| Co-Investigator(Kenkyū-buntansha) |
TOKITSU Kousuke Faculty of Medicine, Osaka Medical College, Assistant, 医学部, 助手 (60257855)
ASADA Kunio Faculty of Medicine, Osaka Medical College, Associate Professor, 医学部, 助教授 (80131316)
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| Budget Amount *help |
¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
The extent of cellular damage in cryopreserved rat aortas, frozen in a programmed freezer and subsequently stored in a vapor-phase nitrogen freezer for various periods, and the usefulness of these aortas as allografts in allogeneic aortic transplantation was evaluated. Cellular damages were quantified by measuring contraction of freezed-thawed aortas in response to various concentrations of potassium chloride or noradrenaline (functional evaluation) and also by comparing the numbers of injured cells, which were fluorolabeled with ethidium homodimer-1, against the numbers of total aortic cells (morphological evaluation) and by measuring supernatant LDH value of stored solution (biochemical evaluation). Experimental tissues consisted of cryopreserved rat thoracic aortas thawed after storage periods of 2 weeks, I month, 3 months, 6 months and 1 year. Rat thoracic aortas removed immediately after sacrifice of rats were used as controls. Functional evaluation in all experimental groups show
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ed decreases of approximately 50% in contraction, decreases that were statistically significant. However, it showed no significant differences in contraction between tissues stored for various periods. Morphological evaluation revealed that 19.2% to 27.4% of endothelial cells and medial smooth muscle sells were damaged, although it failed to reveal any differences in rates of damaged cells between groups. Biochemical evaluation showed the upward tendency of supernatant LDH value at only 3 months-stored period. As a next step, histological alterations over time in cryopreserved aortas having been stored for 6 months and used in allogeneic aortic transplantation were observed and compared with those of unfrozen control aortas. In both aortas, endothelial regeneration and intimal smooth muscle cell proliferation occurred immediately after transplantation. However, more rapid endothelial regeneration occurred in cryopreserved aortas than in control aortas, and the extent of intimal smooth muscle proliferation was less in cryopreserved aortas than in controls, which resulted in better patency of transplanted aortas in cryopreserved aortas than in controls. From these findings, it is concluded that cryopreservation using a programmed freezer and subsequent storage in an evaporated layer of liquid nitrogen makes 1-year cryopreservation possible with no serious damage to frozen aortas. In addition, cryopreserved aortas are considered to be useful as grafts for rat allogeneic aortic transplantation. Less
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