 Exercise science and cardiac rehab from the Dr. University of Lithuania. He has been a visitor in the U.S. Army of Affairs for the last 15 years, where he worked in the biological research of the U.S. Army of Research of Physiology at the University of Iowa for David Carter at the Department of Physiology at the Medical College of Wisconsin. Dr. Erick has remained at MCW and is currently an assistant professor in the Department of Pediatrics Division of Critical Care. His research focuses on regulation of cerebral workflow and how the loss of important regularly mechanism results in greater mortality and morbidity following a stroke and traumatic brain injury. Today, he's going to talk about vascular cup. He has to go on with the talk, so sorry, we are short on the time. And I mean, I understand if we don't have this talk on the record, but you know, the students have to go after this and everybody's on a schedule. So I guess we kind of skip this one in terms of being online. Okay, sounds good. So today, Kevin is going to talk about vascular complications leading to end organ damage, role of oxidative stress, and elevated glucose. Kevin, please. Very diverse crowd today. I was told to kind of keep it somewhat educational. So this idea of the barrel reflects within your carotid arteries and even the aorta, you have these sensors, the barrel receptors that respond to stretch, they'll sense changes in blood pressure and they'll act accordingly then to send signals up to your brain, then back to your heart and blood vessels to kind of compensate and then change the pressure, hopefully bringing it back to normal. So when you have a rise in your blood pressure, you'll see a drop in your heart rate and ultimately this brings your blood pressure back and your system is very efficient at doing this. So like if you stand up, normally you'll see that your heart rate rises. It's because your blood pressure drops a little bit. So in a healthy situation, this barrel reflects, helps to maintain your blood pressure nice and stable throughout the day. You don't really see a lot of variability in it. There are several disease conditions, even just aging itself along with different cardiovascular diseases or diabetes, kidney disease. These different conditions could affect this function in this system, so it will impair its response. And if you impair your barrel reflex, it leads to more increases in variability in your blood pressure and those fluctuations in blood pressure then have big complications to damage your blood vessels and other organs. So there are some old studies that looked at what happens when you increase this variability in blood pressure. This graph on the right shows in the white bars in all the conditions is just kind of a controlled animal that has increased blood pressure variability, hypertension, high blood pressure. This is a total damage. This relates to your heart and this one over here is just your kidney or your renal system. What this study was looking at was the gray bar in the middle and then the black bar on the end were two different treatment groups. The one in the middle, they gave a drug that would decrease the blood pressure and this variability and they saw that reduced damage. The other bar, the black bar here on the end, they gave a drug that did decrease the hypertension. So these animals were no longer, they no longer had high blood pressure, but this drug was unable to decrease that variability. So they still had these large fluctuations in their blood pressure. So even being normal-attensive, still having these large fluctuations, they still see this damage occurring. So along with these fluctuations, there's some evidence to suggest that it's really this force going through the vessels, depending on if it's a constant force, the continuous, say, flow or continuous force on the vessels, you don't see much of a change. What happens when you have more of this fluctuating change, if you can induce large increases or decreases in these forces, like the blood pressure going through the vessels, you see this slow rise in these oxidative stress markers. So these oxidative stress indicators, things that you take like vitamin C or vitamin E, all these antioxidants, these are things that would inhibit some of these dangerous markers and prevent some of the increase and then resulting changes in your blood vessels or your other organs. So there's some nice evidence to suggest that variability in flow or pressure leads to this sort of pro-oxidative damaging environment. It's kind of a busy slide, but I wanted to show, this was kind of a cool experiment that we had done to induce variability. So we took some rats and we were able to pace their heart. So we would pace the heart rate at different frequencies and by changing the heart rate, we could change the blood pressure. So if we increase heart rate, you see a decrease in blood pressure. So what we were trying to do was find that actual frequency of the blood pressure variability that would lead to more damage. This works really well acutely. We could do this by pacing the animal and get any sort of blood pressure change that we want. The problem was doing it chronically in these animals. We were having trouble pacing their hearts for long periods of time. Long enough, that would actually allow us to cause some of the heart and organ damage. So there's another model then that we started to use instead of the pacing. We would actually go in and go into that carotid artery around the carotid sinus and we would denervate the barrel reflex. We would take the nerves and do a surgical procedure to just kind of remove all those nerve connections. So when you remove the nerve connections, it can no longer sense the changes in blood pressure. So when you do that, you go from the control condition, nice stable pressure to this very variable pressure because you no longer have that barrel reflex feedback. So this is the model that I'll use and describe for some of the results then. So when you induce this, what is that going to do to your vessels or your heart function? So this is just some other data showing that and so the SAD, this is the denervated rat group. When you do that denervation, you see this increase in these, this is a marker of oxidative stress, this MDA. These ones down here, these are beneficial type of markers. Enos produces a nitric oxide which vasodilates and protects your arteries. DDH2 is another sort of vasodilating sort of enzyme. So when you see decreases in these sort of markers, that's a negative type of effect. So the general idea that we're going with for this study, when you increase the blood pressure variability, you see this change in these mechanical stresses on the vessels. Goes through this cascade, leading to this increase in ROS. That's a reactive oxygen species or the increase in this oxidative stress environment. When you have that increase, you get these downstream dysfunctional vessels, heart and some structural changes that you see, hypertrophy, all these things that could lead to say heart failure. So what we wanted to do was create this model and then see if we could treat it by blocking the oxidative stress with a drug called Tempol. It's kind of an antioxidant. It'll go and it'll get rid of some of these negative oxidative stress markers. So there's been a fair amount of research that looked at this sort of model. A lot of it focused on much larger arteries like ureorta, some of the conduit arteries. So we were interested in looking at some smaller resistance arteries where a lot of the actual function and reactivity is occurring. So these smaller resistance type arteries are ones that really have more control over the flow and the circulation going to your organs. And then the other thing just to see if our treatment to prevent oxidative stress could change and help this condition. So these were the two protocols that we're going to go through. We're going to use some different animal models and different imaging techniques to look at endothelial function within some of these small resistance vessels and then use our drug to treat and see if we could prevent some of the vessel or heart changes. So this was a long protocol. We started out before any of the surgery. We would get some in vivo measures that I'll describe in a couple of slides. Doing some echo data for looking at the aorta. Then we go in and do our protocol to... We would insert some telemeters to measure blood pressure as well as do our denervation to induce the increase in barrel reflex impairment. And then we'd follow these animals over a six-week period and then do some final endpoint measures of heart function. So this is one of our recordings. The top one is the control group. This is just a blood pressure recording. The bottom one is what happens when you denervate the barrel reflex. Hope you could appreciate that. This nice sort of stable blood pressure, it goes away. So this is over an hour recording. You'll see points where you have pretty large drops in blood pressure as well as some spikes in blood pressure. So these animals, it's kind of interesting. So over a 24-hour period, they're still considered normal-tensive. The mean blood pressure still... It's not elevated at all. You have these periods where they kind of look like a normal animal. But then there's other periods just throughout the day where they become potentially ischemic. They have large drop in blood flow. They could have then low perfusion to different organs. So this is some data showing that... Yes, even though we have these large fluctuations, over time they really are normal-tensive. You don't have any sustained periods where they're hypertensive or hypotensive. There's a little bit of an increase at the very first week, but that goes away. That's probably just some of the stress from the surgical procedure. So over the first two, three days, it's a little bit elevated. Then by the end of the week, it comes back. So it's not significantly different between the groups. So this graph shows the... Here's the barrel reflex sensitivity. So this measures how much your blood pressure might change and then how the heart rate changes if it's according or not. So this would just demonstrate that, along with the variability we saw in the blood pressure, this demonstrates that the barrel reflex sensitivity, a lower number would mean less sensitive. So it's not functioning as well. So our denervation surgery worked pretty good and it stayed consistently down, decreased during the whole six-week period. So this one is a measure of the variability in the blood pressure. It's actually quantifying one of the first graphs that we saw. So we can look at the variance within the systolic blood pressure. And you can see that it's quantifies clearly significantly different. And over the course of the experiment, it did come down but it still stayed. This is from about 30, still about 60. So it's still about two-fold higher or more throughout the entire experiment. So this is one of the fun experiments we did. This was a non-invasive way to look at vascular function that we were developing in the lab. We would take some rats and while they were conscious, we're basically just using a camera, something similar that an eye doctor would use and we would be taking pictures of their eyes. By doing this, we could actually start to look at some of the arteries within the eye. So this is an artery within the iris muscle. You can see a zoomed in picture here. We can look at the lumen or the wall of the artery as well as apply different eye drops to cause these vessels to dilate or constrict. So we were able to look at their endothelial cell function or their smooth muscle cell function. I was usually the guy working the camera. I would make the students hold the rats because they were conscious. So it's nice to do experiments if you can in a conscious animal as opposed to an anesthetized animal. So some of the data I have later is in anesthetized animals and we could see that some of those effects are, they just kind of mess up your experiment. So anytime you could do anything in a conscious animal or less invasive, it's a lot better of an experiment. So let's show some data that using that technique, these are in hypertensive rats now. So we gave a drug here that would work on the endothelial cells to cause a vasodilation. So you could see a nice rise in the white bars that control normal tensive group. You see a smaller rise in their hypertensive type of rat. And then this other drug would cause a constriction blocking that endothelial cell function. So we're able to use this in vivo technique to show that hypertension. This is about 10 points of measurement from 1 to 10, right? Why are you imagining it's not in one position? To get an average, if you... So what we tried to do is... I'll show a picture, see? This one here, this is because of the structure of the vessels, we can come back week after week and get to the exact same spot. The one thing you do have to watch is because of different vasomotion characteristics, you might have some constriction here and a little bit of dilation down here. So we would always do our before and after drug kind of in the same spot just to try and control for some of those changes. And then it was, yeah, 10 points kind of an average just because you do have spots where it's more or less constricted. So, yeah, trying to be as... trying not to persuade the data to go one way, I guess, trying to get a more overall picture. What is the distance between these points? So there, you know, that's kind of just... I would probably actually take over that whole area for most of the experiments. This was a diagram, I think we put it in that manuscript just to show where we would place the dots around the wall. So we're able to measure the wall thickness as well as the lumen diameter. And you could basically... the program will let you put them anywhere you want. This picture shows 10. We could do more. Sounds. So, yeah, this was... we would go through, do our baseline imaging before any drugs, give this pylocarpene which acted on the endothelial cells cause a nice endothelial cell regulated sort of dilation. The L name, this is a drug that blocks nitric oxide and blocks the effect of endothelial cells. And then there's another drug that we gave. It's a nitric oxide donor. So it just causes a dilation without the endothelial cells even needing to do anything. So it works directly on the other cells in the vessel, the smooth muscle cells. So a lot of times you'll see in diabetes or other diseases that you lose the endothelial cell function, but the smooth muscle cells can still dilate on their own. So most of the experiments you always have to do is kind of these two different measures to look if it's endothelial or smooth muscle cell related. So we're saying we can measure the wall thickness using this technique. So we didn't see any changes in wall thickness or lumen diameter. So suggesting there really wasn't any hypertrophy or structural changes within the eye vessel. We did start to see some, as opposed to the structural changes, we did see some functional changes. So in the SAD group, the group that lost the barrel reflex and had the increase in variability, over the course of the six-week experiment, we saw this loss in this endothelial dependent dilation. So we put the pile of carpene on. The vessel would not respond anymore. So that was a vessel in the eye. We also wanted to move a little bit more towards the brain. So we started to, so since we couldn't image any vessels in the brain, we would wait till the end of that six-week period and then take vessels. We'd take the brain out of the rat and take vessels off of the brain and do some experiments. So we were using the middle cerebral artery, which is coming off of the circle of Willis right here. The vessel from the eye would actually be a branch of this ophthalmic artery. So they were actually pretty closely related as far as their structure or size of vessels. So similar type of vessel between the iris vessel and the MCA. And so this would be taking a vessel off of the brain and doing kind of an ex vivo experiment in a little vessel bath in a chamber in our lab. So we would do a similar thing. This is the change in diameter in response to another type of drug that would also act on the endothelial cells. And we see that similar impairment in dilation following the denervation. Okay, but is lumen diameter the only quantitative margin that is important? How long for the density of the vessels? So why only the diameter? That's the most important in terms of... As far as a functional measure, the structural remodeling normally goes to more towards like a hypertrophy or change in that lumen, let the wall to lumen ratio. So thicker wall, smaller lumen of the vessel, maybe less blood being able to flow through. And what is important about six weeks? The six weeks was... It's a lot of work to keep them alive. Well, they were very healthy throughout the whole thing, but to do all these measurements every week. It was based on some of the older literature as well showing structural changes that occur by the fourth week. So a lot of the previous literature would look two, four, six, ten. Probably no one did much more than ten weeks. So we kind of went right in the middle looking for where we assumed there should be structural changes already, but we weren't sure about the functional changes if they would be present or not. And so the SAD group is a denervated group. The blue line shows the temple treatment here. So as we saw before, the temple treatment, it didn't affect the barrel reflex sensitivity. That was still depressed. The variability was still present as well. So these brass that were treated with the temple to remove the oxidative stress, they still had that insult of the variability and the loss of barrel reflex sensitivity. But by giving this oxidant, the antioxidant, we were able to restore this vascular function. So that was one of the smaller vessels. We also did some echo data looking at the aorta coming from the heart. So we were looking at kind of the ability of the vessel to relax and if there were any structural changes in like the size of the aorta. So this was another vessel that also showed a, this is a measure of distensibility, kind of how stiff or how much tone there is on the vessel. So a smaller number, less distensible, it means it's a stiffer vessel. So it's not as flexible. So normally when the blood comes out of the heart, it would stretch the aorta and then it would relax and come back. A stiffer aorta like this one in the denervated group doesn't have that ability to stretch. So you get larger pressures that are affecting the heart as well as going downstream, affecting the vessels. And again, we saw some of the ability of the temple to improve this function as well. Skip that one. We'll go, this is the, so... So this is the structure of the aorta. We did some histology looking at the aorta. So we saw that it was stiffer. We wanted to know if there was any remodeling that was going on. And the SAD group in the middle was showing a slightly thicker wall. It was a thicker wall suggesting hypertrophy, kind of increased. You could see the yellow is this, like the smooth muscle layer of the blood vessel. So some hypertrophy of the vessel then that was reversed with the temple treatment. It's just a quantification of those vessel pictures. This one then is the heart muscle itself. This is a histology looking at the collagen or sort of more hypertrophy and fibrosis of the heart muscle. So again, we saw an effect on the vessel. Now we're seeing some effect on the heart. So the increase in the heart weight, hypertrophy, remodeling of the heart. So all these things are kind of suggesting that this variability is leading to a heart failure type of condition in these animals. So the last thing we wanted to do then was to actually look at the heart function. We're able to put a catheter into the heart. So you could see this is the pressure. Pressure catheter showing the left ventricle function. We could look at the pressure at the end of diastole as well as during systole. And then the slope of this line with the rise from the acolyte to systole gives us a measure of the contractility or the function of the heart. And so this was suggesting that we are seeing some diastolic dysfunction. This is kind of what happens when there's more blood that gets left in the heart. It doesn't get pumped out. So it's kind of what happens during early stages in heart failure. But we weren't really seeing any changes in contractility at this point. So we're seeing some early remodeling of the heart and early loss of heart function. So just to summarize some of those results, we're seeing that six weeks in the increased blood pressure variability, we were losing some functional measures of the vessels as well as there were some structural remodeling within the heart and the vessels. And our temple treatment, it was able to reverse a lot of that. It didn't return everything back to normal. So it was somewhat improved, but there was still some room for improvement. So that was suggesting that oxidative stress does play a role, but there were still some other variables that we would need to look at and see what might be there to help fully return function. Okay, so for the second part then, going to switch a little bit towards the brain and some of the effects of glucose. So we've been studying diabetes because it has a lot of the same, there's a very strong oxidative stress component to diabetes as well as a lot of vascular changes that occur. So a lot of the complications that diabetics have with their heart or kidney or brain, a lot of it comes down to these sort of vascular changes that are shown here on the right side. There's just a list of different structure or functional sort of changes that are known to occur in diabetes. So one of the things our lab is interested in is the effect of what happens during strokes. There's some interesting literature that would suggest that the timing, how long you have diabetes or are exposed to the high glucose, as well as the severity, if it's a moderate or more severe case of hyperglycemia, you might actually have different outcomes based on the stroke. They're showing that some of the chronic or moderate hyperglycemia might actually result in less of an infarct. It's almost protective in some cases. It's going to depend on the model and so it's almost like you have this storage of, storage of glucose that helps you during the ischemic portion but then the reperfusion injury tends to be a lot worse. So regardless of the timing or the severity of the glucose, they still have these vascular related reperfusion type of injuries. So this edema or hemorrhage is a large complication in a lot of the diabetic populations. So this is some of the angiogenesis or new blood vessel growth that isn't seen to occur. So in a control versus the diabetic brain, the diabetic brain tends to have more vessels. There's like more collateral that will start to form. And that's in response to the diet, in response to the glucose and an increase in some of the vascular growth factors. So this is one of the concepts that they think might actually help protect against the ischemia. So you have more vessels. So if you have a block, you could have blood flow coming through one of these collaterals and help to restore the flow there. That's what this one paper here showed. They caused the occlusion of the middle cerebral artery. So a similar sort of drop in flow or occlusion. The very next day, the diabetic rats here had more flow on that occluded side even though they still had the MCA blocked. So it was a permanent occlusion. They would go in, tie off the crowded artery. And without restoring flow, the collaterals in the diabetic brain started to restore the flow on its own. I'll be looking at MRI. Um, these are, it's like a speckle imaging. And is it going to be for animal model? Yeah, these were, uh, it's like, uh, similar to the speckle contrast imaging. This was, there's a company that has, uh, it's a pyramid company. They have their own. It's kind of like a microscope giving you the whole image. So, so when you see that restore and so more flow the very next day, what they did was then take those brains after the 24 hours and they're showing that the increase in flow related to a smaller infarct size within the diabetic rat. Although even with a smaller infarct size, they still have more edema and more permeability. So there was kind of this hemorrhage that was occurring within the brain, more blood flow, uh, some of the fluid leaking out into the tissue. So this was after 24 hours only. If you do have the edema and this permeability, if you would let that animal survive longer, some of these factors now are going to, uh, sort of continue to damage the brain. So most likely if you looked at this rat, uh, the 48 hours or three or four days later, this might be flipped. So it's really more about the, uh, reperfusion injury within the diabetic animal or diabetes. So, so diabetes affects the vessels in the brain. It's also going to affect different cellular components within the neurovascular unit. So the neurovascular unit's made up of, you can see down here, you have your blood vessel. You also have some cells within the brain, uh, different glial cells, uh, neurons, other cells related to that are found around blood vessels, this parasite. So not only will the vessel structure function be affected in diabetes, but the high glucose will have an effect on all these other cells that are there and that help to mediate or control blood flow as well. So the one cell that we look at in our lab is this astrocyte. It's kind of right here in the middle of everything. It's one of the schematics you see a lot. You have neurons and the astrocyte is kind of poised in the center there. So it could sense the activity or the communication between the neurons. So when neuroactivity increases, the astrocyte here will get activated. It goes through a whole bunch of different signaling cascades in the middle and on the other side, then it would release different factors to, uh, control or change blood flow. So it would release some of the things we look at or like this one, the eats. These are different things that I'll call this, uh, cause a vasodilation or so when you have an increase in neural activity, you get more of the vasodilator and you get more blood flow. So there's some literature then showing that this DCF is just a measure of that oxidative stress. So high glucose has been shown to increase oxidative stress similar to the model we used the first time and it also is shown to cause a decrease in these vasodilators. So it's impacting the astrocyte. The astrocyte, its ability to control blood flow as well in a negative way. The other kind of opposite of that. So people are now finding out that the astrocyte can also release another factor. Known as 20 heat, which is kind of the polar opposite. It's actually a vasoconstrictor. It's more of a negative type of factor. So even though high glucose decreased the other one, we're now seeing that high glucose probably increases this vasoconstrictor or negative sort of protein. So it's not exactly sure what causes this switch from the antioxidant to the pro-oxidant into the oxidative stress, but there's a bunch of different cytokines as well as these oxidative markers that are increased suggesting a different inflammatory type of processes that are occurring in response to the high glucose environment. So we're doing a cell experiment to kind of look at some of the signaling. We take our astrocytes and we were growing them in a lower glucose media and then we would switch them, either switch them to the high glucose to represent a diabetic condition or keep them in the low glucose media. And then we would do some measures looking at the ability of the cell viability or its health as well as some calcium signaling and then changes in some of the receptors that would be receiving the messages from the neurons. So if it goes in parallel with the tissue? We have not. This other paper, this was some of that here. A lot of it is like some of the fluorescence signaling looking at either hydrogen peroxide or superoxide. So they do show when you have cells in a higher glucose, it does go up. Some of it was kind of time-dependent, like in these graphs with the cytokines. There's some other data showing that the longer they're in, it just kind of does keep accumulating. These are our astrocytes. One of the interesting things we found out, so a lot of research is done on what is considered a neonatal astrocyte. So you take those cells out of like a one, two or three-day-old animal. It's just because it's a lot easier to get them out. People don't usually use cells from adult animals because the brains are so much more developed it's harder to kind of break up those connections and get the cells back out. But what we're seeing is there's a big difference in the effects of the neonatal versus the adult cells. So if you're going to do something like studying Alzheimer's or some of these more chronic disease conditions, it's probably better to use cells from that sort of correct point in development. This is one of the other differences we see. So glutamate is a neurotransmitter that is commonly seen to cause changes in astrocytes. So this is a calcium signal. So most people consider, if you have a rise in calcium in the astrocyte, that's kind of this activation. It tells you the cell is working. So we're looking at the response to glutamate, which is often used to stimulate the astrocytes, and you don't really see any change in the adult cell, but you do see kind of this is a classic response in the neonatal cell. So it's again one of these developmental differences that could really sort of impact your experiments. Again, so this is the effect of glucose. So even though we didn't see, this is a receptor for glutamate, but we didn't see a change in that receptor. We still, or there was no change in the signal, we still can detect the receptor and we can detect that glucose has a large effect within the astrocyte, but maybe only on the adult astrocyte. So this was one other marker we used. So we can't get a nice rise in calcium or activate adult astrocytes, not with glutamate, but other factors such as ATP. So different metabolic factors. So we can't show nice activation of these cells. And this is one that we saw was, had a pretty good effect from the glucose. So when we would switch the cells to a higher glucose, we saw this rise in calcium. So anytime you have an increase in calcium within the cell, that could be this condition of calcium overload, which affects the mitochondria and affects the health of the cell. So that's usually a negative type of situation. So you get this rise in the baseline calcium and then when you try to stimulate the cell, we're seeing less of an activation as well. So this is kind of, then the last part of the talk, try and get through this kind of quicker. So we were looking at the neurovascular unit, some of the smaller blood vessels, some of the cells. Glucose also has an effect then on some of your larger arteries that control the blood flow. So going into some of what is known as the cerebral auto-regulation or blood flow auto-regulation. So cerebral auto-regulation, there's a whole bunch of mechanisms that control it. We still don't know everything. I like this figure legend down here. It's a simplified diagram. And it's not very simple, but this is my simplified diagram over here. So within a certain range of blood pressure, your blood flow should be pretty stable. If you go outside of that range too low of a pressure or too high of a pressure, and then you're going to have these large fluctuations in the blood flow that's going to your brain. So this is a model that we use to study blood flow regulation. So we have some catheters that we have. These are actually in one of the arteries. We just kind of tunnel them under the skin and bring them out to the back so we can access them during our experiment. We have a, this is a Doppler device. This gives us this line down here. So it's kind of a way to quantify the perfusion to the brain. So we developed this sort of technique with the catheters and the Doppler device. So now we can do our experiments in a conscious rat. He's kind of tethered. He's got these things coming off of him. But he's in a cage. He could walk around. He could be active. He could do all these different things. And we have the catheters. We could infuse drugs or measure the blood pressure while we're doing our experiments. So this is one of the main reasons we wanted to go towards this conscious preparation. You see, this is your blood pressure. If we give a drug to rise and increase blood pressure, your auto-regulation should keep flow the same. You get this nice barrel reflex. Heart rate goes down. The flow stays the same. If you have an anesthetized animal, though, the whole thing gets messed up. In that auto-regulation capacity, the normal function kind of goes away. So you have to be able to control the animal so you go back to this sort of normal response. So that last one showed a giving a drug to increase pressure. We also have ways that we can decrease the blood pressure in a nice controlled manner. So we could look at that lower side of the graph to the left of the smiley, green-faced guy. So as we lower blood pressure, you should also maintain your perfusion until you get to a certain point. Just somewhere around this 40 millimeters of mercury here, you get to a point where the blood pressure's so low that eventually the blood perfusion to the brain has to go down. It will decrease. So this is in an animal under room air. And then what we wanted to do was have the carbon dioxide go increase to the animal. What that causes then is this rise in blood flow and a kind of a loss to this auto-regulation function. So then this is our diabetic rat. So we have these nice tools to study auto-regulation. Now we're looking at what happens in a diabetic situation. We have our controlled rat. We decrease the blood pressure. The blood flows maintain pretty well unless the animal's now been exposed to high glucose. This was only even after one week of high glucose exposure. So even after just one week of diabetes, this rat lost all of its auto-regulatory capacity. And then the other component of this, if you restore the blood pressure, you see on the right here the blue line, the control rat, you should be able to control that reperfusion period. This is what we were saying is the worst part for the diabetic rat or the diabetic condition. It's that sort of this larger increase in perfusion. So this is where a lot of the vascular and organ injury comes from. So that goes back to this reperfusion period. So leading to more edema or more hemorrhage within the diabetic condition. This was just a quick showing of some of the different stroke models we can do. But I think I'll stop there because we're about out of time. So this is going to be the next step now is to actually take that diabetic rat. We know it has no auto-regulation capacity. So now we're going to go through and we're going to give that diabetic rat a stroke and kind of see if what we see with the auto-regulation, if that leads to more of this infarct, this damage, and then how can we attack sort of this reperfusion period here to prevent that either in a diabetic or a non-diabetic animal. Great. Thank you so much for that. Pass this microphone around. It's for the equity. Okay. I have one question. You mentioned that there's a separate mechanism like endothelium dilation and smooth muscle dilation. I thought if smooth muscles don't let any dilation, then the cell doesn't dilate, right? I didn't know that endothelium can also dilate the vessel. So it's sending the endothelial cell will send, say, the nitric oxide or the dilating message to the smooth muscle. It will tell it to dilate. So that's kind of that normal response. Even just that shear rate of the blood flow causes nitric oxide release and sort of that dilation of the vessel. So if you damage the endothelial cell, though, you're removing that nitric oxide component. So now you have that shear flowing through. There is no nitric oxide release. So you don't get a dilation unless you come at the smooth muscle cell with something else. And that was what our other drug did, the SNP, that is basically nitric oxide. So we're stepping over the endothelial cell and we're just giving nitric oxide to it, to the vessel, and causing a dilation. So it's possible to have the situation where smooth muscles just relax but the vessel doesn't dilate because of the endothelial. Right. There could be less dilation. In the diabetic condition you see this increase in tone. There is more vascular tone. It's like there's more, say, calcium buildup maybe. That's causing this constriction all the time. That could be due to endothelial dysfunction. It could also be due to this depolarization that's directed at the smooth muscle. Any other questions from you guys? I have a question. So what I understood or concluded is that you said the infarct size in the scabial perfusion model is smaller in diabetes due to endogenesis. Is that right? And some of that is, it's like that other experiment that showed that was, it was more of a, there was some re-perfusion, but it wasn't, say, like they got rid of the occlusion. It's like they didn't treat them, remove the occlusion. It was still ligated. It was still ischemic. There was some flow from the collateral. So part of the smaller infarct size could be that collateral flow from other areas. So it's going around the occlusion. Otherwise, it could be some of what we're seeing now with our astrocytes is, so the brain doesn't normally store energy. The astrocyte may actually store some glycogen. So it might have a small component where it could have some energy substrate around. And in the diabetic condition, there could be an increase in this glycogen content. So that could be part of it, as well as this collateral flow. But it's actually the re-perfusion that causes more damage. I think so, yeah. Especially in the diabetic condition, because you have this sort of change in the blood-brain barrier. You have change in a lot of the vascular properties that would protect, like you see this large sort of re, like when we cause... This is our normal baseline flow. This is a two-hour period where we've actually gone in and blocked the flow to the MCA. And then at this point, I could remove that suture and restore the flow. And you see this huge sort of re-perfusion period. And this is even higher in our diabetic rats. And this is a stoke model, right? Right. Any other questions for Kevin? I guess Romina and I have still a lot of questions. We can do it offline. I think I'll leave if you have to. All right, so let's tank our speaker again.