Ann Thorac Surg 1997;63:408-413
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Addition of Calcium to Euro-Collins Solution Is Essential for 24-Hour Preservation of the Vasculature
Richard Ingemansson, MD, PhD,
Ramunas Bolys, MD,
Algimantas Budrikis, MD,
Anna Lindgren, BSc,
Trygve Sjöberg, PhD,
Stig Steen, MD, PhD
Department of Cardiothoracic Surgery, University Hospital, Lund, Sweden
Accepted for publication August 13, 1996.
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Abstract
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Background. Genuine Euro-Collins solution is calcium free. The aim of this study was to investigate whether the addition of calcium would improve its capacity to preserve the vasculature.
Methods. The infrarenal aorta of Sprague-Dawley rats was investigated in organ baths: as fresh controls, after 24 hours of cold (4°C) storage in Euro-Collins solution, or in Euro-Collins solution with the addition of calcium in amounts ranging from 0.05 to 1.5 mmol/L. The thromboxane analogue U-46619 was used to investigate contractility. Endothelium-dependent relaxation was tested by cumulative addition of acetylcholine. Papaverine was used to elicit endothelium-independent relaxation. Investigation by transmission electron microscopy was also performed.
Results. Storage of rat aorta for 24 hours in genuine Euro-Collins solution almost abolished smooth muscle function, and severe edema was found in the endothelial cells. However, if calcium was added, the rat aorta could be stored for 24 hours without affecting smooth muscle function, and endothelium-dependent relaxation was only slightly reduced. Furthermore, only slight edema could be demonstrated in the endothelial cells.
Conclusions. If calcium is added to Euro-Collins solution in amounts ranging from 0.4 to 1.5 mmol/L, it allows good preservation of rat aorta for 24 hours. Without calcium, this solution destroys both the function and morphology of the vessels.
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Introduction
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The most commonly used preservation method in clinical transplantation today is flush perfusion of the organ with a cold preservation solution to remove blood and obtain quick core cooling. The preservation solution is left in the organ until transplantation can be performed. The tissue most exposed to the preservation solution is the vascular tissue, particularly the vascular endothelium, which is in direct contact with the solution during the storage period. It is of great importance to preserve the vessels well, because without good preservation of the vascular endothelium, endothelial swelling may lead to occlusion of the small capillaries. This will cause difficulties in the redistribution of blood to the organ during the reperfusion period (the no-reflow phenomenon), which may lead to cell death and tissue necrosis. Furthermore, damage to the endothelium can cause spasm, thrombosis, intimal hyperplasia, and exposure of subendothelial receptors, which may increase the risk of graft rejection [1].
In 1988, Locke and co-workers [2] concluded that flush perfusion with Euro-Collins solution gives good lung preservation for 6 hours. Since then, Euro-Collins has been the solution used at most centers for preserving lungs for clinical transplantation. We have stated previously that calcium-free preservation solutions are not optimal for long-term preservation of the vasculature [3, 4].
The aim of the present study was to investigate the functional and morphologic effects on smooth muscle function and endothelium-dependent relaxation after 24 hours' storage of rat aorta in genuine Euro-Collins solution and in Euro-Collins solution with added calcium.
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Material and Methods
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We used ring segments from the infrarenal aorta of 16 male Sprague-Dawley rats, each weighing about 300 g. Ten animals served as fresh controls; these samples were investigated after 24 hours' cold (+4°C) storage. Genuine Euro-Collins solution (Fresenius AG, Bad Homberg, Germany) and Euro-Collins solution with the addition of calcium (0.05, 0.1, 0.2, 0.4, 0.7, 1.0, and 1.5 mmol/L) were used as storage solutions. Six animals served as donors for transmission electron microscopy. The animals were treated in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).
Harvesting and Preservation Procedure
All animals were anesthetized with ether. A dissecting microscope (Leika Wild M 619; Wild Leitz Ltd, Heerbrugg, Switzerland) was used for visualization. After exposure of the infrarenal aorta, the part of the aorta between the renal arteries and the iliac bifurcation was dissected free from the inferior vena cava. Two microvascular clamps were placed proximally and distally on the dissected aorta, isolating a segment 12 to 15 mm in length. The aorta was extirpated, and intraluminal blood was removed by gently dripping room-temperature Krebs solution through it. The segments were immediately investigated in organ baths as fresh controls or were transferred to Euro-Collins solution, with or without added calcium, and stored at 4°C for 24 hours, after which they were taken out and investigated in organ baths. We have shown previously that infrarenal rat aorta can be handled in this way without disturbing the endothelium or the smooth muscle function [5, 6].
Recording Contractility
Isometric tension was measured using a myograph consisting of a chamber with a volume of 5 mL, water-mantled to control the temperature of the bath solution (37°C). Krebs solution, the medium used in the organ baths in all experiments, was bubbled with 95% oxygen and 5% carbon dioxide, which gave a pH of approximately 7.4. The composition of the Krebs solution was (in mmol/L): NaCl, 119; NaHCO3, 15; KCl, 4.6; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 1.5; and glucose, 11. Each ring segment was suspended between two metal holders (0.2 mm in diameter). One holder was attached to a Grass FT 03 transducer (Grass Instrument Co, Quincy, MA), connected to a Grass polygraph for continuous recording of isometric tension. The other metal holder was fixed to an adjustable unit, by means of which the vessel segment was repeatedly stretched until a basal tension of 8 mN was reached (in earlier experiments, we found that the maximum response is obtained at this tension). A first contraction was then induced with the thromboxane A2 analogue U-46619 (The Upjohn Company, Kalamazoo, MI), added at a concentration of 10-6.5 mol/L. In a previous study [7], concentration-response curves with U-46619 showed that a concentration of 10-6.5 mol/L induces a contraction that is 95% ± 1% of maximum. In the same study, we removed the endothelium from some segments and clearly demonstrated that U-46619 is an endothelium-independent vasoconstrictor in this model. The bath medium was then changed by repeated washing with Krebs solution to bring the curve back to baseline.
Recording Endothelium-Dependent Relaxation
A second contraction was induced with 10-6.5 mol/L U-46619. When the contraction had reached a stable plateau, increasing concentrations of acetylcholine (acetylcholine chloride; Sigma, St. Louis, MO) were added cumulatively to the baths. Acetylcholine elicits the release of nitric oxide through receptors on the endothelium. In each segment, the response to the different concentrations of acetylcholine was expressed as a percentage of the U-46619–induced contraction.
Recording Endothelium-Independent Relaxation
If complete relaxation was not obtained with acetylcholine, the endothelium-independent vasodilator papaverine (10-4 mol/L) was added to the bath to investigate whether complete relaxation could then be obtained.
Morphology
The infrarenal aortas of 6 male Sprague-Dawley rats (each 300 g) were harvested and cut into nine segments, each 1 mm in length. Three of them were transferred immediately to 2% glutaraldehyde for prefixation, to serve as fresh controls. The remaining segments were randomly placed into genuine Euro-Collins solution or Euro-Collins solution with 1.5 mmol/L added calcium and stored for 24 hours at 4°C before prefixation in 2% glutaraldehyde.
After prefixation in 2% glutaraldehyde for 5 to 7 hours, the specimens were washed with 0.1 mol/L sodium cacodylate buffer (containing 0.1 mol/L sucrose; pH 7.2). Postfixation was carried out with 1% osmium tetroxide, 2% OsO4, and S-Collidin (2, 4, 6 trimethylpyridine) buffer in equal amounts. After this, the specimens were washed with 0.1 mol/L sodium cacodylate buffer (containing 0.1 mol/L sucrose; pH 7.2). They were then dehydrated in ethanol, in rising concentrations from 70% to 99.5%, and with 99% propylene oxide. Before embedding in Agar 100 (containing 47% Agar 100 resin, 18.6% dodecenyl succinic anhydride, 32.4% methyl nadic anhydride, and 2% N-benzyldimethylamine), impregnation was done with equal amounts of propylene oxide (99%) and Agar 100. Sections 1 µm thick were prepared and stained with methylene blue. Sections with surface endothelial layer were identified, and ultra-thin (approximately 60 nm) sections were made. These were contrasted with 4% uranyl acetate for 30 minutes at 40°C and with lead citrate for 2 minutes. The sections were examined under a Philips CM 10 transmission electron microscope (Philips Co, Eindhoven, Holland). A series of photographs was prepared at magnifications ranging from 8,320 to 45,600.
Data Analysis
Results were expressed as mean ± standard error of the mean, with n representing the number of animals used in each group. The functional part was evaluated statistically by one-way analysis of variance, using Dunnet's test for multiple comparisons. A p value less than 0.05 was considered statistically significant. Morphology was evaluated using a four-step scale, with changes graded from normal to severe compared with fresh controls, and statistical calculation was performed with the marginal homogeneity test. Statistical calculation was done by Clinical Data Care AB (Lund, Sweden).
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Results
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All results are depicted in Figures 1 to 4
and Table 1.
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Fig 1. . Contractile response to the thromboxane analogue U-46619 (top) and the maximum endothelium-dependent relaxation elicited by acetylcholine in rat aorta precontracted by U-46619 (bottom). Bars show the results from fresh controls and from vessels preserved for 24 hours in genuine Euro-Collins solution at 4°C and in Euro-Collins solution with added calcium. Each bar represents the mean ± standard error of the mean; n = 10 rats in all groups. (A = endothelium-dependent relaxation could not be investigated because the vessels lost almost all ability to contract; *p < 0.05; **p < 0.01; ***p < 0.001.)
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Fig 2. . Transmission electron microscopy of endothelial cells investigated immediately after harvesting (A) and after 24 hours' storage in genuine Euro-Collins solution (B) and in Euro-Collins solution with 1.5 mmol/L added calcium (C). The homogeneous bandlike structures next to the endothelial cells represent the internal elastic lamina, and arrows indicate the endothelial cells. (All x8,320 before 35% reduction.)
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Fig 3. . Transmission electron microscopy of the nuclei of smooth muscle cells investigated immediately after harvesting (A) and after 24 hours' storage in genuine Euro-Collins solution (B) and in Euro-Collins solution with 1.5 mmol/L added calcium (C). The arrows indicate the smooth muscle nuclei. (All x10,560 before 35% reduction.)
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Fig 4. . Transmission electron microscopy of mitochondria investigated immediately after harvesting (A) and after 24 hours' storage in genuine Euro-Collins solution (B) and in Euro-Collins solution with 1.5 mmol/L added calcium (C). The arrows indicate a typical mitochondrion. (All x45,600 before 35% reduction.)
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Table 1. . Results of Morphologic Examination Comparing Vessels Stored for 24 Hours (4°C) in Genuine Euro-Collins Solution and in Euro-Collins Solution With 1.5 mmol/L Added Calciuma
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Preservation of Contractile Function
Genuine Euro-Collins solution was able to preserve very little contractile function after 24 hours' storage at 4°C. Vessels preserved in Euro-Collins solution with 0.05, 0.1, or 0.2 mmol/L added calcium showed a significant decrease in contractile capacity compared with fresh controls (see Fig 1
). The addition of 0.4, 0.7, 1.0, or 1.5 mmol/L calcium to Euro-Collins solution resulted in no significant decrease in contractility after 24 hours' storage at 4°C compared with fresh controls.
Preservation of Endothelium-Dependent Relaxation
Vessels preserved for 24 hours at 4°C in genuine Euro-Collins solution were not investigated because the contractions were too weak. Vessels preserved in Euro-Collins solution with 0.05, 0.1, 0.2, 0.4, 0.7, 1.0, and 1.5 mmol/L added calcium showed a loss of endothelium-dependent relaxation of 21% (p < 0.01), 18% (p < 0.05), 16% (p < 0.05), 13% (p < 0.05), 11% (p < 0.05), 13% (p < 0.05), and 19% (p < 0.01), respectively, compared with fresh controls.
Preservation of Endothelium-Independent Relaxation
For cases in which full relaxation was not obtained with acetylcholine, 10-4 mol/L papaverine, an endothelium-independent vasodilator, was added to the baths; complete relaxation was then elicited in all cases.
Morphologic Changes After 24 Hours' Storage in Genuine Euro-Collins Solution
Vessels preserved in genuine Euro-Collins solution for 24 hours at 4°C showed markedly swollen endothelial cells with loss of most organelles, including the chromatin of the nuclei (see Fig 2B). The nuclei of the smooth muscle cells were severely edematous, and disintegration of the chromatin was seen (see Fig 3B). The mitochondria were swollen, and most cristae were destroyed (see Fig 4B).
Morphologic Changes After 24 Hours' Storage in Euro-Collins Solution With Added Calcium
Vessels preserved in Euro-Collins solution for 24 hours at 4°C with 1.5 mmol/L added calcium showed only slight endothelial cell swelling, and the nuclei of these cells seemed almost intact (see Fig 2C); the nuclei of the smooth muscle cells were slightly swollen but also appeared intact (see Fig 3C). The mitochondria were swollen, but most cristae were intact (see Fig 4C).
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Comment
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Recently, we demonstrated that Krebs solution with a calcium concentration of 1.5 mmol/L is able to preserve vascular contractility in rat aorta after 36 hours of cold (4°C) storage [3, 4]. However, the more sophisticated organ preservation solutions-Euro-Collins, University of Wisconsin, and Perfadex solution, none of which contain calcium-were not able to preserve contractility in rat aorta for 36 hours of cold storage. We therefore proposed that prolonged storage of vascular smooth muscle cells in solutions lacking calcium is harmful to contractile function. However, regarding endothelium-dependent relaxation, the University of Wisconsin solution was found to give good preservation; with Krebs solution, this endothelial function deteriorated over time and was gravely disturbed after 36 hours of storage [3]. Could the addition of calcium be deleterious to endothelium-dependent relaxation during prolonged cold storage? Abebe and co-workers [8] studied rat aorta with scanning and transmission electron microscopy after 24 hours' storage at 4°C in genuine Euro-Collins solution. They found the endothelial cells to be markedly swollen with loss of intracellular organelles, including most mitochondria. There were breaks in the cell membrane and granularity in the chromatin of the nuclei. The smooth muscle cells had lost most organelles and were noticeably edematous, as was the interstitium. In a previous study by our group [9], we found that after 24 hours of storage at 4°C in genuine Euro-Collins solution, almost no contractility was left in the vessels; evaluation of the endothelium-dependent relaxation therefore could not be performed.
What is the optimal concentration of calcium in Euro-Collins solution for the preservation of vascular endothelial and smooth muscle function? In an interesting article, Burgmann and co-workers [10] argued that if calcium is added to Euro-Collins solution, the free calcium concentration will be about 0.3 mmol/L regardless of whether the total calcium concentration is 1 or 4 mmol/L. They were also able to show that this considerable calcium complex–binding capacity of Euro-Collins solution is mainly attributable to the high concentration of phosphate in this solution.
In the present study, we demonstrated that the optimal total calcium concentration for 24-hour preservation of vessels with Euro-Collins solution is between 0.4 and 1.5 mmol/L. In this range, there is no significant decrease in contractility and only a slight decrease in endothelium-dependent relaxation after 24 hours of cold (4°C) storage, compared with fresh controls (see Fig 1). We propose that this decrease in endothelium-dependent relaxation is due to the hypothermic storage conditions. It is known that exposure to low temperatures can impair the basal and stimulated release of endothelium-dependent relaxing factor [11]. Studies on cultured human endothelial cells have shown that structural changes are induced by hypothermia, but that rewarming elicits a rapid and nearly complete reversal of these changes [12]. According to an earlier study by our group [7], this small, probably cold-induced decrease in endothelium-dependent relaxation disappears after 2 hours of in vivo reperfusion. The morphologic results from the present study demonstrate that the endothelial cells, smooth muscle nuclei, and mitochondria of vessels preserved in genuine Euro-Collins solution are more swollen and disintegrated than those stored in Euro-Collins solution with 1.5 mmol/L added calcium (see Figs 2 to 4).
There are several reports [13, 14] showing that the extracellular calcium concentration is crucial in the regulation of endothelial permeability. Studies in human endothelial cell systems indicate an increased endothelial permeability after the depletion of calcium [15–17]. We suggest that hypothermic storage in a calcium-free solution results in increased permeability, with an influx of fluid and cell swelling as a result. Furthermore, a decrease in free calcium concentration due to diffusion out of the cell along its concentration gradient might occur. This increase in endothelial permeability and calcium outflow might be stopped by adding calcium in proper concentrations to the storage solution. The specific interaction between calcium and magnesium has not yet been properly evaluated. However, it has been proposed that magnesium acts as a physiologic calcium channel blocker [18]. Genuine Euro-Collins solution contains 0.8 mmol/L magnesium, and this concentration seems to be too low to be an effective calcium channel blocker and thereby to stop calcium leakage.
In conclusion, the addition of calcium to Euro-Collins solution seems to be essential for good 24-hour preservation of vascular endothelial and smooth muscle function. The presence of calcium in concentrations ranging from 0.4 to 1.5 mmol/L prevented any substantial decrease in contractility, and the morphology was almost intact after 24 hours of storage. In contrast, without calcium, almost no contractility was left after 24 hours' storage, and morphologic examination revealed severe edema in all cell components.
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Acknowledgments
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This study was supported by grants from the Swedish Heart Lung Foundation, T Westerströms stiftelse, and the Medical Faculty at the University of Lund.
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Footnotes
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Address reprint requests to Dr Steen, Department of Cardiothoracic Surgery, University Hospital, S-221 85 Lund, Sweden.
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References
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N. J. Odom and S. Steen
Addition of Calcium to Euro-Collins' Solution: A Word of Caution
Ann. Thorac. Surg.,
November 1, 1997;
64(5):
1520 - 1522.
[Full Text]
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Abstract
Introduction
