keratinocyte growth factor (palmiferin; DeltaN23-KGF) for the prevention of oedema in lung transplants. Intratracheal instillation of 5 mg x kg(-1) DeltaN23-KGF was performed in Lewis rats on days 3 and 2 before explantation. Control animals obtained an equivalent volume of vehicle. Left lungs were isogeneically transplanted and the graft recipients were sacrificed 1 day later for stereological analysis of intra-alveolar oedema and bronchoalveolar lavage. The total protein and phospholipid content, as well as surfactant proteins, were measured. Surfactant activity was analysed with a pulsating bubble surfactometer. In grafts from control treated donors, the fraction of intra-alveolar oedema amounted to 3.4+/-1.1% of the total parenchymal volume. Treatment of donor lungs with DeltaN23-KGF reduced oedema to a fraction of 1.6+/-0.8%. In the lavage fluid of pulmonary grafts from DeltaN23-KGF-treated donors, the total protein content was decreased compared with vehicle-treated lung transplants, whereas phospholipids did not differ. The protein fraction contained increased amounts of surfactant protein-C after DeltaN23-KGF treatment and surfactant function was improved. Treatment of donor lungs with palifermin protects against intra-alveolar oedema formation upon transplantation. This effect appears to be mediated by an improved surfactant homeostasis.
European Respiratory Journal 02/2008; 31(1):21-8. · 6.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantitative (immuno) transmission electron microscopy using design-based stereology was performed on specimens collected by means of systematic uniform random sampling of rat lungs, which were fixed by vascular perfusion to stabilize intra-alveolar surfactant in situ. This procedure ensures that the data recorded are representative of the whole organ. Ultrathin sections of specimens embedded at low temperature in Lowicryl HM20 were labeled by indirect immuno-gold staining for surfactant protein A. We observed that, 3 days after treatment of lungs in vivo with truncated keratinocyte growth factor (DeltaN23-KGF), a potent mitogen of alveolar epithelial type II cells, surfactant protein A associated with the tubular myelin fraction of intra-alveolar surfactant was increased by 47% in comparison with buffer-treated control lungs. Despite the marked type II cell hyperplasia, the relative amount of ultrastructural surfactant subtypes was not significantly affected. Because surfactant protein A reduces the sensitivity to inhibition of the biophysical activity of surfactant by exudating plasma proteins, we propose that pretreatment of lungs with DeltaN23-KGF ameliorates adverse effects observed in acute lung injury following, for example, ischemia and reperfusion.
The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology 09/2007; 290(8):974-80. · 1.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis of alveolar septal cells has been linked to emphysema formation. Nitrogen dioxide, a component of cigarette smoke, has been shown to induce alveolar epithelial cell apoptosis in vitro. It is hypothesised that exposure of rats to nitrogen dioxide may result in increased alveolar septal cell apoptosis in vivo with ensuing emphysema-that is, airspace enlargement and loss of alveolar walls.
Fischer 344 rats were exposed to 10 ppm nitrogen dioxide for 3, 7, 21 days or 21 days followed by 28 days at room air. Age-matched control rats were exposed to room air for 3, 21 or 49 days. Lungs fixed at 20 cm fluid column, embedded in paraffin wax, glycol methacrylate and araldite, were analysed by design-based stereology. Alveolar septal cell apoptosis (transferase dUTP nick end labelling assay, active caspase 3) and proliferation (Ki-67), airspace enlargement, total alveolar surface area, and absolute alveolar septal volume as well as the ultrastructural composition of the alveolar wall were quantified.
Nitrogen dioxide resulted in an eightfold increase in alveolar septal cell apoptosis at day 3 and a 14-fold increase in proliferation compared with age-matched controls. Airspace enlargement, indicated by a 20% increase in mean airspace chord length, was evident by day 7 but was not associated with loss of alveolar walls. By contrast, nitrogen dioxide resulted in an increase in the total surface area and absolute volume of alveolar walls comprising all compartments. The ratio of collagen to elastin, however, was reduced at day 21. Lungs exposed to nitrogen dioxide for 21 days exhibited quantitative structural characteristics as seen in control lungs on day 49.
Nitrogen dioxide exposure of rats results in increased alveolar septal cell turnover leading to accelerated lung growth, which is associated with an imbalance in the relative composition of the extracellular matrix, but fails to induce emphysema.
[Show abstract][Hide abstract] ABSTRACT: Bronchial asthma is characterized by chronic airway inflammation and airway remodelling which occurs in both proximal and distal airways. These changes are associated with development of airway hyper-responsiveness and airflow limitation.
This study was aimed to analyse whether chronic inhalative allergen challenges in mice lead to morphological and physiological changes comparable with this phenotype.
For this purpose, BALB/c mice were systemically sensitized to ovalbumin (OVA) followed by aerosol allergen challenges on 2 consecutive days per week for 12 weeks.
In chronically challenged mice, tissue inflammation in proximal as well as distal airways was observed with a predominance of lymphocytes within the cellular infiltrate. In contrast, inflammation in the airway lumen decreased over time. These changes were associated by a shift in bronchoalveolar lavage-cytokine levels from IL-4, IL-5 and TNF-alpha production (during the acute phase) towards markedly increased levels of TGF-beta during the chronic phase. Goblet cell hyperplasia and subepithelial fibrosis occurred throughout the airway tree. In terms of lung function, chronically challenged mice developed persistent bronchial hyper-responsiveness and progressive airflow limitation. Six weeks after OVA aerosol discontinuation, airway inflammation still persisted although lung function was normalized.
These data indicate that our model of chronic aerosol allergen challenges leads to a phenotype of experimental asthma with participation of distal airways and persistence of inflammation thereby resembling many morphological and physiological aspects of human bronchial asthma.
[Show abstract][Hide abstract] ABSTRACT: BackgroundOptimal preservation of allograft integrity is essential to reduce post-ischemic organ dysfunction after lung transplantation. Retrograde organ preservation leads to homogeneous intrapulmonary distribution and eliminates intravascular thrombi. So far, no comparative studies exist with regard to preservation quality following retrograde preservation with Perfadex and Celsior after extended cold-ischemia intervals.MethodsIn an in vivo pig model, 5 lungs each were preserved for 27 hours using antegrade or retrograde perfusion techniques with Celsior (Ceant/CEret) and Perfadex (PERant/PERret). After left lung transplantation and contralateral lung exclusion, hemodynamics, oxygenation and dynamic compliance were monitored for 6 hours and compared with sham-operated controls. Pulmonary edema was determined stereologically. Statistics consisted of analysis of variance (ANOVA) with repeated measures.ResultsMortality of all Celsior-protected lungs was 100% due to severe reperfusion injury with profound lung edema. In contrast, organ preservation with PERant led to sufficient graft function without mortality. Preservation quality after retrograde administration of Perfadex resulted in optimized oxygenation capacity compared with PERant (p = 0.046). Furthermore, intra-alveolar edema was reduced and generally comparable with sham controls. In general, retrograde preservation led to continuous washout of small blood and fibrin clots from the pulmonary capillary system.ConclusionsPerfadex solution provided sufficient lung preservation for 27 hours of cold ischemia, and its retrograde application led to significant functional and histologic improvement compared with antegrade perfusion. In contrast, preservation with Celsior solution resulted in lethal post-ischemic outcome, regardless of the route of administration, and therefore must be considered unsuitable for extended lung procurement.
The Journal of Heart and Lung Transplantation 09/2005; · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ischemia-reperfusion injury accounts for one-third of early deaths after lung transplantation. To expand the limited donor pool, lung retrieval from non-heart beating donors (NHBD) has been introduced recently. However, because of potentially deleterious effects of warm ischemia on microvascular integrity, use of NHBD lungs is limited by short tolerable time periods before preservation. After intravenous prostanoids are routinely used to ameliorate reperfusion injury, the latest evidence suggests similar efficacy of inhaled prostacyclin. Therefore, the impact of donor pretreatment with the prostacyclin analogue iloprost on postischemic NHBD lung function and preservation quality was evaluated.
Asystolic pigs (5 per group) were ventilated for 180 minutes of warm ischemia (Group 2). In Group 3, 100 microg iloprost was aerosolized during the final 30 minutes of ventilation with a novel mobile ultrasonic nebulizer. Lungs were then retrogradely preserved with Perfadex and stored for 3 hours. After left lung transplantation and contralateral lung exclusion, hemodynamics, rO2/FiO2, and dynamic compliance were monitored for 6 hours and compared with sham-operated controls (Group 1). Pulmonary edema was determined both stereologically and by wet-to-dry weight ratio (W/D). Statistics comprised analysis of variance with repeated measures and Mann-Whitney test.
Flush preservation pressures, dynamic compliance, inspiratory pressures, and W/D were significantly superior in iloprost-treated lungs, and oxygenation and pulmonary hemodynamics were comparable between groups. Stereology revealed a trend toward lower intraalveolar edema formation in iloprost-treated lungs compared with untreated grafts.
Alveolar deposition of Iloprost and NHBD lungs before preservation ameliorates postischemic edema and significantly improves lung compliance. This easily applicable innovation approach, which uses a mobile ultrasonic nebulizer, offers an important strategy for improvement of pulmonary preservation quality and might expand the pool of donor lungs.
The Journal of Heart and Lung Transplantation 05/2005; 24(4):371-8. · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major features of chronic obstructive pulmonary disease (COPD) comprise a not fully reversible airflow limitation associated with an abnormal inflammatory response, increased mucus production and development of emphysema-like lesions. Animal models that closely mimic these alterations represent an important issue for the investigation of pathophysiological mechanisms. Since most animal models in this area have focused on specific aspects of the disease, we aimed to investigate whether exposure of C57BL/6 mice to nitrogen dioxide (NO2) may cause a more complex phenotype covering several of the characteristics of the human disease. Therefore, mice were exposed to NO2 for 14h each day for up to 25 days. Initial dose response experiments revealed the induction of a significant inflammatory response at a dose of 20 ppm NO2. Mice developed progressive airway inflammation together with a focal inflammation of the lung parenchyma characterized by a predominant influx of neutrophils and macrophages. In addition, goblet cell hyperplasia was detected in the central airways and increased collagen deposition was found in the lung parenchyma. NO2-exposed mice developed emphysema-like lesions as indicated by a significantly increased mean linear intercept as compared to control mice. Finally, the assessment of lung functional parameters revealed the development of progressive airway obstruction over time. In conclusion, our data provide evidence that the inflammatory response to NO2 exposure is associated with increased mucus production, development of airspace enlargement and progressive airway obstruction. Thus, NO2-exposed mice may serve as a model to investigate pathophysiological mechanisms that contribute to the development of human COPD.
Experimental and Toxicologic Pathology 05/2005; 56(6):341-50. · 2.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Declining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.
Using immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.
After IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.
We suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).
Respiratory research 02/2005; 6:60. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A model of inducible expansion of the gas exchange area in adult mice would be ideal for the investigation of molecular determinants of airspace regeneration in vivo. Therefore, the post-pneumonectomy (post-PNX) compensatory lung growth in adult C57BL/6 mice was characterised in this study. Mice underwent left-sided PNX. Right lung volume was assessed on days 1, 3, 5, 7, 10 and 21 after PNX, and total DNA and cellular proliferation of the right lung were determined. Lung histology was studied using immunohistochemistry and quantitatively characterised by detailed stereological investigations. Pulmonary function was assessed using a mouse body-plethysmograph. Following PNX, right-lung volume rapidly restored the initial volume of left and right lung. Total DNA increased significantly over 21 days and equalled the total DNA amount of both lungs in the control mice. Septal cell proliferation significantly increased after PNX, and included endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. Stereological investigations of left and right control lungs versus right lungs 21 days after PNX indicated complete restoration of body mass-specific alveolar surface area. Pulmonary function testing showed marked alteration at 3 days and normalisation at 21 days post-PNX. In conclusion, well reproducible reconstitution of alveolar gas-exchange surface based on septal tissue expansion may be provoked by pneumonectomy in adult mice.
European Respiratory Journal 11/2004; 24(4):524-32. · 6.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lung transplantation is limited by scarcity of donor organs. Lung retrieval from non-heart-beating donors (NHBD) might have the potential to extend the donor pool and has been reported recently. However, no studies in NHBD exist using the novel approach of retrograde preservation with Perfadex solution.
Asystolic heparinized pigs (n = 5/group) were continuously ventilated for 90, 180 or 300 min of warm ischemia. Lungs were then retrogradely preserved with Perfadex and stored at 4 degrees C in inflation. After 3 h of additional cold ischemia, left lung transplantation was performed. Hemodynamics, pO(2)/F(i)O(2) and dynamic compliance were monitored for 5 h. Intrapulmonary lung water was determined by both global wet-to-dry lung weight ratio (W/D ratio) and standard stereological examination of relative volume fractions of intraalveolar edema. All results were compared to sham-operated controls and to lungs obtained from standard heart-beating donors after retrograde preservation with Perfadex and 27 h of cold ischemia. Statistics comprised ANOVA analysis with repeated measures and Mann-Whitney tests.
No mortality was observed. During flush preservation of NHBD lungs, continuous elimination of blood clots via the pulmonary artery was observed. Oxygenation, compliance, intraalveolar edema fraction and W/D ratio were comparable between groups, whereas PVR was significantly lower in sham-controls.
Use of NHBD lungs is feasible and results in similar postischemic outcome when compared to sham-controls and standard preservation procedures even after 5 h of pre-harvest warm ischemia. Especially, the NHBD with high-risk constellations for intravascular coagulation might benefit from retrograde preservation by elimination of thrombi from the pulmonary circulation. This innovative technique might also be considered in situations, where brain-dead organ donors become hemodynamically unstable prior to onset of organ harvest. Further trials with longer warm and cold ischemic periods are initiated to further elucidate this promising approach of donor pool expansion.
European Journal of Cardio-Thoracic Surgery 08/2004; 26(1):144-50. · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Optimal preservation of postischemic organ function is a continuing challenge in clinical lung transplantation. Retrograde instillation of preservation solutions has theoretical advantages to achieve a homogeneous distribution in the lung due to perfusion of both the pulmonary and the bronchial circulation. Thus far, no systematic screening studies followed by in vivo large animal reevaluation including stereological analysis of intrapulmonary edema exist concerning the influence of retrograde preservation on postischemic lung function after preservation with low potassium dextran (LPD) solution (Perfadex).
For initial screening in an extracorporeal rat model eight lungs, each, were preserved for 4 h using antegrade or retrograde preservation with LPD solution (Perfadex; PER(ant)/PER(ret)). Respiratory and hemodynamic results after reperfusion were compared to low-potassium Euro-Collins (LPEC). For systematic reevaluation, five pig lungs, each, were preserved correspondingly for 27 h, and results were compared to sham-operated control lungs. In both models, edema formation was quantified stereologically. Statistics comprised different ANOVA models.
In both models, use of PER(ret) resulted in significantly higher oxygenation capacity, lower inspiratory pressures, and lower amounts of intraalveolar edema as compared to PER(ant). Results of PER(ret) were not different from sham controls in the in vivo model; furthermore, a continuous retrograde elimination of blood clots from pulmonary microcirculation was noticed.
Retrograde application of LPD solution (Perfadex) results in significant functional and histological improvement as compared to antegrade perfusion. This innovative technique can be applied very easily in clinical practice and might be an ideal adjunct to further optimize the results after lung transplantation with LPD-based graft protection.
Journal of Surgical Research 05/2004; 117(2):239-48. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Keratinocyte growth factor (KGF) is a mitogen for pulmonary epithelial cells. Intratracheal administration of KGF to adult rats results in alveolar epithelial type II and bronchiolar epithelial cell proliferation. While cellular responses to KGF have been intensively studied, functional consequences regarding lung function are unknown. Therefore, in this study, we sought to investigate whether KGF alters pulmonary function variables. Rats received either recombinant human KGF (rHuKGF) (5 mg/kg) or vehicle intratracheally. Before and on days 3 and 7 after treatment, pulmonary function was determined by body plethysmography. Subsequently, lung histological changes were quantified. rHuKGF induced a transient proliferation of alveolar and bronchiolar epithelial cells. The extent of type II cell hyperplasia was significantly correlated with a transient reduction in tidal volume and an increase in breathing frequency. In addition, quasi-static compliance, total lung capacity, and vital capacity were reduced after rHuKGF instillation, suggesting the development of a transitory restrictive lung disorder. Moreover, reduced expiratory flow rates and forced expiratory volumes, as well as increased functional residual capacity after rHuKGF but not vehicle, suggest obstructive lung function changes. In conclusion, the induction of alveolar and bronchiolar epithelial cell proliferation by KGF is paralleled by moderate functional consequences that should be taken into account when the therapeutic potential of KGF is tested.
Journal of Applied Physiology 03/2004; 96(2):704-10. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lung transplantation is limited by the scarcity of donor organs. Lung retrieval from non-heart-beating donors (NHBD) might extend the donor pool and has been reported recently. However, no studies in NHBD exist using the novel approach of retrograde preservation with Perfadex solution.
Heparinized asystolic pigs (n = 5, 30-35 kg) were ventilated for 90 min. The lungs were retrogradely preserved with Perfadex solution and stored inflated at 4 degrees C for 3 h. Left lung transplantation in the recipient was followed by exclusion of the right lung. Results were compared to sham-operated animals. Oxygenation, hemodynamics and dynamic compliance were monitored for 4 h. Infiltration of polymorphonuclear cells (PMNs) and stereological quantification of alveolar edema was performed. Statistical analysis comprised Kruskal-Wallis and Mann-Whitney tests and ANOVA analysis with repeated measures.
No mortality was observed. During preservation, continuous elimination of blood clots via the pulmonary artery venting site was observed. Oxygenation and compliance were similar between groups, but sham controls showed significantly lower pulmonary vascular resistance. Stereological quantification revealed higher volume fractions of intra-alveolar edema in NHBD grafts, while PMN infiltration was comparable to sham controls.
Use of NHBD lungs results in excellent outcome after 90 min of warm ischemia followed by retrograde preservation with Perfadex solution. This novel approach can optimize lung preservation by eliminating clots from the pulmonary circulation and might clinically be considered in brain-dead organ donors who become hemodynamically unstable prior to organ harvest. Further trials with longer warm and cold ischemic periods are necessary to further elucidate this promising approach to donor pool expansion.
European Surgical Research 01/2004; 36(1):1-7. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After clinical lung transplantation, the amount of vascular endothelial growth factor (VEGF) was found to be decreased in the bronchoalveolar lavage from lungs with acute lung injury. Since Type II pneumocytes are a major site of VEGF synthesis, VEGF depression may be an indicator of pulmonary epithelial damage after ischemia and reperfusion.
Using an established rat lung model, we investigated the relationship between VEGF protein expression, oxygenation capacity and structural integrity after extracorporeal ischemia and reperfusion (ischemia 6 hours at 10 degrees C, reperfusion 50 minutes) and preservation with either low-potassium dextran solution (Perfadex 40 kD, n = 8) or Celsior (n = 6). Untreated, non-ischemic lungs served as controls (n = 5 per group). Perfusate oxygenation was recorded during reperfusion. An enzyme-linked immunoassay (ELISA) for VEGF protein and reverse transcription-polymerase chain reaction (RT-PCR) for mRNA splice variants were determined on tissue collected from the left lungs, whereas the right lungs were fixed by vascular perfusion for VEGF immunohistochemistry as well as structural analysis by light and electron microscopy. Tissue collection by systematic uniform random sampling was representative for the whole organ and allowed for quantification of structures by stereological means.
After ischemia and reperfusion, the 3 major VEGF isoforms, VEGF(120), VEGF(164) and VEGF(188), were present. VEGF protein expression was reduced, which correlated significantly with perfusate oxygenation (r = 0.736; p = 0.002) at the end of reperfusion. It was inversely related to Type II cell volume (r = 0.600; p = 0.047). VEGF protein was localized by immunohistochemistry in Type II pneumocytes, alveolar macrophages as well as bronchial epithelium, and staining intensity of Type II cells was reduced after ischemia and reperfusion. Alveolar edema did not occur but significant interstitial edema accumulated around vessels and in the blood-gas barrier, which showed a higher degree of epithelial damage after preservation with Celsior compared with the other groups.
Depression in VEGF protein expression can be considered an indicator for increased alveolar epithelial damage. Preservation with low-potassium dextran solution resulted in improved oxygenation and tissue integrity compared with Celsior.
The Journal of Heart and Lung Transplantation 10/2003; 22(9):967-78. · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Optimal preservation of postischemic graft function is essential in lung transplantation. Antegrade flush perfusion with modified Euro-Collins solution represents the standard technique worldwide. However, growing evidence suggests the superiority of extracellular-type Perfadex solution (Vitrolife AB, Gothenburg, Germany) over Euro-Collins solution. During ischemia and reperfusion, endogenous pulmonary nitric oxide synthesis is decreased, and therefore therapeutic stimulation of the nitric oxide pathway might be beneficial in ameliorating ischemia-reperfusion damage. However, research mainly focuses on nitric oxide supplementation of intracellular solutions, and no studies exist in which the effect of nitroglycerin on Perfadex preservation quality is evaluated.
Eight rat lungs each were preserved with Perfadex solution with or without nitroglycerin (0.1 mg/mL) and compared with low-potassium Euro-Collins solution. Postischemic lungs were reventilated and reperfused, and oxygenation capacity, pulmonary vascular resistance, and peak inspiratory pressures were monitored continuously. Stereological analysis was used for evaluation of pulmonary edema and assessment of the vasculature. Statistics were performed by using different analysis of variance models.
The oxygenation capacity of the Perfadex-preserved groups was higher compared with that of the low-potassium Euro-Collins solution group (P <.03). By using nitroglycerin, flush-perfusion time was reduced, and Perfadex solution with nitroglycerin-protected lungs showed superior oxygenation capacity compared with that seen in Perfadex solution-protected organs (P <.01). Furthermore, pulmonary vascular resistance and peak inspiratory pressures were improved in the nitroglycerin group (P <.01). Stereology revealed comparable intrapulmonary edema between groups and a trend toward less vasoconstricted vasculature in Perfadex with nitroglycerin-protected lungs.
Perfadex solution provides superior lung preservation in terms of postischemic oxygenation capacity than Euro-Collins solution. Supplementation of the nitric oxide pathway by nitroglycerin further enhances functional outcome of Perfadex-preserved organs and might be an easily applicable tool in clinical lung transplantation.
Journal of Thoracic and Cardiovascular Surgery 07/2003; 125(6):1208-16. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Keratinocyte growth factor (KGF) induces transient proliferation of alveolar type II cells (AEII) associated with surfactant alterations. To test the hypothesis that homeostasis of intracellular phospholipid stores is maintained under KGF-induced hyperplasia, we (1) collected tissue from adult rat lungs, fixed for light and electron microscopy 3 days after intratracheal instillation of 5 mg recombinant human (rHu) KGF/kg body weight or phosphate-buffered saline (PBS), and from untreated control animals (five animals/group) for design-based stereology of AEII and lamellar body (LB) ultrastructure; and (2) we analyzed uptake and distribution of instilled radiolabeled phospholipids. After rHuKGF, AEII-coverage of alveolar walls (PBS:8.3 +/- 3.0%; rHuKGF:30.6 +/- 4.8%) and number of AEII/ml lung volume (PBS:28.5 +/- 6.5 x 10(6); rHuKGF:48.2 +/- 5.8 x 10(6)) were increased (p < 0.008). Number (PBS:97 +/- 25; rHuKGF:54 +/- 7) and volume (PBS:45.3 +/- 13.8 microm(3); rHuKGF:21.0 +/- 4.7 microm(3)) of LBs per cell were decreased (p < 0.008), but not total amount/ml lung volume (PBS:128 +/- 46. 4 x 10(7) microm(3); rHuKGF:103 +/- 34. 7 x 10(7) microm(3)). This was paralleled by a shift to larger LBs. After rHuKGF, radiolabeled phospholipids accumulated in whole lung tissue relative to lavage fluid (p < 0.01). However, less radiolabel was incorporated per cell (p < 0.01). We conclude that under rHuKGF-induced AEII proliferation intracellular surfactant was decreased per single cell, whereas a constant amount was maintained per unit lung volume. We suggest that surfactant homeostasis is regulated at the level of phospholipid transport processes, for example, secretion and reuptake.
American Journal of Respiratory and Critical Care Medicine 05/2003; 167(9):1264-70. · 11.04 Impact Factor