Preservation by Desiccation of Isolated Rat Hearts ... - IngentaConnect

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placed them into a specially made chamber, filled the chamber with a gas mixture of PCO (4,000 hPa) ..... Blackstone, E.; Morrison, M.; Roth, M. B. H2S induces.

0963-6897/12 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368911X605547 E-ISSN 1555-3892 www.cognizantcommunication.com

Cell Transplantation, Vol. 21, pp. 609–615, 2012 Printed in the USA. All rights reserved. Copyright  2012 Cognizant Comm. Corp.

Preservation by Desiccation of Isolated Rat Hearts for 48 Hours Using Carbon Monoxide (PCO = 4,000 hPa) and Oxygen (PO2 = 3,000 hPa) Naoyuki Hatayama,* Munekazu Naito,* Shuichi Hirai,* Yu Yoshida,† Tomohiro Kojima,† Kunihiro Seki,‡ Xiao-Kang Li,§ and Masahiro Itoh* *Department of Anatomy, Tokyo Medical University, Tokyo, Japan †Resonance Club Co., Tokyo, Japan ‡Faculty of Science, Kanagawa University, Kanagawa, Japan §National Research Institute for Child Health and Development, Tokyo, Japan

It is currently said that CO has anti-inflammatory and antiapoptosis effects and it has attracted attention as a medical gas. We used CO for rat hearts and conducted a preservation experiment. We isolated rat hearts, placed them into a specially made chamber, filled the chamber with a gas mixture of PCO (4,000 hPa) and PO2 (3,000 hPa), and preserved the hearts in a refrigerator at 4°C for 48 h. We then performed a heterotrophic transplantation on the neck of each recipient rat and resuscitated the preserved hearts. We herein report our findings. Key words: Isolated rat heart; Perfluorocarbon (PFC); Preservation; Desiccation; Resuscitation; Heterotrophic transplantation

INTRODUCTION

very long periods, including such problems as damage to the cell membranes caused by various factors such as the low temperature of 4°C and ischemia (8,15,16). However, it is believed that current systems of supplying organs can be considerably improved if organs can somehow be preserved for longer periods in a manner similar to that presently used to preserve blood, and there is thus an urgent need to establish new and effective techniques for long-term organ preservation (4). Until now, 4–18 h has been the preservation limit, even in the cryopreservation and resuscitation of isolated rat, rabbit, baboon, and human hearts using University of Wisconsin Solution (UW) that has been clinically applied based on the simple method of immersion in a preservative solution (13,24). Both tissues and cells in organs decompose slightly even at low temperatures, but there have been reports of trial examinations in which the preservation times were extended by supplying oxygen to the preservation solution and perfusion solution (22). Seki (one of the authors of this report) and Toyoshima focused on cryptobiosis, which slows the metabolism by decreasing the moisture content within an organism and thus allowing it to adapt to extreme environments such as by desiccation or low temperatures. Seki and Toyoshima

Currently, organ transplantation is an established medical treatment for patients and it has already spread throughout the world. However, every country is facing the reality regarding the fact that there is difficulty in securing a sufficient number of donors to meet the demand, and thereby most countries suffer from a serious shortage of donors. Therefore, there is no assurance that patients can always undergo transplantation, and it is not uncommon to have to wait for a long time, thus resulting in a substantial economic burden on patients and their families as well as the death of many patients who could not receive a transplantation, wherein the deficiency in the number of available organs has become a serious problem. The major reason for this is that organs cannot be preserved for a long period of time. Clinical transplantation therapy for human lungs, heart, liver, and kidneys has become widely used and it has now become a common practice (9). Currently, the main method of preservation of human organs for transplantation is cryopreservation, which has a time limit of from 4 to 24 h for preservation (4). Many reasons have been identified as to why organs cannot be preserved for

Received March 31, 2010; final acceptance July 18, 2011. Address correspondence to Naoyuki Hatayama, Department of Anatomy, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 1608402, Japan. E-mail: [email protected] or [email protected]

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suggested that tardigrades with this cryptobiosis could be resuscitated even after having been exposed to 600 megapascals (MPa) of pressure or lower (20). They applied the method of desiccation and low temperature to this organism in order to preserve a rat organ, decreased the moisture content using a perfluorocarbon (PFC) solution, and then performed resuscitation experiments; however, no significant reproducibility was observed. This marked the beginning of our unique method of preservation by desiccation, which is a completely new organ preservation method. Subsequently, Seki and Yoshida focused on carbon dioxide (CO2) gas, which has an anesthetic action and metabolic suppression action in the organism, performed heart preservation experiments under the environmental conditions of decreased moisture content and high concentration (20%), thus preserving the heart for 24 h, and a cervical ectopic heart transplantation performed, and thereafter consistently successful resuscitation was obtained with 100% reproducibility (21). Yoshida (also one of the authors of this report) and colleagues added carbon monoxide (CO), which has a reversible relationship with oxygen, exposed the heart to carbon monoxide gas at high partial pressure, PCO = 400 hectopascals (hPa), preserved the heart for 24 h, performed a cervical ectopic heart transplantation, and obtained successful resuscitation with 100% reproducibility (25). In this experiment, we placed hearts isolated from rats into a chamber and filled the chamber with high-partial-pressure carbon monoxide (PCO = 4,000 hPa) and oxygen (PO2 = 3,000 hPa). After preserving the hearts in a refrigerator at 4°C for 48 h, we performed a heterotrophic transplantation on the neck of each recipient rat and thereafter performed resuscitation. We then recorded an electrocardiogram and demonstrated that the transplanted hearts continued to function. We herein report the new findings made based on the results of our study.

solution. For the KH solution, we dissolved glucose at three times the normal levels. We suspended each isolated heart in a chamber that was cooled to 4°C. Subsequently, we filled the chamber with a gas mixture of PCO (4,000 hPa) and PO2 (3,000 hPa) (Fig. 1). After preserving the hearts in a refrigerator at 4°C for 48 h, we removed the isolated heart from the chamber, performed heterotrophic transplantation in the right side of the neck of each recipient rat, and performed suturing after the heartbeat was stabilized. We provided the recipient rats with drinking water in which antibiotics had been dissolved, conducted follow-up observations in a feeding room, and then recorded the heartbeats of the recipient rats and donor rats using electrocardiograms (Fig. 2). RESULTS After preserving the hearts in a gas mixture of CO and O2 (PCO = 4,000 hPa and PO2 = 3,000 hPa) for 48 h, we performed heterotrophic transplantation on the

MATERIALS AND METHODS In this experiment, we used inbred LEW/SsN Slc rats (male, 6 weeks old) that were developed for transplantation to prevent the expression of rejection at Japan SLC, Inc. The experimental protocol was approved by the Animal Research Committee, Kanagawa University. The animals were maintained under standard conditions and given rodent food and water under the supervision of the Institute of Laboratory Animals, Kanagawa University. All experimental procedures were performed in accordance with the NIH guidelines for the care and use of laboratory animals. We isolated the hearts under ether anesthesia, severed the aorta and pulmonary artery, removed the blood using Krebs-Henseleit (KH) solution, and injected additional KH solution as a preservation

Figure 1. The conditions during the experiment in which the isolated rat heart was placed into a preservation container and preserved in a refrigerator at 4°C. The preservation container was filled with PCO (4,000 hPa) and PO2 (3,000 hPa) and the heart was preserved for 48 h. During the preservation, a container with a small amount of water was placed in the chamber and the relative moisture in the chamber was maintained at 90% or higher. Cannulae were attached to the arteries of the heart, which was preserved by being suspended from a wire.

PERSERVATION OF RAT HEARTS FOR 48 H USING CO AND O2

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Figure 2. Electrocardiogram (ECG) of a donor heart and a recipient heart after preserving the isolated rat heart for 48 h, performing cervical ectopic heart transplantation on March 18, 2009, and resuscitating the rat. The smaller electrical potential is the recipient rat’s ECG, and the larger electrical potential is the donor rat’s ECG, wherein pulsing at a constant interval can be observed in both.

neck of the each recipient rat. After transplantation, the heartbeat could be detected in six cases. Among these cases, the number of donor hearts that survived after 6 weeks was five cases (83%). Both the resuscitation rate and the survival rate after preservation for 24 h using this method were six of six cases (100%). In addition, as a control experiment, we conducted preservation by decreasing the amount of CO and increasing the amount of O2. After preservation at PCO = 3,000 hPa and PO2 = 4,000 hPa, the resuscitation rate was two of six cases (33%) and the survival rate was one of six cases (16%). Furthermore, we also conducted preservation using helium (He) instead of CO. After preservation at PHe = 4,000 hPa and PO2 = 3,000 hPa, both the resuscitation rate and survival rate were zero of six cases (0%). Moreover, we also conducted preservation using a simple immersion preservation method by using a UW solution for 4 hours. According to the results, both the resuscitation rate and survival rate were six of six cases (100%) (Table 1, Fig. 3). DISCUSSION Seki and Toyoshima stated that tardigrades, which were in a state of decreased moisture content within the organism, could be resuscitated even under the super-high

pressure of 6000 atmospheres in PFC (20). The phenomenon of living organisms reducing their decomposition by decreasing the amount of water in their bodies in order to adapt to extreme environmental conditions, such as dryness or low temperatures, is called cryptobiosis and this phenomenon can be found in many forms in the natural world, such as the drought dormancy of plants in the Arctic Circle or the phenomena in which many bacteria enter a dormant state when the relative humidity is 60% or less, and such bacteria and tissues are now being developed for practical use in preserving dryness (5). It is believed that one of the characteristics of this phenomenon is associated with the fact that part of the free water in the cells is lost but the bound water protecting the biopolymer surface remains, and decomposition in the cells is thereby reduced to the regenerable limit as the amount of free water decreases. Accordingly, it is believed that if the structured water within the tissue or cell remains and free water is removed without damaging the tissue or cell, the metabolism would slow and the tissue could be preserved for a long period. As a result, Seki and colleagues worked on an experiment for preserving an isolated mammalian organ. At the beginning, they performed an experiment in which they decreased the moisture content and then

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Table 1. Results of the Resuscitation Status for Each Preservation Method

Group A1 A2 B C D

Preservation Method

Preservation Times (h)

n

Revival Rate (%)

Survival Rate (%)

Stop (Days)

PCO = 4000 hPa + PC2 = 3000 hPa (CO = 57% + O2 = 43%) PCO = 4000 hPa + PC2 = 3000 hPa (CO = 57% + O2 = 43%) PCO = 3000 hPa + PC2 = 4000 hPa (CO = 43% + O2 = 57%) PHe = 4000 hPa + PC2 = 3000 hPa (He = 57% + O2 = 43%) UWS

48 24 48 48 4

6 6 6 6 6

6/6 (100) 6/6 (100) 2/6 (33) 0/6 (0) 6/6 (100)

5/6 (83) 6/6 (100) 1/6 (16) 0/6 (0) 6/6 (100)

1, >42 × 5 >42 × 6 1, >42 — >42 × 6

A to C show preservation conducted by filling gas into special chambers. D shows preservation conducted using a simple immersion preservation method with a University of Wisconsin solution (UWS). The donor hearts were considered to have survived after 6 weeks (42 days) after transplantation.

Figure 3. Graph of the data in Table 1. The horizontal axis is divided by each preservation method. The vertical axis represents the resuscitation rates and survival rates of the donor hearts based on the number of individuals. A1 represents preservation at PCO = 4,000 hPa and PO2 = 3,000 hPa for 48 h, wherein the resuscitation rate was six of six cases (100%) and the survival rate was five of six cases (83%). A2 represents preservation at PCO = 4,000 hPa and PO2 = 3,000 hPa for 24 h, wherein both the resuscitation rate and survival rate were six of six cases (100%). B represents preservation at PCO = 3,000 hPa and PO2 = 4,000 hPa for 48 h, wherein the resuscitation rate was two of six cases (33%) and the survival rate was one of six cases (16%). C represents preservation at PHe = 4,000 hPa and PO2 = 3,000 hPa for 48 h, wherein both the resuscitation rate and survival rate were zero of six cases (0%). D represents preservation using a simple immersion preservation method with a University of Wisconsin (UW) solution for 4 h, wherein both the resuscitation rate and survival rate were six of six cases (100%).

PERSERVATION OF RAT HEARTS FOR 48 H USING CO AND O2

carried out resuscitation by using PFC solution in rats and swine a number of times, wherein they occasionally obtained good results, but the reproducibility was found to be insufficient. Subsequently, Seki and colleagues focused on the fact that carbon dioxide gas has an anesthetic action and metabolic suppression action in an organism. They performed an experiment in which they preserved and resuscitated a heart in an environment of reduced moisture content and a high concentration of carbon dioxide gas, preserving the heart for 24 h, and performed an ectopic heart transplantation on the recipient rat, after which they stated that resuscitation was possible with reducibility (21). These results were considered significant, and it was suggested that the method of preservation by desiccation could therefore be used as a method of preserving organs. CO has about 250 times more affinity with hemoglobin than oxygen and it forms carbon monoxide hemoglobin (COHb) by binding with the hemoglobin. In addition, it disturbs the bond between hemoglobin and oxygen, inhibits the transportation of oxygen into the tissue, and causes the suffocation of the tissue. As a result, toxic symptoms are expressed, including headache, dizziness, tinnitus, vomiting, and/or other symptoms in mild cases, and if exposure is prolonged, it may result in death. CO has no color and no smell and so there are many accidental deaths related to CO. Although CO is a gaseous body with toxicity such as that described above, some have reported that usage at low concentrations has beneficial effects for protecting tissue and organs (10,14). We used a unique method of preservation by desiccation by exposing the organ to a gas (gaseous body) by applying CO. Yoshida et al. used CO for preservation. They exposed the organ to high-partial-pressure CO at PCO = 400 hPa, preserved the organ for 24 h, then resuscitated it by performing heterotrophic transplantation on the neck, and obtained results yielding a reproducibility of 100% (25). In this experiment, we filled the chamber with a gas mixture (PCO = 4,000 hPa, PO2 = 3,000 hPa), preserved the organ in a refrigerator at 4°C for 48 h, performed heterotrophic transplantation on the neck of each recipient rat, and then succeeded in resuscitating the preserved hearts. Based on some reports that CO has anti-inflammatory and antiapoptotic effects (3,11,17), it is believed that these effects were adopted by the preserved hearts. Furthermore, CO has an action of binding with the Fe2+ of cytochrome oxidase, which is an essential enzyme for the production of energy from glucose by organisms, and so acts to suppress the activity of this enzyme (23). It is believed that, because CO suppresses this enzyme,

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the metabolism itself within the cell was suppressed, and necrosis was thereby prevented. Furthermore, it is believed that, because the isolated heart was exposed to an environment with a high pressure of 7 absolute atmospheres (ATA), CO increases its ratio of binding with cytochrome oxidase, thereby further suppressing the metabolism and increasing in the ratio of preventing necrosis. Moreover, it is believed that, in both cardiac cells and tissue under high pressure, a dynamic equilibrium between O2 and CO was established, components to be destroyed were dissolved preemptively via the law of entropy increase, reconstruction became possible before disarray accumulated, and necrosis was thereby prevented (6,7,18). It is believed that preservation was possible at such high concentrations of CO because bonding between the CO and the hemoglobin was avoided by conducting preservation in a state in which the blood was removed. Furthermore, the use of CO as a gas in this method of preservation by desiccation made it possible to preserve the hearts at concentrations that normally would not be considered possible. Methods that apply CO directly to a living body or dissolve CO in a solution inevitably keep the concentration of CO low. It is believed that the high concentration of 4,000 hPa of CO enabled the preservation for the long period of 48 h. After preservation conducted as a control experiment by decreasing the CO and increasing O2 (PCO = 3,000 hPa and PO2 = 4,000 hPa), the results tended to worsen, wherein the resuscitation rate was 33% and the survival rate was 16%. Furthermore, after preservation in CO at 100% (PCO = 7,000 hPa) to increase the amount of CO to an extreme level, both the resuscitation rate and survival rate were 0%. These results showed that the notion that using CO is inherently good is shortsighted and thus suggested that a balance with O2 was important. In addition, in this experiment we used the KH solution rather than the UW solution. This is because when performing a comparative experiment with a UW solution the KH solution showed better results, but there is no particular reason to designate the KH solution as the best available, so we will continue to try and identify the optimal preservation solution in future studies. Recently, we have also been performing a method whereby Yoshida et al. desiccated and preserved an organ with a PFC solution (26) and a two-layered method (TLM) whereby Salehi et al. evaluated the human pancreas (19). Furthermore, in addition to basic studies regarding the effects of CO and CO2, ATP concentration within the tissue of the preserved organ, microscopic tissue images, and the isolated heart of large animals have also been used in the application of preservation and resuscitation experiments. With the aim of achieving longer periods of preservation and better

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conditions, we have been attempting to select and create an optimal gas partial pressure and preservation solution in order to maintain the dormant state of the cardiomyocytes. These experiments were aimed at suppressing the metabolism of the heart with either a decreased moisture content or CO and CO2 gas in order to extend the preservation period. Other concepts for preserving organs include temporarily stopping the vital activity completely through low-temperature exposure (1) and inhibiting electron transfer enzymes (2) in a way that allows for resuscitation, and this method is called suspended animation. Both cases involve the application of the dynamic equilibrium theory that aims to make it possible to switch between a living state and a material state (12). We would like to propose that this technique be called semibiology. An automobile can be repaired in order for it to be able to be driven again when it breaks down, because there are design drawings, parts, and repair techniques. In the case of humans, design drawings (anatomical drawings) and repair techniques (surgery) have already been completed, but there are unfortunately still very few spare parts available (organs). These spare parts depend on a supply from brain-dead human donors, but the maximum preservation limit for such organs continues to be 24 h. Our organ preservation and resuscitation technique has extended the preservation time of these parts (organs) from 24 to 48 h. If the preservation time of organs can be extended to 1 year or more, then human life could potentially be made semipermanent, much like a semiconductor. It is believed that human life can someday be made semipermanent, much like automobiles, if semibiology techniques can be successfully developed in the future. The natural phenomenon of plants and animals in the natural world entering drought dormancy almost every year and then awakening in the following spring has already been applied to the cells and tissues of plants and animals, and advances are now being made toward the practical use of these same phenomena. In this experiment, we were able to show that this natural phenomenon, which is constantly repeated in the natural world, can also potentially be applied to isolated mammalian organs, and moreover, we were able to verify its reproducibility. This experiment is a completely new method of desiccating and preserving an organ by exposing it to the air for preservation. We believe that our presentation of the possibility of applying the desiccation and preservation method to the organs of other mammals in this experiment will make it possible to expand the future framework of “organ preservation.” The current organ preservation method is mostly an immersion and preservation method, and research on organ preservation

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mostly focuses on the preservation solution. When asked to provide examples, this is a study that takes a branch from the trunk of the tree of the immersion and preservation method. We believe that we were able to grow a completely new trunk of the tree by means of the desiccation and preservation method with organ preservation methods. On the tree of the desiccation and preservation method, no branches have yet been grown, and there are abundant new developments and possibilities for growing a number of important branches. ACKNOWLEDGMENT: The authors declare no conflicts of interest.

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