William Rymer, M.D. Ph.D., Milap Sandhu, Ph.D., Arun Jayaraman, Ph.D., Gordon Mitchell, Ph.D. and Randy Trumbower, Ph.D. present at the Johns Hopkins Department of PM&R’s Grand Rounds on December 13, 2017
I need to use a microphone now. Hello there. So um hello, I don't know if good morning, good afternoon, whatever you had to say, good noon. Um So thanks everybody for coming. This is uh an a as a special time and, and, and lecture. So it's a, a great uh pleasure uh for me to introduce uh Seb who is visiting us uh today from uh in of Chicago, I think um uh most of you uh know or have heard of. Um So is um originally from Australia and uh he's a physician, although he didn't practice much medicine after doing residency and so on, he came for a post dog here to the US um uh many years ago uh who works uh at the time he worked at the NIH and he also, this is a post dog here at Johns Hopkins. So he's been in Hopkins long ago. We were talking about Baltimore Park uh a few decades ago. Um And then is uh I think very known, at least uh because he was uh the engine perhaps behind all the research that was happening in Inti of Chicago. Uh He, he's been the director of the Sensory Motor performance program and a lot of the work that R IC has created the space of rehabilitation. And he's, he's been behind that. So he's uh a very uh well known uh and very important um character in uh developing a lot of the research and concepts uh behind rehabilitation. So, for us, it's a fantastic uh opportunity and great pleasure to, to, to have him here today and to share some of uh his current work. Um And thank you everybody for, for attending. So, so, so it's a great pleasure to be back. I was, I've been back to Baltimore periodically, but usually for conferences and things. But to be back in the Hopkins areas, especially a pleasure to see what's the same and what's different. So, so I want to talk a little bit today about uh a spinal cord injury, about neuroplasticity. And as it turns out, I'm not an evolutionary biologist, but what's become apparent along the way is that actually we are tapping into some fundamental by, by some of the things that I'll talk about, we are probably tapping into some, some fundamental characteristics of the way in which uh different kinds of animals protect themselves from hypoxia. And what you'll see as this evolves is that um the nervous system protects itself rigorously from low levels of oxygen. And it turns out this is a theme that goes through many different animals uh in including mammals and somehow or another, we may potentially have found a way to um to tap into this in a useful way. Um And seekers were appointed, ok. They want to be here. I want to, especially these are my colleagues uh is a research scientist who works along the spinal injury. A is a person uh scientist involved in some of the early studies. Uh The key person driving our work is Gordon Mitchell. Um Gordon Mitchell is a neuroscientist. He was a chair in Neuroscience at the University of Wisconsin in Madison. And he just moved down to the father a year and a half ago lead to a department of physical therapy. He is the main staff at the meeting talks. Are this been for year? He said you so that microphone, that's actually, so I'll use that one for a better. So I do. Yeah. OK. So, so it's hard to see around corners. That's why it was uh maybe this one is functioning. He was, oh that's I see. OK. So we can make this up to you if this is for the camera, this is for the the room. Oh I wanna be able to see. So I do, I heard this up. Do you wanna put this uh in the pocket on it? Yeah. And put it to your table thing. Is that OK? Suffering. Can you hear me stand up anyway? Uh sorry to stop and start uh a special uh mention for about Gordon, because his basic animal work drives this story and without having a rich uh sort of uh catalog of how animals respond to hypoxia, especially the boundaries between uh safety and and potential adverse effects is key. So we are very grateful to uh to Gordon and he remains an active collaborator. So, rehabilitation of the nervous system paraphrased is actually a search for neuroplasticity and by neuroplasticity. Uh I mean, I'm speaking in a temple of neuroplasticity, but neuroplasticity is as uh as part of rehab is an attempt to change the structure and function of the nervous system. So that it avoids the need for people to develop special compensatory responses. If you have a stroke or a spinal cord injury, you develop alternative strategies to do the work uh that you try to do. But Neil says maybe we can change structure and function if not permanently, at least in a long, a long term way. So we know there are established and known sources. These include of course spontaneous recovery, you get a lot of sprouting as a result of neural damage. And we know that exercise and rehabilitation can help to sculpt those sprouts to make the neural recovery more effective. We know or believe that a repetitive task performance, especially if you're paying attention and have strong cognitive engagement helps. We know that there are electrophysi physiological methods, including transcranial magnetic stim coupled with peripheral stimulation. If you do the timing right, you can produce uh a paired pulse facilitation which can be quite striking. But then the last one option, which is basically what I want to talk about is hypoxia. And the bottom line is it's easy, it works quite strongly, it has very strong effects and if done appropriately, it seems to avoid any significant side effects. So it's quite promising. This is not a story about a successful validated therapy. This is a story now about things all being well may turn out to be that in a, in a few years. So, so the story begins with respiratory uh with your control of respiration. And one of the lessons that's emerged over the last couple of years is our friends and colleagues who have been studying your control of respiration, new stuff that we didn't and we didn't talk to them enough because it turns out that a lot of these long-term changes that follow certain protocols of hypoxia have been, have been known and studied for a long time. And one of the early illustrations, this is a famous animal prep. I'll explain at the minute. Recently, Harry Gosh and people in, in Detroit have been studying the. But the point of this prep is that um if you test the neural control of breathing in certain ways, you can show enormous resiliency and the ability to change pathways very swiftly when hypoxia is present. So this is a uh it's called a cross prep. It's a a rat preparation in which uh two things are done. One is to cut the spinal cord on one side of the body in the cervical region. And that interrupts the sending projections from brain stem to phrenic nuclei on one side. So there's no discharge visible on the frantic nerve here on the other side, uh the uh the uh the nerve stays in intact and you can see the periodic discharge on the nerves of the neurogram. But this Nove is then cut. So that diaphragm is not moving. If you do those things closely in time, the animal is at risk to die. I mean, it has no way to breathe. The diaphragm can't function. So what we've known for many years is that over a period of a minute or two, the animal figures out how to breathe. And the only way it can have done that is by taking what was evidently a a coes pathway on this side, on the left and using it to activate the frantic nerve nuclei on on this side of the, of the body. And this happens within a very short period of time, a minute or two. So there's no time for any radical restructuring of neural connectivity. This has to be facilitation of existing pathways. This pathway was there before somehow it has been encouraged, augmented to put out uh a larger synaptic drive to the, the phrenic nuclei. So this is the model uh that sort of provoked the idea that hypoxia can change the way the nervous system functions. So just to set the story up, if you cut phrenic nerve and cervicals, heise section and animal models, hypoxia seems key driver of neuroplasticity. These effects on breathing recovery are manifested very swiftly and must be due to some type of synaptic facilitation. Uh We believe that this infrastructure that sets up this recovery of breathing is also present not just in the respiratory system and your control of respiration in humans, but also in limb circuitry. The same synaptic structure, the same transmitters, everything else seems to be analogous. So that's what's driving. Uh That's the motivation behind some of these studies and that's where we sort of didn't do our homework because as I said, respiratory people have known this effective hypoxia for a long time, but we didn't talk to them enough. So the message is that facilitation and plasticity induction using this a ah which stands for uh acute intermittent hypoxia is is now looking as if it could be a useful intervention. So um I mean, I I'm saying something which annoys my evolutionary biology friends through a fluke of evolutionary biology. This neuroplasticity mechanism could be extended to other circuits. The reason it annoys them is because the separation between breathing and movement is arbitrary. In, in, in mammals, humans, we've got like separate uh anatomical structures and physiological control. But if you're a salamander or something living in murky water where the oxygen concentration may fluctuate and sometimes be extremely low. The act of breathing can be inseparable from the the action of movement. So the the distinction that we draw in ourselves is somewhat arbitrary. Um these rescue mechanisms are are available, as I said, and they can be activated rather readily by uh modest levels of hypoxia. So I'm just gonna review briefly some of the animal models that have driven uh their work in this area. As I said, a lot of it comes from, from uh breathing literature. This comes from uh group in Wisconsin some years ago. And they have developed preparations and equipment which allows them to cycle different concentrations automatically through the day. And this is an example in which a uh a rat has had a um heise section uh of the spinal cord sufficient to drop its breathing. They're measuring tidal volume here and this is baseline level um before the uh the lesion, then they expose the animal to this 10 episodes of hypoxia per day uh uh with each 15 minutes long and they do this automatically for seven days and over a period of time, you get this progressive increase in uh the ability to ventilate. So this animal is able to recover the neural control of breathing quite readily. This is a very high level of CO2 here and not normally what we would see, but the CO2 isn't mandatory to be there but this is using a oxygen concentration of just uh 10.5%. That's about half of what we have in, in the atmosphere. Um And it's roughly equivalent to being on a very tall mountain. It's equivalent to Kilimanjaro. Kilimanjaro is about 19,000 ft. So you can pre climb it. You don't need to get, you know, uh oxygen to do it, you know, when you're working up there because you do get short of breath. But this is a relatively modest level of hypoxia. But in this case, five minutes long, that's not what we do in man, we go much, much shorter. So this is the kind of thing you can see in the animal model, here's an animal model in which uh we're recording from the Frantic Nerve. Uh And there is a exposure of hypoxia dropping down 11% oxygen inspired air. This again is relatively long periods, five minutes. And then, and here is the periodic response on the neurogram of the Frantic Nerve. And then over a period of time, you see this progressive slow increase in background discharge, which is uh a form of long-term facilitation. And it's key that it be done intermittently. Uh So, for example, if you drop it down for 25 minutes, you get uh a sustained increase during the hypoxia period, but you don't get the same augmentation in background discharge, you don't get the facilitation uh that seems to be important. Uh And it, it is important also apparently that the the reduction in oxygen be relatively sharp. Um So um his other tests now relating not to respiratory function but to uh motility as a test of fall in function in AAA Rat, who has trained to do a ladder climbing task. This comes from uh Julian Moore in ca in Canada. And his animal received a sort of baseline performance on this um ladder then has a uh uh spinal lesion in the cervical area affecting fall in function uh that begins to make a lot more errors in the first few uh assessments. And then over a period of time, two things can happen if it's a a sham without getting hypoxia, the performance on the ladder task stays poor. If you expose the animal to hypoxia, using one of those uh uh enclosures that I showed you, it gets progressively better and almost gets back to baseline. So hypoxia can help with recovery of breathing, but it can also help with uh locomotion. So, without going into all the background details, we now have know partly because of basic science. Colleagues, we know quite a lot about the different um metabolic and and biochemical pathways that are involved. We know that serotonin is pivotal, it has to be released early as a result of the hypoxia and that's picked up by chemosensitivity, afference. We know that the serotonin binds and gives rise to uh synthesis and release of BDNF brain derived neurotrophic factor. And then we go through a series of alternative pathways and there are at least now four or five of them which result in facilitation at uh at different synapses including the motor neuron. Um And these uh the sequence can be activated very swiftly within, within a few minutes. And we've now seen similar effects in at least three places. One, as I've shown you in breathing, the second one was in the ladder locomotion, but not surprisingly. Now, you, you can guess it's also found in man. Um So our protocol is a little bit different than the animal model. Uh We use 15% e uh 15 episodes of 9% oxygen using AAA special mask, which allow you to um uh non ox, you know, room air for hypoxia, we use only 60 to 90 seconds. So, I mean, half of that time the, the uh oxygen saturation has not dropped very far. So it only starts to go down towards the 10.5% level. Uh I mean, it drops down to about 85% towards the mid point of this and then it lags and comes back rather quickly. And then we alternate that with a 62nd exposure to room air. And we do 15 episodes of this over about 30 to 45 minutes. And interestingly and importantly from both experimental standpoint and from a, a human health standpoint, most subjects don't know what they're breathing. The effect is so short and so mild that frequently they don't know they're breathing reduced oxygen mixtures. Some people acknowledge or complain that uh the the pulse rate may go up a little bit, but usually they don't know. And then what we do in the earliest experiments is simply to look at a uh a, a rather straightforward outcome variable which is a test of isometric strength. Um This is uh just to back up some of the uh anatomical and chemistry studies showing that you, you get increases in uh BDNF concentration after animal exposure to hypoxia and also increased concentrations of uh of regional serotonin uh as well. So this is the first uh series of studies we did in man some years ago in 2012 and it had a person with um or, or a, a group of patients, I should say with uh incomplete uh uh lumber, uh cord injuries so that they were paraplegic and we put them in the set up here is the, the uh device is called the hypoxia generator. It's made by a company called hypoxic. Um A person has got uh their foot in a uh a foot against the foot plate and we can measure the plan inflection torque that they are producing with the load cell. We are recording electromyogram activity from uh plan of flexes and, and we are also doing pulse, low oxygen and normal concentration. And we can also do sham studies where the person is, is just getting room error all the time and doesn't know it. And we can do a series of outcome assessments including muscle strength and then later on walking speed and, and then endurance in different studies, not all in the same one. And this is what we see. It's fairly representative. So this is baseline before we started the hypoxia uh plan a reflection torque of about 56 Newton meters uh and background emgs. And then at 60 minutes and 30 minutes after completing that sequence of hypoxic episodes, the force has gone up substantially 30 to 40%. And then at the 60 minute mark, it's gone up even further. So we haven't got quite double, but we've got at least 40 to 50% increase in isometric talk. Um And uh we have seen this almost in everybody. Um It's a, it's a side topic as to uh why not exactly in everybody. And we don't really know that we don't know if it's a genetic predisposition or whether there's some other factors that limit everybody's response, but it's in the vast majority, we see it in 60 70% of our patients with incomplete uh paraplegia that they will respond to hypoxia. So this shows you difference between sham who does nothing, uh a population and then uh different points in time following uh this uh sequence. So, um it's very striking, it's very powerful and uh the use of the, of the, of protocol is very easy. It may be deceptively easy because we want to make sure that we are covering, uh, potential adverse events, et cetera. But it's very attractive for all these reasons and it happens so fast. Those of you who are working clinically, um, is that a question? No? Ok. Huh. Oh, well, I'll, I'll show you, uh, an example of that in a little while. We don't know exactly in the legs how long it lasts. But we've got good data on the, the hands and arms. So um hm for those, for those of you doing uh therapy to get 40 to 50% increase in strength alone, you'd be training for months literally. And sometimes you'd never get there in a in somebody with a spinal cord injury. So to, to have a technique which is giving you that rapid change in voluntary force is, is very promising. Now, we all know in rehabilitation that the intervention needs ideally a drug or in this case, a hypoxia ideally will work best if it's coupled with training of other kinds. So this is example where we've trained people with overground walking and I want to show you all the data, but this is a a 10 m walk test. So if you're doing better in this study, the time and the 10 minute walk is a 10 m walk is gonna go down. So here we are looking at after a period of overground training where we've combined the hypoxia and walking and then looked at the effect uh over time, over several weeks on the speed of walking, which is what the 10 m test. And you can see if you get the hypoxia, some people bounce around. But basically the trend is towards more rapid walking. If you give just people sham where they don't get hypoxia, it, it doesn't have a systematic and there's one person who gets very much better, most mostly people do not. So the effect of hypoxia seems also to be present uh following a period of training of of in this case of of of loco locomotive training. So, so the there are now quite a few studies, you always worry that this is something unique to your place, your methods, uh you know, your patient populations. Um And there are now actually six studies that announcement. So this was the first one and we had another one done combined with uh with colleagues at Emory and showed more or less the same thing a resident actually at RC in my study, looking at the effects of anti inflammatory agent with an ibuprofen ibuprofen. That was a different story. But along the way, she also verified that this protocol produced sustained increases in ankle torque. And we've had uh a colleague, one of physician in in Chile. Uh the the the tough work of showing that there was no associated at least short term changes in cognition or in this case, word retrieval. So so far it's pretty safe so that, you know, this is looking a bit more promising other people are seeing it and and the effects are broadly comparable. So the next question is, well, something special about the legs or can it also be seen in the arms which you'd expect? Uh So we tried now several studies not yet published on upper extremity. So we've done incomplete cervical injuries. So quadriplegia, but with uh uh some use of uh hands, Asia CD classification. So we begin to do a preliminary evaluation, we expose people to that similar protocol of intermittent hypoxia and we then do a series of evaluations going afterwards. And we do at this point um uh grip strength, which is the easiest one to do, but we also do uh pin strength, lateral pinch, we do and other measures, coordination including box and block and nine hole peg test as well. And I won't show you all of that. Um But this is where the, the duration of the of the effect is now something we can start to comment on. Uh So here we're looking at a uh somebody classified as between C four C 7, 13 years after the injury. And here we're tracking grip strength uh every hour or two following the exposure to the hypoxia. And you can see sham, which is basically breathing room air without knowing it doesn't change over that period and the grip strength in the hypoxia, uh, this is one person changes substantially roughly the same as what we saw in the legs. But there's some interesting thing now, about time course, this, we brought the person back the next day, 24 hours later and it's still way different and that's been true now, for a substantial population. Um, so this is a, uh, uh, both arms of a person, a group of nine or 10 patients with uh a cervical incomplete injury. And that, and this is the, the, the main uh values again, taken over time, also going out to 24 hours. So you'll see the sham people didn't change much. In fact, they went down a bit and I think that may be uh fatigue, uh most likely. Um whereas the um hypoxia showed a sustained increase, the mean value is not quite as large, it's maybe 2025% but it's clear and these are obviously uh very dif different populations. So, yes, the uh arms and uh and hands appear to show the same uh benefit. Um So because the arms, arms and hands were a little bit different and because there is a, I think a little bit of skill, even though a grip test is not very difficult, you, you have to have some coordination in a wrist and hand to be able to do it properly. We thought it might be useful Yes. Can you clarify what's special about the protocol versus something like intermittent, holding your breath? For example, I tried it and it didn't work. You can't get there. Maybe I don't have the stamina anymore to hold on for long enough. But, uh, it would be nice if you could and you, you could, uh prepare, uh I'll come back to that in a minute. But, so this is now a test of proximal muscles uh to see if we could start to understand a bit better what was going on. Although I have not broken that down to any extent here. But this is a test of biceps. And we're now using high density grids emg recordings uh to record over media, uh long and short haired biceps. Uh and there's a load cell now, so we're measuring uh elbow flexion talk quite precisely. So here is the map of the, of the biceps color coded for intensity uh on the left. And this is sort of background. Uh this is now max at 30 minutes after and then high intensity later. Now you kind of there is actually some force traces in here. It's hard to see. Uh but they're up above uh a 60 Newton which which is not that high. But um uh this in indirectly addresses the question. Well, how do you know uh doctor Reimer that this isn't the muscle changing alone? But the neural signal as reflected in the electrogram is also going up. So there's at least a strong muscle, I mean, at least neural component there remains a possibility that there's also a, a muscle contribution to this. Uh which if I have time, I'll, I'll, I'll chat about in a minute, but the levels of uh to increase at the elbow were much closer to what we saw at the ankle. So there may be something special about large muscles with uh a lot of uh you know, so switch fibers in them. So anyway, um we see both of these effects in uh uh hypoxia effects in both upper and lower extremity. So to, to sort of parallel what we did with training in the lower extremity, we started to train people. This is ongoing using our mayor spring my spring for most of you, it's not a robot. It's a, a spring mounted device which allows somebody with upper extremity impairment to play computer games. It supports the weight of the arm. It has sensors at, at different joints including uh linked to grip strength. And I it's nn almost certainly not better than an expert therapist, but it's a good way for us to quantify exactly what the person was doing. So we can look at repetitions, we can look at errors and we can also change the level of weight support that the person has to provide. So it gives us a a controlled environment and in early results, as we might expect the combination of upper limb training with acute intermittent hypoxia so far is significantly better than sham uh plus upper limb training. So, so the message hopefully is starting to convince people uh that um this may be promising. So this is kind of what we think now that uh acu hypoxia provides. Um I think an extraordinary opportunity to look at a new type of therapy for incomplete spinal cord injury. Uh and that's because there is no therapy currently available that comes even close to uh generating this change in muscle strength in uh such a short period of time. Rather importantly, uh A H is rather simple to implement has relatively low cost. One of these uh hypoxi machines is about $4000. It does not yet include control of co two concentration, which we should probably do. So, one of the things that probably needs to happen if this is to find widespread use is that we need a bit more sensors and other control mechanisms inside the machine. So it may turn out to be a bit more than 4000, but it's not an expensive machine. It's not a lock on the locker costs $350,000. So this is somewhere probably around between five and 10. Um And the whole issue now is is is well, does it last long enough to be useful? And most of all, is it safe? Um So the safety issue is ongoing so far, it's I mean, it's hard to prove a negative, not seeing a problem is reassuring, but you don't know if you just keep going another week or a month or something that you wouldn't see some kind of adverse effect. So that, that's not something that one can answer readily. It has to be accumulated painstakingly over time. So one thing I mentioned just now, we say it's nerve meaning CNS, but we cannot say that some of this improvement in strength is not due to muscle. And there are a number of athletic training protocols around the world where people use roughly similar protocols and do change muscle strength. This is not the same. However, as training at altitude training at altitude, you have to be there for a long time. Uh and it uses different mechanisms than what we see. But there may be some uh protocols in intact people that have em emulated this in the past. This is not what most exercise scientists tell you about. When they're training their athletes, they tend to use much more prolonged exposure to hypoxia than we have. So there's something special about the short bursts, which seems to be important and there's relatively little literature in intact subjects on that story. In fact, we're looking at that now we sort of went backwards, we started on spinal cord injury when if we'd have had a head screwed on, right, we would have gone backwards and done it in, in intact persons, risk factors. We know that hypoxia can produce uh endothelial adverse effects can promote neural inflammation if it's severe. And long enough, we do not know yet how to optimize the hypoxia dosage. Right now, we pretend that humans are slightly larger rats. We're following exactly the rat protocol and it's proved to be safe and effective. We don't know how to combine the training with hypoxia. Fortunately, the animal model promoted the view especially again from the respiratory literature that there was a period where things had to gel 30 to 60 minutes before the training for uh certainly for breathing was useful and also in the rat working on, on a treadmill or something. So we have a period in which we can finish the hypoxia and then start some kind of additional training. But we don't know if that's optimal. As I said, we, we're following the rat. We don't do not yet know or are starting to get a better idea about time to peak and the forgetting curve. If you took, if you think about hypoxia as a medicine as a pill, you would look at those response curves, you would look at how long it takes to reach peak value and you would track how long it takes to decay because that will then give you some hint about how to stack it in time. Uh And the nice thing about OXY is it's relatively easy to do that much easier than to do that with most physical therapy protocols, we do not know what the dose response curve is for most ptoot interventions. Uh This is a lot easier to do when we are doing it right now. And also, as I said, we copy the rat and uh and it has proved to be a safe base to use to design the protocols, but it may not be optimal. I mean, you know, we may do better with longer or deeper uh exposures than we're currently using. OK. So just to sum up uh that's kind of where we are, it's work in progress. And this is uh some of the funding sources that uh we used to support the work. So I I welcome uh comments and criticisms, et cetera. Thank you. So maybe it sure, I don't know if it's working. Good question. So do you think, I mean, I have a few but um let you go. So one is uh any theory or knowledge about where this is happening? Is it a spin level or is it uh more of uh upstairs to frame mechanisms that engage? And the other question that I have is is your strength changes versus uh it also, do you see any evidence of behavior, evidence of control uh the point? So most of the uh so most of the basic work has been done in spinal cord uh and it started with breathing and we are sort of replicating the breathing work to show similar effects in other uh spinal systems in including fall in control in the rat for that ladder climbing task, et cetera. So it is affecting uh serotonin BDNF and spinal cord at least. Uh there is no reason that it should not also be working in cortex. Uh But we don't, I don't know how to quite yet how to track that. The problem about. One of the reasons we started to do pinch strength as well as nine hole peg and, and block and block was to see if there was some demonstrable change in coordination. But the problem with that is you have to be able to extract what was coming from strength alone. If you get stronger, you get better at many of these tests. And we, we need some other test which can separate at least to some extent the skill and coordination from the strength. I I think it can be done, but we don't know the answer yet. So we, we could just be seeing uh strength mediated effects. Yeah, I'd like to hear some of your comments about possible. Um My hearing is not great. So can I come like to hear some of the comments about the effects of this treatment on the developing brain? Um Whether or not you have any data about the effects on uh normal plasticity that occurs in a immature brain. Uh Being a pediatric doctor, I'm very interested in um non traumatic forms of spot injury such as transversal my and acute my, which is something that we see in uh kids. So for something like this, so that that is a concern, there are issues both positive and not so much negative. But concerning we have a lot of people now, I mean, uh the uh Gordon who's sort of been the centerpiece of all of this work has, has had interest from people studying multiple sclerosis and especially a LS and uh her injury, spastic diplegia. There are a lot of uh human illnesses that may also be of value. We chose spinal cord injury because they were chronic and stable and uh hopefully without potential harm. Uh you know, in terms of, of cortical function, uh there, there is a serious concern about adverse effects in the developing brain. And that one of our colleagues David Gazal, who's a pediatric pulmonologist from the University of Chicago. He acts as an advisor for us. He shows us scary pictures of uh kids with prolonged sleep apnoea and they show significant differences in cortical thickness on MRI scans, et cetera. And now that's severe repet, I mean, I'm not a pulmonologist but kids having repeated sleep apnea, you know, as a result of, of tonsils or something or, or other reasons. I mean, they can, their, their, the, the depth of hypoxic can be much deeper and the episode can be much more prolonged than what we see. So hopefully we're not in that neighborhood. But we do worry, you know, if we do small doses repeatedly, will we start to look like sleep apnea? But that's why we're, we're becoming uh experts on cognitive testing, et cetera. And we will do some radiologic studies as far as uh other developing uh brain work. I don't know. There may well be because, I mean, hypoxia and utero is a big problem. So there may be uh some studies there, most of those would be sustained hypoxia. So we sort of clutch onto the idea that 90 seconds at relatively modest level is probably safe, but that has to be proven. So, have you seen a deficit in the effect of A IH based on time? Say that again, sorry, this time since injury influence the effect of A I. So we've only looked at chronic uh and that was again for experimental reasons, if you use some intervention and they're too close to the time of injury, there's some hypothetical capacity to improve. So we wanted really stable. Uh but it may well work better earlier on because I assume your ability to uh release BDNF and to, you know, synthesize new, new fibers is probably better or closer to the time of injury. But we don't know, we've all, we've only stuck to relatively uh long term uh spinal cord injury. Yes. Any change in your life based on your uh that's been, we have not done that. Uh because it's, but it's been suggested that we should because we should, if there is a region of impairment immediately adjacent to the cervical injury, for example, we can test the benefits of hypoxia by looking at say hand function. Uh you know, someone sitting, you know, in, in, in that region, but we haven't done it yet, but it has been suggested as a, a useful test. Oh, I, I have not yet. Uh Monica Perez has, you know, was uh was brought to this religion by Mitchell who's not that far away. He's in uh in Gainesville. And I think she's been doing uh the, the chairs and I think she had something almost ready for submission. I'm not sure if it's gone in. But I mean, I, I would expect to see changes, commensurate with changes in, in strength, more or less, maybe not linearly, especially the strength starts to climb. But we have not, I also would expect to see changes in spasticity which will look the same, but we haven't tested that yet. But uh hm. Yes, I wonder your job to actually think about a patient. But also when I go to the gym, I'm seeing people are wearing or working out with a resist of their, uh what are your thoughts on that? So can you repeat the question with microphone? Repeat the question. Well, my question is I, I, I'm a patient, I'm thinking of but um in the gym, I'm seeing athletes wearing face masks that, of making it more difficult to breathe for part of the training program. Well, I think uh it would probably be equivalent to limb training with hypoxia. If you did resisted training and breathing coupling it with hypoxia probably would get, you know, increases in total volume, et cetera. But I'm not aware of any drug test of that. I had two questions. Um One was, do you suspect that perhaps with ridiculous, we may be able to see a similar effect. And then the other question that I wanted to ask was about uh you focused on motor pathways. But I was curious if maybe anecdotally, some of these patients had pain, for example, which is very common for a spinal cord injury if you in your lab have seen any effects of that as well. Um I don't know if it would work on Meath or there are, I mean, there are sodium channels of different kinds on nerves and they are responsive to uh monoamine and neuro modulators. So, but it wouldn't have the same a synapsis out there. Um No, no much sprouting. So I, I think those things would not not be helpful. Um What was the second? Sorry, the other pathways besides? Oh yes, we've only anecdotally. I mean, I think it's highly likely that it's happening. But uh well, I didn't ask for pain specifically, but we have sort of asked about sensation, but we didn't do proper control I mean, some, they, they, they're in a complicated protocol and, you know, squeezing load cells and things. So it, it would have to be set up more carefully than we have done. But we've certainly asked about it, you know. Thank you. Mhm.