Antonia Fehrenbach

Stereological estimation of the volume weighted mean volumes of alveoli and acinar pathways in the rat lung to characterise alterations after ischaemia/reperfusion

The aim of this study was to characterise pulmonary reimplantation injury in isolated, perfused rat lungs following 2 h of cold ischaemia, and 50 min. of in vitro reperfusion. The effects of 2 differently composed lung preservation solutions (low potassium Euro‐Collins and Celsior; each n=5) were examined in comparison with untreated, nonischaemic control lungs (n=3). After fixation by vascular perfusion and tissue collection by systematic random sampling, the volume weighted mean volume (v) of alveoli and acinar pathways was estimated by light microscopic stereology using the method of point sampled intercepts in plastic embedded, Azan‐stained material. Significantly higher v of alveoli and acinar paths was found in the Celsior group than in Euro‐Collins preserved lungs. However, in the controls the size of acinar pathways was similar to Celsior preserved lungs whereas alveolar size was comparable to preservation with Euro‐Collins. The between‐animal coefficient of variation of alveoli was very low in controls and Celsior preserved but higher in the Euro‐Collins group. Size distribution of alveoli and acinar paths in 15 size classes was largely homogeneous in all groups tested. In the Euro‐Collins group the fractions of both class 1‐alveoli and class 1‐acinar paths significantly exceeded those of the other groups. Widely expanded alveoli (size classes 13–15) only occurred after preservation with Celsior whereas wider acinar paths (size class 15) were found in the Celsior group and in the controls. It is concluded that lung preservation with low‐potassium Euro‐Collins and Celsior solutions may act differently on distinct spaces in the distal gas‐exchange regions of lungs. This may be due to selective effects on pulmonary surfactant activity and on elastic tissue elements in the alveolar ducts, respectively. Additionally, the method of point sampled intercepts is considered to be an efficient tool to evaluate the effects of different preservation solutions on lung parenchyma.

Reduced vascular endothelial growth factor correlates with alveolar epithelial damage after experimental ischemia and reperfusion

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Combined use of prostacyclin and higher perfusate temperatures further enhances the superior lung preservation by celsior solution in the isolated rat lung

BACKGROUND The poor tolerance of the lung to ischemia and reperfusion (IR) still represents one of the limitations in clinically successful lung transplantation. Modified Euro-Collins (EC) is routinely used in lung preservation, but alternative solutions have been developed for improvement of pulmonary preservation. Celsior is an extracellular solution that has significantly reduced the IR-induced pulmonary damage in animal studies. So far, no extensive experimental studies exist concerning the influence of Celsior on pulmonary gas exchange following IR. METHODS In an extracorporeal rat lung model 10 lungs, each, were preserved with Celsior (CE) and Celsior/prostacyclin (CEPC, 6 microg/100 ml) at 4 degrees and 15 degrees C, each, and compared to low-potassium Euro-Collins (EC-40, 40 mmol/liter potassium). After 2 hours of ischemia lungs were reventilated and reperfused using a roller pump. Oxygenation in terms of oxygen partial tension in the left atrial effluent, pulmonary vascular resistance (PVR), peak inspiratory pressure, and wet/dry ratio were monitored for 50 minutes. Furthermore, edema formation was evaluated by light microscopy. Statistical analysis was performed using ANOVA models. RESULTS Compared to the EC-40 group, oxygenation was increased and amount of edema was reduced in most Celsior-preserved organs (p<0.032) with exception of the CEPC group at 4 degrees C (p = 0.06). Additional application of prostacyclin did not have any significant effect on oxygenation in the Celsior group. However, after temperature elevation of the CEPC perfusate to 15 degrees C, a superior partial tension of oxygen was observed (p<0.023) in contrast to the 4 degrees C groups CE and CEPC. The lowest PVR was found in the CE 4 degrees C group (p<0.02). CONCLUSIONS Celsior provides better lung preservation than EC-40 solution. Application of prostacyclin at higher perfusate temperatures results in additional functional improvement. In vivo experiments and ultrastructural analysis are warranted for further evaluation of Celsior in lung preservation.

Evaluation of pulmonary edema : Stereological versus gravimetrical analysis

Assessment of lung edema by gravimetrical analysis is a standard method to evaluate the severity of experimentally induced ischemia/reperfusion (IR) injury. The aim of this study was to compare gravimetrical assessment of pulmonary edema with a stereological approach which allows for qualitative and quantitative distinction between intravascular and edematous fluids by light microscopy. Eight experimental groups which differed in mode of preservation, ischemic storage and pharmacological treatments were studied in an extracorporeal rat lung model. Analysis of the pooled data showed that the wet/dry ratio values mainly reflected the amount of intra-alveolar edema (rs = 0.442; p = 0.0057) but only stereological assessment of edema formation revealed differences depending on the treatment used. Only stereological data correlated significantly with oxygen tension measured at the end of reperfusion (rs = –0.530; p = 0.0009). We conclude that gravimetry is of minor functional importance compared to assessment by stereological methods which prove to be a reliable and efficient tool for the evaluation of IR injury in the different experimental settings.

Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A.

BackgroundDeclining 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.MethodsUsing 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.ResultsAfter 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.ConclusionWe 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).

Experimental lung preservation with Perfadex: effect of the NO-donor nitroglycerin on postischemic outcome.

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Nitroglycerin alters alveolar type II cell ultrastructure after ischemia and reperfusion.

BACKGROUND Although administration of nitric oxide (NO) has been suggested to reduce pulmonary reimplantation response, concerns remain about cytotoxic side effects. METHODS Using light and electron microscopy, we examined the effects of the NO donor nitroglycerin (NTG) (0.1 mg/ml) as a supplement to the preservation solution Celsior on the structural integrity of rat lungs after extracorporeal ischemia (4 hours at 10 degrees C) and reperfusion (50 minutes) (IR). We performed evaluation in comparison with Celsior alone after IR using either standard antegrade perfusion through the pulmonary artery or retrograde perfusion through the left atrium as an alternative way to improve the preservation quality. Untreated, non-ischemic lungs served as controls (n = 5 per group). We recorded respiratory and hemodynamic parameters during reperfusion. Tissue collection using systematic uniform random sampling was representative for the whole organ and allowed stereologic quantification of structures. RESULTS After IR, histochemistry revealed no breaks in the alveolo-capillary barrier and we detected no alveolar flooding. Edema formed in the peribronchovascular cuffs, of which the volume fraction was increased (p =.008). Vasoconstriction of the smaller arteries accompanied antegrade flush, which occurred neither after administration of NTG nor after retrograde flush, as shown by immunostaining for alpha-smooth muscle actin. Treatment with NTG was associated with focal disintegration of Type II cells, which displayed edematous swelling of distinct cell compartments and lysis of mitochondria and cells. Nitroglycerin prevented alveolar collapse, which was increased in the other IR groups (p = 0.013). We observed alterations in intra-alveolar surfactant components. CONCLUSION These findings indicate pathologic effects of NTG treatment on alveolar epithelial integrity. Therefore, we suggest further critical evaluation of NTG/NO for therapeutic use in lung transplantation.

Exogenous surfactant application in a rat lung ischemia reperfusion injury model: effects on edema formation and alveolar type II cells.

BackgroundProphylactic 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.MethodsRats 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°C and 50 min of reperfusion at 37°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.ResultsSurfactant 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 μm3(0.10)) and CE+S (481 μm3(0.10)) compared with controls (323 μm3(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).ConclusionIntratracheal 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.

Nitrogen dioxide induces apoptosis and proliferation but not emphysema in rat lungs

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Lung retrieval from non-heart-beating donors: First experience with an innovative preservation strategy in a pig Lung transplantation model

Objective: 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. Methods: 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°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. Results: 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. Conclusions: 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.