The reanimators: Giving dead pig brains new life

In a US lab, scientists are giving dead pig brains new life — a discovery that in time may challenge our understanding of what it means to die, writes Matthew Shaer.

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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 centred on the mammalian brain. More specific, it centred on the tree-shaped neurons that govern speech, motor function and thought — the cells, in short, that make us who we are.

In the 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 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 keep the cells alive for several weeks at a stretch.

When I met with Sestan this spring, at hisNew Haven lab, he took great careto 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 anentirely 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 90billion neurons and hundreds oftrillions 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, OK , 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 labouring in time they stole from other projects, managed to develop a customised fluid that could preserve centimetre-thick chunks of mouse, pig and human brain for long periods.

“Six days was our record,” Sestan recalled. But there was a hitch: The tissue stayed intact only when the samples were stored in a fridge.

Once they were brought to room temperature (any accurate modelling of neuronal function would have to occur at 37C), 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 slicingan entire brain into extremely thin leaves of tissue, the delicate interior architecture was decimated.

One afternoon, Sestan 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 enoughpost-mortem, it can prevent cellular decomposition.

“Everything just sort of gets put on pause,” Belanger told me recently.

Sestan stopped in front of thegravity feed, eyes wide. Maybe the solution didn’t lie in slices of brain, but in an entire brain, perfused the way Belanger was perfusing this one, with haemoglobin-rich fluid standing in for a preservative.

“It was my light-bulb moment,” he said. However, 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”.

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.

“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.

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.”

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.

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.

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 anaesthetic in his lab.

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, Bio MedInnovations, 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; a majority of the test subjects were pig organs obtained from a nearby slaughterhouse.

Sestan was intrigued, and when he travelled to the Washington area that February to present a paper on his gene-expression research, he arranged a side trip to Blacksburg to meet Robertson in person.

“I couldn’t get there fast enough,” Sestan told me.

Back in New Haven, Sestan showed pictures of the machine to his colleagues. Some questioned his sanity. Others, busy with their own projects, were wary of getting involved.

“And that was when I found Zvonimir,” Sestan recalled.

Zvonimir is Zvonimir Vrselja, afellow Croat who was 28 at the time.

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 withParkinson’s disease.

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 cauterised 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 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 whirr, 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 thatremoves any naturally occurring bubbles.

Over that spring, Vrselja and Daniele fixed brains from separate specimen sets and delivered them to Sestan.

“It was the mostastonishing 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.

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. 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 categorised 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 cerebralfunction in a living human.

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 been tirely inactive. Dead.” And yet as low as the score was, it wasn’t zero.

“I just thought, Yeah, OK, 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’.”

For Sestan, the thorniest issue centred 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 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.

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 afeverish nightmare, perpetually reliving the very moment of its slaughter.

“Imagine the ultimate sensory-deprivation tank,” a member of the NIH’s Neuroethics Working Group told me. “No inputs. No outputs. In your brain, nobody can hear you scream.”

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 over-interpretation”.

“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 totalbaloney. 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 yetcomplete. 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-optimised 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.

“There’s a lot of research left to be done,” sais Sestan.

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.

Greely and Nita Farahany of Duke, along with the young Duke scientist Charles M Giattino, recently published an 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.”

In the paper, Greely, Farahany and Giattino advocate the adoption of guidelines modelled 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 agrees. “Every one of these decisions shouldn’t be up to me alone,” ,” he told me.

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 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,” he said.

Adapted from an article in The New York Times Magazine.c.2019 The New York Times