Ann Thorac Surg 1998;65:24-27
© 1998 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Metabolic Detriment in Donor Heart Valves Induced by Ischemia and Cryopreservation
Jen-Her Lu, MD, PhD,
Yen Chang, MD,
Wen-Hu Hsu, MD,
Betau Hwang, MD,
Chuh-Khiun Chong, MS,
Chi-Chin Wu, MS,
Ping-Zen Yang, VD, PhD,
Hsung Hsing-Wen, PhD
Division of Pediatric Cardiology, Section of Thoracic and Cardiovascular Surgery, Veterans General Hospital–Taipei, National Yang-Ming University, Taipei, Taiwan, Republic of China
Department of Comparative Medicine, Pig Research Institute, Miaoli, Taiwan, Republic of China
Accepted for publication May 15, 1997.
Dr Lu, Division of Pediatric Cardiology, Veterans General Hospital, National Yang-Ming University, Taipei, Taiwan, ROC.
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Abstract
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Background. The injury resulting from postmortem ischemia is a critical deterrent to the availability of donor valves. Using the reduction of XTT-tetrazolium salt as a marker of metabolic sequelae, we assessed the injurious effect of ischemia and the metabolic sequelae in 156 porcine semilunar leaflets.
Methods. The leaflets were randomly allocated to noncryoprocessed (n = 72) or cryoprocessed (n = 72) groups. At each preservation temperature of 4°C, 24°C, or 37°C, 24 leaflets each were exposed to one of four storage periods of 9, 17, 30, or 60 hours. Twelve fresh aortic leaflets served as baseline reference samples.
Results. There was a progressive loss in the metabolic functioning of valve leaflet cells in both noncryopreserved and cryopreserved tissue as the storage times increased. Cryopreserved tissue showed a greater loss of function than noncryopreserved tissue did. The metabolic injury was mainly a consequence of cryoprocessing. The greatest loss in metabolic functioning occurred in the valves stored for 60 hours. The least favorable combination of variables was cryopreservation and a precryopreservation storage time of 60 hours.
Conclusions. We conclude that 30- to 60-hour delays do not have a significant metabolic effect on cardiac leaflets. Thus it may be possible to safely extend the permissible ischemic periods after organ harvest.
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Introduction
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In the current protocol for aortic allograft cryoprocessing, either the beating heart of a brain-dead donor or an arrested heart must be harvested within 12 to 24 hours of donor death [1][2][3]. However, the availability of cryopreserved allografts is limited by donor supply. Should the restrictions regarding the ischemic time and the storage temperature of allograft preservation be safely extended, this limitation to allograft availability would be eased. Armiger and associates [4][5] have suggested that the ischemic interval could be extended up to 30 hours after death. Further, Meissier and colleagues [6][7] demonstrated high levels of adenine nucleotide pools in cryopreserved allografts after prolonged ischemia. Kadoba and associates [8] confirmed that a 48-hour delay from donor death to graft harvest did not have a considerable effect on conduit functioning.
No matter which processing method is used, however, a period of ischemia is unavoidable. Such periods occur between cessation of the donor heartbeat and harvesting (cadaveric recovery) as well as during preservation in tissue storage solution. These intervals are critical determinants in leaflet injury [9][10][11][12]. The injurious effects and their metabolic sequelae differ at varying storage temperatures [13][14]. However, most studies have examined only one of the multiple contributing factors. The variability in the results of these studies therefore indicates the need to study the effects of a single factor in a systematic fashion.
The tetrazolium salt reduction technique involves the use of quantitative assays to evaluate the activity of mitochondrial dehydrogenase enzymes within living cells [15][16]. In this experiment, XTT testing was used to evaluate cardiac leaflets preserved at different postharvest temperatures and involving different ischemic times. The synergic changes in metabolic tolerance in response to delayed cryoprocessing were also assessed.
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Material and Methods
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One hundred fifty-six fresh semilunar leaflets from 26 pigs were obtained from a local slaughterhouse immediately after sacrifice. An obligatory 60-minute period of postmortem ischemia was allowed for preparation of the valves. During heart procurement, the heart cooled from normothermia (37°C) to an ambient temperature (20° to 24°C). After cardiectomy the aortic and pulmonic roots were rapidly dissected from the hearts. Leaflets were put in bottles containing a culture medium consisting of minimal essential medium and Earle balanced salt solution, supplemented with 2.2 g/L of sodium bicarbonate, 0.1 g/L of streptomycin, and 200 units/L of penicillin; 2.5 mg of amphotericin B; and 20% (vol/vol) fetal calf serum.
Reference Groups
Twelve fresh leaflets served as baseline reference samples. The colorimetric assays were promptly performed on six fresh leaflets. Another six fresh leaflets in 15 mL of minimal essential medium supplemented with 20% fetal calf serum and 10% dimethylsulfoxide were cryoprocessed at a rate of -1°C/min to -70°C and then immediately immersed in liquid nitrogen (-190°C) until analysis. The cryopreserved grafts were thawed immediately before colorimetric assay.
Experimental Groups
The remaining 144 leaflets were randomly allocated to the noncryoprocessed (n = 72) or cryoprocessed (n = 72) groups. Each group was then subdivided into three groups of 24 leaflets each, which were stored at one of three different preservation temperatures of 4°C, 24°C, or 37°C. These subgroups were further divided into four groups of six leaflets each, which were kept under ischemic conditions for 9, 17, 30, or 60 hours. After this, the colorimetric assays were promptly performed. The leaflets in the cryoprocessed groups were cooled to -70°C at a controlled rate of -1°C/min in the presence of a cryoprotectant (10% dimethylsulfoxide) and then immediately immersed in liquid nitrogen (-190°C) until analysis. The cryopreserved grafts were thawed immediately before colorimetric assay.
XTT-Tetrazolium Colorimetric Assay
Colorimetric assays of cardiac leaflets could only be performed at 37°C. Therefore all cryopreserved leaflets were rapidly thawed in a 50°C water bath containing an isotonic saline solution according to a stepwise dilution procedure. The dimethylsulfoxide was removed in a gradual manner, with stepwise reequilibration of the allograft to isotonicity over a 10- to 15-minute period. To prevent errors related to the differential diffusion of XTT in large tissue, the leaflets were cut into three pieces (weight, 154 ± 32 mg) and homogenized to prevent the localization of enzymes and coenzymes. These pieces were then placed into flat-bottom, 96-well microliter plates (Greiner, Frickenhausen, Germany) prefilled with 100 µL of minimal essential medium. Fifty microliters of freshly prepared XTT (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)beneze sulfonic acid hydrate in RPMI 1640 [1 mg/mL], premixed with N-methyldibenzopyrazine methylsulfate; Boehringer Mannheim, Germany) was added to each well (final XTT concentration, 0.3 mg/mL) and incubated at 37°C in 5% carbon dioxide for 60 minutes. To extract the formazan, which remained trapped in the valvular tissue, 150 µL of octylphenolpoly(ethyleneglycol-ether)n (Triton X-100; Boehringer Mannheim, Mannheim, Germany) was added to each well, and the wells were then incubated for another 90 minutes. Optical density for the formazan was read at 490 nm, with a reference wavelength of 650 nm.
Statistical Analysis
The optical density values were first analyzed using KineticCalc software Version 2.03 in a Bio-Tek Reader (Bio-Tek Instruments, Winooski, VT). All measured optical densities were converted to percentages of XTT reduction after being standardized according to tissue weight. Analysis of variance and nonparametric Wilcoxon tests were used to analyze these values in relation to the ischemic intervals and preservation temperatures as independent variables.
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Results
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Absorption Spectrum
The wavelength used for the maximal absorption of formazan is 490 nm. The absorption spectrum of the formazan extracted by Triton X-100 showed optical densities of between 490 and 510 nm for the maximal absorption of the formazan. The addition of Triton X-100 did not affect the maximal chromatogenicity of the formazan. In this study, 490- and 650-nm optical density measurements were used. The weight of the porcine semilunar leaflets was 416 ± 42 mg (n = 156). In this experiment the optimal range of optical density was limited to between 0.5 and 2.5 and a linear correlation existed between XTT reduction and leaflet tissue weighing 40 to 180 mg (Fig 1).
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Relationship between cardiac leaflet tissue weight and XTT-tetrazolium reduction. The optimal absorbance was limited between 0.5 and 2.5. A good correlation was established between XTT reduction and fresh cardiac valve leaflet weighing 40 to 180 mg.
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XTT Reduction in Leaflets Without Cryopreservation
No distinct difference in XTT reduction was found in leaflets exposed to preservation temperatures of 4° and 24°C. After 60 hours of ischemia the XTT reductions were 71.1% ± 4.9% and 86.8% ± 6.8%, respectively, in the leaflets maintained at 4° and 24°C, but the XTT reduction was decreased to 39.5% ± 7.4% in leaflets preserved at 37°C (Table 1).
After 30 hours of ischemia the XTT reduction was still high at 86.9% ± 8.3% and 89.6% ± 7.4% in leaflets exposed to 4° and 24°C, respectively, whereas it was 51.9% ± 9.4% in the leaflets exposed to 37°C. There was a significant loss of mitochondrial dehydrogenase activity in valves preserved at 37°C in comparison with the enzyme activity in those preserved at 4°C and 24°C for all four ischemic times (p < 0.05).
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Results of XTT-Tetrazolium Salt Assay in 78 Noncryoprocessed Cardiac Leaflets Stored at Various Temperatures and Times
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XTT Reduction in Leaflets with Cryopreservation
Cryoprocessing induced an additional decrease in XTT reduction to 78.5% ± 4.5% in fresh leaflets at starting time 0. It was consistently demonstrated that cryopreservation itself led to an abrupt decline in XTT reduction in leaflets preserved at all three temperatures (Table 2).
Enzyme activity in leaflets maintained at 24°C remained relatively stable at 52.3% ± 3.6% after 60 hours of preservation, whereas enzyme activity in leaflets stored at 4°C was 42.4% ± 4.6% and it was 12.1% ± 2.5% in those stored at 37°C. After 30 hours of ischemia, there was a 64.7% ± 6.6% XTT reduction in leaflets stored at 24°C. Leaflets stored at 24°C showed the highest XTT reduction among the three cryoprocessed groups for all four ischemic periods.
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Results of XTT-Tetrazolium Salt Assay in 78 Cryoprocessed Cardiac Leaflets Stored at Various Temperatures and Times
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Comment
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The viable matrix cells of cryopreserved aortic allografts may mediate a cell-dependent mechanism of collagen turnover and matrix restoration after transplantation [17][18]. The retained matrix cells also represent a marker for the preservation of the critical acellular leaflet components after complete processing [19][20]. Although there is no consensus regarding the relationship between the durability of the homograft and matrix cellular viability in the existing literature, the minimization of tissue injury is a common important focus.
Livi and associates [3] studied the cell viability of human homografts in vitro using an autoradiographic technique. They calculated the probability of graft viability to be 85.0%, 76.6%, and 68.1% at 1, 24, and 48 hours, respectively, after donor death. A significant association between the reduction of XTT and progressive tissue ischemia through 60 hours was also found in this study. The cellular damage represented by the reduction in the mitochondrial dehydrogenase activity begins shortly after death and progresses incrementally. A high rate of metabolic injury begins very early after heart procurement, but the rate of enzyme activity loss levels off after 30 hours of ischemia. Although the rate at which tissue autolysis occurs at various temperatures remains unclear, the present study showed that, when valves are maintained at 4°C or room temperature, the thermal setting of a cooling cadaver from which valves are harvested, dehydrogenase enzymes are only minimally depleted. The interval between donor death and the processing of the valve at either temperature had only a minor influence on cellular metabolism. The incubation of leaflets at 37°C before cryoprocessing has been recommended as a way to "revitalize" metabolic activity [12][13][14]. However, our results revealed a significant loss of XTT-tetrazolium salt reduction, particularly in those stored at 37°C. The rapid acidification of the culture medium at 37°C may only further reduce mitochondrial activity or even increase the injury to the matrix cells.
Ways to measure the viability of a cardiac leaflet include the quantification of cellular function in tissue. Previous efforts to assess cellular viability have primarily focused on the use of autoradiography after radioactive amino acid or thymidine incorporation by individual cells [19][20]. Autoradiography assesses DNA synthesis, protein metabolism, or amino acid uptake. Cellular viability in cardiac valves can also be assessed by direct measurements of metabolite concentrations at the cellular level, such as the measurement of the adenine nucleotide pools by high-performance liquid chromatography [6][7][9] or the colorimetric quantification of enzymatic activity [21][22]. The loss of mitochondrial dehydrogenase activity is indicative of irreversible cellular death and tissue infarction [15][16]. The XTT-tetrazolium test is based on the metabolic reduction of (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate, resulting in the formation of a colored, water-soluble formazan dye, which can be quantitated through the measurement of its optical density [23]. The cleavage of XTT-tetrazolium salts into formazan is mediated by mitochondrial dehydrogenase enzymes in living cells. Therefore the amount of formazan generated is directly proportional to the activity of cellular mitochondrial enzymes in the metabolically active cells. Although there are many types of dehydrogenases in cells (some are soluble and present in the cytosol and mitochondrial matrix and others are membrane bound), the XTT test is a specific indicator of the activity of mitochondrial dehydrogenase enzymes in living cells [15]. Hence, the XTT test provides a representative view of the fluctuations in mitochondrial enzyme activity. However, in dense collagenous tissue such as valve leaflets, where living cells may be trapped in situ, it is essential to take into account the possibility that the XTT reaction and formazan salt dilution may be restricted, thereby interfering with colorimetric measurement. This is a critical difficulty to overcome when performing XTT tests in dense collagenous tissue. The XTT reaction may also be restricted, and the formazan salt may even be prevented from being dissolved for further colorimetric measurement. Homogenization can ensure a total enzyme reaction and minimize these restriction. Triton X-100 or dimethylsulfoxide should be added to extract the formazan that remains entrapped in the valvular tissue. In this experiment, the entrapped formazan was dissolved and extracted by Triton X-100.
On the basis of the results of the present study, we conclude that there is progressive loss of metabolic functioning in valve leaflet cells in both noncryopreserved and cryopreserved tissue as storage times increase. However, cryopreserved tissue showed a greater loss of function than noncryopreserved tissue did. Although all valves were exposed to amphotericin, which is known to damage the cells in valves that are cryopreserved, the metabolic injury occurred mainly as a consequence of cryoprocessing. The least satisfactory metabolic function was seen in leaflets stored for 60 hours. Because no significant adverse effect on allograft metabolism was seen in leaflets stored at room temperature, the current restrictions on cadaver harvest can be eased. Although the hypothesis that the successful maintenance of cell viability is essential to the good long-term functioning of allografts remains controversial, the information in this report may cause allograft availability to be expanded and thereby ease the shortage of donor valves. This study did not assess allograft valve durability, the risk of bacterial contamination, or long-term cellular viability. Because our results showed the presence of important metabolic sequelae in leaflets stored at 37°C, it is important that a study be undertaken to further assess the merits of the "revitalization" of cryopreserved allografts at 37°C.
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Acknowledgments
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This research was supported by the National Scientific Council (NSC 83-0412-B-075-063), Taiwan, ROC.
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Abstract
Introduction
