Hi everyone, Joe for jaskiescasebreaks.com coming at you with 2020 Gold Rush autograph football jerseys This is a three-box half-case break random letter break number five from jaskiescasebreaks.com We sold 16 spots and we were going by the first letter of their first name first letter of their first DK Metcalfe goes to D. Kyler Murray goes to K. So on and so forth. There's also a Multi-sign Jersey spot So there's some cool multi-sign jerseys that could be possible out of this. So one spot will get any of those All right, so big thanks to all of these folks for getting into it. There are the letters right there. Let's roll it Let's randomize it snake eyes two times for each list easy one two bam Gerald down to Kurt two times for the letters one and and two Easy K down to T. All right, so Gerald with K Aaron with G and H Gerald with B Sean with NOP. No, it looks like New Orleans Palakins right there Kurt with M Logan with R Joe with C Tyler with J Dennis with S Debra with V plus W Dan with E plus F Oliver with a Aaron with D and L and the Multi-sign Jersey spot and Kurt with T So let's sort this by column B by letter right there and feel free to trade if you would like I don't know if Joe burrows in this stuff like this. Usually normally doesn't they don't have checklists So they really just kind of give you an overview of what you could possibly get Doesn't doesn't look like it, but that doesn't mean it's not there could be there who knows All right, so we got the case back here Let me open up that case these Let's try to get my mouse Let's get it all on one screen right here Or at least on the top camera in the main cameras. Here's what we're gonna do These three right here will be one two three on the dice roll and these three down here Will be four five six on the dice roll Let me pick a die. Let's pick that one So once again one two three for the top three and then four five six for the bottom three and it's six So it's going to be the bottom three right here. So for the top three Big sharpie here there is will be for break number six, which will be which will be posted soon You can run this back pretty easily tonight. I would think all right, so you might be able to see on the top camera I've got them marked six. It'll be all three boxes will be mark number six. All right, looks like no trade So let's print let's rip And let's play a couple a few quick rounds America's favorite game who am I I give you the clues and you guess the players for bragging rights good old bragging rights you can Bragged to your friends. Hey, I won a game of who am I? What did you do today? Try to turn us away from the camera as much as possible top camera the Main camera Who am I? I'm a retired quarterback Played for one two one two three teams in my entire career One time Super Bowl champion and a Super Bowl MVP Two-time NFL most valuable player four-time pro bowler two-time first-team all-pro two-time passing touchdowns leader three-time NFL completion percentage leader. I'm a Hall of Pro football Hall of Famer and I was also in the Arena League for a little bit with the Iowa barnstormers and then I played in Europe With the Amsterdam Amarals. No one's got it yet Those players didn't play for the Amsterdam Amarals Yep, Riffer's got it. It's Kurt Warner. I think they're making a movie on Kurt Warner, aren't they? Yeah 208 career touchdowns Pretty pretty great story You can see Kurt Warner in his Rams gear. You can see the yellow trim right there the Beckett sticker right here Where's his autograph? There it is There's his auto right there. He's got a good-looking autograph I'm pretty sure these I'm not gonna take these out because I think they're custom jerseys says Warner on the back of course Yeah, these are just custom jerseys so it really won't it won't have any of the logos or anything like that on there But it does have the the proper color scheme and the numbers stitched on they're perfect for framing actually All right, so there you go. Who has a the Iowa barnstormers part did it for you? I Don't even I didn't remember he played for the Iowa barnstormers. That's for Gerald. There you go, Gerald letter K For Kurt Warner hurt with a K Not to be confused With Kurt Warner Kurt with a C. Let's bring number five right there next one trying to think of Jane Hames That's one like one of the more common ones Joe John Jim James Jack is it that person? I think it is Bryce is correct Bryce was like I feel a running back coming up. This is a running back an active running back in the NFL I'm in my second year in the NFL that should give it away pretty quickly 23 years old from Woodland Hills From Pittsburgh, Pennsylvania, that is Second round pick part of the pfw all rookie team last year So far in my career. I have a 818 rushing yards 4.6 rushing average three rushing touchdowns 50 receptions 509 receiving yards three receiving touchdowns and 314 return yards. I've got a I've got a pretty bright future ahead of me Not Jacobs juju is a right receiver not Jacobs not Barkley not Conner. There you go sky highs got it miles Sanders David's got it too. Yeah, Miles Sanders From Pittsburgh went to Woodland Hills High School and PA and then went to Penn State It's a nice looking jersey here Feels a little known as a custom jersey as well, but they got they put they got some shadowing on the letters here There's his big number 26 right here the silver ink autograph right there There you go, there's good shot at it right there nice number inscription 26 the number looks really nice They got they got nice numbers for this Got the JSA card here, and there's the JSA sticker right there. We'll leave it nice and fold it in here, but It's a custom jersey, so they won't have any logos or anything like that. All right Who's got mild Jason Jaspi might be interested in this one Kurt with the letter M. He's Collecting a lot of miles Sanders stuff All right second half of this will be in the store. So check it out. Jasper's case greats calm We have I mean throughout all the breaks. We've done of these These have been selling out quickly. We've seen a seen a lot of nice jerseys All right, let's see what we got for the last one hide this a little bit here. What do we got here? All right, I'm another active running back another running back Who's in the second year of my career? first round running back this time I Was the Pepsi NFL rookie of the year and NFL offensive rookie of the year a pro bowler of PfWA all rookie team consensus all-american back in college 400 2344 rushing yards in my career 1159 receiving yards in my career 17 rushing touchdowns six receiving touchdowns 143 receptions. Yeah, he's pretty good He's pretty good and and teammates college teammates with miles Sanders Yeah, Riffer's got it. Yeah, say it's a quant now. So you go another number 26 What numbers were they in college then if they both like 26? There's a say quant Barclay There's his autograph right here There's the JSA card right here in the JSA sticker Right down there Poor say qualm Barclay on on the on the IR Which which sucks, I thought he was gonna have a really really big year this year All right. Well, there you have it boys and girls Thanks for watching. Thanks for bringing one. Who's that go to s? Say one Dennis Dennis Knox with the letter s you get the say qualm Barclay. Thanks for watching everybody I'm Joe for jazz piece case breaks.com. I will see oh, I guess take one was two years ago miles Sanders Lester Anyway, thanks for watching everyone. I'm Joe for jazz piece case breaks calm Thanks for watching. I'll see you next time. We got the second half in the store. Check it out. Bye. Bye


I make a goal to find something at each one of these expos that I haven't before seen. So we found a strapping mechanism that someone had invented that can strap a palette of cans. So these guys have engineered something really neat that we discovered and didn't even know we needed. This is my first expo that I've been to, but I came to actually see and touch different products and see what different vendors have to offer. Because it's hard to do the research online, so seeing it in person is helpful. I highly recommend the conference. Oftentimes as the end user of all these different machines, you don't actually get to interact day to day with our machine builders. Anytime that you can have a one-on-one in a relaxed environment, you are able to talk to them and learn from them. Because a lot of times they come to you with applications that you're not aware of. And if you listen, you'll find out what the next trend is and be ready for the future. We've been in existence since the early 70s and our best customer that we ever got came from PacExpo East. So it's a significant event and convention for us.  And I think a show like this gets you out of the box, so to speak. It really makes you reassess if your company is really being innovative. It's an easy word to say, but when you see it here, you learn about innovation.


Hey, I'm going on tour and I'm coming to your city When in March of 2022 Wow, it's coming up fast. Make sure to go to I have to do this dot show and get your tickets now I'll see you there.  I'm about to get my holes Absolutely ruined. Well, baby.  You have a mount in this game. His name is torrent. Look at this guy Look at him I'm so sorry. I'm so sorry. You can also craft Whoa He was roll.  So this is a field boss Or actually this isn't a field boss. I think about it Absolutely obliterated and I said that don't make a sand. Everything's fine You know gonna be okay, let's see what's down here. I bet none here yet. I don't think so. Oh Traverse the mist Well Let's try it Beast man Farum Azula Well, there it is There I go. Ashley. Just for you.       It's just chillin here This is a friendly nice castle that I can go in. This is a cool nice friendly peaceful castle I bet you it absolutely totally is Wait, I was invaded. What are you doing? Oh? My god invaded and raided kind of funny games What the hell I was I'm gonna die hold on hold on hold on hold on hold on hold on hold on hold on hold on hold on Hold on Because I've my what? Huh, oh To the game crash yep Wait the game didn't crash it's still running okay up. I'm in the game. I'm in the game. Okay.  I completely forgot that it came back up. Whoops. Oh, I Got invaded again.  Sorry There's a big golden boy over here. Yeah, that dude. I don't think that I can kill him But should I try it? This is a field boss Can I get a single hit?  I want to see what's up in this castle a little guy You can't get away from me He missiles at me.    I do absolutely nothing to this dude Wow, I Could maybe take on one of them. Oh He yelled at me. He yelled at me Okay his big screen is so large My guy you need to heal you see that dodge though That's good. I want to um, I want to see what this guy's about. Well first Okay, so those are easy to kill. What about this dude?  He's too powerful too weird-looking.  I put myself over oh my god. Oh my god. Thank you Let's touch this thing. I don't think I need to rest there necessarily. Oh Oh my god, this looks amazing.  Yeah Bell I think he's so much for the five Bella stopped gifting subs Mods should not give subs feel good about this one. I feel bad About this. Oh, I do nothing. I do nothing to him Margaret the fell. Oh my god.  Oh my god guys. We're holding hands Round table hold Round table holders a place where tarnished gather guided by grace combat is prohibited on the premises due to the pact of non-aggression Would you allow me to hold if you might share with me some of your life?  Do you think it vulgar perhaps where I come from? It is a sacred. Whoa You can hold me my thanks great champion now sometimes it's nice to be out be a little spoon The most awkward weird Am I through the metal I got ballchins blessing what you felt like though it is but a fleeting thing I am afraid is this back to me. What should you require another? I will take you in my arms and as you need Did I get a boner?


Good morning, everyone. It is my pleasure to introduce Kate Keller this morning. So Kate completed her grad school at the University of Edinburgh, my side of the pond. Kate completed grad school at the University of Edinburgh before going on to do her postdoc on excecellular matrix biology at Shriners Hospital for Children in Portland, Oregon, where she has remained. She joined the KCI Institute in 2003 and is now a professor there doing her research on the trabecular meshwork, which she's a leader in the field of the excecellular matrix biology in the trabecular meshwork tissue. And her goal really is to discover the various ECM components involved in the trabecular meshwork and determine how they can be leveraged to increase outflow through the TM. And from there, Kate, I'll let you take it away. Well, thank you Fiona for your kind introduction and thank you for the moron eye centre for inviting me to give this talk. I'm hoping that you can all hear me okay. And the title of my talk today is trabecular meshwork, intraocular pressure, and glaucoma. I do have one disclosure. I'm going to show one experiment. I'm using Natacidal, which was sponsored by Erie Pharmaceuticals. So here's the outline of my talk today. Today I'm going to talk about the intraocular pressure, glaucoma. We're going to talk about methods to study the IOP in vitro and in vivo. We're going to talk about the Morrison controlled elevation of IOP rodent model. And then I'm going to switch gears completely and talk about targeting the actin cytoskeleton to reduce intraocular pressure. So I don't really need to introduce glaucoma to this group, but here we go with this irreversible blinding eye disease and it affects over 60 million people worldwide in an aging population. This is probably going to increase significantly in the next 20, 30 years. Glaucoma is a group of diseases. The main one that most people study is primary open-angle glaucoma, but there's normal tension glaucoma and glaucoma congenital and many other forms. The main risk factors are age, increasing age, race, family history, and of course elevated intraocular pressure. So normal pressure is around 15 millimeters of mercury and high elevated IOP is anything over 20. And it's due to buildup of the aqueous fluid in the anterior chamber, due to blockage in the aqueous drainage pathways in the trapecula mesh work. When this happens, it pushes the lens and the vitreous all back onto the optic nerve at the back of the eye and this causes axon degeneration and vision loss. So intraocular pressure, the aqueous is continuously produced by the ciliary body here. It flows into the anterior chamber through the pupil and it drains out through the trapecula mesh work to Schlem's canal and the venous system. The trapecula mesh work is a small triangular piece of tissue that not many people work on, but I find it very interesting. It builds a resistance to aqueous outflow and this generates the IOP. And once that IOP is high enough aqueous flows out to Schlem's canal and it passes through the TM as a bulk flow and it's driven by that pressure gradient and it's there's no active transport that is involved. So when you have when aqueous human production by the ciliary equals the drainage through the TM, you get a normal IOP of about 15 millimeters mercury and here's the typical goldman equation. IOP is the rate of formation divided by the outflow facility plus the episclerous venous pressure. And we know the outflow resistance in the TM increases with age, but it increases a lot more with COAG and this results in the elevated IOP. So IOP is not actually constant throughout the day. There's been new studies done with this contact lens sensor called Trigafish. It measures pressure throughout the day for 24 hour period and you can see in the daytime most of the IOPs are lower than in the nighttime. And then if we look at the three groups of patients here, they've have healthy subjects in blue, COAG patients in red and normal tension glaucoma in green. You can see that the pressures in the COAG patients come up earlier in the day and the peak higher in the nighttime than the normal subjects in blue. And this can also be seen in the normal tension group as well. And so the peak times for the peak IOP occur earlier in the nighttime, excuse me, compared to the normal subjects. And this is, you know, this is very interesting because when patients come into the clinic, they usually have one IOP test during the day sometime. It just gives a snapshot. So this is really giving interesting information, especially for those normal tension glaucoma group. And then another group has shown the circadian IOP in the normal tensions patients, which are in the black circles here. Anything towards the outer edge is the higher pressure compared to the lower pressure to the center here. And you can see that all the non glaucoma patients have their peak IOPs during the nighttime, whereas a significant portion of the normal tension patients have them during the daytime. So even though they're called normal tension, they're not actually normal IOPs. And as a clinical strategies, the only therapy that is available to glaucoma patients right now is lowering their IOP. And how did we do this? Well, pharmaceuticals have been developed and they can target different paths of the outflow pathway. So there's a aqueous humor suppressant such as the beta blockers and the alpha 2 agonists. And these target the ciliary body and they suppress production of aqueous humor. About 20 to 30% of aqueous actually exits the anterior chamber through this uveol scleral pathway and increasing the outflow through this pathway via the prostaglandin analogs and the alpha 2 agonists. It's also been used as a clinical therapy. And then this targeting the traditional conventional pathway through the trapeculomesh work. This is where 70 to 80% of aqueous flows out of the anterior chamber and the new class of rokinase inhibitors target the TM and there's also cholinergics such as pylocarpin. And then this is the first line of attack and this doesn't work in the clinic. Then the glaucoma surgeons get pretty nasty to the tissue which is in my opinion not nice because I like the trapeculomesh work. So they make holes in the sclera and this helps drain aqueous out of the anterior chamber. Even more macabre is that they put a suture in through the hole of the trapeculomesh work and then they just rip it out. And then they burn holes in it. They can use laser trapeculoplasty using ALT or SLT. These differ slightly in the energy and laser power that they burn the holes and also how far circumference how many holes you go around with circumference of the TM. So I'm here to convince you that the TM is actually a very interesting tissue. And this is a nice summary of all the therapeutics that are currently on the market for glaucoma. So this is from Miriam Culco's group in Denmark, a review article here. And you can see that the main state of glaucoma therapies is IOP lowering. There's a couple of vascular therapies out there and a few neuroprotective therapies. But as a basic research scientist my goal is to understand the TM molecular or cellular level and understand how this TM regulates IOP in order to develop new glaucoma therapies. So here is just a H&E section or the cross section of TM showing the uveal and conial scleromesh work. Down here is what's known as the juxtacanalicular region or JCT and that's right next to Schlem's canal. And this is just a schematic of the same thing. Aqueous is going to flow through around all these fenestrated beams out through the juxtacinicular region to Schlem's canal. And in this JCT region there's abundant extracellular matrix and that is what composes the outflow resistance. In glaucoma's TM, there's a much more disorganized extracellular matrix. So they found thickened extracellular sheaths surrounding the elastic fibres and this causes narrowing of the outflow channels so the aqueous can't flow through and out to Schlem's canal. And then the other phenotype that they found is the reduced cellularity. So in normal aged TM's there's a fewer cells as you get older but in POEG this is much more pronounced and there's much fewer cells there. So then I want to move on to how can we study intracial pressure in the lab. So the main stage that I've been working on is anterior segment perfusion culture. This is an organ culture system and here we take a human eye from we get them from cadaverized from the local eye bank and we bisect them. We take out all the lens, the iris, the ciliary and so we're left with a cup basically of anterior tissue and this contains conia sclera trabecular mesh work and we leave a little bit of sclera on there so that we can clamp it into this chamber and then we can perfuse serum-free media up into the anterior segment and it flows out and drains out through the trabecular mesh work. And according to the height of this bottle we can mimic just normal tension so normal tens of about 15 millimeters of mercury but of course we don't have the epiphenous pressure there so it works out to about 8 millimeters of mercury but if we raise this bottle we can actually give a 2x pressure and that mimics what was happening when IOP is elevated to McLaughlin patients. And so what happens when we do this? So we can flow at a certain constant flow rate to give that 8 millimeters of mercury and then if we double the pressure immediately the flow rate increases but over time it actually comes down and at homeostasis down back to normal over you know two to three days. So IOP or homeostasis is defined as the corrective adjustments of the aqueous humor outflow resistance which occur in direct response to sustained pressure changes and which maintain IOP with an acceptable physiological ranges. So how does this happen with molecules that are happening in the TM? So in the dark line here this is just again showing that you can double the pressure and it comes down over the next couple of days but at the same time if we look at gelatinase A which is also known as matrix metalloproteinase 2 this immediately goes up and becomes activated and these are the enzymes that chew up the extracellular matrix. And this is another matrix metalloproteinase called Adams TS4 and in control sections you can see that there's small punctic dots throughout the TM and very few along the juxtacana ubiquular area whereas if this section is from an eye a human eye that has been confused at 2x pressure for 48 hours and you can see that the Adams TS4 is highly increased right along the JCT area right where we would expect it to be if it's chewing up the extracellular matrix in response to pressure. Other labs have looked at genes that are altered in this perfusion system in response to the high intracranial pressure so this is worked by Tourette Boris's group she perfused at 15 millimeters of mercury or 50 and then at different time points she looked at the genes that were changing in response and this has been very effective at looking at increased genes some of the extracellular matrix and she's looked at this one called matrix gloprotein which is very interesting. The other thing that we can look at is segmental outflow. Now what is segmental outflow? Doug Johnson back in the late 80s noticed that pigment granules would be collected in certain regions around the circumference of the TM and Dave Epstein's group went on to use catanized ferritin to study this further and they called regions of high and low outflow segmental outflow so this is a study that we've done relatively recently and this was with fluorescent nanoparticles we add them to the perfusion system and again they flow into all areas around the circumference of the TM but there's regions of where they go faster so that's the high flow of regions and the low flow regions where there's relatively few fluorescence nanoparticles and then you can cut these sections out and in this case we're looking at genes so we isolated the RNA and found the extracellular matrix genes are change expressions within these high and low flow areas so certain collagens, integrins, laminins, matrix metalloproteinase are enriched in the high flow regions where others are relatively enriched in the low flow regions and this can have clinical implications so this is work done with Alex Wang's group and actually Fiona was on this paper which was very interesting they've been using aqueous angiography to find out these high flow and low flow regions so again here's a high flow region and a low flow regions and then Alex was doing trabecual bypass surgery and they found that if you put your surgery in the low flow regions you actually get a greater outflow facility when targeting those low flow regions so this the segmental outflow you know studying it at the basic research side of it can have clinical implications of where would be the best place to do your surgeries so the summary of the anterior segment they advantages that is a really easy system to set up you can use human eyes but if you don't have access to those you can use pig eyes or bovine eyes it's very easy to manipulate so you can change the pressure you can add drugs you can do it for different time points up to about two weeks it's very easy to harvest the cells and tissues at specific time points in the experiment and they've been really helpful to identify genes and pathways involved in IOP homeostasis but the major benefits advantage is that they don't fully recapitulate the in vivo environment so people have used in vivo models and many people have used mice because they're very small and easy to use and they've done various methods to increase um intracular pressure one of the main ones that people use is called this micro bead occlusion model so they inject polystyrene microbeads into the anterior chamber and as the aqueous flows down through the TM it takes the beads and it blocks all outflow channels and as that happens the intracular pressure increases and then they've been looking at the optic nerve injury and found like after about 10 days to two weeks they find some degeneration other groups have used various viruses to transduce molecules into this so AAVs and adenoviruses for instance this TGF-8-2 when you put that into and jink back into camera Lee it can produce the IOP increase of 10 to 15 millimeters of mercury and of course there's the jet genetically modified my mice one of the the main ones here is the DBA-2J mice they have a spontaneous mutation that causes sloughing of pigment granules from ancillary and again that um goes into the trabeculine ashwork blocks the outflow channels and it causes IOP increases um and there's another one called myosilin um his transgenic mouse has um the human myosilin with one of the mutations that they found in glaucoma patients and again that causes um exondegeneration in the posterior pole other people have been using dexamethasone it's a quote-to-quote quote-to-quote steroid and this induces extracellular matrix production which hinders the aqueous outflow much like what we saw in the glaucoma and then finally they use laser treatment again to burn away the um tm and photo coagulation so again these um mice models um are really good and have helped a lot of um basic researchers um they're relatively cheap you can um there can be easily genetically modified uh very useful for studying the effects of specific genes proteins um or signaling pathways and they're very very good for looking at the optic nerve responses but the several disadvantages um there's little control of the level or the onset of IOP elevation so when you put like beads in there um we don't know exactly if they're not if the IOP is immediate if it stays up um or if there's fluctuations and spikes are happening um many of these models the IOP is only monitored intermittently like like how you do with the glaucoma patients in the clinic so they maybe you'll be only being monitored maybe once twice a week and then nearly all of these models the tm cell function is disrupted or destroyed or compromised in some way so we can't use these models to look at the tm cell function so other models are needed to identify genes related to IOP or homeostasis so now I'm going to talk about the Morrison controlled elevation of IOP so John Morrison is one of my colleagues at the KCI Institute he's a glaucoma clinician but he's had a basic research lab um for many many years now and he uses rats um and the choice of rats is that the eyes are bigger than mice and so they're just slightly easier to handle the rat model is still relatively cheap and the angle and outflow anatomy is very similar to the human so this is just um a cross section of rat eye um with the anterior chamber iris ciliary body um here's the angle the trabecula mesh works right there and then this is a collector channel there's still two shlems canal and that's just a more higher magnification of the trabecula mesh work I will say that it is slightly different and that is not as big as the human um trabecula mesh work there's only maybe one or two of those penetrated beams but it still has the jc team area so in 2016 um John and his team um did this um this publication and called it the controlled elevation of IOP and here the anterior chamber is cannulated and um you can do this on four rats at a time and you continually monitor the blood pressure the oxygen the heart rate and the breathing rate and um the the duration of the experiment which is usually eight hours these rats do absolutely fine so this is let me just turn off my point a second so this is a little video showing how to cannulate the rat's eye um use a 31 gauge needle to poke a hole through the cornea and then they thread in um a small tube to cannulate the the anterior chamber and the tip of the tube um it doesn't touch the iris it doesn't um it just sits in the anterior chamber and this tube is connected to a larger tube that goes up to um a bottle full of um balanced salt solution and again like that anterior segment perfusion model um by varying the height of the bottle um it gives the pressure that the eye will receive so the major advantages of this model is that you can set IOP to define pressure um you know exactly when you're actually applying that pressure to the eye and then you can use it for various defined um IOP durations so we decided to use this model and I will say that tube because it's not um it doesn't touch the TM um we can now go ahead and look at um what's happening in the TM in response to elevated IOP in Vivo for the first time so what do we um when we're designing our experiment what do what things did we have to consider when we have to consider the duration of the experiment the IOP level and then what controls can we use so for duration um we chose eight hours so we know that the eight hours um the rats all survive um anesthesia and um they sit there quite happily and um from our anterior segment work we know that a homeostatic response is launched after six hours so we expect to find um genes involved in the homeostatic response if we give them a pressure for eight hours and it has no effects an eight-hour pressure has no adverse effects on heart rate or any of the other physiological readings that we're taking but what is normal tense of IOP and rats um again rats show a circadian IOP much like the humans it's higher at the night and lower during the day but the mean IOP over the 24 hour period is about 21 millimeters of mercury um and the peak in the night time is about 30 so for designing our experiment we chose to have our normal tens of IOPs at 20 millimeters of mercury our hypertensive group um is going to be 50 which is about 2x what the peak IOP is and then we kind of have a group of animals that have are naive so they've had no surgery or no anesthesia um they've just been running around in their cages so we performed the CAI on um brown no-way rats um these are typically used um and actually have a bit advantage over other rat species because um um the TM is pigmented in these ones compared to say albino rats they're retired breeders so they're aged about six to nine months um and we used equal numbers of males and females and each group had um um n of 11 or 12 per group we monitor IOP carefully with a thermometer so we might measure it every 30 minutes over that eight hour period and um the mean IOP for each of the animals over that time in the CEI 20 which is right about 20 and those in the hypertensive group were right around 50 and at the end of the experiment we enucleate the eyes and dissect the TM and we came out with a paper um just earlier this year describing this method in detail um there's a nice video if you want to go and have a look at the video um but we bisect the eye we cut it into quadrants the ciliary and iris is removed um we put pins here just to give it a little bit tension to pull it off this one shows just a need a small pin that's been pushed up through the um TM so that you can see that pigmented TM there and then we use um jeweler's forceps to pick it out and just peel it back and here's a section here that has TM and you can see the slight pigmentation um and in the white arrow um you can see the translation translucent translucent area of the outer wall of shalem's canal right now so then we did um histology and immunostaining on some sections but most of the eyes were um the TM was taken out and um the RNA was isolated from the cells in the TM so we first assessed whether we were doing good job of dissecting the TM's here are H&E sections um before we dissected and after we dissected here's the TM and post dissection we've done a pretty good job of removing all of the TM and again this is just immunostaining with a smooth muscle actin and green um to show that it's pretty much removed from these eyes and then we also wanted to make sure that we didn't really have much contamination from the ciliary so we used um three genes matrix gla protein mycelin and chitonase three like proteins these are biomarkers of TM um and in TM tissue these were um high as expected compared to ciliary um body and then ciliary markers were desmond mycelin heavy chain 11 um and in the TM samples these were relatively low so we were fairly sure that um we're getting quite clean dissections here so then we went ahead and isolated the RNA and um we didn't have um enough to do RNA seek analysis so we chose this um technology called nanostring um it uses RNA um to directly you don't have to make cDNA um so we used RNA directly and they um have probes um that you isolate the you incubate with the RNA so there's a capture probe um it has half the target gene sequence on it with a biotin and um the three prime repeat and then you have your reporter probe that has the other part of the um target sequence and then it has these fluorescently labeled RNA segments and in four different colors and the combination of all of these um these colors you can get 700 no sorry 972 unique barcode fluorescent signals um onto your genes so once you've incubated it with your RNA samples um you bind these probes to streptapidin that's coating the surface of the imaging surface um you flow some fluid over them then knock small flat in order for the um machine the encounter machine to actually be able to count these and one reporter barcode is um equivalent to one messenger RNA molecule so obviously the more um counts that you get um or more barcodes you get the more abundant those RNA molecules are they have pre-designed panels that you can choose from and so we chose this pan-cancer panel it has 770 genes and um they're all involved in inflammation or microenvironment and tumor biology and because cancer involves many extracellular matrix genes there was many genes on this panel that were of interest to us and again we have um our three group comparisons so we have um our cei 50 millimeters of mercury um compared to 20 um we have 50 versus a naive and these two groups we're calling um i should give genes that are iop related whereas the other group which is the cei 20 versus a naive this is probably you're going to give genes that are more related to um the cannulation procedure or um anesthesia so looking at the three different groups this is the 50 versus 20 the 50 versus naive and the 20 versus naive so these two groups here um we found that there was together there was um 46 genes that we're calling the iop related genes and the the blue and the red um highlighted genes here were found in both of these groups so we're quite confident that they are iop related and then we found um 55 genes that may be more cannulation or anesthesia related when we put this iop related genes into um bioinformatics software um called shiny go um we did a kick pathway analysis and the iop regulated genes um included um the ubiquitin mediated proteolysis um and notch signaling pathway this is um involved in cellular communication and then the down regulated genes um very interestingly um pulled out the tgf beta signaling pathway and you know when you treat um tm cells with tgf beta they actually produce more ecm um so we're kind of thinking that that might um be a protective measure that um and when when the tm is exposed to these higher pressures it's actually down regulating tgf tgf pathway genes and suppressing that extracellular matrix production so um in summary for this part of the talk um the cei rap model um it's very useful for studying iop and vivo this is the first time it's been done um in vivo for the tm um the cannulation procedure is relatively simple and we're monitoring iop regularly so we know exactly what's happening in these animals um but one of the limitations is that um eight hours seems to be about a maximum time point for the animals um um anything further than that is not it can be very harmful so um tm can be dissected from the nucleated rat eyes but it needs skill surgeons um that's done under a dissecting microscope we have managed to isolate RNA from the tms um but they're in small quantities um and it's not been enough yet to do our full RNA seek analysis so we've used nanostring technology and this has been um useful um because we've identified iop related um genes but this panel that we chose was a mouse panel um they don't have a um pre-designed rat panel yet um and so um there's a good homology but there's some of those genes that are on that panel that are not completely homologous okay so now i want to shift gears completely and um talk just a little bit about the actin cytoskeleton and how that um has been targeted to reduce intraclopulopressure so um this is just a schematic of the actin cytoskeleton um the um actin within the cell can assemble into different structures including phylopodia um lamellopodia or stress fibers um my talk um later today we'll be talking a lot about um the phylopodia and these things called tunneling nanotubes which will be very interesting um but right now i'm going to focus on the row kinase pathway so this um once this is activated it leads to formation of um stress fibers and um if you inhibit this pathway um we know that um it increases outflow and this is the um this was the development of opressor which is one of the FDA approved um glaucoma drugs that's on the market these days so what are actin stress fibers well they're contractile actomyosin bundles and they're anchored at each end by focal adhesions and when we take um normal TM cells or um TM cells from a glaucoma eye and we place them in culture and we label the actin we can see that um glaucoma T M cells have much thicker and more prominent stress fibers than their normal um TM cells and if you measure the width of these things um you show it um significant increase in um the filament width in glaucoma cells compared to the normal cells we also did live um imaging let me turn off my laser pointing um of normal and glaucoma um so if we start up this movie so these are the stress fibers here in the normal um TM cell and you can see that they're um turning over it's called tread milling and they're foaming disassembling there's little um dots punctic dots of actin rich vesicles and these are moving around quite happily in these um normal TM cells but when we do live cell imaging of glaucoma TM cells you can already see that they're much more um prominent stress fibers they're not being tread milled as fast or in fact hardly at all and then these little actin vesicles some of them are moving around but many of them are just sitting there doing nothing and so um we can see by live cell microscopy that the actin dynamics in normal and glaucomatous TM cells um are highly different so um back in the um mid 90s um they started looking at the actin cytoskeleton and then targeting it with the actin myosin drugs like latronculin and this was pioneered by um David Epstein and Paul Kaufman um and when they put some of these drugs into the anterior segment perfusion culture system um they showed a nice increase um in outflow facility over time and then over the years um they um they refined this slightly and um looked at that pathway and realized it was the rokinase um pathway that was um very interesting and um Vasant Rao has um a good um review article here um telling you all about um the effects of roa inhibition um both on TM and Schlimm's canal cells um but also in the optic nerve I'm thinking that it might be slightly um near protective and then also um for use in glaucoma filtration surgery so Dave Epstein um and Casey Kopinski in um North Carolina um they they developed um the company Aerie Pharmaceuticals and this was their um rokinase inhibitor that they developed it's called metacital so it inhibits rokinase but it also inhibits the norepinephrine transporter and teamed up with Casey um and tested the effects of metacital on normal and glaucoma TM cells so this was just still images of um live cell imaging zero minutes or 120 minutes so you can see um TM cells have these stress fibers and after two hours they basically disappear um after metacital treatment and then here's the same set of experiments with glaucoma TM cells again they start off with um prominent stress fibers and after two hours like for this cell they're more or less all gone which is fine but there's many cells that you still have some stress fibers left um so metacital isn't quite as good and effective on um reducing stress fiber um in the glaucoma cells the actin cytoskelin is also very important for um phagocytosis um TM cells are highly phagocytic so when um aqueous flows in through the TM on its way out to Schlem's canal um the TM cells in Gulf particles and debris um that are collected in the aqueous humor um and when they're forming these phagocytic pits actin is used to polymerase out into a little cup that goes and gulfs the particle um apoclox groups shown that phagocytosis is um impaired in glaucoma TM cells um in the right here compared to normal and then we found we were doing this experiment within that metacital with live cell imaging and so these are actin rich um particles that have been secreted and deposited onto the surface and this cell here it's going and um engulfing them but the minute that we add um metacital it highly it becomes highly phagocytic and I look at this it feels like it's like a little Roomba vacuum cleaner going around and um sucking up all these um particles off the surface of the um the the dish here so this was unexpected we didn't really expect to find this but the metacital apparently it stimulates phagocytosis and in the TM um this could aid clearing of the debris from the out outflow channels and that may be an additional way that this drug is acting in the TM so in summary for the actin cytoskeleton um shown you that glaucoma TM cells in culture have less dynamic actin cytoskeleton um and it has some have thicker stress fibers than normal TM cells um metacital is effective for depolymerizing these stress fibers but it takes much longer in the glaucoma TM cells and then um metacital treatment activates phagocytosis and this potentially helps clearing of the TM outflow channels so even though you know repressors is now on the market um we're still finding out new um things about it and how it's acting um on the TM and both um in situ and also in cell culture and then finally I just want to finish with a couple of slides about um what new glaucoma therapeutics are in development so again this is um Marian Kalko's um review paper here there are still um IOP lowering drugs that are being developed including here's metacital um and they're they're linking it up with for instance latanoprost you know so they're doing these combo drugs um they're also looking still at vascular therapies and um several new neuro-protective therapies um that are in clinical trials right now including this one called nicotin temomide so just a supplement and then finally um there's this group um in the Mayo Clinic including Mike Feist and um you to you to your Roy Chowdhury they've been looking at this um ATP sensitive potassium channel opening pro drug um it's called QLS 101 um and they made a company called QRAUS to study this um and instead of acting on the aqueous um suppressants or increasing the out uveal scleral or conventional outflow it actually acts on the distal outflow pathway so this is anything further on than um the travecal mesh work and they're thinking that this could be used in combination with some of the other drugs um it's currently going through maybe phase one or phase two um and they're finding quite interesting effects so in mice um I've once daily topical treatment um was shown to lower the IOP um five millimeters mercury which is quite a lot in the mouse um and that was good for 24 hours so that would be the first one that targets um a different pathway than the conventional um drugs that being used and then finally I just want to thank um the members of my lab so Yunfeng Yang, Yang Yang Sun, Paul Holden, Fontein Chen, Fontein is a very talented medical student who joined our lab for um a year so she was great um and then Teddy Cox lab um Jan Sprank and Mary Kelly, John Bradley, Mini Aga, John um he set up the um CEI mouse model and members of his group Deanna, Elisa and Bill um I've been very good at characterizing that model and then the last um part of my talk was in collaboration with Casey Kupcinski at Erie Pharmaceuticals um he's now an Alcon because Alcon just took over Erie and of course my funding sources um the NEI and research to prevent blindness so um with that I will stop and I will happily answer any questions okay if no one else has any questions um Kate for that rat model um have you looked at segmental flow do rats have segmental flow the way humans do um and do you think the ECM would be different in that same way that's a great question um we haven't actually looked at segmental outflow in the rat yet um I would imagine that it does exist um because they've shown in mice that um segmental outflow exists um and in the mouse um studies they have shown that there are differences in the um extracellular matrix around the circumference of the eye so we expect that the rat would show um the similar thing but we just haven't done it yet thanks yeah great talk so one thing that was interesting for me was the circadian control of IOP and if I understood correctly initially you showed that during the day it looked pretty similar even in some uh glaucoma patients but then you had much bigger difference during the night so should we actually do these measurements during night I I think so um I think that you would get much more information um that is clinically relevant if you did your measurements in the nighttime but I don't think you would get much compliance with the patients I don't think they'd probably be willing to come in and maybe you wouldn't be willing to come in to test their IOPs in the middle of the night um but yeah I mean I think having that one snapshot IOP measurement you know a couple of times a month maybe um is not giving you a full picture of what is happening with um IOP um both on a circadian 24 hour time or over like a week or months um and so I think these these contact lenses I think they've still got ways to go you know um but I think they're providing providing some very interesting information about actually what what a patient is feeling so uh this nocturnal pressure elevation at night has been known for a long time actually and uh patients who have very good pressures otherwise during the day when when they can come in and be seen that are progressing there have been multiple studies that have gone to the trouble of looking at night is their pressures out of control at night surprise surprise and that's why they're advancing so um you've got the problem that patients aren't really happy about coming in at two o'clock in the morning and providers are not really happy about measuring them so I think the secret is going to be the kinds of things like trigger fish other simple ways of remote monitoring and clearly we need to understand that and handle it but it's just been difficult other than in you know specific circumstances but it is a major problem and often we're missing the most important point of the day for sure yeah I agree um and you know I mean I think you know with all this you know I've heard before about like turning your iPhone into a um iop monitor you know so I think um you know there's opportunities to develop new kind of devices and um and um technology to help us um self-testing there's some self-testing coming on patients and do it which I think will also be helpful yeah absolutely so I think um a combination of all of these things will be really useful for a glaucoma clinician um to get a much better idea of what that patient is actually um um being subject to pressure-wise okay if there are no more questions I think we'll end it here thank you for your time Kate