 Hello everybody. Welcome to the Ethics in Research and Biotechnology series. This is a monthly series that brings together bioethicists, scientists and other researchers to talk about ethical issues of the cutting edge of science. And today's topic is absolutely at the cutting edge of science. The topic today is called experimenting with large mammalian brains X vivo. Let me just go over a little bit of the ground rules for today. Before we get started with introductions. This event is being recorded and it is live streamed on Facebook. The event video will be posted on the center for bioethics Facebook and YouTube pages later. We encourage you to submit questions along the way at any time. And to do that you have to use a Q&A feature at the bottom of your screen. Don't use a chat box use a Q&A feature. So we have a lot of questions at the end to pose to our participants for discussion. And I really look forward to that interaction with you, our audience. If you have any technical issues, then you can use a chat feature to send a message to the panelists or to the staff that could help you. Upcoming events are listed on that website, bioethics.hms.harvard.edu. And with that, let me go ahead and introduce our speaker and the topic. So today we have our first speaker, who is Nanad Sistan. He is an MD PhD. He is professor of neuroscience comparative medicine genetics and psychiatry at the Yale School of Medicine. Back in 2019, he was actually named one of the 10 people who mattered in science by the journal Nature. And this topic today will take us through his research that led to that that that designation by the journal Nature of being the ones to watch in 2019. My name is since you can, I'm director of research ethics and I'm a faculty member in the Center for Bioethics at Harvard Medical School. I'm also a professor of bioethics and philosophy at Case Western Reserve University. So we're going to start with Dr. Sistan, and he will take us through a discussion of his research for about 30 to 40, 45 minutes or so. Then we have a special discussant after that Dr. Robert True, who I will introduce at that time. And after some thoughts from Dr. True, we will then open it up for the Q&A session. So again, I encourage you to ask questions along the way by using the Q&A box. And with that, let me turn it over to Dr. Sistan. Thank you so much for joining us. Thank you for your kind introduction. And you can see my presentation. Yes. Sorry. For some reason it's forcing me to leave. I don't understand what's happening. Okay. Okay, so thank you so much for your kind introduction. Thank you and all the members of your center for inviting me. Of course, it will be much nicer if we were in one of my favorite cities. We are experiencing a global crisis and health crisis. Of course, more than health crisis. And I hope we'll have another opportunity to do this in person. And we can see your presentation view. So just FYI. Sorry, we can see your notes in your presentation. I don't know what's happening. Can you see now? No, now we don't see your screen. Because I am having a really difficult because I'm using, can you? Okay, let me just try again to share. Sorry about this because I've been experiencing difficulties with my screen. Oh, I know what happened. Okay. Okay, excellent. Can you see it now? Yes. Yep. I apologize for this. Okay, now you should, you see my screen now? Yes. And you see my laser pen? Yes. Okay, thank you so much. So, so, so as Institute said, I will share a little bit about our work that started several years ago and really on X people. And it's really that I get to continue to talk to the audience that to the colleagues outside the neuroscience community so especially do that I just buy ethical aspects of the study and so I really look over to your feedback. So, I would like to say that I have no commercial support for this presentation but however I am listed by colleagues on the IP that is helped by the university. So just let you know. And so before I dive into the meat of the presentation, really, I would like to provide a brief outline of what we are going to discuss today. Also, I would like to say that it's my hope that by the end of this presentation. I may convey and potentially convince you that the brain and specifically cells in the brain can demonstrate a higher tolerance to skin injury than is widely accepted. And the way that I will be arriving there is by starting off with a little bit of background information on the brain. And what is really important in context of this presentation, it's high metabolic demands. And then I will examine some brief data on brain tissue responses following circulatory arrest and global is himia. And then I will describe the central research question and hypothesis that our team has tested. And really showing how we tested this show with the design and establishment of technology we named brain X brain. And this stands for brain X vivo. And which consists of three core critical component I would like to introduce immediately. One is a surgical approach by which we actually isolate the brain and its vasculature and then component or technology by itself has two components that are critical. One is the extracorporeal profusion device. And the third is a synthetic a cellular cytopathic perfusion. So just basically the perfusion box and a solution. And then just be able to do this x we will be also had to really design a way how to extract the brain from a big brain and from the big scale actually and then I will describe how we validated this technology as a research platform and using post mortem and big brains I'll explain how what was the motivation to do this because I think it's probably interesting to all of you by how we came about this and how we did it. And, and, and finally, of course, nothing is created in the watching and so I would like to acknowledge two amazing colleagues who have done most of the work that I will describe today. I am a neuroscientist who is still in the lab and step on a Daniela who is now back in the medical school he actually successfully finished his PhD by its back medical school. And so I am a neuroscientist and until recently I've been really exclusively studying basically develop them in specifically I was interested in understanding how different cell types and and their synaptic circuits are generated in the developing brain. And this is really what we have been doing for years and recently. Actually, we came up on something that I will describe how we came up this idea, and it led us to really surprising discovery about brains resilience. And that's what I will talk about today I will not talk about the brain development but I really wanted to mention that to you to get understanding how we came to what I'm going to talk to you about it today. But before I do that, let me tell you about, about the brain. And so, as you probably already know the brain is the most complicated complex structure we know in known universe. What is important at least in the context of what we study in the lab is at the root of what makes a unique as species. There are approximately 170,000 billion neurons in the center nervous system of which 86 billions are neurons. And these neurons need to be wired very precisely for brain to function properly and to explain this complexity and understand why brain is so unique among organs is not just associated with us as a species but it also it's really when you think about an energy demand is really unique. And the reason is that if you take this 86 billion euros, they need to be connected precisely and they need to maintain those connections and they connect using has been estimated this is not our work 850,000 kilometers of fibers which is more than twice the distance from here to the moon. And around 600 trillion synopsis and this is really crazy numbers and the pattern of this incredibly complex connectivity is what provides the basis for cognitive functions and behaviors that distinguish individuals and species. And this is what we are trying to understand how this wiring diagram is formed during the development and what is important but what is important to know for this presentation is that the brain is highly metabolically active. And while it presents only 2% of the body weight, the brain consumes 20% of oxygen and 25% of glucose, which is more than any other order, even if you adjust for the number of cells. And so high energy requirements of the brain is is fulfilled by a constant transport into the brain with the blood and so what blood does it actually brings oxygen brings nutrients and and and removes the base. That's basically what blood does. And the main reason this gets disrupted is is that the flow of blood to the brain can be slowed down and in completely stop and the most common reason for that is cardiac arrest. And as all you know very well cardiac arrest is one of the leading causes of death and disability and basically I think I read somewhere that it's almost around 20 million people died a year in and it's number one cause of that actually United States and most of other developing countries and around 20 million people die worldwide. From the cardiac arrest and and and this is and the survival rate of cardiac arrest is not that great is somewhere between. I think seven to 10% survival rate. And this is further complicated by the fact that up to 50% of surviving individuals go to on to display persistent neurological many neurological deficits. And of course this needs to significant impairment. The overall quality of life. And this is largely due to the fact that brain has very limited energy reserves. I mean that is no fact that I mean there's not bad but it's used for my life. And the brain cells, some of which I will simplify here and illustrated here have high basic metabolic rates. And the reason for that is just think about 86 billion neurons. They are constantly firing I mean they cannot be actually they cannot rest and they cannot hibernate and basically they have to constantly fire and send up some potentials. And that requires a continuous flow of blood to the brain to supply of course oxygen and nutrients, while also carrying away metabolic based product that these cells produce during their normal function. And thus you would not be surprised and probably all know this that actually ancient Greeks told that basically brain is a radiator because when they look at the brain there were so many blood vessels. And the reason you have so many blood vessels they need to bring nutrients and oxygen and move the metabolic waste. And so we recently became very interesting understanding what happens to the brain. When blood flow stops completely. Okay. Such in the case of cardiac arrest and, and, and all that and importantly, whether we can develop a technology to better study cellular processes and including connections and really potentially delay stuff, or maybe even reverse them to salvage the tissue. And as I mentioned to you earlier, this is not something that you were working on. And it's really I would say was a serendipitous discovery and observation and I'll take you in the next couple of slides how we get to that. Before I do that let me just tell you a little bit about. Sorry, I think my slides in advance. And so let me tell you a little bit about what happens to the brain in, in, in, in, in example of cardiac arrest or you know when you stop blood flow or or in this case. And now, you know, what are the responses of the brain and outcomes following this. And what it happens that when you stop blood flow or when you die basically is that you have something that is basically globally seen. Your cells are now deprived completely of oxygen. There is no blood flow. There is no removal of theabolic waste. And so what it happens is so to really use that to keep the simple. I'm going to show really a couple of really groundbreaking studies that really dive into this one time ago, almost 60 decades ago, and some little more recent and so what happens at the molecular level is that the key metabolites such as glucose. Sorry, ATP and fossil creating. And so here I did. Basically, they do precipitously within the first minute of interrupted blood flow. These are components that you need for minerals basically work not just for every other cells. On the other hand, some other metabolites such as a lactate, which is actually produced in without oxygen actually increased steadily soon after, and really, you know, as substantiating my earlier point is that the energy reserves within the brains are highly limited. And this is what the problem is, is that you have all of these 86 billion neurons, they need these critical component glucose ATP and fossil creatinine some others, and then basically that depleted immediately there are no reserves in the metabolic brain, and the brain is really basically. So, but what happens with you know and so what is the consequence of that the consequence of that is that global electrical activity and here is a study done by horseman and say to 1970s basically what it happens is that global electrical activity which is what we need to think most deeply about the entire study is what is required for higher order cognitive function is just normal functioning of the brain, and most importantly, in the context of bioethical consideration consciousness drops to a completely flat line. And this happens basically within 14 seconds of the onset of global is him so basically in 14 seconds and this is done actually cat that they have done the studies in other large mammals and and and really tells you about how sensitive brain is to lack of oxygen and basically in 14 seconds, you lose your e g signal. Basically, there is no global coordinated global electrical activity. Basically, you lose your consciousness and and as you would not be, you know, of course, not surprising as is that, you know, so what is the correlate of this course studies, you know, and these are the pioneering studies by Paul and Corda in 1950s, and what they publish their seminar findings in just one but actually serious of the paper on the outcome of precipitation from the cardiac arrest and established the so-called four minutes rule, and they found that those patients who were assassinated within four minutes had much better outcome. Okay, so this is under four minutes, then those that were resuscitated or four minutes and if you put together this molecular. I have two screens that's what I realized now it's happening on my monitor. And so, when you look at what happens to molecular reagents that are required for neurons to survive, it's basically they are lost within basically minutes to electrical activity and then what happens in something that is critically relevant, which is cardiac arrest. So, all these together studies really demonstrate and indicate is something that actually we all know from history and and that the brain is highly vulnerable to interruption of blood flow, an ultimately brain cells are universally damaged or died within a narrowly defined time point after cessation of blood circulation. So, that is really and a clinical really work over the last, you know, more than, you know, not just in our sense and just both historically as well as the in the last probably two centuries really support this view that brings it's really sensitive and raise it. However, that was, you know, I went to medical school that was my, you know, really what I accepted and really clinical work really show that that is true. However, over the years, there has been multiple lines of observation that call to question finality of several viability minutes or even after after that. Okay, and I'm going to show you two papers that really helped us form this hypothesis and try to pursue this question. And so one important study among these is from Dixbab and his colleagues in the Netherlands and actually I was following his work because he's an important neuroscientist who had done really fundamental work and I remember this, this paper when it came out, you know, like I would say it was maybe, yeah, 18 years ago. And we show that viable tissue cells can be harvested from adult human brain up to eight hours after that and maintain ex vivo or in this case the new cell cultures for prolonged period of time. But actually what they show that that space in these cultures can be maintained. I think that please forgive me if I'm not correct but I put it here on the slides, like 78 days. So the bottom line of this story is they took post mortem brain from donors that died, you know, eight hours and this is an experience, to some extent, probably combination of warm and little bit cold is he me I'm because most of these cases bodies are refrigerated. They harvest the brain this is an adult brain that's also not an important thing to keep in concert, and they make tissue organotypic tissue slices and they show actually really remarkably surprising preservation of tissue. I mean you don't survive there was some cell that they could study connections and then they, you know they even did EM show that actually synapses are preserved. And again, this is eight hours and this is really gains goes against what we know in clinical cases as well as what we know about really tremendously. Quick depletion of metabolites as well as the most local electrical activity. Well, you know, you know, we were thinking about this and but also really what really really stimulate us to really take this question more seriously and test this hypothesis was that our own lab observed similar also several years ago. And in our studies of brain development what we were doing we have been collecting postmortem tissue, and we observed viable tissue and cells can be harvested from brain that they're, you know, getting combination of short warm is he me but mainly cold store up to 49 hours after that and and and actually this is really not it's anecdotal we actually see two brains that were shipped from the brain bank to us, and they fortunately the courier did this, you know, I missed the airplane and and and the brains stood on wet ice and were delivered to us. This this action case that you are looking here is from the brain that was 49 hours PMI that came to our lab. And actually I did not expect this to work and actually I asked somebody who was in the lab to really okay take the brain, you know, see, try to learn a little bit this person was doing a bit more computational. It's good to know and you know we should fix it and you know, but maybe you can practice to make organotypic tissue slides so this is something similar to what the swap that and he's calling but this is a commonly used technique that we used in the lab using, let's say mice and other species. And, and, and, you know, his name is Andre came to me after several weeks and he said, man, you know, you have to look at these cultures and they are really growing. And, and, and again, this is not just dissociated cultures I mean this is really so the cells still have some, you know, their bend rights their synapses, and they were incredibly healthy baby grew them for several weeks, and you know really made me think about not just me but the entire lab think about how this possible, you know, and and again, this is not something unique and almost everybody has observed this who worked in neuroscience lab. If you take, you know, like the sec mouse brain you cannot dissect all of them at once. So you can keep them on ice for several but quality of tissue. And on the other hand, we were actually collecting, you know, bioptic tissue, and the tissue was maybe not even better than this and so really really made us to think about it. And basically what we, we concluded from these studies that not all is lost, at least immediately, and that the cell that in the post mortem brain follows more a gradual and stepwise process, instead of occurring in a very narrow defined time window, immediately after cessation So basically is your cells are not going to die. And you know there is a break it supported this even before we started to think about this. And it's normal that people made cultures in the brains that you know, had several hours post mortem delay. And that means that basically that post mortem brain cells, and in this case, it's a post mortem brain but think about this is such as project arrest and stroke where there is a to some extent focal or global is himia. And so the capacity, at least in the cases of two studies I mentioned this capacity has been maintained ex vivo to fully recover at when it comes to their cell function in tissue cultures and again, under appropriate experimental conditions. And we were really struck by these observation and big question started to really question, whether we could translate these findings from a small tissue specimens and dissociate the cells to fully interact break. And this is an important point that I would like you to convey at the moment is, is, is that the reason we're interested in doing this in the whole brain is that, even if you do organotypic tissue cultures, you lose connectivity. You maintain local connections, but the connections that make the white matter is lost. And this is what our lab was interested in studying is how genes control the formation of those long range connections in the silver quote. There's no way to study them because either you have to dissociate the cells, or you have to make organotypic slices cultures, or you use an animal to trace connections and stuff like that. Well, while you can trace connections in, let's say, mouse or even some non female primates, most of animals are actually not, you know, this is not applicable. And, and, and we really were thinking, okay, maybe we can really capitalize on these observations in cultures and really try to do this with a fully intact brain. And so basically hypothesize basically that that under again, appropriate experimental conditions, circulation and cell functions in that large brain may remain, you know, I have the same capacity for restoration even multiple hours after that. And not just by dissociating them and culture, but maybe doing this in a whole. And, and, and again, just to remind you and refresh you by really testing this hypothesis, instead of like taking cultures in a fully intact brain. We thought that we might be able to better understand how cells in the brain reactive circulatory unless because that was also not well understood. And if we could somehow intervene and to revive salvage or maintain these cells. And, and, okay, and so what you know so we started very naively in the beginning. We actually thought, okay, we can take the post mortem range. And maybe we can find a way to study cells and connections and preserving them for, sorry, I'm like a little parrot to preserve a 3D organization, which was never possible before. And that was our really initial idea how to do this. Okay. And, and after brainstorming about how to test this, we chose pigs for multiple reasons. And, and, and, and also thinking about this and really having the medical background and several members of our team that, you know, physicians, I'm not one. And you know we realize that actually we should really talk to somebody and actually one of the first people we talked about was Tim Layton, who was, who has the Yale at the biotech center here at Yale, trying to really get insight into this. We also talk to colleagues who are really experts in this. And including Steve Waxman and other colleagues who really were studying, you know, regeneration recovery from cardiac arrest or hypoxia and ischemia try to understand how to do this and whether it can be even done. And one thing that came out of these discussions is that we should do this in pigs. Okay, and we chose pigs for, for multiple reasons. But first, and we really, you know, we were thinking, okay, what if you write a protocol you want to do this. I mean, you know, just, it has not been done and I really wanted to be sure again, we didn't want to, we wanted to test ischemia so this is why we chose post mortem. You are not interested in keeping the brain alive. That was not our intention just to make it also clear. We wanted to see if we can develop a technique that will help us recover this and restore cells in intact but like what we have done and others have done many, many other loves have done in tissue cultures. Okay. And so thinking about this how to do this and of course the first choice was to do this in rodents. And I think I was the one against doing this in rodents and the reason for that. And so after really brainstorming and talking to some colleagues at other university, it came to us obvious that actually we should do this in pigs. And so first things are processed every day for food production. And their brains are routinely discarded. And in the USA, I think several years ago that there are, I think, over 120 million pigs were processed that year. And, and, and we, the previous users 120 million missed opportunities to study the brain. And we wanted to take advantage of this procurement method. And, and because it has an important not just ethical consideration, you know, really trying to do something without using an animal, you know, if you can do this, that's, I mean, that's a plus but also important experimental implication because they're not obvious to us in the beginning, you know, and as I said, it's better, not me, but many people say it's better to be lucky that smart. And I'll get point to the advice that's in there. And so, again, great no animals were sacrificed solely for this research. We basically what we did is actually called only local food production facility and and and we asked them, you know, what do you do I mean they use meat, you know, and and they did take advantage of the brain and they basically gave us a scout that was really removed and and base. And, you know, so, you know, we had now opportunities to really think about this and practice this. Okay, the second reason we also chose pigs is that, you know, I was surprised when I look at the pig brain I mean I have seen pictures, but it's really, and it's more than human brain but it's actually the same size as resus macaque brain, which is the most commonly studied non criminal primates. And it's actually, I like macaque has more gyri and and and I, and it has a really large right matter and we felt this might be important, you know, eventually to translate the actual treatments directly to humans and again nothing has been done so far in humans. And, you know, but we felt whatever you do you should do it in a mammal that you can get an access to the brains that you that the brain is large and this really big ended up a really good model and again just not availability but it rates around I think 800 grams, which is like like 7.5 times smaller than human brain, which is like around 1400 grams. And again, but it was I was totally surprised the same sizes as the reasons macaque brain it's complicated which is really make it and it has a similar white to green ratio to the brain. And this is an important thing because if you look at the rodents white matter is like maybe 5% it's small and hammer dynamically it's a different problem. Well, when you take a pig brain that is almost 100 times larger has a large percentage almost 30% of white matter. And all of this made us really think that we should use pig rather than let's say mice or rats, you know, most commonly studied mammals. And again just to repeat myself, because they have small and less complex brains. Okay. Anyway, so normally, as I mentioned to you is earlier is that normally in life people brain maintains homeostasis to a coordinated function of variety peripheral organs. Remember, you need blood to bring nutrients to bring oxygen, but also remove metabolic waste and that metabolic basis process by order such as liver, you need oxygen to get to the lab. And, and, and outside the body this is not simply not the case and, and therefore in order to maintain viability of intact post mortem brain x vivo. We needed to engineer extra corporal perfusion system that pumps in this case solution into the brain, but also mechanically mimics many of those organs. An important point that I would like to convey here is that we decided not to use blood in the beginning, and there were many reasons for that. So again we set up a committee of colleagues who we felt are experts in the field. And, and, and we thought about how to do this now if you want to do and, and so blood will be, you know, some suggest that it's easy, you know, there is nothing to think about. We really use some of the features that that that were created in in in culture systems. And we also wanted to create something that is synthetic and it's under that so that each brain can be perfused with identical solution and then this really I think it's important in creating a process where you can iterate iterate and learn from the existing and if you use a blood you have to gain. Which animal is slightly different so actually that was also one other reason and ended up being really important feature, which took into consideration just to make sure that that we think all ethical aspects of the study. Anyway, and so, okay, now we have come up and took us almost three years to develop the solution just that you know this was not overnight. Okay. And I'm going to talk a little bit about how we created this, but we also had to pump this solution and and and we talked to companies that have been doing this by the way, you know, action. And, and, and, and, you know, we realize that their machines that features cannot do this okay and and for the reason that I'll try to explain. So, in doing this actually wanted to mechanically and as well as physiologically meaning some of the key peripheral organs because, again, brain cannot. It does not function in market. And so in doing so we incorporate the components of the heart. And where we can control flow rate possibility and pressure, you know the blood is not continuously pumped through the organs it's actually creates the veins. And that ended up being an important I'm not going to talk much about it just because I think it's a little bit more technical, but I'm happy to be invested after the questions. And also, we also mimic the lungs so that we can modulate the gases and get and also we finally mimic functions of kidney and liver so that we can control a couple of lights, metabolites and especially pH and and and And so we assemble all these components and and and and so and then we did this with isolated brain and doing this with isolated brain was also an important thing. And in planning phase became clear to us because when we talk to colleagues who actually use pigs for their research it became obvious that actually this will be almost cost prohibited to do in pigs. And we also created by extracting the brain actually this is a whole probably presentation how is one in a step on and call it succeeded in extracting the brain and maintaining the bascular. And actually, so we created a close temperature control loop. Okay. And so this way we control what comes, what goes into the brain and what comes out of the brain. And the volume of that is substantially smaller than you would have that with the whole P. And so this allowed us to basically really test conditions of the perfusion system and and and now one of the is that. And so this is how we did them and if you have questions. So we created this solution and normally this. So the solution has components. So the way we did that we had to have oxygen carrier so we use a in in collaboration with the HBO therapeutical leader in the field actually they created a polymerized version of the bovine amoglobin which is exchange gas exchange in the state. Then we tested that we tested the target in metabolic components sell that inhibitors inflammatory modulators. And then secondly we also thought about it, we weren't sure how successful we will be we have no idea what will happen. And so we really wanted to be sure that we don't do anything stupid and so basically also we wanted to include your own inhibitors, and also there is a second reason for that is accepted city has been known as one of the main mechanism that neurons get damaged in the stroke, or other examples of ischemia, basically, and so we wanted also to target that we also created negative stress inhibitors and exoxidants and pre radical scammages over the rationally designed this package of tested it over almost 300 brains. Okay, in order not to only promote the recovery following prolonged period of interrupted blood flow, so we could not just pump the blood, actually cannot do this with the blood, you cannot and we wanted to be basically sure that we can do this, recover the cells but also maintain that ability, while really making sure that they do not create a global activity. Also, through the entire course of the experiment, we actually measured the activity using the ecosystem, which was a clinical system done by somebody who does this, and was actually in the center of the imaging center. And so basically we decided to use a four hours PMI multiple reason why four hours again we did not want to hook up the live brain to the machine that was not our intention wanted to test for hours was two reasons one we wanted to test the prolonged ischemia but also basically we wanted to practically we could not do it faster because we also get these brains from the slaughterhouse. And so we had the poor group we had a group that we call one PMI hour where we flushed we also flushed the brain actually at the slaughterhouse actually you know we could move it. And then we would extract this brain bring it to Texas one hour get back to the lab. Then we had another group which was a 10 hour PMI where the brains were left untouched at room temperature, then we have a control perfusion where we try to produce the brains using actually perfusion was lacking key components and then also we have what we call backs or brain x shorter perfusion and we started perfusion four hours after that was killed or sacrificed and also it takes a lot of long time to extract the peak brain from the scalp that was probably the peak is probably not good model for that it has a very thick but poor scalp and and just was really, I would say complicated. And so basically these are our and then we refuse for six hours and the total perfusion total period after that was 10 hours, we stop after six hours for two reasons, we could not produce control brains and they were basically degree dating and after six hours of perfusion resource tremendous differences, and also for cost and many other reasons we actually decided to stop with that okay. So, and indeed so first of all, can we produce the brain and indeed, as you can see here with ultra Doppler antra ultrasound. Sorry, when I press this without controlling, you can see that basically. So our perfusion is ecogenic so basically you can see it on ultrasound Doppler and you can see reveal robust flow, red color means a flow through internal carotid anterior all major blood vessels both in anterior posterior parts of the brain. And importantly, you can see that that our waveform waveform waveform analysis show through this is a through pericolosal artery reveals the cardiac like biphasic waveforms indicating a low vascular resistance, and also suggested that downstream vascular blood cells are functional or patent. And then also to assess we can also show this is a brain being produced, and you can press this cortical vein, this is on the surface of the cortex. And basically you can deplete it and you can see that that blood, in this case it's not blood it's our perfusion which is actually red as blood. Okay. And, and, and you can see that it's the thing means that the capillary is going from anterior from arteries to vein is functional. And then also the blood vessels, we saw the blood vessels are also functional that we tested, basically wanted to see by administrating nymodyping, which is a cerebral cerebral vasodilatator and and and basically indicating that basically that if you inject the bolus you should have dilatation of the blood vessels that that's exactly what we so basically to make story short of all of this and finally sorry I forget, you also added this with nano particles and then the image the entire brain using a CT scanner, and you can see beautiful really the entire fusion of the entire brain. We also checked in an MRI how brains look and in 10 hours in our 10 hours there is a decomposition of the degradation of the brain. You can see collapse of the ventricles collapse of the tissue, you see even gas pockets which are formed when the brain is degrading. You can control perfusion, basically the brain goes in a demon I cannot be perfused even if you want to force me to use it. This is exactly what happens also skin that brain goes into diva. And in our brain. This is a brain MRI under the perfusion of our technology. You see really beautiful contrast between white and gray matter. You also measure the, and there is no decomposition. The actual radiology study was a live brain. And this is a veteran radiologist and basically we also measured the water content and show that actually water content in our produce technique but not in in the control one is similar to normal indicating that it prevents rebranding which is one of the key problems in brain injuries and schema. And just so I next three slides I'll show you what happens to neurons, what happens to glial cells, and what happens to function of the new. Basically, one of the areas that has been known, most known for being susceptible to ischemia is a hippocampus C1 field. This is a hippocampus in one hour PMI. Control perfusion and backs perfusion you can see that the red color of our perfusion in the in blood vessels. You can see that in one after one hour cells become swollen that's from edema and then while in 10 hours and control pressure that cells are washed out basically they are dead. You can see in our control you can see beautiful cells that are not all well as you can see here. We see the same number of cells as we would see to one hour control again this is not a zero hour control just because at that time we could not get in zero hours. You can also observe a this is a staining with any of its defined your own on market, you can see preservation of neurons, comparable to one hour PMI, but not in the two 10 hours controls. And you can see that basically there is this activated caspase staining you can see that there is a significant down regulation of activated caspase which is a proxy of some that. And so basically when it comes to this technology preserves your own organization and reduces a activation of caspase, which is a known to be activated or sell that. You can also test what happens to to to to glial cells and and glial cells in the brain are astrocytes oligodendrocytes and microglia. And this is a control brain you can see. This is a control brain, and you can see. So in the red are gfap astrocytes and green are microglial cells and you can see beautifully to entire cortex and the white mirror you see both astrocytes and microglia. You don't see that after 10 hours there is a significant loss of glial cells also perches a basic brain becomes like yogurt. You have to understand we do this at 37 degrees. Okay, and this is our backs per music and and you can see similar distribution of astrocytes and microglia they are not dying, or at least they are not disappearing. We see that they are a little bit thicker process which is a sign of activated astrocytes and microglia nothing surprising. And then we wanted to know, are they functioning and previous study have shown that actually in vitro in, sorry, in this case, in vivo sorry injections of LPS that's like a lipo polysaccharine, which is a whole like receptor in activates in a response by glial cells and induces a production of cytokines and inflammatory molecules, and to test this reaction during the perfusion at the end of the perfusion, we inject LPS in the dorsal part of the frontal cortex, and then basically wait half an hour and then remove that piece of the cortex and basically do a LISA test and using a standard kit and we can see induction of cytokines and inflammatory responses in control in backs per fusion, sorry backs per fusion, but not in the control and indicating that for this to be able the cells have to be viable. Of course the question that probably everybody was interested in us. Okay, can we use this technology to study to study connections and cells. Here are the EM examples from CA1. Again, this is the part of the brain that is most susceptible to ischemia. It has been known that neurons die here. It's selective vulnerability of this and it's not yet known why. And so actually most of our studies were focused on this region because if we can save these cells that means that we are doing something that hopefully it's good. We can save these cells and their survival and so this is a synopsis, and you can see that actually even after 10 hours of PMI, as well as in control, you can see synopsis because the synopsis actually very rugged structure in the brain and that has been known that you can see that this presynaptic butons are a very few vesicles, unlike a short PMI as well as our per fusion, and you can see that so basically, and you can even if you look carefully you can even see that some of these vesicles are using. Of course, we wanted to see what happens with the global activity. All experiments were done with measuring global activity using either base system and more risk and then using an ECOG. So the way we put electrodes on the surface of the brain, we observe the A on that isoelectric so there is no global synchronized activity which is required for brain to have awareness or even consciousness. And then this is a very important, this is where when the study leaked out, most of the public will be got confused. So there is no activity in the brain when it's produced because we also have inhibitors just to let you know, however, when we stop the per fusion washed out the perfusion, we make organotypic tissue slices move those organotypic slices to a controlled medium which is a cell culture, it has no inhibitors, no our perfusion, the cells can are electrically active, and you can see here in the sea, you see voltage traces as well as inward and outward currents mediated by voltage dependence sodium and potassium can indicating that these cells are at least some of them again, we don't know how many are active. And of course, since what is lastly, since now we have evidence that at least some of the features of neurons and glial function are preserved. Not all of them. We can now test what happens with the global metabolism, and the good thing about it is remember it's isolated brain so we know what goes in, and we can measure what goes out. So we're collecting every half an hour, what the perfusion comes out, and then we could see what happens to consumption of oxygen, the brain is consuming oxygen as you can see arterial versus venous gradient. It's consuming glucose and actually these numbers look very similar to what has been reported in vivo. It also produces CO2 as well it stabilizes many of the metabolites are important. So indicating that we can now test that brain is metabolically active. So the bottom line in all of these implications that we have a technology that has two implications one is a research platform. We think that has a potential to change the way we study cells and then circuits in post mortem brain, including maybe one day disease brain we have never done any of this in human brain. We have done it all in pig brain so just like so far. And, and, and also, we do think that our technology is show signs of your protection. First, that anoxic cell that is not immediate actually that sells in an oxy, maybe in your brain can be revived under certain conditions. So we really use this technology to better understand how cells in the brain react to circulatory arrest or anoxia global is himia, and really try to develop this technology to the point where hopefully we could intervene and solve it regarding ethical consideration and from the beginning we actually included two important things which really help us guide us to this time is first we actually had a, we will call it I think embedded why it is this that was late them and actually, we actually took him to lunch. And then he said, okay, that's interesting. And then also we set up a committee as well advisory committee. In the beginning was local I actually also contacted my couple of program officers at NIH so we set up also advisory board that we had actually for meeting before the paper was published. We monitor a protocol, a global activity using clinical instruments that are using clinics, you know, most advanced ECOG, which is grids on the surface you also in G by the way. And what I want to say and that you know what clinically defined this is not the leading brain at least to my definition, this is basically salary active. And also we, we had also an aesthetic so which we want to monitor activity because we didn't want brain to get into activity that was not our goal. And really, I feel thinking about this for last almost eight years I think we need to have strict ethical guidance about possibility, what it happens and, you know, and we do think our technology may be able to recover the brain fully we haven't done that. And we still keep it and really an anesthesia and potentially apply this brain to human brains I think that we haven't done it. We don't have a plans and I do think it's important to really have a discussion about whether ethically and morally is justifiable. At the end, I would like to thank all my colleagues here listed including the PI these are PIs at the end that are part of this and effort and we are testing this technology. And this is a really team effort so thank you for your attention and I look forward to your questions. Thank you so much. And Dr says that that was a terrific, you know, you're absolutely right that this study raised a whole series of ethical questions and lots of commentary around it in fact nature published not one but two ethics commentaries in alongside your paper and me and my colleague at Case Western Reserve Stuart Youngner and another commentary was written by Hank Greeley and Neeta Farahani. So now I want to turn it over to our discussant Dr Robert Trugue. Dr Trugue, he is the Francis Glussler Lee Professor of Medical Ethics, his professor of anesthesiology and pediatrics and director of the Center for Bioethics at Harvard Medical School. Dr Trugue also practices pediatric intensive care medicine at Boston Children's Hospital. And among the specialties are ethical issues around brain death and organ transplantation I'd really like to hear some of your thoughts on this work and let me turn it over to you Dr Trugue. All right, thank you. In Sue. Are you seeing me and presentation view presentation view. Yeah, you're seeing one slide right. I'm seeing slides and future slides. Oh, let me just let me just try again and just see if I can correct that. Oh, okay. Why are we having so much trouble today with our slides. We give from laptops and this is my first time from my office. My screen is so big so it splits it into two that's probably your problem too. So you have that. Did I fix that. Yes. Okay. All right, well, wow, what a what a fascinating presentation. Dr system thank you and this has certainly been something we've talked a lot about within the bioethics community. So let me share some reflections on what I see as the ethical implications of this work. And I'm going to divide my comments into those related to animal welfare, and those related to human well being. We can see your speaker view back again. You can. Yeah. Yes. Now it's fine. Okay, thanks. Thanks. Okay, so for animal welfare. I think the ethical issues will discuss are based on the assumption that the methods used to kill animals for food production are humane that sort of taken as a given. You know that being said I think that we would probably all agree that being slaughtered is probably not a pleasant experience. And so I think that that translates into a commitment not to completely eliminating anything that might be painful, but reducing pain as much as possible consistent with the goal of producing meat for our consumption. I think in that sense the ethical standards of the investigators were exemplary. Also want to revisit here the emphasis that was placed on several distinctions, looking at cellular activity in isolation, looking at what might be organized electrical activity of the brain, and then looking at what might be truly functioning such as the presence of consciousness. And in the paper it said it is important to distinguish between resuscitation of neuro physiological activity and recovery of integrated brain functions. And so to this end inhibitors were used to describe to minimize neuronal interaction, again focusing primarily on cellular activity and isolation. And then a little bit about the use of the EEG to detect organized activity. As the authors acknowledge this is an unreliable indicator of consciousness. We know that patients who are anesthetized or those that we believe to be unconscious and a persistent vegetative state do retain EEG activity. And that's the activity measure is activity that's at the surface of the brain so you don't know what might be going on, deeper down in the tissue, but I was struck, Dr. System as you went as you went through it, how much you emphasize that you saw no EEG activity. And what is so important and if you did how you would react. And so it got me to thinking a little bit about well what if you did see some EEG activity, and what if it closely resembled a human EEG. And that made me think a little bit about a paper that I know in Sue is very familiar with work from the lab of Allison low tree and UC San Diego. And let me just say a word about what they did. They took human neural stem cells and they grew them in a dish for for many months actually up to nine months. And as the cell grew, they self organized, and they form layers like we see in the human cortex, and they eventually grew to be about the size of the P. But what I wanted to point out is that they also recorded EEG activity from these tiny little, you know, pea sized clumps of cells, and it developed over time. And you can see it becoming more organized, more complex, more rhythmic. And by eight months they were seeing organized electrical activity that they reported to be indistinguishable from what you would see in a premature baby. That is the time not to take anything away from your emphasis to find not having EEG activity, but like even if you did what would we make of it. So we know if this were happening in your lab, would you also feel compelled for example to anesthetize these little clumps, or you know what do you do when you're done with the experiment. And they die do you do you do you bury them I mean are they like, you know little humans that died or what do you throw them out with the other laboratory stuff at the end of the day. I commend you on what you did with the EEG but I think when we're worried about things like like consciousness, it gets to be kind of complicated how how we actually determine whether consciousness is present or not. And in this regard I think there might be some interesting lessons that we can learn from the clinical diagnosis of brain death. So when we diagnose brain death, we also have to determine that the patient is irreversibly unconsciousness so unconscious so we want to be sure that no consciousness exists. And so that brings up the question of can we reliably diagnosed unconsciousness. This has been a problematic area in neurology. So for a long time we thought that we could and so when neurologist would go to the bedside of a patient who was thought to be unconscious in a persistent vegetative state. They felt, again, pretty confident that they could do a physical exam and say whether or not this person is conscious. We now know that that's not a very reliable determination and in fact when neurologists do this as a bedside exam, they're wrong about 40% of the time. And we know that because we've taken some of the patients that we that they thought were unconscious and when you put them in an fMRI scanner. They're actually able to answer yes or no questions by lighting up differential parts of their brain. So, you know, when it comes to many patients it's very uncertain as to whether we're able to tell whether they're conscious or not. But I would say that the situation is a little bit different when we diagnose patients as being brain dead. And in this situation. I think we actually have a way of being very confident. And it is because we focus on the functioning of the brainstem. I would say a word about why I think that matters. So here, you know, looking at a human brain here. And we know that, you know, most of what we regard as our thinking and our cognitive activity is happening up here in the cortex. But actually there is this system of neurons here in the brainstem called the reticular activating system and network of neurons. And it is responsible for maintaining awakeness for making us awake. And if that's not functioning we can't be awake. If we can't be awake, we can't be conscious. Now, we can't actually test the functioning of this group of neurons directly. But we do know that this network is in close proximity to a number of other brainstem nuclei, such as those that control constriction of the pupil, or our gag reflex or, or, you know, other brainstem reflexes and so our brain death testing looks at the function of these other nuclei very carefully determines that they're not working. If they're not working we infer that the RAS, which is right next to them also isn't working. And if the RAS isn't working, then we infer that the patient is unconscious. And this is important because it allows us to be highly certain when we, when we diagnose brain death that the patient is not conscious, without having to look at all of this complicated structure up here, we're determining the lack of function is much more problematic. And so, you know, I'm not sure maybe it's just on you or not, you've talked about this but I'm wondering if in your work if removal of the pig brainstem could actually be a way of really providing assurance that your pigs are unconscious, even, you know, even if you were to very successfully necessitate other parts of the brain show integrated activity and function, if they didn't have this part of the brainstem I don't know how the anatomy of a pig necessarily relates to that of a human. But if a pig has a reticular activating system eliminating that could be a way of being more confident that you're not crossing any ethical barriers there. And so, in terms of animal welfare, I think your plans to use cooling or anesthetic in the event that organize electrical activity is seen I think that's wise and prudent and I think you got great advice from from Steve Latham about that. I also think let's let's be aware we shouldn't fool ourselves into believing that we actually have a good understanding of the definition or the neurological substrates of the phenomenon of consciousness. This remains a lot of uncharted territory so I think what you did was good, but we shouldn't just go oh yeah absolutely we know what was going on in those pig brains or even in human brains in a similar condition. And then the second thing I want to address is this issue of human well being and talk a little bit about what I would imagine to be the benefits of this kind of research. And I see, I hope I'm right about this that the benefits of the research could be understood, in terms of improved understanding of the neurophysiology of the injured brain, and that that will lead to improved outcomes for patients suffering from brain injury. And particularly the type of injury that we call hypoxic ischemic injury a lack of blood flow and oxygen to the brain, as opposed to example, or from traumatic injury where the brain is actually physically, physically injured. And my presumption again I hope I'm right here is that since the brain next technology requires an isolated brain for at least a near future this these benefits would likely be indirect I don't imagine that you're a man you know hypothesizing that you would remove a brain from a brain injured patient, resuscitate it with brain X, and then put it back into the patient that yes you're shaking your army, of course that's not going to be the case. But what you are going to learn is what what can we learn about the cellular physiology so that we when we do see patients with brain injury will have a better understanding about how to resuscitate those brains in vivo. And so this sort of raises the question about how we look at the ethics of neuro resuscitation research in general. And in that sense, I would say the risks of doing this research would be very similar to other types of neuro resuscitation research. And the question that comes up is what's the potential that we will do more harm than good. And let me give a couple of examples. So I do critical care medicine and one of the treatments that we now use routinely in newborns with hypoxic ischemic injury is hypothermia to cool the body down and there's currently research that's going on in adults for this as well. Similarly in patients who have traumatic brain injury, there's works work that has been done on decompressive craniectomy, which is where you remove a section of the patient's skull to relieve the pressure in the brain and allow the brain to expand. There's a lot of work that's been done here and, and, you know, some of it's been highly successful but along the way, it's often come under scrutiny, because for many of these resuscitative techniques, you can oh, you can improve overall survival in the patients, but this comes at the cost of increasing survival with with in patients with neurological disability. So you know anytime you use I think one of these neuro resuscitative approaches, you run the risk that patients who otherwise might have died are going to end up surviving but in a terrible neurologic state. And you know so that's something that I think we have to think about. Two examples there another slightly different but I think dramatic one was one that ethicist Joe fins describes in his book rights come to mind. Where he talks about a research subject Greg Pearson, who entered a study in a minimally conscious state so often completely unconscious sometimes with just a little bit of consciousness, and he had an electrode placed for deep brain stimulation. And it worked. It restored him to a state of consciousness. I mean, the research was successful. And now that he was conscious they asked him do you want us to keep doing this should we keep stimulating your brain and he said no this is terrible. I hate living in a case in a state like this and so they stopped. And you know it's pretty remarkable when you think about it that this was really the first time that personal agency had been restored to a person. It was an aesthetic device, such that the person could actually then competently refuse to have the intervention. So, no I guess to maybe summarize this it would it would be that you know, efforts to improve neuro protection obviously a good thing I mean the morbidity that comes with with neurological injuries profound in our society but it's it's not, it's always with with great outcomes, or without risks I should say. Finally, the last thing I want to say is is the impact on organ procurement. And commentators and including in Sue and his colleague, Dr. Younger have worried quite a bit that techniques to restore neurological function to patients with profound in brain injury could negatively impact organ procurement and transplantation. And so as they wrote about in nature the idea would be that those who would otherwise have agreed to donate organs in other words diagnosis of brain death and organ donation would opt instead for experimental neuro resuscitative efforts. And therefore they would never be able to be diagnosed as brain dead and then you know we would have the loss of these organs. Not saying that this is an unreasonable concern, but I think that it's very unlikely. So let me just explain why is that I think that we are rapidly advancing the capability to develop transplantable organs from pigs. So here was an article in the New York Times a while back on the use of gene editing and there's many variations of this that are being done experimentally now. One that I'm maybe more familiar with is being done at Harvard Medical School and George Church's lab actually now in a spin off company from his research where they're editing pig genomes to make it so that humans don't have an immunologic reaction when you transplant the pig organ into a human, and also to get rid of all the infectious viruses that pigs tend to be tend to have a lot of. And now, you know, maybe this is over optimistic with the various companies that are doing this are actually estimating that they could be in clinical trials within a matter of a few years. This would be absolutely revolutionary because it would create basically an unlimited supply of transplantable organs and we could talk about that. I don't think that would be an unadulterated good either, you know, suddenly everybody could have as many organs as they wanted that would come with its own problems. But as I look and that this time tell me if it's different. I mean, I see your work is really progressing over a longer time frame than this and my guess would be is that before we have to worry about reducing the number of brain dead patients and therefore the availability of transplantable organs. I think we are likely going to have alternatives to people as as the source of organs for transplantation. So my last slide my conclusions would be, I think the risk of restoring consciousness and resuscitated pig brains is small consciousness is a very complex activity I think all of the things that you've done to, to reduce concerns about better great. But I think actually there's the potential for you to develop this research much more and start to look at communication between neurons and even neuronal function in ways that would avoid risks of the brains becoming conscious and suggest I don't know maybe that you know removing the brain the brain stem from the pig preparation could be one way of doing that. I would say that improvements in neural resuscitation could benefit many who suffer from hypoxic ischemic or traumatic brain injury, but that these efforts don't always have a happy ending, and there's there's bad outcomes as well as the potential for wonderful outcomes. I am less concerned that these technologies will reduce the supply of transplantable organs. I think that pigs are more likely going to be the solution to the organ shortage, and not part of the problem. I don't stop. Thanks very much. Thank you so much for your perspective on all these issues before we turn to questions that are now starting to trickle in Dr. Sesson, do you want to respond to anything that Dr. True just said. First, I agree with everything Bob said, I just want to say these are all important questions that require careful total consideration. I wish everything in the lab I give moral consideration to everything everything I do in life from plastics to to to everything. And so, so that's so we don't know how useful, or even at all would be useful our technology we don't know that we are progressing relatively slowly for one is just the nature of doing research but the second one is. As I said we have advisory committee of really I felt top experts that NIH has picked up for us. And we want to be sure we actually carefully discuss every experiments we have done with them. So that's just. I don't have a formed opinion on one of those things that's organoids because I do organoids, but I just, this is not something I do a lot in the lab or it's not so it's very hard. I mean, and, and, and the question now is the activity. What does it represent. I mean, because I see in the Q&A somebody wrote a really excellent David wrote a question and, and, and, you know, the brain is not isolated organ and I agree, you know the question is, can you recover the brain. Hypothetically x we will without being peripheral input. I don't know that, to be honest. Another thing that occurred to us and we have, we made our technology available to everybody. So we shared everything we ever wanted to come, even during COVID we opened the lab for them. And one of the uses that other groups have tried and want to use in X vivo or the generation of organs so basically from the same patients and some physicians are very interesting in like when you have a liver cancer. When you have a liver out treated with chemotherapy without treating the whole body put the same liver back on its clean. That's one use that others are investigating and using our technology so we still don't know what will be useful. And again, we don't know how useful would be. I agree with you that you have to be very careful with everything you do in life because you don't know whether you are doing more good or good. It's just the way I've been thinking about it and this is somebody who actually has been thinking about this longer than me says it's like a CPR we give CPR to the patient, you know, should we give everybody CPR, and we do give him it's our own. And it's important to really be very thoughtful and careful about where everything you do and especially assigned so let me turn to some audience questions that have come in and I'm going to continue on with the question you started with from David Jones. And this is related to a suggestion that Bob truth actually just had with removing that. Do you have to, to prepare the brain for brain X the big brain you have to sever the medulla or the spinal cord and what damage this is to the system and as a corollary what if you were to remove a part of the brain system is Dr. So just the one one important thing or so so the way we do this this was designed also by how the food was how the thing is processed. So unfortunately, we are fortunately I don't have any interference it's used USDA approved facility and actually do it on Tuesday and then there is a USDA agent. And, and so they process animal we don't enter the facility so we don't know how they process the animal just to let you know okay, and, and they sever it so we cannot, you know, ask them to remove the full CNS so we never tried to do that just so the reason we did it this way was necessary. Okay. And, and, and so just that explains why we did it. We have never removed the brain stem. The problem is that you cannot do this by removing brain stem because you have to have blood vessels intact and removing the brain with blood intact is an incredibly difficult problem. Okay, so basically we use colleagues that work in neurosurgery to help us do this in a dead pig. In this case, what we get is a really scow we get scow. And we bring it in into the lab and then try to use the tool so because you don't want to sever the blood vessel just to let you know so again, this is dictated how this is done, and just cause we would never be able to do this with a whole pig. It's just, and I feel that that was justifiable, at least at that time. I just, if you can do something without sacrificing the animal, I think it's over. And it's the route to take. So one thing I want to say it's in the paper. I study long range connectivity and one of the tags that we are very interested in and have been working on this corticospinal tract. So when you said they're severe cuts, seven, the brains then you completely cut axoprolyze corticospinal tract axon. And so the first thing I wanted to know actually when they show me sections I was is that the cells are called bad cells that are largest neurons in the cerebral cortex, they are actually very easy to identify the only existing primary I mean I shouldn't say they look like they are from the, you know, live animal. While the one in the control they actually are dying. That is, you know, they basically you cannot even find control. So, and again, we haven't went beyond that point just just and so but just preserving these cells to study them just morphological is what we were hoping to do, just to let you know. And that I think would be, you could consider that some level of success. We don't know whether we are preserving them and freezing them in time, or really reviving them, because we haven't, you know, really tested this, but we think that they, they are not dead. And for several reasons is they look really remarkably healthy, you know, in essays that healthy cells will do. Okay. We have never made slices for motor cortex just practically there is a limit we focused on hippocampus because we knew that. But if and again this is a very huge if these cells are not dead, even though their axles are cut, that will be tremendous success. That means that something in our per fusion technology is helping the cells not to die. One other important thing that also I want to make clear everybody. This is not really neurological function. You know, and, but another thing is that we don't know to what level do we even save the cells for this to be really something you would need to really do this for a long period and try. You know, maybe we are just preventing inevitable, you know, as I tell everybody, it's like finding somebody on the street. So let's say they had a cardiac arrest and their heart stopped, let's say four hours earlier, I don't think this could be done. Just, just my honest opinion, just make it, you know, I really have been having conversations with this low period of time. And I do think that this will not even if they they will be neurological. So going back to what Bob said, you have to, there has to be strict regulations about this just by this is my imperative. But imagine that you give somebody CPR and they open their eyes and that's where we stop the experiment. So now the question is this person going to survive, or they're just going to close the eyes and so this is analogous just make everybody clear we haven't done any of those experiments. So we don't know to what extent we are saving the cells and this requires a lot of work and it's just not easy designing the experiments to be very thoughtful. So there's so much to talk about because we have questions that we can always ask about detail. Let me ask a question about some people have already asked this in the chat. How do you see this translating into human clinical work, if at all, is it going to be indirect the way that Dr. True suggested or do you have a more direct application of this technology. So, and one of the biggest problem we are experiencing now is that we have so many requests from colleagues that are doing this and the problem is this is not how to say something that is easily translatable into somebody's lab because the technology you know it's just complex technologies not like buying a new antibody just to explain so but we actually have built machines for other labs and stuff like that. And I can tell you what others have been thinking about using this I want to stick to the brain. So seeing how well this is working with other organs that has been under that's undergoing current risk by other groups and we know what they are having as a result. So the question is to what extent can you use principles that we have identified here. Other organs also testing other models of the scheme of cardiac arrest, as well as a stroke. That's also is undergoing. And then, I'm not sure anybody's doing that, but really thinking about long lines we have oncologist really talking to us. Could you use some of these to keep organ sex vivo from the patient and instead of like, like if somebody has a kidney cancer. Unfortunately, you have to give them systemic chemotherapy, which will be good if you could avoid person has to give me so you can take one out, treat it, help it heal, make sure that they don't have and give it put it back. I had a cardiologist and cardio surgeons and actually we actually some of them came to the lab asking, okay, what if somebody has dilatated heart that has some kind of heart malformations we take the heart out, give them put them on a pump and discard the heart. Maybe we can recover that heart and really put it back in the same person and again there is no immunosuppression needed because it's the same person. We also get a lot of inquiries actually from both labs and companies and again we are, we have shared the treatment as well as as as agents labs and companies that are doing 3D organs. They, okay, we need a technology that can maintain these 3D organs. And, you know, the problem is, is you have to develop something that is not blood, that is not packed blood cells and what we have is a really can work as a blood. And so we have provided our reagents to everything to these companies and so far there seems to work. I don't know how well because of course they don't tell you everything but considering that they are still talking to us and asking, you know, so, so there are many applications and and so some of them really have very low concern as as as as as you know societal and public health but some of course have. I also have a lot of requests from colleagues all over the world to use this in a human brain. I refuse to be part of those studies and I don't think that should be done, just to mention. Maybe, maybe and again three times maybe one day, but I think before we ever come to that that has to be a lot of work done to make sure that we don't do any harm. And so I have a standard answer to them, you know, you have rights to whatever is published but I really refuse to share equipment and agents for those. And unfortunately, we had those requests, I am sure, considering how aggressive they were in requesting and I felt bad because I like to share everything but I really didn't feel comfortable sharing something at this moment. Maybe in 10 years that we have enough knowledge that this could be used for, let's say, somebody undergoing stroke or something like that but I do think that, again, what Bob said, it's very important to come to the realization that the chance that we do more good is, you know, better odds. And this is why we have cell cultures, this is why animal research, you know, the reason we give animals cancer is not to torture animals and give them cancer is to really test this before we use it in humans. And so here is an example of something that actually should be really carefully, you know, so control and really, really ethical guidance and some kind of consensus. Right. So I have a question for Dr. True. So what if, you know, if we were to define death as irreversible and, and so organ procurement is ethical only if the patient is dead. Doesn't this kind of research upset a little bit of the assumptions made that, you know, after cardiac arrest, you sort of give, give some time, a few minutes, maybe 30 minutes, 20 minutes before you procure the organ. If in principle, Dr. Assistant's work suggests that maybe the brain is much more resilient. And maybe I'm not all functioning is lost of course there's a whole question of how much functioning is necessary for life and for consciousness but but does that throw into doubt some of the sort of assumptions that people have made about when it would be ethically appropriate to procure organs is curious about your thoughts on that point. Any great questions ensue, I guess to respond in a couple of ways one is that brain death does not require the death of all the cells in the brain it requires the the irreversible loss of all functions of the brain. That in itself is a bit problematic because our current criteria don't actually guarantee the loss of all functions but I won't go there. There's also a distinction made between permanence and irreversible. And let me describe it as follows is that we consider the loss of a function to be permanent. If it will become irreversible, assuming that nothing is done to to attempt to reverse it, which is the case when people will normally die. So, you know the loss of consciousness would become permanent before it would actually irreversible because we've made a decision not to attempt to resuscitate the brain in the same way that we say cardiac arrest is permanent, but not irreversible if it's a DNR order we've made a decision that we're not going to attempt to resuscitate the heart. And I think that that's kind of core and how we think about issues of brain death so that we would say in order to be brain dead your brain would have to permanently have lost functions. But whether that would be irreversible is going to depend a lot on what you choose to do and as technologies develop what we think of as being irreversible could very well change. Thank you for that very clear answer that was very helpful. I mean just to really make all of this even more complex one thought experiment just think about if somebody is declared brain dead. What if you put the stem cells into this person's brain and and these stem cells will grow and create activity. I mean is this now reversal of that. That's the answer to that. And, and, and, you know, because even if you reverse some function this person may not remember anything might be just a bullaraza. So I do think that the technologies are here. Not yet. That would really make all of this incredibly difficult to answer and I think we'll have such great areas that there's a society will be divided what to do. So, you know, there are companies that are developing these technologies and, you know, to me, I just to me this is not even thought experiment this will be done, and it will work. And I just don't know what to be outcome, you know, is the person going to have any memory any knowledge who they are and I think they will not. I mean probably they will have something very fragmentary now you have to decide is this a new person, or this is a, you know, I think you know that that this is really. And an important time in our history of society, where, you know, science, you know, it's like, it's like almost to me it's like a right brothers, you know, like, you know, we can fly. But not everybody is realizing what fly can do this or auto honey and this on the scripting the atom, you know, and you know you could use it for good things and all technologies can be abused I mean CRISPR was a good example how was abused. And but also CRISPR is something that can really help society and, and so I think that that that this is really incredibly hard and complex questions that will not be resolved. And I don't think that whatever guidelines we put as a society and ethicist scientists will will need to treat them because something new will come, you know, and really made us think about it. Well, I think those are perfect comments to conclude this session on we're out of time. I want to thank our guests and assistant and our commentator Dr true for joining us today. I want to thank everybody on behalf of the Center for bioethics that sponsored the series. Thank you for joining us today. I also want to especially thank Ashley Troutman and Angela Alberti who helped me organize this series I could not do this without your help. Please join us next month for our very last session for the series. It will be on April 16. My guest will be Magdalena Zernicka gets and she'll be talking about our groundbreaking work, culture and human embryos up to the 14 day limit. So we're really looking forward to that presentation. So thank you for joining us have a great weekend. Goodbye. I'm sorry for not answering all questions. I see that I'm one of questions. Thank you everybody. Thank you. Thank you. And thank you, Ashley.