Multimodal Characterization of the Late Effects of Traumatic Brain Injury: A Methodological Overview of the Late Effects of Traumatic Brain Injury Project

Abstract

Epidemiological studies suggest that a single moderate-to-severe traumatic brain injury (TBI) is associated with an increased risk of neurodegenerative disease, including Alzheimer's disease (AD) and Parkinson's disease (PD). Histopathological studies describe complex neurodegenerative pathologies in individuals exposed to single moderate-to-severe TBI or repetitive mild TBI, including chronic traumatic encephalopathy (CTE). However, the clinicopathological links between TBI and post-traumatic neurodegenerative diseases such as AD, PD, and CTE remain poorly understood. Here, we describe the methodology of the Late Effects of TBI (LETBI) study, whose goals are to characterize chronic post-traumatic neuropathology and to identify in vivo biomarkers of post-traumatic neurodegeneration. LETBI participants undergo extensive clinical evaluation using National Institutes of Health TBI Common Data Elements, proteomic and genomic analysis, structural and functional magnetic resonance imaging (MRI), and prospective consent for brain donation. Selected brain specimens undergo ultra-high resolution ex vivo MRI and histopathological evaluation including whole-mount analysis. Co-registration of ex vivo and in vivo MRI data enables identification of ex vivo lesions that were present during life. In vivo signatures of postmortem pathology are then correlated with cognitive and behavioral data to characterize the clinical phenotype(s) associated with pathological brain lesions. We illustrate the study methods and demonstrate proof of concept for this approach by reporting results from the first LETBI participant, who despite the presence of multiple in vivo and ex vivo pathoanatomic lesions had normal cognition and was functionally independent until her mid-80s. The LETBI project represents a multidisciplinary effort to characterize post-traumatic neuropathology and identify in vivo signatures of postmortem pathology in a prospective study.

Keywords: MRI; dementia; neurodegeneration; neuropathology; traumatic brain injury.

FIG. 1.

Overview of study design. The Late Effects of Traumatic Brain Injury (LETBI) study integrates in vivo data (left) and ex vivo data (right) to comprehensively characterize post-traumatic neurodegeneration. LETBI participants undergo extensive cognitive, behavioral, and neurological examinations. In addition, structural and functional magnetic resonance imaging (MRI) are performed and blood samples are drawn for proteomic and genomic analysis to identify in vivo biomarkers of post-traumatic neuropathology. Autopsied brain specimens undergo gross pathological analysis, histopathological analysis including Histelide and whole–mount analysis, and ultra-high resolution ex vivo MRI on 3 Tesla and 7 Tesla MRI scanners. Co-registration of ex vivo and in vivo MRI data enables determination of whether pathologically relevant ex vivo lesions were present during life. In vivo signatures of postmortem pathology are then correlated with cognitive and behavioral data to characterize the clinical phenotype(s) associated with pathological lesions. Color image is available online at www.liebertpub.com/neu

FIG. 2.

White matter lesion identification on in vivo 3 Tesla magnetic resonance imaging (MRI) and ex vivo 7 Tesla MRI. An in vivo 3 Tesla MRI performed at age 88, after the patient's fifth TBI, revealed bihemispheric corona radiata and internal capsule white matter hyperintensities, including a punctate lesion at the posterior aspect of the putamen (arrow). The same lesion was identified by ex vivo 7 Tesla MRI (arrow), which was performed on the formalin-fixed right hemisphere. A zoomed view of the lesion is shown in the right panel (arrow) to demonstrate the increased anatomic precision provided by ex vivo MRI (200 μm resolution) for delineating the borders of the lesion with respect to nearby anatomic structures. Cd, caudate; GP, globus pallidus; Ins, insula; Put, putamen.

FIG. 3.

Ex vivo 7 Tesla magnetic resonance imaging (MRI) reveals focal lesions not detected by in vivo 3 Tesla MRI. Axial T2*, proton density (PD), and flash20 images synthesized from the multi-echo flash sequence are shown at the level of the superior border of the thalamus. These three datasets reveal two lesions that were not visualized on the in vivo MRI dataset (lesions 3 and 4) and confirm the presence of a lesion that was seen in vivo (lesion 2). Lesion 2 is a periventricular hyperintensity in the frontal lobe, suggesting leukoaraiosis. Lesion 3 is a curvilinear hypointensity that traverses the superficial layers of cerebral cortex in the frontal operculum, suggesting hemosiderin deposition. Lesion 4 is a punctate hyperintensity in the periventricular white matter of the occipital lobe, consistent with leukoaraiosis or possibly chronic traumatic axonal injury. Zoomed views of each lesion are shown on the inset panels, with red arrows indicating the lesions. Color image is available online at www.liebertpub.com/neu

FIG. 4.

Correlative analysis of 7 Tesla ex vivo magnetic resonance imaging (MRI) and histopathology for the periventricular lesion. (A) A 7 Tesla ex vivo MRI coronal image of periventricular signal abnormality (red arrow) is shown at the level of the nucleus accumbens (Acc). The image shown here is a flash20 synthesized image from a multi-echo flash sequence. (B) A coronal histopathological section at the same neuroanatomic location is shown (red arrow), stained with amyloid-precursor protein and counterstained with hematoxylin. (C) Microscopic analysis of the periventricular region revealed disrupted axonal transport, as evidenced by positive amyloid precursor protein staining (red arrow). The degree to which chronic microvascular disease and chronic traumatic axonal injury contributed to this axonal pathology cannot be definitively determined, but the perivascular location suggests a vascular etiology. Neuroanatomic landmarks: CB, cingulum bundle; Put, putamen. Color image is available online at www.liebertpub.com/neu

FIG. 5.

Correlative analysis of 7 Tesla ex vivo magnetic resonance imaging (MRI) and histopathology for the curvilinear cortical lesion. Zoomed views of axial, sagittal and coronal flash20 images synthesized from the 7 Tesla multi-echo flash sequence reveal a curvilinear hypointense signal abnormality (red arrows) in the subpial region of the frontal operculum. Hematoxylin and eosin (H + E) staining of this region revealed atherosclerosis of a vessel within the subarachnoid space. Staining with glial fibrillary acidic protein (GFAP) and Perls' iron stain revealed gliosis and hemosiderin deposition, respectively, in the underlying cerebral cortex. The inset in the bottom right panel shows a high-powered view of hemosiderin-containing microglia. These findings suggest the possibility that a healed contusion accounts for the signal abnormality seen on ex vivo MRI. Color image is available online at www.liebertpub.com/neu

FIG. 6.

Ex vivo 3 Tesla diffusion tractography reveals regional white matter injury. Transcallosal fiber tracts generated using a corpus callosum seed region are shown from a left lateral perspective for the index patient (left) and a representative control subject who died of non-neurological causes (right). Tractography analysis revealed a decrease in the relative density of transcallosal fiber tracts in the parietal region, compared with the corresponding region of fiber tracts in the control dataset (white arrows). Fiber tracts are color-coded according to their direction (top right inset). Color image is available online at www.liebertpub.com/neu

FIG. 7.

Co-registration of ex vivo and in vivo magnetic resonance imaging (MRI) data. Precise anatomic alignment of in vivo and ex vivo MRI datasets from an individual patient is challenging because postmortem fixation causes nonlinear deformations. A deformation is seen by comparing a sagittal image from the in vivo T1 multi-echo Magnetization Prepared Rapid Acquisition Gradient Echo dataset (top left) with a sagittal image from the ex vivo multi-echo flash dataset (top right). We developed a combined volume- and surface-based co-registration technique to address this challenge and obtain precise voxel-to-voxel match between the in vivo and ex vivo datasets. The inflated cortical surfaces generated from the in vivo and ex vivo datasets are shown in the bottom left and bottom right panels, respectively. Given that the topology of the cortical folds is invariant, this topology can be used to initialize a biomechanical nonlinear deformation. Color image is available online at www.liebertpub.com/neu

FIG. 8.

Co-registration-based lesion localization on in vivo magnetic resonance imaging (MRI). Using combined volume- and surface-based co-registration (as shown in Fig. 7), the punctate hyperintense lesion seen in the occipital white matter on the ex vivo MRI dataset (red circle, right panel) is colocalized to its precise anatomic coordinates in the in vivo MRI dataset (red circle, left panel). The ex vivo MRI dataset used in the co-registration was the flash20 parameter map synthesized from the multi-echo flash 7 Tesla acquisition (200 μm spatial resolution), and the in vivo dataset was the T1 multi-echo Magnetization Prepared Rapid Acquisition Gradient Echo dataset (1 mm spatial resolution). Although no lesion was detected in the occipital white matter during the initial review of the in vivo MRI dataset, there appears to be a punctate hypointensity on the in vivo MRI dataset (red circle, left panel) that was only appreciated after the co-registration procedure. This observation suggests that ex vivo MRI may reveal lesions that were initially undetected on in vivo MRI. Color image is available online at www.liebertpub.com/neu

FIG. 9.

Targeted whole–mount histopathological analysis based on ex vivo magnetic resonance imaging (MRI) lesion localization. The punctate hyperintense lesion in the occipital white matter identified on ex vivo 7 Tesla (7T) MRI is first localized in the coronal plane (A, white arrow), because this is the plane in which the gross pathological specimen is cut into ∼1.5 cm thick slabs. During an audiovisual consensus teleconference, the precise anatomic coordinates of the lesion seen on ex vivo 7T MRI are compared with surface landmarks seen on the serial coronal slabs using high resolution photographs of the tissue slabs. The investigators of the Late Effects of Traumatic Brain Injury study select a slab for whole–mount analysis (B) based upon its correspondence with the coronal MRI image that contained the lesion. Whole–mount analysis is then performed on this coronal slab (C), enabling targeted histopathological analysis (D) of the lesion identified on ex vivo MRI. This histopathological analysis revealed enlarged perivascular spaces (arrows), corpora amylacea, and white matter rarefaction (asterisk). A single pathological process, ischemia, is the likely cause of these pathological findings. Color image is available online at www.liebertpub.com/neu