EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Methods for conservation and preservation of vascular grafts are often controversially discussed. Furthermore, immunologic monitoring or immunotherapy for allogeneic graft is not considered necessary in many cases. The present study was initiated to examine the cellular vitality and functional efficiency of vein transplant during preservation.

Materials and Methods: Twenty-seven human vein segments (vena saphena magna) were stored after explant in University of Wisconsin solution or histidine-tryptophan-ketoglutarate solution at 4°C. After 3, 24, 48, 72, and 96 hours, vein functionality was tested. Ring segments were fixed by triangles in Krebs-Henseleit buffer. Contractile function was measured after addition of potassium chloride solution (80 mM) and phenylephrine (0.2, 2, or 20 μM). To investigate endothelium-dependent vasorelaxation, 1 μM acetylcholine was added.

Results: Of 27 segments, 5 showed endothelium-dependent relaxation. Vasorelaxation continued for up to 48 hours after administration of acetylcholine in University of Wisconsin solution and for up to 24 hours in histidine-tryptophane-ketoglutarate solution. At 48 hours, potassium chloride solution-induced vasocontraction was 17% more effective than phenylephrine in University of Wisconsin solution. University of Wisconsin solution was significantly more effective than histidine-tryptophane-ketoglutarate solution in terms of preservation of phenylephrine (0.2, 2 μM)-induced vasocontraction. Phenylephrine (2 μM)-induced contraction was retained in University of Wisconsin solution after 24 hours by 81% and after 48 hours by 55%, with comparable results in histidine-tryptophane-ketoglutarate solution of only 62% and 34% after 24 and 48 hours.

Conclusions: At 48 hours, human saphenous vein transplants had better endothelium and smooth muscle function when preserved in University of Wisconsin solution versus histidine-tryptophane-ketoglutarate solution.

Key words : University of Wisconsin solution, Vascular function, Vena saphena magna graft

The principles of organ preservation by cold storage with different storage solutions were primary discussed in the late 1980s.^1,2 However, because long-term results have been unsatisfactory for the most part, the procedure has been frequently adjusted. In addition to technical complications, other complications include the ability to preserve the allograft and adequate immunosuppression. The use of allografts that have been cryopreserved for 1 to 2 days is not frequently used in transplant procedures because cryopreserved material can add risk of endothelial necrosis, intima defacement, and fibrotic degeneration.³ Other transplant preservation procedures such as radiotherapy, lyophilization, or formalin fixation are not efficient and/or cause tissue toxicity and thus, have only historical significance.⁴ Previously, studies have shown that conservation solutions with low concentrations of sodium ions can provide proper preservation conditions for the vascular vitality of veins.^1,5 Both the University of Wisconsin (UW) solution and the histidine-tryptophan-ketoglutarate (Bretschneider’s) solution have low sodium concentrations.

In this study, our aim was to determine a conservation procedure for human veins that would best preserve cellular vitality and functional efficiency. We analyzed the morphologic conditions of the endothelium with scanning electron microscopy after vein preservation and tested the functional vascular contractility and relaxation of the vessels while immersed in special organ baths.

Materials and Methods

Vein segments
All experiments were performed with segments of human saphenous veins, which were obtained after bypass surgery. After explant, all vessels were perfused with isotonic NaCl solution at room temperature. Veins were stored until the cardiac surgery procedure in physiologic NaCl solution supplemented with heparin (5000 U/mL). Vein segments that were not used for coronary bypass (2-7 cm in length) were obtained 2 to 3 hours after explant and immediately placed in 150 mL of cold (4°C) sterile conservation solution. Veins were kept in these solutions for 3, 24, 48, 72, and 96 hours. During storage, veins were maintained at 4°C and protected from daylight.

Organ bath studies
After storage, veins were cut into small ring segments (5 mm) for organ bath investigation. Care was taken to preserve the intimal endothelium. The ring segments were fixed between stainless steel triangles in a water-jacketed organ bath (37°C) filled with Krebs-Henseleit solution (pH 7.4) that was composed of (in mM) 143.07 sodium ion, 5.87 potassium ion, 1.6 calcium ion, 1.18 magnesium ion, 125.96 chloride ion, 25.00 bicarbonate ion, 1.18 dihydrogen phosphate ion, 1.18 sulfate ion, and 5.05 glucose, as described previously.⁶ Resting tension was 1 gram vascular tone was measured isometrically by means of a Statham force displacement transducer (Statham Instruments, Inc., Los Angeles, CA, USA).

After equilibration (1 h), contractile function was tested by addition of potassium chloride (80 mM). The presence of intact endothelium was verified by relaxation of phenylephrine (0.2 μM)-preconstricted segments after application of 1 μM acetylcholine. Finally, the concentration-dependent contractile response to phenylephrine was tested by cumulative application of the drug (0.2-20 μM).

Scanning electron microscopy
The conserved vessels were fixed with glutaraldehyde and prepared for scanning electron microscopy. Tissue sections were analyzed at ×625 and ×1025 magnification by Dr P Reinecke (Institute of Pathology, Heinrich-Heine University, Dusseldorf, Germany). We made a global assessment of the condition and function of the endothelium using a 4-point scale: (1 = from 0% to 25% of initial endothelium, 2 = from 25% to 50% of initial endothelium, 3 = from 50% to 75% of initial endothelium, and 4 = from 75% to 100% of initially endothelium).

Substances and solutions
Vascular tissues were stored in 2 commercially available solutions. The UW solution (Viaspane, DuPont Pharma GmbH, Germany) is a sterile, pyrogen-free solution with an osmolarity of approximately 320 mOsm, a sodium concentration of 29 mmol/L, a potassium concentration of 125 mmol/L, and a pH of 7.⁴

The HTK solution (Custodiol, Köhler Pharma GmbH, Alsbach, Germany) has a sodium concentration of 15 mmol/L and a potassium concentration of 10 mmol/L. All other chemicals (analytic grade) were obtained from Sigma (Deisenhofen, Germany) and Merck (Darmstadt, Germany).

Statistical analyses
Results are presented as mean ± standard error of the mean. We compared results between groups of storing solutions using either t test or analysis of variance (GraphPad Prism version 3.0, La Jolla, CA, USA). P < .05 was considered significant.

A total of 49 segments of saphenous veins were collected and investigated. Of these, 27 were suitable for the experiments (55.1%). The other segments did not show any vasocontractile response to either potassium chloride or phenylephrine, which was most likely the result of damage during the explant procedure.

Endothelium-dependent vasorelaxation
We assessed endothelium-dependent vasorelaxation by adding 1 μM of acetylcholine to equilibrated venous segments precontracted with 0.2 μM phenylephrine. Overall, we found vasodilation to be weak, reaching a maximum of only 39% ± 21.6%. Directly after preparation of the segments at the 3-hour time point, segments stored in the UW solution showed a trend toward improved endothelium-dependent vasodilation (Figure 1) versus segments stored in HTK solution. A similar situation was found after 24-hour storage. This trend became statistically significant at 48 hours. With prolonged storage in HTK solution, endothelium-dependent vasodilation decreased, whereas some segments still relaxed to acetylcholine after 72 hours of storage in UW solution (Figure 1). At 96 hours of storage, the endothelium-dependent vasodilator response discontinued in all segments and in both baths.

Vasoconstriction to potassium chloride
The storage solution had little effect on vasoconstriction induced by potassium chloride (Table 1). There was a trend to improved vasoconstriction at 3 hours, but this was not statistically significant (P = .53, unpaired t test). Likewise, we found no differences related to storage solution (Figure 2). Analysis of variance showed that the decrease in contractile response was dependent on storage time (P < .001) but not on the type of the storage solution (P = .91). Therefore, it appears that the contractile apparatus of the vascular tissues is increasingly damaged by prolongation of the storage time, whereas the type of storage solution seems to be of minor importance in this process.

Vasoconstriction to phenylephrine
We investigated α1-receptor-dependent vasoconstriction after different times of storage of venous segments in either HTK or UW solution (Tables 2 and 3). Phenylephrine concentration dependently constricted venous segments. Vasoconstriction of segments subjected to HTK solution tended to be smaller; however, no statistically significant difference was shown between storage in HTK and UW solution. This situation changed after storage time was increased. Low (0.2 μM) and moderate (2 μM) concentrations of phenylephrine resulted in significantly better preserved vasoconstriction of venous segments stored in UW solution (Figure 3), whereas a maximal concentration of phenylephrine (20 μM) resulted in nearly similarly preserved vasoconstriction in both storage groups (Figure 3). These data suggest that storage of venous allografts in HTK solution may result in desensitization against α-mimetic stimulation.

Morphologic analyses with scanning electron microscopy
After the functional tests, we used scanning electron microscopy to examine 8 vein segments. The segments had been previously stored for 3, 24, 48, 72, and 96 hours, with 4 preserved in UW solution and 4 preserved in HTK solution (Figure 4). After 3 hours of storage in either solution, we observed that nearly 75% of the preserved vessels showed small endothelial damage (Figure 5). In addition, after 3 hours of storage, vessels showed an endothelial status of 81.3% with a score of 3.25 ± 0.25. After 24 hours of storage, the endothelial status at the 3-hour time point was completely preserved in both solutions (3.25 ± 0.25 in UW solution, 3.25 ± 0.48 in HTK solution). At 48 hours, both storage solutions resulted in significant endothelial damage, with a score of 2.75 ± 0.45 in UW solution (endothelial status of 68.8%) and a score of 2.50 ± 0.50 in HTK solution (endothelial status of 62.5 %). After 72 hours of storage, segments in UW solution showed an endothelial status of 37.5% and segments in HTK solution showed 43.8%. Toward the end of the storage time (96 h), endothelial status was dramatically decreased in all segments, with a score of 1.5 ± 0.5 in UW solution (status of 37.5%) and a score of 1.25 ± 0.25 in HTK solution (status of 31.3%). The results at 96 hours were not statistically significant as measured by 2-way analysis of variance between the solutions (P = .69). Both solutions resulted in significant damage to the endothelium of all segments during storage (P < .0001).

The potential antigenicity of allogeneic vein transplant has been described periodically.⁷ The patency rates of this procedure are 60% to 80% after 1 year and are compatible with autologous system transplants. It has been shown that it is important to extract the transplant vessels extremely carefully to avoid traumatic endothelial lesions. Vein walls are extremely vulnerable and are sensitive to traction or mechanical compression. This explains why in our experiments, from 49 examined vein segments, 22 were unsuitable and 17 of these a priori showed only minimal or no functional reaction.

A so far unresolved complication of allogeneic vein transplant is proper preservation. In the present study, the preservation of vitality and functionality was a major goal. Damaged vessel transplants are often fibrotically reorganized, which are functionally intact only for a limited period until they undergo reparative occlusion.

The high number of such early occlusions could not be prevented in the past by cryopreservation.^8-10The main reason seems to be that this kind of preservation is often associated with devitalization of intima and fibrotic wall degeneration.^3,11Other methods of preservation such as irradiation of the transplants, lyophilization, or formaldehyde/glutaraldehyde fixation are, because of their limited efficiency or tissue toxicity, only of historical importance.⁴ The aim of future allogeneic vein transplants is the use of fresh tissue. Generally, these are vessels from donors after brain death, although in some cases from living donors. Because of time consumed during handling and the necessity of immunologic monitoring (cross-match), preservation plays an important role.

Earlier studies verified on isolated veins segments noted that, in addition to heparinized autologous blood, only conservation solutions with low concentrations of sodium ions (intrinsic solutions) guaranteed a satisfactory conservation status of transplants.¹² Among these solutions are HTK (Bretschneider) solution and the frequently used UW solution. Santoni and associates¹² and Anastasiou and associates¹³ verified that other media, such as heparinized sodium chloride solution, evoked acidosis and initiated the denaturation of cellular proteins with terminal coagulation necrosis of the endothelium. The consequences are leukotaxis of inflammatory cells to the intima and adherence of fibrin and thrombocytes to endothelium-free areas of the vessel wall. Subsequently, this leads to occlusion of the lumen.

In the present study, we tested the functionality (contractility and relaxation ability) of the vein segments after preservation in UW solution and HTK solution. We were able to show that endothelium-dependent vasorelaxation in both solutions decreased within a few hours, with acetylcholine-dependent vasorelaxation still measurably after 48 hours in segments that had been stored in UW solution. In contrast, we could not verify a similar vasorelaxation after 48 hours of storage in HTK solution.

In their study with heparinized autologous blood and UW solution, Santoni and associated demonstrated in 1993 a predominance of UW solution regarding preservation of the endothelium for up to 5 hours.¹² However, the role of high potassium concentrations (125 mmol/L) on the endothelium has not been clarified, because Anastasiou and associates¹³ showed a decrease in endothelial-dependent vasorelaxation with UW solution and suggested that high potassium concentrations was responsible. In contrast, Cavallari and associates^14,15 determined, as suggested by our results, that the endothelium was functionally preserved for many hours. Even after 1 month of preservation, there would be a functionally intact endothelium; however, it is more likely that this is due to reendothelialization of the intima. The quick downfall of the endothelium in venous vessel segments seems to be tissue specific, since venous endothelium is significantly more vulnerable than arterial endothelium. Luscher and associates16 investigated endothelium-dependent vasorelaxation induced by acetylcholine in 58 patients, comparing between arterial (arteria mammaria) and venous (vena saphena) vessels. The group demonstrated 86% vasorelaxation in arteries and in veins, as suggested by our results, only 39% vasorelaxation.16 Moreover, an additional role is that the venous vessel walls have a thinner intima and fewer smooth muscle cells, and thus the weaker vasorelaxation and contraction may be anatomically substantiated.

The contractile apparatus was tested with the vasoconstrictor potassium chloride, which has receptor-independent effects, and with phenylephrine, which has receptor-dependent effects (α1-mimetic). In recognition of these facts, the receptor-independent contractility function of the vessels was preserved superiorly in both solutions versus the receptor-dependent function. Therefore, we verified that the potassium chloride-induced vasoconstriction of vessels stored in UW solution after 24 hours was at 93% and after 48 hours at 72%. For vessels stored in HTK solution, vasoconstriction was at 92% after 24 hours and at 55% after 48 hours. These levels subsided with phenylephrine-induced (20 μM) contraction in the UW solution to 74% after 24 hours and to 54% after 48 hours, with levels subsided in HTK solution to 89% after 24 hours and to 53% after 48 hours. Therefore, the originating damage during preservation may have also affected signal transduction between the smooth muscle cells, owing to the effect of adrenergic phenylephrine on α1-receptors and gap junctions. With 0.2 and 2 μM phenylephrine, there was functional loss in both solutions compared with that shown with potassium chloride, although still more aggravating (see Figures 2 and 3 and Tables 2 and 3). Vasoconstrictions at low (0.2 μM) and moderate (2 μM) concentrations of phenylephrine were significantly better preserved in venous segments stored in UW solution (Figure 3), whereas maximal vasoconstrictions to 20 μM phenylephrine were nearly similarly preserved in both groups (Figure 3).

Our results suggest that it is more appropriate to use UW solution to preserve the contractile apparatus during the first 48 hours versus using HTK solution. Receptor-dependent contraction, induced by phenylephrine, and receptor-independent contraction by means of potassium chloride pointto better conservation results. The differences between the solutions fall out perspicuously by the phenylephrine-induced vasoconstriction and less perspicuously by the potassium chloride-induced vasoconstriction. Preservation of the smooth muscle contractile apparatus in UW solution has also been confirmed in studies from Santoni and associates,¹²and Mingoli and associates,¹⁷ and Vischjager and associates.¹⁸

Vessel transplants should be preserved, as suggested by our results, in UW solution because biostability can be guaranteed for up to 48 hours. Preservation for more than 48 hours is not suggested.

Volume: 14 Issue: 1 February 2016

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