Function and Viability of Vessels in Different Preservation Solutions - An Experimental Study on Human Great Saphenous Veins

There are necessarily certain limits to applying the results of organ bath experi¬ments to in vivo reality. Nevertheless, the results of earlier studies as well as those pre¬sented here clearly show that the results of animal experiments can be applied to human blood vessels.

Although Vasa Vasorum (VV) in saphenous grafts are needed for the nutrition of the vessel wall, the presence of the VV in our three groups was not assessed. However, the density and the distribution of VV had shown to be uniform and not to differ in great and small saphenous veins [22]. Thus, any damage on VV of our saphenous grafts would have affected the results within the three groups to a similar extent, i.e., differences in VV very unlikely can explain the observed differences described for the three different protection solutions.

Because of an average age of 67.1 ± 15.4 years, presence of varicose veins was to be expected and could have affected the results. For such veins, differences in the orientation of the Smooth Muscle Cells (SMC) in the inner and outer wall layers were reported, but the SMC orientation was uniform along the varicose saphenous veins [23] Thus, we do not consider changes in the SMC orientation responsible for the differences found for the protective solutions, in particular, because veins with clearly recognizable varicose had been discarded.

A final drawback of the study could have resulted from the conventional technique of harvesting the vein grafts in this study opposed to the no-touch technique that has been recommended by some groups, because it provided a significantly higher patency [24] and left ventricular ejection fraction [25] at a mean time of 16 years than the conventional technique.

Kopjar and Dashwood [26] focused on a critical aspect of the no-touch vein harvest that presents a major barrier to wide-spread adoption, which being the morbidity of the leg incision. Because all vein grafts in this study were harvested using the conventional technique, different solution-dependent results cannot be explained by different techniques.

Veins are rather small, and during storage they are bathed both inside and out by preservation solution, which means that both inner and outer surfaces are subject to the same oxygen partial pressure [27]. Damage due to hypoxia is thus less pronounced than for blood vessels within large organs. On the other hand normoxia promotes oxidation processes and the formation of free radicals.

Solutions for preserving blood vessels are now expected to maintain good function and morphology for about two weeks. This length of time is required in order to perform the microbiological tests prescribed by the European guidelines and the German tissue bank law [16].

In the present study the ability of a newer extracellular solution (TiProtec) to preserve human Saphenous Vein (SV) was examined for up to 72 h after removal. TiProtec was compared with the clinically established, intra-cellular University of Wisconsin (UW) solution. The controls were veins stored in Krebs-Henseleit (KH) buffer. Two independent parts of the study were per¬formed to evaluate preservation ability: in the functional part (organ bath) receptor independent and receptor contraction as well as endothelium dependent relaxation were measured. In the morphological part of the study (confocal laser microscopy) cell viability was investigated.

In the following some of the peculiarities of the preservation solutions used in this study are discussed. The functional and morphological results are viewed in con¬nection with the current literature.

Krebs Henseleit (KH) buffer is frequently employed in experiments using organ baths, cell culture or perfusion. Its pH value depends on continuous bubbling of 95% O2 / 5% CO₂ through the solution. Without this precaution the pH becomes alkaline. The poor results found in this study with this solution might have been due to this factor, since alkaline pH causes serious damage to stored organs [27]. However, the time course of pH was not measured in this study.

The morphology, too, gives an indication of the importance of gassing the solution. Cultured hepatocytes have been preserved in KH buffer both with and without gassing [28]. Without gassing 4 h in cold storage induced con¬siderable cell damage, as documented by the massive increase in the number of necrotic cells seen using Propidium Iodide (PI).

A further reason for the poor results with KH buffer might have to do with the high concentration of redox-active metals [29], which contribute to the development of Reactive Oxygen Species (ROS) during cold storage and subsequent rewarming. This damage mechanism is not observed with normothermic storage at 37°C, such as is required for cell cultures.

Even on day 1 of preservation veins showed slight morphological changes, such as loosened structure and a somewhat higher number of PI-positive cells. On day 10 there were almost no viable cells and necrotic cells dominated in all cell layers. Thus, KH buffer appears unsuitable for the cold storage of veins.

The University of Wisconsin (UW) solution is frequently used for the preservation of abdominal organs. The UW solution belongs to the intracellular solutions owing to its high K⁺ (125 mM) and low Na⁺concentration (29 mM). Disadvantages of this solution include endothelial dysfunction [5] and in the case of lung transplants vaso¬constriction and reduced compliance.

A major concern in the case of UW solution is the prevention of hypothermia induced cell swelling, which presumably results from inactivation of the Na⁺-K⁺-ATPase. The ensuing influx of Na⁺ and Cl- ions draws water into the cells osmo¬tically. To prevent this, UW solution contains lactobionic acid, raffinose and Hydroxyethyl Starch (HES). Lactobionic acid also acts as a chelator of Ca²⁺ and iron, which has been sug¬gested to contribute to its effectiveness in preserving cell integrity during hypo¬thermic sto¬rage. UW solution also contains glutathione, which as an anti¬oxidant has a pro¬tective effect on liver transplants [30]. On the detrimental side, how¬ever, glutathione has been shown to cause increased endothelial dysfunction in cold storage [31].

The UW solution has proven useful for the preservation of liver, kidney and pancreas among other organs for 8 to 12 h, sometimes as long as 24 h. It is also used for heart transplants. The clinical importance of this solution is thus apparent.

The protective properties of the UW solution are temperature dependent. An inherent toxicity has been observed during the rewarming phase [5-7], which appears to be the result of a cold induced mechanism mediated by ROS [32]. This cold induced damage has been studied morphologically [32]. One hour after rewarming DNA fragmentation and apoptotic changes were observed, along with a 2.5-fold increase in PI uptake, a sign of more necrotic cells. Such damage was alleviated by adding an iron chelator [32].

In the present study, a stable contraction and relaxation behavior was observed up to 48 h after 10 days of cold storage, a result that was likely owing to the well maintained morphology.

The Tissue Protection (TiProtec) solution was specially developed on the basis of the Custodiol-N solution [33], which in turn was derived from the HTK solution (Custodiol) for liver preservation.

As hypoxic damage is of little importance in the case of thin-walled vessels, avoi¬dance of other damage mechanisms was of primary concern in developing TiProtec. One concern was histidine, which serves as buffer in Custodiol but enhances iron formation at high concentrations [2]. For this reason, in TiProtec histidine was replaced by N-acetylhistidine (pK = 7.2), which has a different affinity for redox-active iron and forms more stable complexes [2].

Another concern was the normoxia during hypothermic preservation, which can lead in particular to ROS induced endothelial damage. In order to avoid this source of damage at most, TiProtec contains two different iron chelators: desferoxamine, which is hydrophilic and does not pass through the cell mem¬brane, and LK 614 [34], which being lipophilic easily passes through the membrane.

Comparatively little solution is required for preserving blood vessels. Hence, amino acids and ions can be used effectively to supply energy and regulate osmolarity. For example, replacing lactobionate anions with chloride led to a significantly increased rate of survival of endothelial cells in segments of pig aorta [3], a result that was confirmed using segments of rat aorta and mesenteric artery [35]. By using a high K⁺concentration (93 mM), the mitochondrial membrane potential was better maintained [14] and vasoconstriction and EDHF-mediated relaxation were better than under the HTK solution, the extracellular-type solution used as a starting point [35].

A final concern in the design of TiProtec has to do with the amino acids glycine and alanine, which had already proven useful in the Custodiol-N solution for pre¬venting Na⁺ influx during hypoxia. These two amino acids were retained in the TiProtec solution for their membrane stabilizing properties, and aspartate and ?-ketoglutarate were added for maintaining the citric acid cycle [36].

Based on its advanced composition, TiProtec has led to excellent experimental results. Segment of pig aorta have been preserved for up to 21 days [3]. With segments of rat aorta and mesenteric artery, potassium induced vasoconstriction, both endothelium dependent and independent relaxation, eNOS expression and endothelial ultrastructure were all significantly better after four days preservation in TiProtec than in HTK solution [34]. Endothelial struc¬ture had already been lost after 2 h in HTK solution, a result that underscores the impor¬tance of employing the most appropriate preservation solution as early as possible.

Studies of human thoracic artery (ATI) [4] yield similarly positive results, as does the present study using human veins. For ATI, too, vascular tone was better preserved after four days in the TiProtec solution than in either 0.9% NaCl or the HTK solution.

The damage caused by cold storage and rewarming has already been studied morphologically [3]. No matter which preservation solution was used, the number of PI-positive cells was reduced when iron chelators were added. Of the solutions tested, only TiProtec contains iron chelators as part of its formula. As shown in the present study, iron chelators appear able to effectively reduce damage due to ROS, in particular the number of necrotic endothelial cells was definitely lower than in the other two solutions, a finding that is in good agreement with a previous study on human SV segments [17]. The present study shows in addition that TiProtec also very adequately preserves contractile function. 

The human great Saphenous Vein (SV) is clearly a representative model of a blood vessel transplant. SV may also be viewed as a simplified model of an organ con¬taining numerous blood vessels, which must be preserved. This is especially true if an intact vascular endothelium is essential to the function of the organ. It should be remembered, however, that veins induce an immune reaction in the recipient, so they must be protected from being rejected after transplantation.

The clinical importance of the TiProtec solution has remained an open question. Currently available results indicate that TiProtec, specifically designed for use with blood vessels, is a good alternative for the short- and long-term preservation of vessels stored in hypothermia. Given the increas¬ing numbers of blood vessel transplantations, not just clinical con¬side¬rations but also economic aspects are gaining greater importance. Thus, a com¬prehensive cost-benefit analysis ought to include both the ease of use of TiProtec and the resulting lower rate of reocclusion and reoperation.