 So we'll go ahead and get started. A quick just pointing information courtesy of Gino, they have a meeting right after Grand Rounds today, so if you would appreciate your expedited leaving of the room so that they can get it cleaned up and turned over. Dr. Hartnett is our speaker today. She's going to be talking to us about ROP and the role of the Jeff in neuro-protection and maybe reminding us of the zones and state disability. Thanks so much for that. Okay, thank you, Russell. And thank you everyone for coming. So I'm just going to speak a little on ROP and some of the work in our laboratory as well regarding VEGF signaling and neuro-protection. So, well that's interesting. Yeah, well let's just go ahead and I'll try to tell you what it says. So we have a 24 and 3 7th week, 580 gram gestational age girl who was a twin. She's twin B. She was delivered by emergency C section due to fetal bradycardia. And her app guards were four and seven, so that's one minute and five minute. She was given surfactant for respiratory distress. So at 32 weeks post gestational age, that's when she had her first eye examination. This is what her rec cam images showed. So again, this is just an image contact for premature infant with optic nerve and vessels. And no ROP stage was present according to the examination. So that was at 32 weeks. And follow up examinations were performed by pediatric ophthalmology. And then at 34 to 35 weeks post gestational age, she had this examination. So what are the zones and stages of ROP in these eyes? So Brian, yeah, can you give me an idea how would you describe this? Left eye? Okay, that's okay. It's hard to tell, but that's good. Good, good. And what's this out here? Probably not fibrous proliferation, but yes, abascular retina. So where the retinal vessels have not yet extended, that's very good. And then what about in the, this is the right eye. Good, good. Okay, so that's a good way of assessing it, right? Great. Okay, good. And then Russell, what would you describe this kind of vasculature here? Good, okay, good. So we'll just go over that. And Brian talked about this already. So zone one is considered if a circle's centered on the optic nerve, and the radius of that circle is twice the distance between the optic nerve and where you think the fovea would be. And of course, the fovea is not mature, so it's hard to do that. And a way to clinically assess it is to center your image of the retina using a 28-diopter lens and an indirect ophthalmoscope on the optic nerve. And if any of the vessels in any clock hour falls inside that circle, it's considered zone one. So you can see there can be a lot of variation in zone one. You may just have one little notch of a vessel, and that can be very different than if you have all the vasculature within zone one. And then zone two is this circle with the radius from the optic nerve to the auricirata, and zone three is the outside crescent left over. And stages, just to go over, stage one is just a faint line. Stage two is a ridge that has volume. If you were able to slow and depress it, you would see that it sticks up into the vitreous cavity. Stage three is extra retinal neovascularization, so blood vessel growth into the vitreous. And in research terms, we call that intravitreal neovascularization, and you'll see that, I'll talk about that a little later. Stage four is a partial retinal detachment, so this is when you start to get fibrobascular changes in the retina, and it can be divided into A or B, depending on whether or not the macula is involved, and B, the macula is involved, and then stage five is total retinal detachment, and I'll show you a picture of that later. So we have the zones and stages of these eyes, so it's probably posture zone two, stage two to three with plus disease. And so what's the broad classification of that? What would we describe that? How about Ron, what would you call that? If you have a kid who's got plus disease in zone two, which stage two to three. Good, okay. So type one ROP, and from the early treatment retinopathy prematurity study, we know that the natural history of type one ROP, and actually it was proven, but there's a 15% risk of an adverse outcome, which can include stage five retinopathy prematurity with total retinal detachment. Now this is a picture of that. So we have clear cornea, we've got some posterior synechia, the iris on the lens, it looks like there's a cataract, but that lens is actually crystal clear, and behind that lens is a total retinal detachment with a fibrobascular membrane. And even with successful surgery of these eyes, and that's very difficult to achieve the visual outcomes poor. So we want to prevent stage five ROP. So what are some of the treatments of type one ROP? The standard to care based on the early treatment retinopathy prematurity study is laser ablation of the peripheral vascular retina. And this is an example of what that can look like. But there was a recent clinical trial that tested an anti-Vedjev agent called Bevacizumab for zone one, stage three plus ROP. And that trial found that in those eyes, there was better, it was less likely that you would need repeat surgery, meaning repeat laser or anti-Vedjev treatment after anti-Vedjev treatment than if those eyes had been treated with laser. So it was only for that subgroup of eyes where they found a significant improvement of anti-Vedjev laser. And actually, why would you consider anti-Vedjev treatment? Well, when early pre-clinical studies were done looking at anti-Vedjev and adult diseases, some of the models that are used, the animal models, are oxygen-induced retinopathy models. And those models have similarities to what we see in retinopathy prematurity. And those models found that VEGF was a causative agent of abnormal pathologic intravitrial neovascularization and that using a number of different methods to inhibit VEGF reduced pathologic neovascularization. We also found that if you can restore VEGF receptor two signaling, so VEGF is a ligand that binds to its receptor, if you can restore the signaling to physiologic levels, you actually cause the cleavage planes of the dividing endothelial cells to become ordered so that it promotes vascularization into the retina rather than having the blood vessels or the endothelial cells divide on top of each other and get access into the vitreous. So that's at least the hypothesis that we had. And there's also, besides that experimental evidence that physiologic retinal vascularization is not inhibited compared to control. So inhibiting VEGF, which is an angiogenic agent, you could say, well, okay, I'm inhibiting pathologic angiogenesis, but in a premature infant, the retinal vascular is still developing. So it's probably wouldn't also inhibit normal vascularization and at least the experimental evidence suggests it doesn't at certain doses. And that may be that if you can restore physiologic VEGF receptor to signaling, you actually can promote normal vascularization and inhibit pathologic. However, since that study, there were a number of reports that were very concerning of persistent vascular retina, recurrent neovascularization, even stage five retinopathy prematurity reported up to a year after treatment with one intravitrile dose of anti-VEGF. And this is unusual because the natural history, after laser treatment, usually we think of kids being in the clear at about 54 weeks post gestational age. So that's much lower than a year after treatment. So people thought, well, maybe it's just that anti-VEGF is somehow changing the natural history. And it's just that in laser, you're already ablating that avascular retina. You're not doing that when you give anti-VEGF. So that avascular retina is still a stimulus that's promoting the production of more VEGF and therefore pathologic angiogenesis. So we just need to change the way that we follow these kids. So that was one thought. However, then there were these studies that show that when you give an intravitrile anti-VEGF agent, you reduce the serum VEGF levels for at least two weeks. And I just came back from a meeting where it was eight weeks that was what was reported in the study. And VEGF is a survival factor, not only for endothelial cells and important in retinal vascular development, but it's also a neuroprotective agent. So there were concerns about this and what effects it might have on a developing premature infants. And then we also looked in an animal model that was representative of our ROP. You know, what would happen if we used a neutralizing antibody to rat, we use a rat model, rat VEGF and follow it over time. So what we found is we could significantly inhibit intravitrile neovascularization, but if we follow those animals at a later time point, they developed recurrent neovascularization into the vitreous and it was a typical looking. It was these plaque-like areas as opposed to the typical intravitrile neovascularization we see. And it occurred in association with like a rebound of increased signaling through VEGF, but also other angiogenic pathways including erythropoietin, suggesting that if we gave more anti-VEGF treatment, like what we do in adults, it wouldn't treat it. We would only be making the situation worse. So I just wanna get back and remind us all how, because I'm gonna be using terminology and I can visualize it. So I think it sort of helps if I can share a picture with everyone. So VEGF has a number of family members. We're gonna talk about VEGF-A. And there actually are at least three different receptors and then co-receptors as well, these neuropylons. The ligand, that's VEGF, binds to its receptor and then it causes these biochemical effects in the receptor that's inside the cell that then can trigger signaling through pathways within the cell and then that leads to biologic effects such as angiogenesis. And we're gonna talk about VEGF-A because that's the one that's angiogenic or thought to be the most is known about the angiogenesis right now. And we're also gonna be probably focusing on VEGF receptor two, which is really the receptor that's thought to be the angiogenic receptor. We also need to remember that there are a lot of different cell types in the retina. And when you're giving an antibody to VEGF, you're binding the ligand so it can't get to the receptor in any of these cell types. So we're not only talking about the endothelial cells which have VEGF receptor two, but VEGF receptor two is also found on Mueller cells, it's found on neurons, it's found on ganglion cells and other cells. So it can have other effects. So we came up with the hypothesis that if we could target the cells that overproduced VEGF in the retina that we would safely reduce intravitrile neovascularization without inhibiting normal vascular development. And that if we knocked down a splice variant of VEGF, VEGF 164, it would be safer than knocking down all of the VEGF A compound. Now, we can't experiment on premature infants and so we must use representative animal models. And the information that we learn from adults is not the same as what happens in a premature infant, especially in the eye and probably also in the body. So we use a representative model of retinopathy or prematurity developed by John Penn. It's called the RAT 5010 oxygen-induced retinopathy model and it looks like type one ROP. It develop, there's a delay in physiologic retinal vascular development and this area of intravitrile neovascularization that occurs at the junction of the vascular and avascular retina. So these are flat mounds taken from rat pup eyes. The optic nerve is in the center because rats don't have maculas. The vasculature is stained with lectin so we can visualize it. And because the eye is round, when you flatten it out, it comes out looking like a clover leaf unlike the circles that we draw. So the other reason that we use, the other reasons why the rat model is representative of human ROP is that the pups and their moms are placed into a cycled oxygen environment and these oxygen levels translate to arterial oxygen levels in the rat pups that are similar to what human preterm infants that develop type one ROP experience. So the oxygen model, the oxygen that the inspired oxygen causes arterial oxygen similar to what preterm infants have. It also uses oxygen fluctuation which is becoming more thought to be important in severe retinopathy prematurity and the model creates extra uterine growth restriction which is also a risk factor for ROP. So historically this has been a great model to use but because it's in rat we can't use transgenic animals easily and so it was hard to study genetic mechanisms but in this we've addressed this problem now. So we previously found out in the rat model the time points when VEGF was increased in the model and we found that it occurred actually earlier around postnatal day 12. So earlier then here's where intravitrial neovascularization occurs and here at postnatal day 25 that's where regression of disease usually starts. So these are the two time points I'll be talking about. So VEGF actually is expressed earlier in the model than when we see the neovascularization. So then we wanted to find out where the VEGF message was. So where the mRNA, what cells were actually producing VEGF? And so we did in situ hybridization which is a way of visualizing where the message is. And so here are the different splice variants of VEGF. This is through the mRNA and we see that VEGF is in the RPE, it's in sort of the photoreceptor outer segments here and in the inner nuclear layer. So the Mueller cells have been discussed by other people that they may be the ones that are expressing pathologic amounts of VEGF as well. And so we wanted to target Mueller cell VEGF and we used a lentivector gene therapy strategy to target over expressed VEGF and Mueller cells only. So this was a novel lentivector that we developed in the lab but we did it through collaboration with a lot of experts. So we designed and tested several SHRNA, short hairpin RNAs to VEGF-A, VEGF-164 or luciferase which is a non mammalian gene and serves as a control. And we tested the efficiency in reporter cell lines that over expressed some of the splice variants and we also test the specificity to Mueller cells in a cell line and also in VEBO. Now we, John Flannery had developed a plasmid that has a CD44 promoter that targets Mueller cells only. And so we, and it has a GFP so it has green fluorescence protein tagged with it so we can see where that lentivector with this plasmid would get into the, where it would go that would go to the Mueller cells. One of the problems or the challenges is that CD44 requires polymerase two to be expressed and to be driven. And the polymerase two is not really that efficient at driving SHRNA. So in order to get by that problem we had the SHRNAs embedded within a micro RNA 30 construct. So it's not a micro RNA but it's within that construct which makes it more efficient to be able to drive the target SHRNA and GFP. So we put this all together, put them into lentivectors and then it took six years. And now the next slide, okay. And then so here are some pictures because pictures I think are more fun than words. And so what we see here is this shows that first the top shows that we are able to transduce Mueller cells. So here's PBS, so this lentivector is given as a subredinal injection at postnatal day eight in the rad pups. PBS we don't see, and these are micron images and live animals. So no GFP but the luciferase which is the control we see good GFP of the Mueller cell and feet and also the VEGF-A SHRNA. Now we want that, we want the control to show GFP so we know that we actually were able to transduce Mueller cells. And then this is a cross section showing a CRLBP labeled Mueller cell co-labeled with GFP. And then here this just shows that when we took the retinas out and measure them for VEGF, this is un-injected RUMIR so this is kind of in development. We see that there's a significant reduction in the VEGF levels by the VEGF-A SHRNA compared to PBS injection or to luciferase injection as a control. So the first thing we wanted to do now that we had the tool was to determine the effect of targeted knockdown of VEGF-A in Mueller cells on the natural, the capillaries, the developing normal physiologic capillaries of the retina and compare it to when we use an anti-VEGF antibody. Because we had found previously that the anti-VEGF antibody had recurrent neovascularization and that it also up-regulated other angiogenic pathways besides the EGF. So we collaborated with Brian Jones here at Moran and we used a synchroscan. So here just gives an example. This is a flat mount of retina that's labeled with lectin. And then we were able, even though it looks like two different colors, this is just dividing out by looking at different planes in the z-axis, the interplexus and the deep plexus. So we were able to actually measure the inner and deep plexi in this model. So then we wanted to study the effect in two different ways. So classically, we just measure the amount of vascular to total retina when we think of the effect on capillaries, on physiologic vascularization. But that doesn't get a capillary density as well as actually measuring the pixels of fluorescence. So we used two different methods. We looked at an extent, we called it extent of vascularization, up to total vascularization. And we looked at the pixels of fluorescence to the total retinal area, okay? And we compared the antivegep to its control and the SHRNA to VEGFA to its control. And Hybel-Wayne really spearheaded this. She's a research assistant professor in my laboratory. And what we found, what she found was that the vascular coverage, the way that we would classically measure the effect on the retinal vascularization, was not affected. There was no difference in each treatment compared to its respective control. However, when we looked at capillary density, the antivegep here significantly reduced capillary density compared to its respective control in both the inner and deep plexus. We also looked at the angles of the cleavage planes and basically when these are two, this is a mitotic figure that it's actually a drawing, but it represents a mitotic figure during anaphase that's been labeled with antifosphohistone H3, which is an antibody that shows us the two dividing in mitosis, the daughter cells. And the angle predicts whether the vessels elongated, widened or irregular or disordered. And we found that the targeted knockdown of VEGF reduced the number of mitotic figures and restored angiogenesis. So it does, it gave us more evidence to support that hypothesis. The targeted knockdown did not have an adverse effect on physiologic vascularization, did not reduce vascular density in the inner and deep plexi, whereas the antivegep did, and it may induce capillary plexus dropout leading to hypoxic stimulus for recurrent intravitrile name vascularization. So that's the hypothesis we developed from this experiment. We also though thought that maybe vascular coverage, like what we see when we look in a premature infant eye may not be sufficient to measure safety what's going on in the retina. So then we look at the second part of the hypothesis, and this is about whether VEGF-164 knockdown would be safer than VEGF-A knockdown. And again, this is just showing micron images that we can target the Mueller cells with our control and the two SHRNAs. And this shows that we can significantly knockdown VEGF in the retina compared to the control luciferase. We looked at two time points, postnatal day 18 when intravitrile name vascularization peaks and postnatal day 25 when we often see regression of neon vascularization. And so this is what we found. So on the top, these are flat mounds stained with lectin, and we see that postnatal day 18, either the VEGF-A SHRNA or the VEGF-164 SHRNA significantly reduced intravitrile name vascularization compared to control. But at day 25, only the VEGF-164 SHRNA maintained that inhibition. And that's what we see in the bottom row and also right here, here. And there was no effect on a vascular area of retina, which is how the readout for the coverage or vascular extent. So as I said, we're knocking down VEGF-A in Mueller cells. And so we were interested in what was going on in receptor 2 signaling and endothelial cells. So we did immunohistochemistry of sections of the retina co-labeled with lectin that stains endothelial cells and phosphorylated VEGF receptor 2, so the activated form of the receptor. And what we found was that P18, postnatal day 18, that each lentivector significantly reduced signaling through the VEGF receptor 2 and endothelial cells. But at day 25, only the VEGF-164, or it did not have an increase in receptor 2 signaling. So this may account for some of that persistent neovascularization that we saw in the previous slide. And this was really done by Yanchao Zhang, who was in my lab and who has now moved on. So it's not surprising to have glial cells produce the ligands that trigger signaling through endothelial cells. And so in nori disease, for example, or norin, which is the protein that's affected by nori disease, which causes blindness in mainly males, norin is produced by Mueller cells and it triggers signaling through receptors in the endothelial cells, Frizzled 4 and LRP5. And this kind of scenario is common in a number of ligand and receptor pairs. But we also have to remember that VEGF can affect other cells. As I said, Mueller cells, ganglion cells, RPE, photoreceptors. And so we looked at safety. Now these are sections, DAPI just labels the nuclei. And then tunnel is a stain that is used to represent cells or to visualize cells that are dying. They can die through apoptosis or necrosis. And what we see here is after sub-retinal injections at postnatal day eight, at postnatal day 18, the VEGF-A SHRNA causes more disruption of the layers of the retina and increased number of tunnel positive cells compared to either the VEGF-164 SHRNA or control. And at day 25, everything looks like it's okay. However, if you look at the thickness of the inner and outer nuclear layer, you found that the VEGF-A actually causes thinning of the outer nuclear layer at both day 18 and day 25, suggesting that VEGF-A in Mueller cells may have an important effect on survival in the photoreceptors and that when you inhibit it, you may be affecting photoreceptor health. But we need to look at function. And then importantly, there were no differences in serum VEGF or body weight gain in these animals. So in conclusion with our research part, we were able to specifically target Mueller cell overexpressed VEGF-A or a slice variant of VEGF in the rat. And that's new, that's novel. We found that intravitrile neovascularization was significantly inhibited by either lentivector SHRNA but only sustained with the VEGF-164 SHRNA and that knocked down a Mueller cell VEGF-A but not 164 caused cell death and thinned the outer nuclear layer. And neither of these affected physiologic retinal vascular development, serum VEGF or body weight. But I think there are some things that we can take away from this and maybe consider when we think about the premature. So let's bring it back to the clinic. So maybe targeted knockdown of overexpressed VEGF-164 maybe safer for the developing rat. You know, we don't know and some of this could still be a dose effect but some of the soluble forms, the secreted forms of VEGF are not affected by inhibiting VEGF-164. So those secreted forms may be able to get to the ganglion cells and the photoreceptors and other cells and maintain their health. Assessing body weight gain, serum VEGF, recurrence of neobascularization or vascular coverage may not be sufficient to test safety because all those parameters were not affected in our study and yet we found significant differences in the effects on the retina. But we need to do studies on structure and function. And so we're hoping that we're funded to continue doing those studies. So we've refined a relevant ROP model and made it rigorous to study molecular mechanisms. And our plan now is actually to start to target the receptors at the endothelial cell level and to try to specifically inhibit the over-activated VEGF receptor to there to normalize it to physiologic states. So let's go back to baby girl, LB then. So she was 34 to 35 weeks post gestational age. The thought was it was post-year zone two with stage two to three plus disease and the broad diagnosis Ron had said was type one ROP. So they're clear media, not stage three plus, not zone one. So the decision was to treat this with laser. And so she had laser treatment of the peripheral vascular retina and then weekly follow-up for progressive stage four ROP or continued vascular activity. So just as a clinical reminder, I guess. So when you have, when you treat with laser, you wanna see that one week after laser that it's not worse. At two weeks you'd like to see some regression of disease. In type one, many times you see regression much earlier than that. But as the baby becomes older and post gestational age to about 38 to 42 weeks post gestational age, there is a change where you start to see fibrovascular change. And at that point, even if there's plus disease, if there's not neovascularization, those cases can go on to develop retinal detachment and they're treated with surgery. Laser won't help that. But if you still have neovascular activity, meaning intravitrile neovascularization of stage three and plus disease, prior to those times that you see the fibrous change, then laser can work. So practically what do we do? If I see vascular activity, so not just plus disease, but I see still stage three present, then I try laser, especially if there's skip laser. Because if you can prevent the need for surgery, that's better. But at some point, you make a decision to go in because as that in stage four ROP, the fibrous tissue comes up to the lens. And you get to a point where it's very difficult to go in and not hurt the lens. And you wanna keep the lens in these kids because they have a better chance for visual rehabilitation. So this was the plan with her. And what happened was, and I'm told she's continuing to regress. So after laser, you can see the laser treatment. You can see that at least in these pictures, there do not appear to be areas of skip lesions. And she's had a resolving plus disease. I'm persistent, some persistent ridge, but as I understand, that's going away as well. Okay, so she was treated with laser. But when do you consider anti-vegeta treatment and what treatments do you use? Because there may be times where this is used. And according to the American Academy of Pediatrics and Ophthalmology, there may be cases where anti-vegeta agents have to be used in order to prevent blindness. And so they came out with a statement in Pediatrics in January of 2013. And these were the recommendations. They recommended Bevacizumab because it's the only anti-vegeta agent that's been tested in a clinical trial so far. So we only have series of cases with Ranibizumab. We don't have controls. Neither, Bevacizumab's not FDA approved for the eye. So it's really important to have a detailed informed consent when you talk with the parents. It's only to be used for zone one, stage three plus ROP, so not zone two ROP. This is, these are from the recommendations. And it's used when other things kind of preclude your ability to treat with laser because laser is the standard of care based on the large ETROP trial. And there were a lot of concerns about the clinical trial with anti-vegeta treatment. Other requirements, weekly examinations until the retina becomes fully vascularized. So this is because the anti-vegeta can cause persistent a vascular retina even year after treatment. It's important to have a log with the dates of treatment and who the treating physician was. And then good communication between the treating and receiving teams upon discharge of the infant or transfer to another NICU. So it's really critical that these kids not be lost because if they have persistent a vascular retina, it is conceivable that that provides a hypoxic stimulus for new VEGF and other angiogenic factors and then that can lead to retinal detachment. We don't know the right dose. We don't know the type of anti-vegeta agent to use or the effects on safety. So those are still questions. And I think it's really important to think about this. These are recommendations based on regions in which oxygenation of the infants is regulated. So we're talking about the United States. There are countries emerging, developing countries that are able to save preterm infants by using 100% oxygen and recreating the ROP that the United States saw in the 1950s and 60s. And those countries, the babies tend to be bigger. They might develop the ROP at 36 weeks gestation, big birth weight babies. Yes, what we need to do is not have them give 100% oxygen. But for the babies that do have that and develop ROP where they don't have people treating with laser, we just don't know. I mean, maybe anti-avastin is the way to do it. We don't want blind babies, but sometimes you can't solve all the problems all at once. So those are things to consider when you're making this decision. So anyway, that is what I wanted to, and then I wanted to talk a little about some of the other studies that we have ongoing in pediatric retina. We have a lot of the team members here, and I'm so glad that everyone could come. But does anyone have any question right now about this on any of the VEGF or the studies that we did? Yes, Leah. We've got it done. Right, you know, that's an excellent question. So I'm told that Mike Tracy did a study with macogen, but I don't think it's ever been reported, but Mike talks about it and he says it doesn't work at all. But he also says that they gave macogen right around 36 to 37 weeks post gestational age. And that's right at the time where you're starting to get the fibrovascular change. So you can get the crunch phenomenon similar to what you see in diabetic retinopathy when you give anti-vegetary treatment. And then the other thing, it's an afterburner. So I'm not sure that it would be, I'm not exactly sure how it works in comparison to other mechanisms to deliver anti-vegetary. Yes, Barb? Right, right, correct. Right, and maybe, I keep thinking it may be that a different way of approaching the drug delivery or the way to target it might be useful. Yes, right. So, yeah, that's a good question. So we have collected the organ, so we haven't looked at it yet, but that is what we wanna do. And also look at the brain, I think with Joanna and maybe Shrana Patel too. We were gonna do sections and also Bob DeGeronimo. I think all that we talked about it, I think it's just a matter of, we have tissue. Do we have, Hibo, do we have tissue that like the brain where we can section it and give to people? We just have the hypothalamus. Should we be doing, getting something else, the whole brain on these? And should we try to do it for sections or should we do it for westerns? Do we know how to do that? Do we know how to collect it? Great. Okay, good. That would be great, Camille. And yeah, great, thanks. So I've always been really cautious because of the effects on the systemic. So I don't think it has, but this is my approach. You know, I try to dilate the baby. If I can't get good dilation, I use atropine as long as it's okay with neonatology. And sometimes I use topical steroids to reduce some of the haze and the angiogenesis that way by reducing inflammation, at least because a lot of times they'll have angiogenesis, as you know, in the tunica vasculosulentus or vascularization. It makes it really hard to see in the back. And then it's like what I say to people when you get a neovascular glaucoma. It used to be where we had to do treatment with laser. It's like you sit down for a long winter night. You just say, okay, we're gonna do the laser and treat. And try to get as much in and sometimes repeat treatment is needed, especially in the zone one cases because there can be flat neovascularization, as you know, that regresses and shows us new skip lesions. But it's very difficult to tell what's flat neovascularization versus what's normal vascularization. Now, if the kid came in that I couldn't treat, I would recommend or consider it, certainly. But I, you know, I think it's a sledgehammer. I think it's more of a sledgehammer than laser, actually, because it affects so much of the kid. Right. And otherwise, those kids would be blind probably or some of them would be, right? So, you know, I hope that they're following the kids and making sure that they vascularize and if they don't, maybe treating with laser. And I hope that they're keeping track. Somebody is keeping track of what's happening as best they can. Some places there aren't that active. It's simply doing it with different injection prints. Yeah. Right. Yeah. Yeah, well. I'd be curious to hear what you find from them. And I want to ask to the neonatologists because one of the questions that comes up when we talk about this, the pediatric retina surgeons' meetings, is how can we tell whether or not a premature infant who, you know, the premature infants who get ROP are the ones who also get cerebral palsy, right? And polyventricular local malacia. So they have a lot of problems going on. How do you tell whether or not the effects on development are from anti-vegeta versus premature birth? I mean, you know, it's, yeah. Well, I think beep-rope, so not beep-rope. What is it called? Block-rope, right? I think there is, David Wallace from Duke is now the chair of PDIC, Bob, tell me if I'm wrong. And he was very interested in the block-rope study which we had submitted to NEI and did not get funding for. But through PDIC, which is a pediatric eye disease, yeah. And is very well respected by NEI. He's organizing a trial. So Bob and I will be, and David and whoever else will be involved, hopefully, with that as well. Yes, Dr. Puff? I mean, it is a good sound. We're going to tell a little bit about the malachite. Thank you. Right. Thank you. So let me just, I'll just move on then and show you some of the other things that we're doing because we've got a wonderful team and it's just such a privilege to work here. I can't tell you how much I've really enjoyed my move. So, you know, we are doing optical coherence tomography and prematurity and retinopathy prematurity and we're doing this as a sort of, as our Moran slash University of Utah slash primary Children's Medical Center kind of group and also as part as one site in a study that's going into NEI as a U-grant, hopefully at the end of the year, to study the hypothesis that we can use OCT images to predict infants at risk of severe ROP earlier than ophthalmologic examinations. And I will also say that this grant right now is also considering looking at OCT images, predicting neural development. So because there are changes that occur in the cupping of the optic nerve, so there are other things that are being seen that may be useful in telling us which infants are more at risk of neural development, neural developmental problems. So the lead is at Duke right now and the goal standard also, my goal too is to understand the relationship of the retinal structure determined by OCT and visual and neural development. So because we're seeing that things are different in premature infants. So these are some images. So Glenn and Siri, Paula and Jim and Mel, our photographers are really, I mean being able to take images of premature infants by handheld OCT in the NICU while the infants are awake. And we also have the whole neonatal nursing group. We have neonatologists that are supporting this. And so here are some images that we see and what we see that are kind of different. So this is the macular region. This is actually the fovea. There's hardly any indentation of the fovea and you can just kind of see some of the cysts here. Now the image quality would be better if we averaged all the images, but this is just raw data. And then here where we do actually see a foveal indentation, you see that there are retained layers of the retina. So during development when you develop the fovea in the interplexiform and other layers kind of move out, they migrate out and you get cone density packing that occurs. So we see these kind of persistent layers here, the interplexiform layer. And so those are the kinds of things that we're looking at. There's also reduced photoreceptor segment length that occurs. So that's one of the things that we're doing and getting on board. We're also looking at genetic and epigenetic associations with prematurity, retinopathy of prematurity and severe RLP in combination with Meg DeAngelis in her lab. We're starting to obtain cords from premature infants and from full term infants through the help and the coordination of the primary children medical center and university nurse practitioners. So we have collaboration there through the help of Brad Yoder, Joanna Bicci, Roger Fakes and Kanyos, Bob DeGeronimo are involved in that as well. And we're collaborating with the Neonatal Research Network nationally to understand genetic variants associated with severe RLP. And then we're looking at the structural features of neural retina by OCT and association with genetic mutations and other pediatric retina conditions and Mimi Young and my fellows Ron Hobb and Nick Batra are working on that. So I'd like to, and those are some of the studies we're also trying to get into developing animation to be able to explain to parents and family members what goes on in some of these complex retinal diseases because when you think about it, we show flat images of a sphere where we have these complex things affecting over time and we explain to people, well, these blood vessels are grown here and these blood vessels are grown here but if we had animation where we could show three dimension over time it may be very useful in improving the education and helping parents understand what's going on. So I wanted to acknowledge my funding sources but especially people in my lab and my collaborators, Hibo here up in front and actually we have to get a new picture because we've had a whole new change in the laboratory and also Brian Jones who's so helpful with the synchro scan and the studies doing that. And also I wanna acknowledge our clinical team with pediatric ophthalmology, the retina clinic photography clinical studies that help us so much with the IRBs, genetic counseling, primary children's medical center, Moran surgery, the teams of the neonatal nurse practitioners at both the UUMC and PCMC, occupational therapy which is now helping us perform examinations on babies without causing them so much stress. All the neonatologists of Utah, Meg DeAngelis in lab and Vanessa Shannon who's my assistant and then Cindy Toath at Duke as well. So thank you very much. And if anyone wants to be involved in any of those just please we're always looking for people to help. Thank you.