Name: whole-brain segmentation and change-point analysis of anatomical brain mri&#8212;application in premanifest huntington's disease
Uploaded: 2018-06-09T13:00:00
Description: Watch this Scientific Journal Video about Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRIâ€”Application in Premanifest Huntington's Disease at JoVE.com

We thank the PREDICT-HD investigators, particularly, Dr. Hans Johnson and Dr. Jane S. Pauslen from University of Iowa, for their generosity in sharing the MRI data and constructive discussion on the data analysis and results.
This work is supported by NIH grants R21 NS098018, P50 NS16375, NS40068, R01 NS086888, R01 NS084957, P41 EB015909, P41 EB015909, R01 EB000975, R01 EB008171, and U01 NS082085.

1. Atlas-based Whole Brain Segmentation
<ol>
	<li>Data preparation
	<ol>
		<li>Convert three-dimensional (3D) T1-weighted images, typically acquired with MPRAGE (magnetization-prepared rapid gradient-echo) sequence, from vendor-specific DICOM (Digital Imaging and Communication) format to Analyzed format. Note that the cloud computation requires users&#39; data to be transferred to remote clusters. According to the Health Insurance Portability and Accountability Act (HIPPA), remove the patients&#39; per...

<ol>
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I., Trouve, A., Younes, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffeomorphometry+and+geodesic+positioning+systems+for+human+anatomy.">Diffeomorphometry and geodesic positioning systems for human anatomy.</a> Technology. 2, (2013).</li><li>Tang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+Parameter+Estimation+and+Segmentation+in+the+Multi-Atlas+Random+Orbit+Model.">Bayesian Parameter Estimation and Segmentation in the Multi-Atlas Random Orbit Model.</a> PLoS One. 8 (6), e65591 (2013).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Atlas+Segmentation+with+Joint+Label+Fusion.">Multi-Atlas Segmentation with Joint Label Fusion.</a> IEEE Trans Pattern Anal Mach Intell. 35 (3), 611-623 (2013).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resource+atlases+for+multi-atlas+brain+segmentations+with+multiple+ontology+levels+based+on+T1-weighted+MRI.">Resource atlases for multi-atlas brain segmentations with multiple ontology levels based on T1-weighted MRI.</a> Neuroimage. 125, 120-130 (2015).</li><li>Mori, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRICloud:+Delivering+High-Throughput+MRI+Neuroinformatics+as+Cloud-Based+Software+as+a+Service.">MRICloud: Delivering High-Throughput MRI Neuroinformatics as Cloud-Based Software as a Service.</a> Computing in Science &amp; Engineering. 18 (5), 21-35 (2016).</li><li>Wu, D., Ceritoglu, C., Miller, M. I., Mori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+estimation+of+patient+attributes+from+anatomical+MRI+based+on+multi-atlas+voting.">Direct estimation of patient attributes from anatomical MRI based on multi-atlas voting.</a> Neuroimage-Clinical. 12, 570-581 (2016).</li><li>Miller, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+Neurodegeneration+in+Alzheimer's+Disease+via+MRI+Based+Shape+Diffeomorphometry+and+High-Field+Atlasing.">Network Neurodegeneration in Alzheimer's Disease via MRI Based Shape Diffeomorphometry and High-Field Atlasing.</a> Front Bioeng Biotechnol. 3, 54 (2015).</li><li>Faria, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Content-based+image+retrieval+for+brain+MRI:+an+image-searching+engine+and+population-based+analysis+to+utilize+past+clinical+data+for+future+diagnosis.">Content-based image retrieval for brain MRI: an image-searching engine and population-based analysis to utilize past clinical data for future diagnosis.</a> Neuroimage Clin. 7, 367-376 (2015).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+order+and+pattern+of+brain+structural+MRI+changes+using+change-point+analysis+in+premanifest+Huntington's+disease.">Mapping the order and pattern of brain structural MRI changes using change-point analysis in premanifest Huntington's disease.</a> Hum Brain Mapp. , (2017).</li><li>Younes, L., Albert, M., Miller, M. I., Team, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+changepoint+times+of+medial+temporal+lobe+morphometric+change+in+preclinical+Alzheimer's+disease.">Inferring changepoint times of medial temporal lobe morphometric change in preclinical Alzheimer's disease.</a> Neuroimage Clin. 5, 178-187 (2014).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+critical+gestational+age+at+birth+that+alters+brain+development+in+preterm-born+infants+using+multi-modal+MRI.">Mapping the critical gestational age at birth that alters brain development in preterm-born infants using multi-modal MRI.</a> NeuroImage. 149, 33-43 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indexing+disease+progression+at+study+entry+with+individuals+at-risk+for+Huntington+disease.">Indexing disease progression at study entry with individuals at-risk for Huntington disease.</a> Am J Med Genet B Neuropsychiatr Genet. 156b (7), 751-763 (2011).</li><li>Vonsattel, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+classification+of+Huntington's+disease.">Neuropathological classification of Huntington's disease.</a> J Neuropathol Exp Neurol. 44 (6), 559-577 (1985).</li><li>Paulsen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Huntington's+disease+decades+before+diagnosis:+the+Predict-HD+study.">Detection of Huntington's disease decades before diagnosis: the Predict-HD study.</a> J Neurol Neurosurg Psychiatry. 79 (8), 874-880 (2008).</li><li>Paulsen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+manifest+Huntington's+disease+with+clinical+and+imaging+measures:+a+prospective+observational+study.">Prediction of manifest Huntington's disease with clinical and imaging measures: a prospective observational study.</a> Lancet Neurol. 13 (12), 1193-1201 (2014).</li><li>Djamanakova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+multiple+granularity+analysis+of+brain+MRI+data+for+individualized+image+analysis.">Tools for multiple granularity analysis of brain MRI data for individualized image analysis.</a> Neuroimage. 101, 168-176 (2014).</li><li>. A package for T1 volumetric analysis of MRIcloud output. R package version 0.0.1 Available from: <a target="_blank" href="https://github.com/bcaffo/MRIcloudT1volumetrics">https://github.com/bcaffo/MRIcloudT1volumetrics</a> (2017)</li><li>Aljabar, P., Heckemann, R. A., Hammers, A., Hajnal, J. V., Rueckert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-atlas+based+segmentation+of+brain+images:+atlas+selection+and+its+effect+on+accuracy.">Multi-atlas based segmentation of brain images: atlas selection and its effect on accuracy.</a> Neuroimage. 46 (3), 726-738 (2009).</li><li>Huntington Study Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unified+Huntington's+Disease+Rating+Scale:+reliability+and+consistency.">Unified Huntington's Disease Rating Scale: reliability and consistency.</a> Mov Disord. 11 (2), 136-142 (1996).</li><li>Liang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Cross-Protocol+Stability+of+a+Fully+Automated+Brain+Multi-Atlas+Parcellation+Tool.">Evaluation of Cross-Protocol Stability of a Fully Automated Brain Multi-Atlas Parcellation Tool.</a> PLoS One. 10 (7), e0133533 (2015).</li></ol>

As demonstrated in this paper, whole-brain segmentation of brain MRI can be conveniently achieved using our online platform MRICloud. T1-weighted MRI based volumetric marker has shown to be robust and sensitive to a range of neurodegenerative diseases1,2,3. The volumetric measures are used for various downstream analysis, such as mathematical modeling, and feature-selection and classification analysis to assist clinical diagnosi...

Magnetic resonance imaging (MRI) has substantially enhanced our ability to examine the brain anatomy and functions in neurodegenerative diseases1,2,3. T1-weighted structural MRI is one of most widely adopted imaging tools in routine clinical practice to assess the brain anatomy and related pathology. Quantitative analysis of the high-resolution T1-weighted images provides useful markers to measure anatomical changes during brain degeneration. In particular, segmentation based quantification approaches effectively reduces the image dimensionality from voxel level (on the order of (106)) to anatomical structural level ((102)) for high-throughput neuroinformatics4,5. Automated brain segmentation can be achieved using atlas-based methods6,7,8,9 that map the pre-defined anatomical labels from an atlas onto the patient images. Among the atlas-based methods, multi-atlas algorithms10,11,12,13,14 have yielded superior segmentation accuracy and robustness. Our group has developed a fully automated T1 multi-atlas segmentation pipeline, with advanced diffeomorphic image registration algorithms15, multi-atlas fusion methods16,17, and rich multi-atlas libraries18. The pipeline has been distributed on a cloud-computing platform, MRICloud19, since 2015, and it has been used to study neurodegenerative diseases, such as Alzheimer&#39;s disease (AD)20,21, Primary Progressive Aphasia22, and Huntington&#39;s Disease23.
Once the high-resolution images are segmented into brain structures, regional features, such as volumes, can be used to establish mathematical models to characterize the neuroanatomical changes. A change-point analysis method was recently established by our group to analyze the temporal order, in which statistically significant brain morphometric changes occur, based on longitudinal and/or cross-sectional MRI data. This statistical model was first developed to quantify shape-based diffeomorphometry over age in AD patients21,24; and it was later adapted to investigate brain structural changes in Huntington&#39;s disease (HD), as well as to describe brain developmental changes in neonatal brains25. In HD patients, the change-point was defined in with respect to the CAG-age product (CAP) score, as an indicator of the extent of exposure to the CAG expansion in HTT 26. It is well-known that striatal atrophy is one of the earliest markers in HD, followed by the globus pallidus27. Yet, the changes in striatum in relation to other gray and white matter structures across the brain remains unclear. Such relation is crucial for us to understand the disease progression. Change-point analysis of volumetric changes in all brain structures will likely provide systematic information of brain atrophy in premanifest phase of HD.
Here we demonstrate the procedures to perform whole-brain segmentation using MRICloud (www.mricloud.org), and steps to perform change-point analysis of volumetric data in premanifest HD subjects. The MRI data were collected from a large population multicenter PREDICT-HD study28,29 with approximately 400 controls and premanifest HD subjects. The combination of atlas-based segmentation and change-point analysis brings unique information about the spatiotemporal order of the brain structural changes and the disease progression pattern across the brain. The techniques are potentially applicable to a range of neurodegenerative diseases with various biomarkers to map the brain degeneration.

<table border="0" cellpadding="0" cellspacing="0" width="849">
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			<td height="40" style="height:40px;width:216px;">MATLAB</td>
			<td style="width:123px;">Mathworks</td>
			<td style="width:344px;">N/A</td>
			<td style="width:167px;">Version 2015b and above</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dell Workstation</td>
			<td>Dell</td>
			<td></td>
			<td>Dell Precision T5500 (Intel Xeon CPU)</td>
		</tr>
	</tbody>
</table>

whole-brain segmentation and change-point analysis of anatomical brain mri&#8212;application in premanifest huntington's disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Using the procedures described in 1.1-1.3, whole brain segmentation maps can be obtained from MRICloud. In the current version of atlas (V9B), 283 parcels are segmented at the finest granularity (level 5), which can be grouped to different levels of granularity, e.g., from hemisphere to lobules and parcels, according to specific ontology definitions. Figure 3 shows two types of multi-level segmentations at five levels, in axial and coronal views. For...

Watch this Scientific Journal Video about Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRIâ€”Application in Premanifest Huntington's Disease at JoVE.com

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

The overall goal of this image analysis pipeline is to perform quantitative volumetric analysis of T1 weighted MR images and to identify critical change-points when brain atrophy begins in neurodegenerative diseases. This method can help answer key questions in quantitative analysis of brain anatomical changes during the course of neurodegenerative disease. For example, the order and the pattern of brain atrophy in the Huntington's disease.The main advantage of this technique is that the combination of Atlas-based segmentation and change-point analysis bring unique spatial temporal information about brain degeneration from large population of studies. The volumetric analysis of brain MRI was achieved by MRICloud, which provides fully automated image segmentation and quantification through remote computation and web-based user interface. Change-point analysis of the brain MRI provides us a systematic view of the disease progression across the whole brain.The applications of this technique extend toward a range of neural degenerative disease. To begin this procedure, convert three dimensional T1 weighted images from vendor-specific DICOM format to analyzed format. Double click to open Dcm2Analyze.exe.A pop up window will open. Specify the input DICOM data directory path as input and analyzed image file path and name as output. Then, click go to complete the conversion.For MultiAtlas-based T1 image segmentation using MRICloud, log in BrainGPS and select segmentation tool from the main menu. There are two options under segmentation, the T1-MultiAtlas for single T1 image segmentation, and T1-MultiAtlas Batch for batch processing. Submit jobs on T1-MultiAtlas Batch API by compressing multiple analyzed image files into a ZIP file, and then click zip to upload the ZIP file.Now, fill in the required fields. For processing server, choose Computational Anatomy Science Gateway. For slice type, choose from Sagittal, Axial, or Sagittal converted to Axial.For MultiAtlas library, choose the Atlas library with the closest age range to the user data to optimize the segmentation accuracy. Check the job status through my job status. Once the jobs are finished, a download results button will appear that allows users to download the segmentation results as a ZIP file.Results will be saved in individual folders. Visualize the results by clicking the view results button. The web page will turn to the visualization interface.The Axial, Sagittal, and Coronal views of the segmentation map are overlapped on the T1 weighted anatomical image. 3-D rendering of the segmented brain structures are shown in the upper-left window. Color of the overlaying segmentation map indicates the z-score of the structural volumes.Adjust the visualization options, including overlay on/off, opacity of the overlay, zoom in and out, and slice positions from the upper right panel. To perform batch processing to obtain brain volumes in a population, use an in-house MATLAB program to extract individual brain volumes and combine them in a spreadsheet. In MATLAB, run main.m, and a GUI will pop out. In the T1 volume extraction from MRICloud panel, specify the inputs, including the study directory where the downloaded segmentation results are saved, and the multi-level lookup table file path and file name. Specify the output directory of the spreadsheet file where the volume data will be written to.Click the extract volume button to run the analysis. A message will show after successful completion. Results can be checked in the spreadsheet, where the rows contain individual subject data and the columns contain the structural volumes of these individuals defined at multiple anatomical granularities.To calculate change-points for individual brain structures, use the same MATLAB GUI. In the change-point analysis panel, specify the spreadsheets that contain the multi-level volume data and the associated diagnostic information. Then, specify the directory of the output text file, which the change-point results will be written to.Afterward, choose the level of granularity and type of ontology definition in the drop down box, at which the change-point analysis will be performed. Also, choose the main repressor to be used in the change-point analysis, for example, CAP score for Huntington's disease. Click the calculate change-point button to perform the analysis and the resultant change-points will be saved in the user-defined output excel file.To perform statistical evaluations of the change-points in the MATLAB GUI, specify the parameters for statistical tests, including the number of permutation and number of bootstrap. Click the statistical test button to run the tests. After this step, the p-values from the permutation test as well as the mean and standard deviation from the bootstrap operation will be written to the output text file as extra columns.Optionally, to map the change-points into the brain and 3-D space, click map change-point. After this step, and change-point map will be generated and visualized by overlying on the T1 weighted image. Shown here are the representative results of the Atlas-based whole brain segmentation at multiple granularity levels.Axial and Coronal views of multi-level segmentation maps are overlaid on T1 weighted anatomical images. The hierarchical anatomical relations between the hemisphere the cerebral nuclei, the basal ganglia of the cerebral nuclei, the striatum and globus pallidus of the basal ganglia, and putamen and caudate of the striatum are illustrated in 3-D. Here, the scatter plots demonstrate the change-point analysis of these structures, where the blue dots denote the z-scores of volumetric data after correcting for age, sex, and intercranial volumes.The black curves are the fitted z-scores, with regression to the change-point dependent CAP scores, and the red lines indicate the positions of the change-points. Here are the whole-brain change-point maps at multiple granularity levels. The regions that show significant change-points are mapped onto a T1 weighed image, and the colors indicate the change-point values in unit of CAP score.Once mastered, this procedure can be accomplished in several hours for the image segmentation part, depending on the availability of the cloud resource. And, another few hours for the change-point analysis, depending on the user-defined level of anatomical granularity. While attempting this procedure, it is important to always check the quality of the original image and accuracy of image segmentation before proceeding to the change-point analysis.After its development, this technique paved the way for researchers in the field of medical image analysis to explore whole-brain anatomical change and its spatial temporal pattern in large MRI studies of neural degenerative diseases. After watching this video, you should have a good understanding of how to perform automated segmentation of T1 weighted images and how to use the MATLAB GUI to run change-point analysis of photometric data.

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Research

JoVE Journal

Medicine

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior Cingulate Cortex (PCC) is a potential imaging marker for monitoring the progression of AD. Previous studies did not focus on the functional connectivity between the PCC and nodes in regions outside the DMN, but our study is an effort to explore these overlooked functional connections. For collecting data, we used functional Magnetic Resonance Imaging (fMRI) and Granger Causality Analysis (GCA). fMRI provides a non-invasive method for studying the dynamic interactions between the different brain regions. GCA is a statistical hypothesis test for determining whether one-time series is useful in forecasting another. In simple terms, it is judged by comparing the &#34;Known all the information on the last moment, the distribution of the probability of X at this time&#34; and the &#34;Known all the information on the last moment except Y, the distribution of the probability of X at this time&#34;, to determine whether there is a causal relationship between Y and X. This definition is based on the complete information source and stationary chronological sequence. The main step of this analysis is to use X and Y to establish the regression equation and draw a causal relationship by a hypothetical test. Since GCA can measure causal effects, we used it to investigate the anisotropy of the functional connectivity and explore the hub function of the PCC. Here, we screened 116 participants for MRI scanning, and after preprocessing the data obtained from neuroimaging, we used GCA to derive the causal relationship of each node. Finally, we concluded that the directed connection is significantly different between the Mild Cognitive Impairment (MCI) and AD groups, both from the PCC to the whole brain and from the whole brain to the PCC.

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer&#039;s Disease and Mild Cognitive Impairment

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer&#39;s Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...