This is a dead sow. It had been dead for an hour, and a special perfusate was being slowly poured into its body, circulating through its veins and arteries, with fresh oxygen flowing through it. Over the next six hours, the researchers quickly found that some cells in the pig’s heart, liver, kidney and brain began to regain function and activity, and the pig’s circulatory system gradually recovered. Although the pig’s heart has not fully recovered, electrophysiological activity can be scanned, indicating that it has the ability to contract. In every vital organ, cells are more responsive to glucose, suggesting they are restarting part of their metabolism. Throughout the process, people were also surprised to observe involuntary muscle movements in its head and neck. Also, as a medically deceased animal, it was not rigid, nor did it develop edema and corpse spots.
It appears that the pig is slowly “resurrecting”.
On August 3, 2022, the team of neurobiologist Nenad Sestein of Yale University School of Medicine published the “resurrection” experiment and its results in the top journal “Nature”, which aroused widespread attention and discussion. The last time a similar discussion took place three years ago, in April 2019, Sestein’s team “resurrected” a pig’s brain four hours after it died, upending years of stereotypes that brain death is irreversible.
From the brain to the organs of the whole body, at this moment, a natural question is: along this technological path, is it possible for human beings to truly come back to life in the future?
“Opening Doors to the Future of Organ Transplantation”
This is the first time that someone has been able to “reanimate” various organs in a mammal’s body at the same time, and some experts have pointed out that this achievement is “subversive”. Mammalian cells require oxygen and blood to stay alive, and within minutes of ischemia, acidosis and edema develop within the cells, leading to irreversible damage. Generally speaking, mammalian organs will die completely if they are deprived of oxygen for about 15 minutes.
In this experiment, Sestein’s team injected pigs with “resurrection agents” one hour after ischemia and hypoxia, mainly composed of pig blood, artificial hemoglobin, nutrients needed by cells, antioxidant drugs and drugs to prevent cell death. mixed with various compounds. Zhang Shupei, one of the co-authors of this study and a doctoral candidate at Yale University School of Medicine, pointed out to China News Weekly that this is an optimization and improvement of the perfusate used in the pig brain experiment in 2019. The oxygen-carrying capacity of artificial hemoglobin is very strong. This time, a system called OrganEx was used for the experiment. In OrganEx perfusate, the ratio of hemoglobin to blood is 1:1, “so that cells receive oxygen with the highest efficiency.” In addition, drugs such as antioxidants in the perfusate can be used to inhibit and alleviate cell damage, “as opposed to targeting a single organ in the brain. Compared with the modified version, it is more compatible with various organs of the body,” she said.
But the secret to “resurrecting” pigs isn’t just in this recipe.
The experiment also set up a control group using an extracorporeal membrane oxygenation device (ECMO). ECMO is often used as an important tool to rescue patients with cardiopulmonary failure in clinical practice, and can partially or completely replace the patient’s cardiopulmonary function, equivalent to an artificial heart-lung machine. However, ECMO generally uses venous blood perfusion. In recent years, the function of ECMO as a transplant organ preservation facility has been gradually developed, because ECMO can provide stable oxygenated blood perfusion immediately after cardiac death, thereby better maintaining the quality of the transplanted organ and improving the transplant efficiency.
A problem that has existed in organ transplantation for many years is “reperfusion injury”, that is, the reinjection of oxygen into cells after hypoxic ischemia will bring about cell damage such as blood vessel collapse. The study found that compared with the ECMO group, the OrganEx group suffered less damage to cells after perfusion, less bleeding and tissue swelling, and maximized perfusion efficiency. Cell function was better restored in all major organs of the OrganEx group of pigs. In pig kidney cells, cell proliferation also appeared.
Zhang Shupei pointed out that in clinical practice, ECMO perfusion efficiency “is actually very low”, and the requirements for use time are very strict. “That’s why we chose to perfuse the pigs an hour after they died, because the average ambulance time in the United States to transport a critically ill patient is about an hour,” she explained.
OrganEx Technical Overview and Schematic Schematic of the Experimental Workflow
In her opinion, the key to bringing the pig back to life is the perfusion system OrganEx, a computer-controlled extracorporeal perfusion system through which perfusate is injected into different organs throughout the pig’s body. During this process, real-time sensors monitor important circulating indicators, metabolites, and signaling pathways “relevant to cellular repair,” allowing researchers to intervene at any time. The system also includes an oxygenator and a hemodialysis unit that maintain stable levels of electrolytes and other essential molecules in the perfusate.
”This is the most difficult part of the experiment,” Zhang Shupei said. OrganEx needs to better simulate the function of organs in the whole body. Taking the kidney as an example, the most important function of OrganEx is “exchange”, expelling toxic substances and leaving hemoglobin behind. , and the system needs to achieve “more efficient exchange” by “well-controlled” the corresponding hemodynamics.
Zhu Tongyu, deputy dean of Shanghai Medical College of Fudan University, is one of the well-known experts in organ transplantation in China. He pointed out to “China News Weekly” that in fact, in the field of organ transplantation, as early as the 1990s, some people began to try to use perfusate in perfusate. Add various ingredients, no longer use pure human blood. Therefore, the main significance of this study is that “there has been a new improvement and progress in the design, procedure and control of the mechanical perfusion system, which is a comprehensive optimization”.
”This discovery solves a major problem in the field of organ transplantation – the preservation efficiency of transplanted organs, and opens the door to the future of organ transplantation.” Sestein said in a news conference released by the study. Gabriel Oniscu, a transplant surgeon at the Royal Edinburgh Hospital in the United Kingdom, also said that given OrganEx’s significantly better results than ECMO, the study could “significantly increase the number of organs available for transplantation” in the future.
According to the latest statistics from the U.S. Health Resources and Services Administration, more than 100,000 Americans are waiting for fresh organs for transplant. Every 9 minutes, a patient is added to the waiting list for donation. Because there are not enough available organs, every day 17 people died while waiting. As of 2021, the most urgent need for organ transplants in the United States is patients with kidney failure, accounting for about 85% of all organ transplant waiting patients.
Recently, the “Report on the Development of Organ Transplantation in China (2020)” (hereinafter referred to as the “Report”) was released. From 2015 to 2020, the number of organ donations and transplants in China ranked second in the world. The “Report” pointed out that although the number of organ donations and the donation rate in China have increased compared with before, but like other countries in the world, they still face the problem of shortage of donors and cannot meet the huge needs of domestic patients.
Zhu Tongyu pointed out that in the next step, the difficulty of perfusion technology in organ transplantation is to increase the degree of fit with different organs, because each organ is different in size and blood vessel shape. For example, the blood vessels of the kidney are “thinner than chopsticks”, the blood vessels of the liver are also different from the hepatic artery and vein, and the heart has large blood vessels, so these different organs should be equipped with more targeted perfusion fluid and perfusion system.
Can death really be reversed?
On the cover of the April 2019 issue of Nature magazine, a giant hourglass is drawn with a brain at the top and bottom. The brain above appears to be clearly textured and still has basic structure, but it is constantly dissolving and breaking down into fine particles of sand and falling into the lower end of the hourglass, suggesting that a healthy brain is dying. At the bottom of the hourglass, the fine sand continued to gather again, forming a brain that was beginning to take shape, indicating that brain death was being reversed. The content of the cover of this issue is exactly the pig brain “resurrection” experiment of Sestan’s team. The system used is BrainEx. The title is quite meaningful, called “Reversing Time”.
Can death really be reversed? The Nature editorial board saw the more controversial and subversive issues behind the experiment. Sestein says previous research has shown that cells go through a process of dying within minutes of an individual being declared dead. But what we’ve shown is that cell death is a gradual process, some of which can be delayed or even reversed.
Arthur Kaplan, a bioethicist at New York University, says it’s therefore difficult to come up with a uniform way of declaring a person dead, which means that as medicine continues to advance, the definition of death will continue to adjust. People now tend to use brain death as the criterion for determining death, but there is not much consensus on when cardiac death occurs. Dan Parent told China News Weekly that this finding raises many questions, the most important of which is that we need to re-examine the criteria for determining medical and biological death.
A single definition of human death, developed by professional representatives, medical experts, and professional societies in several countries, states: “Death is the permanent loss of consciousness and all brainstem functions that does not recover spontaneously and cannot be recovered by intervention.” Loss of function.”
Now, the first challenge is “permanence”. Experts pointed out that if OrganEx is used clinically in the future, after blood and oxygen deprivation, human cells will take longer to reach the level of “irrecovery”, and the time point at which an individual is declared dead will also change.
Parente pointed out that determining a new death point in the future needs to be based on two considerations: First, clinically, in the case of a person losing blood and oxygen, what is the probability of using the OrganEx system to restore the “functional ability of the organ” ? What is the probability that the person will never regain “full consciousness” or “never get off the life support machine” if only partial ability is restored? Second, in terms of values, after using OrganEx, if the probability of recovering “good function” is 90%, but there is a 10% probability that life support equipment will never be separated after “resurrection”, does the patient want the clinical team to try save his life? If the most likely scenario is to regain only partial consciousness? “Will the patient want this?”
However, Zhu Tongyu pointed out that the current determination of brain death requires that two EEGs show a straight line at an interval of 24 hours, that is, the patient’s brain waves disappear and the brain function is in complete and irreversible loss. The experiment only verified the “cell changes within seven hours” after death, and it is “still a long way from it to be enough to revise the standard of brain death”.
In addition to potentially “re-understanding death”, several experts pointed out that in the future, surgeons will also face more contradictory trade-offs between saving and giving up, between saving lives and organ transplantation. After the results of the pig brain experiment in 2019 were released, Stewart Youngner, professor emeritus of the Department of Bioethics at Case Western Reserve University School of Medicine, and Ren Soo-hyun, director of ethics research at Harvard Medical School, jointly wrote in Nature that BrainEx may would intensify the debate about when to “remove an organ from a donor and transplant it.”
There are currently two main organ donation agreements, one is donation after brain death; the other is donation after circulatory death, which is for people who have been declared dead after cardiac arrest, especially those who have suffered severe brain damage but are not brain dead individuals, the latter has become more common in recent years.
Typically, with the patient’s consent, doctors turn off the patient’s ventilator and any other life-support equipment, and pronounce the patient dead 2 to 5 minutes after the patient’s heart stops beating. There is a “race to the clock” lurking here. Younger and others point out that it is often impossible to adequately test for brain death in order for the organ to be removed from the donor as quickly as possible and as healthy as possible.
Youngner et al. argue that surgeons sometimes use ECMO to perfuse the donor organ after cardiac arrest, allowing the donor organ to restore oxygen and blood flow after death to keep the organ healthy. The point of contention here is that brain function may recover in some patients following ECMO perfusion. In short, when should doctors switch from saving one person’s life to saving organs for another’s survival? And “BrainEx may complicate matters,” wrote Youngner et al.
Currently, there are “considerable differences” between countries in the ethical and legal considerations of whether and how ECMOs are used. In France and Spain, ECMO devices can be transported by special ambulances to wherever the patient is; but in the United States, the technology is controversial and rarely used in organ transplants.
Experts believe BrainEx and subsequent research need to be accompanied by a “more open discussion”: What criteria qualify someone for organ donation? How to ensure organ donation can be integrated into end-of-life care with “minimal controversy”? Parente emphasized that in any case, the values and rights of organ donors should be given higher priority than the possibility of organ donation.
“Resurrection” is a very distant plan
BrainEx is a “new beginning” for brain research.
Dedicated research on the human brain has been difficult for years for ethical reasons, and pig brain experiments have allowed the brain to “live” intact outside of the body. Zhang Shupei pointed out that previous studies were limited to brain slices from dead animals, so humans lacked understanding of the fine structure of the brain. For example, in what way are the neurons in the brain connected to each other, and what are the specific neural circuits? And now, for the first time, humans can study the brain in three dimensions.
A deeper understanding of these complex neural networks will help scientists to further study the principles and test drugs for brain diseases such as Alzheimer’s disease and autism. “We are equivalent to providing a platform on which we can do some drug screening.” Zhang Shupei said.
What’s more, the BrainEx experiment may help scientists break through the most sensitive question in brain research: “consciousness.” So far, no one has created consciousness in the lab, and some experts believe the BrainEx experiment “is the most likely one.” However, Sestan’s team is very cautious about this, repeatedly emphasizing in public that “the restoration of consciousness has never been the goal of this research.”
Zhang Shupei explained that due to ethical considerations, the BrainEx experiment isolated the “generation of consciousness” from the root, because the researchers injected the pigs with neuron activity blockers, which can block any neural interactions that may lead to consciousness, and match them with There is an EEG to monitor brain activity at all times. If any conscious activity was detected, i.e., the EEG showed that it was not in a straight line, the experiment was terminated immediately.
Under this premise, in order to test the activity of brain cells, the researchers cut the brain into slices of about 300 microns after perfusion, and observed whether there were signal fluctuations by stimulating the neurons in the slices. Neurons are alive.” But Zhang Shupei pointed out that consciousness must be born from “the overall linkage between neurons in the whole brain”.
So the paradox is that if humans want to further understand the nature of the brain, the field that must be touched is: how do millions of brain nerve cells and the trillions of connections between them work together? One day in the future, human beings will not stop at “reviving” brain cells, but thoughts and characters. Are we ready for this? If humans could “create consciousness”, what kind of world would that be?
One of the ethical guidelines for pig brain experiments, Nita Farahani, a professor of law and philosophy at Duke University, suggested in her 2019 paper “The Moral Dilemma Raised by a Partially “Resurrected” Pig Brain Just like the ethical guidelines for research, in the future, related research on “brain resurrection” also needs to obtain the same scientific and ethical guidelines, and form a consensus at the international level. Moreover, citizens must be involved in the process, and ethical boundaries set by scientists alone “do not guarantee future public acceptance.”
Parente pointed out that if the technology is to be developed to the point where it can help humans, we will have to look at “what happens to the brain without neuroblockers?”
For OrganEx technology, these Discussion is still far away. Zhang Shupei pointed out that, from the level of cells to tissues, organs, and the function of the entire body, to achieve “resurrection from the dead”, “this is a very, very long-term plan.”
It is uncertain whether the OrganEx technology can achieve a functional fix if the “resurrection” is extended to the organizational level, she said. For example, the kidney, one of the most important organs in the human body, its main function is to produce urine, but the experiment may have an impact on the urine due to hypothermic perfusion. “In fact, we did not collect a lot of urine in the experiment. liquid”. This also means that the kidneys are “not necessarily really functional” at this point, just “active” at the cellular level. Therefore, after the organ is perfused, whether the organ itself can restore its own function, “is the most important thing we will demonstrate in the next step.”
In fact, the two important organs of the pig heart and brain in the experiment only recovered “very small” functions, and other important functions such as cardiovascular and cerebrovascular circulation did not recover. Zvonimir Verseria, a neuroscientist at Yale University School of Medicine and one of the other co-authors of the study, made this very clear. The research just shows that when animals die, we can get cells to do “something they wouldn’t otherwise be able to do,” he said, but the activity of those cells “is not clinically relevant to life or death.” That said, this result doesn’t suggest that pigs are somehow resurrected after death.
Zhu Tongyu also pointed out that an important premise of this study is that heparin was injected within one hour after the pig died to keep the blood from coagulating, which is the basis for ensuring the “resurrection” of cells. “Without heparin, there is no back story.” So this study is just a proof-of-concept. It mainly simulates the physical conditions during organ transplantation, not the real state after death. “The blood will coagulate within an hour of natural death in animals and people.” The
study The researchers admit that, aside from the near-sci-fi idea of ”resurrection from the dead”, even in organ transplantation, this research is still far from clinical application. One of the first hurdles to overcome is time. The study only perfused pig whole body organs for 6 hours. What about after 6 hours? How long can OrganEx keep these cells alive? “We only flew a few hundred meters, but can we really fly?” Sestan sighed.
Zhang Shupei explained that the perfusion time limit is due to the ethical review of animal experiments and other reasons, and will consider extending the perfusion time in the future. “If the repair of cells can still be promoted after prolonged perfusion, clinical trials in humans may be advanced, and we need more data.” She also revealed that in the shorter term, the research team plans to use pig organs in the next step. “Transplant into another pig” to see if the organ function is restored. “As for experiments on humans, it may be at least 10 to 20 years away.”