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Ann Thorac Surg 2001;71:1140-1145
© 2001 The Society of Thoracic Surgeons

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Original article: general thoracic

Raffinose improves 24-hour lung preservation in low potassium dextran glucose solution: a histologic and ultrastructural analysis

^a Thoracic Surgery Research Laboratory, Division of Thoracic Surgery, Toronto General Hospital Research Institute, Toronto, Ontario, Canada
^b Department of Pathology, Hospital for Sick Children, University Health Network, Toronto, Ontario, Canada
^c The Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada

Accepted for publication December 20, 2000.

Address reprint requests to Dr Keshavjee, Toronto Lung Transplant Program, Division of Thoracic Surgery, Toronto General Hospital, 200 Elizabeth St, EN 10-224, Toronto, Ontario, Canada, M5G 2C4
e-mail: shaf.keshavjee{at}uhn.on.ca

	Abstract

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Background. We have previously shown that the addition of raffinose to low potassium dextran (LPD) preservation solution improves transplanted rat lung function after 24 hours of storage. The mechanisms by which raffinose acts are unclear. The aim of this study was to examine the histologic and ultrastructural correlates of this enhanced pulmonary function after preservation with raffinose.

Methods. In a randomized, blinded study, rat lungs were flushed with LPD, or LPD containing 30 mmol/L of raffinose, and stored for 24 hours at 4°C. Control lungs were flushed with LPD but not stored (n = 5 each group). Changes in postpreservation edema were determined. In addition, lungs were flushed with a trypan blue solution to quantify cell death, and examined using both light and electron microscopy.

Results. The LPD lungs gained significantly more weight (25.5% ± 5.5%) compared with raffinose-LPD lungs (5.2% ± 5.3%; p < 0.0001). There were higher percentages of dead cells in the LPD lungs (29% ± 0.3% of total cells) compared with raffinose-LPD lungs (14% ± 1.4%; p < 0.001) and control lungs (0.2% ± 5%; p < 0.001). Control lungs maintained normal ultrastructure, whereas LPD lungs showed a decreased number of intact type II pneumocytes and significant cellular necrosis. Interstitial and alveolar edema with interstitial macrophage infiltration was also observed. Alveolar capillaries were collapsed. In contrast, raffinose-LPD lungs showed only mild alterations such as minimal interstitial edematous expansion, fewer damaged cells, and minimal capillary injury.

Conclusions. Raffinose exerts a cytoprotective effect on pulmonary grafts during preservation, which explains the previously documented improved function. This simple modification of LPD with raffinose may provide clinical benefit in extended pulmonary preservation.

	Introduction

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Lung transplantation has become a standard treatment modality for end-stage lung diseases [1]. Approximately 15% of patients die within the first 3 months after lung transplantation of severe organ dysfunction, and injurious processes caused by preservation and ischemia–reperfusion are believed to be a major contributor [2].

Currently, preservation times of approximately 6 to 8 hours are accepted in clinical lung transplantation [2]. A recent report reaffirmed the occurrence of diminishing graft function after periods of storage in excess of 4 hours only [3]. The strategy used for preservation of lung grafts is important, and has been the subject of much experimental research during the last 10 years. In particular, low potassium dextran (LPD), University of Wisconsin, EuroCollins, and Wallwork blood-based solutions have each been tested in conjunction with a variety of pharmacologic adjuncts [4].

We have previously described that the addition of the trisaccharide raffinose to LPD improves the function of transplanted rat lungs after 24 hours of cold storage [5]. Raffinose is included in University of Wisconsin solution as an impermeant solute and has been shown to be largely responsible for the efficacy of University of Wisconsin as a lung storage medium [6]. In addition, it has been shown to be superior to a variety of other saccharides for this purpose [7]. The precise mechanisms by which raffinose acts are unknown. However, it clearly improved the physiologic outcome in transplanted marginal lungs in our earlier study. Previous studies on mechanistic actions of raffinose, however, have yielded conflicting results [8].

This study was performed to examine the ultrastructural and morphologic effect of raffinose on preservation injury in lungs stored for 24 hours in LPD. We hypothesized that raffinose has a cytoprotective effect on pulmonary graft cells during the prolonged hypothermic storage phase, which decreases cellular injury and cell death.

	Material and methods

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Preparation of the raffinose-modified low potassium dextran preservation solution
The raffinose-modified LPD (R-LPD) solution that was used in this study was equal to that in our previous study [5]. Briefly, raffinose (17.86 g = 30 mmol/L) pentahydrate (Sigma Chemical Co, St. Louis, MO) was dissolved in 500 mL LPD solution (Perfadex, Biophausia, Uppsala, Sweden) at room temperature, and then reintroduced into the 1000-mL bag through a 0.22-µm filter (Millex-GS, Millipore Corporation, Bedford, MA). Tromethamine (THAM) was added to adjust the pH to 7.5. The resultant solution was colorless and indistinguishable from standard LPD solution.

Graft harvesting procedure
The Institutional Animal Care and Use Committee of the Toronto General Hospital reviewed and approved the protocol for this study. All animals received humane care in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).

Fifteen isogeneic Lewis rats (Charles River Inc, Montreal, Quebec, Canada) with an average body weight of 299 ± 34.3 g were anesthetized by intraperitoneal injection of 1 mL of sodium pentobarbital (Somnotol, MTC Pharmaceuticals, Cambridge, Ontario, Canada) and intubated through a tracheostomy with a 14-gauge intravenous catheter. Animals were connected to a volume-controlled ventilator (Harvard Rodent Ventilator, model 683, South Natick, MA), and ventilated with an inspired oxygen fraction of 1, at 75 breaths/min, a tidal volume of 10 mL/kg, and a positive end-expiratory pressure of 2 cm H₂O. After this, a median laparosternotomy was performed, and 300 USP of heparin (Hepalean, Organon Teknika, Toronto, Ontario, Canada) was injected into the inferior vena cava. For the retrieval of the heart-lung block, the inferior vena cava was incised, the left atrial appendage truncated, and a 14-gauge cannula placed through a right ventricular outflow tractotomy into the main pulmonary artery. The lungs were flushed through this cannula with 20 mL of either LPD (n = 10) or R-LPD (n = 5) at 4°C. The investigators were blinded as to the flush solution used in each animal. The flush solution also contained 500 µg/L of prostaglandin E₁ (Prostin VR, Upjohn, Don Mills, Ontario, Canada) in accordance with clinical practice. Immediately after flushing the lungs, the intratracheal tube was clamped to keep the lungs inflated for the period of storage, and the heart-lung block was excised and stored for 24 hours at 4°C.

Control lungs
Five lung blocks of the 15 served as controls. These were flushed with LPD and prostaglandin E₁ and immediately prepared for microscopic assessment with no cold ischemic preservation period.

Assessment of postpreservation pulmonary edema
Pulmonary edema is a morphologic feature of pulmonary preservation injury representing fluid uptake during storage [9]. To evaluate the degree of edema in our experimental lungs after the preservation period, the organs were weighed immediately after flushing and extraction using a digital scale (model DI-100, Denver Instrument, IES Corporation, Portland, OR) and then stored in 40 mL of LPD or R-LPD. At the end of the 24-hour cold ischemic period, the organs were reweighed. The gained weight was calculated and expressed as the percent weight gain of the pre-preservation graft weight. The following formula was used for the calculation: (Gained weight during preservation x 100)/ Pre-preservation weight = percentage weight gain during preservation.

Histologic assessment of cell death
Immediately after resecting the right middle lobe of the lungs for electron microscopy examinations, all study lungs were flushed for 5 minutes with 20 mL of a 500 µmmol/L trypan blue (Sigma Chemical) solution through the main pulmonary artery. This flush was followed by 20 mL of 0.9% normal saline solution and 10 mL of 4% paraformaldehyde. Trypan blue was dissolved in Krebs-Henseleit buffer (pH 7.4; Sigma Chemical). The lungs were then fixed in 10% formalin. The middle third of the left lungs was used for histologic examination as it is representative of peripheral and central parenchymal areas. The transvascular trypan blue flush has been validated and successfully used in other studies on cell death in lung transplantation–related models [10, 11]. After paraffin embedding, 4-µm sections were cut and mounted on glass slides. An eosin counterstain was used to identify all viable cells, which do not pick up the trypan blue as dead cells do by losing their ability to exclude the dye actively. Slides were then photographed at x400 magnification on a color slide film using a light microscope. Cells were counted from six randomly chosen fields per slide. All viable cells were counted first, followed by the count of all trypan blue–stained cells. Results for dead cells (trypan blue–positive cells) are expressed as a percentage of total cells (viable cells + trypan blue–positive cells). Two individuals blinded to the identity of the specific specimens performed the counting.

Transmission electron microscopy
After the completion of the 24-hour cold preservation period (LPD group and R-LPD group) or immediately after flushing the lung (controls), the right middle lobe of all study lungs was resected by simple ligation and prepared for ultrastructural examination. Small pieces of fresh tissue were immersed in universal fixative (1% glutaraldehyde, 4% paraformaldehyde, pH = 7.4) immediately after biopsy, postfixed in 1% osmium tetroxide, dehydrated in graded acetones, and embedded in an epon-araldite mixture. One-micrometer-thick sections were stained with toluidine blue, and ultrathin sections from randomly chosen blocks were stained. The grids were examined in a Philips 201 electron microscope (N.V. Philips, Gloeilampenfarbrieken, Eindhoven, The Netherlands). The pathologist was blinded to the study groups.

Statistical analysis
To evaluate statistical differences among groups regarding the weight gain, analysis of covariance and paired and unpaired Student’s t tests were used. Regression lines were fitted to scatterplots of pre-preservation weights versus postpreservation weights. Descriptive statistics in the form of percentage weight gain were also used. To evaluate differences between the two preservation groups and the control group regarding cell death, an analysis of variance was performed. A p value of less than 0.05 was considered statistically significant. SPSS Release 9.0 (SPSS Inc, Chicago, IL) and SigmaStat Version 1.0 (Jandel Scientific, San Rafael, CA) were used for the statistical analyses.

	Results

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Increase in lung weight during preservation
The mean initial weight of lungs in the LPD group was 3.8 ± 0.2 g. The mean weight of these lungs after 24 hours of preservation was 4.7 ± 0.4 g. A paired Student’s t test revealed that the increase of 0.9 g was statistically significant (p = 0.001). For the R-LPD group, the weight changed from 3.7 ± 0.3 g before preservation to 3.8 ± 0.2 g after preservation. A paired Student’s t test showed that the increase of 0.2 g was not significant (p = 0.084). All data passed tests of homogeneity of variance and normality before the Student’s t tests were performed. Descriptively, during the 24-hour preservation period, lungs in the LPD group gained significantly more weight compared with lungs in the R-LPD group (25% ± 5.5% versus 5.2% ± 5.3%, respectively; p < 0.001). By using the analysis of covariance, the pre-preservation weight was found to affect the postpreservation weight (p = 0.001) as did the treatment (LPD or R-LPD; p = 0.027). A scatterplot of pre-preservation weights versus postpreservation weights for each treatment is shown in Figure 1.

View larger version (14K): [in this window] [in a new window]   Fig 1. Changes in lung weight (postpreservation edema) after 24 hours of cold ischemic preservation in low potassium dextran (LPD) or raffinose-modified LPD (R-LPD). The correlation coefficient for the LPD regression line was 0.61 (p = 0.073) and for the R-LPD regression line, 0.98 (p = 0.004). The scatterplot demonstrates that the addition of raffinose to LPD contributes significantly to reducing weight gain during preservation as compared with LPD alone. Moreover, the effect increases with initial lung weight.

View larger version (10K): [in this window] [in a new window]   Fig 2. Proportions of dead cells in lungs after 24 hours of cold ischemic preservation in low potassium dextran (LPD) or raffinose-modified LPD (R-LPD) and in control lungs. Data are expressed as mean ± standard deviation. The numbers of dead cells are expressed as a percentage of total cells. However, the R-LPD group showed significantly lower numbers of dead cells (14% ± 1.4% of total lung cells) than lungs in the LPD group (29% ± 0.3%; p < 0.001). The R-LPD lungs also contained a significantly higher percentage of dead cells than control lungs with 0.2% ± 5% (p < 0.001). (One-way analysis of variance, *p < 0.001 among all groups.)

View larger version (85K): [in this window] [in a new window]   Fig 3. Representative images of lung tissue sections after using the transvascular trypan blue flush and an eosin counterstain. Dead cells lose their ability to actively exclude trypan blue and consequently stain dark blue. Each dark blue dot represents a dead cell. Control lungs (A) showed none to very little cell death, whereas lungs flushed with and stored in raffinose-modified low potassium dextran solution for 24 hours (B) demonstrated moderate cell death and lungs that were preserved with low potassium dextran solution only (C) showed high numbers of dead cells.

View larger version (154K): [in this window] [in a new window]   Fig 4. Representative medium-power electron micrograph from a control lung that was flushed with low potassium dextran solution without cold storage. Note the overall good preservation of the cells, including the type II pneumocyte (P2) containing multilamellar surfactant bodies (S). Capillaries (c) within the interstitium are normal and show intact endothelial lining (arrows) and well-preserved tight junctions (arrowheads). (x7,450.)

View larger version (108K): [in this window] [in a new window]   Fig 5. Representative electron micrographs from the lungs flushed with low potassium dextran solution and stored for 24 hours. (A) Low-power view demonstrates portions of several alveolar spaces and the lining alveolar cells. Note the cellular debris (arrowheads) and macrophage (M) within the alveolar spaces. Type I and type II pneumocytes are less distinguishable. The interstitium is of variable thickness, shows edema (*), and contains necrotic cellular debris (D). Capillaries within this interstitium are poorly defined. (x4,630.) (B) High-power view shows an apoptotic cell (A) within an alveolar capillary. Note that this capillary is distorted and contains edema and cell debris (arrowheads). The type I pneumocytes (P1) lining the alveolar space are swollen and edematous in appearance, representing severe cellular injury. (x13,440.)

View larger version (120K): [in this window] [in a new window]   Fig 6. Representative electron micrographs from the lungs flushed with low potassium dextran solution containing raffinose and stored for 24 hours. (A) Low-power electron micrograph demonstrates the overall good preservation of the lung architecture and ultrastructure. Alveolar spaces are lined with a usual complement of type I and type II pneumocytes (P1, P2). The interstitium looks similar to the interstitium of control lungs and contains well-defined capillaries (c) with intact cell junctions (arrowheads). Note the absence of alveolar macrophages, apoptotic, or necrotic cells. (x3,850.) (B) High-power view illustrating the well-preserved ultrastructure of type II pneumocytes (P2) with multilamellar surfactant bodies (S). Alveolar capillaries (c) are within normal limits. The interstitium (I) shows no abnormality. (x14,560.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Previous studies have suggested that saccharides of higher molecular weight may have a stronger preventive effect on cellular edema during cold storage than those of lower molecular weight. The theory is that saccharides of lower molecular weight are more easily taken up by graft cells, which is associated with increased fluid uptake into the cell, leading to cellular edema [12]. A simple approach to measuring preservation-related pulmonary edema is to weigh the lung before and after storage. This does not distinguish among interstitial, alveolar, and cellular edema, but it is a marker for increased lung water and pulmonary injury in general.

To evaluate the extent of lethal cellular injury, we used a transvascular trypan blue flush. Trypan blue is a dye that is usually actively excluded by living cells [10, 13]. As we have shown previously, cell death is the pathophysiologic consequence of irreversible cellular injury and, therefore, an important end point in lung preservation [14]. In another study we have shown that, dependent on the mode of cell death, a significant cellular loss in the graft is a major contributor to lung dysfunction after transplantation [15].

To examine preservation injury in our study lungs, we chose to use transmission electron microscopy, as it is the gold standard for detecting injury at the cellular and subcellular level and it has proven to be helpful in previous studies of lung injury [9, 16]. Using electron microscopy, different features of pulmonary injury, including smaller alveolar surface fractions of normal type I pneumocytes, higher volume density of type II pneumocytes, interstitial and intraalveolar edema, pericapillary edema involving the blood–gas barrier, increased basement membrane thickness, breakage of the intact alveolar capillary endothelial cell sheet, and aggregated lamellar bodies of type II pneumocytes, have all been noted [9, 17]. Some of the above findings, including cell death (apoptosis and necrosis), macrophage infiltration, and reduction in the number of type II pneumocytes, were noted in the LPD group in this study, representing the severity of preservation injury in this group. In comparison, examination of the lungs in the R-LPD group showed only minor alterations and overall good preservation of the lung architecture and ultrastructure after 24 hours of storage.

In conclusion, raffinose appears to exert its cytoprotective effect on pulmonary grafts during the cold ischemic preservation period, as a significant improvement in pulmonary edema and ultrastructure is seen after preservation only, as demonstrated in this study.

	Acknowledgments

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The authors acknowledge the professional assistance of Peter Lewycky, PhD, who reviewed statistical analysis of this study, and the assistance of Joan Mates, PhD, Senior Research Technician, and Baljid Kalirei in conducting the experiments. Julia Hwang (medical electron microscopy technologist) prepared tissues for electron microscopy examinations. Doctor Liu is a Scholar of the Medical Research Council of Canada. The LPD solution (Perfadex) was generously provided by Biophausia, Uppsala, Sweden. The authors also acknowledge the financial support of the National Sanitarium Association of Canada and the Canadian Cystic Fibrosis Foundation.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): David Hopkinson Shaf Keshavjee Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fischer, S. Articles by Keshavjee, S. Search for Related Content PubMed PubMed Citation Articles by Fischer, S. Articles by Keshavjee, S. Related Collections Lung - transplantation