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Ann Thorac Surg 1995;59:1177-1181
© 1995 The Society of Thoracic Surgeons

Long-Term Preservation of Vascular Endothelium and Smooth Muscle

Department of Cardiothoracic Surgery, University Hospital, Lund, Sweden

Accepted for publication January 31, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This study was performed in organ baths on 400 ring segments of infrarenal aorta taken from 40 Sprague-Dawley rats that had been randomized into five groups. Contractility was tested with the thromboxane analogue U-46619. Acetylcholine was used to elicit endothelium-dependent relaxing factor (EDRF). The results obtained from vessels preserved at 4°C for 6, 12, 24, and 36 hours were compared with those from autologous vessels studied immediately after harvesting. Vessels preserved in Euro-Collins solution showed a 46% (p < 0.01) decrease in contractility after 12 hours of storage; after 24 hours only weak contractions could be elicited, and after 36 hours they had lost their ability to contract. The EDRF function was slightly reduced after 12 hours and could not be investigated after 24 and 36 hours. With the University of Wisconsin solution (UW) and the low-potassium–dextran–glucose solution Perfadex no decrease in contractility was seen in the first 24 hours, but at 36 hours the vessels preserved in UW had lost 40% (p < 0.01) and those preserved in Perfadex 30% (p < 0.05) of their contractility. The EDRF function was significantly reduced by about 15% after 6, 12, and 24 hours in both the UW and the Perfadex groups. At 36 hours, vessels stored in Perfadex had lost 41% (p < 0.001) and those stored in UW 17% (p < 0.01) of their EDRF function. Vessels stored in Krebs solution (the only solution containing calcium) manifested no reduction in contractility throughout the 36-hour test period, but a marked decrease in EDRF function was seen at 6 hours (12%; p < 0.05), 12 hours (31%; p < 0.05), 24 hours (68%; p < 0.001), and 36 hours (86%; p < 0.001). Vessels stored in heparinized blood maintained good contractility the first 24 hours, but after 36 hours a 30% (p < 0.05) loss of contractility was seen; the EDRF function was good for a period of up to 12 hours, but showed a decrease in relaxation capacity after 24 hours (39%; p < 0.05) and after 36 hours (70%; p < 0.001). To conclude, UW and Perfadex gave good preservation for 24 hours. After 36 hours, UW was slightly better for the endothelium whereas Perfadex was slightly better for the smooth muscle function. Euro-Collins solution was not a suitable solution for long-term preservation of blood vessels. Considering the excellent results obtained with Krebs solution regarding contractility, we suggest that the addition of calcium to UW and Perfadex will improve their ability to preserve smooth muscle function during prolonged storage.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The preservation method most commonly used in clinical organ transplantation is to flush the organ with a cold preservation solution to remove all blood and to obtain quick core cooling of the organ; the preservation solution is left in the organ, which is submerged in cold solution until the transplantation can be performed. The tissue most exposed to the preservation solution is the vascular tissue, particularly the endothelium, which is in direct contact with the solution during the storage period.

The aim of the present study was to investigate organ preservation solutions in current use with regard to their capacity to preserve endothelium-dependent relaxation and vascular smooth muscle function after long-term storage. Four preservation solutions were studied: Krebs, Euro-Collins, University of Wisconsin, and Perfadex. Heparinized cold blood also was studied, as this would be the medium in contact with the endothelium if a perfect flush out were not obtained or if topical or body core cooling was used as the only preservation method.

The infrarenal aorta of rats was selected for this study because the use of this preparation enabled the investigation to be standardized. Earlier we have shown that this vessel can be handled without disturbing endothelium-dependent relaxation or smooth muscle function [1]. We also have used this model to study short-term preservation of blood vessels [2].

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The infrarenal aortas of 40 male Sprague-Dawley rats were used. The rats, weighing about 300 g each, were randomized into five groups, each consisting of 8 animals. Four preservation solutions were tested: Krebs (fresh solution was made before each experiment), Euro-Collins (Fresenius AG; Bad Homburg, Germany), University of Wisconsin (ViaSpan; Du Pont Pharma, Hertfordshire, England) and Perfadex (Medisan, Uppsala, Sweden). Fresh blood was obtained from separate rats after heparinization (3 mg/kg) and immediately cooled to 4°C. The animals were treated in compliance with the ``Guide for Care and Use of Laboratory Animals'' published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Harvesting Procedure
The animals were anesthetized with ether. A dissecting microscope (Leika Wild M 691; Wild Leitz Ltd, Heerbrugg, Switzerland) was used for visualization. After exposure of the abdominal aorta, the segment between the renal arteries and the iliac bifurcation was freed from the inferior vena cava. Two microvascular clamps were placed proximally and distally on the freed part of the aorta, thus isolating a segment about 12 to 15 mm in length, which then was harvested. Ten segments, 1.1 ± 0.1 mm long, were cut from the vessel, and two of them were transferred immediately to tissue baths (fresh controls). The remaining segments were kept in the different preservation solutions or in blood at 4°C for 6, 12, 24, and 36 hours before being investigated.

Recording of Endothelium-Dependent Relaxation
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). This was bubbled with 95% oxygen and 5% carbon dioxide, giving a pH value of approximately 7.4 in the Krebs solution. Each ring segment was suspended between two metal holders (0.2 mm in diameter). One holder was attached to a Grass FT 03 transducer connected to a Grass polygraph for continuous recording of the isometric tension. The other metal holder was fixed to an adjustable unit by means of which the vessel segment was stretched repeatedly until a basal tension of 8 mN was reached. (In separate experiments, it was found that the maximum response was obtained at this tension.) Contraction then was induced with the thromboxane A₂ analogue U-46619 (The Upjohn Company, Kalamazoo, MI) added at a concentration of 10^-6.5 mol/L, giving a stable and strong contraction in the range of 7 to 14 mN. In separate experiments, concentration-response curves showed 10^-8.5 mol/L U-46619 to induce half maximum contraction and 10^-6.5 mol/L U-46619 to induce contractions in the range of 80% to 100% of the maximum. In separate experiments it also was shown that the contractile force elicited by 10^-6.5 mol/L U-46619 was the same regardless of the presence or absence of endothelium in the investigated ring segments. After repeated washes resulting in a return to the basal tension, contraction again was induced with 10^-6.5 mol/L U-46619. When this contraction had reached a stable plateau, increasing concentrations of acetylcholine (acetylcholine chloride; Sigma, St. Louis, MO) were added cumulatively to the bath. For each segment, the response to the different concentrations of acetylcholine was expressed as a percentage of the U-46619-induced contraction. One segment was not given any acetylcholine; this segment served as an internal control to ascertain that no spontaneous relaxation occurred. If complete relaxation was not elicited by acetylcholine, the endothelium-independent vasodilator papaverine (Kabi Pharmacia, Uppsala, Sweden) was added to the bath to ascertain whether complete relaxation then could be obtained.

Data Analysis
Results were expressed as the mean ± the standard error of the mean; n is the number of animals used in each group. The data were analyzed statistically comparing the results obtained after storage with those obtained with fresh autologous vessels (fresh controls) immediately after harvesting. One-way analysis of variance with repeated measurements was used. Differences were considered statistically significant when p was less than 0.05.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Preservation of Endothelium-Dependent Relaxation
Figure 1 shows the concentration–response curves of acetylcholine obtained at different times in vessels preserved in the different solutions. In the fresh controls, acetylcholine was able to elicit a maximal relaxation of 90% to 92% of the contraction induced by 10^-6.5 mol/L U-46619. Regardless of preservation solution used, a significant impairment in endothelium-dependent relaxation was seen after 6 hours of cold storage (Fig 2, lower panel). Vessels stored in Krebs solution showed a progressive decrease in endothelium-dependent relaxation (6 hours, 12%, [p < 0.05]; 12 hours, 31%, [p < 0.05]; 24 hours, 68%, [p < 0.01]; 36 hours, 86%, [p < 0.001]) compared with their fresh controls. Vessels stored in Perfadex and University of Wisconsin solution showed a significant decrease in relaxation capacity after 6 hours, but after that time the endothelium-dependent relaxation was not further impaired in the period up to 24 hours. After 36 hours, the loss of endothelium-dependent relaxation was significantly greater in the segments stored in the Perfadex solution than in those stored in the University of Wisconsin solution, when compared with the fresh controls (41% [p < 0.001] versus 17% [p < 0.01]). Vessels stored in heparinized blood still had well-functioning endothelium-dependent relaxation factor at 12 hours, but they showed a decreased relaxation capacity after 24 and 36 hours (39% [p < 0.05] and 70% [p < 0.001], respectively). Regarding the vessels stored in Euro-Collins solution, evaluation of endothelium-dependent relaxation was not carried out at 24 and 36 hours due to the near absence of contractility at these times. However, at 6 and 12 hours the Euro-Collins solution was as capable of preserving the endothelium as were the other solutions.

View larger version (42K): [in this window] [in a new window]   Fig 1. . Effects of cumulative addition of acetylcholine (Ach) to rat aorta precontracted with the thromboxane analogue U-46619 in (A) fresh controls (nonpreserved), (B) vessels 6 hours after preservation, (C) vessels 12 hours after preservation, (D) vessels 24 hours after preservation, and (E) vessels 36 hours after preservation. Open symbols show vessels where no acetylcholine was given. Each point shows the mean ± the standard error of the mean (n = 8 animals in each group). Endothelium-dependent relaxation could not be investigated in vessels stored for 24 and 36 hours in Euro-Collins solution as they had lost almost all contractility. (EC = Euro-Collins; PERF = Perfadex; UW = University of Wisconsin.)

View larger version (60K): [in this window] [in a new window]   Fig 2. . Contractile response to the thromboxane analogue U-46619 (upper panel) and the maximum endothelium-dependent relaxation elicited by acetylcholine in rat aorta (lower panel). The bars show the results from fresh controls and vessels preserved for 6, 12, 24, and 36 hours in the different solutions. Each bar represents the mean ± the standard error of the mean (n = 8). Endothelium-dependent relaxation could not be investigated in vessels stored for 24 and 36 hours in Euro-Collins solution as they had lost almost all contractility. (*p < 0.05, **p < 0.01, and ***p < 0.001 compared with fresh controls.)

Preservation of Contractile Function
Krebs solution proved best with regard to preservation of contractility; after 36 hours of preservation there was no significant decrease in contractility. Perfadex, heparinized blood, and University of Wisconsin solution were almost equally effective in preserving contractile function; compared with the fresh controls, there was no significant decrease in contractility during the first 24 hours (except for heparinized blood at 12 hours; p < 0.05) but after 36 hours the vessels had lost 30% (p < 0.05), 27% (p < 0.05), and 40% (p < 0.01) of their contractile capacity, respectively. Vessels preserved in Euro-Collins solution showed no significant decrease in contractility after 6 hours of storage; after 12 and 24 hours they had lost 46% (p < 0.01) and 91% (p < 0.001) of their contractile capacity, respectively, and after 36 hours of storage the vessels were unable to contract.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
The University of Wisconsin solution is used widely for preservation of the kidney, liver, and pancreas in clinical transplantation. The scientists behind this solution claim that cold-induced cellular swelling is controlled (with the cell membrane–impermeable molecules lactobionate and raffinose), free radical damage is minimized (with allopurinol and glutathionate), adenosine triphosphate synthesis is aided (with adenosine), and pH stability is maintained (with phosphate buffer) [3]. The Euro-Collins solution is used widely in clinical lung transplantation. Like the University of Wisconsin solution it is a buffered solution with intracellular concentrations of potassium and sodium. Perfadex is a buffered dextran solution with extracellular concentrations of potassium and sodium. Recently, safe lung preservation for 24 hours was demonstrated using this solution [4]. Krebs solution is a classic organ-bath solution with extracellular electrolyte composition. Heparinized cold blood is of interest when topical or body core cooling is used as the only preservation method and in cases where a perfect flush perfusion is not accomplished. In these cases, blood is the medium in direct contact with the vascular endothelium during the storage period [5].

With all solutions studied, we obtained a small but significant reduction in endothelium-dependent relaxation after 6 hours (see Fig 2). It is known that exposure to low temperatures can impair the basal and stimulated release of endothelium-derived relaxing factor [6]. Studies on cultured human endothelial cells showed that structural changes were induced by hypothermia, but rewarming elicited a rapid and nearly complete reversal of these changes [7]. Earlier we have shown that 20°C tends to be more beneficial for the endothelium than 4°C for the short-term preservation of vessels [2]. We therefore suggest that the small but almost equal reduction in endothelium-dependent relaxing factor function seen in all groups after 6 hours of cold storage in the present study is caused by the low temperature. Investigation of the vessels after reimplantation is necessary to know if this reduced endothelium-dependent relaxation is reversible, and this is the topic for a coming study by our group.

According to the present study, the University of Wisconsin solution was the most effective in protecting the endothelium after 36 hours of preservation. One explanation for the effectiveness of the University of Wisconsin solution may be the optimal osmolarity created by the cell membrane–impermeable molecules lactobionate and raffinose, which prevents cell edema during hypothermic storage when the efficacy of the sodium pump is reduced [3]. Regarding endothelium-dependent relaxation, Perfadex was as effective as University of Wisconsin solution for 24-hour preservation, but after 36 hours of storage University of Wisconsin solution was slightly better.

Due to its high concentration of glucose, Euro-Collins solution has a high osmolarity compared with the other solutions (Table 1). Stringham and co-workers [8] found that hearts stored in either hyperosmolar (357 mOsm/L) or hyposmolar (277 mOsm/L) University of Wisconsin solution functioned poorly compared with hearts stored in an almost isosmolar (297 or 327 mOsm/L) solution. One reason for the poor results obtained with Euro-Collins solution, therefore, could be that it has too high an osmolarity. Morphologic evidence for the superiority of the University of Wisconsin solution over Euro-Collins solution has been presented by Abebe and co-workers [9], using scanning and transmission electron microscopy. They found that rat aortas stored in Euro-Collins solution at 4°C for 1 hour revealed the presence of marked swelling of the endothelial cells with occasional large vacuoles and separation of the cells from the basal lamina; there was also mild interstitial edema and smooth muscle cell swelling. The mitochondria were swollen, and striking calcium deposits were observed. After 24 hours of storage in Euro-Collins solution, the endothelial cells were found 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 showed loss of most organelles and were markedly edematous as was the interstitium. However, after 1 hour of storage in University of Wisconsin solution at 4°C they found that the individual cells were almost intact and after 24 hours of storage in this solution only slight endothelial cell swelling and some swelling of the mitochondria were seen, whereas the nuclei of the cells appeared normal. The smooth muscle cells were slightly swollen but the myofilaments were intact. Functionally, Abebe and co-workers [9] could demonstrate a significantly reduced contractility in the rat aortas stored for 24 hours in Euro-Collins solution but not in those stored in University of Wisconsin solution, confirming the findings of the present study.

Krebs solution, although unable to preserve endothelium-dependent relaxation, was the only solution able to preserve full contractile capacity after 36 hours of storage (see Fig 2). What could be the reason for this superior capacity of Krebs solution to preserve smooth muscle function after prolonged storage? As seen in Table 1, Krebs solution is the only solution that contains calcium, and we suggest that prolonged storage of smooth muscle cells in solutions containing too little calcium or none at all is harmful to contractile function. Decreasing the calcium concentration in University of Wisconsin solution from 1.0 to 0.1 mmol/L had a deleterious effect on myocardial function in rabbit hearts preserved for 30 hours [12]. Weyland and co-workers [13], using Euro-Collins solution in heart preservation, found that the modification of genuine (calcium free) Euro-Collins solution by addition of 0.0225 mmol/L of calcium was essential. In the present study, both Perfadex and University of Wisconsin solutions were able to preserve the contractile capacity for 24 hours, but after 36 hours the contractile capacity was significantly reduced in both groups, slightly more in vessels stored in University of Wisconsin solution. We suggest that addition of proper amounts of calcium to the calcium-free solutions Euro-Collins, Perfadex, and University of Wisconsin will increase their abilities to preserve vascular smooth muscle function after prolonged storage, but this hypothesis has to be proved in future studies.

The contractile capacity and the endothelium-dependent relaxing factor function of vessels preserved in heparinized cold blood underwent significant reduction during the storage period, although good results were obtained after 12 hours of storage. We have no good explanation for this, but we suggest that toxic products from deoxygenated blood cells might have affected these functions negatively.

To sum up, University of Wisconsin solution and Perfadex solution resulted in good preservation of blood vessels for 24 hours. After 36 hours University of Wisconsin solution was slightly better for the endothelium, whereas Perfadex was slightly better for the smooth muscle function. Euro-Collins solution and Krebs solution were not suitable for long-term preservation of blood vessels, whereas cold heparinized blood resulted in good preservation for 12 hours.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This study was supported by grants from Swedish Heart Lung Foundation, Westerströms Stiftelse, and the Medical Faculty, University of Lund.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Steen, Department of Cardiothoracic Surgery, University Hospital, S-221 85 Lund, Sweden.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract 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 Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ingemansson, R. Articles by Steen, S. Search for Related Content PubMed PubMed Citation Articles by Ingemansson, R. Articles by Steen, S.