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In a stunning scientific breakthrough, Yale School of Medicine researchers restored brain activity in pigs that had died hours before. The finding could revolutionize the neuroscience field and how scientists conceive the boundaries between life and death.

By circulating a cocktail of cell-rejuvenating compounds throughout the pigs’ brains, the researchers prevented tissue decomposition and restored some cell function. If replicated, their technique could be used as a model for drug testing and has implications for how scientists understand brain plasticity after traumatic events such as strokes.

But the researchers stressed that they did not restore consciousness to the brains. Although the group observed activity in individual cells, the brains lacked widespread, coordinated activity — a requirement for consciousness.

“We wanted to investigate what happens in the brain following global injury … and whether we could restore certain cellular and molecular functions in the whole, intact brain four hours after death,” co-first author Stefano Daniele GRD ’21 told the News. “We were successfully able to do so.”

Daniele and co-first author Zvonimir Vrselja, research scientist in neuroscience, headed the study under the supervision of School of Medicine neuroscience professor Nenad Sestan.

It took Vrselja and Daniele several years of trial-and-error to refine their research protocol. In the end, each successful trial required a 10-hour sprint.

“We have an entire dance that we do,” Daniele said.

Typically, scientists will purchase test animals from speciality research companies. But Vrselja and Daniele took a different approach: Each iteration of the experiment began at a local food processing plant where the duo would wait for new specimen to become available.

“We did not want to kill any animals for this research,” Daniele explained. “ So we just simply made use of tissue that was going to be discarded.”

After securing the tissue, the two rushed back to the School of Medicine and removed the pig’s brain from its skull within four hours.

Vreslja and Daniele designed a system to pump and circulate their solution throughout the brain. For six hours, the contraption acted like an artificial circulatory system — pushing solution around the brain while simultaneously removing waste from the tissue.

“A lot of these components are readily available and no one really thought to put them together in a particular manner that could result in this type of study,” Daniele said.

Miraculously, following the treatment, the team was able to repeatedly show that cells in the brain could respond to stimuli. Drug treatment expanded the brain’s blood vessels. And individual neurons gave bursts of electrical activity.

By all accounts, a brain deprived of oxygen and other nutrients for four hours is considered dead. But the activity described in the study shows that brain cells are more robust and are able to survive longer than previously thought.

Still, for now, clinical definitions of brain death are intact.

“I don’t see anything in this report that should undermine confidence in brain death as a criterion of death,” Winston Chiong, a neurologist at the University of California at San Francisco told The Atlantic.

In 2016, when the team grew concerned that their experiment would cause neurons across the brain to fire together — the marker for consciousness — they recruited Yale Interdisciplinary Center for Bioethics Director Stephen Latham, who became a co-author of the study.

In all studies involving human or animal subjects, federal law mandates that scientists must consult their institution’s ethics committees before starting research.Though the present study passed the bar, future studies may enter an ethical gray area.

“A disembodied brain that’s obtained from a pig that’s already dead is not an animal and it’s not a human subject,” Latham explained. “Nobody has jurisdiction to ask questions like ‘is this brain suffering? Can it suffer if it’s sitting alone in a vat unconnected to a body? Even if we see EEG [signal], does that indicate consciousness? Does it indicate pained consciousness in any way?’”

Following the landmark study’s publication, Sestan, the senior author, is calling for independent groups to replicate the findings.

Others in the medical community agree. Yale New Haven Hospital Chief of Neurology David Hafler, who was not involved in the study, told the News that the work needs replicating as the “natural process of science” drums onward.

“Until the work is replicated independently by another laboratory,” he said, “in my mind it doesn’t exist.”

Still, Hafler said that the results of the Yale study are “one of the most remarkable findings I’ve seen in my career.”

“I really congratulate them for just a game-changing paper,” Hafler told the News. “They should be proud to be from Yale.”

As of Wednesday night, Nature reported that 132 news outlets have picked up the study.

Marisa Peryer | marisa.peryer@yale.edu

Matt Kristoffersen | matthew.kristoffersen@yale.edu