Name: three-dimensional shape modeling and analysis of brain structures
Uploaded: 2019-11-14T10:00:00
Description: Watch this Scientific Journal Video about Three-Dimensional Shape Modeling and Analysis of Brain Structures at JoVE.com

The work was funded by the National Research Foundation of Korea (JP as the PI). JK is funded by Kyungpook National University Research Fund; and MCVH is funded by the Row Fogo Charitable Trust and the Royal Society of Edinburgh. The hippocampal segmentation was adapted from in-house guidelines written by Dr. Karen Ferguson, at the Centre for Clinical Brain Sciences, Edinburgh, UK.

Brain MR images were acquired as per the protocol approved by the local institutional review board and ethics committee.
NOTE: The tools for shape modeling and analysis can be downloaded from the NITRC repository: <a href="https://www.nitrc.org/projects/dtmframework/" target="_blank">https://www.nitrc.org/projects/dtmframework/</a>. The GUI software (DTMModeling.exe) can be executed after extraction.<!--The publication list on the shape modeling tools can be found in the proje...

<ol>
	<li>Costafreda, S. G., 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=Automated+hippocampal+shape+analysis+predicts+the+onset+of+dementia+in+mild+cognitive+impairment.">Automated hippocampal shape analysis predicts the onset of dementia in mild cognitive impairment.</a> NeuroImage. 56 (1), 212-219 (2011).</li><li>Platero, C., Lin, L., Tobar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+Neuroimaging+Hippocampal+Markers+for+Diagnosing+Alzheimer's+Disease.">Longitudinal Neuroimaging Hippocampal Markers for Diagnosing Alzheimer's Disease.</a> Neuroinformatics. , 1-19 (2018).</li><li>Vald&#233;s Hern&#225;ndez, M. D. C., 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=Rationale,+design,+and+methodology+of+the+image+analysis+protocol+for+studies+of+patients+with+cerebral+small+vessel+disease+and+mild+stroke.">Rationale, design, and methodology of the image analysis protocol for studies of patients with cerebral small vessel disease and mild stroke.</a> Brain and behavior. 5 (12), e00415 (2015).</li><li>Kalmady, S. 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=Clinical+correlates+of+hippocampus+volume+and+shape+in+antipsychotic-na&#239;ve+schizophrenia.">Clinical correlates of hippocampus volume and shape in antipsychotic-na&#239;ve schizophrenia.</a> Psychiatry Research: Neuroimaging. 263, 93-102 (2017).</li><li>Kim, J., Vald&#233;s Hern&#225;ndez, M. D. C., Royle, N. A., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+Shape+Modeling+Based+on+a+Progressive+Template+Surface+Deformation+and+its+Verification.">Hippocampal Shape Modeling Based on a Progressive Template Surface Deformation and its Verification.</a> IEEE Transactions on Medical Imaging. 34 (6), 1242-1261 (2015).</li><li>Kim, J., 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=3D+shape+analysis+of+the+brain's+third+ventricle+using+a+midplane+encoded+symmetric+template+model.">3D shape analysis of the brain's third ventricle using a midplane encoded symmetric template model.</a> Computer Methods and Programs in Biomedicine. 129, 51-62 (2016).</li><li>Kim, J., Ryoo, H., Vald&#233;s Hern&#225;ndez, M. D. C., Royle, N. A., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Ventricular+Morphology+Analysis+Using+a+Set+of+Ventricular-Specific+Feature+Descriptors.">Brain Ventricular Morphology Analysis Using a Set of Ventricular-Specific Feature Descriptors.</a> International Symposium on Biomedical Simulation. , 141-149 (2014).</li><li>Vald&#233;s Hern&#225;ndez, M. D. C., 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=Hippocampal+morphology+and+cognitive+functions+in+community-dwelling+older+people:+the+Lothian+Birth+Cohort+1936.">Hippocampal morphology and cognitive functions in community-dwelling older people: the Lothian Birth Cohort 1936.</a> Neurobiology of Aging. 52, 1-11 (2017).</li><li>Lee, P., Ryoo, H., Park, J., Jeong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+Microstructural+Changes+of+the+Hippocampus+in+Early+MCI:+A+Study+Utilizing+the+Alzheimer's+Disease+Neuroimaging+Initiative+Database.">Morphological and Microstructural Changes of the Hippocampus in Early MCI: A Study Utilizing the Alzheimer's Disease Neuroimaging Initiative Database.</a> Journal of Clinical Neurology. 13 (2), 144-154 (2017).</li><li>Cox, S. R., 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=Associations+between+hippocampal+morphology,+diffusion+characteristics,+and+salivary+cortisol+in+older+men.">Associations between hippocampal morphology, diffusion characteristics, and salivary cortisol in older men.</a> Psychoneuroendocrinology. 78, 151-158 (2017).</li><li>Sled, J. G., Zijdenbos, A. P., Evans, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonparametric+method+for+automatic+correction+of+intensity+nonuniformity+in+MRI+data.">A nonparametric method for automatic correction of intensity nonuniformity in MRI data.</a> IEEE Transactions on Medical Imaging. 17 (1), 87-97 (1998).</li><li>Tustison, N. J., 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=N4ITK:+improved+N3+bias+correction.">N4ITK: improved N3 bias correction.</a> IEEE Transactions on Medical Imaging. 29 (6), 1310-1320 (2010).</li><li>Zhang, Y., Brady, M., Smith, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation+of+brain+MR+images+through+a+hidden+Markov+random+field+model+and+the+expectation-maximization+algorithm.">Segmentation of brain MR images through a hidden Markov random field model and the expectation-maximization algorithm.</a> IEEE Transactions on Medical Imaging. 20 (1), 45-57 (2001).</li><li>Wardlaw, J. M., 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=Brain+aging,+cognition+in+youth+and+old+age+and+vascular+disease+in+the+Lothian+Birth+Cohort+1936:+rationale,+design+and+methodology+of+the+imaging+protocol.">Brain aging, cognition in youth and old age and vascular disease in the Lothian Birth Cohort 1936: rationale, design and methodology of the imaging protocol.</a> International Journal of Stroke. 6 (6), 547-559 (2011).</li><li>Morey, R. 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=A+comparison+of+automated+segmentation+and+manual+tracing+for+quantifying+hippocampal+and+amygdala+volumes.">A comparison of automated segmentation and manual tracing for quantifying hippocampal and amygdala volumes.</a> NeuroImage. 45 (3), 855-866 (2009).</li><li>Boccardi, M., 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=Survey+of+protocols+for+the+manual+segmentation+of+the+hippocampus:+preparatory+steps+towards+a+joint+EADC-ADNI+harmonized+protocol.">Survey of protocols for the manual segmentation of the hippocampus: preparatory steps towards a joint EADC-ADNI harmonized protocol.</a> Journal of Alzheimer's Disease. 26 (s3), 61-75 (2011).</li><li>Winterburn, J., 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=High-resolution+In+Vivo+Manual+Segmentation+Protocol+for+Human+Hippocampal+Subfields+Using+3T+Magnetic+Resonance+Imaging.">High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging.</a> Journal of Visualized Experiments. (105), e51861 (2015).</li><li>MacLullich, 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=Intracranial+capacity+and+brain+volumes+are+associated+with+cognition+in+healthy+elderly+men.">Intracranial capacity and brain volumes are associated with cognition in healthy elderly men.</a> Neurology. 59 (2), 169-174 (2002).</li><li>Gower, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+Procrustes+analysis.">Generalized Procrustes analysis.</a> Psychometrika. 40 (1), 33-51 (1975).</li><li>Lorensen, W. E., Cline, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marching+cubes:+A+high+resolution+3D+surface+construction+algorithm.">Marching cubes: A high resolution 3D surface construction algorithm.</a> ACM Siggraph Computer Graphics. , 163-169 (1987).</li></ol>

In summary, we have described the software pipeline for the shape analysis on brain structures including (1) MR image segmentation using open tools (2) individual shape reconstruction using a deformable template model, and (3) quantitative shape difference measurement via transitive shape correspondence with the template model. Statistical analysis under the false discovery rate (FDR) correction is performed with the shape deformity to investigate the significance of morphological changes of brain structures, associated ...

Shape analysis of brain structures has emerged as the preferred tool to investigate their morphological changes under pathological processes, such as neurodegenerative diseases and aging1. Various computational methods are required to 1) accurately delineate the boundaries of target structures from medical images, 2) reconstruct the target shape in the form of 3D surface mesh, 3) build inter-subjects correspondence across the individual shape models via shape parameterization or surface registration, and 4) quantitatively assess the regional shape differences between individuals or groups. Over the past several years, many methods have been introduced in neuroimaging studies for each of these steps. However, despite the remarkable developments in the field, there are not many frameworks immediately applicable to research. In this article, we describe each step of the shape analysis of brain structures using our custom shape modeling tools and publicly available image segmentation tools.
Here, we demonstrate the shape analysis framework for brain structures through the shape analysis of the left and right hippocampi using a dataset of adult controls and Alzheimer&#39;s disease patients. Atrophy of the hippocampi is recognized as a critical imaging biomarker in neurodegenerative diseases2,3,4. In our shape analysis framework, we employ the template model of the target structure and the template-to-image deformable registration in the shape modeling process. The template model encodes general shape characteristics of the target structure in a population, and it also provides a baseline for quantifying the shape differences among the individual models via their transitive relation with the template model. In the template-to-image registration, we have developed a Laplacian surface deformation method to fit the template model to the target structure in individual images while minimizing the distortion of the point distribution in the template model5,6,7. The feasibility and robustness of the proposed framework have been validated in recent neuroimaging studies of cognitive aging8, early detection of mild cognitive impairment9, and to explore associations between brain structural changes and cortisol levels10. This approach would make it easier to use the shape modeling and analysis methods in further neuroimaging studies.

three-dimensional shape modeling and analysis of brain structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

The shape modeling process described here has been employed for various neuroimaging studies on aging6,8,10 and Alzheimer&#39;s disease5,9. Especially, this shape modeling method showed its accuracy and sensitivity in the shape analysis on the hippocampus for an aging population of 654 subjects8. A quantitative ana...

Watch this Scientific Journal Video about Three-Dimensional Shape Modeling and Analysis of Brain Structures at JoVE.com

Three-Dimensional Shape Modeling and Analysis of Brain Structures

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Accurately recovering the shape features against rough and noisy segmentations is critical to achieving good anatomical correspondence between individual brain shape models. Our framework provides various tools for individual shape modeling, group-wise template construction, and shape deformity computation. And it has been used for large datasets of the human brain.Demonstrating this procedure will be Dr.Jaeil Kim, a former grad student from my laboratory who developed the software for the brain shape modeling. For manual editing of the hippocampal segmentation, open the T1 weight magnetic resonance image and the automatic hippocampal segmentation results in the graphic user interface software. Click the icon in the display window to select the coronal view and scroll through the volume until the uncus is located.Including the uncus in the hippocampal mask where it is present, use the add and subtract functions to edit the mask of the hippocampal body after the uncus has receded. Continue editing the hippocampal mask until the hippocampal tail is found. As the pulvinar nucleus of the thalamus recedes superior to the hippocampus, the fornix will emerge.Finish editing the last coronal slice of the hippocampus in which the entire length of the fornix is visible, but not yet continuous with the splenium of the corpus callosum. Then, save the masks for both the left and right hippocampi in NifTI format. To construct a group template model load the shape modeling plug-in and click Open Directory to open a directory containing the binary masks of the study population of interest.Input the desired number of vertices and click Template Construction for the group template construction. Then check the mean shape mesh. For individual shape reconstruction, load the T1-weighted magnetic resonance image of interest and its corresponding segmentation mask and select the working directory to save the files.Select a template model for the individual shape modeling and check and modify the modeling parameters in the shape modeling plug-in as necessary. So our modeling framework is almost automated, however, some steps require user confirmation. For example, if the value for the hippocampal regions is not one users must change the intensity parameter.Then, check the result in the 3D view of the toolkit workbench. To perform a shape and deformity measurement select the shape model of the subject of interest in the data manager of the software and click Select Template to select the template of interest to obtain the measurement. Here, a representative deformation of the hippocampal template model for individual shape reconstruction can be observed.The method induces a large to small scale deformation of the template model to minimize the distortion of its point distribution while restoring the individual shape characteristics. In this figure, reconstructed shape models from two subjects with their segmentation masks are shown. In these images aligned individual shape models, their average model, and the shape difference vectors with an individual shape model can be observed.These data represent average shape deformity maps projected onto the average model. For one group with a small brain tissue volume, and one group with a large brain tissue volume. The shape deformity maps of the two groups present opposite patterns of hippocampal shape difference at corresponding regions.Check the segmentation mask and the individual or either the shared model together. If the model is not fitted to the image boundary, adjust the modeling parameters to achieve better results. Statistical analysis using the shape deformity can be performed to investigate the group-wise shape We have also provided MATLAB code for the analysis at our project page.This robust method has been applied to a number of clinical studies, not only involving critical structure modeling such as Alzheimer's disease or aging study, but also foot-bone disorders which require the analysis of compound bones.

three-dimensional-shape-modeling-and-analysis-of-brain-structures

Research

JoVE Journal

Neuroscience

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...