April 17, 2019

expert reaction to study on restoring cellular functions in the pig brain after death

Research published in Nature describes a system that can restore brain circulation and some cellular functions in a pig brain hours after death.

Dr Martin Monti, Associate Professor in the Departments of Psychology and Neurosurgery, University of California Los Angeles (UCLA), said:

“Death is a process and it takes time, not just seconds or minutes — we knew that.  The advance here is that, with the right technology, we might now have more time to recover some molecular, cellular, and microvascular function before these are completely compromised in the non-human animal model, a prospect which might some day extend the potential timeline for restorative interventions in human tissues.  However, this should not be confused with acts of magic restoring any function in one’s favourite long-gone hero, which is not going to happen any time soon, and it should also not be confused with re-infusing cognitive processing or, much less, sentience in the decapitated head.  Despite the impressive result, no global electrical activity could be detected, not even a remote precursor to the complex interactions needed for any cognitive function to arise.

“Whether this finding moves the goal-post with respect to issues of organ transplant or determination of brain death is not clear.  First, as exciting as the result are, the animals remained in a state of complete neural silence, thus leaving untouched one of the core criteria of brain death.  Second, since these results say very little about the possibility of undoing the neural silence once it has taken hold of the brain, they say even less about the possibility for the recovery of any semblance of cognitive function and (self)awareness.  Finally, whether this result in the swine model will ever translate to human beings is also unknown.  So, for now, the cautious interpretation of this work is that, with this technology, the window for rescuing from the process of death profoundly damaged neural tissue, in the pig model, might be larger than we thought.  Already a remarkable achievement.”

Prof Derek Hill, Professor of Medical Imaging Sciences, UCL, said:

“The received wisdom is that, within minutes of cutting off the blood supply, the brain of humans and other advanced mammals suffers irreversible damage.  This paper from researchers at Yale University builds on other recent research to challenge that view.  They have developed a machine called ‘Brainex’, and have shown that this can restore some aspects of brain biology hours after the pig brain had literally been cut off the body.  This research was not done on research animals specifically reared to have their brains studied in a controlled setting, but on brains from pigs raised for food, and obtained from an abattoir around four hours after the pigs had been slaughtered.  The brains were removed from the carcass, and then attached to BrainEx.

“The researchers used some established clinical methods including ultrasound imaging, MRI and CT scanning to look at how the blood flow and brain structure changed over time, and at how the cells were functioning.  Those pig brains connected to BrainEx showed the recovery and then preservation of some aspects of brain biology for many hours, whereas the control brains that were not connected to BrainEx rapidly degenerated as expected.  BrainEx most certainly didn’t bring the brains back to life – and in particular there was no evidence of any electrical nerve activity in the brain.

“But this research raises some fascinating questions.  Firstly, was this somehow a chance finding, or can it be reliably replicated?  Secondly, could brains maintained by BrainEx help scientists discover new treatments for brain diseases like Alzheimer’s and Parkinson’s disease, which are proving really hard to treat?  And thirdly, what are the ethical implications for the way we treat animals after slaughter, and humans after accidents.  This ingenious experimental work provides challenges and opportunities both to brain scientists and for science policy makers.”

Prof Konstantin-Alexander Hossmann, former Director and Emeritus Professor at the Max-Planck-Institute for Metabolic Research, said:

“The publication in Nature takes up an observation that 50 years ago had already caused considerable attention in the international press.  At that time it could be proven for the first time in animal experiments that the brain of cats and monkeys can be revived after a complete circulatory arrest of one hour and that the nerve cells do not die already after 8 to 10 minutes^1,2 – as is still often assumed today.  The current investigations confirm this finding in pigs and suggest that brain resuscitation may be possible up to 4 hours after the onset of circulatory arrest.  The prerequisite, however, then as now, is that the brain is completely and uniformly perfused with oxygen and the nutrients necessary for the brain metabolism already at the beginning of the attempted resuscitation.  In the current Nature publication, this is achieved by using a hemoglobin-based, cell-free, cytoprotective nutrient solution that is flushed through the brain vessels with a high-performance perfusion system.

“It remains to be seen whether this method can also be used in clinical reanimatology.  Earlier resuscitation experiments with extracorporeal pumps were not very successful.  However, this may be due to the fact that normal blood and no blood substitute solutions were used in most cases.

“Whatever the outcome of these new experiments, they will not be able to solve the fundamental problem of clinical resuscitation.  The most common clinical cause of an arrest of the brain circulation is cardiac arrest.  It differs from the experimental studies in that not only the brain but also the heart is damaged by the circulatory arrest.  However, since such a damaged heart is not able to supply the brain with sufficient blood during the resuscitation phase after only a few minutes, the prerequisite for a successful resuscitation of the brain is not given in clinical everyday life.  In real life, the most important window for cerebral resuscitation is still much narrower than the hours looked at in this experiment – and the worldwide initiative for immediate bystander resuscitation (The ‘World Restart a Heart’ initiative 2018³) within the first four to five minutes after cardiac arrest is still important.

“This also puts into perspective the concerns expressed in the comments to the Nature paper about the reliability of the clinical guidelines for the detection of brain death in organ harvesting.  According to the current state of knowledge, it has not been possible to achieve neurological resuscitation under clinical conditions if the EEG or other functional vital signs of the brain did not return within the first hour after the start of resuscitation.

“However, it is to be expected that stroke research will be boosted by the new findings.  Even the early resuscitation attempts of the 1970s refuted the hypothesis that the nerve cells die within the first 10 minutes after circulatory arrest and helped to overcome the nihilism of stroke treatment that was prevalent at the time.  With the new findings, the therapeutic windows of the already propagated delayed recanalisation therapies in stroke may be extended even further, for example thrombolysis with drugs in stroke units or thrombectomy, a kind of stent for brain vessels.

“However, it should not be forgotten that success can only be expected if the brain is actually sufficiently reperfused by these measures.  Whether this succeeds remains to be seen – but the hope for it is strengthened by the new research results.”

1 Hossmann KA, Sato K, (1970): Recovery of neuronal function after prolonged cerebral ischemia. Science. Apr 17;168(3929):375-6. DOI: DOI: 10.1126/science.168.3929.375

2 Hossmann KA, Schmidt-Kastner R and Grosse Ophoff B (1987): Recovery of integrative central nervous function after one hour global cerebro-circulatory arrest in normothermic cat. Journal of the Neurological Sciences. 1987, 77:305-320

3 Böttiger et al. (2019) Over 675,000 lay people trained in cardiopulmonary resuscitation worldwide — The “World Restart a Heart (WRAH)” initiative 2018. DOI: 10.1016/j.resuscitation.2019.02.033

Dr Mark Dallas, Associate Professor in Cellular Neuroscience, University of Reading, said:

“The brain is acutely sensitive to changes in oxygen levels and a reduction in oxygen supply leads to nerve cell death.  This study looked at the ability to revive brain cells in pigs following reconnection to an artificial blood supply with a cocktail of active ingredients.  Their experimental intervention highlights a previously undetermined level of resilience within the brain and also the ability to restore limited functional responses after being disconnected from a living blood supply for a period of time.  This did not however culminate in restoring higher brain function, such as consciousness, but raises interesting questions about the cellular processes surrounding brain death.”

Prof David Attwell, Professor of Physiology, UCL, said:

“This paper reports that, after pigs have been killed in slaughterhouses, connecting the pigs’ brains to a pump providing a solution that mimics oxygenated blood, which also contained some factors that can protect nerve cells, led to the artificial blood going through the brain’s blood vessels, and to improved survival of the brain’s nerve cells, which showed some electrical activity when stimulated.

“As the authors state in their Discussion: “The observed restoration of molecular and cellular processes following 4 hours of global anoxia or ischaemia [i.e. loss of blood supply after the pigs were killed] should not be extrapolated to signify resurgence of normal brain function”.  Indeed, there is no way that this approach is going to allow us to save the injured brains of our loved ones or even pets, in the near future.  Indeed, almost all drugs that have been tried in humans, in efforts to prevent the deleterious effects of loss of brain blood flow (as causes stroke), have failed to show a useful improvement.

“Nevertheless, this paper does offer some hope for the distant future.  It should be considered in three parts:

“1. Restoration of blood flow.  The authors appear to be able to get their synthetic blood to flow through even the smallest blood vessels in the brain (capillaries), allowing brain metabolism to at least partly occur (at about 2/3 of the normal rate judging by the oxygen consumption).  This is encouraging because previous work has found that, for stroke, even after a blood clot is removed from a large artery going to the brain, the capillaries remain poorly perfused, in part owing to the contraction of small muscle-like cells (called pericytes) which wrap processes around them.

“2. Reduction of cell death.  The authors found less nerve cell death when perfusing their artificial blood, but it is not clear whether this means that the cells will not die later.  After a loss of blood flow causes a stroke, the nerve cells normally take hours to die, so it is quite possible that the cells have been triggered to die even though they have not died yet, or that they are significantly damaged so that although they look alive they do not work properly.

“3. Preservation of electrical activity.  The paper shows that some neurons can generate electrical signals when stimulated.  This work is reminiscent of a paper from 20 years ago by Charpak & Audinat (ref 14 of this paper) which found a similar long-term survival of the capacity for electrical activity of nerve cells.  Despite this, the EEG displayed a flat line in that paper and the current one, indicating that normal brain function was not restored.  A particular problem may be maintaining the propagation of signals through nerve cell connections which are wrapped with myelin, which confers a high signal speed, because the myelin is particularly sensitive to loss of blood supply.

“Overall, it is very difficult to assess what ‘mental state’ a brain maintained in this way would have: just as we do not know what attributes of brain function give us consciousness, we are uncertain which ones need to be removed to lose consciousness and (for practical purposes) make the brain die.”

Prof Tara Spires-Jones, Deputy Director of the Centre for Discovery Brain Sciences, and UK Dementia Research Institute Programme Lead, University of Edinburgh, said:

“This paper by Prof Nenad Sestan and team at Yale shows brain cells in pigs can retain some of their basic functions hours after death if the brain is provided with artificial blood flow.  They did not see any evidence of the type of brain activity that is needed for perception or thought.  For neuroscientists this paper is important as it provides another tool for studying the brain.  We do not fully understand how the millions of brain cells and the trillions of connections between them work together to allow cognitive function.  A better understanding of brain function is important for understanding what makes us human and will also help us treat devastating diseases of the brain like Alzheimer’s disease.  This paper is a step in that direction.  However, this study is a long way from preserving human brain function after death as portrayed in the cartoon Futurama where heads were kept alive in a jar.  It is instead a temporary preservation of some of the more basic cell functions in the pig brain, not the preservation of thought and personality.

“Although we are a long way from being able to restore function of brains of people who are declared dead, this work has fuelled the ethical debate about how to balance when someone is declared dead for the purposes of organ donation.  In my view, the evidence in this paper does not raise any concerns for people considering organ donation today.  An average of three people die every day in the UK waiting for an organ transplant, and this new paper does not suggest people should stop donating as their brains cannot be kept fully functional after death.”

Prof David Menon, Head of Division of Anaesthesia, University of Cambridge, said:

“We have known for a long time that if there is a temporary arrest of circulation, complete resuscitation of brain function is possible with rapid restoration of brain blood flow.  However, we know that increasing the delay to such restoration of blood flow results in increasing amounts of damage to nerve cells, supporting tissues (called glial cells), and the blood vessels that supply the brain.  Where the cessation in blood supply is prolonged, and affects much or all of the brain, clinical experience is that extensive damage to brain cells and systems results.  However, we have known for some time that even prolonged and severe deprivation of blood supply may leave some islands of brain active, and that some cell types (nerve cells) are more sensitive to these insults than other cells (such as the blood vessels).

“In this paper, Vreselja and colleagues show that the use of novel resuscitation fluids and perfusion devices, applied to an isolated pig brain, four hours after death, can potentially extend the window for successful restoration of function in all cell types from minutes to hours, and do this in the intact brain up to a duration of perfusion that lasts six hours.  They show that such restoration of function is possible in many cell types and brain regions.  However, they have not succeeded (in this paper at least) to restore function of ‘the brain as a whole’, nor indeed do they demonstrate any integrated brain function that provides evidence of functional communication between nerve cells – and they explicitly state this.  Further, they do not show (or claim) that such restoration of cellular function is long lasting – it is impossible, at present, to exclude the possibility that given a longer period of observation, the process of cellular injury may be reinitiated, merely having been postponed by their intervention.  Finally, it is important to observe that these results were obtained in isolated brains – an important fact, since restoration of blood flow to the rest of the body (following a cardiac arrest, for instance), may activate injury processes in non-brain tissues which produce substances that damage the brain (for example, through activation of inflammation).  Allowing for these caveats, the science in this paper appears to be of high quality.  While the requirement to replicate results is a fundamental tenet of scientific progress, there seems no reason to doubt their methods or results.

“The authors are careful to limit discussion of broader conclusions from their data that might be relevant to the clinical context.  The National Institutes of Health (NIH) press release that accompanies the paper accurately reflects the work as published, and highlights its scientific and conceptual importance.  The applications of these methods and results include the understanding of the process by which brain cells (and brains) die, and providing a framework to examine the benefit of new therapies in this novel model of resuscitation.  Indeed, some of the additives in the special resuscitation fluid that was used in these studies may, in the future, lead to potential new therapies for testing in clinical practice.  In contrast, the NIH press release makes only a very brief mention of the ethical and clinical implications of this work.  However, the commentaries that accompany the paper in the journal address important questions about the conceptual and scientific importance of these findings, and raise questions about their ethical and clinical implications.  The first of these is the issue of animal research regulation.  While this is an important consideration, I would expect it to be most relevant to the experiments that attempt such advanced brain resuscitation, rather than to broader animal research, since there are already stringent guidelines in most countries to ensure minimisation of suffering and the use of anaesthesia and pain relief (as discussed in one of the commentaries).

“The implications for clinical practice are more difficult to summarise.  In one sense, these experimental findings corroborate a direction of travel in clinical practice, particularly in the context of aggressive management of stroke, where clinical evidence suggests that increasing intervals (of several hours) may still result in useful benefit following aggressive therapies such as thrombectomy.  Further there is an increasing acceptance that in many conditions with potentially devastating brain injury, we need to be cautious about early prediction of outcome in the absence of incontrovertible structural evidence of extensive brain injury from trauma or intracranial bleeding.  In conditions such as cardiac arrest, these trends have meant that we often delay prognostication and testing for irreversible brain injury well beyond the 24 hour delay that was common as recently as a decade ago.  Also, as outlined above, the components of the novel resuscitation fluid that was used in these experiments, and the platform that these experiments provide for testing novel therapies may, in the future, lead to therapeutic advances that can be applied in far less severe brain injury (perhaps in combination with interventions such as thrombectomy), and well before the stage at which a devastating diagnosis of brain death is being considered.

“The direct therapeutic relevance of these findings to the situation in which devastating brain injury (from severe brain trauma, for example) has resulted in established brain death is less certain.  Not only has the brain been deprived of blood supply for a considerable period of time but severe brain swelling in these settings makes restoration of blood supply a far greater problem.  This problem, in the current clinical context is insurmountable, and while improvements in technology and systems of healthcare delivery may change this, there seems no obvious prospect of such improvements at present.  It is also worth pointing out that testing for brain death is usually undertaken after far longer intervals after the insult than the four hour time point in these experiments – often more than a day later.

“However, despite these caveats, the more immediate challenges of these data are ethical ones, which medicine cannot address without involvement of society.  These challenges involve a range of issues and uncertainties – death of the ‘whole brain’ versus death of ‘the brain as a whole’, individual acceptance of disability, religious and cultural views, and the fact that resources are never unlimited.  These are difficult questions – and they have no best answers, only least worst ones.  Scientists and clinicians need to articulate these emerging concepts honestly to patients and the public, be open to the fact that the parameters that we operate under are constantly changing, and be aware that these changes need to inform practice and a need for professional prudence, humility and caution.  Perhaps most importantly we need to find ways to explain the translation of such science to medicine for patients and society – a process which is often imperfect and uncertain, but is always essential.”

Prof Dominic Wilkinson, Professor of Medical Ethics, Oxford Uehiro Centre for Practical Ethics, University of Oxford, and Consultant Neonatologist, John Radcliffe Hospital Oxford, said:

“After someone dies, their brain normally deteriorates and disintegrates within a matter of hours.  This intriguing research demonstrated in pigs that it was possible to halt the progressive cellular damage that normally occurs in the tissue of the brain after death.  The researchers connected the disconnected brains of pigs, who had been dead for 4 hours, to a pump with an artificial preserving fluid, and showed that they could maintain some of the microstructure and even some of the cellular function for a period of up to 10 hours after the pig had died.  However, there was no evidence of return of global function of the brain.  The brains of the pigs remained electrically silent.

“What does this mean?  1. This research reminds us that ‘death’ is less an event, and more of a process that occurs over time.  Cells within the human organism may be alive for some period of time after the human person has died.

“2. This research might pave the way to important research using the human brain.  There are, obviously, real limits to the research that can be done on human brains – because of the risks to research participants.  However, research on brains after death is also limited – because brains normally deteriorate so quickly after someone has died.  This sort of technique might mean that someone could donate their brain to research after they had died, and researchers might be able to gain crucial insights into some of the microscopic structure and functions of the brain.

“3. This research might pave the way to future techniques to prevent deterioration in the brain – for example after a stroke or a head injury.

“4. More speculatively, it is possible that the research might lead to future techniques to attempt resuscitation of the brain after very severe damage.  However, we are a long way off from that.  The research did not show any evidence that their technique could restore meaningful function to the brain after death.

“5. At present we should be clear that this research does not have any implications for brain death or for organ transplantation.  Nor does it mean that there is a realistic prospect, any time soon, of bringing back people from the dead.  ‘Brain death’ refers to the irreversible loss of the capacity for awareness and consciousness.  Once someone has been diagnosed as ‘brain dead’ there is currently no way for that person to ever recover.  The human person that they were, has gone forever.

“If, in the future, it were possible to restore the function of the brain after death, to bring back someone’s mind and personality, that would, of course, have important implications for our definitions of death.  That might be possible one day.  But it is not possible now, and this research does not change that.”

‘Restoration of brain circulation and cellular functions hours post-mortem’ by Zvonimir Vrselja et al. was published in Nature at 18:00 UK time on Wednesday 17 April 2019.

Declared interests

Dr Martin Monti: “I have no conflict of interest to report.”

Prof Derek Hill: “No conflicts of interest.”

Dr Mark Dallas: “No conflicts of interest to report.”

Prof David Attwell: “None at all.”

Prof Tara Spires-Jones: “I have no conflicts of interest with this paper.”

Prof David Menon: “I have no interests to declare.”

Prof Dominic Wilkinson: “No conflicting interests to declare.”

None others received.