 Now I have to behave and fabric disease. In the lab, we have several projects which all come together and they're under the umbrella of inflammation and fiber and formation. Even fabric disease has some inflammatory stuff going on after one second to me is the one I'm going to talk about today. I go in the clinic, I see patients and then I go back to the lab, and I look at some of the basic science behind what I see in the clinic, and sometimes some of the things I see in the lab, go back to the patients as well. Now this doesn't work and goes back to patients as well. So I'm always thinking on both sides of the fence and hopefully creating things and finding new information that can help patients down the road in the future. That's my favorite disease rat. So I'll speak about cataract and cataract surgery today. It's the most common procedure in the United States that affects all ages, and some are at a higher risk of complications, particularly children and patients with EVitis to the point where it changes our surgical plan and management. This is something that really got to me when I was a fellow where I had these little babies and they had cataracts and we had to operate right away. But then it was really just really complicated afterwards and that's why people didn't want to do it. So the key here is children are not just small adults. It's not just actually their eyes are almost the same size as an adult at birth that grows a little bit in that first year, but pretty much the same. But they behave differently. One big thing that's different about children is amblyopia. So grandma and grandpa has a pretty gnarly cataract. They don't need it out ASAP because they aren't going to lose their vision and have physiologic and anatomic changes that could be permanent. Children yes. So that's why when a child is born and they have a dense cataract in one eye, we want to do it before six weeks or both eyes. We want to do it before two to three months to prevent nystagmus because the longer you let that vision be low in that eye, the more solidified that poor vision is going to be. Then you have to deal with the amblyopia. So that's half of the challenge, probably more than half. That's because children have a critical period of visual development. If you don't correct the problem and rehabilitate the vision by about eight years old, you're never going to get it back. And that's something that parents don't understand in the post-op period. And the younger the age, the faster it changes. So if you have a two-year-old with a globe trauma and one eye has reduced vision, you're actually racing to rehabilitate that vision. If they have a cataract or something, because the longer that sits there, the faster those changes are going to happen. So same with pediatric intraocular surgery. Cataract is pretty common for intraocular. And if you don't treat it, they get amblyopia and you're fighting against that. The earlier you remove it, the better off you are. The problem is you're trading time and the physiologic and anatomic changes for amblyopia with that exaggerated post-operative response. So that's why we try to wait till six weeks to operate on a child, even with being a lateral cataract. Because if you do it earlier, you could have more complications. And in fact, we don't, I don't know why this keeps going randomly, but in fact, we don't even put in a lens in the eye at MCW, we don't even think about it before the age of one. And between the age of one and two, we think hard about it depending on the compliance of the family. So it would be nice to have an intraocular lens in the eye, but we're left with two options with these newborn cataracts. One, we take the lens out and we leave them a fake it and give them a contact lens for unilateral. At this point, we've been giving glasses for bilateral more often, partly cause it's difficult with the lens fitting and lens loss. There's a risk of infection. It could be completely impractical in rural areas of the world or certain situations where they can't come back and forth very easily. And if you don't use that contact lens or some sort of refractive correction, they get amblyopia. So you're trading off one for the other. The other option is to put in an IOL which we don't do under the age of one because they could get severe inflammation. They could literally have a fibering cocoon around the lens. The membrane conform and change the location of the pupil where it's not in the center at all and give them cartopia. And one thing we learned during COVID is that the pandemic really highlighted the need to address this and be able to put IOLs and babies. I swear to God, I don't know why this is happening. Maybe it's from the light. Oh, okay. I'll just keep doing it. But it highlighted that we do need to work towards getting IOLs in younger ages cause the soul soft lenses for the A-fakes were not manufactured for quite some time. And we had a lot of trouble getting contact lenses for babies even now it's still difficult to get them. So we had to go to RGPs and that's a little harder to teach parents to use. So lots of issues in the pandemic has even highlighted even more that we need to get this taken care of. So postoperative issues that we see are that membrane formation. This is actually a picture from a 10-year-old child. So it's not just babies. The synechia and pupil displacement can be highly relevant. We have several that we try to re-op and then they just scar again and all sorts of issues and intraocular pressure could go up. And we watch that for their entire lifetime. In the infant apacheal treatment study where they looked at kids who had an IOL put in versus no IOL, they found the IOL group under six months old had a five times higher rate of needing follow-up surgery. And that's just not a good thing. So that's why we lean towards not putting in an IOL under a year of age but it would be nice to not need to do that. So the rationale for our lab is if intraocular surgery is so high-risk due to this inflammation and fibrosis making us delay surgery, sometimes changing the surgical management not putting in an IOL. Maybe we could find a way to not have that happen. And in order to do that you have to understand why it's different. It doesn't make sense that a newborn baby with no immune system has an overactive immune system in the eye. It just doesn't logically make sense. So we really wanted to understand the scientific reason for these differences and see if we could target new therapies to prevent or treat these complications. And this is highly applicable to children and patients with EVitis which is what this picture is from. So the nice part about IOLs and kids would be constant refractive correction, reduced risk of corneal infection which actually happens quite frequently. Even with the infant AFAKIA treatment study that was an ideal situation where they had access to lenses all the time, lots of teaching. That's not the real world. The family is highly burdened and hopefully in the end it could improve their vision. So we looked for a good animal model to look at the post-operative response and we chose New Zealand white rabbits. They're nice to handle and they're often used in industry to evaluate new intraocular lenses. So they already have that mechanism to look at how things do after implanting an IOL. And the enter chamber is similar to humans. So we use the same instruments, we use the same materials actually. Everything's all the same. We use the fake machine, even the same IOLs. They have a seriously exaggerated post-operative response or our thought was if we could get a rabbit to have a good post-op outcome, children should be almost a slam dunk and it's a very controlled environment. You sending a child home with whatever you're doing, you don't know what's being done. Rabbits, animal models are great because it's a controlled environment. We don't give post-op steroids and we look at their post-op response and it creates a lot of scientific rigor which makes it easier to translate to people down the road. So the way we did this was we had the hypothesis that some proteins go up after surgery and there are probably some that go down or become undetectable after surgery. And we did this by sampling the aqueous humor before and after a lensectomy and looking at clinical exams. So just keep in mind that the aqueous, the enter chamber pretty much holds 200 microliters on a good day if you could get that much. So that limits what we could do. So this is actually a video of our rabbit surgery. So we do a paracentesis before to get the fluid and then we do use an MBR to make the incision. Oops, why is it doing this? Oh no. So we just make a small incision because we expand it later. So we put a viscoelastic and then we open up the enter capsule with the cystitone needle and micro forceps. And then I actually use Simcoe irrigation aspiration to remove the lens because it's so soft and easy in little baby rabbits. This is actually my old grad student. We pre-placed the suture because it could get... There's a lot of posterior pressure. We put in the IOL and then we take out the viscoelastic and close the wound and bury it. And if we put in medications, we put it in once the suture is buried and everything's stable. And then we do slant exams post-operatively. So that's a nice... I had to borrow this from the web but that's a nice example of the kind of sewing flare we could see in our bunnies. But these are bunny eyes. This is a normal eye without surgery. And this is one of our rabbits post-op and you see that huge chunk of stuff. We used to call it a clot because we weren't sure what it was made of. So that's pretty dramatic. And so we could literally look with the slip beam and do the exams just like you do in clinic. Same slip lamp. So we just first wanted to make sure it was an appropriate model. So we looked at older rabbits and younger rabbits and the older rabbits were much less responsive post-op than the young rabbits. So here on the left is a young rabbit with a huge chunk in the anterior chamber and on the right is an older rabbit with the same surgery. We did it with FACO and it looked pretty good comparatively. So it's a good model. First, our first question was, what are the different surgical stages do to the proteins in the anterior chamber? So we actually broke it down step by step. We did just paracentesis, then just the incision. Then what happens when you just take the lens out and leave them a fake it? And then what happens when you take the lens out and put it in an IOL? And we wanted to know what the protein changes were before and after. So clinically, just a paracentesis, really no difference. It's a nice clear lens. Incision with suture, also nice clear lens dilates well. After lens activity without an IOL in the juvenile rabbits, there's some fibrin in the anterior chamber, but not nearly as much as when you put in an IOL. And it's really, really reactive across the board. We see this so often that our number of rabbits to achieve statistical significance is only six, so it's pretty dramatic. So your question is the fibrin on the lens, is it on the posterior capsule? Yeah. It's where the lens was. So it's in that, I didn't do a posterior capsulotomy and a vitrectomy in the e-fake it, because I just wanted to know the lens removal. So that's in that capsule area. So it's not on it, it's just kind of in there. And we did remove the lens a lot. There weren't lens particles remaining, so that's just the inflammation. In the lensectomy with the IOL rabbit, you could actually see turbidity of the anterior chamber fluid before we were done, because it's so reactive. Yeah. Yep. Yeah. So the, so the age of the rabbits were six weeks, exactly six weeks, because that's when they could be weaned. And the weight was usually, it was under a kilogram, usually it's around 700 grams or so, 600, some of them were smaller, it was variable. So, and then the heparin in the irrigation fluid, we didn't use it, and you'll see my NFD slides. It actually, I know that they use it in the veterinary world, and that was part of the reason why I wasn't surprised at some of the results we got. Stay tuned, right here. So one thing that we did see, which would be a surprise to some clinicians, but not to Dr. Werner, is that there is a large increase in coagulation factors, but there's no blood. It's not like it's, I call it a vascular coagulation in the anterior chamber. So there's a large increase in coagulation factors and fibrin. So that goes to the heparin in the irrigating fluid because one of our targets is using for preventive therapy is using low molecular weight heparin. So, but we didn't use heparin itself, we used a different one. The other thing we saw was complement components also increased dramatically, especially in the Lensectomy with Iowa Lensectomy groups. So we knew there was inflammatory components, that there's a blood coagulation cascade activation. And then another thing we saw was some immunosuppressive proteins really dropped in these young rabbits, even just a parasyntesis. So that told us that perhaps the young rabbits or the young children also, we still have to do some of those studies, perhaps the reason they're so overactive isn't because their immune response is hyperactive, it's more that they haven't developed inhibition or immune privilege. So maybe that explains why an immature immune system results in these overactions, it's like a double negative. So they haven't developed immune privilege is our gas, but we're still working on that. So we actually mapped out with the coagulation cascade all the things that increased. And so everything in red is increased postoperatively and that gave us an idea of where we could target our therapies. Also in the inflammatory cascade side, we saw that the classical lexin pathway was pretty activated in using the proteomics. So, and some of them talk to each other, which makes sense with COVID too, where you have a high inflammatory response and you also have a lot of coagulopathies going on in these patients. So once COVID hit, I just said, oh, they have to be anti-coagulated too. And look at what we do now. So the question for us was, now we know all these things increase, but where is it coming from? Is it from the structures in the eye or is it from the blood? And we did that with RNA sequencing and I won't go into too much detail because this is a clinical talk, but we've looked at the RNA sequencing changes in the iris and ciliary body after surgical intervention. And it turned out that all these pathways increased as well. So perhaps it comes from the iris and ciliary body, but we also looked at the cornea and these RNA transcripts also increased. So that tells us that the eye is a very dynamic system and that perhaps a lot of this development of the post-operative immune and scarring response may come from the eye itself and maybe some of the coagulation activation may also be from within the eye. So in conclusion with our surgical step-by-approach, the fibrin formation goes up at each surgical step clinically, but really after lens removal and especially with increased implantation of the IOL and the juvenile rabbits. The proteins detected, we have some that increase, but also some interesting ones that decreased following the surgical intervention at all steps. And we did this with mass spectrometry, which was nice because it's a small volume. We tried ELISA and it was just, we would have had to use so many rabbits that it wouldn't have been a good way to do it. And that the ocular structures likely contribute to these changes. Yes. Hi, I can't see anything because it's all bright. Some aspects of rabbits, kids are like rabbits in some ways, not all. Do you use heparin in the kits? Oh, okay, yeah, yeah. Yes, they do. That would be great. Yeah, I actually, a lot of our animal model development came out of scientists from Utah. So I used a lot of your papers. I was walking around and was like, I know who that is. So thanks for your pre-work, helping us develop this model. And so you'll be happy to hear about the prevention side is to, so knowing the coagulation cascade and the complement cascades are activated, we wanted to inject candidate medications at the end of surgery and see if we could prevent these responses. Right now, all these in the real world is steroids, right? Try and send a loan intraocularly if we get really worried systemic steroids, topical steroids. And that's really not ideal in kids because kids tend to have steroid response glaucoma. So I had a child, actually one of the first pictures is the patient that inspired me. And he had increased an IO, he came to us for surgery. The attending didn't want to do the surgery. So we did the surgery, sent him back but she wanted to handle the post up. He was on steroids four times a day, which I usually do a lot more than that. And then his pressure started to climb. And so she stopped the steroids because she thought of a steroid response. And then it really went up. And then he ended up with surgery. And then he had glaucoma surgery because it was all just stuck down. And then he had a retinal detachment and he had all sorts of complications after that when if we could have just prevented this, the inflammation and scarring post up, perhaps he would have done a lot better. Maybe there wouldn't be this confusion about whether a steroid response glaucoma or a fake glaucoma. So he's my inspiration. So now looking at these cascades, instead of heparin, I thought, let's put something in the eye that's gonna stick around post up and stick around for as long as we can. So I chose anoxaparin or lovinox. It's already used in children. So I love that part of it. And we still use trial and send alone. So I use that as one of our controls or standards of care. And then I actually put them in combinations to see if they have a synergistic effect. So this is our untreated as you have seen multiple times again and again, it's just a big chunk in the anterior chamber. This is with anoxaparin. So it acts on the coagulation cascade and prevents that fibrin clots. And we actually did some immunohistochemistry and proved that it was fibrin. So now I call it a fibrin clock because I know it has fibrin in it. So that's really nice. It doesn't have that nice red reflex that we like to see but it's definitely a huge improvement. Eight milligrams is a huge dose. We didn't see bleeding post up because we did it when the anterior chamber was stable. So I think by not having those changes in the IOP we didn't have bleeding events. So that was, and it was pretty consistent. Trinocinolone, I used a very low dose intentionally because I wanted to see just with a low dose and then in combination, that white stuff is the trinocinolone painting, the inflammatory stuff in the anterior chamber. That's not vitreous like you see in the OR. So that's just fibrin that's being highlighted. So we actually got a little confused because the cell was more in the trinocinolone but that's because we were seeing trinocinolone. So I'm not gonna present that data. And then when we put them in combination that's when you see that nice red reflex. So we were pretty excited. So I wish someone would put this in a kid on tube chicken to do it. So we need more dose response curves and a little more science behind it before we translate it to patients. But this is really exciting because if we could prevent things an ounce of prevention is worth a pound of cure. So we also looked at some of the inflammatory and coagulation cascade changes post op with targeted mass spectrometry. And this is just a heat map. So you don't get into the weeds of the details. But if you look at the combination, black is closer to zero, red is increased. Some things don't look any different or better with the combo treatment but there's a lot more dark. So the compliment, some coagulation factors are significantly closer to pre-op compared to untreated or an oxaparin alone or even just try and sit alone alone. So that shows that there are definitely proteomic reasons for the clinical exam we see. And we could actually map it out and quantify it with the way we did our mass spectrometry. And it kind of correlates with what we see that the combination is closer to the preoperative proteomics in the anterior chamber of the eye. So we're pretty hopeful that this may be something to target in the future to help our kids. Now, the question is what happens further down the road and you do well in that first week close up in the rabbits but what does that mean in a child? Does that prevent it way down the road months, years from now? So there are a lot of questions between the rabbit and children. So the other thing we thought about is treatment. Cause we have a lot of patients who have scarring or UVA just patients with a big fiber and membrane and we don't know what to do. So we thought let's target the coagulation cascade again and look at our clot buster tissue plasminogen activator. So we injected it at the peak of that postoperative response and examined it 24 hours later and we used a dose of 25 micrograms. So what it does is it targets plasminogen to plasmin and increases the degradation products and breaks it up. So this is our classic chunky, chunky interchamber of the eyes before. Our controller's just balanced salt solution says you'd expect it really doesn't do much. Maybe it clears up a little bit but that's just the natural way things happen. Pretty nasty looking. And then one day after tissue plasminogen activator injection there's a nice breakup of that fiber and clot. Didn't always go away but I'd be pretty happy if I had a patient with a nice cocoon around their lens and lots of problems and it looked like that. So we're also pretty hopeful with that. We did look at the cell and flare and in the TPA it looked to go up but that could be because those fiber and chunks are breaking up into little pieces and that's what we're seeing. So we did look at coagulation cascade and compliment changes as well because we didn't want something that would make it even worse and make it more inflammatory because it looks good but who knows? Maybe the eye is gonna get bad CME or something like that. So we did look and it doesn't look nice the way the preventative therapies does where it goes back closer to normal but it wasn't much worse. So we're still looking at this and seeing if it's a viable target. So it doesn't look like it reduces the inflammatory or coagulation cascade components but it doesn't increase them significantly but that might be just because there's kind of high to begin with. So still more work to be done on that but also promising. So in conclusion, anoxaparin is a hopeful use for preventing this postoperative complications especially with trienceanolone. It prevents and fiber and formation and then the trienceanolone kind of works in synergy to decrease some inflammation. TPA is also hopeful to improve the clarity of the visual axis after scarring has already occurred and one of my fellowship mentors has actually used this in desperation in one of her UVitis patients and she said it worked pretty well but the guy was blind already. So she just didn't want him to have complications but I'm not that bold. Future directions are looking at these eyes longer term. Really these experiments are done in one to two weeks. I wanna look months out and see other things especially the PCO. That's the poster capsular classification is totally different field that I do not have the time to attack. So we could always talk and see if there's other things that you could work on based on this research and all our data is publicly available. So if you wanna look at other proteins it's on our public data sharing website. And then our next goal. So we did the rabbits and this is a clinical talk and I haven't talked about a patient really very much. Our next goal is what about humans? So rabbits, we have a nice story with the rabbits but who cares if humans aren't the same? It's hard to do human subjects because there's so much variability in the population genetically, environmentally. Pediatric surgery is pretty rare overall. We see it a fair amount but that's just selection bias. And then the pre and post-op regimens are different depending on the surgeon and the compliance. So it's really hard to gather data in just an end of sex in humans. And we need a lot more numbers. So this is where we're still trying to work out a lot of the methods. And our main question is are we similar in animals versus humans? And this is where Dr. Huang comes in is what about the vitreous? The aqueous is not an isolated system. The vitreous is also very dynamic. And how can that help with PVR and post-operative fibrosis in kids? Because in an adult, they get a retinal detachment. There's some standard if it's mac-off you could wait a little bit. But in kids, even if they have mac-off their entire retina is just gonna be one big vibrant folded mess. So maybe children can, we can take advantage of some things in the posterior chamber as well. So what we're doing now is we have a relatively large sample bank with about a hundred aqueous and vitreous samples of all ages, some surgically naive, some not surgically naive. It's hard we can't get pre and post-op samples because our post-op samples and the rabbits are three days after that's not ethical in human subjects. So we really have to take advantage of surgically naive and patients who are re-ops for whatever reason. So it's a little statistically more complicated but we are building the sample bank at this point. And we did do some preliminary runs. We really have to alter our method because humans are so variable and very different from each other. So one is there's just a pediatric trauma patient here. So it's hard to parse out whether it's a pediatric or the trauma, but their complement was way higher and some of their coagulation proteins were elevated as well, particularly the fibrinogen so that we're still trying to get more pediatric samples to run together because we need a higher end but it was just a method run to see if it works. But definitely there are some proteins that go down and some that are lower and some that are higher because we don't know if it goes down or up because we don't have a pre sample. So we're looking to collect a lot more samples for more analysis and also the vitreous as well. So in another interesting patient we had a pan-UV-itis patient and looked at sample versus the pool. So we had to look at controls which were just adults that were having cataract surgery and nothing else going on. And it looked compared to our controls that the pan-UV-itis had elevated fibrinogen which is that coagulation fibrotic cascade, RBP3 which may be an immune suppression protein was decreased, which makes sense in UV-itis because they're very immune activated. So maybe that's an immune privilege issue and their immune inflammatory proteins were really high, really high compared to our controls. So we're still looking at this and just trying to get more samples and run them together. So lots of interesting things with our pan-UV-itis sample with our pediatric sample, which I pointed out already the fibrinogen was really high and the immune suppressor protein was low even though it was just a trauma patient we, they, she hadn't had surgery. The surgery was for cataracts. So the other thing that was interesting about her or about the sample was that it was a trauma patient we didn't see a frank tear in the eye in the lens capsule or even during surgery we didn't see a tear in the lens capsule but there are lens capsule proteins were super high. So that tells me that there's probably a micro tear that created the hydration of the lens and created the cataract. So why does this matter is pretty obvious but we do have patients that have had gene therapy and in pediatric patients who have had lexterna they are known to be at a very high risk of cataract and we're not sure if it's an inflammatory thing or if it's related to lens protein release. So this would be really helpful in those patients or those subjects where they develop a cataract if we'd go do the cataract surgery perhaps we could parse out if it's truly inflammatory or if maybe there are micro tears in the capsule. So lots of potential here. So we wanna increase sample size use our surgically naive in different age groups as the control we kind of throw them all together and look at the differences as a whole group. We realize that's not good to do in humans that are in rabbits and we really need to focus on reoperative samples because that's what we're looking for is why they have post-op issues. So that's requiring collaborative sites. So Dr. Huang is a collaborative site, Duke and Iowa might also join in as well and we're expanding our vitreous. So I just wanna say most of this work is not done by me directly. I was very hands-on but Jonathan Young depended his thesis based on most of this work. So I have to give kudos to him as well as Joe Bogard who's now doing his retina fellowship and the collaborators I have are straight-up biochemists and other really important people in my world. And this is John's cover of one of the journal articles he published that on the cover of a journal. So this is really pretty, I thought he did a great job. And then just in terms of hobbies, I love traveling. So this is my family and the reason why I have so many pets. Any questions? I see one from, should I read it? So Marissa LaRochelle said, we routinely inject TPA in the AC after you've had a cataract surgery cases at the end, 25 micrograms. So sounds about right. I would be curious to know if that really prevents the post-operative scarring that you see especially if you use IOLs. And I would be curious to see if anoxaparin would help as well. So anoxaparin, why do we use anoxaparin for prevention and TPA for treatment? We actually use several different drugs for the preventative side because I wasn't sure what would work. So Heparin was just too short. We did lobenox because it's really cheap and already approved in kids. And then I used our Gatcherban and that was complete disaster. The eye was just full of blood. So we abandoned that really early. So we did try several based on the science. And so we kept going with anoxaparin because it has a pretty good safety profile too. You don't, you inject it at home. You know, you don't have to have it infused. And so I don't think systemically it would be a good idea to use it but intraocularly it's really isolated. And then TPA that was pretty much the only clot breaking drug that I knew was available. And we were very careful with our choice of the TPA. We use rabbits recombinant TPA because we didn't want an inflammatory response from like recombinant human TPA. So we were very careful in what we chose. Yeah, so anoxaparin acts in several sites to inhibit that end production of fibrin and TPA breaks up the fibrin which is probably why we saw more cell in the TPA. We didn't do it immediately post-op. I didn't even think of that. We just wanted to see if it would work in that huge fibrin trunk. So that's a good thought to use that immediately post-op and see. That looks pretty good. Yeah. Yeah. The half-life of TPA I think is not terribly long. So it's good. I do think there is a lot of value to preventing or to not having this fibrin scaffold in the beginning. And I think it has long-term benefits. So I think whatever method you use to not allow fibrin to form even microscopically because we don't always see it at the beginning. I think it's probably gonna have benefits months and years down the road. That's why we need to do longer-term studies. Yeah. Yeah, so good control prior to surgery. We do have like two or three patients that have been stuck who are in that amblyogenic range and have new diagnoses you guys can't see anything. And we're just in this horrible cycle of when do we intervene? What do we do? So if we had a little more in our armamentarium to prevent these complications like TPA. Yeah. Yeah, I hate AFIP, I think kids. It's better than nothing. Yeah, absolutely. I agree. But that's why if we could have a couple of more things that are non-steroidal in our armamentarium, I think it would be super helpful. So I'm excited to hear that TPA is being used at the end. And we are in the rabbits, we use it the same way right at the end of surgery, everything stable. Because if you had the pressure go up and down you're probably gonna get some bleeding. So, and then is there any concern with retinal toxicity with lobenox? I did. So that's a great question. And we had the same question in the beginning and we actually did ERGs on the rabbits after the lobenox and the steroid and the control and the ERGs were completely normal. So we didn't see anything, any signs of retinal toxicity but that the more studies would need to be done. Yes. So your question is if we saw Hyfema post-op TPA or lobenox, because you saw it in the TPA in that study, we actually didn't. I think TPA is a fibrin breaking drug. So it's very possible that it's opening up some of the blood vessels. And some of that has already happened because you're using iris hooks and whatnot. So that's I say potential for health and UVitis because it's a much more complicated surgery and you're definitely touching the iris and tearing it. We didn't see it with either one but we were very careful when we did it. Not to, we didn't have as much trauma because they're not UVatic rabbits. So, but I'd be curious to see what lobenox if you get the same problem. Was the Hyfema significant to create problems for you? Yeah. So that's helpful. Yeah. Yeah, I think we could deal with Hyfema pretty well. Yes. Thanks. Yes. So in children today, what would I suggest to minimize the inflammation with or without what has already been done? I think in general, I think surgery is getting better in a lot of our instruments are better. I feel like the more you touch the iris, the worse the post office. And in children particularly, the darker eyes do worse than the lighter eyes. So we actually are parsing out our dark-eyed versus light-eyed subjects for the human samples. So I think the less manipulation, the better. We actually recruited an amazing adult surgeon to do the surgery itself. And then we deal with the post-op because he doesn't touch anything and his post-ops are amazing. But for children, this is not even close to ready for prime time. You have to do things in adults first and then march back. I wouldn't use lobenox or TPA in any child right now. My chair, old chair at some point, we had a UBI just child who had a post-op mass. So can you inject TPA? I was like, no way. And it turned out they had an ophthalmitis. So I was glad I did it because I would have been in a lot of trouble. So I think there have to be those finding studies, a lot more work until you even put it into adults and then march it back to kids. The adults have to prove that it's good enough. It's just huge, pediatric IRBs are really tough. Yeah, sorry. Yeah, it's vague. I'm very vague with the slide. It's not that specific for trying similar. I'm being vague. Yes, post-operatively is upregulated. Yes, so because of the, I'm gonna go back to the pathway. Can we take advantage of the classical pathway activation to find something that is non-steroidal? Yes, I'm very interested in that. The more new drugs come out, the more excited I get. You just have to be careful. We actually targeted prothrombin inhibition and it was a mess also. There's a lot more to a drug than just the drug. There's the vehicle, there's a preservative free. So you can't just go willy nilly. I think that's what animal models are great for because you can kind of shoot a little bit in the dark and hope something sticks. But yeah, I would be really interested to see some of the new anti-inflammatories. We have to get off of the steroids. It's too vague, especially in children. If they have it systemically, it could hurt bone growth. So there are lots of issues with steroids. Yep. I think there's a lot of crossover with Andy as well. I think a lot of stuff in the eye is immune dysregulation. And just to be clear, I know there's a lot of cytokines and chemokines that are really heavily researched, especially in uveitis and other inflammatory eye disorders. We did not detect any, but that's most likely because we can't detect it because it's such a low concentration. So we haven't been able to get at that really well. We do see some changes in the RNA sequencing with the chemokines and cytokines, but we just can't detect it. And that doesn't mean anything. We even looked at doing human protein analysis using those antibodies. And it's not worth doing that because the crossover isn't good enough. So we haven't chased that lately. There are so many things to do. Yes. Yeah. So your question is, have we taken advantage of the immune privilege? Am I interpreting that right? Investigate. Yes. So we are really trying to get more in the specifics of the test of the immune privilege question, but that's a really broad thing and it's pretty messy. So grant funding is a little difficult when you go for something that's very new and unknown. So we're trying to get some more baseline data before we really go after it. But right now it's so fruitful going after the coagulation cascade and the inflammatory cascade that seems to be the closest immediate low hanging fruit. And that's also something that we do kind of on the side. That's where grateful patients come in really important because then I could explore quite a bit. Great. Thank you. Again, at noon to the research faculty and personnel in person here. Thank you.