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Ann Thorac Surg 1996;61:1158-1162
© 1996 The Society of Thoracic Surgeons

Original Article: Cardiovascular

Importance of Calcium in Long-Term Preservation of the Vasculature

Richard Ingemansson, MD, Trygve Sjöberg, PhD, Stig Steen, MDPhD

Department of Cardiothoracic Surgery, University Hospital of Lund, Lund, Sweden

Accepted for publication November 12, 1995.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background. The aim was to investigate the effect of calcium in organ preservation solutions with respect to 36-hour preservation of vascular smooth muscle function and endothelium-dependent relaxation.

Methods. The infrarenal aortas of 60 Sprague-Dawley rats were studied in organ baths as fresh controls and after 36 hours of cold (4°C) storage in different preservation solutions with and without calcium. The thromboxane A2analogue U-46619 was used to study contractility. Endothelium-dependent relaxation was tested by the cumulative addition of acetylcholine. Papaverine hydrochloride was used to elicit endothelium-independent relaxation.

Results. Krebs solution was the only solution able to fully preserve contractility. Krebs solution without calcium gave poor preservation. After the addition of 1.5 mmol/L of calcium to University of Wisconsin solution and to Perfadex, both these solutions became fully able to preserve contractility. None of the solutions (with or without calcium) were fully able to preserve endothelium-dependent relaxation, although University of Wisconsin solution gave good preservation and Perfadex, fair preservation. Euro-Collins solution and K+ (124 mmol/L)-enriched Krebs solution were not able to preserve smooth muscle function or endothelium-dependent relaxation.

Conclusions. Calcium is essential for long-term preservation of vascular smooth muscle function but not for long-term preservation of endothelium-dependent relaxation.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 References
 
The efficacy of different solutions in preserving myocardial function during cold ischemic storage has been studied extensively over the last 25 years. Only recently, however, have systematic studies been published regarding the preservation of vascular smooth muscle and endothelial function [1–5]. Without adequate preservation of the vasculature, a progressive deterioration of the blood flow during reperfusion may ultimately lead to the no-reflow phenomenon, even though the function of the other cells in the organ may be adequately preserved. Further, poor preservation of the endothelium may lead to thromboembolic complications, spasm, and intimal hyperplasia [6]. Damaged endothelium may also expose complement receptors on the endothelial cell surface, and it has been suggested that the subsequent binding of complement to the endothelium increases the risk of graft rejection after transplantation [7]. Thus, if you cannot preserve the vasculature, you cannot preserve the organ!

Recently we [2] 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. The more sophisticated organ preservation solutions, ie, Euro-Collins, University of Wisconsin, and Perfadex, none of which contains calcium, were not able to preserve contractility in rat aorta after 36 hours of cold storage. We therefore proposed that prolonged storage of vascular smooth muscle cells in solutions lacking calcium is harmful to the contractile function of those cells. However, regarding endothelium-dependent relaxation, 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 [2]. Could calcium be deleterious to the endothelium during prolonged cold storage? The aim of the present study was to investigate the effect of calcium in organ preservation solutions with respect to prolonged preservation of vascular smooth muscle function and endothelium-dependent or endothelium-independent relaxation.


   Material and Methods
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 Footnotes
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Ring segments from the infrarenal aorta of 60 male Sprague-Dawley rats, each weighing about 300 g, were used. Five different solutions were tested with and without calcium: Euro-Collins solution (Fresenius AG, Bad Homburg, Germany), University of Wisconsin solution (Du Pont, the Netherlands), Krebs solution and high-potassium Krebs solution (both made in our hospital pharmacy), and Perfadex solution (Medisan, Uppsala, Sweden). For the composition of the different solutions, see Table 1Go. 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 85-23, revised 1985).


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  		Table 1. . Composition of Preservation Solutions
 
Harvesting and Preservation Procedure
All 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 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, thus isolating a segment 12 to 15 mm in length. The aorta was extirpated and sliced in segments about 1 mm long. These were kept in the different preservation solutions at 4°C for 36 hours, after which they were taken up and transferred to organ baths. In 10 rats, segments were cut and immediately transferred to organ baths to serve as fresh controls. We [8] have previously shown that the infrarenal rat aorta can be handled in this way without disturbing the endothelium or the smooth muscle function.

Recording of 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). This was bubbled with 95% oxygen and 5% carbon dioxide, which gives a pH of approximately 7.4 in Krebs solution, the medium used in the organ baths in all experiments. 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 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 separate experiments, we found that the maximum response was 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 separate experiments, concentration–response curves with U-46619 showed that a concentration of 10-6.5 mol/L induces contractions in the range of 90% to 100% of maximum.

Recording of Endothelium-Dependent Relaxation
A contraction was induced with 10-6.5 mol/L U-46619. After repeated washes resulting in a return to the basal tension, a new contraction was induced with the same concentration of U-46619. When the contraction had reached a stable plateau, increasing concentrations of acetylcholine chloride (Sigma, St. Louis, MO) were cumulatively added 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 of Endothelium-Independent Relaxation
If complete relaxation was not obtained with acetylcholine, the endothelium-independent vasodilator papaverine hydrochloride was added to the bath to investigate whether complete relaxation could then be obtained.

Data Analysis
Results were expressed as the mean ± the standard error of the mean. Student's t test for unpaired data was used for evaluation of data. Differences were considered significant when the p value was less than 0.05.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
The results are depicted in Figures 1 and 2GoGo. As seen, calcium played an essential role in the preservation of contractile function but not in the preservation of endothelium-dependent relaxation.



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  		Fig 1. . Contractility and endothelium-dependent relaxation in rat aorta after 36 hours of storage at 4°C with and without 1.5 mmol/L of calcium. Each bar represents the mean ± the standard error of the mean. There were 10 rats in each group. (UW = University of Wisconsin solution; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.)

 




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  		Fig 2. . Effects of cumulative addition of acetylcholine (Ach) to rat aorta precontracted with thromboxane A2 analogue U-46619 in fresh vessels (Control) and in vessels stored for 36 hours at 4°C in (A) University of Wisconsin solution (UW), (B) Euro-Collins solution, (C) potassium-enriched (K+) Krebs solution, (D) Krebs solution, and (E) Perfadex without or with 1.5 mmol/L of calcium (Ca2+). The shaded symbols at the top of each panel show the contractile condition recorded in vessel segments where no acetylcholine was given. Each point represents the mean ± the standard error of the mean. There were 10 rats in each group. Where the standard error of the mean is not shown, it is so small that it is hidden within the symbol. (conc = concentration.)

 
Preservation of Contractile Function
Krebs solution was the only unmodified preservation solution that was able to preserve vascular smooth muscle function after 36 hours of cold storage, but without calcium, it was not able to accomplish this. When 1.5 mmol/L of calcium was added to the calcium-free University of Wisconsin and Perfadex preservation solutions, they became as effective as Krebs solution in preserving muscle function. The amount of calcium in University of Wisconsin solution with benzylpenicillin (10-6.4 mol/L) affected contractility favorably but not enough to transform genuine University of Wisconsin solution into a fully effective vascular smooth muscle preservation solution comparable to Krebs solution or to University of Wisconsin and Perfadex solutions with 1.5 mmol/L of added calcium. The addition of 1.5 mmol/L of calcium to Euro-Collins solution or to potassium-enriched Krebs solution without calcium increased their ability to preserve contractility, although not to the same level as University of Wisconsin and Perfadex solutions with calcium.

Preservation of Endothelium-Dependent Relaxation
No solution was able to fully preserve endothelium-dependent relaxation, although University of Wisconsin solution gave good preservation. Krebs solution, which was able to preserve muscle contractility, was not able to preserve endothelium-dependent relaxation. The presence or absence of calcium in the solutions did not affect their ability to preserve endothelium-dependent relaxation.

Preservation of Endothelium-Independent Relaxation
The endothelium-independent vasodilator papaverine at a concentration of 10-4 mol/L elicited complete relaxation in all segments.


   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
This study demonstrates the need for calcium in the preservation solution if long-term preservation of vascular smooth muscle function is to be obtained during cold storage. However, in regard to the long-term preservation of endothelium-dependent relaxation, the presence or absence of calcium in the preservation solution seems to be of no importance.

What is the optimal concentration of calcium for the preservation of vascular smooth muscle function? To answer this question, it is necessary to know the calcium-chelating capacity of each specific preservation solution under investigation. It is the concentration of free ionized calcium and not the total calcium concentration in the solution that is important. Burgmann and co-workers [9] demonstrated that the free calcium concentration in University of Wisconsin solution and Euro-Collins solution is around 0.3 mmol/L regardless of the total calcium concentration between 1 and 4 mmol/L. St. Thomas' solution, Bretschneider solution, and normal saline solution, however, have virtually no calcium complex–binding capacity, ie, if 1 or 4 mmol/L of calcium is added to these solutions, the concentration of free (ionized) calcium will be 1 to 4 mmol/L, respectively. This group [9] was also able to show that the considerable calcium complex–binding ability of Euro-Collins and University of Wisconsin solutions is attributable mainly to the high concentration of phosphate in these solutions and, in University of Wisconsin solution, to lactobionic acid. Both Krebs and Perfadex solutions contain small amounts of phosphate, but not enough to have any substantial calcium complex–binding capacity [9].

What should the concentration of free calcium in a preservation solution be to achieve optimal preservation of vascular smooth muscle function after prolonged cold storage? The concentration of free calcium in University of Wisconsin solution (with added calcium in the range of 1 to 4 mmol/L) is around 0.3 mmol/L [9]. This concentration of free calcium seems to be adequate, as no significant reduction in contractility was seen in the vessels stored in University of Wisconsin solution with 1.5 mmol/L of added calcium. However, the concentration of calcium in University of Wisconsin solution containing 120 mg/L of benzylpenicillin is 10-6.4 mol/L, and this is too low because a significant reduction in contractility was seen in this group. Considering the excellent results obtained with Krebs solution (containing 1.5 mmol/L of free ionized calcium), it seems that prolonged storage of vascular smooth muscle can successfully be carried out in solutions with an ionized calcium concentration in the range of 0.3 to 1.5 mmol/L.

A comparison of the results obtained with Krebs solution (extracellular) and high-potassium Krebs solution (intracellular) regarding contractility after 36 hours of storage shows that Krebs solution is the more effective, a finding indicating that high potassium concentrations are not essential in long-term preservation of vascular smooth muscle function. Euro-Collins solution was the only one that was not able to preserve any vascular smooth muscle function at all after 36 hours of cold storage. However, the addition of 1.5 mmol/L of calcium to Euro-Collins solution had a strikingly positive effect, ie, the vessels retained almost 50% of normal contractility.

With all solutions studied, we obtained a significant decrease in endothelium-dependent relaxation after 36 hours of storage. In an earlier study, we [2] found a significant decrease in endothelium-dependent relaxation already after 6 hours of storage in all solutions studied. For University of Wisconsin and Perfadex solutions, this loss in endothelium-dependent relaxation did not increase over time. The storage temperature in that study was also 4°C, and we suggested that the loss of endothelium-dependent relaxation could be due to the low storage temperature. It is known that exposure to low temperatures can impair basal and stimulated release of endothelium-dependent relaxing factor [10]. Studies [11] involving cultured human endothelial cells have shown that structural changes are induced by hypothermia, but rewarming elicits a rapid and nearly complete reversal of these changes. Further studies are needed to elucidate the effect of low temperatures on the endothelium.

If 4°C is injurious to endothelial function, will the endothelium recover after reperfusion or will it be further damaged? The endothelium-dependent relaxing factor function in porcine lungs preserved for 24 hours with cold Perfadex solution was found to be significantly reduced, and the greater the reduction, the higher the pulmonary vascular resistance [4]. After 24 hours of reperfusion, the endothelium-dependent relaxation neither recovered nor deteriorated further in that study. In a study [3] comparing Euro-Collins solution with Perfadex solution in terms of 24-hour preservation of rat aortas, the endothelium-dependent relaxation, which was significantly reduced after 24 hours of cold storage, had fully recovered after 7 days of reperfusion in the Perfadex group but not in the Euro-Collins group.

Thus, with an adequate preservation solution, we suggest that a reduction in endothelial function after prolonged cold storage is reversible. However, considering that a transplanted organ needs an adequate homogeneous circulation of oxygenated blood right from the start of warm reperfusion to avoid ischemic injury, which otherwise will set in within minutes, further studies should be carried out with reperfusion models including the whole microvasculature. Only then will it be clear how dangerous is the certain degree of reduction in endothelial function that is the consequence of prolonged cold storage. In porcine donor lungs, storage at 8°C in Perfadex for 24 hours does not lead to poor reperfusion of the capillaries, as the blood gas exchange in such transplanted lungs is excellent from the very beginning of warm reperfusion [12].

No morphology was included in this study because Abebe and co-workers [5] have already studied rat aorta by scanning and transmission electron microscopy after 1 hour and 24 hours of cold storage in Euro-Collins and University of Wisconsin solutions. 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 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, the individual cells were almost intact; 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 these cells appeared normal. The smooth muscle cells were slightly swollen, but the myofilaments were intact.

In conclusion, calcium is essential for long-term preservation of vascular smooth muscle contractility but not for long-term preservation of endothelium-dependent or endothelium-independent relaxation. With the addition of 1.5 mmol/L of calcium to University of Wisconsin or Perfadex solution, both solutions were able to preserve vascular smooth muscle contractility for 36 hours. However, none of the solutions tested in this study were able to fully preserve endothelium-dependent relaxation, although University of Wisconsin solution gave good preservation and Perfadex, fair preservation of this important function. Euro-Collins solution was not able to preserve either vascular smooth muscle function or endothelium-dependent relaxation regardless of the presence or absence of calcium.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 
Address reprint requests to Dr Steen, Department of Cardiothoracic Surgery, University Hospital of Lund, S-221 85 Lund, Sweden.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

  1. Massa G, Ingemansson R, Sjöberg T, Steen S. Endothelium-dependent relaxation after short-term preservation of vascular grafts. Ann Thorac Surg 1994;58:1117–22.[Abstract/Free Full Text]
  2. Ingemansson R, Sjöberg T, Massa G, Steen S. Long-term preservation of vascular endothelium and smooth muscle. Ann Thorac Surg 1995;59:1177–81.[Abstract/Free Full Text]
  3. Ingemansson R, Massa G, Pandita RK, Sjöberg T, Steen S. Perfadex is superior to Euro-Collins solution regarding 24-hour preservation of vascular function. Ann Thorac Surg 1995;60:1210–4.[Abstract/Free Full Text]
  4. Kimblad PO, Sjöberg T, Steen S. Pulmonary vascular resistance related to endothelial function after lung transplantation. Ann Thorac Surg 1994;58:416–20.[Abstract/Free Full Text]
  5. Abebe W, Cavallari N, Agrawal DK, et al. Functional and morphological assessment of rat aorta stored in University of Wisconsin and Euro-Collins solutions. Transplantation 1993;56:808–16.[Medline]
  6. O'Connell TX, Sanchez M, Mowbray JF, Fonkalsrud EW. Effects on arterial intima of saline infusions. J Surg Res 1974;16:197–203.[Medline]
  7. Ryan US. The endothelial cell surface and response to injury. Fed Proc 1986;45:101–8.[Medline]
  8. Sjöberg T, Massa G, Steen S. Endothelium-mediated relaxation in transplanted aorta. Ann Thorac Surg 1992;53:1068–73.[Abstract/Free Full Text]
  9. Burgman H, Reckendorfer H, Sperlich M, Doleschel W, Spieckermann P. The calcium chelating capacity of different protecting solutions. Transplantation 1992;54:1106–8.[Medline]
  10. Bodelsson M, Arneklo-Nobin B, Törnebrandt K. Cooling augments contractile response to 5-hydroxytryptamine via an endothelium-dependent mechanism. Blood Vessels 1989;26:347–9.[Medline]
  11. Solberg S, Larsen T, Lindal S, Prydz P, Jörgensen L, Sörlie D. The effects of two different crystalloid cardioplegic solutions on cultured human endothelial cells. J Cardiovasc Surg (Torino) 1989;30:669–74.[Medline]
  12. Steen S, Kimblad PO, Sjöberg T, Lindberg L, Ingemansson R, Massa G. Safe lung preservation for twenty-four hours with Perfadex. Ann Thorac Surg 1994;57:450–7.[Abstract/Free Full Text]



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