**Transcript of Cyberseminar**

**Timely Topics of Interest**

**Diagnosis of TBI: Biomarkers**

**Presenters: Fiona Crawford, PhD; Elaine Peskind, MD; Robert Ruff, MD, PhD**

**October 23, 2012**

**Diagnosis of TBI: Biomarkers**

**October 23, 2012**

Moderator:Well, it looks like we are at the top of the hour so at this time I would like to introduce our three speakers. Speaking first we have Dr. Elaine Peskind. She is the codirector of VISN 20 MIRECC and Friends of Alzheimer’s research professor in the Department of Psychiatry and Behavioral Sciences at the University of Washington School of Medicine, and staff psychiatrist at the Joint Base Lewis McChord in Tacoma, Washington. Speaking next, we have Dr. Fiona Crawford who’s the vice president and associate director of the Roskamp Institute and a VA scientist at Tampa VA Healthcare System. And finally, we have Dr. Robert Ruff, the national director of neurology for VA Central Office and he is located at the Cleveland VA Medical Center. So at this time we are going to turn it over to Dr. Peskind. Also, just to let all of our attendees and presenters know if you would like to minimize the go-to webinar dashboard located on the right hand side of your screen simply hit the orange arrow in the upper left hand corner. And you can get that out of your viewing screen. Thank you. I’ll turn it over to you now Elaine. 

Dr. Elaine Peskind:Okay, thank you and I apologize to everybody but I’m going to have to step  off the call as soon as my presentation is over because today I’m getting the army commander’s award for public service. It's a big deal today here. So can I have my first real slide please on the nature of an emerging and unprecedented problem. So roughly three and a half million service members have been deployed to Iraq and Afghanistan. And depending on your source, approximately nine to eighteen percent of those return with persistent post concussive symptoms. Next slide please...

And I’m sure you’ve all heard a lot about the role of repetitive head trauma in professional athletes such as boxers and football players, which has been associated with a rare dementing disorder called chronic traumatic encephalopathy. It's a mid life dementing disorder characterized by pure tauopathy or neurofibrillary tangled pathology. Next slide please...

And this recent paper by Anne McKey’s group, Lee Goldstein the first author, demonstrating chronic traumatic encephalopathy in four veterans of Iraq veterans with either repetitive blasts or combination blasts and impact, TBI, has really raised an additional paper by Bennett O’Molly that have really raised concerns that the effects of repetitive blast mTBI, impact TBI experienced by our service members and veterans may also be putting them at risk for chronic traumatic encephalopathy. Next slide...

On this slide we see the typical pathological picture of this tauopathy dementia. This is in a twenty-seven-year-old Iraq veteran and showing the typical neurofibrillary tangles, all those little black things you see. And these tangles are identical with those seen in Alzheimer’s disease as well. And next slide please...

But again, to point out that chronic traumatic encephalopathy is a rare, mid-life dementing disorder. Not so rare in professional athletes with repetitive head trauma but rare certainly in the general public. But however, head trauma that is severe enough to give you loss of consciousness is the best characterized environmental risk factor for developing Alzheimer’s disease, the common late life dementing disorder. Next slide...

There has been controversy about the etiology and the course and what we should be doing about the persistent, somatic, cognitive behavioral symptoms in our OIF, OEF, OND veterans following repetitive mTBI. An early study done by Charles Hoag at Walter Reed in active duty army soldiers suggested that the chronic, but post concussive, symptoms were more attributable to PTSD and depression than they were to TBI itself with the exception of headache. And I believe at this point that is a minority opinion. Next slide...

And you know, I and many other skilled clinicians, I think, Dr. Ruff would agree with this, are convinced that our service member and veterans chronic symptoms of mTBI reflect real, although possibly subtle, persistent brain damage. And so we set out to ask the question whether these chronic symptoms reflect persistent changes in either brain structure or function and in cerebrospinal fluid biomarkers of neurodegeneration. Next slide please...

Participants on whom we have full data to date are thirty-four male Iraq and/or Afghanistan veterans who have had at least one blast induced mild traumatic  brain injury. But as you’ll see some may have had a lot more than one. Their mean age is about thirty-one and a half and there are three control groups. The first is sixteen non-blast exposed Iraq/Afghanistan veterans. So these are veterans who were deployed to Iraq and/or Afghanistan but who have never had lifetime head trauma of any sort. In addition we have two groups of civilian controls. There were twelve who underwent FDG-PET imaging only and an additional fifty-five different civilian controls, all males, who underwent spinal fluid collection for CSF biomarkers. Next slide...

Seventeen of the thirty-four mTBI veterans also met the DSM-IV criteria via the clinician administered PTSD scale interview for combat operations PTSD. And they had higher scores for depression and alcohol use and had poorer sleep. Alcohol abuse or dependence was an exclusion factor so none had alcohol abuse or dependence. And nearly all the mTBI veterans had persistent post concussive symptoms. Next slide please...

And here is a – on this table on this next slide you see – what we put on this table was – were the symptoms from the neurobehavioral symptom inventory that’s a sort of laundry list of potential post concussive symptoms that’s used by the VA and their secondary screen for TBI. And we only put on this table symptoms that were rated as at least moderate, severe, or very severe, and as you can see the frequency of how symptomatic these veterans are. And there are a number of these, and I don’t have the colors in front of me because I’m working on hard copy, but there are a number that could easily be attributed to PTSD or depression. Feeling anxious or tense, difficulty falling or staying asleep, irritability, but there are also a number that I think are very specific for TBI. And when I say forgetfulness I mean day to day, senior moment forgetfulness, ringing in the ears, headaches, sensitivity to noise, and hearing difficulty, and slowed thinking. Next slide please...

And let’s talk a little bit about the blast exposure history. We took a very careful blast exposure history for, not only blasts, but also for lifetime exposure to any head trauma. The average time in our mTBI veterans since their last blast exposure was four years. The average number of blast exposures that resulted in loss of consciousness was one. However, the average number of mTBIs that resulted in acute symptoms consistent with the American Congress of Rehab Medicine criteria for mild traumatic brain injury including loss or alteration of consciousness was fourteen. And you can see from this list here exactly how common multiple exposures was with the vast majority having greater than two and then a substantial number even having between fifty-one and a hundred blast mTBIs that meet those criteria. Next slide...

We then went on to do multimodal neuroimaging using two structural techniques, diffusion tensor imaging and macromolecular proton fraction mapping, and a single functional neuroimaging technique with a fluorodeoxyglucose PET. Next slide please...

This slide literally lists all the papers, the manuscripts, that have been published in blast mTBI in veterans and active duty service members. And they’re really a mixed bag. Levin’s study was negative. All the other studies were positive in one way or another but differed substantially with respect to methods and with respect to results. Next slide...

I’m going to be presenting a number of images and the thing I want you to understand about how the images are presented is that they’re all subtraction images so that what you’re seeing is the mTBI group minus the Iraq and Afghanistan deployed controls unless I say otherwise. And the differences are shown. The brighter they are the more different they are. And there’s a Z-score scale on the side, on the right side, of the image. And by the time we get to the kind of the orange red, reddish, almost a yellow, it is statistically significant at four standard deviations. So these are the results of the diffusion tensor imaging. And what we found was decreased fractional anisotropy in genu of corpus callosum in the mTBI veterans compared to the veterans who had no head trauma history. Within the TBI group there were no differences between veterans who had or did not have PTSD. And again, as I said, the results vary between labs both in methods and results. Next slide...

And this led us to want to try to come up with a technique that was more sensitive and more reliable. So this is a new technique called macromolecular proton fraction. And it is a magnetization transfer structural imaging technique and it provides an index of the macromolecular composition of neurons, and the brain in general. And in human and animal studies, especially in studies where you can do clinical pathologic correlation, these indices – the MPF has correlated with indices of myelin disintegrity. So it may be a good measure of myelin integrity. Next slide please....

And here are the findings of our MPF mapping, macromolecular proton fraction mapping. We found a reduced MPF in numerous, very numerous, at least twenty, subgyral, cortical, subcortical, and longitudinal white matter tracts. So in fact, it was much more sensitive. Within the TBI group, again, there were no differences between veterans with and without PSTD and we felt that these findings were consistent with the mechanism of diffuse axonal injury and suggest that there may be alterations of myelin structure in vulnerable white matter tracts. And we feel that this technique may have potential as a perspective quantitative biomarker of blast induced mTBI. Next slide...

Our last imaging technique we used was the functional technique which fluorodeoxyglucose PET which measures glucose metabolism in the brain. And again, this is a subtraction Z-score map and what the results show is that there is regional glucose hypometabolism in the parietal lobes bilaterally in addition to the left sensory motor cortex and the right visual cortex in the mTBI veterans compared to the Iraq or Afghanistan deployed veterans who had never had a TBI. And again, within the control group there were no differences between veterans with and without PTSD. Next slide...

But we also had some unexpected findings of two kinds both in the FDG-PET imaging and in the spinal fluid biomarkers. And I’ll try to get you oriented to the slide. Again, these are subtraction Z-score maps. So it's only showing the differences between two groups. The top panel is just a template of MRI to orient you to the anatomy. The middle panel is our blast mTBI veterans versus our twelve civilian community controls. These were the results we published a couple years ago in neuro image which showed differences in our blast mTBI veterans with glucose hypometabolism in the cerebellum, the pons, the thalamus, and the medial temporal lobe. However, when we then collected a group of non-blast veteran controls, so these are controls again, deployed to Iraq or Afghanistan, never had a head trauma, and compared those to our civilian controls we found similar – not quite as severe but similar hypometabolism in the cerebellum, pons, thalamus, and medial temporal lobe. You can imagine this was a bit of a surprise. And so it appears that our compared to civilian controls our Iraq and/or Afghanistan deployed controls also do not have a normal brain metabolism. Next slide please...

We then did spinal fluid biomarkers neurodegeneration and we did this in three groups. On the far right is our TBI group, in the middle is our veteran controls who were deployed to Iraq or Afghanistan but who did never had a TBI, and on the left are a different group of fifty-five community controls in whom we had CSF collected, and who have never had a history of TBI. As you can see, both the TBI group and the TBI controls who have never had a head trauma have an elevation of the ratio of phosphorylated Tau to total Tau in CSF consistent with insipient tauopathy. Obviously, this was very concerning. Next slide please....

We made a number of complete speculations about why this might be the case that we have abnormalities of both FDG-PET imaging and CSF biomarkers in our Iraq and Afghanistan deployed controls. So obviously there may be selection bias with pre-military vulnerabilities, military “lifestyle.”  Many of our – even though none of our controls had reported a history of head trauma many were high school athletes. There may have been some alcohol misuse. They may have taken body building supplements. We were concerned about the effects of military training particularly combatives, Pugil sticks, being choked out in the marines, the new phenomenon of mixed martial arts, the hyperthermia in the Iraq combat operations environment, a nice day in July in Baghdad is about a hundred and thirty degrees. And then just the whole concept of the stress of, first of all, being deployed to the combat zone, and also possible environmental exposures. We took an index of environmental exposures and they are, in fact, exposed to a lot of things, the gamut from flea collars, insecticide treated uniforms, depleted uranium, anthrax vaccinations, and there’s a whole laundry list of things that could be potential environmental exposures. Also, incidentally, a high percentage of our non-blast veterans, and we think this is purely chance, had a higher than expected frequency of the apolipoprotein e4 allele which has been associated with bad outcomes from head trauma as well as Alzheimer’s disease. Fifty-eight percent of veterans of whom we had a genotype rate where we had the APOE e4 allele and you would expect about twenty percent to. So as I said, again, I think that’s a chance alone. Next slide please...

So in summary and in conclusion, so both the – I’d like to focus first on the two differences between the TBI veterans and the TBI controls. They’re both structural and functional imaging modalities that suggest a very coherent picture of diffuse axonal injury and that doesn’t appear to be attributable to PTSD and specific for TBI. And also we have these unexpected abnormalities in our veterans who had no head trauma but had been exposed to the Iraq and/or Afghanistan combat zone, both glucose hypometabolism on the PET scan, and in these areas listed, and elevations in the CSF p-tau tau ratio. And both these things suggest neurodegenerative changes. It's going to be essential to have long term follow up to determine the consequences of these brain abnormalities. Next slide please...

Of course, there is a cast of thousands to thank.  Next slide please...

Including our Army collaborators and you can go on to the next slide...

And on the two – the two guys on the left are command sergeant major, retired, Thomas Adams and command sergeant major Rob Prosser. And on the right is first sergeant Creed McCaslin. And the other guy, I think, is Bruce Willis. I’d like to thank everybody and if I have a few minutes for questions I’d be happy to take them. And if I don’t...

Moderator:Thank you Elaine. We do want to leave plenty of time for our other two presenters but a few questions have come in for you so we’ll get through those real quick. Why did you exclude individuals with alcohol use disorder given its prevalence in this population? 

Dr. Elaine Peskind:Well, that’s a very good question. I think that’s a future study but we wanted to for the initial investigation – we want to figure out the effects of TBI specifically are when we made this an exclusion. But that’s a good question and that’s another group we should be doing in the future. 

Moderator:Thank you. Could the similarity between the non-mTBI versus mTBI vets be due to long term adrenaline stress of deployment/war? 

Dr. Elaine Peskind:I think that’s one of the possibilities among others. I think that just the stress of the combat environment absolutely could be. A fair number of our control group, our veteran control group, were non-combat MOS’s, military occupational specialities. So I think that remains to be seen. We’re certainly going to work hard on trying to figure that out. 

Moderator:Thank you. That attendee also followed up with a few comments. With my deployments it seems that tours longer than six months are associated with burnout effects and also consider testing older vets, Vietnam or Korean vets for comparison. 

Dr. Elaine Peskind:Right and we’re planning on submitting a Gulf War grant to do the same, a very similar study in Gulf War veterans. 

Moderator:Great! We’ll take two more questions and then we’ll move on. How do the tau and p-tau CSF results differ among groups before you take the ratio? Was this the NO test kit for p-tau-181? 

Dr. Elaine Peskind:Okay, I can tell you, well, first of all we did all the samples in the same assay to not have batch effects and that’s very important in measuring these difficult to measure proteins. There were no absolute value differences in any of the biomarkers, a-beta-42, total tau, or p-tau-181. What differed was the ratio of p-tau to tau. Oh and also the assay we used was the aluminex X map platform using the [inaudible] antibody. 

Moderator:Great! The last question we have, are you planning a long term follow up study to see if the cerebral metabolic changes in deployed veterans persist? 

Dr. Elaine Peskind:Yes, in fact, we are now collecting our two year data on many of these veterans and we hope to be able to continue this very long term. And these were great questions and I really appreciate them. They’re really good questions. 

Moderator:Thank you so much. Well, we do congratulate you on the award you’re about to receive and thank you for sharing your expertise and we bid you adieu. 

Dr. Elaine Peskind:Thank you. 

Moderator:Thank you. 

Dr. Elaine Peskind:Bye-bye. 

Moderator:Okay, Dr. Crawford, are you prepared to show your screen? 

Dr. Crawford:Yes, I am. 

Moderator:Great! You’ll see a popup now. Go ahead and accept it.  Perfect. Thank you. 

Dr. Crawford:Okay. Well, good afternoon. I’m going to take things in a little bit of a different direction focusing very much on basic research and I’m going to be discussing our biomarker discovery approaches with mice models of TBI. And this is all focused on blood biomarkers as a readily accessible, easily available biomaterial in which to hopefully identify markers, not just of diagnosed symptoms but also potentially prognosis of TBI. So at the Roskamp Institute our research program involves a multidisciplinary approach. We have mice models of TBI both the CCI model and a new head injury model that I’ll discuss in a moment. And with those mice models we perform a detailed characterization, neurobehavioral characterization, a molecular level characterization, and neuropathological characterization. On the human side we have two human populations. One is the VA patient population of the James A. Haley VA in Tampa and that, of course, is a very diverse population of folks who have sustained TBI through a wide variety of means and maybe at extended time points after their injury at the time of our recruitment. The other population is an active military population and I’ll be discussing some very exciting data from that population at the end of the talk. 

So the two mice models and two studies I’m going to focus on, the first is using the controlled cortical impact model of head injury. This is a well established animal model of head injury. It involves performing a craniectomy, we did a craniectomy over the right hemisphere and then we used electromagnetic device shown in the slide here to impact directly on the surface of the brain. And depending on the depth of injury we can control the level of severity of the injury that the mice received. So we use a one point eight millimeter depth as a severe injury. If we went any higher than that we got significant mortality and that wasn’t a goal of our studies. So one point eight is severe and one point three millimeter depth is a relatively mild injury by comparison. That’s the first injury model I’m going to discuss. The second is our newly developed model that we just published in J Neurotrauma. This is a closed head injury model that we specifically developed in order to address the increasing awareness of the significance and consequences of mild TBI and, in particular, repetitive TBI in the human population. So this is a hit using the same electromagnetic device. A hit directly on the midline, closed skull, but we specifically set up the parameters so that there’s no fracture and no bleeds. So it really is a mild injury and we either give a single injury or a repetitive injury which is five hits with a two day interval between each hit. And then, of course, there are the appropriate anesthesia controls, either one anesthesia or five anesthesias. 

But the first study I’m going to discuss involved the CCI model. And, as Elaine mentioned, APOE is a known risk factor for poor outcome after TBI. And because of the nature of our work we actually focused on APOE as a way to hone in on our datasets. We have been using proteomic and lipoidemic approaches in order to characterize at a very high level the molecular profiles in response to head injury. So we use liquid chromatography and mass spectrometry approaches to compare the brain and the plasma from different mice models of head injury. And what we’re able to do using that is identify qualitatively a very large number of proteins that are present in the brain. We’re able to identify somewhere between one and three thousand proteins, in blood, anywhere between five hundred and a thousand. So as you can imagine this generates huge datasets and one of the ways that we use to try and make sense of those data is to employ additional filters. So we approach this using APOE transgenic mice. So we used mice expressing either human E3 or human E4 as a way to try and discriminate between the favorable and unfavorable responses to head injury. 

So our hypothesis was that if we looked at the profiles of the head injured mice and how they differed in E3 versus E4 it would give us some insight into the most damaging pathways that were being triggered in response to TBI. We looked at three different time points, twenty-four hours, one month, and three months after injury and we looked at mild and severe up to twenty-four hours in one month and we looked at severe injury only at three months. And in addition to the qualitative analysis with our O mixed approaches we used a technique called I track labeling which I won’t get into the details of now but essentially this enables us to carry out quantitative analyses so we are actually able to look at particular proteins in each sample and see how they change relative to one another whether they’re increased or decreased in response to injury and/or in response to APOE genotypes. We also use something called ingenuity pathway analysis which is a software. This is a knowledge base that links proteins and genes based on their known cellular and molecular interactions in cells. It's a way to take these huge datasets and try and attribute some functional significance. And one of the things that we’re doing as regards to the blood biomarker profiling is that although in the blood we’re really looking for particular proteins or particular lipids that could be put together to create a biomarker panel. One of the things we anticipate is that in the translation from mouse to human it won’t necessarily be exactly the same markers that are present in humans as we see in mice. But it may well be proteins that are related to those we see in mice. So ingenuity pathway is a way to look at the proteins that are related and then you can target those for investigation in the human sample. 

This table is pretty horrible. I do apologize but it's from a paper that we published in J Neurotrauma reporting the plasma profiling in this study. And what you can see here, these are all the proteins that were shown to be significant across the different datasets. These are the proteins that were significant after correction for multiple testing. So this is the most stringent level of analyses. And you can see that we are looking at proteins here that are involved in inflammatory responses. We got compliment proteins. We have a number of immunoglobulin proteins. We have proteins that are involved in iron handling. I think one of the most important messages from this dataset is that even as late as three months after injury in the mice, which we expect translates to a much longer period in humans, even at that late time point we are seeing proteins over in the right hand column here. These are proteins that were significantly different at three months after head injury and they’re discriminating, not just between injured and control mice, but also between the different APOE genotypes. So these are proteins that are not only diagnostic but they’re also prognostic for head injury. And I think this is very encouraging when we think of translation to humans because this does suggest that even at extended time points after injury there will be biomarkers accessible in the blood that will tell us whether an injury has occurred and may be able to tell us something about the anticipated path forward, the prognosis. 

Just to give a snapshot here, this is an example of the sort of molecular pathway and the links that can be created using the IPA software. So what we’re looking at here are all the proteins that were identified. This is actually from our brain datasets but these are all the proteins that were present significantly modulated across all of the datasets and the particular snapshot we’re looking at here is from the three month severe time point. And the proteins shown in red are those which were upregulated in response to injury and green are those which were down regulated in response to injury. And what we’re showing here is the E3 response, and if you look, there’s a protein with little boxes next to them, and that shows you what the swap was with E3 versus E4. And in many cases the difference between the E3 presentation and the E4 presentation was a complete flip. The proteins that were upregulated in response to injury in the E3 mice were down regulated in the E4 mice. So this highlighted particular pathways that we think are probably going to be most amenable to therapeutic intervention and we’ve already carried out some studies that validate these findings. 

We can take these to higher levels, as I mentioned, from the list of proteins, you can then group them into these [inaudible] pathways shown here such as due to [inaudible] metabolism or a key phase response, and then we can go a higher level again up to things such as lipid metabolism being impacted. And I focus on lipid metabolism because this is something this result prompted us to move forward into some lipidomic analyses which I will discuss in a moment. So just to recapitulate, our approach is that if we have a good animal model of head injury and can carry out a detailed characterization at a range of time points after injury in a manner that is, of course, not possible in the human population then the molecular responses and molecular profiles that are detected in those temporal profiles of mice models will be able to be used to map back from humans back onto the mice profiles. So, for example, if we see particular profiles in the plasma of humans, we can see where in the mice pathway those are most common. And that will also potentially give us an idea of what’s going on in the brain of the humans at the same time because it's much like what’s going on in the brains of the mice at the same time as we see those particular plasma profiles. 

So our new model, as I said, we think it is of greater relevance to the human condition. We see a mild, but progressive, pathology in this mice model of mild injury. We see axonal injury, inflammation, and rather excitingly we do see developing tau pathology. Elaine mentioned the significance that is now attributed to tau in terms of TBI pathogenesis. And we see tau pathology increasing in these wild type mice. And we also see acute cognitive deficits in both single and repetitively injured mice, but interestingly the persistent cognitive deficits only occur in the mice that got repetitive injury. 

And this next slide shows you the results from testing cognitive function in the Barnes Maze. So this is where mice are tested for the time it takes them to escape the maze and they’ve already been trained in how to escape the maze. And you can see if you look at the first chart here the singly injured mice in black and repetitively injured in orange, both of these are taking longer than their respective controls to find a way out of the maze at the twenty-four hour time point. However, if we look at six, twelve, or eighteen months after injury it's only the repetitively injured groups shown in orange that are still showing significant cognitive deficits. So we think this is a very nice model. We’re very pleased with these data because we think that this new model we’ve come up with is actually going to be a very nice model for the human condition. 

And that’s further underscored by what we see in the pathology on the right hand side here. We see tau pathology in normal, wild type mice six months after the last injury. This brown staining here is tau in the hippocampus of the repetitively injured mice compared to their sham controls. On the left hand side we are looking at mice that are transgenic for the human form of tau. And you can see that in these mice we actually see the tau pathology very acutely. This is twenty-four hours after the last injury. So this we think is going to be a very nice model moving forward and we are carrying out a very detailed characterization. 

We’ve looked as well at lipid data in this mouse model. Based on the fact that we did see that lipid metabolism was significantly modulated in the earlier study, we probed the plasma of these mice using similar liquid chromatography and mass spectrometry approaches. And what we see here is that at twenty-four hours after injury when we look at total [inaudible] choline on the left or total [inaudible] on the right, we do see some significant effects. You can see here on the left that the singly injured mice show a significant decrease in total PC and you can see that the repetitively injured mice show a significant increase compared to their sham controls in total SM. But the really exciting data, I think, are on this next slide. 

This is twelve months after head injury. And you can see here that in both total PC and total SM that the repetitively injured mice showed significantly higher levels of PC and SM. I think this is remarkable the fact that we are seeing changes like this twelve months after injury. Again, this is very encouraging from the point of view of being able to detect biomarkers at extended time points after injury. And to whatever extent this is a reflection of ongoing processes in the brain I think that’s also encouraging that there may be a greater window for therapeutic intervention than was originally anticipated. 

Based on these data we moved forward with our active duty troop study. And this is in collaboration with Dr. Mike Dretsch at USAARL in Alabama. In this study we have recruited four hundred and seventy troops, pre and post deployment to Afghanistan. And these troops are getting a full neuro psychological evaluation and are providing blood samples for genetic and for omic analyses. And so these troops, each person is their own control. We’re comparing their post deployment profiles with their pre-deployment profiles. And that means we're taking out a lot of the variation that is inherent in any sort of human study. 

We have pilot data this time from fifty troops and I think this is very exciting. If we look here at the bar shown in green, this is the TBI group. So these are folks who came back from their deployment with a diagnosis of TBI. And we’re looking at individual lipid species here. These are all phosphatidalcholine species. And you can see we’ve got many more charts like this one just showing four here, that these four phosphatidalcholine species we see a significant increase in the TBI population compared to controls and, in many cases, compared to some of the other diagnostic groups. And here we’re looking at PTSD and we also have a TBI plus PTSD group. This is obviously very preliminary. It's just fifty soldiers but we have four hundred and seventy to look at. And these are significant after correction from multiple testing. So again, very encouraging that we will be able to have readily accessible blood biomarkers that will tell us what has happened to the soldiers and what will help us with medical management going forward. 

In conclusion, we believe that these multidisciplinary approaches are really critical to tackle the complexity of TBI, that the detailed characterization of lab models will provide a better understanding of TBI and a platform for us to translate from mice to human. Our omics technology appears to be a very powerful tool to identify molecular signatures. And we’ve already shown that immune and inflammatory and lipid pathways are significantly disrupted in response to TBI. And the plasma and brain responses that we see do appear to progress and persist for lengthy periods after the injury in the mice models and perhaps suggest that there are larger detection windows and larger therapeutic windows than originally anticipated. So this really was to just give you an idea of the scope and potential of the laboratory modeling and what we can derive from that. I’d like to acknowledge everybody at the Roskamp Institute who has contributed to this work. Many of us also had appointments at the Tampa VA and here are also some of our key collaborators at the Tampa VA. And, of course, Dr. Mike Dretsch and his team in Alabama. And of course, we can’t forget the funding. Thank you very much. 

Moderator:Thank you very much Dr. Crawford. And we are running a little bit over on time so I’d like to encourage Dr. Ruff to take as much time as he needs for his presentation and we can always begin the Q&A a little bit late. So with that Dr. Ruff are you prepared to show your screen? 

Dr. Ruff:Yes, it's on. 

Moderator:Okay, you should have a popup. Just click show my screen. There you go. 

Dr. Ruff: Okay, I can’t get rid of this thing in the upper left hand corner. I can make it bigger but not smaller. 

Moderator:Oh, you can get the hit orange arrow in the upper left hand corner and that should shrink it down out of your viewing screen. 

Dr. Ruff:That actually makes it bigger. 

Moderator:You should be able to get it down to just a small strip of icons. 

Dr. Ruff:Yeah, that’s all I can get it to do. 

Moderator:Yeah, it won’t completely disappear. 

Dr. Ruff:Alright, thank you. So I wanted to talk about an approach that really began with coming back from Washington in 2006 and realizing that I had to take care of patients as well as deal with people in D.C. And we were confronted at that time with a large number of people coming into the VA system to get healthcare. And one of the issues was how do you go about evaluating people. And what I’d like to report on is the use of olfactory testing as a simple and easy to accomplish screening technique and relate the findings on olfaction possibly to the genesis of PTSD. Can you go to the next slide please? 

Moderator:Oh, you can just click anywhere on your slide and it should advance to the next one. 

Dr. Ruff:Okay. 

Moderator:There you go. 

Dr. Ruff:I have, and my colleagues, have nothing to disclose. The questions we had were, that we’re going to address, is what is the most sensitive physical exam test? This is a test that can be done in the setting of a clinical exam room. And is it possible to relate CMS damage to a predisposition to develop PTSD? 

So the group of people during a two year period, there is a progressive screening of an initial group of almost twenty-one hundred veterans down to a study group of a hundred and six veterans. And these were people who had clinician-confirmed mild TBI with at least one episode of loss of consciousness. So all these people had loss of consciousness. Many had multiple episodes of loss of consciousness. And they had more episodes, more total episodes of mild TBI. We didn’t have numbers as high as some of the people in Dr. Peskind’s group but we were otherwise comparable. 

And there were also other – so the study group was the group of a hundred and twenty-six. There were also other control groups that we were able to test including olfaction. And they were people who had mild TBI without loss of consciousness, people who were veterans who did not have TBI, civilians who had mild TBI without, with loss of consciousness, and a group of twenty-one civilians who did not have TBI. These – the VNE were age matched to each other. 

The neurologic exam again included a quantitative test of olfaction. This is one of several different tests that are available from a company Sensonics. I have no commercial interest in this company. I’m not endorsing their particular product but they are one of several companies that makes qualitative olfactory tests. And their tests are being used by the Department of Defense in long-term studies of people for predisposition to Parkinson’s Disease and Alzheimer’s disease. Olfaction is being used as a presumptive screen for elevated risk for both Parkinson’s Disease and Alzheimer’s disease. We tested cognition, sleepiness, pain, and the important feature was that, of the people, and this will – the bottom point will be repeated several times, that of the people who had neurologic deficits on examination the most common, the most frequently recognized were deficits in olfaction. And I want to emphasize that these were people who did not have total loss of smell. They almost always did not note, did not recognize that their olfaction was compromised. And this is very different from prior studies that looked at olfaction as really the presence or absence of the ability to smell anything, really markers for anosmia, which so the testing that you can do with a quantitative olfactory test kit is much more sensitive than just a simple test for the presence or absence of olfaction. 

The other things that proved to be useful were eye movement testing particularly looking for [inaudible]. And this was a test done in a clinic not using quantitative recording devices which would have been more sensitive, and balance using a simple Romberg test, again, not as sensitive as one could obtain using a balance platform. But neither a balance platform nor eye movement recordings are possible in the clinic setting. The findings were that neurologic deficits were present on the people who had mild TBI with loss of consciousness, also seen in a few civilians with mild TBI with loss of consciousness, but not seen in the other groups. And that’s true for both olfaction and non-olfaction neurologic deficits. PTSD was much more frequent in the group of veterans who had mild TBI with loss of consciousness. And their performance on a MOCA scale, on the MOCA test, which is a test for cognitive function that goes from zero to thirty. The scoring is similar to the mini mental state exam. And twenty-six is usually considered a cutoff for normal, so these people as a group were having problems and it was mostly with immediate memory and trails testing. 

This slide is complicated, and I hope that you can see it clearly. The X axis in all cases is the number of episodes of loss of consciousness for that veteran group of a hundred and twenty-six people. And what you can see is that in the people who had impaired olfaction that there was a decline in function, a worsening, if you would, or a decline in olfactory function with an increasing number of episodes of TBI with loss of consciousness. For the PCLM is a measure of PTSD severity. It's one of several possible measures that a person could use. And what you see is that the PCLM scores for people who had PTSD and a threshold score of fifty, the higher the score the more severe the PTSD symptoms are. Again, this – there was a tendency for PTSD to be more severe with increasing number of episodes of loss of consciousness. MOCA scores showed a gradual decline with increasing number of episodes of loss of consciousness. And this bottom panel, panel D, the open circles are neurologic deficits that includes olfaction. The plus signs are neurologic deficits other than olfaction. The filled squares are the presence of PTSD. And you can see that as the number of episodes of loss of consciousness increases, the presence of impaired olfaction increases. The presence – the likelihood of an individual having PTSD increases. And even if you don’t look at olfaction there is still an increase in the likelihood of other neurologic deficits with increasing numbers of episodes of loss of consciousness. Again, consistent with the prior two speakers in terms of an effect of episodes of TBI on function. 

So what – so a little bit more about this test. Olfactory dysfunction has been recognized for a long time as being associated with traumatic brain injury. And there are several mechanisms of injury. The first to remember is that the brain is a structure that has a consistency somewhat like jello. If the head suddenly stops, the brain will continue to move forward. That can result in a shearing effect of the olfactory nerves going through the cribriform plate, but also this has been demonstrated on MR studies. Damage to ventral medial frontal cortex, or olfactory cortex, and hence the combination of these two effects may explain the relatively large number of neurologic deficits picked up in olfaction. The test itself that we used was a scratch and sniff tester, a person that scratches a site with a pencil that releases an odor and they have to make a forced choice between the number of possibilities as to what the odor is. And they do this multiple times. 

What may or how may the ventral medial frontal cortex be related to PTSD? If we go back, we see that PTSD was a frequent accompanier of mild traumatic brain injury particularly with loss of consciousness. And this is something that the association of PTSD with TBI has been found in multiple settings, even Dr. Charles Hoag. If you look at his data, the likelihood of an individual having PTSD was increased fourfold if they had mild traumatic brain injury versus other trauma and increased even further compared to groups who suffered no injury whatsoever. The amygdala and ventromedial prefrontal cortex have been strongly associated with PTSD. Current working model that’s used by NIH is that PTSD is associated with dysregulated excessive activity of the amygdala, that ventromedial frontal cortex serves to regulate amygdala activity. And this regulation is complex. It's largely but not purely inhibitory. Damaging the interaction between the amygdala and ventromedial prefrontal cortex may facilitate PTSD generation. And in studies from the Vietnam era if you obliterate the right amygdala or large areas of the frontal lobe including prefrontal cortex, you can protect against PTSD, again, indicating that these areas are associated with the presence or genesis of PTSD. 

So again, the amygdala shown here has input from several areas including the hippocampus and prefrontal cortex shown here. There are other areas as well, the thalamus, sensory cortex, that feed into the amygdala. This is just to emphasize the association of olfactory function with ventromedial prefrontal cortex. This panel illustrates some of the connections between the prefrontal cortex and the amygdala. Again, most of these are inhibitory but not all are inhibitory. This is showing the connections in another perspective. 

An interesting finding is that animals exposed to chronic stress, these are animals put into situations such as might occur if one were to put an individual into VACO and ask them to exist in central office. And what you see is that there’s alteration both in prefrontal cortex and in the amygdala neurons. And the panel to the left shows that in association with stress, this is control, this is a stress situation, that in the presence of chronic stress that there is a simplification of and loss of neurons in prefrontal cortex. In contrast, in the amygdala – so in prefrontal cortex chronic stress leads to neuronal loss and neuronal simplification. In the amygdala the opposite occurs. There is an elaboration of the dendritic tree in the presence of chronic stress and an increase, what’s shown over here is an increase in the number, not only of secondary branches, but also of synaptic boutons on a particular branch in association with the situation of chronic stress. So this is the control situation for an amygdala neuron. And in the presence of chronic stress the number of synaptic boutons increases both on the primary branch and on secondary branches. So you get an increase in number of synaptic – an increasing ability of these amygdala neurons to be excited. They have more dendritic branches, they have more connections per dendritic branch. 

What do you see in individuals who have PTSD? In the amygdala and prefrontal cortex this is a study from women who developed PTSD in association with partner violence. You see exaggerated activity in the region of amygdala, decreased activity in the region of prefrontal cortex. Even in a pediatric study you see exaggerated, you see injury. This is an MR study. You see loss or damage to prefrontal cortex in children who develop PTSD often in association with a trauma that was incurred. This is a very important study by Koenig, this is the lead article that appeared in Nature Neuroscience in 2008. And this was a study looking at a large number of individuals who suffered focal brain damage. These were usually due to penetrating wounds, often bayonet wounds. And what is shown is the areas that the closer to red the color the more overlap occurred, the more innate individuals it was likely that this area was going to be injured in people who were exposed to combat trauma who did not develop PTSD. So if you wiped out an area including the right amygdala you were not likely to develop PTSD. In contrast, if you wiped out a large area of prefrontal cortex, anterior prefrontal cortex, on both sides you were also not likely to develop PTSD. 

So I’d like to summarize. First, that olfaction is a test that can be done in a clinic setting. It takes a few minutes to do, and it is, at least in my experience, the most sensitive test that can be done to look for neurologic deficits in people following traumatic brain injury. It's important – the importance of damage to the prefrontal cortex may be that it alters an individual’s susceptibility to develop PTSD following exposure to a traumatic event. And that’s what this top panel is supposed to show. So an individual is exposed to a traumatic event. They can go on to develop or not develop PTSD. And the likelihood of going this pathway versus not developing PTSD is going to be influenced by many things, as we’ll talk about in the next panel, but one of them is that presence of mild traumatic brain injury may enhance the likelihood of a person developing PTSD. The things that are known to, that lead to, recovery without PTSD, that is reduce the likelihood of PTSD versus having an individual develop PTSD, include as shown before destruction of the amygdala, good pain control. This was based on an excellent study from the military, a strong support system. This is based on studies, several studies, and something that is being explored more extensively, which is psychological resiliency. Things that may develop, may lead to or enhance the likelihood of PTSD developing are soft neurologic signs. This was a series, a twin study from the Vietnam era, and those soft neurologic signs included signs referable to ventromedial frontal cortex, mild traumatic brain injury, and obviously, the nature of the psychological trauma. 

There’s a list of some references that are attached. And I would like to thank you. We have my email, Ron Riecher’s email, a colleague from Walter Reed, who’s joined the VA in Cleveland, and Suzanne Ruff, a psychologist who worked on this study as well. And I don’t know if there’s time for questions or not. 

Moderator:Thank you very much Dr. Ruff. Actually, I’d like to ask you and Dr. Crawford if you have time to stay on and answer a few questions. 

Dr. Ruff:Yes, I have time. 

Dr. Crawford:Yep. 

Moderator:Okay great! We’ll get right to it then. We’ll just alternate back and forth between questions that were addressed to each of you. So this first one was for Dr. Crawford. Did you find mitochondrial damage in the TBI mice? 

Dr. Crawford: We haven’t specifically looked for indicators of mitochondrial dysfunction but I’m fairly sure just thinking back to the datasets that we did see certain proteins that did suggest a mitochondrial dysfunction was ongoing. And I think some of the higher level functions that we saw impacted did suggest that as well. Certainly it was something that was a feature in earlier work that I did with genomic analyses. And I believe it was part of the proteomic datasets too. 

Moderator:Thank you for that response. And one for you Dr. Ruff. Can you describe further the components of the BSIT?

Dr. Ruff:So the components are basically a person is presented repeatedly with a scent and the individual has to, under controlled conditions, has to identify what that scent is from a list of choices. So for example, they may be asked to identify the smell of a rose, the smell of – hold on, I’m sorry I’m blanking on the components – hand soap, the smell of smoke. And all of these are tests of chemicals associated with scent that are not irritants. So, for example, when I was a resident we used to sometimes stupidly use an alcohol pad and that really doesn’t test for olfaction. There’s scents of other things like coffee, for example. And...

Moderator: Thank for that reply – go ahead. 

Dr. Ruff:Okay, I was just going to say the scores are normalized based on response from a large control population of people and it's normalized based on an individual’s age. So as people get older, olfactory sensitivity normally declines. 

Moderator:Thank you. This next question is for Dr. Crawford. What is – sorry, one moment. Okay, what is the predictive validity of the mouse model for human mild TBI, mouse models in Alzheimer’s are notoriously non-predictive of human AD, how good is the mouse model, or is this just a starting point? 

Dr. Crawford:Well, the mouse Alzheimer models with which I’m very familiar also, all these things are just models. They model some of the aspects of the pathology, perhaps some of the aspects of the behavior. They are a starting point, they are a tool for us to screen therapeutics, explore targets that then advance into clinical trials. And yes, of course, there are many examples of being able to cure Alzheimer mice and the same approaches can’t work in humans or are less successful in humans. It's a starting point. I think right now we don’t have that direct correlation available because the work is relatively new. I think one of the areas of interest to me in TBI is the possibility that although the immediate consequences of TBI will be, to a certain extent, determined by the nature of that initial injury. I’m hoping that one of the things that we might see is that as you look at the later time points after injury there may be a convergence of neurodegenerative processes so that regardless of whether we see a blast – whether we’re using a blast injury model, a closed head injury model, a fluid percussion injury model, we may see a convergence of processes which may mean that there might be common areas for therapeutic intervention to look at in humans. But for sure the mice are just a starting point. It's certainly better than in vitro because we’re able to capture all of the possible cellular responses that might be present in the human brain. But there’s certainly a long way to go from preclinical to clinical. 

Moderator:Thank you very much for that reply. This next one is a comment directed at Dr. Ruff. Your PCI scores are very high. The range of PCL scores is seventeen to eighty-five with fifty being the discriminate score cutoff that separates PTSD from non-PTSD groups. Scores in the eighty to eighty-five range are often exaggerated and more a reflection of the distress because this is a self report measure versus a diagnostic interview and the response scale asks how bothered have you been by this symptom in the past month. 

Dr. Ruff:So I think that that’s entirely true. And it's – I agree with the person who made the comment that it is a self report index, and it has to be regarded in that context. 

Moderator:Thank you for that response. And the final question we have is for Dr. Crawford. What is the rationale for examining the specific lipids in the mTBI mouse model, PC and SM? 

Dr. Crawford:Well, lipoidemic technology is really expanding and growing at the moment. One of the areas that we saw when we performed our proteomic analyses was evidence that lipid metabolism was disrupted in response to TBI both in the plasma and in the brains of the CCI injured mice because what prompted us as we got a lipidemic platform to look at those, and it's certainly relatively easy to hypothesize how lipid disruption may play a role in head injury. So that was really one of the areas that we were interested in pursuing. We do a lot of work on membrane integrity. We do a lot of work on blood brain barrier function and dysfunction. And the thing that is true of lipidemics right now is that we can generate these really detailed datasets telling us the families of possible lipids that are being affected and we can get into the nitty gritty and look at the individual molecular species, the individual lipid species that are being affected. What that actually means is still a little bit of a mystery at the moment. And we actually need to go piecemeal going back and looking at these individual species to try and see how they may have an effect. We don’t have the same databases and knowledge bases available just now to attribute a functional significance. But the significance for us of seeing these lipid changes, first and foremost, a biomarker can just be a biomarker. First and foremost, we would like to have something that we could use from the blood, a panel, as we expect it will be. It won’t be a single biomarker but we expect to be able to have a group of proteins and lipids that collectively could form a biomarker panel predictive of TBI. And there’ll be a particular algorithm associated with that and we will be able to apply that in order to determine whether somebody’s had a TBI, potentially what sort of severity they had, and potentially even what their prognosis might be going forward. That is almost distinct from what the functional significance is because we are just looking for a marker. 

If we take it to the next level, however,  where we’re making any kind of assumptions or have any expectation of what we see in the blood is whether directly or indirectly a reflection of what’s happening in the brain. That’s something we can really start to get into some exciting areas because if changes in lipids in the blood are a reflection of changes of lipids in the brain then we already have a precedent whereby we know that there are potentially relatively straightforward ways to address changes in lipids in the brain. There’s been work done in the past with oral administration of particular phospholipids and the effects that that can have. So I think it's a very promising area. The idea though, first and foremost, is for a biomarker panel, and I think the lipid data are also nice. So this is a very long answer to your question here. But the other reason that I’m very excited about the lipid data is that these are absolute values. This is all measured against internal standards whereas the proteomic data, those were relative in mice compared across injured and non-injured animals. With the lipidemic analyses, it's absolute values, and I think again that will lend itself to the development of a biomarker panel that may be useful for humans. 

Moderator:Thank you very much for that reply. That is the final question that has come in. Well, I’m sorry a few more. Dr. Crawford what role, if any, does a glutathione - I’m sorry I know I’m butchering that – supplementation play in protection from mTBI? 

Dr. Crawford:We haven’t actually looked at that ourselves. Certainly, glutathione supplementation is something that has been explored by others. We haven’t reached a stage yet of trying any specific therapeutic approaches in this mice model. We have looked at anti-inflammatory and anti-amyloid approaches in the CCI model based on the proteomic findings. And both of those showed an improvement in the mice who were treated compared to those that were untreated. Glutathione obviously is tackling any of the sort of oxidative stress issues and oxidative lipidemics is another area of interest. So that’s certainly something that we may be considering looking at as we go forward. 

Moderator: Thank you and at this time I’d like to give you and then Dr. Ruff a chance to make any concluding comments to our audience. 

Dr. Crawford:Well, I would just like to thank all of you for attending. I hope I was able to provide – I know I try to pack a lot in but I hope I was able to provide a sort of broad overview of the capability and the application of mice models in TBI and the scope of what we are able to dig into these days with some of the remarkable technology that’s now available to us. And certainly I would be happy to address any further questions if anyone wants to communicate offline I’d be happy to do that. Thank you very much. 

Dr. Ruff:So I would like to also echo my appreciation for the audience and the last slide contains email contacts if there are other questions that people think of later. 

Moderator:Great! Well thank you both once again for lending your expertise and thank you to our audience for joining us today. Please do join us for the third of this four part miniseries on TBI. The next presentation will be this Thursday the 25th at 2:00 pm Eastern and it will be on the management of TBI and rehabilitation. Please do fill out our survey that will populate on your screen after you exit today’s presentation. We do like to offer subjects that are of interest to our audience. So thank you once again to everyone and this does conclude today’s cyber seminar.  

Page 17 of 17