The Truth about Animated Brains

Brain separately, mice separately

Polina Loseva, N+1

Last week, many Russian news outlets talked about how "Japanese scientists revived a dead mouse brain." The title sounds like a sensation: until now, no one has been able to resurrect a single animal brain. These notes are based on an article that appeared in Analytical Sciences, a little–known journal with an impact factor of 1.618. Does this mean that larger journals ignored the scientific revolution? Or that the authors of the work set up a dubious experiment, the results of which were not accepted anywhere else for publication? Let's figure out what really happened to the brains of Japanese mice after death.

Either the patient is alive or he is dead

When we hear about the "revival of the brain", something like a head separated from the body appears to the imagination, which by itself begins to growl, squeak or clap its eyes. Something similar allegedly managed to be carried out by the Soviet surgeon Sergei Bryukhonenko, although now his reports are considered falsified, and no one has managed to repeat his experiments so far (read more in the article "Patient without a head").

From the point of view of a medic, "brain revival" is the disappearance of signs of brain death. In Russia, for example, there is a separate order of the Ministry of Health with a list of clinical criteria, the presence of which indicates that the brain is dead, and the absence – on the contrary, that it is alive. All these criteria are a test of various kinds of reflexes – from eye movement to reaction to pain, that is, they are designed to show how the brain copes with its main function – the coordination of body movement.

But what if the brain is separated from the body and is "in a test tube"? Or if scientists concentrated on reviving the brain alone and ignored the state of the other organs? In such a situation, the brain technically cannot control any muscles. Then another criterion comes to the rescue, which the order of the Ministry of Health prescribes to be carried out if the brain is dead according to all other signs – electroencephalography.

If the EEG shows no signs of activity (electro-cerebral silence) for at least 10 minutes, it can be assumed that the nerve tissue has stopped doing its job of transmitting signals and the brain is completely dead (except in rare cases of reversible coma, when the patient returns to life within a few hours). Thus, in order to talk about the "revival of the brain", it is necessary to demonstrate evidence of its electrical activity.

The story of one pig

The previous high-profile story with the separation of the brain from the body occurred in the spring of 2019. Then scientists told that they managed to "restore blood circulation and cellular functions of the brain" of the pig four hours after the death of the animal, and some journalists also started talking about "revival".

In fact, the following happened: researchers came up with a way to keep the brain in a viable state. To do this, they placed the organ in a chamber with high humidity, connected it to the blood circulation system, and the blood plasma itself was replaced with a liquid with a complex composition – in order to avoid clotting and blockage of blood vessels. In this state, they managed to keep the pigs' brains for up to 10 hours.

The state of the nervous tissue was then assessed by scientists on several grounds. First, they made sure that the size of the brain and its contours did not change, that is, the structures did not begin to collapse. Then they began to look for traces of neurodegeneration at the cellular level and found that not all parts of the brain were preserved well: in the areas most sensitive to oxygen deficiency (hippocampus, neocortex and cerebellum), they managed to find decaying cells.

Then the researchers checked the state of the circulatory system and confirmed that the vessels of the isolated brain can contract in response to vasoconstrictive drugs. Finally, they measured the electrical activity of the nerve tissue. Despite the fact that individual neurons remained capable of generating impulses, scientists found no traces of global activity in the whole brain.

That is why the researchers were wary of making far–reaching conclusions and stated only that their system gives brain tissues a chance to hold out until activity is restored - if we ever figure out how to do it.

Was there a brain

Japanese scientists, who gave the reason for the "revival of the brain" to get into the newspaper headlines again, do not say a word about it in their article. Their work is devoted to the cultivation of explanted tissue – that is, how to prevent a section of the organ from collapsing after removing it from the animal's body.

This problem does not arise with individual cells, most of them feel at ease in laboratories. But when it comes to a complex structure, such as the nervous system, it is important not only that the cells remain alive, but also that they do not lose their functions and continue to work smoothly.

Japanese biologists have chosen the suprachiasmatic nucleus, a tiny part of the brain responsible for biological rhythms, as the object of research. This is a closed structure of tens of thousands of neurons, each of which has its own circadian rhythm, but at the output this neural network generates one powerful signal – the "time on the biological clock" of the body.

It is quite difficult to cultivate the suprachiasmatic nucleus in vitro: with a lack of water, the tissue dries quickly, but if you cover it with a solution and rinse it, as is done with conventional cultures, the fluid flow destroys the connections between neurons. Therefore, the authors of the work designed a microfluidic device that allowed to keep the fluid pressure constant and not injure the tissue.

Here and below are pictures from an article in Analytical Sciences.

In order to confirm that the suprachiasmatic nucleus remained intact and functional, scientists used transgenic animals. The luciferase gene was triggered simultaneously with the PER2 gene, one of the key biorhythm genes, in the tissue donor mice. Therefore, at the peak of PER2 expression, the nucleus cells glowed synchronously. The researchers measured the glow in the suprachiasmatic nucleus, which was cultured in the usual way, updating the medium from time to time, and in samples placed in a microfluidic device.

It turned out that the cyclicity of the glow and, consequently, the functional activity of neurons fades much more slowly with a new, microfluidic method of cultivation. In static cultures, fluctuations disappeared by the fifth day, and in microfluidic cultures they could be observed on the 25th day. Researchers believe that the lifetime of such a culture of the suprachiasmatic nucleus can be much longer – up to hundreds of days.

Fluctuations in the intensity of bioluminescence in relative units. The upper row is microfluidic cultures, the lower two rows are control cultures.

Thus, Japanese biologists have developed a new method by which it is possible to keep the nerve tissue in working condition. It can certainly be useful in practice – for example, to study the structure of nervous tissue and the mutual arrangement of cells, which is not always possible to do in a dead brain.

However, scientists worked only with a small area, without claiming to maintain life in the whole brain. In addition, they did not even aim to preserve the electrical activity of cells, but measured only gene expression – an indirect indicator that the neural network remained intact. Therefore, there can be no question of any revival of the organ, only about the viability of individual cells.

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