Impact of preservation solution on the extent of blood-air barrier damage and edema formation in experimental lung transplantation
Article first published online: 21 MAR 2007
Copyright © 2007 Wiley-Liss, Inc.
The Anatomical Record
Volume 290, Issue 5, pages 491–500, May 2007
How to Cite
Mühlfeld, C., Müller, K., Pallesen, L.-P., Sandhaus, T., Madershahian, N., Richter, J., Wahlers, T., Wittwer, T. and Ochs, M. (2007), Impact of preservation solution on the extent of blood-air barrier damage and edema formation in experimental lung transplantation. Anat Rec, 290: 491–500. doi: 10.1002/ar.20518
- Issue published online: 13 APR 2007
- Article first published online: 21 MAR 2007
- Manuscript Accepted: 12 FEB 2007
- Manuscript Received: 16 JAN 2007
- Deutsche Forschungsgemeinschaft. Grant Numbers: Oc 23/7-3, Wi 1625/1-4
- lung transplantation;
- electron microscopy
A major aim in lung transplantation is to prevent the loss of structural integrity due to ischemia and reperfusion (I/R) injury. Preservation solutions protect the lung against I/R injury to a variable extent. We compared the influence of two extracellular-type preservation solutions (Perfadex, or PX, and Celsior, or CE) on the morphological alterations induced by I/R. Pigs were randomly assigned to sham (n = 4), PX (n = 5), or CE (n = 2) group. After flush perfusion with PX or CE, donor lungs were excised and stored for 27 hr at 4°C. The left donor lung was implanted into the recipient, reperfused for 6 hr, and, afterward, prepared for light and electron microscopy. Intra-alveolar, septal, and peribronchovascular edema as well as the integrity of the blood-air barrier were determined stereologically. Intra-alveolar edema was more pronounced in CE (219.80 ± 207.55 ml) than in PX (31.46 ± 15.75 ml). Peribronchovascular (sham: 13.20 ± 4.99 ml; PX: 15.57 ± 5.53 ml; CE: 31.56 ± 5.78 ml) and septal edema (thickness of alveolar septal interstitium, sham: 98 ± 33 nm; PX: 84 ± 8 nm; CE: 249 ± 85 nm) were only found in CE. The blood-air barrier was similarly well preserved in sham and PX but showed larger areas of swollen and fragmented epithelium or endothelium in CE. The present study shows that Perfadex effectively prevents intra-alveolar, septal, and peribronchovascular edema formation as well as injury of the blood-air barrier during I/R. Celsior was not effective in preserving the lung from morphological I/R injury. Anat Rec, 2007. © 2007 Wiley-Liss, Inc.
Primary graft dysfunction (PGD) remains a major cause of early morbidity and mortality after lung transplantation. This is mainly due to ischemia-reperfusion (I/R) injury caused by suboptimal lung preservation measures. The clinical spectrum of I/R injury ranges from mild acute lung injury (ALI) to severe acute respiratory distress syndrome (ARDS) (Trulock, 1997; Arcasoy and Kotloff, 1999; de Perrot et al., 2003; Van Raemdonck et al., 2004; Mulligan, 2006). In addition to the early effects, several studies suggest that I/R injury also significantly impacts the long-term outcome after lung transplantation (Fiser et al., 2002; Christie et al., 2005a; Daud et al., 2007).
Clinically, I/R injury is characterized by hypoxemia, increased pulmonary vascular resistance, decreased lung compliance, and pulmonary edema formation (de Perrot et al., 2003; Van Raemdonck et al., 2004; Christie et al., 2005b; Ng et al., 2006). On biopsy, nonspecific diffuse alveolar damage is present (Howell and Palmer, 2006). In experimental studies, to study pulmonary edema and the fine structure of the blood-air barrier in detail, a formal quantitative (stereological) analysis in conjunction with the resolving power of transmission electron microscopy is required (Ochs, 2006; Weibel et al., 2007). Only at the electron microscopic level is it possible to distinguish permeability edema due to primary blood-air barrier injury in ALI/ARDS from the lesions in hydrostatic edema (Montaner et al., 1986; Bachofen and Weibel, 1998; Martin, 2002). Moreover, edema fluid is not homogeneously distributed in the lung. We have shown previously that pulmonary edema, which is commonly assessed globally by the lung wet/dry ratio, can be quantitated and differentiated into intra-alveolar, septal, and peribronchovascular edema by using a stereological approach after lung fixation by vascular perfusion (Fehrenbach et al., 1999, 2001; Ochs et al., 2000).
Current clinical and experimental evidence suggests that extracellular-type (i.e., low potassium) preservation solutions such as Perfadex or Celsior are superior to intracellular-type (i.e., high potassium) solutions such as Euro-Collins or University of Wisconsin solution (Van Raemdonck and Steen 2003; Van Raemdonck et al., 2004; D'Ovidio and Keshavjee, 2006). In a previous study (Wittwer et al., 2005), we have shown that Perfadex, when compared to Celsior, results in superior postischemic function in a pig lung transplantation model after prolonged cold ischemia. Moreover, retrograde application of Perfadex further improved lung preservation quality. Here, we extend these observations by providing a detailed stereological light and electron microscopic analysis of edema formation and blood-air barrier damage in order to characterize the ultrastructural correlate for differences in lung preservation quality between Perfadex and Celsior.
MATERIALS AND METHODS
An established in vivo pig lung transplantation model was used as described in detail previously (Wittwer et al., 2004). Pigs were randomly assigned to a sham (n = 4), Perfadex (PX; n = 5), or Celsior group (CE; n = 2). An initial number of five pigs were assigned to CE; however, three animals in this group died within 15 min in the early posttransplantation period due to an ischemia-reperfusion-triggered lethal right heart failure, and only two animals survived 45 or 60 min, respectively. These two only were used for the morphological analysis and it was considered unethical to operate more CE pigs since the disastrous results were obvious.
In short, all donor animals received ketamine 10% (20 mg/kg), atropine (0.04 mg/kg), and propofol (3 mg/kg) for premedication. Mechanical ventilation was performed with 50% oxygen in a pressure-controlled mode (PIP [positive inspiratory pressure]: 20 mm Hg, 18/min; PEEP [positive end-expiratory pressure]: 8 mm Hg). Anesthesia was continued with fentanyl (i.v., 0.3 μg/kg/min), midazolam (20 μg/kg/min), and pancuronium (10 μg/kg/min). Pigs were heparinized intravenously (200 IE/kg). After a median sternotomy, a perfusion cannula with a side port was placed into the left atrium to measure perfusion pressure. After cardiac inflow occlusion, the inferior caval vein was incised and the whole blood volume evacuated. At the same time, the pulmonary trunk was incised and retrograde perfusion with either Perfadex or Celsior was started via the left atrium and maintained at a maximum pressure of 14 mm Hg for 6–10 min. This time was sufficient to perfuse 2,000 ml of preservation solution. Ventilation was continued throughout the perfusion period. After preservation, the heart-lung block was excised with both lungs inflated in an end-inspiratory state and stored at 4°C for 27 hr.
For recipient and sham animal preparation, the same anesthetic and ventilatory protocol was used as for the donor pigs. After a left thoracotomy in the fifth intercostal space, pneumonectomy was performed in PX and CE groups as described previously (Wittwer et al., 2004). Implantation of the left donor lung was performed and, prior to reperfusion, the donor lung was carefully deaired. After declamping of the pulmonary artery, the graft was ventilated at a PEEP of 10 mm Hg. The right pulmonary artery and bronchus were clamped after 15 min of reperfusion and reperfusion was maintained for 6 hr. The experiments were terminated by an intracardiac injection of magnesium sulfate.
Fixation, Sampling, and Processing of Lung
After the reperfusion was terminated, the entire left lung was fixed by perfusion with 2 l of a fixative containing 1.5% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.35, buffer osmolality 300 mOsm/kg). Perfusion pressure was 15 cm H2O. During fixation, inflation of the lung was maintained at a constant pressure of 12 cm H2O. Afterward, the fixed lung was excised, the volume was estimated by fluid displacement (Scherle, 1970), and the lung was then cut into 2 cm thick slices. To ensure that every part of the lung had the same chance of being included in the analysis, systematic uniform random sampling was performed by projecting a transparent uniform point grid on each tissue slice (Ochs, 2006). Whenever a point hit the surface of a lung slice, a tissue block of approximately 1 cm3 was excised at the given location and stored in fresh and cold fixative (Ochs et al., 1999). After storage of the tissue blocks in the fixative for at least 24 hr, the tissue blocks were further sampled systematically for light (LM) or transmission electron microscopy (TEM). The samples were subsequently washed in 0.1 M sodium cacodylate, postfixed in osmium tetroxide, washed again in sodium cacodylate and distilled water, stained en bloc in half-saturated watery uranyl acetate overnight, dehydrated in an ascending ethanol (LM) or acetone (TEM) series, and finally embedded in a glycol methacrylate resin (Technovit 7100; Heraeus Kulzer, Wehrheim, Germany) or in an epoxy resin (Araldite; Serva Electrophoresis, Heidelberg, Germany). For LM, sections of 1 μm thickness were cut from the glycol methacrylate-embedded tissue blocks and stained with methylene blue. For TEM, ultrathin sections of 40–70 nm thickness were obtained from the araldite-embedded tissue blocks and stained with lead citrate and uranyl acetate using an Ultrostainer (Leica, Bensheim, Germany).
All investigations at the LM level were carried out using an Axioscope light microscope (Zeiss, Oberkochen, Germany) and a computer-assisted stereology system (CAST 2.0; Olympus, Ballerup, Denmark). Test fields for further analysis were gathered by systematic uniform random sampling from at least three different tissue blocks per animal. A point grid with an adjusted number of test points was projected onto each test field and the volume fraction of a particular structure of interest per unit volume of the reference space (VV(str/rv)) was estimated by counting points hitting the structure of interest (Pstr) and those points hitting the reference volume (Prv). The volume fraction was estimated by VV : = Pstr/Prv and converted to the total volume by multiplication with the reference volume (Weibel, 1979; Weibel et al., 2007). Table 1 gives the parameters and the objective lens magnifications at which the estimations of the volumes of different pulmonary structures were performed.
|V (par, lung)||Volumes of pulmonary parenchyma and nonparenchyma||4×|
|V (nonpar, lung)|
|V (airwa, lung)||Volumes of nonparenchymal compartments (airway wall, vessel wall, airway lumen, vessel lumen, peribronchovascular space)||10×|
|V (veswa, lung)|
|V (airlum, lung)|
|V (veslum, lung)|
|V (pbv, lung)|
|V (air, lung)||Volumes of parenchymal compartments (airspace, airspace filled with edema fluid, alveolar septa)||20×|
|V (ed, lung)|
|V (sep, lung)|
|τ (epi)||Artihmetic mean barrier thickness of alveolar epithelium, interstitium and capillary endothelium||12,000×|
|SS (normal/bab)||Surface area fractions of normal, swollen and fragmented blood-air barrier in relation to total surface area||12,000×|
Transmission electron microscopy was carried out with an EM 900 (Zeiss) equipped with a digital camera (Megaview III; Soft Imaging System, Münster, Germany) and an image analysis software (AnalySIS 3.1; Soft Imaging System). Systematic uniform random sampling was performed at a primary magnification of 12,000× on the ultrathin sections of three different tissue blocks per animal. Whenever a test field included thin parts of the blood-air barrier, a digital micrograph was taken, which was superimposed with a test system consisting of parallel line segments and points (Weibel, 1979). The arithmetic mean barrier thickness of the alveolar epithelium, interstitium, and capillary endothelium was estimated by point and intersection counting and using the equation τ : = (lT/2) × (Pb/Ib), with τ being the mean barrier thickness, lT the length of a test line, Pb the number of points hitting a barrier profile, and Ib the number of intersections of the test lines with the reference surface of the barrier (Weibel, 1979). Since the barrier thickness alone does not provide information about the preservation state of the blood-air barrier, we set up a semiquantitative characterization of the blood-air barrier (Velazquez et al., 1991) according to the following ultrastructural appearance. One, normal. All parts of the blood-air barrier show normal ultrastructure with electron-dense alveolar epithelium and capillary endothelium and small interstitial space. Two, swollen. If one or more compartments of the blood-air barrier appeared swollen, this category was used. Three, fragmented. In severe cases of blood-air barrier injury, the alveolar epithelium and/or the capillary endothelium showed disruptions causing discontinuities in the blood-air barrier. Intersections of the test lines with the blood-air barrier were counted and grouped into one of the categories described above. By relating the number of intersections of a certain category to the total number of intersections with the blood-air barrier, we estimated the surface fraction of normal, swollen, and fragmented blood-air barrier in relation to the total surface (Fehrenbach et al., 1999, 2003).
The groups were compared with the nonparametric two-sided Whitney-Mann U-test. Differences between groups were regarded as statistically significant at P < 0.05. Due to the small number of animals reaching the endpoint in CE, only very few P values reached significance level. Therefore, scatter plots are provided for better comparability of the results.
All LM and TEM sections showed lung tissue with widely opened blood vessels and only little erythrocytes left in the capillaries as well as open airspaces apart from a few focal areas of atelectasis in PX and CE. These structural characteristics indicated a well-performed perfusion fixation; thus, further structural changes could be attributed to the experimental procedure. In the lungs of the sham-operated pigs, pulmonary ultrastructure was widely normal. In a few sections, a thin film of intra-alveolar edema fluid was infrequently observed and, at the TEM level, the cytoplasm of some parts of the alveolar epithelium was more electron-lucent than normal, indicating a slight swelling of type I pneumocytes. However, no further pathological changes were observed. In PX, the intra-alveolar edema was more pronounced and the peribronchovascular space appeared less tight than in the sham group, although clear signs of edema in the peribronchovascular space were missing. Generally, signs of severe I/R injury were missing. In particular, the preservation of the blood-air barrier did not differ from the sham group. In the two lungs of CE, however, we observed severe intra-alveolar and peribronchovascular edema that was much stronger than in PX. At the TEM level, large parts of the blood-air barrier showed a loss of integrity with swelling or fragmentation of the alveolar epithelium and increased interstitial space. Only in very few areas did the blood-air barrier appear normal in CE (Figs. 1 and 2).
Due to the small number of animals in CE, most parameters did not show significant differences (Table 2). However, the scatter plot analysis clearly shows that, in general, PX and sham animals were in a similar range and CE showed more severe signs of injury due to the experimental procedure.
|V (lung) [ml]||343 (78)||516 (126) §||740 (113)|
|V (nonpar, lung) [ml]||79.92 (7.87)||90.35 (23.31)||120.82 (22.91)|
|V (par, lung) [ml]||262.58 (70.83)||425.64 (103.71) §||619.18 (90.22)|
|V (air, lung) [ml]||200.18 (62.84)||330.24 (111.48)||317.57 (133.56)|
|V (ed, lung) [ml]||4.41 (0.81)||31.46 (15.75) §||219.80 (207.55)|
|V (sep, lung) [ml]||57.99 (8.18)||63.95 (13.77)||81.56 (15.83)|
|V (airwa, lung) [ml]||6.41 (1.41)||12.58 (3.60)||15.17 (2.46)|
|V (veswa, lung) [ml]||3.95 (0.95)||4.41 (1.86)||5.66 (1.74)|
|V (airlum, lung) [ml]||28.46 (7.13)||38.53 (18.49)||48.64 (4.80)|
|V (veslum, lung) [ml]||27.94 (7.91)||19.28 (7.56)||16.23 (3.08)|
|V (pbv, lung) [ml]||13.20 (4.99)||15.57 (5.53)||31.56 (5.78)|
|τ (endo) [nm]||381 (36)||403 (32)||434 (9)|
|τ (int) [nm]||98 (33)||84 (8)||249 (85)|
|τ (epi) [nm]||310 (9)||302 (25)||367 (16)|
|τ (bab) [nm]||789 (24)||789 (18)||1049 (59)|
|SS (normal/bab) [%]||58.06 (5.72)||57.11 (23.88)||15.04 (0.91)|
|SS (swollen/bab) [%]||40.83 (4.98)||41.42 (22.66)||70.54 (2.11)|
|SS (frag/bab) [%]||1.12 (0.83)||1.45 (2.36)||14.48 (3.10)|
Intra-alveolar edema was observed in all three groups. In the sham group, a very small volume of edema was observed, whereas in those animals treated with Perfadex, a significantly higher volume of intra-alveolar edema was present. In both CE animals, volume estimations of intra-alveolar edema were considerably larger than in PX (Fig. 3). The volume of the peribronchovascular space was enlarged in CE, too, while in the other groups, similar values were estimated, indicating a peribronchovascular edema in CE (Fig. 4). The volume of the alveolar septa was measured at the light microscopic level to see if an increase in septal interstitial volume was present. PX and sham groups did not show significant differences in this parameter, and in CE, the increase was less severe than in the former parameters (Fig. 5). A more sensitive parameter to look at the septal interstitial volume increase at the electron microscopic level is the arithmetic mean barrier thickness, which was similar in PX and sham, whereas both estimations in CE exceeded PX and sham values (Fig. 6). The severe injury in the CE group was also reflected by a larger surface fraction of swollen and fragmented blood-air barrier than in sham and PX, which exhibited similar surface area fractions (Fig. 7).
Despite advances in clinical management, early graft survival after lung transplantation is still worse than for other solid organs. Thus, further research to optimize lung preservation and thereby to prevent I/R injury is needed (de Perrot et al., 2003; Wilkes et al., 2005). The pathophysiological and clinical characteristics of I/R injury reflect damage to both the epithelial as well as the endothelial side of the blood-air barrier. Consequently, an important strategy is to preserve the integrity of both epithelial and endothelial cells (Novick et al., 1996; Steen 2001).
A combined stereological and microscopic approach allows for quantitative pulmonary edema analysis in its preserved micro-organization and localization within the organ and for the quantitation of the particular contributions of intra-alveolar, septal, and peribronchovascular edema (Fehrenbach et al., 1999, 2001; Ochs et al., 2000). At the level of alveoli, the fine structural details of the blood-air barrier can only be resolved by transmission electron microscopy. Therefore, the aim of the present study was to investigate qualitatively and quantitatively edema formation and blood-air barrier damage after lung preservation by electron microscopy and stereology. In a pig lung transplantation model of extended ischemia, retrograde preservation with Perfadex resulted in improved oxygenation, decreased pulmonary vascular resistance, and increased lung compliance (Wittwer et al., 2004, 2005). Using this model, the present results show that retrograde preservation with Perfadex, when compared to Celsior, leads to less severe edema formation and better fine structural preservation of the blood-air barrier. In particular, Perfadex attenuated the extent of edema formation in all three compartments investigated (intra-alveolar, peribronchovascular, septal). Moreover, the ultrastructural appearance and thickness of all components of the blood-air barrier (alveolar epithelium, interstitium, capillary endothelium) was similar to the sham group in Perfadex-treated lungs, whereas severe swelling and fragmentation of the blood-air barrier components occurred after treatment with Celsior.
The development of pulmonary edema follows a sequence of fluid accumulation in various compartments of the lung (Staub, 1974; Albertine, 1998). Fluid appears first in the connective tissue compartment around bronchi and larger blood vessels: the peribronchovascular space. This is followed by the alveolar septal interstitium. Finally, alveolar flooding occurs after the two interstitial compartments (which are connected) are filled. In rats, it has been shown that intra-alveolar edema is the functionally most significant type of edema in I/R injury (Fehrenbach et al., 1999). Although differences between rats and pigs with respect to lung edema formation during preservation have been reported recently (Wierup et al., 2005), the present results demonstrate that severe I/R injury in the pig lung is mainly reflected by intra-alveolar edema formation. In addition, the present study has shown that, for the detection of septal edema, electron microscopy is more sensitive than light microscopy.
The blood-air barrier of the mammalian lung has to face a dilemma in that it has to be thin and strong at the same time (Weibel, 1984; Maina and West, 2004). According to Fick's law of diffusion, oxygen flow rate across a tissue barrier is directly proportional to the cross-sectional surface area and inversely proportional to the thickness of the barrier. Intra-alveolar and interstitial edema thus increase the effective diffusion barrier thickness, thereby leading to decreased oxygenation. Thus, there is a rationale for preserving the components of the blood-air barrier, which is exposed to a variety of detrimental influences from the airspace as well as from the vascular side during I/R. Reactive oxygen species (ROS) are known to play a significant role in the pathogenesis of I/R injury (Novick et al., 1996; Heffner, 1998). ROS can damage epithelial and endothelial cells lining the blood-air barrier by lipid peroxidation (Kinnula et al., 1995; Heffner, 1998). In addition, the permeability of the blood-air barrier is augmented in I/R injury as a result of basal lamina disruption, most likely mediated by matrix metalloproteinases (Soccal et al., 2000).
Perfadex is the only preservation solution developed specifically for lung transplantation (de Perrot et al., 2005). Both the low potassium concentration as well as the presence of dextran individually contribute to the beneficial effects of preservation with Perfadex (Keshavjee et al., 1992). Perfadex contains dextran 40 as an oncotic agent in order to decrease edema formation. The present study demonstrates that Perfadex indeed effectively reduces the formation of intra-alveolar, peribronchovascular, and septal edema. In addition, Perfadex also better preserves the fine structural integrity of the components of the blood-air barrier than Celsior. This beneficial effect might be attributed to reduced lipid peroxidation, which has been reported for Perfadex (Sakamaki et al., 1997).
In conclusion, the present study, using a pig lung transplantation model, has shown that the superior outcome of Perfadex-treated donor lungs is related to improved fine structural preservation of the blood-air barrier and to less severe intra-alveolar, septal, and peribronchovascular edema formation. These results further emphasize that optimized lung preservation should aim at preserving both alveolar epithelial and capillary endothelial cell integrity. In this regard, a combined stereological and electron microscopic approach is useful in assessing the quality of lung preservation techniques in experimental studies.
The authors thank S. Freese, A. Gerken, H. Hühn, S. Wienstroth (Göttingen), and B. Krieger (Bern) for expert technical assistance.
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