 Good morning, everyone. I'll just do a quick introduction of our speaker, Dr. Tatiana Jacobs, who is an MD, if I understand correctly, with a degree from Germany, Wordsburg. And then went to Harvard training with Dr. Dick Maslund, who is really one of the authorities and pioneers of retinal circuits, and in particular, tracing and categorizing amicron cells and ganglion cells. That was really, he has retired, just, I believe, but he has been doing phenomenal work on RGCs and amicron cells. And she started to collaborate with Simon John there, who is one of the pioneers in glaucoma, and then charted her own independent, syncretic way of looking at glial cells in glaucoma. And when I started to slowly dip my toes into this field, I immediately realized that her studies are the most detailed and beautiful and kind of insightful in the whole field. So it gives me a great pleasure. I can sort of introduce her here and look forward to her talk. Thank you very much for that introduction. Thank you very much for the invitation. And the title of my talk is Age versus Glaucoma Related Changes in the Optic Nerve. So I have no financial relations to disclose. So as you undoubtedly know, the prevalence of glaucoma increases with age, and in particular, after a person turns 50, 55. And that is true for all ethnic groups. The curve starts getting steeper and steeper and the incidence of glaucoma increases. However, age, of course, is a glaucoma risk factor that we cannot do all that much about. We cannot reverse the arrow of time. So the reason why we study age-related changes in the optic nerve is two-fold. Well, first, one cannot do anything about time itself. But maybe by finding age-related changes, we could directly address these and hopefully save some ganglion cells. And the second thing is age, of course, is also a confounding factor. So whenever we look at an optic nerve that has glaucoma or had glaucoma, we are presumably also looking at an aged nerve. So it is important to see exactly which changes are related to age, which are glaucoma or which occur in both. So I will focus on the optic nerve for the rest of the talk. However, I would like to add a little bit of a disclaimer here. Age affects many parts of the eye, not just the optic nerve. And I'm not at all implying that the changes in other tissues are not important. So let me briefly introduce the structure of the optic nerve in mice. Mice do not have a laminar fibrosis that is made of collagen and as it's shown in the lowest right panel, which would be a monkey laminar fibrosis. However, they have a structure that looks relatively similar. Now the axons of retinal ganglion cells, as you see in the panel A, are unmyelinated until they cleared a laminar fibrosis-like structure. So that gives the optic nerve in that region this glassy appearance. And then in the myelinated region, it becomes opaque. So this unmyelinated portion contains a region that we call the laminar fibrosis, as shown in panels A and B in the lower panels A and B, where the indicated in red are optic nerve astrocytes that form a sieve-like structure, which very much looks like a laminar fibrosis. However, it doesn't contain any collagen, or at least no other than collagen four, which is associated with blood vessels. In the monkey or in humans too, the ganglion cell axons, which also you see indicated in green in panel A, are directly in contact with astrocytes that line the laminar fibrosis pores. So it's not dissimilar only, of course, it is much bigger. So if you look at the individual astrocyte that makes up the laminar fibrosis, here we're looking at a mouse-grain that has several astrocytes, but not all, labeled with GFP. So it appears like a life-goldy state. So indicated on the bottom are the plane of sections. So in the left-hand panel, you're looking at a cell that is cut in a longitudinal optic nerve. So you can see that it spans with its processes. It spans at least half of the optic nerve. And as you see in the face-on image in the right-hand panel, that the astrocyte touches with its end-feed both the blood vessel plexus inside the optic nerve, indicated in red, and the peel wall that surrounds the optic nerve. So for the rest, I would like to focus on transmission electron microscopy rather than light microscopy to look at age-related changes in more detail. So we had several mice, a naive mice and glaucomatous mice, that from three months to over two years old, mice can live to about three years, at least in captivity. And our glaucoma group was done with microbead injection. That's what is shown on the left-hand panel. So it's an injection of little polystyrene micro particles that get trapped in the trabecular mesh where it can drive up the pressure to about 20 millimeters of mercury. And then it slowly comes down. The normal IOP in mice is around 10. So then we make cross-sections through the glial laminar, as indicated in that image. And we sample the optic nerve with several high resolution images. And that's what it looks like. So since this is a bit difficult to read, I false-collar some of the components for you. Outlined in red are the optic nerve astrocyte processes that intersect into the optic nerve and organize the retinal ganglion cell axons into bundles. Some of the axons here, not all, but some of the axons here are indicated in yellow. And at that level of magnification, you can also start seeing mitochondria, which are here indicated in pale green. So that's what it looks like in a young optic nerve. And also, you see that the axons are, of course, unmyelinated. So tracking cohorts of mice from three months to 30 months, you see that there is no disruption of the general outlier of the optic nerve. But you see that the axons start looking odd. I mean, at least some of them. So we see in the 18 months and the 30 month old mice that we get these big and swollen axons that lose their internal structure. It's pretty obvious in that axon in the 30 month old mouse on the upper half of the panel. So these axons lose their internal structure. I mean, these little dots are in the normal axons. These are microtubules and intermediate filaments. They seem to disappear. And also, you can see at that level of magnification that in the older mice, the mitochondria start swelling up and looking somewhat unhealthy. The same can be seen in longitudinal section. This is a longitudinal section. And if you look at the 18 and 30 month old nerves, you see that ganglion cell axons, again indicated in yellow, have relatively normal or slim looking segments. And then they have these bulbs where you can almost always find abnormal looking mitochondria inside. So this is what it looks like in normal aged mice. We haven't done anything bad to these mice. In glaucoma, glaucoma is optic nerves. And again, I false colored the astrocyte process. These axons in mitochondria. But if I didn't know what was wrong with that mouse, or if there was anything wrong with that mouse, I would say, yeah, it was old. But it wasn't. This mouse was three month old. But from the two month to three month, it had a microbeet injection at two month. And it had an elevated IOP for just one month. However, in many ways, the changes that we're seeing, these huge irregular and swollen axons, very much resemble changes that we would also see in age. So we can use these images to quantify many of our findings. Now, what we really care for is axon loss. Because this is what essentially drives visual defects. And mice, at least C57 black 6, start out with around 50,000 or so axons. And in a linear fashion, as a function of time, axons are lost in normal mice at about a rate of 7,000 per year. In human beings, this is fairly similar, though the numbers are apparently somewhat lower, at least overall, about 4,000 per year. However, when you look at the glaucomatis optic nerves here indicated with red symbols, they lose axons at a far higher rate. Well, that is to be expected. And thus, that would look like they are much older than they actually are. Now, counting axons is really not that trivial. And it's a lot of work, and it's a lot of boring work. So we were looking at some measurements that are maybe easier to do and not quite so time intensive. And the parameter we hit on was axonal diameter variance. I mean, you remember that there are some axons that still stay small and apparently healthy, and these axons that are really large in caliber and seem to be swollen. And so with age, the axonal diameter variance is what increases, and that correlates really well with age, if we're r square of 0.89. And here again, the glaucomatis optic nerves are indicated with red symbols. They appear to be at least one and a half to two years old. So changes that we would see within a year or one and a half years of normal aging when the mice are just sitting on the shelf, the same changes will occur in glaucoma if it just was going on for one month. So the axons itself is not the only thing that change with age and glaucoma. We try to quantify the changes in axonal mitochondria a bit more rigorously by coming up with a grading scheme for axonal mitochondria. And we came up with a grading scheme where a grade one mitochondrion in the top panel is basically one where we can't see anything wrong at all. Grade two are mitochondria which have apparently lost some of their cristae, but I would still think that they were probably pretty normal. And then we have grades of severity. Till in grade five, we pretty much can't see any cristae anymore. The only reason why I can still tell that it is a mitochondrion is because it has a double membrane. So the number of normal or near normal mitochondria in young mice is over 70%. And then it drops throughout a mouse's lifetime. And that is, again, not unexpected. And in glaucoma, we see, that is the bar on the far right, in glaucoma, we see, again, changes that would indicate that the mouse was actually much older than it was older than it actually was. So again, these changes can be quantified by measuring the mitochondria and measuring the variance of mitochondrial diameters. And these two correlate very, very closely with age. So we have a series of very predictable age-related changes. And I can show how glaucoma compares to those. Now I should make one more comment to mitochondria. So when I say the mitochondria swell and they change and you see abnormal and bad mitochondria, this is true only for the axonal mitochondria. So if you look at astrocytic mitochondria, they start out bigger and they accumulate more dye than the axonal mitochondria to begin with. But they seem to stay completely normal, even in very old and in glaucomatous mice. So when I say there are profound changes in their pathology in mitochondria, I mean axonal mitochondria, not mitochondria in the astrocytes or other components of the optic nerve. So now there are, of course, other things that we can quantify in the optic nerve. This image on the left shows a montage of about 40 individual images. You can count the astrocytic nuclei to see if there is a indication of astrocyte hyperplasia. There is not. And finally, other components in the optic nerve, in this case the blood vessels. We can also quantify the changes that you can see in blood vessels in the old mice and in glaucomatous mice. So panels A and B show blood vessels in mice that are essentially normal. 10 months is relatively old for a mouse. You can see lumen and a little bit of mouse. Really, I would need a mouse for that. But this is collagen in the basement membrane of the blood vessels in the optic nerve. And S is true for many other systems, too. In an aged blood vessels, the basement membrane becomes thicker. You get more collagen. You get more fibrils. This is quite statistically significant if you look at aged mice. And in glaucoma, something similar seems to happen. Though it's not quite as pronounced. So if you quantify the basement membrane thickness and the collagen in the basement membrane in glaucomatous mice, it falls kind of between young mice and aged mice. And I think that maybe if had we waited a little longer that one month, it might have been more pronounced. So we see glaucomatous changes in the blood vessels in the optic nerve, too, that resemble those of normal aging. So coming to the summary, we do see axon loss, as expected in glaucoma, of course. During normal physiological aging, mice lose about 7,000 axons a year. In glaucoma, it's about 11,000 per month. This is based on about 20 aged mice and four or five glaucomatous mice. We see an increase in axonal diameter variance. We see signs of mitochondrial pathology. Astrocyte hypertrophy, that is, astrocyte processes becoming thicker. I didn't show the quantification of that. Astrocyte hyperplasia, we don't see that in contrast to some other descriptions in the literature, but I've never seen that in the optic nerve. And in the blood vessel basement membrane with aging, we see the characteristic and expected changes in basement membrane thickness and basement membrane collagen. And in glaucoma, I think we see similar changes, though they somewhat fall into the middle, so I put it in brackets. And finally, I would like to come to the acknowledgments. I would like to acknowledge all the people who helped to work on that project. And in particular, the young person in the top panel, that's Ying Zhu, who did the beautiful EM work. And I also thank our support from the Chinese Scholarship Council and the NIH, mainly. And thank you very much for your attention, and I think I can take some questions. Elevate IOP and then lower it back. Do you see changes that are reversible or? Yes. If you lower, I mean, it depends on, I mean, for how much and for how long. So what we have done is 30 millimeters of mercury for an hour or two hours. And for one hour, you do see changes in the astrocytes. I mean, you see astrocyte reactivity, the processes become thicker. But no axon loss. And after about, I think, two weeks or so, the astrocytes return back to normal. Now, if the injury is more severe, if you wait longer or increase the pressure more than that, then one starts seeing some ganglion cell axon drop out. And it's not very pronounced, but you see some axonal drop out. And then the astrocytes do not entirely return to normal either. But in this, I mean, here we had about one month of relatively not, I mean, it's like 20 millimeter and 15 millimeter of mercury. That's kind of realistically increased IOP not like on an ischemic level. But that causes quite dramatic changes in the optic nerve already. Are there any characteristics in the glaucomatous nerve that aren't present in an aging nerve, or do they? Yes. There are some changes in the astrocytes that are difficult to see in the EM. So these are more easily seen in light microscopy. So the astrocytes start growing out kind of new processes and change their morphology. I didn't have the time in half an hour to go over all that in detail. But there are decidedly changes in the astrocytes that we don't see that are not just occurring as a function of aging. I know your study didn't really look at this, but I know that there's an age-correlated loss of axons in the brain. Do you think there's a direct correlation between the retina and the brain as far as age-related axon loss? I mean, we really haven't looked at the brain. So I'm not sure. So in another thing that we haven't in this part of the study looked at is factors that are intrinsic to the ganglion cells that lead to a ganglion cell loss. Or whether this ganglion axon loss is caused by something other than the ganglion cells itself that goes on in the optic nerve and might be mediated by microglia or by astrocytes. But for that, we would probably have to do like a time series of RNA sequencing of ganglion cells and of the glia, and that's how to do. But not how to do is expensive. But we haven't really looked at factors that are intrinsic to the ganglion cells that might explain their progressive loss. And that would, I think, be similar in the brain. I mean, they are fairly similar to other projection neurons. Sleep apnoea, I'm not sure. Normal tension glaucoma. I mean, all mouse models of glaucoma, as far as I know, are ocular hypertensive models. There are some genes that have been associated with glaucoma by GWAS studies. One of them is CDKN2BAS, which is this long non-coding RNA. And we don't really understand why that predisposes to glaucoma. There is a mouse that has a deletion of the CDKN2BAS or some of its region. These mice are more vulnerable to glaucoma, but there is a whole lot of other pathology in the eye, so they are very difficult to study. But that would be one that comes relatively close. As far as I know, the other models are all ocular hypertensive models, even though, in some cases, the ocular hypertension isn't very pronounced. These, and they're across a different functional plan. How that would suggest that the dendritic somatodendritic compartment is also playing a role in the disease, but it's often ignored, right? What do you think about that? I mean, that there are changes in the dendritic structure of ganglion cells is undeniable. I mean, I think this has been shown by many people. I mean, these changes, too, are very predictable. They kind of retract, they lose higher order dendrites. And there's no doubt about that. Now, the question is, what exactly is what happens first? And here, I mean, probably most people right now would say whatever happens first is in the optic nerve head. Not everybody. But I mean, we have never really tried to dissect that, but it's very difficult to see wherever you look first. Wherever you look, you can find subtle changes long before you find, let's say, changes in the pattern ERG.