The pigs came straight from the slaughterhouse, where workers had severed their jugular vein and carotid artery and then decapitated them. But starting four hours after the animals’ brains had flat-lined and for the next six hours, the brains became … well, a little less dead, scientists at Yale University reported on Wednesday.
By attaching the brains to a specially constructed device and running souped-up artificial blood through them, the researchers said they were able to restore some of the brains’ molecular and cellular functions, including spontaneous electrical activity in neurons and such signature metabolic functions as consuming oxygen and glucose.
Although the pig brains showed none of the organized, cortex-wide electrical activity associated with consciousness, sensory perception, pain, distress, or other higher-order functions, the experiment challenges long-held medical dogma and is likely to reignite an impassioned debate about what constitutes brain death, especially for the purpose of organ donation. Less controversially, the restoration system, called BrainEx, promises to give neuroscientists a better way to study brain wiring and function.
Scientists not involved in the study, who had heard the lead researcher present preliminary results at closed-door meetings in 2017 and 2018, described it in almost awestruck terms, calling it “incredible,” “very impressive,” and “extremely significant.”
The Yale team “showed that, at least at the cellular and molecular level, things are not as irreversible [after the brain is deprived of blood and oxygen] as we thought,” said neurologist Dr. James Bernat of Dartmouth College. “I think it’s remarkable: They were able to restore some brain activity hours after death and the cessation of [blood] circulation, which was previously thought to cause irreversible damage and loss of function.”
Like every scientist familiar with the study, Bernat quickly added that “there is a major difference between the restoration of cellular and molecular activity in a brain and the resuscitation of overall brain function, which they were not able to achieve.” Or as Yale neuroscientist Dr. Nenad Sestan, who led the study, told reporters on a conference call, “This is not a living brain. This is a cellularly active brain.”
Still, Andrea Beckel-Mitchener of the National Institute of Mental Health called the technology “a real breakthrough for brain research.” Restoring any cell function “has never been done before” in a large, supposedly dead, mammalian brain, she said.
By keeping cells alive and working, she added, the system promises to let scientists study complex circuit connections “and functions that are lost when specimens are preserved in other ways.” Neither isolated brain cells grown in culture nor slices of post-mortem brain tissue reveal much about the neural circuitry and activity that underlies thinking, for instance.
Further development of BrainEx might one day support “neuroresuscitation,” reversing the damage cause by strokes and other brain injuries. “If the system is found to be safe in a living person, there is the potential for improvements in the recovery of brain function,” Bernat said.
Beyond any eventual scientific or medical applications, however, the work raises profound philosophical questions. In an essay accompanying the paper, published in Nature, three bioethicists wrote that it “throws into question long-standing assumptions about what makes an animal — or a human — alive.”
For their experiment, Sestan and his colleagues obtained some 300 pig heads from an abattoir near Yale’s New Haven, Conn., campus, over nearly 10 years as they worked to optimize BrainEx — the “ex” stands for ex vivo, or “out of the living.” Eventually the team connected 32 pig brains to the system — a Rube Goldberg-esque blur of software, blenders, fluid holders, pumps, heaters, filters, and more, whose function was to mimic pulsating blood flow using a special artificial blood — via the carotid artery four hours after the animals had been killed. They kept the brains connected for six hours and at normal temperatures, not cryogenic ones.
Other brains were hooked up to BrainEx but did not get the perfusion solution, or were not hooked up at all. By comparison, the brains connected to BrainEx and perfused showed “dramatic differences,” Sestan said. Brain cells metabolized oxygen and glucose much like living cells. Neurons fired information-carrying electrical bursts called action potentials both spontaneously and in response to an electrical stimulus, as those in living brains do. Blood vessels dilated in response to drugs that cause that response in living brains. Glia, the brain’s immune cells, revved up a lifelike inflammatory response when provoked. Mitochondria, which supply energy to cells, were intact inside neurons, as was myelin, the fatty sheath that provides insulation to neurons so they can conduct electrical signals.
Overall, many more cells remained alive, the circulatory system worked, and the size, shape, and arrangement of neurons and other cells looked normal compared to brains that didn’t get the BrainEx treatment.
In a model of scientific understatement, the authors write that large mammalian brains have “an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.” In other words, in some cases a brain’s death may be neither permanent nor irreversible.
“We never imagined we would get to this point, … restoring cells to this level” of functionality, Sestan said. Neurological dogma has long held that brain cells die irreversibly and within minutes after blood stops circulating, as the pigs’ did. “But we were able to restore some cellular and molecular function” after four hours of oxygen loss, he said. “We were really surprised.”
Although he was emphatic that the absence of brain-wide electrical activity meant the pig brains had not regained awareness and therefore could not feel pain or distress, that was not a foregone conclusion. The scientists were concerned enough about that possibility that they were prepared to administer anesthesia to the brains, or drastically cool them, to shut off any such activity.
“That’s the elephant in the room: consciousness,” said philosopher Jonathan Moreno of the University of Pennsylvania, who reviewed the paper for Nature and heard Sestan describe his findings last year at a meeting at the National Institutes of Health, whose BRAIN Initiative partly funded the research. “There is a huge fight in neuroscience about what people mean by consciousness and what criteria should be used. Is this [electrical activity in the dead pig brains] a little piece of consciousness? A small degree of consciousness?”
Complicating the question of whether consciousness can be restored to a dead brain is that the BrainEx fluid included a chemical that suppresses neuronal activity. Without that, might the pig brains have shown the sort of brain-wide electrical activity associated with consciousness? “They might have,” Moreno said. “That has to make you ask, what would be the moral status” of a once-dead but (partly) “restored” brain?
Beyond raising such philosophical and ethical concerns, BrainEx could complicate the already-fraught debate over brain death and organ donation, experts said.
By challenging the assumption that large mammalian brains irretrievably lose neural activity and consciousness within minutes of when blood flow stops, the findings raise “the possibility that researchers could get better at salvaging a person’s brain even after the heart and lungs stop working,” bioethicists Stuart Youngner and Insoo Hyun of Case Western Reserve University wrote in a commentary on the study. If the results hold up, they “could exacerbate tensions between efforts to save the lives of individuals and attempts to obtain organs to donate to others. As the science of brain resuscitation progresses, some efforts to save or restore people’s brains might seem increasingly reasonable — and some decisions to forego such attempts in favor of procuring organs for transplantation might seem less so.”
The meaning of brain death is sufficiently controversial that ethicists, transplant surgeons, other physicians, and emergency responders wrestle with when to abandon efforts to save someone’s life and instead try to save their organs for donation. Most organs for transplant come from people who are declared brain dead. But if BrainEx is improved and shown to work on human brains, people who are declared brain dead could qualify for brain resuscitation, not organ donation.
Asked if the United Network for Organ Sharing, the private group that manages organ donations in the U.S., had any comment on the study, a spokeswoman said, “We do not. This is way removed from organ donation.”
Experts disagreed. The study “makes it even less clear that the brain is actually dead under certain circumstances,” said Dr. Kathleen Fenton, a bioethicist and pediatric heart surgeon in Maryland. “It should inform the discussion surrounding brain death, [since it] shows that some cellular life and function loss that we have all thought was irreversible appears to not be irreversible. That’s important.”
Or as Duke University ethicist Nita Farahany and two co-authors said in another commentary, a line from the 1987 film “The Princess Bride” notes that “there’s a big difference between mostly dead and all dead. Mostly dead is slightly alive.”