Roel Klaver

Grey matter damage in multiple sclerosis: a pathology perspective.

Over the past decade, immunohistochemical studies have provided compelling evidence that gray matter (GM) pathology in multiple sclerosis (MS) is extensive. Until recently, this GM pathology was difficult to visualize using standard magnetic resonance imaging (MRI) techniques. However, with newly developed MRI sequences, it has become clear that GM damage is present from the earliest stages of the disease and accrues with disease progression. GM pathology is clinically relevant, as GM lesions and/or GM atrophy were shown to be associated with MS motor deficits and cognitive impairment. Recent autopsy studies demonstrated significant GM demyelination and microglia activation. However, extensive immune cell influx, complement activation and blood-brain barrier leakage, like in WM pathology, are far less prominent in the GM. Hence, so far, the cause of GM damage in MS remains unknown, although several plausible underlying pathogenic mechanisms have been proposed. This paper provides an overview of GM damage in MS with a focus on its topology and histopathology.

What drives MRI-measured cortical atrophy in multiple sclerosis?

Background: Cortical atrophy, assessed with magnetic resonance imaging (MRI), is an important outcome measure in multiple sclerosis (MS) studies. However, the underlying histopathology of cortical volume measures is unknown. Objective: We investigated the histopathological substrate of MRI-measured cortical volume in MS using combined post-mortem imaging and histopathology. Methods: MS brain donors underwent post-mortem whole-brain in-situ MRI imaging. After MRI, tissue blocks were systematically sampled from the superior and inferior frontal gyrus, anterior cingulate gyrus, inferior parietal lobule, and superior temporal gyrus. Histopathological markers included neuronal, axonal, synapse, astrocyte, dendrite, myelin, and oligodendrocyte densities. Matched cortical volumes from the aforementioned anatomical regions were measured on the MRI, and used as outcomes in a nested prediction model. Results: Forty-five tissue blocks were sampled from 11 MS brain donors. Mean age at death was 68±12 years, post-mortem interval 4±1 hours, and disease duration 35±15 years. MRI-measured regional cortical volumes varied depending on anatomical region. Neuronal density, neuronal size, and axonal density were significant predictors of GM volume. Conclusions: In patients with long-standing disease, neuronal and axonal pathology are the predominant pathological substrates of MRI-measured cortical volume in chronic MS.

Increased cortical grey matter lesion detection in multiple sclerosis with 7 T MRI: a post-mortem verification study.

The relevance of cortical grey matter pathology in multiple sclerosis has become increasingly recognized over the past decade. Unfortunately, a large part of cortical lesions remain undetected on magnetic resonance imaging using standard field strength. In vivo studies have shown improved detection by using higher magnetic field strengths up to 7 T. So far, a systematic histopathological verification of ultra-high field magnetic resonance imaging pulse sequences has been lacking. The aim of this study was to determine the sensitivity of 7 T versus 3 T magnetic resonance imaging pulse sequences for the detection of cortical multiple sclerosis lesions by directly comparing them to histopathology. We obtained hemispheric coronally cut brain sections of 19 patients with multiple sclerosis and four control subjects after rapid autopsy and formalin fixation, and scanned them using 3 T and 7 T magnetic resonance imaging systems. Pulse sequences included T1-weighted, T2-weighted, fluid attenuated inversion recovery, double inversion recovery and T2*. Cortical lesions (type I-IV) were scored on all sequences by an experienced rater blinded to histopathology and clinical data. Staining was performed with antibodies against proteolipid protein and scored by a second reader blinded to magnetic resonance imaging and clinical data. Subsequently, magnetic resonance imaging images were matched to histopathology and sensitivity of pulse sequences was calculated. Additionally, a second unblinded (retrospective) scoring of magnetic resonance images was performed. Regardless of pulse sequence, 7 T magnetic resonance imaging detected more cortical lesions than 3 T. Fluid attenuated inversion recovery (7 T) detected 225% more cortical lesions than 3 T fluid attenuated inversion recovery (Z = 2.22, P < 0.05) and 7 T T2* detected 200% more cortical lesions than 3 T T2* (Z = 2.05, P < 0.05). Sensitivity of 7 T magnetic resonance imaging was influenced by cortical lesion type: 100% for type I (T2), 11% for type II (FLAIR/T2), 32% for type III (T2*), and 68% for type IV (T2). We conclude that ultra-high field 7 T magnetic resonance imaging more than doubles detection of cortical multiple sclerosis lesions, compared to 3 T magnetic resonance imaging. Unfortunately, (subpial) cortical pathology remains more extensive than 7 T magnetic resonance imaging can reveal.

Neuronal and Axonal Loss in Normal-Appearing Gray Matter and Subpial Lesions in Multiple Sclerosis

Abstract Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease of the CNS. Multiple sclerosis lesions include significant demyelination of the gray matter, which is thought to be a major contributor to both physical and cognitive impairment. Subpial (Type III) lesions are the most common demyelinated cortical lesions. We investigated neurodegenerative features of subpial lesions in cerebral cortex samples from 11 patients with MS and 6 nondemented non-MS controls. There were no significant differences in neuron and axon density between normally myelinated normal-appearing gray matter (NAGM) and Type III MS lesions. Neurons were 11.2% smaller in Type III lesions than in NAGM in the cingulate cortex only; Type III lesions contained 25.4% fewer NeuN-positive neurons compared with control cortex. Neurons in MS NAGM were 13.6% smaller than those in control cortex. Finally, the same regions, immunostained with anti-SMI312 antibodies, showed reduced axon densities in Type III lesions (−31.4%) and NAGM (−33.0%) compared with controls. In conclusion, both NAGM and Type III lesions showed neurodegenerative changes, but they had no consistent differences in neuronal and axonal alterations. This suggests that neurodegeneration in the cerebral cortex of patients with MS may be independent of cortical demyelination.

Postmortem validation of MRI cortical volume measurements in MS

Grey matter (GM) atrophy is a prominent aspect of multiple sclerosis pathology and an important outcome in studies. GM atrophy measurement requires accurate GM segmentation. Several methods are used in vivo for measuring GM volumes in MS, but assessing their validity in vivo remains challenging. In this postmortem study, we evaluated the correlation between postmortem MRI cortical volume or thickness and the cortical thickness measured on histological sections. Sixteen MS brains were scanned in situ using 3DT1‐weighted MRI and these images were used to measure regional cortical volume using FSL‐SIENAX, FreeSurfer, and SPM, and regional cortical thickness using FreeSurfer. Subsequently, cortical thickness was measured histologically in 5 systematically sampled cortical areas. Linear regression analyses were used to evaluate the relation between MRI regional cortical volume or thickness and histological cortical thickness to determine which postprocessing technique was most valid. After correction for multiple comparisons, we observed a significant correlation with the histological cortical thickness for FSL‐SIENAX cortical volume with manual editing (std. β = 0.345, adjusted R2 = 0.105, P = 0.005), and FreeSurfer cortical volume with manual editing (std. β = 0.379, adjusted R2 = 0.129, P = 0.003). In addition, there was a significant correlation between FreeSurfer cortical thickness with manual editing and histological cortical thickness (std. β = 0.381, adjusted R2 = 0.130, P = 0.003). The results support the use of FSL‐SIENAX and FreeSurfer in cases of severe MS pathology. Interestingly none of the methods were significant in automated mode, which supports the use of manual editing to improve the automated segmentation. Hum Brain Mapp 37:2223–2233, 2016.

The substrate of increased cortical FA in MS: A 7T post-mortem MRI and histopathology study.

Background: Using diffusion tensor imaging (DTI), it was previously found that demyelinated gray matter (GM) lesions have increased fractional anisotropy (FA) when compared to normal-appearing gray matter (NAGM) in multiple sclerosis (MS). The biological substrate underlying this FA change is so far unclear; both neurodegenerative changes and microglial activation have been proposed as causal contributors. Objective: To test the proposed hypothesis that microglia activation is responsible for increased FA in cortical GM lesions. Methods: We investigated post-mortem cortical DTI changes in hemispheric, coronally cut sections and investigated the underlying histopathology using immunohistochemistry. Results: Overall, there were few activated microglia/macrophages, and no difference between GM lesions and NAGM was observed. However, cell density was increased in GM lesions compared to NAGM (309.67 ± standard deviation (SD) 124.44 vs 249.95 ± SD 56.75, p = 0.002). Conclusion: FA increase was not due to lesional and non-lesional differences in microglia activation and/or proliferation. We found an increase in general cellular density without a notable difference in cellular size, that is, tissue compaction, as a possible alternative explanation.

Ultra-High-Field MRI Visualization of Cortical Multiple Sclerosis Lesions with T2 and T2*: A Postmortem MRI and Histopathology Study

At 7T, 2D multiecho spin-echo T2WI and 3D gradient-echo T2*WI were acquired from 27 formalin-fixed coronal hemispheric brain sections of 15 patients and 4 healthy controls. Proteolipid-stained tissue sections were matched to the corresponding MR images, and lesions were manually scored on both MR imaging sequences and tissue sections. The T2WI sequence detected slightly more lesions than the T2*WI sequence (28% and 16%). When histopathologic information (type, location) was revealed to the reader, the sensitivity went up to 84%. Many lesions are still missed prospectively. BACKGROUND AND PURPOSE: At 7T MR imaging, T2*-weighted gradient echo has been shown to provide high-resolution anatomic images of gray matter lesions. However, few studies have verified T2*WI lesions histopathologically or compared them with more standard techniques at ultra-high-field strength. This study aimed to determine the sensitivity of T2WI and T2*WI sequences for detecting cortical GM lesions in MS. MATERIALS AND METHODS: At 7T, 2D multiecho spin-echo T2WI and 3D gradient-echo T2*WI were acquired from 27 formalin-fixed coronal hemispheric brain sections of 15 patients and 4 healthy controls. Proteolipid-stained tissue sections (8 μm) were matched to the corresponding MR images, and lesions were manually scored on both MR imaging sequences (blinded to histopathology) and tissue sections (blinded to MR imaging). The sensitivity of MR imaging sequences for GM lesion types and white matter lesions was calculated. An unblinded retrospective scoring was also performed. RESULTS: If all cortical GM lesions were taken into account, the T2WI sequence detected slightly more lesions than the T2*WI sequence: 28% and 16%, respectively (P = .054). This difference disappeared when only intracortical lesions were considered. When histopathologic information (type, location) was revealed to the reader, the sensitivity went up to 84% (T2WI) and 85% (T2*WI) (not significant). Furthermore, the false-positive rate was 8.6% for the T2WI and 10.5% for the T2*WI sequence. CONCLUSIONS: There is no strong advantage of the T2*WI sequence compared with a conventional T2WI sequence in the detection of cortical lesions at 7T. Retrospectively, a high percentage of lesions could be detected with both sequences. However, many lesions are still missed prospectively. This could possibly be minimized with better a priori observer training.

Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis

Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models.

Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains

Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia–axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia–axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte–axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process.

Cortical pathology in multiple sclerosis detected by the T1/T2-weighted ratio from routine magnetic resonance imaging

In multiple sclerosis, neuropathological studies have shown widespread changes in the cerebral cortex. In vivo imaging is critical, because the histopathological substrate of most measurements is unknown.