April 22, 2019
Written by: Carolyn Keating
It’s been all over the news. “Scientists bring pig brain cells back to life.” “Pig brains survive after death.” “Researchers ‘reboot’ pig brains hours after animals died.” The headlines have been sensational. But what did scientists actually do, and how did they do it? Let’s break down what the researchers really showed and how they accomplished this remarkable feat.
A few days ago, scientists from Yale reported that they restored blood circulation and cellular activity in the brains of pigs that had been dead for 10 hours1. How is this possible? Conventional knowledge was that if blood flow to the brain was interrupted and not quickly restored, unconsciousness and cell death occurred within seconds to minutes. But the researchers had a hunch that under certain conditions, brain cells might be able to be restored at longer time points.
To test this idea, the scientists decided to use brains from pigs that were slaughtered for meat. They carefully removed most of the skull from around the brain, making sure to leave the blood vessels that enter and exit the brain intact. They then created a special sort of artificial blood to pump through the brains. This fake blood was based on hemoglobin, the protein in your blood that carries oxygen. However, their substitute didn’t contain any cells, since white blood cells present in the blood can create an inflammatory response that further damages tissue when blood flow is restored, or clump together to block blood vessels2. They also added a variety of chemicals that protect cells from damage and death, as well as a compound that would allow the scientists to detect the artificial blood in the brain noninvasively using ultrasound. To get this artificial blood, called BrainEx, into the brain, they created a custom perfusion device. Perfusion refers to the passage of a fluid through the circulatory system (heart and blood vessels) to an organ or tissue, the brain in this case. In their device, the BrainEx fluid gets heated to body temperature and oxygenated before being pumped into the arteries that send blood into the brain. Importantly, their device didn’t just continuously deliver fluid, but instead sent it in pulses, resembling a pumping heart. An ultrasound was positioned above the brain so the scientists could watch the BrainEx fluid to see if it traveled to all parts of the brain and through all sizes of blood vessels. The fluid then exits the brain through veins, and the waste is filtered out and fluid is prepared to re-enter the brain, just like what happens in our own bodies.
Now that researchers had this BrainEx fluid and perfusion system, they wanted to test a few different situations. In two conditions, brains were perfused: in one case with the BrainEx artificial blood, and in the other with a control fluid without any added protective substances. In two other conditions, brains weren’t perfused at all: in one case the brains were just left in the skulls for 10 hours (representing regular tissue deterioration after death), and in the other the brains were flushed of blood 1 hour after death (representing the least amount of tissue damage that could occur during their tissue-processing procedure).
The authors first wanted to see if their BrainEx fluid and perfusion system could reintroduce blood flow within the brain. Using ultrasound, they showed that brains perfused with the control fluid had poor flow that stopped altogether after 6 hours (10 hours after death, since removing the brain and hooking up the pump took 4 hours), while BrainEx fluid strongly flowed through blood vessels large and small at 6 hours. This successful restoration of blood flow was seen in many brain regions such as the hippocampus, prefrontal cortex, occipital cortex, and cerebellar cortex. In addition to passive blood flow, the blood vessels were actually still active and responded to drugs that cause dilation.
Not only were the researchers able to restore blood flow, but the brain tissue itself didn’t look like it had been dead for 10 hours. Using magnetic resonance imaging (MRI) to visualize the whole brain, they saw that the anatomical structures of brains receiving the BrainEx fluid looked like brains from live animals. In contrast, brains left sitting in the skull for 10 hours had decreased water content and showed signs of decomposition, while brains receiving the control fluid showed swelling, indicating that excess fluid had leaked from the vessels and became trapped in the brain. To look more closely at individual cells in the hippocampus, cortex, and cerebellum (brain regions particularly vulnerable to lack of blood/oxygen), the scientists cut brain sections and labeled them for different substances. They saw that cells in brains receiving BrainEx fluid were still arranged normally, had the same density of cells as the 1 hour after death control brains, and didn’t display signs of structural damage, whereas cells from brains receiving control fluid or sitting in the skull for 10 hours showed signs of deterioration. In addition to cell bodies appearing normal in brains receiving the BrainEx fluid, the arrangement and number of axons were also undisturbed. And this preservation wasn’t just limited to neurons either. Glial cells called astrocytes and microglia were still present in the BrainEx brains in patterns and numbers similar to the 1 hour control brains. Besides just being present, they were also functional: in response to injection of a toxin into the brain, these glia released inflammatory molecules as they would in a live animal!
So far the authors have shown that with their BrainEx fluid and perfusion system blood flow is restored, cells seem to be intact, and brain cells called glia retain at least some function. But the big question is whether the neurons—the cells that use electricity to communicate with each other and control our thoughts, actions, and feelings— are functional. Under the microscope, the specialized regions where neurons communicate with each other (synapses) appeared to be intact in brains receiving BrainEx fluid and in 1 hour control brains, but not brains receiving control fluid or sitting in the skull for 10 hours. But the true test of function is whether there is any electrical activity. The brains receiving control fluid or sitting in the skull for 10 hours were too damaged by the end of the experiment to prepare them to test for individual neuronal electrical activity. But the scientists were able to prepare the BrainEx brains. In the hippocampus, they found that neurons had normal electrophysiological properties, and could even fire signals when stimulated! However, EEG leads on the brain did not show any sort of global electrical signal; the animals were still brain dead. It’s possible that the activity from individual neurons wasn’t strong enough or organized enough to produce network activity across the whole brain. But it’s important to keep in mind that one of the BrainEx fluid components to prevent cell damage is an anti-seizure drug, which dampens that ability of neurons to fire. This compound washed out of the hippocampal brain sections they prepared to test neuronal electrical properties, perhaps explaining why they were able to see a signal there. Nevertheless, together all of these results indicate that under the right conditions, the structure and function of the brain can be restored after long periods without blood flow and oxygen.
Many articles have already started to delve into the ethical implications of restoring activity to brains after death (I personally liked the ones here and here). But the authors emphasize that it’s important to distinguish between the restoration of neuronal activity and the recovery of integrated brain functions; that is, the difference between the ability of an individual neuron to fire and the organized firing of a network of neurons to produce thought, feeling, or movement. And restoring integrated brain functions was not the goal of the study; in fact, the researchers had anesthetics on hand to administer at the first sign of global network activity so as not to cause inadvertent suffering. Instead, the scientists believe that this system could be an important platform to perform functional studies in intact brains that otherwise would be limited to structural or biochemical experiments; and in the distant future perhaps lead to treatments for people experiencing stroke or traumatic brain injury. While standard procedures need to be implemented to address the ethical questions that have arisen, this achievement still represents a remarkable feat.
- Vrselja, Z. et al. Restoration of brain circulation and cellular functions hours post-mortem. Nature 568, 336–343 (2019).
- Bai, J. & Lyden, P. D. Revisiting cerebral postischemic reperfusion injury: new insights in understanding reperfusion failure, hemorrhage, and edema. Int. J. Stroke 10, 143–152 (2015).
Cover image from Gaetan Lee via Wikimedia Commons, CC BY 2.0. https://commons.wikimedia.org/wiki/File:Human_brain_in_a_vat.jpg
Nicely done !
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