Scientists have restored cellular function in 32 pig brains that had been dead for hours, opening up a new avenue in treating brain disease—and shaking our definition of brain death to its core. Announced on Wednesday in the journal Nature, researchers at the Yale University School of Medicine devised a system roughly analogous to a dialysis machine, called BrainEx, that restores circulation and oxygen flow to a dead brain.
The researchers did not kill any animals for the purposes of the experiment; they acquired pig heads from a food processing plant near New Haven, Connecticut, after the pigs had already been killed for their meat. And technically, the pig brains remained dead—by design, the treated brains did not show any signs of the organized electrical neural activity required for awareness or consciousness.
“Clinically defined, this is not a living brain,” says study coauthor Nenad Sestan, a neuroscientist at the Yale University School of Medicine.
The new system instead kept the brains in far better shape than brains left to decompose on their own, restoring functions such as the ability to take in glucose and oxygen for up to six hours at a time. Researchers say that the technique could give a major boost to studies of human health by providing a rich testbed for studying brain disorders and diseases.
“We're really excited about this as a platform that could help us better understand how to treat people who have had heart attacks and have lost normal blood flow to the brain,” adds Khara Ramos, director of the Neuroethics Program at the U.S. National Institute of Neurological Disorders and Stroke. “It really enhances our ability to study cells as they exist in connection with each other, in that three-dimensional, large, complicated way.”
Even so, the finding opens up considerable ethical questions, a conversation that the researchers themselves welcome.
“This is an extraordinary and very promising breakthrough for neuroscience. It immediately offers a much better model for studying the human brain, which is extraordinarily important, given the vast amount of human suffering from diseases of the mind [and] brain,” says Nita Farahany, a bioethicist at the Duke University School of Law who wrote a commentary about the study for Nature.
“It [also] challenges a lot of the fundamental assumptions that we had in neuroscience, like that once there is a loss of oxygen to the brain, it’s an irreversible march toward organismal death,” she adds. “That turns out not to be true—and because that’s not true, there's some pretty profound ethical and legal issues that are raised as a result.”
Death is final, but the number of truly irreversible medical outcomes has shrunk over time. For millennia, people were considered dead when they stopped breathing and their hearts ceased to beat. But then modern medicine intervened. The invention of mechanical ventilators allowed failing bodies to be kept alive for longer, and decades of improvements to heart surgery and transplants mean that even a stopped heart might not necessarily be the end.
But the brain remains a finicky patient. Mammal brains such as ours are high-performance machines; they demand a constant stream of oxygen-rich blood to work to their fullest. If blood flow is cut off, we lose consciousness after just a few seconds. Within five minutes, the brain's stores of vital molecules such as glucose and ATP—the body's universal currency for chemical energy—run out.
The brain then enters a death spiral that, up to now, scientists considered irreversible: Nerve cells' delicate chemistries get thrown out of whack, a buildup of carbon dioxide makes the brain's blood more acidic, and leaks of a powerful neurotransmitter called glutamate quickly become toxic. Soon, enzymes that break down nerve tissue come online, and the brain's smaller structures and blood vessels rupture and break.
The more researchers understood this process, the more they incorporated it into the definition of death itself. In 1968, a committee of doctors assembled by Harvard University put forth a landmark definition of “irreversible coma,” what we now call “brain death”: a total lack of responsiveness, the inability to breathe on one's own, a total lack of reflexes, and no signs of large-scale electrical activity in the brain. Now, the American Academy of Neurology maintains a checklist that clinicians use to judge brain death in patients.
But there have been hints of greater brain resilience. Some parts of brain cells, such as the mitochondria that process chemical energy, still work up to 10 hours after death. In cats and macaques, researchers have successfully made brains recover after a full hour cut off from blood by carefully restoring circulation. And in humans, some medical case studies point to a brain that can bounce back. In 2007, researchers reported that a woman suffering from acute hypothermia—with a body temperature less than 65 degrees Fahrenheit—made a full neurological recovery.
Working, but not aware
Sestan and his colleagues, led by Zvonimir Vrselja and Stefano Daniele, resolved to test a complex mammal brain's ability to recover, so they devised what they call the BrainEx system.
BrainEx consists of computer-controlled pumps and filters that send a nourishing solution through a dead, surgically exposed brain, with an ebb and flow that mimics the body's natural circulation. The proprietary solution is based on hemoglobin, the oxygen-ferrying protein in red blood cells, and is made to show up in ultrasound scans, so researchers can track its flow through the brain. Yale University has filed a patent for the system on behalf of its creators, but all of BrainEx's parts and procedures will be freely available to nonprofit and academic researchers.
The team took steps to ensure that the brains would not “wake up” in any way, let alone have awareness of the procedure's trauma. Though none of the brains in the experiment showed any sign of awareness, researchers stood at the ready to administer anesthesia and lower the brains' temperatures, just in case. What's more, the team added compounds in the solution to block neural activity, which served the extra goal of resting the brains' cells to give them better odds of healing.
“It was in fact never a goal—and even sort of the opposite of a goal—of the research to have consciousness restored,” says study coauthor Stephen Latham, director of the Yale Interdisciplinary Center for Bioethics.
First, the team checked to see whether BrainEx could restore circulation in the brain, even in its tiniest blood vessels. It does. Researchers also confirmed that the brain's blood vessels were in good enough shape that they could dilate in response to medications. Next, the researchers checked how well BrainEx preserved the overall structure of brain tissue. For the most part, BrainEx-treated brains looked comparable to brains in living animals or untreated brains an hour after death, and they were far more intact than untreated brains examined 10 hours after death.
Brain areas that are especially sensitive to oxygen loss, such as the hippocampus, also preserved well under BrainEx, as did the structures of individual neurons. And as they monitored the chemical differences in the solution flowing into and out of the brain, researchers found that the brain was making CO2 and using up glucose and oxygen—signs of brain-wide metabolism restarting.
Though researchers ensured that the experimental brains wouldn't have large-scale activity, they took small slices of brain tissue to test whether individual hippocampus neurons could still fire after treatment. They could.
“[That result was] the most surprising aspect to me as a working neuroscientist,” says Allen Institute for Brain Science director Christof Koch, who wasn't involved with the study. “They were still capable of generating the spikes that are the universal idiom of fast electrical communication. It means that in principle, those neurons seem capable of neural activity.”
Ethics of animal research
The BrainEx team is acutely aware of the ethical implications of its work, which is why they have consulted with leading neuroscientists and ethicists for years. The Neuroethics Working Group, a consortium convened by the U.S. National Institutes of Health's BRAIN Initiative, which funded the research, has been consulting with Sestan since 2016. The researchers also presented their work at a 2017 bioethics conference at Duke University and at a 2018 NIH workshop.
“Cutting-edge science needs cutting-edge ethics,” says Ramos, who serves as the Neuroethics Working Group's executive secretary. “There is an existing, robust framework of laws and policies that our funded researchers are expected to follow, but the development and application of new neuro-technologies may require us to examine those ethical standards, and for those standards to evolve.”
For one, the technique opens up questions about the ethical use of non-human animals in experiments. As it stands, two sets of rules apply, one for live animals and another for dead animal tissues, since live animals can experience pain or distress. But which rules apply to BrainEx-treated brains from dead animals, especially if there is a chance they could be partially reawakened?
“There's this kind of gaping hole in our protections of animal research subjects, [since] we now have this part-revived, slightly-alive category with the potential—and, as of yet, not fully understood potential—for recovery of function,” says Farahany, who is also a member of the Neuroethics Working Group. “If you're seeking to revive pig brains, or other animal brains, does that mean that that becomes an animal research subject, rather than dead tissue?”
Experts add that the ethical tradeoff here hinges on BrainEx's ability to further research into human disease—or even save people from brain death.
“We cannot willy-nilly impose, just for our curiosity, pain or agony on another creature unless there's a very good motive and the appropriate experiments,” Koch says. “Can this be used to rescue brains? Not just gee-whiz, let's see what happens here.”
Experts also say that BrainEx's ethical implications extend to the next logical question: Would it work on humans? On a technical level, Koch says that would not be a major leap, since both pigs and humans have large, complexly folded brains. But Koch and every other outside expert contacted by National Geographic urged caution in moving toward human trials.
On even broader horizons, future versions of BrainEx could complicate the process of organ donation by blurring the lines of brain death, note Case Western Reserve University bioethicists Stuart Youngner and Insoo Hyun in an accompanying commentary published in Nature. But Kevin Cmunt, CEO of Gift of Hope, one of the United States's largest organ donation networks, doesn't see BrainEx as a major disruption. He says that in many cases, organ donors who are declared brain-dead have suffered oxygen loss well beyond the study's time window, or substantial physical trauma. (Other researchers are creating human-pig chimeras to advance organ transplant options.)
“I think that in the vast majority of brain-dead donors, this intervention would not be material,” he says. “There may be a small subset of cases where [BrainEx] could impact the opportunity for donation, but I think it's relatively small.”
And if BrainEx does appear in clinics, Cmunt adds that it would be incorporated into the list of interventions before declaring someone brain-dead or deciding to end life support. The promise of brain recovery could even improve organ donations by giving medical professionals an even greater imperative to maintain circulation. Then, if the patient is declared brain-dead even after treatment with BrainEx, their organs could be more viable for donation than they would be otherwise.
“I don't necessarily see this as a conflict,” Cmunt says. “These treatments would certainly be a part of care, just like hypothermia protocols are a part of care, and other things that we try to do to stop damage to organs and brains.”
Only the beginning
At its most profound, the discussion around BrainEx shows how gains in knowledge and improvements in treatments have shifted the definition of death itself.
“Imagine you're standing in the clinic, your dad is declared brain-dead, and you've just read this paper. You ask the surgeon, Well, what does brain-dead mean? He says it's irreversible loss of brain function, and you say, Well, wait a minute, there was this paper—doesn’t that mean that 'irreversible' today may not be 'irreversible' tomorrow?” Koch says.
BrainEx's space on the border of life and death echoes science fiction—and at its most lurid, people may well think of Frankenstein and the prospect of resurrecting the dead. But Farahany cautions that we are still many miles away from that feat.
“It is definitely has a good science-fiction element to it, and it is restoring cellular function where we previously thought impossible. But to have Frankenstein, you need some degree of consciousness, some 'there' there,” she says. “They did not recover any form of consciousness in this study, and it is still unclear if we ever could. But we are one step closer to that possibility.”