**Transcript of Cyberseminar**

**Timely Topics of Interest Series**

**mTBI: Technology Advances in Diagnosis: DTI (Diffusion Tensor Imaging)**

**Presenters: Carlo Pierpaoli, M.D., Ph.D.; Rajendra Morey, M.D., M.S.**

**May 13, 2013**

**mTBI: Technology Advances in Diagnosis: DTI (Diffusion Tensor Imaging)**

**May 13, 2013**

*This is an unedited transcript of this session. As such, it may contain omissions or errors due to sound quality or misinterpretation. For clarification or verification of any points in the transcript, please refer to the audio version posted at www.hsrd.research.va.gov/cyberseminars/catalog-archive.cfm or contact: Carlo Pierpoali: cp1a@nih.gov or Rajendra Morey: morey@biac.duke.edu*

Dr. DePalma:It is a pleasure to introduce Carlo Pierpaoli, who is a pioneer in TBI at the NIH. Really, one of the first papers in 1998 was published by him and his group. And, Rajendra Morey from Duke, who is going to present an update on NTBI, the use of BTI for diagnosis. It is a pleasure to welcome these gentlemen. Thank you.

Molly:Thank you very much, Dr. DePalma. And at this time, I would like to turn it over to Carlo. Are you ready to share your screen?

Dr. Pierpaoli:Yes. Can you remind me how I will do it? Is it automatic?

Molly:You will have, there is a button that says “Show my screen.” Just click that button.

Dr. Pierpaoli:Okay. Okay. Done. Okay. Yes.

Molly:Great. We can see it. Now just open the slide show mode.

Dr. Pierpaoli:Okay. I do not know why I had a little conflict here, but anyway. Okay, thank you. Thank you for the invitation...

Molly:Perfect. Thank you.

Dr. Pierpaoli:...and I hope you were able to see my monitor and enlarged also the point.

Molly:Yes.

Dr. Pierpaoli:I will be talking about sort of, maybe a rather methodological aspect and I think it is very important, because we now hear – the field of diffusion MRI has become increasingly complicated – and now with really not only DTI, but many other methods that goes under the general unifying terms of HARDI, which stands for High Angular Resolution Diffusion Imagery methods. And, some of these methods are [inaud.] and so on. And, they are all proposed as methods to be used in traumatic brain imaging [inaud]. I would like to go through the pros and cons of using these methods versus DTI. 

So, my first slide here shows essentially the terminology. The literature is very confusing. I think it is very important to sort of go back to what we measure and we measure water diffusion we use MRI to order to measure water diffusion within the tissue. And, essentially water probes the tissue microstructure at the distance of microns when we do diffusion in the MRI. But, then, the diffusion information is sort of integrated over the entire voxel, so we go from microns to millimeter. And, what we have describing essentially this method, they essentially describe the diffusion displacement  profile of water molecules in the box And, it is not difficult to understand how, you know, a tissue microstructure may affect water diffusion for this is very well known that in the white matter we tend to have a diffusion dispersion profile with instead of being isotropic or spherical, tend to be elongated or ellipsoidal, okay? But, we always had to keep in mind that we are sort of measuring the like scale of microns and then we are integrating the information at the level of our voxel. So, now we have the first poll question, which, Molly am I supposed to read the question, or you do?

Molly:You can read the question. That is fine. I will go ahead and launch it now, so for our 10Bs please take just a second to click the circle next to the answer that best describes your answer. And, actually, I will read it aloud for you, Carlo. So, anisotropy, sorry if I said that wrong, is related to which feature of the water displacement profile? And the answer choices are size, shape, or orientation. And, we have already had half of our audience respond, so we are going to give people just a few more seconds. 

And, while you are responding to that, I just want to make a quick announcement that I was not able to beforehand. You are muted and we will remain muted, so please submit all questions in writing using the dashboard on the right-hand side of your screen. 

And, we have now reached 66% response rate, so I am going to close the poll and share the results. And, Carlo you should be able to see those, if you would like to talk through them real quick.

Dr. Pierpaoli:Actually, I do not. I do not know why I do not see them, yet. No, I do not have the...

Molly:No problem.

Dr. Pierpaoli:So, what are the...

Molly:Okay. The answer is 7% responded with size, 49% said shape, and 44% said orientation.

Dr. Pierpaoli:Okay. So...

Molly:And, I will turn it back to you.

Dr. Pierpaoli:Yes, thank you. So, indeed the correct answer is shape. So B is the correct answer to the thing. And, that is very often confusing, so, we may have an anisotopic structure and we have the, certainly the orientation in space, but what anisotropy tell us about it, just think of the eccentricity of the diffusion displacement profile. It does not tell us anything regarding the orientation of these diffusions. 

Anyway, if we go then quickly on how we compute these images we start from the diffusion weighted images that people rarely see, because we generally see the outcome of all the processes that we do on the diffusion weighted images. But, we rarely report on the quality of the pictures and weighted images. And, here from the diffusion weighted images we compute the diffusion tensor and then we can display the diffusion tensor in this way, which is not very common. But, this is what essentially the diffusion tensor MRI is all about. So, all most historical paper and we have essentially this inset shows larger diffusion displacement profiles of spherical shape in the ventricles than in the gray matter sort of, spherical displacement profile are smaller, then we have elongated diffusion displacement profiles in the corpus callosum. And, what everybody knows about tractography and the use of diffusion methods to investigate brain connectivity that you connect the dots. So, you tend to follow the orientation of [inaud.] create a pathway.

So, when we go from here to HARDI, so you could use High Angular Resolution Diffusion Imaging method, what changes? The fundamental thing that changes is that we do not constrain the diffusion displacement profile to be ellipsoidal, okay? We can add more certain complex features of the diffusion displacement profile. As you can see here, you know we tend to sort of this bumpy type of diffusion displacement profile. And this, from the standpoint of tractography it is very helpful, because if we have a different type of populations with different orientation in the voxel, and we are here able to extract them. So, I could potentially, if you can follow my cursor, I could follow fiber along these green bumps and it will be would color here, or I can follow fiber along these purple bumps and I will go this way. So, it enables to either enrich your representation of water diffusion, which is a positive thing.

So, essentially, what are the fundamental differences from the acquisition. Is like diffusion tensor imaging is already a challenging technique, so we need to acquire diffusion weighted images with different directions. We need to add diffusion sensitization which is usually expressed in b-value and with this type of b-values we already have about half of the signal left in our images. We rarely get to very high spatial resolution in the chemical environment. We have a type of acquisition. The problem is that we need – to do proper HARDI we need to acquire a much larger number of directions.  We need to go to higher b-values, so essentially it is another thing that we know has becomes _____ [00:09:53]. And, then generally we get the way by using another spatial resolution, because we need to compromise from somewhere. But, the fundamental point is that High Angular Resolution Diffusion Imaging is more demanding?

So, here I have some examples of the diffusion and we will go to now maybe to some of the issues that we may be encountering with HARDI data. So, here I have two HARDI scans. This is the magnification of the corpus callosum. Here again is the singulum, some gray matter region in this part, and here is the longitudinal data. Sorry for the standing state. It is essentially the same subject, tissue DNA, the same subject scanned on two different occasions. And you can probably, you know, clearly see that although the underlying [inaud.] seems quite similar, the diffusion has been profiled are different between Case A and  Case B. It is a much richer representation of the diffusion displacement profile here are much bouncier in this situation and this other situation. 

And, so, the next question is, and I will read it, because it is probably a little bit long, but essentially, how would you try to interpret, if you are essentially a clinical general scientist does a study like this one and then after a while maybe you know what happened, but it is kind of like _____ [00:11:39] you have to decide what happened, okay. And, one of the options is just tell me what you feel is the mostly likely in your view. At least, I work in the child _____ [00:11:52] so one possibility is that in Scan A the subject was younger and the brain of the subject was more immature so it had less connections and less rich bumpiness of the diffusion displacement profile. The second one is that Scan A was in the acute phase of trauma and Scan B essentially is showing us brain reorganization, a sort of sprout in the _____ [00:12:19] due to brain plasticity. The third one is that the pattern is compatible with all of the above or either of the above, essentially, depending on what really, you know, the history of the subject and you will be confident in the, you know, drawing this type of conclusion. So, you are sort of a grumpy person and you think that is some experimental artifact. So, now, send the screen back to Molly for the poll, but which of this poll possible options do you think is the most likely.

Molly:Thank you very much. I can see people are a little more shy to answer this question. We only have 50% that responded, but that is okay. I am going to go ahead and close the poll now and share the results. So, we have 2% that responded with the first answer choice, a subject is younger and an immature brain and has less connections. 33% say A is in acute phase of trauma and Scan B is later showing brain reorganization. 63% say pattern compatible with all of the above, and 2% say it is an experimental artifact. So, thank you for those replies and I will turn it back to Carlo.

Dr. Pierpaoli:Okay. I do not know why it goes back to, it takes it to view. Okay. So, indeed I am glad we have optimistic person in this audience and I feel that it is essentially motivated by a logical factor, but, unfortunately, is the correct answer is number four. It is some strange experimental artifact. And the strange effect of the experimental artifact that is determining the different appearance that in this case _____ [00:14:34] noise, just white noise to the computation. So, if I would have done the scan in a good _____ [00:14:41] with less noise I would have had these results, and in a _____ [00:14:46] with more noise I would have had these results. But, it is not related to a biological change. And, these fundamental aspects that we need to keep in mind that we tend as a researcher always to accrete the findings of the sort to, you know, underlying biological phenomenon we are studying, but we need to be very alert that it may be related to experimental processes.

So, the first point I want to make is that proper HARDI requires a larger amount of high quality data than DTI, and that in a clinical setting artifacts can be easily mistaken for biologically meaningful findings. And, again, I do not, I stress this point a little bit. 

Okay, now I am going to a dataset that is one of the poorest dataset that we receive from a motor center study, and you can see, and this was not even a collected property for HARDI data, but it was, as you can see, a single _____ [00:15:52] and you have a sort of very bad artifact in _____ [00:15:58] motion and the distortion. If I throw into an HARDI software, it will start to look pretty good, pretty nice, very appealing. So, we need to be, and that is a fundamental problem of HARDI data. The more flexible model accommodate _____ [00:16:23] very well. The other aspect is big one very quickly I want to point out that the problem of going to high b-values we never see the raw images when people represent their results, but I want a b-value typical of DTI is this level. The image is still can be corrected for motion, you know we can realign the images properly. This is the appearance of images collected with a b-value of 9000 which is used for DSI or other of these HARDI type of _____ [00:17:00] and you can see how _____ [00:17:03] the signal we have. So, if the subject move, if we have a _____ [00:17:08] we will not be able to correct for this artifact. 

The second point I would like to make is that HARDI is almost _____ [00:17:17] that you use for tractography and connectivity. I personally, and here I am in the minority. I do not believe that diffusion MRI is that helpful to study brain structure or connectivity, but I will not go into this discussion, because it would take hours. My point here is that we tend, when we do HARDI we tend to extract the three things that we discussed before, shape, size and orientation. And, we tend to extract mostly the orientation, so we have a reduction approach. And, those again, I find it very difficult to extract even _____ [00:18:02] if even two or three fiber population from this type of _____ [00:18:02]. It is still a very difficult _____ [00:18:11] problem, okay.

The third point that I want to make is that there is no free lunch. So, with HARDI one has to trade off resolution to characterize a more complex displacement profile. So, HARDI requires mining more data. So, what we need to do is to work with lower spatial resolution to maintain the same _____ [00:18:35] And, there are alternatives what we can do with DTI is to use the time that we have available to go into much higher resolution and there are some examples of _____ [00:18:50] resolution acquisition of DTI in which we can notice here from _____ [00:18:56] of the anterior limb of the internal capsule. And those we can investigate the structures like, you know, the _____ [00:19:09] and very tiny structure with very complex architectures and then come to some conclusions about the arrangement of white matter structure within the voxel, thanks to fact that with DTI we can go at a much higher resolution. With HARDI, we will not be able to do that.

The final point I want to make is that the actual clinical usefulness. So, HARDI is again the new game in town, but it has not really shown a pedigree of clinical usefulness. DTI in that respect is much better. And, again, I want to point your attention to sort of neglected ones that we tend to neglect just to look at tractography and this type of thing. But, for instance, _____ [00:20:08] which is also a trace of the _____ [00:20:09] or it is also called ADC or many, unfortunately many, many terms in the literature, which seems so uninteresting as an image. In reality it is great to certain forms of pathology. For instance, here in trauma, and similarly to what is done in stroke, we see in an almost normal appearance of the T2 weighted image that the Trace the _____ [00:20:40] image show _____ [00:20:42] of the brain with reduced _____ [00:20:44] which are essentially regions that have going through a metabolic process and metabolic suffering of the tissue. 

And, it is also very nice to see after many years of people that were attributing these things to just _____ [00:21:07] or other things there are now interesting interpretations about what we are seeing when we see these reduction diffusing _____ [00:21:17] into this, you know, paper by Matt Budde essentially it is that reduction in the _____ [00:21:27] is related to beading. Essentially, here you have _____ [00:21:31] image and you can see the neurites here, and then during the _____ [00:21:39] what happens is that the neuritis shows this beading process. So, you can see back and forth, okay. And this beading process essentially water diffusivity as sort of cracked by this beading, and so we see in according to Matt Budde, this why we see a reduction in the diffusion. And, then after successfully _____ [00:22:06] we know that diffusion can indeed go back to normal. So, these are all very clinical, very interesting, you know, tools that we should be using. 

Anisotropy, again related to the shape of displacement profile. A normal brain is very sensitive to white matter architecture. I do not like the fact that now people say, it is white matter integrity. Not really integrity, in particular in the normal brain is related to white matter architecture and it is very sensitive to white matter regeneration. You can see here for instance in the chronic stroke case at the level of the cerebral peduncle, but we can look at the _____ [00:22:58] almost, tractographic regeneration of fibers in the _____ [00:23:04] map comparing left and right. Really, _____[00:23:10] reformation. Then we have this orientation information. Again, without doing any tractography with DTI, here this is not a case related to DTI, but just to show that in a _____ [00:23:25] it is well known that this regeneration of the tranverse pontine fibers and you can clearly see the regeneration here. A normal pulse with the right fibers, the tranverse pontine fibers and even without the tractography we can really see the degeneration of these _____ [00:23:48] in the color maps, okay.

So, eventually this concludes my talk, and this are again I would like to go to quickly the four points that I tried to make, so that HARDI requires a large amount of high quality data than the DTI. And, I think I show you some example that when we do not control for artifacts it is very easy to be mistakenly attributing to biological findings things that indeed are artifacts in the HARDI acquisition. That also HARDI puts so much emphasis on tractography that we are missing out on very interesting clinical features of the diffusion process. The third point is that there is no free lunch, that we need HARDI, we can then mimic high definition tractography, but in reality our original data collected was very poor resolution. So, we can then play with our computers and get very fancy graphical representations. But, we need to look at the resolution of the original data we have. And, then the clinical usefulness, I really would hope that, again, HARDI has some theoretical, interesting advantages, but in the clinical field we do not know if we will deliver what it promises. So, we need to eventually be wary of this aspect. And, with this, I will conclude and thank you.

Molly:Excellent. Thank you so very much. I am going to turn it over to Dr. Morey now. Are you ready to share your screen, Raj?

Dr. Morey:Yes, I am.

Molly:Great. There you are.

Dr. Morey:Okay, so, my talk today is on White Matter Damage in Blast-exposed Veterans without Mild TBI, and I will begin with some background. So, as most of you may know that exposure to explosive forces from bombs is common in Veterans of the recent military conflicts. And, the majority of traumatic brain injury at forward deployed medical facilities involves exposure to an improvised explosive device, grenades, RPGs or mortar fire. And, damage to brain tissues, that is white matter, using diffusion tensor imaging is now fairly well established in cases where there is clinical symptoms that are observed or reported that meet the criteria for mild TBI. And, the last point, which I am going to use as a stepping point, is that there is a couple of recent studies, I should say, of sports or athletes showing compromised white matter integrity, even when they have an absence of any concussive symptoms. There are no clinical symptoms, but yet there is evidence of changes with diffusion tensor imaging.

So, this is actually one of those reports that came out late last year in JAMA, and this is a study, a small study of elite soccer players in Germany. And, they compared elite soccer players with stub concussive hits to the head to another group of athletes which were elite swimmers, who obviously did not have impacts to their heads. And, this study reports that there are white matter changes in the group of elite soccer players. 

This is another study by some of the same people, some of the same investigators as the JAMA study. This study was done in a Canadian ice hockey players at the collegiate level. And, again this was a longitudinal study design where the ice hockey players were imaged before and after season of ice hockey play, and showed white matter change, or DTI changes, FA changes, fractional anisotropy changes before and after one season of play, but without any clinical symptoms of concussion.

So, I am going to go to this poll question, and...

Molly:Would you like to read it aloud, Raj?

Dr. Morey:This is a very sort of mainstream question. How many of you are familiar with the Junior Seau case? Yes, no, or not sure.

Molly:Thank you. I knew I was not going to pronounce that last name correctly, so, I thought I would...

Dr. Morey:I have a hard time with it myself.

Molly:[Laughs] All right. We have already had 71% of our audience vote and the answers are still coming in, so we will give people just a few more seconds. We do appreciate you taking the time to respond. All right. And, the answers have stopped streaming and now we got about three-fourths of our audience to reply. I am going to go ahead and close the poll, and I will share the results.

So, we have 49% that answered yes, 39% answered no, and 11% said not sure. Thank you.

Dr. Morey:So, just for those of you who are not familiar, Junior Seau is an NFL player who committed suicide and was after post-mortem examination was found to have very pronounced chronic traumatic encephalopathy. So, his brain was full of _____ [00:31:23] protein and he is an NFL player who purportedly didn’t have any concussions, but after his suicide one of his teammates said that he estimated that Junior Seau probably had 1500 grade one concussions in his career. 

Molly:I am sorry for interrupting. I am sorry for interrupting. Can you press the button, Show My Screen, again? We cannot see your slides at the moment.

Dr. Morey:Thank you. So, this is just an image of, it is a picture of Junior Seau, the NFL player. So, the second poll question, which is really the topic of my presentation, is similar to the DTI changes we have now seen reported in elite athletes. It is a question about, is there injury to brain tissue in Veterans with blast exposure, even when they do not have any clinical symptoms of TBI, and this may be a bit of a rhetorical question, but the options are yes, no and not sure. And, I will let Molly take over.

Molly:Thank you very much. We have had about three-fourths of our audience reply and we will give people just a few more seconds to get those answers in. Okay, looks like they have stopped rolling in, so I am going to go ahead and close the poll for now and I will share the results. We have 61% replied yes, 12% replied no, and 27% replied not sure. And, I will turn it back to you.

Dr. Morey:So, I will just take the rest of the time, the slides to kind of address this question. But, we did a study, a very basic pilot study. We had six Veterans from recent conflicts in Iraq and Afghanistan, six Veterans with primary blast injury. That means no secondary or tertiary mechanisms of injury. We had sixteen blast exposed Veterans without mild TBI. And, we had twenty-three blast unexposed control subjects and all of them had DTI scans in the chronic stage. 

And, this was our results, comparing the blast unexposed and the blast exposed group. So, that this is the blast unexposed, which is the last one, I am sorry, the sixteen blast exposed and the twenty-three blast unexposed. And, this was done with what I would call a conventional whole brain voxelwise analysis, and that basically means that FA values were averaged between, within the two groups and FA values, the mean FA values of one group were compared to the mean FA values of the other group. But, when I say conventional, basically, if you think of the brain as, if you think of DTI images, a three dimensional grid, the whole brain voxelwise analysis basically compares corresponding points of that grid between the two groups. However, it seems to be becoming more clear that the way blast injury and even impact injury in athletes, the blast injury is, that much of the damage is happening from secondary mechanisms of injury, which are neuro-inflammatory and neuro-toxic processes. And, that the injury may not have any spatial correlation with the actual events around the injury, so that essentially the damage is spatially heterogeneous is what I would call it. 

And, so we used a different analytic strategy and rather than comparing corresponding voxels across brains, we actually looked at, we tried to identify how many voxels in the white matter have abnormally low or unusually low FA values. And, we did this by calculating the mean and standard deviation of the FA values at every voxel in the reference group, which is the unexposed group, and this is just an illustration that shows the data in this graph at one particular voxel location. And, this would be an example of an FA value of an individual subject in the blast exposed group, who did not have TBI symptoms. And, in this example we see that the test subject has an FA value that is lower than two standard deviations below the mean of the reference group. And, so we can actually do this process at every single voxel that represents white matter, and actually, those voxels are highlighted here in green, and the voxels that are actually two standard deviations below the mean are highlighted in red. 

And, we can actually do this for every subject in the blast exposed group, and then we can look at clusters of voxels. And, we looked at three sizes of clusters, so we looked at small, medium and large clusters. And, so small cluster were plus twenty-five or more voxels that were localized together that had abnormal FA. Medium potholes, or clusters were fifty or more voxels and large clusters were seventy-five or more voxels. And, what this graph is showing is the number of low FA clusters, or potholes, in the three groups. So, here the blast unexposed group has the lowest number of small, medium and large clusters. The blast exposed group in green has significantly more clusters of abnormally low FA. And, also, the blast-mTBI in red also has significantly more clusters or potholes. And, so we see across the small, medium and large that the blast exposed and blast-mTBI are more similar to each other and they are significantly different than the blast unexposed. So, that basically gives us the answer to that question.

This is leading to some of our other data. So, fractional anisotropy is really gives us, as Dr. Pierpaoli highlighted in the shape of our diffusion anisotropy. But, we can also look at two different measures, which is the radial diffusivity, which is this arrow pointing downward and we can also look at the axial diffusivity which is this arrow along the access of the myelin fibers. This is a very simplistic kind of interpretation that we have to be careful about – that axial diffusivity represents axonal integrity and radial diffusivity represents myelin integrity. But, that is a different and very complicated topic. I am just going to skip past that and just say that when we looked at radial diffusivity, we also found that the blast exposed and the blast-mTBI group had significantly more potholes or low FA areas than the blast unexposed. That was true for the small potholes, the medium potholes and the large potholes. And, when we looked at axial diffusivity there was a trend level, but this was not statistically significant. And, finally, we wanted to actually confirm that the damage was indeed spatially heterogeneous and, as we hypothesized, because the idea that secondary mechanisms of injury like neuro-inflammation caused this kind of spatially heterogeneous type of damage.

So, this figure is basically showing all the white matter voxels, it is highlighting all the white matter voxels where at least one subject had a pothole present at that location. So, all the voxels in this lime green represent voxels where there was at least one blast exposed subject with a pothole at that location. The violet color represents voxels where there were at least two subjects with potholes present at that location, and the blue represents three subjects having potholes at that location. So, as you can see, and it is hard to see it in these coronal sections, but I have this larger coronal section, which is taken from these other, this other montage. But, you can see that the majority of voxels where there were potholes were only present in one subject out of twenty-three, and there were a few with two subjects, a few with three. There were actually, in rare instances, four or five, six, but those are not even shown here, because there are so few. Similarly, in the blast-mTBI group, most subjects only had potholes at one location, or most voxels only had one subject with potholes at any given location. So, this really underscores that the damage is spatially heterogeneous.

After looking at white matter, we thought, well, if this damage is spatially heterogeneous and also that secondary mechanisms of injury like neuro-inflammation are causing this to happen, we might also look at gray matter, not just white matter, and we did this analysis in a separate group. This is not a group that had a blast exposed without TBI group. It simply had a control group and a TBI group. But, we do see, when comparing this control and TBI group, that indeed, there are more potholes, which would be really regions of altered gray matter intensity in the TBI group, compared to the control group. And, this is present at small, medium, large and even bigger than large voxel clusters, or potholes.

So, the conclusions, I will just highlight three main conclusions. So, blast exposed Veterans experience white matter damage that is comparable to mild TBI, even in absence of clinical symptoms, at least in our preliminary data. The lack of clinically observable TBI symptoms following blast exposure may lead to the erroneous conclusion that little or no damage has occurred to the brain tissue, and consequently may go unnoticed. And, if these findings are confirmed in a large and definitive study, our findings would argue for an overhaul of the established approach for making diagnoses based on clinically observable symptoms of mild TBI in favor of a more novel neuro-imaging based diagnostic criteria that looks below the surface for pathology.

And, these are just references for the audience, and I will actually open it up to questions and answers to both doctors.

Molly:Great. Thank you very much. Yep, we will take questions now for either presentation, so go ahead and write those into the control panel and press Send. We will get to them in the order that they were received. We do have some great pending questions. 

So, this one I am going to start with is for you, Dr. Morey. What does pothole mean pathologically?

Dr. Morey:Well, I think the pothole, I will just make one disclaimer that it is, perhaps, a kind of an unfortunate term. It actually was referred to as a pothole by a paper by Jorge, et al. I am sorry, yeah, I believe it was the second paper here in the references section in the American Journal of Psychiatry. But, essentially, pothole in terms of diffusion tensor imaging, is a cluster of voxels that are, that have abnormally low FA, and they occur in a cluster of predetermined size. We used 25 voxels. But, I think I can actually ask Dr. Pierpaoli to comment about, maybe about what FA means in terms of brain pathology. It certainly means that white matter is affected, but in terms of the exact architecture, I will defer on that.

Dr. Pierpaoli:Yeah, it is unfortunately not a simple answer, and that is indeed quite common for mainly MRI matrix, and we sometimes tend to translate MRI findings into biologically meaningful thing, but it is not an automatic thing. And, so what is FA? FA is influenced by many things. In normal brain, one of the most important factors is probably the organization of the fibers will be in the voxel. That is the most resilient feature. Generally, one believes edema FA tends to be reduced. When fibers are destroyed also FA is reduced, but generally more severely. In the past, there has been a lot _____ [00:49:03] chance, so for a certain period we could see in the literature any change in the _____ [00:49:09] was related to _____ [00:49:13] and is not actually the case. _____ [00:49:17] is largely influenced by _____ [00:49:19] but, one beautiful aspect of diffusion MRI is that you can look at white matter even in the mature brain without any _____ [00:49:29], and you can really see the very fiber. So, _____ [00:49:37] paper by _____ [00:49:39] I think he is publishing neuro-imagery _____ [00:49:44] but also this concept of relating the FA to white matter integrity. But it is worthwhile reading. So, it is not a simple answer essentially, and many factors may affect FAs, including noise. And, that is a danger _____ [00:50:03] a little bit as I show you before, in the HARDI thing. Noise also can change FA, can bias FA. So, that has been one known for a long time. So, we need to really be very careful in assigning the biological meaning to these changes in the diffusion matrix.

Molly:Thank you very much for both those replies. We do have fourteen pending questions. We will go ahead and move right along. A follow-up to that last one. So, are potholes the same as cavitation? 

Dr. Morey:I am actually, I am sorry, off the top of my head, I am not really confident about the term cavitation, but I think the answer is no. I would have to look that up and get back on that.

Molly:No problem. That is perfectly fine. The next question, and this is to both of you. When, in your opinion, will DTI be used for definitive diagnosis of MTBI.

Dr. Morey:Well, I hope soon. Actually, there is a number of efforts to create national multi-site studies that will try to test approaches to using, with the goal of translating DTI into the clinical setting. These multi-site studies would still be definitely in the research phase, but this is something that is a very active area of research. In terms of specifics of time, it is hard to say. I certainly do not think it will happen in the next one or two years, but it is possible in the foreseeable future.

Dr. Pierpaoli:And, again, I do not want to sound like a broken record. I think that, I love diffusion MRI my entire career, in case there are walls around this technique. I think that, and I like it because it is a quantitative technique, so essentially, we can compare matrix from an absolute scale. We are not dealing with single intensities that essentially cannot be compared across subjects reliably. So, it has a tremendous potential. The point is that also experimental factors, _____ [00:53:04] related things, calibration of your instrument, modalities in which the images are acquired are very important. So, you cannot compare, for instance, data that are acquired two and a half millimeter resolution with data acquired at two millimeter resolution. The X-ray would be different, even if you would scale all of the images at the same thing. So, in the twenty unit acquisition would be different from the six unit acquisitions. So, it is very important, in my view the future of DTI in historical studies and in the clinical environment is related to strong efforts for _____ [00:53:46] and a very careful assessment of potential confounds that may be introduced by various experimental practices.

Molly:Thank you both for that reply. The next question. Was your control group from community or military settings. And, I believe this came in during Raj’s.

Dr. Morey:There are all three groups were military, and they were all from recent military deployments, or military, yeah, conflicts, which is Iraq, Afghanistan, and so forth.

Molly:And, were the findings controlled for PTSD?

Dr. Morey:Yes, the findings were controlled for PTSD, though the graphs that I showed of the small, medium, large potholes with the sort of modified bar graphs were, those were controlled for PTSD, presence of PTSD, yeah. And, actually this is a comment about that, is that if, in situations where a Veteran does have exposure to, especially exposure to repetitive sub-concussive blasts, so they were exposed to blasts several times, but they never had symptoms of mild TBI. In other words, they did not have a loss of consciousness, they did not have an _____ [00:55:44] or feeling dazed or confused, or seeing stars, or any of those symptoms. They may assume that none of their symptoms could be related to blast, and I think that is maybe, potentially taking us down the wrong path. It is potentially an incorrect assumption, because if blast exposure without MTBI symptoms is causing white matter damage, then symptoms that they are experiencing, clinical symptoms later on like headache and depression and memory problems and attention problems, might be not really because of PTSD, but they may be because of this sub-concussive exposure to blasts. But, I think that is an important implication of the findings that I presented, if they hold up to be true.

Molly:Thank you for that reply. This next question, it is a little incomplete, so please bear with me. Time from exposure to this study, also repeat studies over time to track recovery changes. Clarification, controls were also combat exposed Veterans, but without blast exposure?

Dr. Morey:Yeah, so, I think the first part of the question, I believe, is asking about were these chronic subject, would the findings, or was the MRI scan acquired at the chronic or in the acute stage. It was definitely in the chronic stage, and yes, so we do not have repeat MRI scans of these subjects, but, yes, that would be nice to, important to assess, and that is something that we will be trying to do in the future. And, I forgot the third part, Molly, if you can...

Molly:No problem. He wanted to know, the controls were also combat exposed Veterans, but without blast exposure, correct?

Dr. Morey:Correct, they are.

Molly: Great.

Dr. Morey:I mean...

Molly:Go ahead.

Dr. Morey:...yes, they were, but I think that is with a caveat, because I would say that with enough combat exposure, you are, the chance of exposure to blasts increases. So, and I do not know the answer to his question, but I am guessing that the unexposed group may have had less combat exposure than the other groups.

Molly:Thank you. This question is for both of you. Can you share with us how we might attempt to run a multi-site imaging trial?

Dr. Morey:Well, there are some already underway. For example, the DoD has a multi-site trial underway, and they use very careful and rigorous methods for trying to calibrate, essentially, the DTI imaging across sites. So, that is a very technical process where, and maybe Carlo can say more about it, but, yes that is something that is happening and actually more multi-site studies are being proposed as we speak.

Dr. Pierpaoli:We have experience with multi-site study and we have been involved in the NIH pediatric imaging study, which we were _____ [01:00:25] imaging center for the diffusion part. My impression is this one. In general, in a multi-center study do lower the bar, it is much more difficult to do a multi-center study, so you have to settle for the minimum common denominator, but you need to be sure that everybody is capable of reaching. And, so sometimes if you have institutions with very different capabilities, you really need to make sure that everybody can actually _____ [01:00:56]. The other suggestion I would give is to have not people collecting and processing their own data, but rather sort of a centralized processing standard, because many things can be corrected during processing and that also ensures a certain uniformity in the data processing. But, _____ [01:01:23] is very important for multi-center studies, not to acquire all the data and then we do not look at the data while it is acquired. The process of quality control is a continual, almost osmotic process, so this data coordinating center should be always active receive each new scan the next day and go through a quick feedback in order to avoid that certain _____ [01:01:57] or things that are deviating from the _____ [01:02:01] would be continued before corrections. So, I think that if this is the case, it will be possible and very desirable _____ [01:02:10]. The final thing is essentially to have a _____ [01:02:15] calibration of the scanners and for us anisotropic _____ [01:02:21]. We have developed a _____ [01:02:27] that we presented with the _____ [01:02:29] which is essentially in the anisotropic _____ [01:02:33] calibration is probably all we need. But, obviously, no one can _____ [01:02:45] anisotropic features, but, for to check that the equipment is working properly, I guess. And, I believe that anisotropic _____ [01:02:54] is enough.

Okay, so there is, again, from the standpoint of risk, there is no reason why HARDI should be more risky than a conventional DTI. It is a longer scan, but essentially there are FDA limitations on this called DBDP, so how quickly _____ [01:03:33] can switch and then generally, and that is really depending on the sequences and then for human studies, generally people stay within this limitations. So, no I do not think that there is an issue. But, in terms of being a true diagnostic technique, then I think it could be a completely different story. But, I am not, I mean I always work with sequences for our research, and so I am not familiar with more of the commercial use of the _____ [01:04:22].

Molly:Thank you for that reply. We will have Dr. Morey answer these in just a few moments. The follow-up to that, is so should we stick to DTI solely? When can we use this for clinical diagnosis?

Dr. Pierpaoli:Well, the point of my presentation was that there are trade-offs, and one needs to ask very clear what are the biological questions and one wants to ask what is the environment in which you ran the acquisition, and I will say, _____ [01:05:04] much time you have. So, if you tell me that you have multiple sites with very _____ [01:05:13] software and hardware, and that you want to do no longer than a ten-minute acquisition, I would definitely go with DTI. If you have maybe fewer institutions and you may do a much longer scan, then you are more in a research environment to definitely one can run a HARDI type of scan. One of the other things that is a compromise, I pointed out, is the issue of you have to decide a priority. If you are more interested in having spatial resolution or having the HARDI type of acquisition, so for the same amount of time, you pay a price in terms of resolution. If you can do DTI, you can go at a higher resolution than if you do HARDI.

Molly:Thank you for that reply. We have just a few more questions to get through. Are there any NIST guidelines on DTI phantoms for machine calibration?

Dr. Pierpaoli:I am not sure that there are. I know that NIST people are working on phantoms and, actually, the compound that we have proposed for use as a phantom has been inserted in their phantom, so, I know that they are working on a phantom. I do know if they have already guidelines or essentially a phantom that can be distributed. On top of that, I think that there are potentially different phantoms for different purpose. So, the phantom that we developed is a phantom that is used to, it is a sphere. It does not have any geometric structure inside the phantom. So, it is not to be used for geometric distortion assessment, mostly for diffusion calibration. The NIST phantom, as far as I know, is a more complex phantom that has also some internal structure. So, depending on the goals, one may want to use a different phantom.

Molly:Thank you very much for that reply. I do believe we just have one question left. This is follow-up to the question about FDA guidelines and limitations for DTI. The second part, I am not sure what RF stands for...

Dr. Pierpaoli:Radio frequency.

Molly:...Okay. RF heating / gradient duty cycles.

Dr. Pierpaoli:Yeah. No, so the RF heating is generally very limited for diffusion data. So, it is generally not an issue, even at the relatively high field. So, the duty cycle of the gradient, which is essentially the heating of the gradient, has no bearing to the well-being of the subject, besides the fact that if the gradients heat up too much, they may break and also the environment may become sort of too warm for the subject. But, the duty cycle of the gradients is mostly an engineering problem. So, essentially, that if the gradients heat up too much, they may malfunction. The thing that has an impact on the subject well-being is the so-called DBDT, so it is the speed at which the gradients gets ramped up and down, and then if it is too hot, one may have peripheral stimulation, nerve stimulation. So, that is the thing one has to keep an eye on. But, again, in general, the diffusion imaging does not require to ramp very fast. One can slow down the ramp. It is important to have strong gradients, and from that standpoint there is no particular concern. So, in general, diffusion sequences are quite safe and one can do sophisticated things within FDA guidelines, without need for pushing too hard.

Molly: Thank you for that reply. These next few questions, just let me know if they are addressed to you. I am not sure. What other conditions besides TBI or blast exposure could cause these findings on VBM?

Dr. Pierpaoli:Okay, so these were mostly for the other speaker, for Dr. Morey. I mean, yeah, the point of the interpretation of the diffusion findings has been, I guess at more than twenty years ago, you know writing papers about interpretation of diffusion findings in _____ [01:11:29] has been an interest of mine since then. In general, we tend to oversimplify what it means, and it can mean several things. Not to exclude potential SNR issues. For instance, SNR stands for Signal to Noise Ratio, so the FA is sensitive to noise. And, unfortunately, noise biases FA, does not simply increase the variance of FA. So, in practical terms, it means that if one has that FA is up, one has to ask first of all if it is up because I have a noise in acquisition than previously. So, this is, for instance a contribution from a potential artifactual source. And, other changes, they can be related. If I have an increasing diffusivity, I may have, it may be related to interstitial edema, it may be related to necrosis, it may be related to immaturity of the tissue. For instance, in a brain of a newborn subject we have a diffusivity that is much higher than an adult brain. In a tumor, certainly we have _____ [01:13:21] may be related to tumor cellularity, and again, to the overall environment of the tumor. So, it is not an unequivocal interpretation in general. So, it is a very, and I guess that is why science is not as simple as other things and it is not just having a matrix that matches something, but also interpreting what it means.

Molly:Excellent. Thank you for that reply. The remaining questions are directed at Dr. Morey, so we will get to those in just a minute. But, I want to thank you very much, Carlo, for addressing the field. Are there any final comments that you would like to make?

Dr. Pierpaolo:No, thank you. It was a pleasure, and I hope that people would like to write to me. I am available to respond or even talk over the phone, so just feel free to contact me if anything was not clear, or there are other issues that you people would like to discuss.

Molly:Excellent. Well, thank you very much and you enjoy the rest of your day. Bye-bye.

Dr. Pierpaolo:Thank you. Bye-bye.

[End of audio]

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