 Well, welcome everybody to Grand Rounds. It's eight o'clock, so we'll go ahead and get started. Our first presenter today is Wyatt Messenger. He is a fellow of Dr. M. Bodies. He went to medical school at OHSU and is originally from Portland. And today he's going to be presenting on COMP and Juan, a novel treatment for CRM. So thanks, Russell, for the introduction. So it's a privilege to be here at the Moran. It's been great working with Dr. M. Bodies this past three months. And it's been great to get to know a lot of you guys, look forward to meeting the rest of you as the year progresses. So like Russell said, my topic is COMP and Juan, the treatment for CREO. Before we get started in the COMP and Juan, let's talk a little bit about the anatomy and the blood supply of the retina. The retina is supplied by two main vessels. The first vessel is the posterior sclerary artery, which supplies the sclerar cord and all the way up to the photoreceptors. And the second main vessel is the central retinal artery, which pierces the optic nerve, travels through the optic nerve, the lamina crevosa, and it becomes the arcuardaries once it enters the retina. So CREO generally presents, with monocular vision loss, it's painless. And it's, but there's actually three different types of CREO, even though we normally talk about the non-artic type, which is the most common. This is from atherosclerotic disease, embolus, thrombus. It can also be from trauma as well. In younger patients, it can also be from a hematologic causes, or systemic or collagen vascular diseases as well. It presents with the cherry red spot, which is the classical presentation of CREO. What you see in the cherry red spot is the kind of the whitening of the ganglion cell layer from edema from where the ganglion cell layer has died. And it's contrasted with the fovea, which doesn't have ganglion cells. And so the pigment is retained and you get that contrast of the cherry red compared to the surrounding retina. On fluorescent angiography, there's a normal coroidal filling, but there's delayed intraretinal filling. And this is due to the reason, to the fact that the central retina artery supplies the intraretinal vessels and then the posterior coroidal vessels supply, or the posterior cellular vessels supply the coroid. And so it looks something like this. So as you can see here, the coroid is already filled with kind of that white background. And then you get kind of a, inside the intraretinal vessels, you get a wave front where you see the dye or the fluorescein entering the vessels and you actually see the leading edge of that front. And that's really specific for a CREO, a non-arturidic CREO. The second type is arturidic. And this is from giant cell arturidus. Giant cell arturidus affects, it's a vasculitis affecting medium-sized vessels. So it also affects the posterior cellular artery. And so you get optic nerve pathology as well. And so you can see the optic nerve here has edema, you can get cotton wool spots, sometimes hemorrhages. And on FAA, it reflects the PCA and the central retina artery is included. And so you get delayed filling of both the coroidal vasculature and the intraretinal arteries. The last type of CREO is a transient arturidic CREO. And this is from multiple different causes. You can get a transient embolus that passes through the intraretinal vessels. And then by the time the patient presents, it's already gone. And so you don't actually see the embolus. Hypotension can also be a cause. So a lot of patients take blood pressure medications at night, so they get hypotensive at night when they're not symptomatic. But when they wake up, they've had decreased perfusion of the retinal vessels and can have a central retina artery. It's not an inclusion, but it presents similar to a CREO. And then vasospasm is another cause. The presentation is variable, but and then there's often normal filling of the on-floor singin geography because the vessels are patent by the time they present. And then going forward, we're gonna be talking about non-arturidic. So everything from here on out, it's regarding non-arturidic CREO. So CREO is relatively rare. It presents about one in 10,000 visits to the ophthalmologist. And so the true incidence isn't fully known because it's fairly rare. It's more common in men than women, like 55 to 45. And then the general age that presents is in the mid-60s. And this is just because it's a reflection of vasosporine patients with atheroesthetic disease present. It's almost exclusively monocular, but there are a few cases where it is bilateral. And then the vision prognosis is really poor. So when they present, they usually present with count fingers to light perception. Some patients, well, maybe we'll get up to 2400. And the majority of patients will get up to maybe 2400. But there's a subset of about 10% of patients that will do better. And this is because they have a cellular retinal artery that is not linked with a central retinal artery. And so in that subgroup of patients, about five or 10%, they will get visual acuity about 20, 50 or better. And so I put a picture of this monkey up here. This is the rousous monkey. This is the model they use. Dr. Hyra from the University of Iowa is kind of pioneer in a lot of the CRIO studies. And this monkey was used as the model for a lot of treatments that we now have for CRIO. And what he, Dr. Hyra discovered was that after 105 minutes, the retina suffers irreversible damage. And at 240 minutes, the damage is massive and catastrophic. And so there are several treatments for CRIO. There's conservative and there's invasive treatments. That going through the conservative treatments, ocular massage has been used for several years. The idea being that when you massage the eye, you can actually dilate the intranetal vessels, move the embolus more distally, and preserve the proximal tissue. Carbogen is a mixture of carbon dioxide and oxygen. It's about 5% carbon dioxide and 95% oxygen. The carbon dioxide dilates intracerebral and intranetal vessels. Hyperbaric oxygen in systemic cases of dilators makes sense when you bring the oxygen and you're dilating the intranetal vessels. Pintoxifilin is actually similar to theophalin where it's adenosine agonist. And again, this also dilates intranetal vessels. Enhanced external counterpulsation is something that was tried about 10 years ago. And before researching this topic, I never heard of this before. But what they do is they inject about 500 milliliters of fluid. And then they put blood pressure cups on the patient's arms and legs. And the blood pressure cups constrict during diastole, moving the blood from the legs and extremities to the organs and to the brain. And it's been shown to increase cerebral perfusion. And lastly, the IOP reducing drugs. The invasive techniques are anterior chamber perisentesis where they pull some aqueous humor off of the anterior chamber, reducing intracerebral pressure and allowing perfusion. And then local intracerebral fiber analysis has also been tried. Obviously, it was like TPA or stuff like that. The problem with these treatments is that it's really hard to randomize these patients and also catch them in time to have a large enough sample size to have two different wings in your study. And so this has kind of been the result. So oculomersage does have indirect evidence, but it has never been shown to actually improve visual acuity in a randomized trial. Carbogen and hyperbaric oxygen, as well as anterior chamber perisentesis have actually failed in clinical trials. Systemic vasodilators pentoxyphilin and enhanced external countable station like oculomersage has indirect evidence that it increases intranetal perfusion, but there's no evidence that actually increases visual acuity. The most controversial is local intracerebral fiber analysis. This is like injecting TPA or streptokinase into the super orbital artery or the retro-bulbary to break up the clot. However, this is the one area that this is the only randomized clinical trial on this topic. And it was from the Eagle trial that was done in Europe about 10 years ago. And what they found was there was no difference between the two treatment arms. They had about 40 patients in both arms and TPA did not make a difference. But there are some retrospective studies that have shown some difference. And so the Cochrane review that was done on this topic kind of reiterates this notion and says there's currently not enough evidence to decide which, if any, interventions for acute non-arturidic central artery, central retinal artery equation would result in any beneficial or harmful effect. So that brings us to our treatment, angioprotein one. So angioprotein one is a, is a molecule that's constituentally released by parasites to endothelial cells where it binds the tied to receptors on endothelial cells and stimulates bibustal stabilization via VE CAD here and which is a molecule that binds endothelial cells together. About a year ago, Judd Kahoon, who's an MD PhD in our lab, came in and spoke about our molecule here, compangioin or angioprotein one. And he, his model was in a chronic disease state. Well, we're now gonna flip it to and look at an acute model and see whether or not it works. And our molecule specifically is compangioin. And the comp part is called, is stands for cartilage of ligomatrix protein. And what the function of this is that we bind it to comp to the angioprotein one and it makes it more stable, soluble and potent. Because if you inject angioprotein one directly into the, into the vitreous, it precipitates and never gets absorbed by the retinal cells. And interestingly, they've used compangioin in stroke models with rats. And they will, there's been studies where they've included the middle cerebral artery and then looked at stroke volume. And compangioin is actually reduced stroke volume, attenuated neurologic deficits and increased viable neural mass. So how do we achieve this, the central retinal artery occlusion in a mouse? We inject something called Rose Bengal, which is a solution that's normally liquid that can be photoactivated where it solidifies. And so we inject it through a tail vein injection. It becomes absorbed and enters, enters the, all the vasculature. And then we shoot a laser at the optic disc and simulate a central retinal artery occlusion where it coagulates and the blood is, or the vessel is occluded. We have four different treatment groups. The first group is compangioin protein, which we inject it triviterally at 15 minutes following CRIO and at one week following CRIO. The second is adeno-associated virus compangioin. And this is a virus that we inject into the vitreous. It gets incorporated into the cells and the cells themselves make compangioin. And we can actually test this and we can measure the levels and we can find that compangioin is produced by these cells. And this is injected one week prior to CRIO. The last two treatment arms are controlled. So PBS is a cell solution, almost like BSS, whereas it's injected into the vitreous to simulate an intravitural injection. The fourth group is adeno-associated virus GFP, where GFP is actually made by the viral cells. This is the control for the adeno-associated virus compangioin group. Right, right. These are all different. They only receive one treatment. They receive either one of these four arms. They don't receive, they don't receive both. So after CRIO, we did fluorescence angiography to confirm that we actually included the vessel. And you can see baseline here is before CRIO. And in six hours and 24 hours, you can see vascular leakage, just to confirm and impaired intranetal vessel perfusion. And that's just to confirm that we actually did this, that Rose Bengal actually did the job and included the vessel. So if we look at our path, there's some market changes. So we have normal on the left, PBS our control in the middle, and then compangioin over on the right. And so remember that the central retina artery supplies the inner retina. And so you can see that there's, in the PBS group, there's ganglion cell loss, there's the inter plexiform layer and the inner nuclear layer. There's vacuolization, there's pleomorphism, there's complete disorganization of the inner nuclear layer compared to compangioin. And even the thickness itself is much reduced compared to the compangioin. And the compangioin, I mean, even just appears to look more like the normal. And this was taken at 10 days, following CRIO. So we wanted to know is there, are there metabolic differences between the compangioin group and the control group? And so what we did is we used the computational molecular phenotyping, which was developed here by the Mark Lab. And what we do is we probe 12 different micro molecules anywhere between these can be amino acids, neurotransmitters, or even markers for specific cell types in the retina. And we get 12 different pictures and then we overlay them and colorize them. And we get these really colorful pictures with different cell types so we can actually identify the different cell types and then we can quantify different metabolic characteristics of these cells. And so this is a really busy slide but I'll walk you through it. There's three different treatment groups by row. There's normal PBS, which again is our control, and then compangioin on the third row. The first column is a picture of what this actually looks like when we put these images together. And right away, you can notice that the PBS group has complete loss of the inner retina. And this was taken at six weeks, so at this point the inner retina has actually had time to shrink down compared to the last slides that we saw, which were at 10 days. And another finding that kind of sticks out in this first column is that you can see the ganglion cell layer is much greener in the compangioin group compared to normal. And so in the last two columns, we actually quantify this difference. And so we can actually measure how much the green stands for glutathione and we can actually quantify this difference. So when we look at glutathione levels, and these markers are actually really specific. So we have, we probably identify about 15 different cell types and all of them are within, are almost right on top of each other, these different columns. And so this difference is actually significant. And you can see an increase in glutathione levels in the Mueller end fee of the compangioin retinas. And we look at Mueller cells, the interest is in Mueller cells because they play a supportive role during ischemia in the retina. So this is all well and good. Like we have these great slides that show that the loss of the inner retina in the PBS group and it's preserved in the compangioin group. But ultimately all that really matters is function. And so to test function in mice, we put them in this box and we trigger the oculosophallic reflex, we put them on this pedestal here and we can actually, we rotate these columns around and you can actually watch the mouse track these columns and we, as the mouse, we can watch this and we titrate the size of the column to the mouse's ability to see the column. And so we can actually make these columns progressively smaller and smaller. And it looks something like this when they begin tracking. And you can see the mouse is tracking the different columns they move around. And this is what we found after we followed them for five weeks with baseline being before central retina artery occlusion. Zero is actually at 24 hours and then we followed them out for five weeks. So in the GFP and the PBS controls there was maybe a little bit of a function that was retained in some of the mice. But then the compangioin virus and the compangioin protein there was a significant increase in function in those two groups. So that kind of leads us to where we're at right now with this study. We have several ongoing projects though with this study we were looking at markers for cell proliferation to see if retinas are treated with compangioin or mice are treated with compangioin. Do you have more markers for cell proliferation compared to controls? We're also looking at mechanisms for neuroprotection. So glutathione levels are increased and right now it hasn't elucidated how compangioin may be increasing glutathione levels. So we're looking into that closer. We're also gonna be using transgenic mice in the Tion lab, Dr. Tion's lab. Looking at retinal ganglion cell density and survival to actually quantify this difference. But probably the most interesting part of all of our future directions is the idea of how we're getting retaining function. All the other treatments for CRIO have focused on reperfusion. Breaking up the clot, dilating the intra-retinal vessels, reperfusing almost like the stroke model where we try to bring oxygen as quickly back to the retina as possible. But compangioin doesn't act that way. It doesn't really act that way. We're not trying to reperfuse the retina. I mean, it does stabilize intra-retinal vessels on the field cells, but it doesn't break up the clot or focus on reperfusion. So it raises this interesting question. Do mammalian muller cells have stem cell potential? So there is a study done about 15 years ago in zebrafish where they show that muller cells can actually become photoreceptors in zebrafish. So the pink is stained for photoreceptors over here to the right, and the green is muller cells. And what they did is they laser the retinas of these muller fish, and they found that these muller cells actually turned into photoreceptors, suggesting that they have stem cell potential. And this has never been shown in mammalian cells despite a lot of efforts. But it does raise an interesting question of how endothelial cells could be impacting this. So 10 years ago, there was a major paper in science that showed that endothelial cells when cultured with neuronal stem cells increased neurogenesis, they were way, several more neurons, or the quantity of neurons grew much more in the cultures with endothelial cells versus the neuronal cells on their own. And it suggests that endothelial cells may be playing a role, and it's a supportive role in neurogenesis. And so that brings us to our project. So we have a marker for, so MCM6 is a marker for neuro-progenitor marker, and in tomato red is a marker for muller cells. And what we did is we overlaid the two so the green and the red would make yellow. And by overlaying the two, we could actually get a sense for whether or not tomato red cells or the muller red cells were becoming neuro-progenitors. And what we found is there's significantly more yellow in the overlay of the CRIO group compared to control. And so in conclusion, we kind of have four major findings. Compange-1 preserves inter-retinal structure in hypoxic controls, in hypoxic compared to controls. Compange-1 increases glutathione levels in muller end-field cells, and glutathione again has a, it's a reactive oxygen sequesterant, so it could be playing a role in neuro-protection. Compange-1 improves optokinetic tracking following CRIO. Ultimately, this is the most important marker because function is what we're all aiming for. And finally, Compange-1 may induce muller cells to de-differentiate into other cell types. And again, this is something that's still kind of speculative, but could be a really fascinating finding of our study. Just a few acknowledgments quickly. So Dr. Arambati, for just his leadership in this study, he's kind of in the brains behind a lot of this. Dr. Felix Vasquez from the Mark Lab, he's been a tremendous help with helping with computational molecular phenotyping and teaching me that technique. The Mark Lab in general has donated their supplies and time to this project. And lastly, Jed Kahoon, who started this project with his diabetic retinopathy mice, it kind of got this project started. To hear my references, I got married three weeks ago, so I had to put a picture of my bride up there. Any questions? Yes? Yeah, it's a great question. So there's different ways to make a CRIO in a mouse. We're using the Rose Bengal technique. It probably lasts about, we're not sure entirely how long it lasts. We've done FA at 24 hours, three weeks and six weeks. And about three weeks, the FA start looking relatively normal. And at six weeks, they're almost, I mean, they're almost like normal, completely normal again. So we are planning to do more FAs to confirm that these models aren't perfect. The other model that they use that Dr. Hyra uses like a glaucoma model where they increase intraocular pressure to the point that there's no perfusion of the retina. But Rose Bengal has been used as pretty standard, this is pretty standard method that's been used for CRIO and other models. For sure. The protein is given at 15 minutes afterwards and then at one week after, there's a retrim at one week. And so obviously the protein is the clinically viable treatment because you can't pre-treat someone for a CRIO. And so mostly the difficulty is trying to find a patient, and I mean, you would never find a patient 15 minutes after a CRIO. You'd find them at probably 12 hours, most likely they were 24 hours. And so eventually, if this is gonna be more clinically relevant, we're gonna probably move back the first treatment to about 12 hours to see if it still preserves. But again, I mean, we're not re-perfusing the retina. So if this is truly working by a neurogenesis, then potentially the timing won't be quite as critical as like the TPA trials. Well, the Rose Bengal should be blocking the flow at the optic, right around the optic nerve, which simulates, I mean, most embolisms lodge in the Lamina Corvosa. And so it simulates a CRIO at that spot. Well, I mean, if the embolism will eventually, it will eventually recede and eventually flow will return. And our model is not necessarily trying to re-perfuse right away, but it might be working by, like I said, through neurogenesis and not actually by re-perfusing the intranetal vasculature. Am I answering your question? Yeah, and I mean, as long as there's re-perfusion eventually, so the hematologic, a clot will eventually break up and there will be re-perfusion, not necessarily, like a calcified embolism might not break up, but some of these will break up and there will be a semi-normal FAA long-term. Dr. Warner? Are you talking about for the histology or the, so that's a great question. I didn't put both up there, but we actually, I can go back. Yeah, so this is the virus right here at six weeks. This is the protein at 10 days. So we are just about ready to harvest the protein at six weeks, so I don't have those pictures yet. And then we have the virus at six weeks, so both are match-up, I mean, both show preservation. So, partly it's because mechanistically, like how can we explain this, both the stroke model and also how can we explain this preservation? And then we also, yeah, understandably, and we're gonna be, we're looking at, we're gonna be using like socks too and some other markers for neurogenesis and then that one overlay that I showed, there was an increase in MCM-6 in those Mueller cells. So, ultimately though, I mean, to preserve, we're trying to find mechanisms to explain function being retained too in light of conference one not really preserving perfusion. No. So thank you, Wyatt. Just a quick announcement that was estimate. So just a reminder, Herbert Fred, who's from UT Health Science Center, will be having a special lecture here on Friday from seven to eight. So, we're encouraged to attend if you're available. The next presenter is Ruju Rai. She is coming to us from the University of Boston and is originally from Chicago. She's actually gonna be presenting on demyelin maybe not the rights.