Using light and X-ray scattering to untangle complex neuronal orientations and validate diffusion MRI.

Disentangling human brain connectivity requires an accurate description of nerve fiber trajectories, unveiled via detailed mapping of axonal orientations. However, this is challenging because axons can cross one another on a micrometer scale. Diffusion magnetic resonance imaging (dMRI) can be used to infer axonal connectivity because it is sensitive to axonal alignment, but it has limited spatial resolution and specificity. Scattered light imaging (SLI) and small-angle X-ray scattering (SAXS) reveal axonal orientations with microscopic resolution and high specificity, respectively. Here, we apply both scattering techniques on the same samples and cross-validate them, laying the groundwork for ground-truth axonal orientation imaging and validating dMRI. We evaluate brain regions that include unidirectional and crossing fibers in human and vervet monkey brain sections. SLI and SAXS quantitatively agree regarding in-plane fiber orientations including crossings, while dMRI agrees in the majority of voxels with small discrepancies. We further use SAXS and dMRI to confirm theoretical predictions regarding SLI determination of through-plane fiber orientations. Scattered light and X-ray imaging can provide quantitative micrometer 3D fiber orientations with high resolution and specificity, facilitating detailed investigations of complex fiber architecture in the animal and human brain.

Automated computation of nerve fibre inclinations from 3D polarised light imaging measurements of brain tissue.

The method 3D polarised light imaging (3D-PLI) measures the birefringence of histological brain sections to determine the spatial course of nerve fibres (myelinated axons). While the in-plane fibre directions can be determined with high accuracy, the computation of the out-of-plane fibre inclinations is more challenging because they are derived from the amplitude of the birefringence signals, which depends e.g. on the amount of nerve fibres. One possibility to improve the accuracy is to consider the average transmitted light intensity (transmittance weighting). The current procedure requires effortful manual adjustment of parameters and anatomical knowledge. Here, we introduce an automated, optimised computation of the fibre inclinations, allowing for a much faster, reproducible determination of fibre orientations in 3D-PLI. Depending on the degree of myelination, the algorithm uses different models (transmittance-weighted, unweighted, or a linear combination), allowing to account for regionally specific behaviour. As the algorithm is parallelised and GPU optimised, it can be applied to large data sets. Moreover, it only uses images from standard 3D-PLI measurements without tilting, and can therefore be applied to existing data sets from previous measurements. The functionality is demonstrated on unstained coronal and sagittal histological sections of vervet monkey and rat brains.

Additional fiber orientations in the sagittal stratum-noise or anatomical fine structure?

The sagittal stratum is a prominent and macroscopically clearly visible white-matter structure within occipital and parietal lobes with a highly organized structure of parallel fibers running in rostro-caudal direction. Apart from the major tract running through, i.e., the optic radiation, the source and arrangement of other fibers within the sagittal stratum is only partially understood. Recent diffusion imaging studies in-vivo suggest additional minor fiber directions, perpendicular to the major rostro-caudal ones, but the spatial resolution does not allow to resolve them, and to unambiguously distinguish it from noise. Taking this previous evidence as motivation, the present study used 3D polarized light imaging (3D-PLI) for micrometer resolution analysis of nerve fibers in postmortem specimens of a vervet monkey brain. The analysis of coronal occipital and parietal sections revealed that the sagittal stratum consisted of an external and an internal layer, which are joined and crossed by fibers from the surrounding white matter and the tapetum. Fibers from different parietal and occipital regions entered the sagittal stratum in the dorsal, ventral or middle sector, as solid large bundles or as several small fiber aggregations. These patterns were remarkably similar to published results of tracer experiments in macaques. Taking this correspondence as external validation of 3D-PLI enabled translation to the human brain, where a similarly complex fiber architecture within the sagittal stratum could be exemplified in a human hemisphere in our study. We thus argue in favor of a dedicated fiber microstructure within the sagittal stratum as a correlate of the additional fiber directions typically seen in in-vivo diffusion imaging studies.

Scatterometry Measurements With Scattered Light Imaging Enable New Insights Into the Nerve Fiber Architecture of the Brain.

The correct reconstruction of individual (crossing) nerve fibers is a prerequisite when constructing a detailed network model of the brain. The recently developed technique Scattered Light Imaging (SLI) allows the reconstruction of crossing nerve fiber pathways in whole brain tissue samples with micrometer resolution: the individual fiber orientations are determined by illuminating unstained histological brain sections from different directions, measuring the transmitted scattered light under normal incidence, and studying the light intensity profiles of each pixel in the resulting image series. So far, SLI measurements were performed with a fixed polar angle of illumination and a small number of illumination directions, providing only an estimate of the nerve fiber directions and limited information about the underlying tissue structure. Here, we use a display with individually controllable light-emitting diodes to measure the full distribution of scattered light behind the sample (scattering pattern) for each image pixel at once, enabling scatterometry measurements of whole brain tissue samples. We compare our results to coherent Fourier scatterometry (raster-scanning the sample with a non-focused laser beam) and previous SLI measurements with fixed polar angle of illumination, using sections from a vervet monkey brain and human optic tracts. Finally, we present SLI scatterometry measurements of a human brain section with 3 µm in-plane resolution, demonstrating that the technique is a powerful approach to gain new insights into the nerve fiber architecture of the human brain.

Distribution and orientation of nerve fibers and myelin assembly in a brain section retrieved by small-angle neutron scattering.

The structural connectivity of the brain has been addressed by various imaging techniques such as diffusion weighted magnetic resonance imaging (DWMRI) or specific microscopic approaches based on histological staining or label-free using polarized light (e.g., three-dimensional Polarized Light Imaging (3D-PLI), Optical Coherence Tomography (OCT)). These methods are sensitive to different properties of the fiber enwrapping myelin sheaths i.e. the distribution of myelin basic protein (histology), the apparent diffusion coefficient of water molecules restricted in their movements by the myelin sheath (DWMRI), and the birefringence of the oriented myelin lipid bilayers (3D-PLI, OCT). We show that the orientation and distribution of nerve fibers as well as myelin in thin brain sections can be determined using scanning small angle neutron scattering (sSANS). Neutrons are scattered from the fiber assembly causing anisotropic diffuse small-angle scattering and Bragg peaks related to the highly ordered periodic myelin multilayer structure. The scattering anisotropy, intensity, and angular position of the Bragg peaks can be mapped across the entire brain section. This enables mapping of the fiber and myelin distribution and their orientation in a thin brain section, which was validated by 3D-PLI. The experiments became possible by optimizing the neutron beam collimation to highest flux and enhancing the myelin contrast by deuteration. This method is very sensitive to small microstructures of biological tissue and can directly extract information on the average fiber orientation and even myelin membrane thickness. The present results pave the way toward bio-imaging for detecting structural aberrations causing neurological diseases in future.

Scattered Light Imaging: Resolving the substructure of nerve fiber crossings in whole brain sections with micrometer resolution.

For developing a detailed network model of the brain based on image reconstructions, it is necessary to spatially resolve crossing nerve fibers. The accuracy hereby depends on many factors, including the spatial resolution of the imaging technique. 3D Polarized Light Imaging (3D-PLI) allows the three-dimensional reconstruction of nerve fiber tracts in whole brain sections with micrometer in-plane resolution, but leaves uncertainties in pixels containing crossing fibers. Here we introduce Scattered Light Imaging (SLI) to resolve the substructure of nerve fiber crossings. The measurement is performed on the same unstained histological brain sections as in 3D-PLI. By illuminating the brain sections from different angles and measuring the transmitted (scattered) light under normal incidence, light intensity profiles are obtained that are characteristic for the underlying brain tissue structure. We have developed a fully automated evaluation of the intensity profiles, allowing the user to extract various characteristics, like the individual directions of in-plane crossing nerve fibers, for each image pixel at once. We validate the reconstructed nerve fiber directions against results from previous simulation studies, scatterometry measurements, and fiber directions obtained from 3D-PLI. We demonstrate in different brain samples (human optic tracts, vervet monkey brain, rat brain) that the 2D fiber directions can be reliably reconstructed for up to three crossing nerve fiber bundles in each image pixel with an in-plane resolution of up to 6.5 µm. We show that SLI also yields reliable fiber directions in brain regions with low 3D-PLI signals coming from regions with a low density of myelinated nerve fibers or out-of-plane fibers. This makes Scattered Light Imaging a promising new imaging technique, providing crucial information about the organization of crossing nerve fibers in the brain.

Anatomy of nerve fiber bundles at micrometer-resolution in the vervet monkey visual system.

Although the primate visual system has been extensively studied, detailed spatial organization of white matter fiber tracts carrying visual information between areas has not been fully established. This is mainly due to the large gap between tracer studies and diffusion-weighted MRI studies, which focus on specific axonal connections and macroscale organization of fiber tracts, respectively. Here we used 3D polarization light imaging (3D-PLI), which enables direct visualization of fiber tracts at micrometer resolution, to identify and visualize fiber tracts of the visual system, such as stratum sagittale, inferior longitudinal fascicle, vertical occipital fascicle, tapetum and dorsal occipital bundle in vervet monkey brains. Moreover, 3D-PLI data provide detailed information on cortical projections of these tracts, distinction between neighboring tracts, and novel short-range pathways. This work provides essential information for interpretation of functional and diffusion-weighted MRI data, as well as revision of wiring diagrams based upon observations in the vervet visual system.

Ex vivo visualization of the trigeminal pathways in the human brainstem using 11.7T diffusion MRI combined with microscopy polarized light imaging.

Classic anatomical atlases depict a contralateral hemispheral representation of each side of the face. Recently, however, a bilateral projection of each hemiface was hypothesized, based on animal studies that showed the coexistence of an additional trigeminothalamic tract sprouting from the trigeminal principal sensory nucleus that ascends ipsilaterally. This study aims to provide an anatomical substrate for the hypothesized bilateral projection. Three post-mortem human brainstems were scanned for anatomical and diffusion magnetic resonance imaging at 11.7T. The trigeminal tracts were delineated in each brainstem using track density imaging (TDI) and tractography. To evaluate the reconstructed tracts, the same brainstems were sectioned for polarized light imaging (PLI). Anatomical 11.7T MRI shows a dispersion of the trigeminal tract (tt) into a ventral and dorsal portion. This bifurcation was also seen on the TDI maps, tractography results and PLI images of all three specimens. Referring to a similar anatomic feature in primate brains, the dorsal and ventral tracts were named the dorsal and ventral trigeminothalamic tract (dtt and vtt), respectively. This study shows that both the dtt and vtt are present in humans, indicating that each hemiface has a bilateral projection, although the functional relevance of these tracts cannot be determined by the present anatomical study. If both tracts convey noxious stimuli, this could open up new insights into and treatments for orofacial pain in patients.

Direct Visualization and Mapping of the Spatial Course of Fiber Tracts at Microscopic Resolution in the Human Hippocampus.

While hippocampal connectivity is essential to normal memory function, our knowledge of human hippocampal circuitry is largely inferred from animal studies. Using polarized light microscopy at 1.3 µm resolution, we have directly visualized the 3D course of key medial temporal pathways in 3 ex vivo human hemispheres and 2 ex vivo vervet monkey hemispheres. The multiple components of the perforant path system were clearly identified: Superficial sheets of fibers emanating from the entorhinal cortex project to the presubiculum and parasubiculum, intermixed transverse and longitudinal angular bundle fibers perforate the subiculum and then project to the cornu ammonis (CA) fields and dentate molecular layer, and a significant alvear component runs from the angular bundle to the CA fields. From the hilus, mossy fibers localize to regions of high kainate receptor density, and the endfolial pathway, mostly investigated in humans, merges with the Schaffer collaterals. This work defines human hippocampal pathways underlying mnemonic function at an unprecedented resolution.

Estimating Fiber Orientation Distribution Functions in 3D-Polarized Light Imaging.

Research of the human brain connectome requires multiscale approaches derived from independent imaging methods ideally applied to the same object. Hence, comprehensible strategies for data integration across modalities and across scales are essential. We have successfully established a concept to bridge the spatial scales from microscopic fiber orientation measurements based on 3D-Polarized Light Imaging (3D-PLI) to meso- or macroscopic dimensions. By creating orientation distribution functions (pliODFs) from high-resolution vector data via series expansion with spherical harmonics utilizing high performance computing and supercomputing technologies, data fusion with Diffusion Magnetic Resonance Imaging has become feasible, even for a large-scale dataset such as the human brain. Validation of our approach was done effectively by means of two types of datasets that were transferred from fiber orientation maps into pliODFs: simulated 3D-PLI data showing artificial, but clearly defined fiber patterns and real 3D-PLI data derived from sections through the human brain and the brain of a hooded seal.

Effect of Intravenous Thrombolysis on the Time Course of the Apparent Diffusion Coefficient in Acute Middle Cerebral Artery Infarction.

BACKGROUND AND PURPOSE: To examine the possible effects of intravenous thrombolysis on the time course of the apparent diffusion coefficient in the patients with acute middle cerebral artery infarct. METHODS: Serial MRI data with all in all 190 MR examinations including diffusion-weighted imaging (DWI), apparent diffusion coefficient map (ADC map) and T2 -weighted imaging (T2 w) of 74 patients with initial intravenous thrombolysis (study group; N = 37) or conservative stroke treatment (control group; N = 37) were retrospectively analyzed. A trend function was fitted to the relative values (rADC, rDWI, rT2 w) to model an objective, general time course. RESULTS: Relative apparent diffusion coefficient (rADC) decreased in both groups to a minimum about 15 hours after symptom onset. Afterwards rADC increased faster in the study group and reached pseudonormalization 5 ± 2 days after symptom onset. In the control group pseudonormalization was determined later at 7 ± 6 days after symptom onset. After pseudonormalization rADC continued to increase in both groups. CONCLUSION: rADC pseudonormalization occurred by trend earlier in the study group. Therefore, intravenous thrombolysis seems to have an effect on the time course of ADC, which is likely to be due to earlier cerebral reperfusion after thrombolysis. In addition, initial stroke treatment as thrombolysis should be considered in radiological rating of stroke MRI time course.

Target sites for transcallosal fibers in human visual cortex - A combined diffusion and polarized light imaging study.

Transcallosal fibers of the visual system have preferential target sites within the occipital cortex of monkeys. These target sites coincide with vertical meridian representations of the visual field at borders of retinotopically defined visual areas. The existence of preferential target sites of transcallosal fibers in the human brain at the borders of early visual areas was claimed, but controversially discussed. Hence, we studied the distribution of transcallosal fibers in human visual cortex, searching for an organizational principle across early and higher visual areas. In-vivo high angular resolution diffusion imaging data of 28 subjects were used for probabilistic fiber tracking using a constrained spherical deconvolution approach. The fiber architecture within the target sites was analyzed at microscopic resolution using 3D polarized light imaging in a post-mortem human hemisphere. Fibers through a seed in the splenium of the corpus callosum reached the occipital cortex via the forceps major and the tapetum. We found target sites of these transcallosal fibers at borders of cytoarchitectonically defined occipital areas not only between early visual areas V1 and V2, V3d and V3A, and V3v and V4, but also between higher extrastriate areas, namely V4 (ventral) and posterior fusiform area FG1 as well as posterior fusiform area FG2 and lateral occipital cortex. In early visual areas, the target sites coincided with the vertical meridian representations of retinotopic maps. The spatial arrangement of the fibers in the 'border tuft' region at the V1/V2 border was found to be more complex than previously observed in myeloarchitectonic studies. In higher visual areas, our results provided additional evidence for a hemi-field representation in human area V4. The fiber topography in posterior fusiform gyrus indicated that additional retinotopic areas might exist, located between the recently identified retinotopic representations phPITv/phPITd and PHC-1/PHC-2 in lateral occipital cortex and parahippocampal gyrus.

Automatic identification of gray and white matter components in polarized light imaging.

Polarized light imaging (PLI) enables the visualization of fiber tracts with high spatial resolution in microtome sections of postmortem brains. Vectors of the fiber orientation defined by inclination and direction angles can directly be derived from the optical signals employed by PLI analysis. The polarization state of light propagating through a rotating polarimeter is varied in such a way that the detected signal of each spatial unit describes a sinusoidal signal. Noise, light scatter and filter inhomogeneities, however, interfere with the original sinusoidal PLI signals, which in turn have direct impact on the accuracy of subsequent fiber tracking. Recently we showed that the primary sinusoidal signals can effectively be restored after noise and artifact rejection utilizing independent component analysis (ICA). In particular, regions with weak intensities are greatly enhanced after ICA based artifact rejection and signal restoration. Here, we propose a user independent way of identifying the components of interest after decomposition; i.e., components that are related to gray and white matter. Depending on the size of the postmortem brain and the section thickness, the number of independent component maps can easily be in the range of a few ten thousand components for one brain. Therefore, we developed an automatic and, more importantly, user independent way of extracting the signal of interest. The automatic identification of gray and white matter components is based on the evaluation of the statistical properties of the so-called feature vectors of each individual component map, which, in the ideal case, shows a sinusoidal waveform. Our method enables large-scale analysis (i.e., the analysis of thousands of whole brain sections) of nerve fiber orientations in the human brain using polarized light imaging.

A novel approach to the human connectome: ultra-high resolution mapping of fiber tracts in the brain.

Signal transmission between different brain regions requires connecting fiber tracts, the structural basis of the human connectome. In contrast to animal brains, where a multitude of tract tracing methods can be used, magnetic resonance (MR)-based diffusion imaging is presently the only promising approach to study fiber tracts between specific human brain regions. However, this procedure has various inherent restrictions caused by its relatively low spatial resolution. Here, we introduce 3D-polarized light imaging (3D-PLI) to map the three-dimensional course of fiber tracts in the human brain with a resolution at a submillimeter scale based on a voxel size of 100 µm isotropic or less. 3D-PLI demonstrates nerve fibers by utilizing their intrinsic birefringence of myelin sheaths surrounding axons. This optical method enables the demonstration of 3D fiber orientations in serial microtome sections of entire human brains. Examples for the feasibility of this novel approach are given here. 3D-PLI enables the study of brain regions of intense fiber crossing in unprecedented detail, and provides an independent evaluation of fiber tracts derived from diffusion imaging data.

Diffusion tensor magnetic resonance imaging of the breast: a pilot study.

OBJECTIVES: Diffusion-weighted MR imaging has shown diagnostic value for differential diagnosis of breast lesions. Diffusion tensor imaging (DTI) adds information about tissue microstructure by addressing diffusion direction. We have examined the diagnostic application of DTI of the breast. METHODS: A total of 59 patients (71 lesions: 54 malignant, 17 benign) successfully underwent prospective echo planar imaging-DTI (EPI-DTI) (1.5 T). First, diffusion direction both of parenchyma as well as lesions was assessed on parametric maps. Subsequently, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values were measured. Statistics included univariate (Mann-Whitney U test, receiver operating analysis) and multivariate (logistic regression analysis, LRA) tests. RESULTS: Main diffusion direction of parenchyma was anterior-posterior in the majority of cases (66.1%), whereas lesions (benign, malignant) showed no predominant diffusion direction in the majority of cases (23.9%). ADC values showed highest differences between benign and malignant lesions (P<0.001) with resulting area under the curve (AUC) of 0.899. FA values were lower in benign (interquartile range, IR, 0.14-0.24) compared to malignant lesions (IR 0.21-0.35, P<0.002) with an AUC of 0.751-0.770. Following LRA, FA did not prove to have incremental value for differential diagnosis over ADC values. CONCLUSIONS: Microanatomical differences between benign and malignant breast lesions as well as breast parenchyma can be visualized by using DTI.

High-resolution fiber tract reconstruction in the human brain by means of three-dimensional polarized light imaging.

Functional interactions between different brain regions require connecting fiber tracts, the structural basis of the human connectome. To assemble a comprehensive structural understanding of neural network elements from the microscopic to the macroscopic dimensions, a multimodal and multiscale approach has to be envisaged. However, the integration of results from complementary neuroimaging techniques poses a particular challenge. In this paper, we describe a steadily evolving neuroimaging technique referred to as three-dimensional polarized light imaging (3D-PLI). It is based on the birefringence of the myelin sheaths surrounding axons, and enables the high-resolution analysis of myelinated axons constituting the fiber tracts. 3D-PLI provides the mapping of spatial fiber architecture in the postmortem human brain at a sub-millimeter resolution, i.e., at the mesoscale. The fundamental data structure gained by 3D-PLI is a comprehensive 3D vector field description of fibers and fiber tract orientations - the basis for subsequent tractography. To demonstrate how 3D-PLI can contribute to unravel and assemble the human connectome, a multiscale approach with the same technology was pursued. Two complementary state-of-the-art polarimeters providing different sampling grids (pixel sizes of 100 and 1.6 µm) were used. To exemplarily highlight the potential of this approach, fiber orientation maps and 3D fiber models were reconstructed in selected regions of the brain (e.g., Corpus callosum, Internal capsule, Pons). The results demonstrate that 3D-PLI is an ideal tool to serve as an interface between the microscopic and macroscopic levels of organization of the human connectome.

Wallerian degeneration of pyramidal tract after paramedian pons infarct.

BACKGROUND: The intention of this study was the prospective analysis of Wallerian degeneration of the pyramidal tract after paramedian pons infarction. METHODS: Patients with paramedian pons infarct underwent MR imaging including diffusion tensor imaging at admission and got 1-3 MR scans up to 6 months of follow-up. Clinical scores and transcranial magnetic stimulation were acquired in the acute phase and 3-6 months later. The pyramidal tracts were manually segmented in fractional anisotropy (FA) color maps after coregistration of all MR datasets of each patient. FA as well as axial and radial diffusivity were measured in the volume of lesioned and contralateral pyramidal tracts distally to the ischemic lesion. RESULTS: From 11 patients studied, 7 developed Wallerian degeneration detected as statistically significant decrease in FA over time in the distal pyramidal tract. Wallerian degeneration could be detected at the earliest between the first and the third days after the onset of symptoms. A continuous decrease in FA and an increase in axial and radial diffusivity in degenerating pyramidal tracts over time were demonstrated. A significant correlation between NIHSS score on admission and the slope of relative axial diffusivity and a significant correlation between motor-evoked potential amplitudes of the arm on admission and the outcome relative FA was found. CONCLUSIONS: The initial MR image cannot predict the following Wallerian degeneration. However, the severity of motor disturbance and the motor-evoked potential of the arm on admission could be possible parameters to predict Wallerian degeneration. For estimation of Wallerian degeneration over time, at least 2 diffusion tensor imaging measurements have to be done at different time points.

Towards ultra-high resolution fibre tract mapping of the human brain - registration of polarised light images and reorientation of fibre vectors.

Polarised light imaging (PLI) utilises the birefringence of the myelin sheaths in order to visualise the orientation of nerve fibres in microtome sections of adult human post-mortem brains at ultra-high spatial resolution. The preparation of post-mortem brains for PLI involves fixation, freezing and cutting into 100-mum-thick sections. Hence, geometrical distortions of histological sections are inevitable and have to be removed for 3D reconstruction and subsequent fibre tracking. We here present a processing pipeline for 3D reconstruction of these sections using PLI derived multimodal images of post-mortem brains. Blockface images of the brains were obtained during cutting; they serve as reference data for alignment and elimination of distortion artefacts. In addition to the spatial image transformation, fibre orientation vectors were reoriented using the transformation fields, which consider both affine and subsequent non-linear registration. The application of this registration and reorientation approach results in a smooth fibre vector field, which reflects brain morphology. PLI combined with 3D reconstruction and fibre tracking is a powerful tool for human brain mapping. It can also serve as an independent method for evaluating in vivo fibre tractography.

Signal enhancement in polarized light imaging by means of independent component analysis.

Polarized light imaging (PLI) enables the evaluation of fiber orientations in histological sections of human postmortem brains, with ultra-high spatial resolution. PLI is based on the birefringent properties of the myelin sheath of nerve fibers. As a result, the polarization state of light propagating through a rotating polarimeter is changed in such a way that the detected signal at each measurement unit of a charged-coupled device (CCD) camera describes a sinusoidal signal. Vectors of the fiber orientation defined by inclination and direction angles can then directly be derived from the optical signals employing PLI analysis. However, noise, light scatter and filter inhomogeneities interfere with the original sinusoidal PLI signals. We here introduce a novel method using independent component analysis (ICA) to decompose the PLI images into statistically independent component maps. After decomposition, gray and white matter structures can clearly be distinguished from noise and other artifacts. The signal enhancement after artifact rejection is quantitatively evaluated in 134 histological whole brain sections. Thus, the primary sinusoidal signals from polarized light imaging can be effectively restored after noise and artifact rejection utilizing ICA. Our method therefore contributes to the analysis of nerve fiber orientation in the human brain within a micrometer scale.

Time course of diffusion imaging in acute brainstem infarcts.

PURPOSE: To study the time course of diffusion imaging at the lesion site in brainstem infarcts. MATERIALS AND METHODS: Sequential MR scans were acquired from 24 patients with brainstem infarcts. Diffusion-weighted images (DWI), T(2)-weighted images (T(2)w), maps of apparent diffusion coefficient, and maps of fractional anisotropy were generated from each MR scan. A trend function was fitted to these measurements to model an objective, general time course of the studied parameters. RESULTS: Apparent diffusion coefficient (ADC) continuously decreased over time until a transition time around 45 hours; afterwards a continuous increase took place. After the 14th day ADC reached values similar to the ADC of the intact contralateral side (pseudonormalization) and then further increased. Fractional anisotropy (FA) decreased continuously over 3 to 6 months. CONCLUSION: Times of transition and pseudonormalization of ADC were longer than described for territorial hemispheric infarcts and describe the acute to subacute phase of brainstem ischemia. In contrast, the continuous decline of FA over 3 to 6 months indicates a chronic process of change of histological structures in brainstem ischemia, and may be regarded as an indicator of the chronic phase.