Is death so irreversible?

From the life of the dead

Fedor Galkin, "Biomolecule"

Any living being is an incredibly complex structure. One would expect that after death this structure would gradually collapse and all life processes would fade away. But it turns out that the cells in the dead body continue to work actively, desperately trying to survive. This article will tell you about the thanatotranscriptome and whether Charon transports in both directions.

What is death?

Today, it is generally accepted that a dead body differs from a living one in the absence of brain activity. At the same time, there are many borderline states for which it is impossible to draw a clear dividing line between life and death.

Now such conditions are called clinical death, but previously no one could have imagined that doctors would learn to bring back to life seemingly already lost people. A century ago, it was not possible to support a patient who had fallen into a coma, and two centuries ago, cardiac arrest meant imminent death. Now everyone knows that a stopped heart can beat again if you massage it before irreversible changes occur. Thus, death is not just a stop of vital processes, but their irreversible termination. How far the "point of no return" is depends on the level of medicine in each historical period.

Until now, despite the triumph of molecular biology, it is almost impossible to reverse intracellular post-mortem changes that occur most rapidly in the nervous tissue. But there is a way to slow down these changes and delay the manifestation of their sad physiological consequences.

The wonders of resuscitation

Since the 1970s, legislation around the world has moved away from the outdated definition of death, in which the main feature was the absence of breathing and pulse, and came to the criterion of lack of brain activity. By the 1980s, it was clarified that activity should be absent in the entire brain, not just the cortex [1]. Since then, the medical community has been particularly interested in the question: how long can a person be in a state of clinical death without irreversible damage to the brain? In the 1980s, it was found out that when the heart stops, irreversible brain changes occur if blood flow is not restored within five minutes [2].

But in the 1990s, the experiments of the American scientist Peter Safar, conducted on dogs, showed that cooling the brain to 30 ° C allows you to extend this time to 10 minutes [3]. After successful experiments on people who were cooled to 32-34 °C during the day after cardiac arrest, brain cooling was included in the recommendations for first aid in cardiac arrest [4]. Medicine has known unique cases when people survived after prolonged circulatory arrest with extreme cooling. So, the Swedish Anna Bagenholm managed to survive after an accident on the ski track in 1999: she fell through the ice, where she spent almost an hour and a half, of which the last 40 minutes – with a stopped heart *. Her temperature dropped below 14 °C, but she survived and was able to fully recover (Fig. 1) [5].

* – The articles "Vitrification – controlled pause of development in a glass-like state" [6], "Suspended animation I." tell about artificial or evolutionarily conditioned stops of "biological machines" with the possibility of restarting. Minimum life" [7] and "Suspended animation II. Death on demand" [8]

Figure 1. "The head should be in the cold..." a – In case of cardiac arrest, it is recommended to place the patient's head in a cold place to avoid irreversible brain damage during ischemia.
 b – There are cases when, after a prolonged cardiac arrest, people remained alive due to hypothermia.
For example, Anna Bagenholm survived a 40-minute cardiac arrest after falling under ice on a ski track. At the same time , her temperature dropped to 13.7 ° C.

Pushing back the definition of death

But what if the brain activity has already disappeared? Is there a way to fix the seemingly irreparable damage? The ability of the mammalian brain to regenerate is extremely low. Despite the presence of nerve stem cells in the brain, their activity is very limited [9]. At the same time, in amphibians and fish, stem cells make it possible to restore the brain even after the loss of entire sections of this organ [10].

But perhaps there are ways to awaken the latent regenerative potential of the human brain. To find these ways, two companies specializing in regenerative medicine – Bioquark and Revita Life Sciences – have joined together in the ReAnima project. In April 2016, ReAnima announced the launch of a proof of concept clinical trial, which will test a set of measures to bring people with recorded brain death back to life. The details of the study were not disclosed, but it is known that it is being conducted at the Anupam Hospital in Rudrapur (India) and 20 patients will take part in it. The therapy will include peptide injections, laser nerve stimulation and cellular technologies. The results of the study will be published in 2017 [11].

The body is dead, but not the cells

From the point of view of individual cells, the death of an organism is only a lack of nutrients and hypoxia, which can stop at any moment. Therefore, even if the damage received by the body is absolutely irreversible, the cells simply do not know this and continue to hope for the best as long as they have the strength. After some time, some of the cells realize that the resources available around them are not enough for everyone, and nobly sacrifices themselves for the sake of saving the rest, launching a self–destruction program - apoptosis.

And yet some cells continue to live, well, or at least support some of the processes going on in living cells. Very little is known about how partially living cells behave. Understanding these processes is important not only for the realization of a distant fantasy about the return of the dead to life, but also for solving more real problems of transplantology. Organs separated from the donor can also be considered alive or dead, depending on whether they can earn money in a new body. To prolong their shelf life, it is necessary to understand what death is at the cellular and molecular levels and how to reverse the processes that ultimately lead to the irretrievable loss of an organ.

Thanatotranscriptome – expression in the next world

An international group of scientists wondered: how does the level of RNA in the cells of a dead organism change? The initial hypothesis assumed that after the death of the organism, the RNA synthesis machine will continue to work for some time, but its activity will gradually fade until the synthesis stops altogether (Fig. 2) [12]. Such a scenario can be compared to a car that has run out of gasoline: the car will continue to move by inertia and will slow down until the pistons complete the last few cycles and the car eventually stops completely.

But it turns out that some genes are actively transcribed days after the death of the organism. If we continue the analogy, then the windscreen wipers would suddenly start working in a stalled car, the radio and turn signals would turn on.

Figure 2. The amount of postmortem RNA released gradually decreases on average. The amount of total mRNA (in conventional units, a.u.) isolated posthumously from two aquarium danio-rerio fish (a) and the brain and liver of one domestic mouse (b). Figure from [12].

One might think that an increase in the content of some RNAs over time is only a consequence of their high stability, however, non–monotonicity and several peaks of concentration for certain types of mRNAs, together with high reproducibility of data, imply that an error of this kind is excluded. In addition, this study confirms the results of earlier and smaller-scale work that revealed the growth of RNA genes of matrix metalloproteases and myosin proteins in human blood and pericardial fluid 12 hours after death [13].

In each of the objects – the fish Danio rerio and the mouse Mus musculus – more than 500 genes were identified, whose expression significantly increases after death (Fig. 3). Among the "finds" (where the increase in synthesis reaches 50-60%) are protein-coding genes and non-coding mRNA. The corresponding proteins are involved in inflammation, immune reactions, cancer degeneration, apoptosis, transmembrane transport, reactions to oxygen starvation and, oddly enough, embryonic development. Most of the genes responsible for all these processes were activated in the mouse within an hour after death, and in D.rerio more slowly – within a day. The expression of some genes in the mouse continued to grow for two days, and in D.rerio – even three days after the death of the organism. This means that during this time the cell retains the remnants of structures that ensure the synthesis of RNA.

Figure 3. Percentage of protein-coding genes with a growing proportion of mRNA in each category, depending on the time after death. Figure from [12].

The growth of transcription of some genes, such as inflammation and immunity genes, has a biological meaning: cells feel a threat to their existence and try to resist it. Similarly, activation of membrane transport genes is a desperate attempt by dying cells to restore homeostasis. At the same time, the expression of embryonic development genes in a dead organism most likely indicates the destruction of complex regulatory pathways, which require a constant influx of energy and certain intracellular conditions to maintain [12].

Criticism of the thanatotranscriptome

It is important to note that the work under discussion contains a number of controversial points.

Thus, only relative concentrations of mRNA were determined in it, which grew even after a sharp drop in the level of total RNA (Fig. 2), that is, RNA degradation is much more intense than synthesis. In addition, the work was carried out with preparations of a mixture of tissues, the cells of which differ in energy needs, and therefore in survival [14]. Because of this, it may turn out that at some point the picture of relative mRNA concentrations changed, because then a certain type of cell died, which increased the relative content of stable mRNAs of this type of cell.

Some morphological postmortem changes can also affect the observed picture: with the outflow of blood from the liver, the level of mRNA of blood cells will fall, and the mRNA of hepatocytes will grow. For greater accuracy, it would be necessary to work with individual tissues and cells removed from animal corpses.

More answers – more questions

The above study is almost unique in its kind. In earlier works, the possibility of active transcription after death was not even considered – it was believed that RNA is not synthesized in a dead body [15, 16]. And although the amount of work done (containing some controversial points) is really great, molecular thanatology still mostly consists of questions, not answers.

The most important of these questions is how do the patterns found in total tissue preparations correlate with data on individual cell types?

But, despite its shortcomings, this study raises a number of practically significant questions. For example, how critical are individual differences in thanatotranscriptomes for successful transplantation? Can the postmortem mRNA repertoire be as important as the compatibility of the immune receptors of the donor and recipient? Are there molecular determinants of successful resuscitation? How will a cocktail of oncogenic, embryonic and apoptotic genes affect the fate of cells that have returned to normal physiological conditions?

It is worth noting that for applied science, it is most interesting to find out all the answers in relation to a person, and this can be very difficult due to ethical restrictions. Without data on the human thanatotranscriptome, any research in this direction is simply doomed to remain without practical application.

Literature

1. What is the Uniform Declaration of Death Act (UDDA)? FindLaw;
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5. Frozen woman: a ’walking miracle’. (2000). CBS news;
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7. Biomolecule: "Suspended animation I. Minimum life";
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Portal "Eternal youth" http://vechnayamolodost.ru  05.07.2016