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Ann Thorac Surg 1996;61:1413-1417
© 1996 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Effect of Temperature in Long-Term Preservation of Vascular Endothelial and Smooth Muscle Function

Department of Cardiothoracic Surgery, University Hospital, Lund, Sweden

Accepted for publication January 25, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Background. In clinical transplantation the donor organ is perfused with a cold preservation solution to obtain quick core cooling and a suitable environment for the tissue cells. Without good preservation of the vasculature, 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. The aim of this study was to find the optimal storage temperature for preservation of the vasculature.

Methods. The infrarenal aorta of 126 Sprague-Dawley rats were studied in organ baths: as fresh controls, after 36 hours of storage at 0.5°C, 4°C, 8.5°C, and 22°C in University of Wisconsin solution, and after 36-hour storage followed by transplantation and a lapse of 2 hours, 24 hours, and 7 days. The thromboxane analogue U-46619 was used to test contractility. Acetylcholine was used to elicit endothelium-dependent relaxation (EDR), and papaverine to elicit endothelium-independent relaxation.

Results. Storing the vessels at 0.5°C proved best regarding preservation of contractility, with a nonsignificant decrease, whereas storage at 4°C and 8.5°C resulted in a significant decrease after 36 hours. The contractility did not recover within 24 hours of in vivo reperfusion, but full recovery was seen after 7 days. Regardless of the preservation temperature used, a significant impairment in EDR was seen after 36 hours of storage. Two hours after transplantation, vessels stored at 4°C and 8.5°C showed no significant impairment in EDR, whereas those stored at 0.5°C demonstrated a significant loss of EDR. After 24 hours and after 7 days, EDR was normal in all groups.

Conclusions. Endothelium-dependent relaxing factor function is best preserved at 4°C and 8.5°C, whereas preservation of vascular smooth muscle function is best preserved at 0.5°C.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
See also page 1445

There is little information published regarding the functional consequences of cold storage on the vasculature. Recently, we demonstrated a significant decrease in endothelium-dependent relaxation after 6 hours of cold (4°C) storage in University of Wisconsin solution, but this loss in endothelial function did not increase any further after 36 hours of storage at 4°C [1]. Could low storage temperatures per se be deleterious to endothelial function? How severe is cold-induced injury? Will in vivo reperfusion cause a quick recovery of vascular function or will it add further damage? The aim of the present study was to consider these questions. The rat aorta was used as a model because we have earlier shown that this vessel can be harvested and transplanted without disturbing the endothelium-dependent relaxation and smooth muscle function [2, 3].

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
One hundred twenty-six Sprague-Dawley rats (275 to 325 g) were used in the study. Fifty-four animals were used as donors and 54 as recipients. Ten animals were used to provide samples for fresh controls and for 36-hour storage. Eight animals were used for controls with and without endothelium. The grafts were stored for 36 hours in University of Wisconsin solution at four different temperatures: 0.5°C, 4°C, 8.5°C, and 22°C. The animals were treated in compliance with the ``Guide for Care and Use of Laboratory Animals'' published by the National Institute of Health (NIH publication 85-23, revised 1985).

Donor Procedure
After exposure of the abdominal aorta, the segment between the renal arteries and the iliac bifurcation was dissected free from the inferior vena cava, a dissecting microscope (Leika Wild M 691; Wild Leitz Ltd, Heerbrugg, Switzerland) being used for visualization. Two microvascular clamps were placed on the freed aorta in such a way as to isolate a segment 12 to 15 mm in length. A 10- to 12-mm-long graft segment was excised, and blood was removed by dropping Krebs solution at room temperature through the lumen. In an earlier study [4] we have shown that Krebs solution at room temperature will not impair endothelium-dependent relaxation or smooth muscle function during 2 hours of storage. The vessel was then transferred within 1 minute to cold (0.5°C, 4°C, or 8.5°C) University of Wisconsin solution, where it was left for a period of 36 hours, after which it was transplanted to isogeneic rats. The temperature was kept between 0.5°C and 1.0°C during storage by putting ice-slush in the solution. Two different refrigerators were used to make sure that temperatures of 4°C and 8.5°C were maintained throughout the experiment.

Recipient Procedure
All animals were given streptocillin vet (Boehringer-Ingelheim, Ingelheim/Rhein, Germany), 0.1 mL subcutaneously, before the operation was started. The aorta was then prepared and clamped in the same way as in the donor and divided midway between the clamps. The graft segment that had been stored for 36 hours in cold (0.5°C, 4°C, or 8.5°C) University of Wisconsin solution was then interposed in its original orientation and sutured end-to-end with resorbable 9-0 Vicryl sutures (Ethicon, Somerville, NJ). Each anastomosis consisted of eight to ten interrupted sutures.

Two hours, 24 hours, or 7 days later, the grafts were removed and immediately placed in warm (37°C) and oxygenated (95% oxygen and 5% carbon dioxide) Krebs buffer solution and freed from any surrounding connective tissue. Two ring segments, each from 1.0 to 1.2 mm in length, were then taken from the midportion of the graft and transferred to organ baths.

Controls
The infrarenal aorta was extirpated and handled in the same way as the grafted vessels. Two segments were immediately transferred to organ baths (fresh controls). The remaining part of the vessel was divided into segments and immersed in University of Wisconsin solution for 36 hours at 0.5°C, 4°C, or 8.5°C and then transferred to organ baths for study. The composition of the University of Wisconsin solution (DuPont Pharma, the Netherlands) was (in mmol/L): Na⁺, 20; Mg²⁺, 5; K⁺, 140; PO₄^2-, 25; raffinose, 30; lactobionate, 100; allopurinol, 1; gluthathione, 3; adenosine, 5; pentastarch, 50 g/L; Decadrone, 8 mg/L (Merck Sharp and Dohme, Haarlem, the Netherlands); Actrapid, 40 IU/L (Novo nordisk AB, Denmark); and Benzylpenicillin, 120 mg/L (Astra, Södertälje, Sweden).

Recording Contractility and Endothelium-Dependent and -Independent Relaxation
Isometric tension was measured in organ baths that were water-mantled to keep the temperature of the bath solution at 37°C. The bath solution (Krebs) was bubbled with 95% oxygen and 5% carbon dioxide, giving it 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 connected to a Grass polygraph for continuous recording of isometric tension (Grass Instrument Co, Quincy, MA). The other metal holder was fixed to an adjustable unit by means of which the vessel segments were repeatedly stretched until a basal tension of about 8 milli-Newtons was reached. In separate experiments it was found that maximum response was obtained at this tension. A stable 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. After repeated washes resulting in the restoration of basal tension, a new contraction was induced with the same concentration of U-46619. When the degree of contraction had reached a stable plateau, increasing concentrations of acetylcholine (acetylcholine chloride; Sigma, St. Louis, MO) were cumulatively added to the baths. Acetylcholine releases endothelium-derived relaxing factor by stimulating receptors in the endothelium. In eight segments the endothelium was removed by gently rubbing the intimal surface over a pair of microtweezers, and in these cases acetylcholine elicited no relaxation (Fig 1). In each segment, the response to the different concentrations of acetylcholine was expressed as a percentage of the U-46619--induced contraction. If the relaxation obtained with acetylcholine in the preserved vessels was impaired, compared with the fresh controls, the endothelium-independent vasodilator papaverine was added to the bath to ascertain whether complete relaxation then could be obtained.

View larger version (23K): [in this window] [in a new window]   Fig 1. . Effect of cumulative addition of acetylcholine to vessels with and without endothelium. Each point shows the mean ± the standard error of the mean (n = 8 animals in each group). When error bars are not visible they are hidden by the symbols.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Contractile and Relaxing Capacity With and Without Endothelium
Concentration-response curves with U-46619 revealed that 10^-7.8 mol/L (pEC 50 = 7.8 ± 0.1) induced 50% of maximum contraction and 10^-6.5 mol/L induced 95% ± 1%. Cumulative addition of U-46619 to segments with and without endothelium showed no significant differences at any concentration (Fig 2). Fresh control vessels with endothelium demonstrated a 90% relaxation after stimulation with acetylcholine, but without endothelium they did not relax at all (see Fig 1).

View larger version (24K): [in this window] [in a new window]   Fig 2. . Effect of cumulative addition of U-46619 to vessels with and without endothelium. Each point shows the mean ± the standard error of the mean (n = 8 animals in each group). There were no significant differences seen at any concentration.

View larger version (57K): [in this window] [in a new window]   Fig 3. . Contractile response to the thromboxane A2 analogue U-46619 (upper panel) and the maximum endothelium-dependent relaxation elicited by acetylcholine in rat aorta (lower panel). Each bar represents the mean ± the standard error of the mean (n = 10 animals in each group, except in the transplanted groups where n = 6). (A = endothelium-dependent relaxation could not be investigated in vessels stored for 36 hours at 22°C because they had lost almost all contractility; UW = University of Wisconsin solution; *p < 0.05; **p < 0.01; ***p < 0.001.)

View larger version (239K): [in this window] [in a new window]   Fig 4. . Effect of cumulative addition of acetylcholine (Ach) to rat aorta precontracted with the thromboxane A2 analogue U-46619. Open symbols show vessels where no acetylcholine was given, to demonstrate that no spontaneous relaxation occurred during the time needed to obtain a full concentration-response curve. Each point shows the mean ± the standard error of the mean. (n = 10 animals in each group except in the transplanted groups, where n = 6). When error bars are not visible they are hidden by the symbols. (UW = University of Wisconsin solution.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
In clinical transplantation the donor organ is perfused with a cold preservation solution to obtain quick core cooling and create a suitable environment for the tissue cells. The organ is then immersed in ice-slush or refrigerator chilled preservation solution until the transplantation can be performed. The temperature in ice-slush solutions lies between 0.5°C and 1.0°C, whereas refrigerator-chilled solutions have temperatures ranging from 4° to 8.5°C.

What is the optimal storage temperature for long-term preservation of vascular smooth muscle function and endothelium-dependent relaxation? According to our study contractility was better preserved at 0.5°C whereas endothelium-dependent relaxation was better preserved at 4°C or 8.5°C. The endothelial dysfunction was completely reversible after 24 hours of in vivo reperfusion in all groups. No significant impairment in endothelium-dependent relaxation was seen after storage at 4°C or 8.5°C followed by transplantation and reperfusion for 2 hours (see Fig 3). It is known that exposure to low temperatures can impair the basal and stimulated release of endothelium-dependent relaxing factor [5]. Studies on 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 [6]. In porcine donor lungs, storage at 8.5°C in Perfadex for 24 hours does not lead to poor reperfusion of the capillaries because the blood gas exchange in such transplanted lungs is excellent from the very beginning of warm reperfusion [7]. The possibility that cold-induced endothelial dysfunction may be rapidly reversible after warm in vivo reperfusion is of the utmost importance because a decrease in release of endothelium-derived relaxing factor combined with mechanical narrowing of the capillary lumen, due to cell swelling, can produce a no-reflow situation with irreversible organ damage within minutes [8, 9].

Abebe and coworkers [10] have studied the rat aorta with scanning and transmission electron microscopy after long-term storage in University of Wisconsin solution at 4°C. After 24 hours of storage in this solution slight endothelial cell swelling and some swelling of the mitochondria were seen; the smooth muscle cells were also slightly swollen, but the myofilaments were intact.

In the present study we demonstrated normal smooth muscle function after 36 hours of storage at 0.5°C. However, vessels stored for 36 hours at 4°C and 8.5°C showed a significant decrease in smooth muscle function. No recovery was seen in these groups after 2 or 24 hours of reperfusion, but after 7 days the vascular smooth muscle function had recovered. This initial reduction in contractile function may not necessarily be a disadvantage: it may protect the vasculature from spasm in the first critical hours of warm in vivo reperfusion. On the other hand, loss of contractile function may be dangerous if the blood pressure is too high: the hydrostatic pressure in the arterial end of the capillaries could rise to such an extent that tissue edema is the consequence.

In an earlier study [1], we have demonstrated that rat aorta may be stored in University of Wisconsin solution at 4°C for 24 hours without a significant decrease in vascular smooth muscle function. The present study shows that if the storage temperature is lowered to 0.5°C, the vascular smooth muscle function can be fully preserved for 36 hours, ie, the colder the storage temperature the better it seems to be for the vascular muscles. However, we have also shown that, by adding 1.5 mmol/L calcium to the University of Wisconsin solution, vascular smooth muscle function can be fully preserved for 36 hours at 4°C as well [11]. The presence or absence of calcium in the preservation solution is of no importance in the preservation of endothelium-dependent relaxation [11]. Considering that the endothelium-dependent relaxation was significantly reduced after storage at 0.5°C, we conclude that the best method of preserving the vasculature for 36 hours with University of Wisconsin solution is to add 1.5 mmol/L of calcium to it and to keep the temperature at 4°C.

	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. 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]
2. Sjöberg T, Massa G, Steen S. Endothelium-mediated relaxation in transplanted aorta. Ann Thorac Surg 1992;53: 1068–73.
3. Ingemansson R, Massa G, Pandita R, 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. 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]
5. 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]
6. 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 1989;30:669–4.[Medline]
7. 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]
8. Fish DR, Nabel EG, Selwyn AP, et al. Responses of coronary arteries of cardiac transplant patients to acetylcholine. J Clin Invest 1988;81:21–31.
9. Ryan US. The endothelial cell surface and response to injury. Fed Proc 1986;45:101–8.[Medline]
10. Abebe W, Cavallari N, Agrawal DK, et al. Functional and morphological assessment of rat aorta stored in University of Wisconsin solution and Euro-Collins solutions. Transplantation 1993;56:808–16.[Medline]
11. Ingemansson R, Sjöberg T, Steen S. Importance of calcium in long-term preservation of the vasculature. Ann Thorac Surg 1996;61:1158–62.[Abstract/Free Full Text]

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