In experiments on pig organs, scientists at Yale made a discovery that could someday challenge our understanding of what it means to die.
By Matthew Shaer, in nytimes.com, July 2, 2019
A few years ago, a scientist named Nenad Sestan began throwing around an idea for an experiment so obviously insane, so “wild” and “totally out there,” as he put it to me recently, that at first he told almost no one about it: not his wife or kids, not his bosses in Yale’s neuroscience department, not the dean of the university’s medical school.
Like everything Sestan studies, the idea centered on the mammalian brain. More specific, it centered on the tree-shaped neurons that govern speech, motor function and thought — the cells, in short, that make us who we are. In the course of his research, Sestan, an expert in developmental neurobiology, regularly ordered slices of animal and human brain tissue from various brain banks, which shipped the specimens to Yale in coolers full of ice. Sometimes the tissue arrived within three or four hours of the donor’s death. Sometimes it took more than a day. Still, Sestan and his team were able to culture, or grow, active cells from that tissue — tissue that was, for all practical purposes, entirely dead. In the right circumstances, they could actually keep the cells alive for several weeks at a stretch.
When I met with Sestan this spring, at his lab in New Haven, he took great care to stress that he was far from the only scientist to have noticed the phenomenon. “Lots of people knew this,” he said. “Lots and lots.” And yet he seems to have been one of the few to take these findings and push them forward: If you could restore activity to individual post-mortem brain cells, he reasoned to himself, what was to stop you from restoring activity to entire slices of post-mortem brain?
To do so would be to create an entirely novel medium for understanding brain function. “One of the things we studied in our lab was the connectome — a kind of wiring map of the brain,” Sestan told me. Research on the connectome, which comprises the brain’s 90 billion neurons and hundreds of trillions of synapses, is widely viewed among neuroscientists as integral to understanding — and potentially treating — a range of disorders, from autism to schizophrenia. And yet there are few reliable ways of tracing all those connections in the brains of large mammals. “I thought, O.K., let’s see if this” — slices of cellularly revived brain tissue — “is the way to go,” Sestan said.
In 2012, Sestan approached two members of his lab, Mihovil Pletikos and Daniel Franjic, and asked them to assist him on the project. Through the spring of 2014, the scientists, often laboring in time they stole from other projects, managed to develop a customized fluid that could preserve centimeter-thick chunks of mouse, pig and human brain for long periods. “Six days was our record,” Sestan recalled. “Six days, and the cells were still culturable.” But there was a hitch: The tissue stayed intact only when the samples were stored in a fridge. Once they were removed and brought to room temperature (any accurate modeling of neuronal function would have to occur at 98.6 degrees Fahrenheit), decomposition rapidly set in.
The primary issue appeared to be one of oxygenation. Mammalian brains are tangled knots of arteries and capillaries, each of which is instrumental in circulating blood (and with it, oxygen and nutrients) throughout the organ. In slicing an entire brain into extremely thin leaves of tissue, the delicate interior architecture was decimated. But Sestan is stubborn, several of his colleagues later told me — in the manner of a dog locking his jaws on a length of knotted rope, he has trouble letting things go. “I get an idea, and I want to finish it,” he admitted. “I have to finish it.” The experiment, he went on, “was constantly on my mind. Like, What is the solution here?”
One afternoon, he dropped by Yale’s pathology department to discuss an unrelated issue with a colleague, Art Belanger, the manager of the university’s morgue at the time. “I look over, and there’s this human brain in a sink, mounted upside-down,” Sestan recalled. As he watched, preservative from a nearby plastic bottle dripped through a few lines of tubing and into the organ’s arteries. The rig, a so-called gravity feed, was being used to “fix” the brain, Belanger explained — to preserve it for further study. Sestan nodded. In his lab, he frequently fixed organs, usually by freezing the specimens or immersing them in formaldehyde. “Trust me,” Belanger told Sestan. “Perfusion is much more effective.”
In contrast to immersion, perfusion leverages the existing vascular network — it mimics the flow of blood through the organ. The resulting fixation is more uniform and drastically faster than traditional methods. And if it’s done quickly enough post-mortem, it can prevent cellular decomposition. “You don’t see any breakdown of tissue; you don’t see any bacterial growth,” Belanger told me recently. “Everything just sort of gets put on pause.”
Sestan stopped in front of the gravity feed, eyes wide. Maybe, he thought, he had been thinking about the problem in the wrong way. Maybe the solution didn’t lie in slices of brain, but in an entirebrain, perfused the way Belanger was perfusing this one, with hemoglobin-rich fluid standing in for a preservative. “It was my light-bulb moment,” he said. (Belanger told me: “For 30 years, I’d waited to see a scientist go screaming down the hallway, screaming, ‘Eureka!’ That was the moment. Finally.”) But soon enough, Sestan’s euphoria was followed by a dawning awareness of where the experiment might take him. If the path to cellular restoration really did lie in the perfusion of a whole brain, his experiment would be entering entirely unexplored territory. “It’s kind of amazing, considering everything that came later, but that was the origin,” Sestan told me. “We didn’t want to restart life, you know?”
As long as scientists have understood the role of the mammalian brain, there have been efforts to reanimate it. “To conduct an energetic fluid to the general seat of all impressions,” the Italian physicist Giovanni Aldini wrote at the turn of the 19th century, “to continue, revive, and, if I may be allowed the expression, to command the vital powers — such are the objects of my research.”
In his 1803 book, Aldini describes decapitating an ox and connecting the head to a rudimentary battery; almost immediately, the head began to violently shiver, as if undergoing some kind of seizure. Later, he moved on to humans. “The left eye actually opened,” he wrote of the murderer George Forster, whose recently executed corpse was provided to him by the British government for experimentation. (When Aldini pressed a conducting rod to Forster’s rectum and ear, the muscle contractions “so much increased as almost to give an appearance of reanimation,” the scientist bragged.)
What Aldini failed to grasp, of course, is that life is not powered by electricity alone. It is powered by blood and oxygen, by gases and acids, by an impossibly intricate symphony of cells that die and regenerate and evolve and grow as we do. And it would be more than 150 years before technology had advanced to the point at which it was possible to observe, let alone duplicate, the most basic of those functions.
In the latter half of the 20th century, a new era of brain research was made possible by inventions like microelectrodes that allowed scientists to listen to neurons communicating and cutting-edge devices like functional magnetic resonance imaging scanners, which allow researchers to track blood flow and neuronal activity in the brain, and to learn how the brain responds to injury. Scientists eventually made great strides on the cellular level: In 1982, Takaaki Kirino, a Japanese researcher, published a groundbreaking paper documenting “delayed neuronal death” in Mongolian gerbils. As Kirino noted, many of the animals’ brain cells apparently remained intact long after blood flow had been cut off to the brain. Later, the same phenomenon was observed in post-mortem human cerebral tissue. And in 1991, scientists discovered that the neurons in the brains of lab rats euthanized up to three hours earlier still retained significant electrical activity. Collectively, the research proved that brain death wasn’t a single event. It happened in gradual steps. And precisely because it was gradual, scientists found that they could delay it or reverse parts of the process altogether — perhaps not as drastically as Giovanni Aldini envisioned, but no less emphatically.
Culturing cells from dead tissue was just a small part of it: Studies showed that the brain was far more resilient than had been understood. It could, for example, recover neuronal function after a half-hour of oxygen and blood deprivation — in other words, it could be taken offline and turned back on again. “What’s happened, I’d argue,” says Christof Koch, the president and chief scientist at the Allen Institute for Brain Science, “is that a lot of things about the brain that we once thought were irreversible have turned out not necessarily to be so.”
In recent years, some scientists have moved from the study of the organic tissue to the wholesale creation of artificial brain matter. Grown from human stem cells reprogrammed to act like neurons, brain organoids, or “mini brains,” can mimic some of the functions of their biological counterparts — last year, for example, the biologist Alysson Muotri announced that his lab at the University of California had grown brain organoids with neurons that fired at a level consistent with that of a preterm infant. Muotri has said he hopes to use the creations to research brain function and formulate disease models without buying lab animals or expensive specimens from brain banks. “The potential uses are vast,” he has said.
So, too, are the ethical quandaries. Writing in his forthcoming book on the biological origins of consciousness, “The Feeling of Life Itself: Why Consciousness Is Widespread but Can’t Be Computed,” Koch argues that the chance that an advanced organoid “experiences anything like what a person feels — distress, boredom or a cacophony of sensory impressions — is remote. But it will feel something.” Ideally, Koch adds, “it would be best if this tissue were anesthetized.”
To Sestan and others, there is a mandate to keep pushing, not least because of what it might mean to the world at large: more diseases combated, more treatments developed, more lives saved and, above all, a fuller glimpse of a dauntingly complex organ. The brain remains “the most mysterious” of all the organs, as Sestan put it to me. “The least — what is the right word? Let’s see — well understood.” He went on: “If you’re nuts enough to make the brain the thing you study, you must accept that there will always be more questions than answers. You’ll always be searching. Always.”
For the past half-decade, Sestan has worked out of the same small office in Sterling Hall, in the heart of Yale’s medical campus. The room has one arrow-slit window, which is almost always shuttered, and a wraparound desk buried under a minor Everest of unread journals. Opposite are his sole concessions to décor: a blue Ikea couch and a pillow for lumbar support, necessities for the evenings he opts to write through the night rather than return to the Madison home he shares with his family. “I’m a naturally restless person,” Sestan told me. “High levels of anxiety, high levels of nervousness. But having that quiet, that peace, it centers me. Focuses me.”
Until this year, Sestan was best known as the senior author of the first full genetic survey of the developing human brain; the paper, published in Nature, earned him a raft of awards, including the prestigious Constance Lieber Prize for Innovation in Developmental Neuroscience, which is given out every two years to a pioneering neuroscientist. “I have rarely seen a scientist be able to identify what the field needs better than Nenad, or to address that void with creative ideas,” one of his colleagues told me. “He takes these seemingly disparate observations and synthesizes them into something completely novel. That, to me, is what constitutes a great thinker.”
Stubbornly, Sestan does not hold himself like a great thinker. He likes fart jokes. He says “Holy guacamole!” more than you’d expect. He drives a rusty Subaru, which he parks on the streets surrounding the lab, declining to pay what he views as the “extortionate” fee for the med-school lots. The first time I met him, he held the Subaru’s key fob up to his head and explained that the fat in his brain, acting as a conductor, would allow him to lock the car from more than a block away. He depressed his thumb; sure enough, the Subaru gave an obliging beep. “I learned about that on YouTube,” he told me. “YouTube! It’s like, Holy guacamole!”
A native of Zemunik Donji, a village not far from the Dalmatian coast of Croatia, Sestan had what he calls “the most perfect upbringing you could imagine.” His father was a sergeant in the Yugoslavian Navy, and his mother was a part-time postal worker; he spent much of his childhood playing in the farmland that surrounds Zemunik. When he was 11, his mother bought him a subscription to a medical encyclopedia series. He was entranced. School had been hard for him: He suffered from what he suspects now were dyslexia and attention-deficit disorder. “But I liked systematic things,” he said. “My brain worked fine that way: From A to B to C.”
Paging through the books, he came across an encyclopedia article by an anatomist named Ivica Kostovic, who would later serve as his mentor in medical school. “Kostovic had done all these dissections, all this work on the human brain, and he wrote that there was no subject more fascinating,” Sestan told me. “I said, ‘I’m going to study the human brain, and I’m going to do it with this man.’ ”
It took him a while to get there. By his own admission, he partied maybe more than he should have; he discovered Iron Maiden, grew his hair long and founded his own band, called Twilight Zone. Before his senior year of high school, as he was planning to eventually leave for college in Zagreb, he learned that his girlfriend, soon to be a sophomore, was pregnant. “I wanted to marry her, but her father goes: ‘She’s too young. Let’s wait,’ ” Sestan said. They dated long-distance for two years; when she graduated, she and their son briefly joined Sestan in the capital, where Sestan set about establishing himself as a neuroscientist. He was a co-author of two papers that were among the first to locate, in the developing human brain, the enzyme that makes nitric oxide, which functions as a transmitter between neurons.The pulse generator, part of the BrainEx that simulates the heart’s rhythm and pulse when pumping the fluid into the brain.CreditThomas Prior for The New York Times
Then came the war years. In the early 1990s, Sestan recalls that his hometown was surrounded by Croatian Serbs and troops from the Yugoslav People’s Army; in the fighting, his childhood home was decimated. “My family, my girlfriend, my son, they fled to Slovenia, became refugees,” Sestan told me. But Zagreb was largely spared, and Sestan was able to stay in the capital to continue his studies. (His family’s home in Zemunik has since been rebuilt.)
In the winter of 1994, Sestan, still several months from earning his medical degree, convened a meeting to discuss his enzyme research. His mentor, Kostovic, who had become the deputy prime minister of the republic, was in attendance. “The conversation was basically: ‘O.K., how do we prove this is correct? We need to conduct molecular tests, and we can’t do it here. We don’t have the equipment we need,’ ” Sestan recalled. But Yale, which offered several fellowships to promising foreign neuroscientists, did. “My colleague, the guy I’d written the paper with, said: ‘I have older kids, and a family. Nenad should go.’ ”
At Yale, Sestan joined a lab run by the renowned neuroscientist Pasko Rakic, then the head of the university’s neuroscience program. “I’m in my 80s now, and I’ve worked with a lot of students,” Rakic told me. “Some of those students, they’re content to sit in place, to do the same thing as everyone else. Nenad was not like that. He always wanted the new thing. The next thing.” Rakic is famous in scientific circles for his research on the cerebral cortex, the center of information processing in the brain. Under his tutelage, Sestan published widely on gene expression and cell development in the cerebral cortex, attracting the notice of the department’s hiring committee. “I think people saw what I saw: how much Nenad was capable of,” Rakic told me. By the summer of 2002, Sestan had been made an assistant professor and given a lab of his own in Sterling Hall, along with a half dozen researchers and postgraduates. He was 32.
The demonstration in the Yale morgue inspired Sestan, and with the help of his team, he set about obtaining all the relevant literature on perfusion, including a 1964 study involving dog brains that had been perfused with whole blood. It wasn’t an apples-to-apples comparison: The animals in those experiments had never been truly dead, and the brains hadn’t been removed from the bodies. But it was something. “If you look back at my notes from that period, you can see a lot of extrapolation,” Sestan told me. “There was no exact precedent, but there was stuff that seemed close. And it kept me going.”
As modern medical technologies go, perfusion is a relatively old one: The first perfusion pump, invented in the 1930s by the Nobel Prize-winning scientist and Nazi sympathizer Alexis Carrel and his close friend, the aviator Charles Lindbergh, was used to maintain blood circulation in cat thyroids during a series of transplant operations. Successive generations of engineers have refined and automated Carrel and Lindbergh’s “artificial heart” — if you’ve had open-heart surgery in the past quarter century, your doctors probably had a perfusion system on hand to keep the blood flowing through your brain.
This type of perfusion, performed on a living organ still housed in the host body, is known as “in vivo.” With current technology, it is relatively easy to achieve. “Ex vivo” perfusion, however, is considered by scientists to be far more challenging, while significant attempts to restore metabolic function through a post-mortem ex vivo perfusion of a whole brain are so rare as to be essentially unheard-of. (The most famous attempt was made by the Soviet scientist Sergei Brukhonenko, who used a circulation machine to “revive” a decapitated dog, as documented in the 1940 film “Experiments in the Revival of Organisms,” though many suspect that the footage was doctored.) “You say to a scientist that you want to do this, they’ll think you developed psychosis,” Sestan told me.
Sestan was determined to think like a scientist, not a philosopher. The existential questions interested him far less than the practical ones. “Our goal, our intention, was to do basic biology,” he told me. “And we had to be focused on what we were doing, because it was so important that everything was done correctly, that all the data was solid. When you let your imagination go berserk, when your mind wanders, you make mistakes, and one thing that I knew was that this was likely going to be the hardest thing, technically speaking, I’d ever done, and we couldn’t afford any mistakes.”
Still, as Sestan acknowledged to me, the project was an outlier for him. He felt compelled to put certain safeguards in place: He added “blockers” to the perfusate, to prevent the rise of electrical activity should the experiment succeed in restoring the neurons to do anything resembling consciousness; later, for the same reason, he began keeping a syringe full of a powerful anesthetic in his lab.Vrselja sketching out the basic design of the BrainEx.CreditThomas Prior for The New York Times
The technical hurdles were immense: To perfuse a post-mortem brain, you would have to somehow run fluid through a maze of tiny capillaries that start to clot minutes after death. Everything, from the composition of the blood substitute to the speed of the fluid flow, would have to be calibrated perfectly. In 2015, Sestan struck up an email correspondence with John L. Robertson, a veterinarian and research professor in the department of biomedical engineering at Virginia Tech. For years, Robertson had been collaborating with a North Carolina company, BioMedInnovations, or BMI, on a system known as a CaVESWave — a perfusion machine capable of keeping kidneys, hearts and livers alive outside the body for long stretches. Eventually, Robertson and BMI hoped, the machine would replace cold storage as a way to preserve organs designated for transplants.
For now, one of the few available machines — the third generation of the CaVESWave system — was in Robertson’s lab in Blacksburg, Va.; a majority of the test subjects were pig organs obtained from a nearby slaughterhouse. Sestan was intrigued, and when he traveled to the Washington area that February to present a paper on his gene-expression research, he arranged a side trip to Blacksburg to meet with Robertson in person. “I couldn’t get there fast enough,” Sestan told me. On Interstate 81, near Roanoke, he was pulled over by a state trooper. “I said: ‘I’ll be honest with you, sir. All my life, I’ve hated driving behind big trucks. They scare me. It’s my paranoia.’ ” The trooper thanked him for his honesty and wrote him a ticket anyway.
Back in New Haven, Sestan showed pictures of the machine to his colleagues. Some questioned his sanity. (“This isn’t a joke, man!” Sestan remembers one telling him.) Others, busy with their own projects, were wary of getting involved. “And that was when I found Zvonimir,” Sestan recalled. Zvonimir is Zvonimir Vrselja, a fellow Croat who was 28 at the time. Angular and bright-eyed, Vrselja specialized in radiology; he has published on the vasculature of the brain and cerebral pulsatility — the way that blood moves through the cortex. In late 2015, a colleague of Vrselja’s in Croatia reached out to Sestan and suggested that the two scientists talk. Zvonimir’s “skill set was exactly what I wanted,” Sestan told me. “Exactly.”
A few months later, Vrselja moved to New Haven; together, he and Sestan reached out to a third scientist: Stefano Daniele, a 25-year-old who had spent years researching brain degeneration in patients with Parkinson’s disease. Daniele was skeptical. In joining what was then still a top-secret project, he would have to rearrange his plans for his dual M.D.-Ph.D. degree. “Nenad took me aside,” Daniele told me, “and said: ‘I have never done anything like this; we have no data. But if it works, it’s going to change neuroscience.’ ”
In the spring of 2015, Sestan made the first of several payments on a BMI machine, which Robertson and the company estimated would take half a year to fabricate. In the meantime, Daniele and Vrselja could test out a version of the system housed at Virginia Tech. (It was decided that Sestan, with his academic commitments, would remain in New Haven.)
All three scientists were adamant that they had never once considered carrying out any tests on human specimens. The regulatory bar was too high, and as Sestan put it to me, when it comes to human tissue, post-mortem or not, “there has to be extreme justification, from an ethical standpoint. Which is exactly how it should be.” To a slightly lesser degree, the same is true of other large mammals. But dead animals are a different matter. And BMI had its relationship with the slaughterhouse near Virginia Tech. It wouldn’t be a problem, Sestan figured, to arrange for the purchase of pig brain tissue — porcine brains, after all, are usually discarded after the animal’s death.
“We knew that with a project like this one, it was going to be trial and error,” Vrselja told me. “Lots of trial and error. And to kill that number of animals, it just seemed absurd.” But if the slaughterhouse would sell them the equivalent of refuse, there would be no ethical quandary at all. “I remember we went to the slaughterhouse manager, and he shrugged. He was like, ‘Are you going to pay me?’ ” When they said they would, the manager replied, “Great, you can work out of my office.”
Almost immediately after arriving in Virginia, Vrselja and Daniele ran into a very big problem. “To perfuse something, you have to know how it works,” Vrselja recalled. “You go to an anatomy class, and that’s what you learn — how an organ functions. But there is not a lot of good literature on pig vasculature. And definitely not about how the blood circulates in a pig’s brain. We had to figure that out from scratch.”
‘We needed to figure out millions of years of evolution in a very short window of time.’
Every morning for several weeks, the scientists woke up around 4:30 to be at the slaughterhouse as the first pigs were led to the killing floor. While they waited, the animals were stunned, killed, eviscerated and stripped of usable meat; later, Daniele and Vrselja would run carrying a bloody pig head in a bag to the manager’s office, where they would use a pump to empty the excess blood from it. Finally, placing the skull on ice, they would drive it back with them to the lab in Blacksburg.
It was difficult not to get discouraged. The architecture of the brains was only half of it: The scientists also had to learn to remove the skull in a way that preserved the organ’s vital architecture, like the arteries. And initially they were working without neurosurgical tools. “We had an oscillating saw from Home Depot,” Daniele said. “It was like sawing into the unknown, because you had to go millimeter by millimeter, and the whole key was to go as close as possible to the brain but not pierce into it, because you actually didn’t know where the floor was, where the brain was.”
With every two steps forward, they seemed to be taking another one back. By running food coloring through the arteries of the brain, Vrselja and Daniele could see how blood traveled through the organ, but the arteries split and joined at such irregular intervals that it took days to figure out how each one influenced the circulation of blood. “Every time we thought that we had it down,” Daniele told me, “a weird branch would come up and steal the circulation from the brain, and then it would leak out that way.”
By the 20th brain, they had a sense of which arteries connected to which; by the 40th, they had worked out what vessels needed to be closed off — and what sections of the skull needed to remain attached. “I remember feeling like shit, physically, because we were up at 4 every morning, going to bed at midnight and doing the same thing again,” Vrselja told me. “But eventually, there was progress.”
Sestan and his team would end up modifying nearly every aspect of BMI’s machine. Still, both the original and the current iteration, which Yale is seeking a patent for using the name BrainEx, work in fundamentally the same way. First, the brain is mostly freed from the skull; all the dangling arteries, save the carotids, are cauterized or sutured. Next, the organ is flushed of residual blood. At the same time, an amount of perfusate equivalent to a bottle of wine is brought to body temperature in the machine’s reservoir and oxygenated — as with real blood, oxygenation turns the perfusate a darker, scarlet red.
Once the fluid — the present form of which includes antibiotics and nine different types of cytoprotective agents — is ready, the brain is lowered into a plastic case the scientists have nicknamed “the football” and connected via the carotids. A small thermal unit (a miniature air-conditioner and heater) sits under the football, controlling the temperature of the organ; the pressure and speed of the perfusate, meanwhile, are governed by a type of pump. With a dull whir, the fluid begins to circulate across the arteries, capillaries and veins of the brain in a loop, exiting on each circuit through a dialysis unit that “cleans” any waste products and through a filter that removes any naturally occurring bubbles.
Perhaps the most innovative modification involved fluid mechanics, one of Vrselja’s specialties in graduate school. As the British mathematician John Womersley managed to quantify more than half a century ago, blood does not circulate through our arteries at a uniform rhythm — it circulates in pulses, in concert with the shudder of our hearts. To account for that dynamic, the BMI unit had shipped with an automated “pulse generator,” a device that replicates the heartbeat’s pulsatility in the organs.
But the pulse generator’s settings proved unsuitable for brains, which have a different flow pattern than the rest of the body. Before Sestan’s team adjusted the settings, the fluid might not completely permeate the vasculature of the organ, leaving parts of the brain essentially untreated. In such tissue, Daniele told me, “you’d end up with this sludgy, white yogurt-ish substance. It was a mess.” Conversely, if the pressure was too high, “the brain could just physically not stand it.” The organ fell apart.
By that summer, Vrselja and Daniele had fine-tuned the pulse generator and attached a number of custom sensors, which ran on software designed by Vrselja; the technology allowed them to experiment more easily, and widely, with different settings. “A good way of putting it,” Vrselja told me, “is we needed to figure out millions of years of evolution in a very short window of time.”
As the weeks went on, Vrselja and Daniele discovered something encouraging: The interior brain tissue had a moist gray hue, as a living organ would — a sign that some cellular function had been restored. But to know for sure, they would have to perform the requisite lab work.
Over the course of that spring, they fixed brains from separate specimen sets and delivered them to Sestan. “It was the most astonishing thing, ” Sestan recalls. Active brain cells can have a variety of shapes, depending on the type and function. But dead or dying or inactive brain cells tend to look alike, as if a bomb has been set off somewhere in the nucleus and the entire structure has imploded from within. In the face of almost everything that was known about the brain — in the face of centuries of scientific research — the cells from the experimental group were metabolically active. Sestan, hunched over the microscope, could hardly believe what he was seeing. “Oh, my God,” he remembers whispering to himself.
Soon, the scientists had ratcheted up the length of the perfusions, from one hour to two or three, and Sestan found himself staring down a fresh and unusual dilemma. In and of itself, he knew, cellular function is not indicative of life, just as Giovanni Aldini’s galvanic experimentation did not amount to the resurrection of an animal’s mind. And yet by all accounts, the longer Vrselja and Daniele perfused the pig brains, and the better they got at the process, the more brain cells were restored.
In 2016, Sestan employed a machine known as a BIS, or a bispectral index monitor, which is used in hospitals to measure how deeply a patient is “under” during surgery. BIS results are categorized on a scale from zero to 100: Zero is the absence of electrical activity — a chunk of wood would score a zero on the bispectral index — while 90 to 100 is consistent with full cerebral function in a living human. (A person between 40 and 60, target numbers for general anesthesia, will be unresponsive to most stimuli.)
That summer, Sestan was preparing a grant application when Vrselja and Daniele summoned him to the perfusion room. The BIS readout had just hit 10 — at the low end of what is called burst suppression, a stuttering pattern often observed in human patients in a deep coma. “That level, it’s not associated with any kind of cognition,” Daniele told me. “The brain is considered to be entirely inactive. Dead.”
And yet as low as the score was, it wasn’t zero. “I just thought, Yeah, O.K., forget it,” Sestan recalled. “I’m not taking any chances. I said: ‘Unplug the machine. Stop the experiment until we can figure out what’s happening.’ ” That same day, he wrote two emails. The first was to a contact at the National Institutes of Health. The second was to Stephen Latham, the director of the Yale Interdisciplinary Center for Bioethics.
Latham is tall and ruddy, with neatly parted gray hair and a big, gaptoothed smile. Trained as an attorney, he speaks precisely, often in complete paragraphs, as if he is weighing each word before it leaves his mouth. “I remember that I shared Nenad’s reaction, which was, ‘No, we can’t have this happening,’ ” he told me recently. “If there is even a possibility of consciousness, yeah, you have got to stop the experiment.”In preparation for microscopic examination, the fixed brains are sliced and dyed and mounted onto slides.CreditThomas Prior for The New York Times
Legally, Latham knew, Sestan and his team weren’t in jeopardy. “The way our existing laws on animal research work — and I’ll stipulate that these are laws that many animal ethics people take issue with — you can kill an animal,” Latham told me. “You can give an animal a disease like cancer, you can let it die and you can dissect that animal to see what happened.” Sestan hadn’t killed any animals; he had merely recycled flesh that would have otherwise been disposed of. “Nenad wasn’t even at the boundary of what isn’t permissible,” Latham said.
And yet in another way, Sestan had long since passed any known boundaries. Cellularly revived dead tissue is “an in-between category,” said Nita Farahany, a law scholar and ethicist at Duke University. “It’s a total gray zone.” There were no rules in place to follow, and no rules to break.
For Sestan, the thorniest issue centered on consciousness and whether the Yale team, inadvertently, might somehow have figured out a way to elicit it from dead flesh. In 2019, brain death — and thus complete loss of consciousness — has become something of a moving target: Research has shown that patients we once thought were in deep comas as a result of a traumatic brain injury are actually able to communicate. As Christof Koch, the neuroscientist, writes, all neurologists agree that electricity in the brain is a prerequisite for thought. But new technologies, including a machine nicknamed the “zip-and-zap,” which uses both EEG monitoring and transcranial magnetic stimulation, have been used to detect brain activity in patients assumed to be in a vegetative state. These machines, Koch writes, challenge “clinicians to devise more sophisticated physiological and behavioral measures to detect the faint telltale signs of a mind.”
As Sestan knew, the chances of real consciousness arising from the perfused brains were slim, thanks to the channel blockers. But there was a worst-case scenario: A partly revived post-mortem brain, trapped in a feverish nightmare, perpetually reliving the very moment of its slaughter. “Imagine the ultimate sensory-deprivation tank,” a member of the N.I.H.’s Neuroethics Working Group told me. “No inputs. No outputs. In your brain, nobody can hear you scream.”
In August, with the experiment now paused, Sestan and his team went to Washington to meet with the members of the N.I.H.’s working group. “It’s fair to say that we were shocked,” the member told me this spring. “Astounded, I suppose.”
The board suggested that Sestan trade in the BIS monitor, which is not made for nonhuman subjects, for a more sensitive electrical monitoring system. And a colleague gave him a name: Rafeed Alkawadri, an expert in invasive intracranial EEG evaluations — a form of electrocorticography, or ECoG, in which the electrodes are connected directly to the brain’s surface rather than the exterior of the head. “Electrocorticography was the way to go, but with ECoG, generally speaking, you’ve got a scalp,” Sestan recalled. “With our experiment, we had no scalp. We said, ‘Let’s get this equipment into the lab.’ ”
A few days later, Alkawadri completed a round of tests on a perfused porcine brain. He saw no “spontaneous global activity” present in the organ or communication between the various parts of the brain; the BIS reading of 10, Alkawadri theorized, was a result of electrical interference produced by the machine.
Still, the incident was not exactly a false alarm. Sestan considered it more like a warning. Remove the channel blockers, he told me, “and you might get a signal” — a real one. I wondered if he had thought about trying it. “No,” he answered quickly. “Not now. And just speaking for myself here, maybe not ever.”
By early 2017, Daniele and Vrselja were again expanding the length of the perfusions, past three hours and then to four. But the brains in the control group couldn’t survive cellularly past 240 minutes, at which point decomposition set in, making comparison to the experimental group impossible beyond that.
After the N.I.H. meeting, Sestan was invited to Duke University to speak with the members of the school’s bioethics faculty and others. “People were aghast,” one person familiar with the meeting told me, “because everyone had this image of a pig’s head on a lab cart, attached to a bunch of hoses and tube, and the pig’s head coming back to life. There was a lot of concern,” the person went on, “that if this was to be made public in the wrong way, it could really be a setback for brain research. Like, decades of setback. It was so easily caricaturized.”
That summer, after a source told me about the Duke meeting, I reached out to Sestan. In a phone call, he called the experiment “the most important thing I’ve ever done, and the most important thing I will ever do,” and mentioned he was preparing to submit a paper to Nature. Once it had been accepted, he went on, he would get back in touch with me; until then, he wasn’t able to comment on the record.
In March 2018, Sestan met again with the N.I.H. Under the impression that everything he said would be kept confidential, he had put together a presentation on his experiment, and while the dozen or so attendees looked on, he clicked through a series of slides showing restored cells from the perfused brains. According to later reports, Sestan, referring to the most recent ECoG data, stressed that he was confident that the brains in his experiment were “not aware of anything.” Still, he went on, he could not speak to what other scientists might do with the research. “Hypothetically, somebody takes this technology, makes it better and restores someone’s [brain] activity,” he said. “That is restoring a human being. If that person has memory, I would be freaking out completely.”
With each meeting, the number of people aware of the project was growing, and Sestan, despite what he described to me as “begging and pleading,” was unable to prevent the publication last spring of an article in the MIT Technology Review, which was apparently based on video of Sestan’s 2018 N.I.H. presentation. Published with a still of a scene from the Steve Martin comedy “The Man With Two Brains,” the article framed Sestan’s work as “a step that could change the definition of death” — a “feat” that “inaugurates a bizarre new possibility in life extension.”
Within hours, the news had been picked up by media outlets around the world. “Scientists keeping pig brains ‘alive’ inside their SEVERED heads in Frankenstein-style research,” read the headline in the British tabloid The Mirror. The conspiracy theorist Alex Jones brought up the experiment on his radio show.
The email flooded in to Sestan’s office. “In case a study comes up and I find myself dying at that time, I volunteer for the brain study,” one read. “That’s right, I give you permission to, upon my untimely death, extract my brain and keep it ‘alive’ as long as you can outside the context of my body.” Another writer chided Sestan for taking measures to prevent the emergence of consciousness. “Progress cannot and should not be held back. … I suggest you seek private research funding from Silicon Valley, there is many a great powers and influential men who would fund this line of research and see it through to its full potential.” Finally, and most tragic, there were the relatives of patients who suffered brain trauma. What Sestan’s project proved, a mother in New England wrote hopefully, was that “there is no way to know when someone is truly dead.” Sestan told me: “You want to respond to each email, you want to try to explain the science, but you can’t. There are just too many.”
This spring, I flew to New Haven to tour Sestan’s lab. In a show of ceremony, he saved a viewing of the BrainEx for last. “You,” he said proudly, throwing open the door to a converted supply closet, “are the first member of the public to see it.” Roughly eight feet wide and mounted on the shelves of a long metal hospital-style cart, the BrainEx was less a single machine than a bristling collection of individual machines, each connected to the next, in a simulacrum of the human body. Here, the pulse generator — the equivalent of a heart. Here, the filters — mechanized kidneys. There, the device that, like lungs, helped oxygenate the perfusate. “We’ll do our dance,” Daniele said, and he commenced a dry run — sans brain — of the process, miming each step.
“Don’t forget the Kanye,” Daniele joked.
“Our soundtrack,” Vrselja said with a grin.
By any measure, the contents of the paper Sestan and his team published in Nature this April were astonishing: Not only were Sestan and his team eventually able to maintain perfusion for six hours in the organs, but they managed to restore full metabolic function in most of the brain — the cells in the dead pig brains took oxygen and glucose and converted them into metabolites like carbon dioxide that are essential to life. “These findings,” the scientists write, “show that, with the appropriate interventions, the large mammalian brain retains an underappreciated capacity for normothermic restoration of microcirculation and certain molecular and cellular functions multiple hours after circulatory arrest.”
When we spoke in June, shortly before this article went to press, Sestan told me he frequently marveled at the direction the experiment had ultimately taken. “You know, I started out hoping to be able to trace connections,” he said. “But in the last three years, what happened was that the project really became more about death than anything having to do with the connectome.”
“The whole project is testament to the fact that the simplest observations can lead to the most exciting findings,” said Stephen L. Hauser, director of the Weill Institute for Neurosciences at the University of California, San Francisco. “It’s the type of finding that after it is made, it might seem obvious. One reaction could be, ‘Hey, why didn’t we think of that?’ But it took creativity, it took doggedness to get to that point.”
In our conversations, Sestan was always happy to expound on the science behind his experiment but cagier when it came to the implications. In the field of modern neurology, Hauser told me, “you’re constantly trying to dampen down overinterpretation. Often, that’s easy, because what’s being written about it is an incremental change over what’s already been established, or it’s just total baloney. Here, though, with this paper, we have something different: These are truly superb scientists. I think there’s a lot more that we still have to learn; the story’s not yet complete. But this is interesting, real science.”
Sestan did acknowledge that, yes, theoretically there is nothing stopping a scientist from immediately building a perfusion machine that could support a human brain. The BrainEx technology is open-source, and pig and homo sapiens brains have a fair amount in common. And there are plenty of conceivable applications for a human-optimized BrainEx. In addition to being an ideal model for testing out drugs, a portable perfusion system might be used on the battlefield, to protect the brain of a soldier whose body has been grievously injured; it might, in some distant future, become standard equipment for first responders. “The thing is,” Sestan said, “there’s a lot of research left to be done.” His focus now is better understanding how brain cells can be saved after major heart events. He sees no path to human tests. “If you could be absolutely certain you could do this on a post-mortem human brain and not get electrical activity of any kind, then maybe, maybe, we talk more,” he went on. “At the moment, I can’t see justification.”
And yet, as the ethicist and Stanford University law professor Hank Greely argued to me recently, we live in a time of breakneck scientific advancements; in 2019, “what ifs” advance more rapidly to the experimentation stage than ever before. Consider, Greely suggested, the case of the Italian neurosurgeon Sergio Canavero and his associate, the Chinese scientist Xiaoping Ren, who claim to have transplanted a head from one cadaver to another. Undoubtedly, a scientist with fewer scruples than Sestan, fewer moral qualms about human experimentation, will emerge. “Somebody will perfuse a dead human brain, and I think it will be in an unconventional setting, not necessarily in a pure research manner,” Greely told me. “It will be somebody with a lot of money, and he’ll find a scientist willing to do it.”
Greely and Nita Farahany of Duke, along with the young Duke scientist Charles M. Giattino, recently published a long essay in Nature on Sestan’s findings. (Their 2,000-word essay is one of two to appear alongside the paper.) “In our view, new guidelines are needed for studies involving the preservation or restoration of whole brains,” they wrote, “because animals used for such research could end up in a gray area — not alive, but not completely dead.” They noted, “We’re reminded of a line from the 1987 film ‘The Princess Bride’: ‘There’s a big difference between mostly dead and all dead. Mostly dead is slightly alive.’ ”
In the paper, Greely, Farahany and Giattino advocate the adoption of guidelines modeled on those established in 2005 on stem-cell usage. “Looking back, those guidelines really helped shape the field,” Greely told me. “Here, we have nothing. We have serious gaps in the regulatory system. We need to be proactive.”
Sestan, for his part, agrees. “Every one of these decisions,” he told me before I left New Haven, “shouldn’t be up to me alone.” In solving countless technical problems, he knew, he had created an entirely new set of implications for the rest of us to wrestle with. “I shouldn’t decide,” he went on, “what we do or what we shouldn’t do. That’s up to you; it’s up to all of us. We make the decision together.”
Matthew Shaer is a writer at large for the magazine. In the fall, he will be an Emerson fellow at New America.A version of this article appears in print on July 7, 2019, Page 29 of the Sunday Magazine with the headline: The Reanimators. Order Reprints | Today’s Paper | Subscribe