REANIMATED PIG BRAINS
https://nature.com/articles/s41586-019-1099-1
https://vox.com/pig-brain-nature-study-revive-cell-death-brainex
Scientists: We kept pig brains alive 10 hours after death
by Brian Resnick / Apr 17, 2019
“Around 15 minutes after a mammal’s brain is cut off from oxygen, the organ is supposed to die. Without life-giving oxygen, the cells of the brain quickly starve. Some of the cells burst open, while the chemistry of others becomes so imbalanced that their membranes break down. This frenzied spiral ends one way: in death. It’s thought that this process is widespread across brain anatomy and irreversible. After brain cells die, they are thought to be impossible to revive. But a stunning new finding published Wednesday in the journal Nature turns that conventional wisdom around. In a paper that reads a bit like an adaptation of Mary Shelley, researchers at Yale University describe how they were able to partially revive disembodied pigs’ brains several hours after the pigs’ death.
First, the researchers took 32 brains from pigs slaughtered for food and waited four hours. Then they hooked them up for six hours to a system called BrainEx, which pumped those brains full of oxygen, nutrients, and protective chemicals. At the end of the 10 hours, the scientists found that the tissue of the pig brains was largely intact, compared to controls. Individual brain cells were up and running, performing their basic duties of taking up oxygen and producing carbon dioxide. To be clear: The neurons in these brains were not communicating, so there was no consciousness. But the cells were alive — and that alone is a very big discovery. “Previously, findings have shown that in basically minutes, the cells undergo a process of cell death,” Nenad Sestan, the Yale neuroscientist who led the effort, said during a press conference. “What we’re showing is that the process of cell death is a gradual step-wise process, and some of those processes can be either postponed, preserved, or even reversed.”
The implications are bigger than just basic science research. The ethics of experimenting on partially reanimated brains is uncharted territory. “My first reaction was holy shit,” says Hank Greely, a Stanford law and ethics professor who reviewed the findings and co-authored a commentary on the paper in Nature. “The conventional wisdom is brain cells die, irretrievably, after about 10 to 12 minutes without oxygen — and that’s part of the scientific underpinning for the definition of brain death, the definition of death in humans. The idea [that] after four hours with no oxygen, or glucose, or anything else, most of the cells in the pig’s brain would start functioning again? That’s astounding.” It’s so astounding, he says, that perhaps we should reconsider the definition of death. And we should definitely consider the unexplored ethical implications of partially reviving a dead animal. Because, what if, even for a moment, that pig brain felt something?
The BrainEx system comes out of the National Institutes of Health’s BRAIN initiative (a.k.a. Brain Research through Advancing Innovative Neurotechnologies). This was President Barack Obama’s “moonshot” project to map the human brain and to create new tools to study it. BrainEx is one of those tools, developed in consult with the neuroethics working group at the NIH. Andrea Beckel-Mitchener, the team lead of the BRAIN Initiative at the NIH, says BrainEx “is really a tool to study basic biology,” to study the three-dimensional structure of a living brain outside the confines of the body. In the past, scientists have been able to culture slices of brains and other tissues from dead animals in a petri dish environment. But never before have they had the chance to study those complex relationships with an entire brain.
“A whole cellularly active organ allows you to study the interrelationship between the parts of the brain,” says Nita Farahany, a professor of law and philosophy at Duke who consulted on the ethics of the study. “You can’t get that with just slices of tissue.” The scientists hope these whole, preserved, somewhat alive brains will also be useful for neuroscience research, allowing others to test drugs and study disease pathways and neuroanatomy. It also raises the far-flung possibility of helping people revive brains damaged due to stroke or other injury. Sestan, the lead researcher on the effort, says he’s not sure if that’s even possible. But if it is, in the much, much longer term, it could force us to reconsider the definition of death itself.
“The part of BrainEx where the pig brain is placed”
Four hours after the pigs were slaughtered in a meat processing plant, researchers hooked up the brains to BrainEx to try to restore and preserve the cellular function of the brains after death. As the researchers stressed many times during a press call, the BrainEx does not have the ability to “reanimate” an entire brain. “That was not the goal,” Sestan says. The goal was to test if individual brain cells could be revived in this manner. Here’s how it worked. (You might have heard of this process before: Last year, the MIT Technology Review reported on this work while it was still preliminary and unpublished.) Four hours after slaughter, the pig brains were attached to the BrainEx device via the carotid arteries. You can think of the device as something like an artificial heart. It pumps a perfusate — a synthetic blood that delivers oxygen and nutrients — throughout the brain via the pig’s own arteries.
“This is a diagram showing the various parts of the BrainEx device”
The perfusate also contains chemicals that stop the oxidation (i.e., breakdown) of body tissue, that stop apoptosis (cells bursting after death). It also contains a neural activity blocker, the purpose of which is twofold. One was to halt excitotoxicity — a process by which neurons become damaged when they are left turned on, creating a chemical imbalance, also leading to cell death. The other was to make sure the brain didn’t “wake up” and regain any level of consciousness. If any consciousness was detected via EEG, the researchers said, they would have shut down the experiment immediately. They were even standing by with anesthesia to administer to the brain in case any widespread neural activity was detected.
After six hours on the BrainEx — 10 hours after slaughter — the researchers didn’t see signs of consciousness in the brains. But they did see signs of life. When hooked up to the BrainEx system, “we see the brains extract oxygen … they use glucose [sugar], and they produce CO2,” Zvonimir Vrselja, a Yale researcher and one of the study co-authors, told reporters. Which is to say: The cells’ metabolism was turned on. That means they were, to some degree, alive. Under the microscope, the cells of the BrainEx brains looked relatively normal, while the cells in the control brains, which were hooked up to the BrainEx but not to the sustaining perfusate, looked damaged. Most compellingly: When the brains were dissected and the neural activity blocker was washed away, the researchers found the neurons retained their ability to communicate with one another. That means the cells retained the function they had when the body was alive. At the end of the six hours, the brains were taken off the BrainEx. By that point, the control brains were too damaged to continue. But the researchers say there’s no reason the brains getting the perfusate couldn’t be left on for longer.
“Immunofluorescent stains for neurons (green), astrocytes (red), and cell nuclei (blue) in a region of the hippocampus of a pig’s brain left untreated 10 hours after death (left) or subjected to perfusion with the BrainEx technology. Ten hours postmortem, neurons and astrocytes undergo cellular disintegration unless salvaged by the BrainEx system.”
At no point during any of the trials, the researchers say, did the pigs’ brains regain consciousness, as measured by EEG (electroencephalogram), which records widespread electrical activity in the brain. Without widespread electrical activity, the brain is not believed to have any ability to think. “This is a clinically dead brain,” Sestan says. But it’s more nuanced than that. It’s dead, but it’s also somewhat alive. What Sestan and his team are claiming is they kept the individual cells of the brain alive, hours after the pig was killed. But those cells, for the most part, weren’t talking to one another. “If consciousness were somehow induced in the brain,” there was no ethics protocol in place, says Stephen Latham, a Yale researcher who served as the team’s bioethicist. “If consciousness was induced by the research, the research would have to stop until we could pull together some kind of ethics and neuro expertise to give guidance to the future conduct of the research.”
“This image shows an analysis of synaptic organisation,
neuronal activity, and wider brain activity”
At the same time, it’s hard to know if EEG is the only indicator of consciousness or perception of any kind. There are billions of neurons in a pig’s brain. How many need to be activated for a pig to “feel” anything — as much as a disembodied pig brain could feel or interpret anything — is not known. “I think they’re right, that given the lack of an EEG, it would be shocking if there were any consciousness there,” Greely says. “But are there parts of the pig brain that are somehow sensing and reacting to pain? … [I]s that something to worry about? The lack of an EEG is very comforting, but what comes next? Will there always be the lack of an EEG? … My honest answer to the question of ‘does this make the possibility of reanimation thinkable?’ is ‘maybe.’”
All of this is so new. There are some other immediate ethical considerations to think through as the science progresses. One is how should scientists think about the ethics of experimenting on partially revived animals? Technically, this doesn’t fall into the category of animal research “because the brain comes to the researchers already from a dead animal,” from a slaughterhouse, Latham explains. That means the researchers don’t have to follow the ethical guidelines that come with animal research, namely that animals should not be subjected to unnecessary harm. But what about partially revived animal brains? There’s no guidance on that. Here’s another. The researchers also used chemicals to block neural activity in the pigs’ brains to try to ensure they never became conscious. But what if the researchers hadn’t done this? They wouldn’t speculate what would happen then. They don’t know if some consciousness could ever be restored after death, and they say they don’t want to try to restore it. But some other research group might take their results and try. And there need to be ethical guidelines in place for that, the ethicists say. “There is, at least, the potential for EEG [brain activity] to be restored,” Farahany says. “And that means the capacity for pain and distress could be restored as well.” Greely adds, “We have an obligation not to be cruel to this former pig.”
In the meantime, Greely and Farahany suggest, the research can continue, but scientists need to keep safeguards — like the neural activity blockers and the anesthesia — in place in the case any consciousness is revived. In a separate commentary in Nature, bioethicists Stuart Youngner and Insoo Hyun warn that the findings could have implications for organ donation. If it’s conceivably possible to keep a brain alive for a period after death, should doctors feel more reluctant to harvest organs for donation? “If technologies similar to BrainEx are improved and developed for use in humans, people who are declared brain dead (especially those with brain injuries resulting from a lack of oxygen) could become candidates for brain resuscitation rather than organ donation,” Youngner and Hyun write. “Certainly, it could become harder for physicians or family members to be convinced that further medical intervention is futile.” Again, that’s far off at this point. But these findings implore us to think a bit more deeply on this question: Where is the line between life and death? It’s less clear than it was a day ago. “We had clear lines,” Farahany says. “There was dead and there was alive. … This calls that into question.” We need to see the study replicated, Farahany says. And there are a lot of unanswered questions about its potential use in other animals, or perhaps in humans. “But at least it opens up the possibility that what once was lost is possibly recoverable.”
“This image shows the restoration of brain circulation
and cellular functions hours post-mortem”
LIFE AFTER BRAIN DEATH
https://news.yale.edu/scientists-restore-some-functions-pigs-brain-hours-after-death
https://newscientist.com/radical-treatments-bringing-people-back-from-the-brink-of-death
Could death be a treatable condition?
by Helen Thomson / 26 November 2024
“The severed pig’s head had come from the local abattoir. It would have typically been discarded, but Zvonimir Vrselja, a neuroscientist at Yale School of Medicine, and his colleagues had other ideas. Four hours after this particular animal was decapitated, they removed its brain from its skull. They then connected the dead brain’s vasculature to tubes that would pump a special cocktail of preserving agents into its blood vessels and turned the perfusion machine on. That was when something incredible happened. The cortex turned from grey to pink. Brain cells started producing proteins. Neurons juddered back to life, displaying signs of metabolic activity indistinguishable from that of living cells. Basic cellular functions, activities that were supposed to irreversibly cease after blood flow stopped, were restored. The pig’s brain wasn’t alive, exactly – but it certainly wasn’t dead. Now, for the first time, the team is using the technique on human brains. “We are trying to be transparent and very careful because there’s so much value that can come out of this,” says Vrselja. Reanimating – in a sense – a dead human brain would have tremendous medical benefits. Researchers could trial drugs on cellularly active human brains, leading to improved treatments. Similar techniques are already being used to better preserve other human organs for transplants, too. And in what is perhaps the most immediately useful application, the resuscitation technology involved raises the possibility of saving people on the cusp of death. The problem is that it is an ethically complicated undertaking, to put it mildly. And by demonstrating the brain’s extraordinary resilience, this work forces us to ask, when is a person really dead?
Vrselja and his colleagues’ work on pigs’ brains sent shock waves through the medical community five years ago. “When I heard, my first reaction was ‘holy shit!’” recalls Hank Greely, a biomedical legal expert at Stanford University in California. He wasn’t alone. The idea that a brain cut from a body, deprived of oxygen and kept at room temperature for 4 hours could be revived “goes against everything we thought we knew about death”, says Lance Becker at the Feinstein Institutes for Medical Research in New York. “We’re at a real paradigm-shifting moment as we redefine what is life and what is death.” Deciding when someone is dead has never been as easy as you might think. Ancient Greeks used cessation of breathing as a sign of death, holding a candle near the mouth to check. Later physicians confirmed death by trying to shock a presumed dead person into life – putting their finger to a flame or poking them with needles. In the early 1800s, after several publicised cases of supposed premature burials, mortuaries and hospitals kept corpses for several days to confirm putrefaction; canny inventors started patenting “safety coffins” with air tubes and bells that could be rung from the inside.
These days, people are typically declared dead when their heart stops beating – known as cardiac arrest – and cannot be restarted. The UK doesn’t have a legal definition of death, instead accepting medical opinion. In the US, on the other hand, doctors in most states rely on the Uniform Determination of Death Act to define death, which states that an individual is dead once “they have sustained either irreversible cessation of circulatory and respiratory function or the irreversible cessation of all functions of the entire brain, including the brain stem”. The key word here is “irreversible”. Until recently, we thought we knew two things for sure about death. First, that when your heart stops beating, the electrical activity in the brain ceases within seconds to minutes. Second, that the brain suffers irreparable damage within 5 to 10 minutes of the onset of oxygen deprivation. But recent studies have upended both of these assumptions.
Let’s start with the notion that electrical activity in the brain diminishes after cardiac arrest. For a study published in 2023, Jimo Borjigin, a neuroscientist at the University of Michigan Medical School in Ann Arbor, and her team analysed brain activity from four dying people before and after the withdrawal of their life support. “Rather than decreasing in activity, the whole brain appeared to be on fire,” says Borjigin. In two of the people, brain regions that were silent while the person was on life support suddenly fizzed with activity after it was withdrawn. High-frequency brainwaves called gamma waves, a hallmark of consciousness, also burst into action. Synchronised activity resembling the kind associated with memory and perception lasted for up to 6 minutes. Some brain areas suddenly tried to strike up conversation with the heart. These waves of activity dampened, then intensified three times; in some areas, the activity was 12 times greater than before the individual’s heart had stopped beating and they were no longer breathing. “The dying brain actually starts this massive rescue effort,” says Borjigin. “If we can better understand what’s going on at this point, I believe we could resuscitate it.”
“This image shows what happened when scientists
pumped chemical solution into dead pig’s brain”
Because the people involved didn’t survive, it is impossible to know what they experienced in those last moments of frenzied brain activity. But the study confirmed that this boundary between life and death isn’t black and white – and it isn’t the only study to do so. “We’re all conditioned to think of death as this binary thing,” says Sam Parnia, a cardiopulmonary researcher at New York University. “You’re alive and then you’re dead. Most people aren’t willing to accept that biology doesn’t work that way.” In 2023, his team recorded brain activity in 53 people who were being resuscitated after a cardiac arrest. Despite almost all of the patients flatlining – meaning their brain activity was silent – 40 per cent later showed spontaneous brain activity associated with consciousness during resuscitation attempts, sometimes up to an hour after their heart had stopped. Parnia’s team spoke to some of the people who survived, as well as others in the local community who had experienced a cardiac arrest, and found that 20 per cent recalled a conscious experience that occurred during the time in which their heart had stopped.
“This image shows magnetic resonance imaging scans of the brains”
Although some question Parnia’s findings, the study nevertheless demonstrates that something is happening at the point of death. “You can’t talk about the dying brain without thinking about the nature of consciousness,” says Parnia. And it isn’t just the dying brain that is making us question our understanding of life, death and consciousness. The dead brain is raising questions, too. We had good reason to believe that the brain couldn’t survive more than 10 minutes without oxygen: trying to revive a person after that deadline is rarely successful and those that survive often experience cognitive problems. However, though oxygen deprivation does initiate cell death, the damage that follows has less to do with the brain not receiving oxygen and more to do with what happens when it is restored. When oxygen-rich blood suddenly starts pumping around a body and brain that have experienced ischaemia, or lack of oxygen, this sudden reintroduction causes what are known as reperfusion injuries. Friedhelm Beyersdorf at the University of Freiburg, Germany, likens it to a broken leg. “If I break my leg and someone helps me to stand, they don’t tell me to walk immediately, because my leg needs treatment,” he says. “It’s the same with our organs. Once they go without oxygen, they are damaged. You can’t just pump blood back in and expect them to work as before – they need to be treated first.”
“ECMO machine temporarily takes over heart and lung function after cardiac arrest”
To prevent reperfusion injuries in his pig brains, Vrselja and his colleagues developed a cocktail of drugs that target the pathways that typically lead to damage after ischaemia. This included molecules that balance cell pH, drugs that prevent an excessive immune response and antibiotics. They modified a dialysis-type machine designed to keep other organs alive outside of the body. Their device, called BrainEx, could perfuse this cocktail through the brain’s arterial network in a pulsating fashion, at just the right pressure to penetrate cells, while also clearing waste products. It worked. Four hours after decapitation, the brains were plugged into BrainEx and revived – albeit not to the point of consciousness. In their 2019 paper, Vrselja and his co-authors were quick to note that they didn’t observe brain activity associated with perception. Indeed, they had made sure this didn’t happen by ending the experiment after 6 hours and including sedatives in their cocktail that inhibited electrical activity. Repeating the experiment on a donated human brain, however, requires a greater level of assurance: if there were any evidence of consciousness-like brain function, that would raise the question of whether you are experimenting on a live person, and what rights they then deserve. “That’s very tricky ethically, legally and scientifically,” says Greely.
Vrselja and his colleagues are treading extremely carefully, noting that they have a broad group of bioethicists and legal and medical experts advising them. “We had to develop new methods to make sure no electrical activity is occurring in an organised way that might reflect any kind of consciousness,” says Vrselja. For now, they aren’t using this technology to attempt to restore consciousness to a dead human brain. Instead, Vrselja and his team are keeping brains cellularly active for up to 24 hours to test treatments for conditions such as Alzheimer’s and Parkinson’s. It is hard to develop drugs for neurological conditions, says Vrselja, because current methods for testing them are insufficient. “That’s why the field is riddled with mystery – often we don’t even know if a drug can get into the brain,” he says. “Now we can address that. It’s research that’s going to make a real difference to humans.”
Vrselja acknowledged, however, that others may attempt to resurrect dead brains in a greater capacity. Trying to bring a brain back to life, consciousness and all, would be nothing less than an attempt at immortality, says Greely. “A living brain in a box seems to me like hell rather than life, but people pay a lot of money to have their heads cryogenically frozen with highly implausible chances of reanimation. I’m a little surprised no quack has tried selling this service yet – as far as I know.” If bringing back an already dead brain is a tantalising if ethically sensitive prospect, Vrselja’s research also suggests we could intervene in brains and bodies on the brink of death. Though he and his colleagues have no intention of plugging anyone at the point of death into their BrainEx machine, they want to use their new knowledge about resuscitation to help save human lives.
In 2022, they developed a similar system, called OrganEx, to perfuse a blood-based cocktail through a whole pig 1 hour after its heart had stopped beating. They found that the reperfusion therapy reduced cell death in various organs and restored function in the pig’s brain, heart and kidneys. The heart began to contract, the brain resumed metabolising and several genes associated with cellular repair were switched on. The pig even started to make involuntary movements, despite being anaesthetised. Similar technology is now being trialled to keep human organs alive for transplant. When a person is pronounced dead, they are attached to a version of OrganEx, with the arteries leading to the brain clamped so that it is shut off from the system. In doing so, organs stay alive for much longer, giving them more time to get where they are needed. “There is a tenfold number more donors than kidney recipients, yet there is a huge waiting list because we can’t get the organs to where they need to be,” says Vrselja. “This can really help save a lot of lives.”
Meanwhile, reperfusion technology is already helping keep more people alive after injury. Researchers at the University of Freiburg, led by Beyersdorf, and German technology company Resuscitec spent more than a decade developing “controlled automated reperfusion of the whole body”, or CARL, which is a modified version of a heart-lung bypass technology called extracorporeal membrane oxygenation, or ECMO. While ECMO temporarily takes over heart and lung function by circulating and cleaning a person’s blood after cardiac arrest, CARL goes further, perfusing the body with a cocktail of haemoglobin – the oxygen-carrying protein in blood – and 13 other components that protect organs from ischaemic damage. It also features two pumps that replicate the pulsing motion of the heart, helping the cocktail reach all areas of the body and brain. Early trial results are promising. Typically, only about 1 in 10 people who experience a cardiac arrest survive. But in a trial run by Beyersdorf, CARL was given to people whose hearts had stopped and who had received CPR for around an hour; of these, 42 per cent survived and 79 per cent of those had normal cognitive function. Early results from another trial of CARL in Germany, in people whose hearts stopped outside of a hospital, showed that it revived five of the first 10 people treated. Resuscitec brought CARL to market in Europe in 2023 and is awaiting FDA approval in the US.
Our greater understanding of the moments after cardiac arrest and the possibilities of restoring life is blurring the line between dead and not dead. “It used to be straightforward to pronounce a patient dead,” says Becker. “I could safely say with confidence that their heart had irreversibly stopped. Now, it’s more ‘they’re dead, nudge nudge, wink wink’.” By that, he means that while they are dead because the treatments available at that moment can’t save them, he can’t be sure that they are irreversibly dead. “If we had the right people in the room, the right machines, I know that the heart and brain might be revived,” he says. “So are they really dead?” At the moment, that question remains unresolved. “As CARL is used more widely, we’ll see how long our organs can survive without oxygen,” says Beyersdorf. “And then perhaps we’ll have to change our definition of death.” For now, we are in a grey zone. “It will take a while, but the field of resuscitation is going to advance so that people who are routinely diagnosed as dead today are not viewed that way any longer,” says Becker. “That’s a pretty remarkable rewriting of human existence.”
PREVIOUSLY
PIG’S BLADDER REGENERATES HUMAN TISSUE
https://spectrevision.net/2008/05/01/pigs-bladder-powder-regenerates-human-tissue/
the ‘MONKEY-FUCKED-a-PIG’ HYPOTHESIS
https://spectrevision.net/2013/12/06/part-pig/
DE-EXTINCTION for BEGINNERS
https://spectrevision.net/2013/05/09/de-extinction/