Your own thank you so I know this is a diverse audience so please don't hesitate to stop me if I'm using lingo that maybe you don't understand so today I'm that talked to about the work that my dad and my lab does using diffuse optics which are kind of the general name that we call the technologies that we use and specifically how we use it to develop noninvasive biomarkers of brain injury. With spin towards Key after complications so the physical principles that underlie the techniques that we use are well known to most of you whether you are familiar not if you've ever taken a class flight and you sign it on your hand what you notice is that your hand glows red So you're sending white light in but only red lights coming out. The other thing that you'll notice is that unlike X.-Ray where you can make out distinct features in your hand like bones instead of your hand glows. Because the light has diffused through the tissue. So the reason that your hand glows is because tissue is highly scattering and it leads to a random walk of photons through the tissue. And here I'm plodding in the. Scattering coefficient for tissue in the near infrared where this reduced scattering coefficient is the inverse of the random walk step size that a photon takes in between scattering events and you can see that in this near infrared region it's on the earth order of ten inverse centimeters so what that means is that a photon is going to. Have an average random walk step size of one millimeter So when your light goes through your hand which is say two centimeters thick it scattering hundreds of times before it makes it out to the other side. In addition to being highly scattering tissue is also highly absorbing so here if we look at the absorption spectrum as a function of wavelength where this absorption coefficient new A is the inverse of the average distance that a photon is going to travel before being absorbed we can see that on average absorption is actually quite high for here I'm putting the two main chroma fours that are the two main things that absorb light in brain tissue which are oxy and D. oxy hemoglobin so absorption is quite high in most of the visible spectrum but in the near infrared there's this window where absorption is low and if we zoom in on that window so just within the near infrared you can see the absorption coefficient is on the order of point one which is about one hundred times lower than the scattering coefficient which means that your photons are going to scatter hundreds of times before being absorbed leading to a deep penetration of light through tissue. So since I apply this principle to the brain I often get asked whether light penetrates through the skull and the answer is Yes So here we took. Laser pointer and put it on the interior of a skull and you can see that that light makes it out through the other side and it's diffused through the skull. So the goal of these diffuse optical spectroscopy techniques in my lab is to develop tools that exploit this unique property of tissue to penetrate. Deeply. And specifically we use two tools called Near Infrared spectroscopy and diffuse correlations spectroscopy talk a little bit more about them and a bit we use these tools we apply them to the brain using just a light source on a detector we characterize the optical properties of the brains the absorption the scattering of as well as a diffusion coefficient which I'll talk about later. And then we relate these optical properties to physiologically relevant parameters that clinicians can use to guide patient care at the bedside so specifically we're able to quantify oxygen saturation blood volume blood flow as well as oxygen metabolism. So today I'm just going to talk to you about one of the diffuse optical techniques that we use which we call D.C.S. or diffuse correlation spectroscopy D.C.S. is used to measure the shear induced diffusion So this is D B parameter. Of the red blood cells that are flowing through the tissue vasculature and this diffusion coefficient is directly proportional to the blood flow in the tissue. So just a little bit about how D.C.S. works basically near infrared coherent light source the light scatters multiple times as it kind of treats the tissue and some of the light is going to make it back to the tissue surface so the electric field at the tissue surface is built from the superposition of multiple scattered light fields that have travelled along different paths to reach the tissue surface then they constructive and destructively to form this interference pattern. If we just look at one of these little speckles and we track its intensity as a function of time if all of the Scouters in your tissue were static that intensity about speckle is just going to be static it's not going to change but if there are moving scatters in the tissue which there are because red blood cells are highly scattering. Then you're going to start to see intensity fluctuations and the faster the blood cells are moving in the tissue the faster the lights going to fluctuate so to characterize these fluctuations in practice what we do is we place a light detector some distance away from our source and we capture just one of these speckles and we quantify in intensity autocorrelation function which just you know from this measured intensity data as a function of time where the decay rate of this intensity autocorrelation function is related to the blood flow in the tissue so just to give you kind of. Intuitive sense of what's going on we measure this autocorrelation function this was data we took on a patient who we had breathed room air for a little bit and then breathe C O two carbon dioxide is a Visa dilator in the brain so it's going to increase your blood flow and you can see that the curve that was taken during the room air period where blood flows lower decays longer than the curve that is taken during the C O two period where we know blood flow is increased so this decay rate of this autocorrelation curve is reflective of the blood flow in the tissue and we have these simple models that tell us how this this current. We fit the curve to simple models in order to extract a blood flow index in the tissue. This measured blood flow index represents a bulk average of the tissue underneath our optical sensor that kind of you can think of it as being a banana shaped region. That propagates from the light source to the light tech detector and the farther apart we place from our source from our detector the deeper on average that banana pattern is going to penetrate into the tissue. So you know in theory we could go all the way through the brain but in reality we're limited by signal noise so for most of our measurements in humans at least probing mostly cortical like this the superficial cortical tissue. So just to give you a sense of what our instrumentation looks like this is our whole little lab on a car we've got our D.C.'s box is measuring blood flow and then we also have another technique that we use to measure several other parameters but I'll focus on this D.C.S. part the important things with these techniques so that they're completely noninvasive So it's red light it's no more exposure to radiation than you would get like on a sunny day outside we're highly portable you know our carts on wheels wheel it all over the hospital. We have very high temporal resolution we can get down right now to about fifty Hertz. And an important distinction is that we're actually sensitive to the microvascular as opposed to the large like feeding arteries in the brain where we're really sensitive to what's going on at the tissue level. And not to make it sound like we're all you know like this is the end all be all technology we do have very low spatial resolution because of this diffuse the nature of the light propagation so we certainly can't compete with something like M.R.I. but because of our portability we're able to do measurements the bedside technique like M.R.I. may not be able to cannot do. So we custom design our optical sensors so the patient interface portion of this device we can make measurements that the sensors that we can use for spot measurements so say we just come and measure a patient on a daily basis or we can make sensors that we strap on to the head and we do laundry monitoring for you know we've we've measured patients for up to seventy two hours so armed with this tool now to measure blood flow we've used to study flow in the brain in both health and health and disease in numerous clinical studies. If they're there mostly pilots we've probably measured maybe one hundred fifty to two hundred patients today across a wide range of disease states so I'm just highlighting a couple results here so we've been able to for example quantify the result the response to different therapeutic interventions that clinicians gives of this data was taken in. Let's go in very very sick babies who were outside too much acid in their blood they give them a drug called sodium by carbonate to neutralize some of that acid and what we saw is that depending on how much of the tread they give you can see profound increases in cerebral blood flow. We've also done a lot particularly have done a lot in healthy neonatal development characterizing how blood flow changes. You know during that neonatal period we see differences between males and females between right and left hemisphere between different brain regions and a lot of this work really hasn't been able to be done before because taking a very healthy baby and sedated them feeding them in them and then M.R.I. is just not ethical right so because we're all noninvasive and can come to the bedside we've been able to glean information about human development that hasn't been seen before. We also are and this is a group where I did my Ph D. Actually that has shown that the blood flow technology can be used as a biomarker of disease severity So this is data taken in acute ischemic stroke patients they basically put two sensors on the brain one on. The stroke come a spear or that the hemisphere that had a stroke and one over the normal It's not really normal because they have a. But they OK side of the brain and they looked at how the brain responded just to a simple postural manipulation because there's a lot of thought that with stroke you have impaired regulation of flow in the brain and what they saw was that with increasing stroke severity you saw. A cemetery and how the stroke versus healthy side of the brain response so suggesting that D.C.S. can be used as a surrogate marker for disease severity or maybe progression. So going back to kind of the main goal slide. We've been able to start giving clinicians the blood flow information with the hopes of helping to guide patient care. But I think this is great in theory but in reality oftentimes when we give clinicians this information they don't really know what to do with it so this is especially true in the critical care arena where all of their patient management is governed by trying to maximize blood flow to the brain in order to maintain adequate oxygen delivery and preserve neuronal health and yet they don't monitor the brain at all so all they do is monitor her grade and blood pressure and oxygen saturation and they've never actually known what's going on the brain so they're so excited when you can tell that the blood flow has actually dropped profoundly in this patient but then when you say OK so now what do we do they're kind of like I don't know. Charted territory. And so one of the things that we've been working on lately is using pre-clinical models of the diseases that we're studying the clinic to try to elucidate mechanisms that are driving changes in these blood flow parameters that we're measuring and then you know with the goal of hopefully better understanding what's going on and being able to give clinicians ideas of how they can normalize blood flow and hopefully improve outcome. So for the remainder of the talk I'll discuss recent work from my lab that's been in the field of mild traumatic brain injury and order to understand how the blood flow data we get can actually be one day used to guide patient care. So just a general overview of mild traumatic brain injury it's caused by rapid acceleration of the head it disturbs brain function in the absence of. Structural debt structural defamations know skull fractures. The cognitive facts of concussion appear immediately and tend to resolve over time and we know that repeated head injuries within some vulnerable window can lead to worsened and longer lasting Coggan of facts. So just you know it's a significant public health problem affects millions of athletes per year just to put it in kind of a local context in two thousand and fourteen I pulled the numbers. Eight hundred diagnose concussions twenty five percent of those kids that had a prior history of concussion and this number is probably a gross underestimation of the actual concussions in the area given how poorly under reported they are as well as you know it's hard to diagnose some of these. So what I'm interested in is although many patients are going to recover relatively clear quickly concussion are to mild T.B.I.. Usually within a week there's about ten to forty percent of patients who are persistent long term consequences. Like cognitive impairments can be seen up to a year after concussion and I'm sure you guys have heard in the news lately we're now learning that repetitive concussive and sub concussive injuries can lead to really long term neurodegeneration So there was the study that was published last year by him as well. They looked at post-mortem analysis retired N.F.L. players and eighty seven percent evidence of basically like Alzheimer's like to mention dementia. So our goal is to determine what is unique about these patients who have these persistent deficits and who go on to develop long term nerve generation so who are they who's most vulnerable to subsequent concussions and and who will have these long term effects why are certain patients more susceptible and. And I guess along why issue we know that biomechanics of injury alone are insufficient to predict outcome so just because two people had the exact same head injury they might not they might have completely different outcomes and then finally we want to help answer what what can we do once we know once we've identified who these most susceptible patients are so to answer these questions we translated to a pre-clinical mild traumatic brain injury model we use this close head injury model that mimics human concussion in that it features kind of rapid head Excel or a. Head about the neck. It's a pretty simple model so if we take. Them. We have this acrylic board that we've cut a hole out in the center and we rested on top of it. We grasp the mouse's tale and then we drop a bolt on this guy to when the bolt hits their head you know I saw your face I know it's really a mild injury. So there and that's by the way so when the ball hits their head they know it breaks the cam way they they wrap and they rapidly rotate. And this induces injury in the brain so the consequences of this model are really subtle we've done lots of them you know has chemistry there's no dear Ronald degeneration there's no arc so no injury there's no D.N.A. damage and there's no blood brain barrier permeable A-T.. Deficit's And just to show that it's not a problem with his chemistry we've done these same studies in more severe T.B.I. model and you can see that we can actually detect these predators It's just that this this injury model is very mild and I should point out that this was actually after five hits we spaced them once daily and we still are seen neuronal injury. However what we do see in this model is the emergence of cognitive deficits so after a single close head injury there's no cognitive deficits the my surf actively find. And we assess cognitive deficits using Morris water mains for those of you who aren't familiar with Morris water maze Basically you take the mouse you put him in a tank of milk. The water so we can't see what's underneath it and you submerge a platform just below the water's surface and mice don't like water so they swim around eventually they stumble upon the platform and they sit on it and you repeat this across multiple trials and eventually they get really good at using visible cues in the room to find that platform even though they can't actually see it. So this is like kind of a typical performance on one of these. Trials So this is the latency is the time to find the platform and you can see as you give them more and more trials in the tests they get better and better at finding the platform. However when you give increasing the number of hits spaced once daily cognitive deficits begin to emerge so this is after five five hits spaced once daily and you can see that the injured group in these open circles takes significantly longer to find the platform they're just not good at it. Remembering what's going on. I just want to point out though that these performance opposites that we see on the group level. You know are clearly statistically significant but within this injured group there's actually a substantial amount of very bill just like in the human condition so even though these mice were subjected to a very consistent mechanical impact you can see that there are some my like this one on the top just like he can't get it. But there's some my super form just like sham So what is the difference between this guy versus the sky. So next thing we done recently is looked at a genetically modified all Summers mouse where they develop Alzheimer's plaques with age and. In these mice we've shown that this injury model accelerates the. Development of beta so here this is a sham injured miles and this is a mouse after three of these injuries spaced once daily and then sacrifice one month later and you can see that there's amyloid beta seen in bread starting to emerge quantifying the amyloid beta deposition across all of our animals we can see that there are significant opposition after a month a month after injury I just lost everything on this screen but this one still here. Can you see my mouse even though it's here we go OK So so again what we want to see is within this injured group you know what's wrong with these three guys who had such bad amyloid beta versus this guy who basically looked like a sham. So now we want to start talking about Who are these mice how do we figure that out so the first thing we did was we started by using D.C.'s to assess flow as potentially a biomarker of these mice there's some literature in the human condition that suggests that blood flow at the time of cognitive testing is a marker of injury so we thought let's see if this is true in mice we designed this custom optical sensor so the great thing that I love about these diffuse optical techniques is I can use them in humans and then I can scale down my sensor and make the exact same measurements that we're making in the clinical scenario in the pre-clinical models. So basically what we do is we rest our little optical sensor over their head everything's noninvasive we briefly anesthetize of the now we're doing away measurements and we validated these measurements in mice as well so here's our study protocol we actually did two we looked at wild type mice in the wild type mice we gave them five closed head injuries spaced once daily and we measured blood flow kind of at a couple of Representative time points across the injury protocol and then we did testing this Morris water maze testing seventy two hours after the final injury and then we had an all time as a group Alzheimer's mice were older they were much more susceptible to injury so we only had them three times and we measured flow before and four hours after the final injury and then we waited a month and we sacrifice them and. That acclamation accumulation of amyloid. So just in the repetitive hit my we can see that. So here and so in the relative change in blood flow from the. Levels so in the injured group four hours after the third injury and after the fifth injury blood flow was reduced but it seemed to return to baseline by seventy two hours later. And like I said these mice are going to develop significant cognitive deficits. Seventy two hours up to that final injury which blood flow has returned to normal so it seems to suggest that these deficits are not due to deficits in blood flow at the time of assessment but we thought that maybe blood flow early on in these acute time points could be a part Bostic marker of outcome. And lo and behold let me just get to this one when we looked at the four hour after the third hit injury time point we saw a significant correlation between blood flow and their average Morris water maze performance so the mice with the lowest blood flow were the ones who did worse on on their cognitive testing. And then also within our all timers My We saw that blood flow at this four hours up to the third hit. Time point was correlated with the amount of amyloid beta accumulation and one month after injury. You know we only did six mice today but I think there's definitely a trend there with the mice who have lower sort of a blood flow having more accumulation of amyloid beta so we seem to have identified this biomarker sensitive acutely sensitive to the long term cognitive effects of and pathological effects of repetitive mild traumatic brain injury and if this finding does translate to humans then we could have an acute marker that could tell you know athletes or soldiers when it's safe to return to play or return to the battlefield. Or potentially identify those who are in risk of having multiple concussions. So next we want to look at why these particular my with low cerebral blood flow are more susceptible to injury so we actually have now have this like pretty unique platform to study mechanisms because we've got this acute biomarker of how my surgeon to do you know a long term cognitively or pathologically. So what we do is we first measure blood flow we predict how they're going to do and then we can harvest the brains of these animals at any time point you know that we're interested in to investigate molecular pathological changes that are going on and we can then relate these. You know underlying physiological changes to blood flow predicted outcomes. So what mechanisms could be responsible for changing cerebral blood flow and ultimately leading to worse outcome because this model doesn't induce significant neuronal injury like I said the next kind of logical mechanism to look at is neural inflammation so in severe traumatic brain injury. Neurons limitation has been shown to. Contribute to blood flow changes and also to be associated with worse outcome and so what we have. What's happening is repetitive. Mild T.B.I. is leading isn't due seen mechanical stress on the neurons release. Or signal or increase. In signal pathways and and increase expression of inflammatory cytokines which lead to micro Clio information and reduce blood flow and ultimately to worse long term outcomes. So I'm going to start by talking about OK. But. Activation so micro Glee are just kind of a gross. Estimator of neurons laboratory response they serve as. Kind of regulators of the immunity response they breathe and they're known to. Promote remodeling and repair after T.B.I.. So at this point out four hours after a third injury we basically measure blood flow and then we looked at the microbial response right there this is a sham animal with the micro clear and red stain with I.B.A. wine and you can see in our two injured animals there's increased Amalie there's increased I.V. A one uptake. It looks like more micro glee as well so within the injured animals we used Western Blot to quantify I.B.A. one and all these animals and what we saw was that the animals with the lowest low again were the ones who had the worsening or inflammation and remember those low C.B.S. animals are going to be the ones who go on to have worse outcomes so it suggests that you know could be playing a role in long term outcome and also is interesting to me just that blood flow is not only a marker of long term outcome but it seems to be a marker of underlying pathological changes. So we think that nitric oxide may provide a functional link between microcode. Elective ation then reduce blood flow nitric oxide plays a well known role in mediating vascular tone and also the inflammatory response. And so nitric oxide in the brain is synthesized by three times. Which. If I was by nitric oxide since which takes the form of three ice of forms I'm going to focus on one of them and to feel ill nitric oxide sent this which has been implicated in more severe T.B.I.. And what we saw basically was that the mice with reduced. Feel and nitric oxide availability were the ones with the lowest cerebral blood flow so again. You know because C.B.S. was correlated with I.B.A. one. Or inversely correlated that. Was also positively or negatively associated with I.B.S. one. We'll see next we can focus on cytokine expression after T.B.I. and specifically what cytokines are expressed acutely in animals with longer worst turnout. So to do this we use Lumina illumine X. assaye to assess twenty seven different cytokines each. Kind of confusing but basically each column denotes a different side a kind that we looked at and each row denotes one of our different animals so we've got our eight injured animals on the top in our sham animals at the bottom you can already see that there are some cytokines that are regulated. In the injured animals. So next we use partially squared regression again through blood flow within the injured animals to identify the top cytokine correlates with. Cerebral blood flow and with worse cognitive outcome and you know the individual. As relevant but basically Randy's was our top correlate so we did him you know his to chemistry looking at Costain with a bunch of different cell types and we actually saw that. Coke lies with the. Neurons suggesting that. This is correct that it's these injured neurons that are releasing these. Cytokines. And finally we wanted to look at interest signaling that could be disrupted after T.B.I. these are the key signaling pathways in the. To be. Katie that regulate transcription of inflammatory factors like cytokines and here in showing just the fossil proteins associated with these pathways which I mean they're like so small and you can't read them but the point is the orange ones are the ones we measured you can see we just kind of took a representative sample across all these pathways. Again another heat which I don't love but it's showing all the fossil proteins that we measured for each of our animals and. Just by I already you can see that the map kinase from the F. Kappa B. pathways have significant regulation in the. Injured animals so similarly we can do a part of partially squared regression within this injured group against cerebral blood flow and find fossil proteins that are highly correlated with cerebral blood flow I mean with worse outcome. We didn't you know his chemistry on a couple of these elevated proteins and again we're seeing them localized to the neuron suggesting this mechanical stress is causing the neurons to really up regulate these signaling pathways and here I'm just highlighting in with yellow stars the top correlates. Here and you can see they mostly fall within the N.F. Kappa B. and map kinase pathways so potentially what I like about this result is that if we could hit these pathways we could potentially improve outcome so there's already. F.D.A. approved blood brain barrier permeable inhibitors of these. Pathways so I think that they have promising therapeutic potential but potentially addressing this question of what can we do. So in summary these results demonstrate how our diffuse optical technique D.C.S. can be a powerful tool to. Are a powerful biomarker not only long term outcome but also of underlying path of physiological changes after T.B.I.. We saw that and T.B.I. leads to. Acute microbial activation cytokine expression and percolation of interest or signaling pathways and through partially regression we write able to identify which of these kind of the hallmarks features are going to be most predictive of outcome. And hopefully the next steps will be to start leading these systems and seeing if we can improve outcome in our mice so with that I'd like to thank you for your attention and my wonderful group members if anybody has any questions I'm open. Just gross like looking at. Not you know nothing nothing really stood out to me these were relatively the. Climbers My but they were a little older and they were like six months as opposed to our wild type mice were two to three months but they weren't really old yet. Yes I'm sorry they were just submerge slightly into the water and the water is like filled with milk basically so you just if it's not transparent they can't see it but they can feel it once they hit it you know but they That's a good question they also tested to just to make sure that the injury wasn't causing some kind of visual impairment and they can see if you raise the platform up they're all very good at finding the. Yes. We didn't. You know we didn't really see that's not my mouse. There it we didn't see many changes in that pathway right there was just not. Exactly and I've got to be. Right. This was four hours after injury and again we chose that time point because we knew that our blood flow biomarker was predictive of outcome at that time point so we thought let's just look at what's going on right at that time point but our next thirty is going to be to really like. The kinetics of all these parameters because. They definitely change over time. Exactly and I think the key then is going to be really hitting how those transients in my translate to humans which is a whole nother can of worms. But you know if we find the blood flow is a nice surrogate of those changes then you know hopefully that will cause blood flow we can measure in humans easily. Yeah. No. I mean there are certainly many things that scatter late into shoe but the moving things are what Worst were sensitive so if you take that away we would. We would have been trouble. If you injected like I see two years. So you. Know what I'm excited to hear you're talkin here about what we could do yeah. Yeah I mean so we we talked. We were thinking about maybe doing that we've actually been talking with Michelle because they have a lot of you know. Once we get the brains they can do a lot. Lifted. The top one likes and stuff like that. So she actually has a bunch of our samples. Well. So. Yes. It's. Sort of those. No I mean you know right so you know no they're the because it diffuses so rapidly. You know you don't get a lot of heat deposition. We are certainly limited by the standards. And I don't know if you're from the government puts standards on you know what safe levels for tissue exposure with D.C.'s we're kind of pushing the boundaries because we are measuring just one single speckle So we're really light starved so we try to pump in as much as we can. I've never had a problem with with burns. Or.