Perspective

Diffusion MRI: what water tells us about the brain

Denis Le Bihan

Diffusion MRI has been used worldwide to water. However, although water is an essen- challenge: How could one differentiate liver produce images of brain tissue structure tial molecule for life, its importance in biol- tumors from angiomas? I had some fuzzy and connectivity, in the normal and ogy has perhaps been often overlooked, if not intuition that, perhaps, water molecular diseased brain. Diffusion MRI has revolu- forgotten. Water diffusion MRI has proven to diffusion measurements would result in tionized the management of acute brain be extraordinarily popular. Its main clinical lower values in solid tumors because of ischemia (), saving life of many domain of application (Fig 2) has been steric hindrance to water molecular move- patients and sparing them significant neurological disease, especially in the ment compared to flowing blood. Based on disabilities. In addition to stroke, diffusion management of patients with acute brain the pioneering work of physicists such as MRI is now widely used for the detection ischemia. With its unmatched sensitivity, Stejskal and Tanner in the 1960s, I knew that of and metastases (breast, pros- water diffusion MRI provides patients with specific encoding of the diffusion process tate, liver). Another major field of applica- the opportunity to receive suitable thrombo- could be achieved using tion of diffusion MRI regards the wiring of lytic treatment at a stage when brain tissue gradients, but the problem lied in the inte- the brain. Diffusion MRI is now used to might still be salvageable, thus avoiding gration of such gradients with those used in map the circuitry of the human brain with them terrible consequences. On the other the MRI scanner to generate images. The incredible accuracy, opening up new lines hand, water diffusion turned out to be aniso- problem was not trivial and indeed many of inquiry for human neuroscience and for tropic in brain , because colleagues at that time thought that diffusion the understanding of brain illnesses or membranes limit molecular movement MRI was not feasible. The idea was to local- mental disorders. Here, as a pioneer of the perpendicularly to the axonal fibers. This ize the diffusion measurements, that is, to field, I provide a personal account on the feature can be exploited to produce stunning map water diffusion coefficients in tissues; historical development of these concepts maps of the organization in space of white this had never been done before, especially over the last 30 years. matter bundles and brain connections in just in vivo. As a junior student in physics and a few minutes, as well as to provide informa- medicine, I was very excited about this mong the sensational 1905 Albert tion on white matter integrity. Diffusion MRI potential, and in a matter of weeks, diffusion Einstein papers, there is one that has been also used in a full-body setting for MRI as we know it was conceived and A unexpectedly gave birth to a powerful the detection and treatment monitoring of implemented. My first diffusion images were method to explore the brain. Einstein lesions and metastases (liver, breast, obtained on a 0.5-tesla MRI scanner from explained molecular diffusion on the basis of prostate), because water diffusion slows the late CGR (Companie Ge´ne´rale de Radiol- the random translational motion of molecules, down in malignant tissues in relation to the ogie, Buc, France). The first trials on liver which results from their thermal energy (Ein- cell proliferation. The versatility and the were very disappointing, as the images were stein, 1905). Moving fast forward, in the mid potential of diffusion MRI, both for research hampered by huge motion artifacts from 1980s, I was able to show that water diffusion and clinical applications, have been respiration. This was due to the fact that the could be imaged in the human brain through reviewed elsewhere (Le Bihan, 2003; Le hardware to generate the magnetic gradi- magnetic resonance imaging (MRI). This Bihan & Johansen-Berg, 2012). Here, I will ents was sub-powered and also that the move was triggered by the idea that water provide a more personal account on the imaging methods at the time were slow diffusion could provide unique information historical development of these concepts and and consequently very sensitive to patient on the functional architecture of tissues, how they inspired my research in the last motion. I thus decided to switch to the since during their random displacements 30 years. brain, as this was my original background water molecules probe tissue structure at a and I must confess that I started scanning microscopic scale (Fig 1). What I did not my own before actually moving to patients! expect was that this pioneering work would The birth of water diffusion MRI It worked beautifully and that move estab- end up some day interesting the readers of a lished the great neurological potential for major molecular medicine journal. Sure, this Back in 1984 (when I was a resi- diffusion MRI; the rest, as the saying goes, story is about an apparently simple molecule, dent), a colleague came to me with a is history.

NeuroSpin, CEA Saclay-Center, Gif-sur-Yvette, France. E-mail: [email protected] DOI 10.1002/emmm.201404055 | Published online 4 April 2014

ª 2014 The Author. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine Vol 6 |No5 | 2014 569 EMBO Molecular Medicine Diffusion MRI: what water tells us about the brain Denis Le Bihan

A IMAGE LEVEL TISSUE/CELL LEVEL MOLECULAR LEVEL Diffusion-weighted MRI image Random walk Hindrance Restriction Barriers

B Diffusion-weighted Standard MRI image MRI image Fast diffusion

Slow diffusion

Magnetic field gradient

Figure 1. Principles of diffusion MRI. (A) Contrast and signal levels in the diffusion-weighted image (left) reflect water diffusion behavior (random walk) (right). Diffusion behavior is modulated by tissue structure at the cellular level (middle): For instance, diffusion can be restricted within cells, water may escape when cell membranes are permeable and might then experience a tortuous pathway in the extracellular space (hindrance). (B) In the presence of a magnetic field gradient (variation of the magnetic field along one spatial direction), magnetized water molecule hydrogen atoms are dephased. The amount of dephasing is directly related to the diffusion distance (a few micrometers) covered by water molecules during measurement (a few tens of milliseconds). Given the great many water molecules experiencing individual random walk displacements, the overall effect of this dephasing is an interference, which reduces MRI signal amplitudes. In areas with fast water diffusion (e.g. within ventricules), the signal is deeply reduced, while in areas of slow water diffusion (e.g. white matter bundles), the signal is only slightly reduced. This differential effect results in a contrast in the diffusion-weighted MRI images, which is not visible in standard MRI images.

The world’s first diffusion images of the 1986) was much better received, with more measurements with MRI in tissues would brain were made public in August 1985 at than 2000 citations up to now (3rd most largely differ from those obtained for water the Society of Magnetic Resonance in Medi- cited paper of all times for this journal). in a glass where water diffuses freely, and I cine (SMRM) meeting in London. My first However, because of the sensitivity of diffu- therefore introduced the apparent diffusion diffusion MRI paper appeared in 1985 in the sion MRI to motion artifacts, many collea- coefficient (ADC) concept to describe diffu- journal of the French Academy of Sciences gues remained sceptical at the time. It was sion MRI findings (Le Bihan et al, 1986). (Le Bihan & Breton, 1985). This paper discouraging, but I kept pressing on and it described all the necessary ingredients to paid off, as diffusion MRI progressively successfully perform diffusion MRI. The gained momentum. Ironically, some of my Acute brain ischemia paper did not receive much attention, possi- early detractors started working full time on bly because it was written in French. My diffusion MRI. At the same time, it was Shortly thereafter, Michael Moseley at UCSF next paper in Radiology (Le Bihan et al, becoming clear that the results of diffusion made an unexpected but crucial discovery in

570 EMBO Molecular Medicine Vol 6 |No5 | 2014 ª 2014 The Author Denis Le Bihan Diffusion MRI: what water tells us about the brain EMBO Molecular Medicine

A B

CD

Figure 2. Main applications of water diffusion MRI. (A) Acute stroke. The angiogram (right) shows an occlusion of the right middle cerebral artery. The diffusion-weighted image (left) clearly shows a bright signal corresponding to a drop in water diffusion resulting from cell swelling (cytotoxic edema) in the tissue undergoing acute ischemia. (B) Brain connectivity. Water diffusion in white matter fibers is anisotropic, faster in the direction of the fibers. By measuring water diffusion in many directions, the orientations of the whiter matter bundles can be retrieved at each brain location. Algorithms are then used to identify bundles, which are represented with arbitrary colors (courtesy CONNECT/NeuroSpin). (C) Cancer. Water diffusion in the glioblastoma has been imaged and measured after the patient underwent a chemotherapy treatment. Areas within the tumor where the treatment has been efficient appear in green (return of water diffusion to baseline values), while areas in red highlight parts of the lesion where diffusion still remains low (courtesy B. Ross, University of Chicago). (D) Functional brain imaging. Areas in color represent parts of the brain (visual cortex) where water diffusion has slightly decreased during activation (visual presentation of a flickering checkerboard for 10 s).

an acute cat brain ischemia model (Moseley gradient coils and power supplies with (Warach et al, 1992). Around that time a et al, 1990): Water diffusion dropped signifi- exceptional performances for the time, but drug company was developing an intrave- cantly (30–50%) during the very early phase we finally managed to obtain diffusion nous recombinant tissue plasminogen acti- of acute brain ischemia. This finding tremen- images in a matter of seconds (instead of vator (rt-PA) drug aimed at thrombolytic dously boosted diffusion MRI, then still minutes) and motion artifacts became therapy for acute stroke patients. Clearly, essentially a pure research tool, by attracting history. The setup was also installed at diffusion MRI could not have arrived at a clinicians and convincing manufacturers to Harvard Medical School in Boston where the better time, and this coincidental match improve their systems. This move to the first patients suffering from acute stroke became a milestone in the history of the clinical field became possible after my (within 3–6 h of onset) were scanned with management of acute stroke patients. encounter with Robert Turner at the whole brain diffusion MRI in just a few National Institutes of Health (NIH) in Beth- seconds. The results immediately and esda, where I had just moved. Dr Turner directly confirmed Dr. Moseley’s observa- The wirings of the brain was an expert on EPI (Echo-Planar Imaging) tions on cat brains: Water diffusion was MRI. With this technique, MRI images could found to be decreased in the infarcted Michael Moseley’s group had made another be acquired as a snapshot in just a fraction areas, where dying brain cells undergo great discovery: In the diffusion images, of a second, virtually freezing patient swelling through cytotoxic edema, clearly contrast seemed to change according to motion: We were thus able to obtain the first highlighting those areas as bright spots, the spatial direction of the diffusion “clean” diffusion images using EPI (Turner while most of the time, standard MRI measurement in white matter (spinal cord et al, 1990). This endeavor was not that images (as well as CT scans) would not and brain). Water diffusion in white matter simple, however, as we had to obtain show any clear sign of abnormality fibers was anisotropic, faster in the direction

ª 2014 The Author EMBO Molecular Medicine Vol 6 |No5 | 2014 571 EMBO Molecular Medicine Diffusion MRI: what water tells us about the brain Denis Le Bihan

of the fibers and slower perpendicularly to proposed, but we always manage to find changes in the neural tissue microstructure them (and no one appeared aware of it until some piece of experimental evidence that to which diffusion MRI is exquisitely sensi- that time). With Philippe Douek, a French questions those models. I have recently tive. Such activation-driven structural student working with me at the NIH, we reviewed these inconsistencies and events, for example cell swelling, have been suggested that this feature could be used to suggested that, beside mechanical or reported in many instances in the literature. determine and map the orientation of white geometric constraints (such as cell As cell swelling in tissues has been shown matter fibers in the brain, assuming the membranes) hindering water diffusion, the to result in a water diffusion decrease direction of the fibers was parallel to the physical structure of water networks in detectable with MRI, we have hypothesized direction with the fastest diffusion. The first tissues, especially close to membranes, that the slowdown in diffusion observed attempt was very crude, with diffusion being might play a role (Le Bihan & Johansen- during neuronal activation could reflect cell measured along two directions only, but the Berg, 2012). The presence (or the amount) swelling (probably at the dendrite and spine concept of white matter fiber orientation of structured water in cells is in itself a levels) occurring within the activated corti- color mapping was born and a proof of prin- subject of great controversy among physi- cal ribbon. Based on this “electromechani- ciple provided (Douek et al, 1991). Progress cists and biologists, and we should be cal coupling,” the thought occurred to me from those basic images to the gorgeous prepared for yet more exciting years of that neural cells could perhaps be seen as fiber tract 3D displays that now make up the brainstorming and great workshops. I piezoelectric sensors: Variations in cell covers of journals and anatomy textbooks, believe we have largely underestimated the shape should, in return, induce cell depolar- implied a big step, which was made possible importance of water in biology, from protein ization, potentially allowing a very fast, by my encounter with Peter Basser in 1990. and membrane dynamics to cell physiology. non-synaptic transmission mechanism Peter was working at NIH on ionic fluxes in This emerging research area should also within neural clusters of the cortical ribbon. tissues. Peter quickly came up with the view greatly benefit from diffusion MRI. Indeed, dynamic changes in neuronal struc- that a better way to deal with anisotropic ture (especially the dendritic spines) are diffusion was to switch to a tensor formalism now thought to play an important role in to properly determine the true direction of Imaging brain function the functioning of such cell clusters, as envi- the highest diffusivity. The problem was to sioned by Crick (Crick, 1982) and even determine each of the terms of the diffusion Another important possible application of Ramon y Cajal (Ramon y Cajal, 1899): “The tensor with diffusion MRI. After some brain- diffusion MRI is the measurement of brain state of activity would correspond to the storming, Peter and I devised a solution in activity. Functional has swelling and elongation of the [dendritic] 1992, which we published and patented become an essential means to study the spines, and the resting state (sleep or inac- under the name of diffusion tensor imaging brain and the mind. Thus far, positron emis- tivity) to their retraction.” Diffusion MRI (DTI; Basser et al, 1994). Experiments were sion tomography (PET) and current func- has the potential to address such questions first conducted on vegetables with fibers, tional MRI (based on the Blood Oxygen and might allow us to further our under- and soon after white matter fiber orientation Level Dependant or BOLD contrast mecha- standing of the biophysical mechanisms could be obtained on a pixel-by-pixel basis nism) have relied on the principle that associated with neuronal activation. within the whole brain in vivo, completely neuronal activation and blood flow are non-invasively and in just a few minutes. coupled through metabolism, and brain acti- Algorithms were developed at the end of the vation can be indirectly imaged through vari- Perspectives and conclusions 1990s to connect those pixels together, ations in local blood flow. With diffusion resulting in the world’s first 3D representa- MRI, a new paradigm has emerged whereby In summary, diffusion MRI has the potential tions of the fiber bundles (with very colorful we can look at brain activity through the to provide, non-invasively and in vivo, infor- representations of the “white” matter) observation of water molecular diffusion. In mation on the cellular organization of the within the human brain. collaboration with my colleagues from Kyoto brain cortex, the connections between University, we have shown that water diffu- regions and the underlying activity. Still, the sion is indeed modulated by brain activity exact mechanisms governing water diffusion Water, the forgotten biological molecule (Le Bihan et al, 2006). The diffusion signal processes in tissues, notably in the brain, response is characterized by a sharp peak, remain unclear. Future research should aim Once back in France a few years later, my faster than the indirect hemodynamic at gathering diffusion data at the individual priority was to understand the basic mecha- response (increased in blood flow) observed and neuron cluster scale to better nisms of water diffusion in biological with BOLD functional MRI imaging (Aso understand water diffusion behavior in tissues, which underlie what we visualize et al, 2009). The diffusion response persists neural tissues. Indeed, a more profound with diffusion MRI. This is a huge and after inhibition of neurovascular coupling understanding of such processes is necessary certainly not a simple endeavor. We still (which suppresses the BOLD fMRI response) to develop the application of diffusion MRI cannot clearly explain why diffusion and shares the features of the underlying further and to directly obtain information on decreases so much in brain acute ischemia, neuronal response (Tsurugizawa et al, tissue microstructure. Access to such micro- how cell swelling leads to decreased diffu- 2013). This “diffusion fMRI” approach thus and mesoscopic scales will benefit from the sion, or precisely why diffusion anisotropy appears to be a paradigm shift in the way we unique ultra-high magnetic field MRI occurs in white matter. Some relevant and visualize brain activity, and more directly systems we have assembled in our institute often sophisticated models have been linked to neuronal function, pointing out to dedicated to ultra-high field MRI (NeuroSpin,

572 EMBO Molecular Medicine Vol 6 |No5 | 2014 ª 2014 The Author Denis Le Bihan Diffusion MRI: what water tells us about the brain EMBO Molecular Medicine

Saclay, France), in particular its preclinical specific cellular arrangements in space along Jelescu I, Ciobanu L, Geffroy F, Marquet P, Le 17.2-T MRI system and an experimental clin- the brain cortex might establish a “neural Bihan D (2014) Effects of hypotonic stress ical 11.7-T MRI system (available from code,” a set of basic functions from which, and ouabain on the apparent diffusion 2015), both unique in the world. Using once connected together on a timely coefficient of water at cellular and tissue magnetic resonance microscopy (MRM), we manner, higher order functions could levels in the Aplysia. NMR Biomed 27: have shown that water diffusion measured emerge. 280 – 290. inside isolated neuronal soma and in the While MRI is merely a means to visualize Le Bihan D (2003) Looking into the functional region of cell bodies of the Aplysia buccal diffusion, molecular diffusion itself (of water architecture of the brain with diffusion MRI. ganglia under exposure to ouabain, resulted or other molecules, metabolites, neurotrans- Nat Rev Neurosci 4: 469 – 480 in a water diffusion increase inside isolated mitters) has a life of its own and remains a Le Bihan D, Breton E (1985) Imagerie de , but a decrease at the tissue level powerful, genuine multidisciplinary concept diffusion in vivo par résonance magnétique (Jelescu, 2014). Such opposite findings at our disposal to understand cell physiology nucléaire. C R Acad Sci ,T.301, Série II, cannot be explained with current “mechanis- and life. After all, all biological processes 1109 – 1112 tic” tissue diffusion models. The scenario require molecules to interact, whether for Le Bihan D, Breton E, Lallemand D, Grenier P, involving a layer of water molecules bound DNA replication, RNA transcription, protein Cabanis E, Laval Jeantet M (1986) MR Imaging to the inflating cell membrane surface could translation, protein and enzyme activity, of intravoxel incoherent motions: application to conciliate this apparent discrepancy. cross-membrane transport, and so on. For diffusion and perfusion in neurologic disorders. The potential of diffusion MRI to probe molecules to interact, however, they must Radiology 161: 401 – 407 human brain connectivity has attracted first meet: Diffusion appears to be the Le Bihan D, Johansen-Berg H (2012) Diffusion MRI worldwide interest and is now widely used universal process through which this occurs. at 25: exploring brain tissue structure and in clinical practice. Recent results from the In a sense, diffusion rates set the speed limit function. Neuroimage 61: 324 – 341 European FP7 CONNECT project (Assaf for life, just as the speed of light sets the Le Bihan D, Urayama SI, Aso T, Hanakawa T, et al, 2013) and the Human Connectome limit in the physical world. Indeed, diffusion Fukuyama H (2006) Direct and fast detection of Project (HCP, USA) have clearly underlined MRI is just emerging from adolescence and neuronal activation in the human brain with the enormous potential of this approach, has a great future. diffusion MRI. Proc Natl Acad Sci U S A 103: yielding insight into how brain connections 8263 – 8268 underlie function and opening up new lines Conflict of interest Moseley ME, Cohen Y, Mintorovitch J (1990) Early of inquiry for human neuroscience and The author declares that he has no conflict of detection of regional cerebral ischemic injury in brain dysfunction in aging, mental health interest. cats: evaluation of diffusion and T2-weighted disorders, addiction and neurological MRI and spectroscopy. Magn Reson Med 14: disease (Le Bihan & Johansen-Berg, 2012). References 330 – 346 The increased spatial resolution expected Aso T, Urayama S, Poupon C, Sawamoto N, Ramon y Cajal S. (1899). Textura del Sistema with the NeuroSpin 11.7-T MRI scanner Fukuyama H, Le Bihan D (2009) An intrinsic Nervioso del Hombre y de los Vertebrados. could allow for the detection of smaller diffusion response function for analyzing Madrid: Imprenta y Librería de Nicolás Moya, white matter bundles. Of special interest are diffusion functional MRI time series. Tome II the short connections between adjacent Neuroimage 47: 1487 – 1495 Tsurugizawa T, Ciobanu L, Le Bihan D (2013) cortical regions, or even within cortical Assaf Y, Alexander DC, Jones DK, Bizzi A, Behrens Water diffusion in brain cortex closely tracks regions. Similarly, there are also hints that TE, Clark CA, Cohen Y, Dyrby TB, Huppi PS, underlying neuronal activity. Proc Natl Acad Sci diffusion imaging could become a very Knoesche TR et al,(2013) The CONNECT USA110: 11636 – 11641 important tool to probe the functional archi- project: combining macro- and micro-structure. Turner R, Le Bihan D, Maier J, Vavrek R, Hedges LK, tecture of the brain cortex. It has been long Neuroimage 80: 273 – 829 Pekar J (1990) Echo-planar imaging of established that cortical cells are not orga- Basser PJ, Mattiello J, Le Bihan D (1994)MR intravoxel incoherent motions. Radiology 177: nized along the brain cortex in a random, diffusion tensor spectroscopy and imaging. 407 – 414 homogeneous way. Cells are rather well Biophys J 66: 259 – 267 Warach S, Chien D, Li W, Ronthal M, Edelman RR characterized (in terms of size, geometry, Crick F (1982) Do dentritic spines twitch? Tins 5: (1992) Fast magnetic resonance receptor type and density) and arranged in 44 – 47 diffusion-weighted imaging of acute human specific patterns, identified by Brodmann in Douek P, Turner R, Pekar J, Patronas NJ, Le Bihan stroke. Neurology 42: 1717 – 1723 1908. The Brodmann areas are deemed to be D(1991) MR color mapping of myelin fiber associated with specific brain functions, and orientation. J Comput Assist Tomogr 15: most functional neuroimaging studies have 923 – 929 relied on the seminal classification of Brod- Einstein A (1905) Über die von der License: This is an open access article under the mann to report the location of activated molekularkinetischen Theorie der Wärme terms of the Creative Commons Attribution 4.0 regions. With diffusion MRI, one may envi- geforderte Bewegung von in ruhenden License, which permits use, distribution and sion that cytoarchitectonic areas could be Flüssigkeiten suspendierten Teilchen. Ann Phys reproduction in any medium, provided the original determined on an individual basis. Such 322: 549 – 560 work is properly cited.

ª 2014 The Author EMBO Molecular Medicine Vol 6 |No5 | 2014 573