 So I'll kind of get us started here this morning. My name is Cole. I'm one of the PGY2 residents. We have two presentations this morning from our PGY3 residents. The first from Dr. Ariane Levin, who is coming to us from New York, undergraduate at Stony Brook and then medical school at Cornell. She is presenting today on kind of a review article of elevation and injector pressure after intravitriol injections, kind of the data surrounding that, as well as a possible prophylactic treatment for that. So without further ado, I'll turn that over to her. Hi everybody. I'm Ariane Levin. Today I'm presenting intraocular pressure spikes after intravitriol injections. We'll start with an example case. A 75-year-old patient with a history of exudative macular degeneration and primary open-angle glaucoma on topical IOP lowering drops comes to your clinic for an intravitriol anti-vegeta injection. After injection, he has an acute onset of throbbing eye pain and decline in vision. His symptoms rapidly resolve. As I speak today, I want you to consider how should this patient be managed now and how about in the future? So first of all, why do intravitriol injections elevate the intraocular pressure? There are a number of theories and we don't have good data to demonstrate which one of these might be true. One idea is that the high volume of fluid in a closed space raises the pressure. Another is that there's injury to the trabecular meshwork from that high volume or that there's a toxic effect on drugs from the intravitriol drugs on that trabecular meshwork, that there's inflammatory damage to the trabecular meshwork, or that it's actually a mechanical block of the trabecular meshwork by a drug. So the question becomes, should we prevent this post-injection intraocular pressure spike? And if we should, how should we do it? Today we'll review a number of papers that discuss the chronicity of post-injection intraocular pressure spikes, the effects of these spikes on ocular health, the efficacy of preventative treatments, and challenges in research in this field. Beginning with the chronicity of post-injection intraocular pressure spikes. We'll look at a number of papers. So this paper studied 188 patients. They excluded patients with glaucoma, which we'll see as a theme here. They had a number of different intravitriol injections which were used, and they did not have a control group. They looked at pressure at one minute after intravitriol injections, 10 minutes, an hour, a day, and a month after. And they determined that there is a transient peak at the one minute mark, but no persistent elevation. They also found a negative correlation with axial length, thinking about that volume issue. This group looked at 120 eyes, including only 20 with glaucoma. They also had a variety of injection drugs and no control group, and they looked at the pressure immediately after injection. They too determined that there's a transient spike, and they found that eyes with a history of glaucoma take longer to recover from that spike. We see in these papers a few interesting ways that the data are presented. So in this graph here, we have the minutes post injection on the x-axis, and then we're looking on the y-axis at the fraction of patients that reach pressure less than 30, so recover from that spike. And we see that by 25 minutes in this group, all the patients have recovered. This group studied 58 eyes, again excluding glaucoma patients with either a flibrecept or vivicizumab, no control, and measured pressure at one day, one week, and four weeks, and determined that there's no long-term elevation of pressure. This group had a larger study of over 1,000 eyes with a flibrecept or vivivizumab, no control. They were looking for pressure spikes greater than six above baseline or a pressure above 24. And they found a few factors that were associated with a sustained pressure rise, which they defined as a pressure rise on two or more visits. They found an association with being male with South Asian ethnicity, older age, a diagnosis of macular degeneration or vein occlusion as the reason for the injections, rhinovisumab, specifically, the number of injections, and also pre-existing glaucoma. In this small study, they looked at whether eyes receiving intravitral injections with prior glaucoma surgery compared to those without prior glaucoma surgery had a difference in this post-injection spike and a difference in the time to return to that baseline pressure. And they determined that surgical eyes, so eyes that were getting injections but had had glaucoma surgery in the past, had a smaller pressure change after injection and a shorter time to return to baseline, so they recovered faster. This study compared different drugs. They looked at eyes that had intravitral rhinovisumab and then switched to a flibrecept and used these eyes as their own controls before and after the switch. The outcome that they looked at was the intraocular pressure. And they found that the intraocular pressure was lower in eyes after switching to a flibrecept from rhinovisumab. In this study of over 500 eyes, they also excluded poorly controlled glaucoma. They did have a control of untreated fellow eyes and they were looking for an elevation of greater than five for at least two consecutive visits. They determined that the total number of injections was associated with increased intraocular pressure. And then finally, I've brought in here one meta-analysis of five randomized controlled trials that supported that there is a sustained elevation of intraocular pressure. So they have a number of interesting risk ratios here for sustained elevation of pressure. They have a significant risk ratio of three in eyes that had injection versus no injection. Of two at six months, a risk ratio of three at 12 months, of three and a half at 23 months. Three and a half in eyes without where the studies did not exclude pre-existing glaucoma. So they included glaucoma. And then a risk ratio of 2.6 in studies that did exclude pre-existing glaucoma. But I want to point out here that the five randomized controlled trials that they used included Restore, Vivid, Vista, Galileo. So almost all the patients that are shown here were getting intravitural injections for DME and not macular degeneration. And the primary outcome of the studies that were used in the meta-analysis was not intraocular pressure. So to sum up this section, the key points looking at the chronicity of this intraocular pressure elevation, we can conclude that there is a transient intraocular pressure elevation after intravitural injection. Most of these studies agree. But that there might or might not be a chronic elevation. And that there might be many factors that are associated, including the specific medication that's injected, the number of injections, the ocular history of the patient. So looking at the effects of intraocular pressure spikes and ocular health, I found just two papers to share with you today. In the first paper, it was a prospective study of 49 eyes with macular degeneration, which were followed for one year. Patients with glaucoma were excluded. The injection was run a busy map and the mean number of injections in this study was 4.8. They did use a control to use the fellow eyes without macular degeneration requiring injection. So they might have had dry AMD. And the outcome that they looked at were RNFLs. They determined that the RNFL thickness was decreased after one year in the injected eyes, but not the control eyes. In the second study here, this was case controlled. They had 75 cases and these were eyes that received the vasizomab injections and then underwent glaucoma surgery. And they compared with 740 control eyes, which were eyes that received the vasizomab injections, but no glaucoma surgery. They were looking at the risk of going on to require glaucoma surgery in eyes that had these injections. And they determined that eyes receiving seven or more injections had a higher risk of going on to get glaucoma surgery than those eyes that received three or fewer injections. The data were not significant or inclusive in the four to six range. So there are a number of ways that people have tried to prevent this transient intraocular pressure spike. And we'll move on now to look at the efficacy of these attempts. This group studied 56 eyes that were randomized. They excluded glaucoma. The injections were intravitrile bavisizomab. And they had a variety of different pretreatments, either oral acetylzolamide and ACTAP or topical bermanidine. They did not have a control group and the pressure was measured at 90 minutes before injection and then a number of time points in the acute phase after injection. They determined that pretreatment is effective. And we're looking at one of their graphics here. Again, on the X axis, we have minutes after injection with the minutes before injection all the way on the left. And then we're looking at the IOP change. And we see that a baseline, all the groups are sort of similar. And then there's this acute spike. And then that pressure comes down. This group looked at 88 eyes. These were randomized. It was a double blinded study placebo controlled with rhinobizomab. And they used a single pretreatment which was a COMBAGAN. They did have a control of artificial tears and they too measured pressure in that acute phase and determined that pretreatment with COMBAGAN was effective at preventing the spike. This group looked at 58 eyes. No glaucoma. They were randomized and cross over. They used bermanidine as well. And then they compared with eyes that received no pretreatment with pressure measurements in that acute phase. They too determined that pretreatment is effective. We're looking at their graphic here. So in the x-axis again, time of measurement and then y-axis is pressure and we see that spike. And then we see that spike resolve. The bermanidine group is the lighter gray line that we see has a lower spike. This group looked at eyes with 250 injections with rhinobizomab. They had a variety of pretreatment, saproclonidine, acetazolamide and then some combo drops as well. They had a control without pretreatment drops and they measured pressure in the acute phase and determined that pretreatment is effective. They saw a correlation with age. They saw no correlation with axial length or lens status. So whether those eyes are fake-ic or pseudo fake-ic. In this group of 175 eyes, they're receiving bubacizomab or rhinobizomab. They used combo drop pretreatment and then they measured at five minutes post injection but also at one day, one week and one month. So we're getting more into this chronic effect. And they determined that pretreatment was only effective at reducing those early spikes at five minutes and 30 minutes. Most of the studies we've seen have shown that pretreatment worked well in their cohorts. In this study, they determined that pretreatment was not effective. They looked at 71 patients. There was a variety of the drug that was injected and there was also a variety in the IOP lowering drop that was used for pretreatment. They did have a control and they measured pre-injection and then in that acute phase up to one week and determined that pretreatment is not effective. So to summarize here, most of these show that pretreatment reduces that acute intraocular pressure spike but there's really no evidence for pretreatment to reduce the chronic intraocular pressure. We can see that there are a number of challenges in this research. There are so many variables that can affect these outcomes. Glaucoma is a big one. It's excluded in a lot of the studies that we looked at. And in the patients who do have glaucoma, there may be different effects from the etiology of glaucoma, their drop history, their surgical history. With the injections as well, there's variety in the drugs that are used and there may be effects from the variety of indications for the injections, whether it's DME or AMD, the number of injections. And then similar to axial length and some studies suggest that there's an effect from the injection fluid dynamics as well. There's a lot of variability in study design. Many of the studies are retrospective. Some of them have no controls. And then between studies or even within studies, there seems to be no standard in the intravitural protocol and also when the pretreatment is given and when the pressure is measured after injection among the study populations. So to summarize, there is an acute elevation in intraocular pressure after intravitural injections probably prevents these early spikes. But we don't know for sure whether there's a chronic elevation, whether pretreatment might have an effect on these possibly chronic effects. And what exactly is happening in these eyes with glaucoma? There are many variables that might influence these outcomes. So I want to open it up now for discussion going back to our original question. Should we prevent post injection intraocular pressure spike? And if so, how? I also want to thank Dr. Chaya, Dr. Schmitz-Walkenberg and Dr. Ron Kealio for their insight on this topic. Thanks very much, Dr. Levin. There's one question that came up in the chat here. It says, Ariana, with what you have reviewed, if someone has primary opening of glaucoma, would you recommend pretreatment ILP lowering drop to prevent even that short spike? You have a different opinion based on what you've reviewed. That's a really good question. And I think in my experience before doing the literature review, I was inclined even to give pretreatment for these pressures, even because emotionally standing in the room, the pressure spike afterwards looks uncomfortable. The patient has a sudden drop in vision. They are having pain. And so even without using data, I feel that there's sort of an emotional component to pretreat and prevent that acute discomfort. Then looking at this literature review, I don't think it convinces me more to pretreat these patients. But at the same time, it seems like a low risk option to prevent something that might or might not be harmful. So I would lean toward pretreating, but I think it still is a very personal decision. And then Dr. Petty brought up another question here with we have quite a bit of data. Millions of injections are given every year. What do you think the primary reason is that we don't have a clear answer on these long-term effects? I think that there are a lot of groups that have tried to study it, but the studies are all smaller. They're retrospective. They're not, I think, rigorously controlled, or there are a lot of confounding variables in them that are not rigorously controlled. And so I agree we have lots and lots of injections. There's lots of opportunity to try to study them. To try to study this. I think that we probably could do it well, but it would take a big and rigorous effort to set it up appropriately. And then one more comment from Dr. Siminett here. Just a practical tip. With pre-filled syringes, there's been more of a kind of with a wider internal diameter than most of the other syringes. The injected volume is really more sensitive to being slightly off when pushing up to that .05 mark in the ilia. And yeah, I've experienced that personally at the VA as well. I have to be kind of very careful that you get that plunger all the way up to the the mark, because even being slightly off can drastically change IOP that I found. And then one more question here. Are IOPs routinely checked for injections here? I can speak best to the VA where we check pressures before injection, but not routinely after. I think that also winds up being clinician specific. If a resident has a suspicion that the pressure is high because the patient like our example case says something changed, then we'll check. I have to ask the retina team to comment on whether they're checked before and after here at Moran. Maybe someone from retina team can respond in the comments. Or we're happy to unmute you as well. So Joe Simonette says no, they are not routinely checked before and after. We do have some time for other questions and comments. If anyone would like to be unmuted before we move on to Dr. Hong. Perfect. A couple more popping up here. Dr. Levin. One of our interns, Dr. Kennedy asks what's the ideal time to check IOP after the injection and Dr. Stagg building on that. How long would you wait after the injection check IOP? So on the same line. And then I'm going to unmute Dr. Warner after you answer those questions. Sure. So the ideal time to check pressure after I think we don't know there was so much anxiety in the studies that were done. Brandon Kennedy is an intern here with us. So Brandon, if you're doing an injection at the VA, sometimes you'll see an acute spike there presenting with that decline in vision, that pain. And so if you notice that in your injections I'd recommend checking then. But as far as studies go, I think we don't know what the ideal time in a future study would be. And then it's like Dr. Stagg has similar question. And then we can go to Dr. Warner. So from my understanding with glaucoma you're looking for loss of function or maybe before that loss of retina and eryloid layer. So the study that I've seen most consistent with was the one that looked at retina and eryloid layer after many injections. However I can imagine that there might be situations where retina and eryloid layer could actually be affected by retinal fluid. If there's fluid that's quite extensive and getting in towards the area around the optic disc do you know if they control for that or were they assessing for that or were they truly seeing retina and eryloid layer thinning impenetrable confounders like extensive edema? I am not sure if they controlled for that. I'd have to go back. I think that I could confidently say that there are a number of factors we could really come up with that could affect RNFL that were not controlled for. I don't recall which they might have controlled for. They weren't doing visual fields or anything. I don't think that visual fields were one of the work. Just one more comment that came up from Dr. Smith-Milkenberg was that in the clinical trials evaluating anti-vegeta agents that the IOP was measured after injection and adverse effects were then followed and he mentioned that no significant concerns were found in those clinical trial and then Dr. Smith-Milkenberg would be kind of following up with that. What was the time period that they were monitored and over how many years we're happy to unmute you Dr. Smith-Milkenberg too. Can you hear me now? We can. I don't have the data by hand but it's typically measured after 30 minutes or 60 minutes and actually the pharmaceutical companies have had always concerns to measure it too early and of course adverse events. Thank you very much for this excellent overview. I totally agree that it makes sense that the pressure rises directly after the injection. I'm much more concerned about the chronic effects in terms of long-term glaucoma development and I think there's a lack of knowledge how significant these pressure spikes are for the patient in the long term. Any other comments? All right, we'll go ahead and move on to Dr. Marshall Huang. He is kind of continuing this discussion and his talk surrounds the current state of optic nerve regeneration after an IOP spike or after optic nerve damage so with that I'll turn it over to you Dr. Huang. Okay. So my name is Marshall Huang. I'm the director of optic nerve regeneration presenting on the current state of optic nerve regeneration. I have no disclosures. As ophthalmologists our ultimate goal is to stave off blindness and as ophthalmologists we know better than most that the irreversibility of true optic nerve or retinal damage as those are direct extensions of the CNS. In our field few conditions are more feared than snuffing out the optic nerve whether from traumatic, ischemic and if I were giving this talk 30 years ago I may have stopped right here. However today I'm here to convince you otherwise. Even though we cannot regenerate CNS damage we also know that it is not impossible in every situation but it's certainly not common. The birds don't do it the bees don't do it heck even the priests don't do it but we know that fish can do it lizards can kind of do it but perhaps most importantly our embryonic cells do it which gives us a hint that it might just be possible. However my goal today is to prove to you that in our lifetimes clinically significant optic nerve regeneration is not only possible but probable. In order to convince you I will briefly go over the obstacles of regeneration and a history of our attempts. I will then explain how we have identified potential pathways to regeneration and our current strategies and finally we'll discuss the future directions and what this means for us as ophthalmologists. There are five primary obstacles to CNS regeneration. The first is the obvious that CNS neurons do not regenerate. The second is that the CNS readily forms glial scars that function as a barrier, physical barrier to axon migration. The next two challenges are for the axon to synapse both in the correct location and the correct topographic orientation in order for there to be visual function. And finally the axon must in order to transduce signals fast enough to be useful. However, despite these long known challenges the dream of optic nerve regeneration is as old as modern neuroscience itself. Ramoni Kahal is often credited as the father of modern neuroscience and in his 1913 volume studies on the degeneration and regeneration of the nervous system. Ramoni Kahal's first true disciple Jorge Teo showed that retinal ganglion cell axons from transected optic nerves can grow into ontologous peripheral nerve graft. Although many attempts were made in the intervening decades the next major breakthrough was not until 1990 when Agayo at all extended this experiment. They transect the optic nerve near the globe and then attached an ontologous peripheral nerve graft to the optic nerve stump on one end and the superior colliculus on the other. They even showed that retinal ganglion cell axons remain responsive to light and that the synapses they form can transmit impulses to the superior colliculus neurons. Although CNS axon growth was shown at this point to be possible through a peripheral nerve graft it was thought that regeneration through a mature optic nerve itself was impossible because of the potent inhibitory environment. It says my screen sharing is paused. We can still see it. Okay, cool. Just a few short years later Barry at all disproved this hypothesis. They showed that implanting a peripheral nerve segment into the vitreous could promote optic nerve regeneration past the crush site. They also found that an A-cellular peripheral nerve implant which was simply a frozen and thawed segment of Cyagnote did not promote regeneration past the crush site. They concluded that peripheral nerves contained cell intrinsic factors that promoted this regenerative effect. Here are some of the potential pathways for optic nerve regeneration with some of the most evidence. Of these I will primarily be focusing on the first three. In the process of studying various agents on axon growth the Benowitz lab discovered serendipitously that intraocular injections that infringe on the lens initiate a cell set of cellular changes that cause retinal ganglion cells to show improved survival and up until this point unprecedented levels of axon growth into the normally prohibitive environment of the mature optic nerve. Here in the upper right corner is a representation of the experiment. After optic nerve crush is performed the lens is either violated with a bent 30-gauge needle in the experiment group or left intact with a straight needle in the control group. Gap 43 is a protein that is expressed only by retinal ganglion cells only during axon outgrowth. Here in A they show significantly increased Gap 43 positive fibers pushing past the crush site compared to controls. With the crush site marked by the asterisks is here. B shows no Gap 43 in normal nerve without injury. C shows very little immunostaining in the distal optic nerve after crush injury alone. D shows little effect after nerve crush with a minimally invasive injection that does not infringe upon the lens. They also found that injection of Zymocin a pro-inflammatory molecule found in the ligand on the surface of fungi had a similar effect. The same lab later identified oncomodulin in combination with cyclic AMP as a growth factor that accounts for the majority of the regenerative effect arising from intraocular inflammation. Here A shows a control with optic nerve crush without treatment. B shows the effect of lens injury or Zymocin injection. C shows the effect of oncomodulin plus cyclic AMP showing basically the same amount of regenerative effect. D shows the effect of lens injury in the presence of P1 which is an oncomodulin receptor antagonist showing significant decrease of this regenerative effect. Moving forward, this is a representation of the general model for the next few experiments. In order to perform genetic manipulation without affecting development into adult hood, the researchers used adeno associated viruses expressing CRE that was injected into adult mice with the appropriate genes flocked. Optic nerve crush is then performed, followed by injection of an enterograde tracer like cholerotoxin beta prior to sacrificing for histology. In 2008, the lab at Harvard showed that deletion of P10 increases RGC axon regeneration in a time-dependent manner. AMB shows optic nerves for 14 days and 28 days after injury respectively. There are significantly more regenerative fibers at 28 days compared to 14 days and virtually no regeneration pass across site without the P10 deletion as shown by C&D. The black and blue lines here represent the numbers of regenerative fibers at 14 and 28 days respectively compared to virtually no regenerative fibers in the controls. Now, we know that this effect is modulated by an activation of the mTOR pathway. P10 acts to dephosphorylate PIP3 to PIP2 and thus acts as an inhibitor of this pathway. Adding rapamycin, a known inhibitor of mTOR, eliminated this effect of the P10 deletion. Now, S6 is a known target of mTOR activation and they found that exotomy abolished nearly all phosphorylated S6 signal. Mice with the P10 deletion maintain this S6 signal despite exotomy. The same lab also found that deletion of SOC S3 had a similar effect to the P10 deletion. On the left, we see how axon regeneration increases steadily from day 1 to day 14 post crush. On the right here is a percentage of renal gingling sounds survival 14 days post crush in control versus SOC S3 deletion mice. And SOC S3 is known to inhibit the jack stat pathway. They showed that this deletion also causes up regulation of mTOR activity like before. However, in this case adding rapamycin does not block the effect of the SOC S3 deletion. This suggests that the primary action was through the jack stat pathway. This was confirmed by deleting the GP130 receptor which blocks the jack stat pathway to regenerate the effect of the SOC S3 deletion. Up until this point studies have only shown the ability to stimulate retinal gingling cells to regenerate axons partway through the nerve. Therefore, it was not known whether mature axons would re-enter the brain, navigate to appropriate target areas or restore vision. But in 2012, the Benowitz labs showed that a combination of zymosin, cyclic AMP, and a P10 deletion cause axons to regenerate the full length of the visual pathway on and into the lateral geniculate nucleus, superior colliculus, and other visual centers. Here in A, we see regeneration of axons through the full length of the optic nerve. In here, the white arrows label regenerating axons in the chiasm with more on the right side which is contralateral to the injury. You also see some axons into the superior colliculus or super chiasmic nucleus. Excuse me. Below that on the left, we have an electron micrograph of an axon that appears to be undergoing real myelination right here. And then in H, we see unmyelinated axons labeled with yellow arrows and an axon with what appears to be thick myelin labeled with the blue arrow. They also show that this regeneration partially restores depth perception, optomotor response, and circadian photo entrainment. Sorry. These charts show that group one, the full intervention mice, spent significantly more time in the shallow end compared to the deep end. Had more robust OMR response, as shown by the blue line here, especially in a time-dependent manner, and behaved more like normal nocturnal mice. As shown here, normal mice are primarily active at night. In group one, they similarly are primarily active at night, whereas group two, which was not treated with all three, the combination therapy, did not show a strong pattern, and the blind mice almost showed a reverse pattern. With multiple pathways that appear synergistic with each other, several combinations haven't attempted in recent years. However, few have shown true visual recovery. Co-deletion of P10 and SOC-S3 appeared promising, especially given the significant axon regeneration, along with formation of functional synapses in the superior colliculus. But unfortunately, no significant recovery was found. However, in 2016, after adding 4AP, a potassium channel blocker used to treat conduction blockade, the mice had improvement in visual activity, suggesting that the problem was lack of myelination. Although significant progress has been made over the past 30 years, these strategies are a long ways away from being ready for prime time. Strategies involving inducing intraocular information or permanently deleting a gene without knowledge of long-term downstream effects is certainly not ideal for our patients. Furthermore, these studies have shown significant improvement in retinal ganglion cell survival and axon regeneration, and even some visual recovery, but we are clearly missing a few pieces to the puzzle. This is particularly obvious when you consider the generative capacity of embryonic cells, which share 100% of our DNA. In 2006, Takahashi and Yamanaka discovered that mature cells can be induced to become pluripotent stem cells by expression of four transcription factors. Oct4, Sox2, Clif4, and Cmic termed the Yamanaka factors. This earned Yamanaka the 2012 Nobel Prize in Physiology or Medicine. The following year, their group finds that the omission of Cmic resulted in more specific induction of adult human fibroblasts to induce pluripotent stem cells. This lowered the efficiency of this induction but decreased tumor genicity. Using this combination of Oct4, Sox2, and Clif4, Lou et al hypothesized that they could potentially treat multiple types of CNS generation and age-related disease by turning back to clack on specific cells. Last July, Lou et al published a preprint of this article. Reversal of aging and injury-induced vision loss by TET-dependent epigenetic reprogramming. Of note, this data has not yet been peer-reviewed, though according to the corresponding author, it has been submitted. Before we dive into this paper, it is important to understand that TET enzymes are instrumental for DNA demethylation. And it is through this action that these transcription factors cause epigenetic reprogram. Here in Figure A is a representation of their TET-ON and TET-OFF model. The TET-ON model will express the OSK transcription factors only in the presence of doxycycline, as you see here. The TET-OFF model will only express OSK when in the absence of doxycycline. The authors begin by showing that their TET-ON and TET-OFF models have no effect on growth using body weight as a surrogate. Here we see co-localization of Clif4 with a marker of a million retinal ganglion cells showing efficient transfection of retinal ganglion cells using adeno-associated virus vectors. Next, they show that the axons are generated significantly when oct4, Sox2, and Clif4 are transfected and expressed on the same vector as shown by the blue line. The green line down here shows that there is no effect with the TET-OFF OSK vector in the presence of doxycycline when OSK is not being expressed. They also attempt in numerous other combinations including the OSK genes in separate vectors that did not achieve the same effect. The bar graph here shows that there is significantly increased RGC cell survival with OSK expression. You can see the blue bar here is with OSK and green is with the OSK-OFF. Here they show a similar effect with the TET-ON vector. Interestingly, they found that the effect is stronger when turning on OSK than after injury in a time-dependent manner. You can see significantly increased regeneration of four weeks compared to two weeks after the injury. Here the blue line shows the four weeks, the red line shows two weeks here. And here you can see the effect if it was turned on before the injury and here if it was turned on after the injury. That's survival of RGCs. In this next figure here, a published epic genetic clock is used to calculate DNA methylation age which is accepted to be a highly accurate molecular correlate of chronological age in humans and other vertebrates. In E, they show that there is a significant increase in DNA methylation age after injury that then comes back towards baseline with OSK expression here. In F, they show that knockdown of either TET-1 or TET-2 eliminates the axon regenerative effect showing that the OSK genes act through the TET-1 and TET-2 enzymes. On the right, they show that OSK expression can protect human neurons from vincristine induced damage and decreased DNA methylation age back towards baseline. Now they move to a glaucoma model induced by injection of microbeads. They first show that this causes an IOP bump that correlates to decreased retinal ganglion cell and axon density here. They then show that there is regeneration of axon density four weeks after expressing OSK right here between no and with OSK in the presence of the beads. Interestingly, they show that the increased axon density was not associated with retinal ganglion cell proliferation as you can see here. Those that are about stable do not return to baseline and certainly do not go above what is normal. They then proceed to show visual recovery in these OSK expressing mice four weeks after treatment in terms of optomotor response and pattern ERG. The gray bars represent undamaged baseline vision here. And the blue bars show an increase in visual acuity in OSK expressing mice after initializing treatment. The blue line shows a significant increase in ERG amplitude only after expressing OSK. Surprisingly, they even show significant improvement of vision in uninjured old mice without significant improvement in young mice. This effect is eliminated by the TET1 and TET2 knockdowns like before. Again, they also show decreased DNA methylation age in these elderly OSK expressing mice without the TET knockdowns. And here again, this is without OSK and young mice with OSK in older mice without OSK and with OSK, the optomotor activity. And here you can see the DNA age of old mice without OSK and here the DNA of the old mice with OSK expression but without any blockage from the TET1 and TET2 enzymes. As a bonus, they show axon regeneration in OSK positive mice here. They also show that phosphorylated S6, which you'll remember as a known target of the mTOR pathway is not increased by OSK expression. The regenerative effects are also not eliminated by rapamycin. This suggests that this uses a separate pathway from mTOR. Overall, here's a summary of the article findings. They found that OSK expression does not negatively impact growth or development in adult mice, can be directly and efficiently expressed in retinal ganglion cells, can be controlled with an oral medication, improves retinal ganglion cell survival and axon regeneration, regenerates axon density and recover vision of glaucoma model as well as in old mice. This is dependent on the TET1 and TET2 enzymes. It is separate from the pathway used by mTOR and it can reverse the epigenic age and epigenic clock. All of these findings fit neatly into the information theory of aging. The core belief behind this theory is that the data stored within DNA remains stable and without loss of information. This is evidenced by the ability to reprogram mature cells into pluripotent cells. Differentiation and aging are the result of epigenetic changes rather than genetic ones. The epigenetic clock, which highly correlates to chronological age can be reversed. Controlled reprogramming of cells can theoretically reverse epigenetic changes associated with age without losing cellular identity or causing tumor and genesis. This could potentially allow cells to function as if they're young again. Now, if we can reprogram specific cells to induce regeneration and return in more youthful phenotype, what other conditions could we tackle? How many other therapies are limited by regenerative capacity of these tissues? So many of the conditions that we battle are, if not inherently caused by aging, are at least age-related. An iPhone 1 cannot be more excited to see where this will lead. In summary, the field of optic nerve regeneration has grown exponentially in recent decades. I've outlined a few of the most promising pathways here, including oncomodulin activation, PTEN or SOCUS 3 deletions, and epigenetic reprogramming. Although none of these pathways may end up being the Holy Grail, the march of progress is unmistakable. In particular, I believe the epigenetic reprogramming has the potential to dramatically alter the landscape of not only ophthalmology, but medicine in general. However, countless great ideas have been lost in translation from animal to human models. So the biggest and most important barriers ahead are human clinical trials. I just want to say a special thanks to Dr. Roscoe and Dr. Katz. Here are some references and if you guys have any comments or questions, that would be awesome. Thank you, Dr. Long. That was excellent. We do have time for any questions or comments, so feel free to put those in the chat or request to be unmuted and we can have a discussion here.