M.S. is grateful for the support from all of the staff in the medical information engineering course in Graduate School of Medicine and Faculty of Medicine, and wants to thank Prof. Tetsuya Takakuwa, Prof. Nobukatsu Sawamoto, and Doris Zakian for their helpful comments. This study was supported by a Grant-in-Aid for Challenging Exploratory Research and by the Leading Initiative for Excellent Young Researchers (LEADER) program to M.S. from MEXT (The Ministry of Education, Culture, Sports, Science, and Technology). The MRI experiments in this work were performed in the Division for Small Animal MRI, Medical Research Support Center, Graduate School of Medicine, Kyoto University, Japan.

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The authors have nothing to disclose.

We developed a new protocol called the 3D-NEO protocol to bridge macroscopic and microscopic spatial scales by overlapping two brain surfaces more accurately than before. Originally, there were two challenges in creating this protocol which made possible the accurate overlapping of two brain surface images and recording healthy neuronal activities from living organisms. First, it was necessary to effectively wipe cutting solution surrounding the extracted brain after extracting it from the skull without injuring the brai...

Nonuniform multiscale architectures at various physical and biological organizations are commonly found1,2. The brain is also a very nonuniform and multiscale network organization3,4. Various cognitive functions are coded in such network organizations, holding temporal changes of electrical spike patterns of neuronal populations in submillisecond temporal resolutions. Historically, the complex networks among neurons were structurally observed in detail using the staining techniques by Santiago Ram&#243;n y Cajal from over 150 years ago5. To observe group behaviors of active neurons, researchers have developed various recording technologies6,7,8, and the recent significant developments of such technologies have enabled us to record electrical activities from huge numbers of neurons simultaneously. Furthermore, from such functional activities, scientists have succeeded to reconstruct networks of causal interactions among huge numbers of neurons and have declared the topological architecture of their complex interactions &#8216;microconnectome&#8217;9. Macroscopic observations of the brain also allow for regarding a whole brain as a network organization because many brain regions are connected by multiple fiber-bundles. The embedding of microconnectomes into the global brain map still has clear limitations within current technological advances, which is why this embedding protocol is so important. However, there are many challenges to the development of the embedding protocol. For example, in order to observe activities of living local neuronal circuits in purely isolated brain regions, brain slices need to be produced for in vitro recordings. Additionally, recordings from brain slices for in vitro recordings are still an important choice for at least two reasons. First, it is still not easy to observe activities of many living individual neurons simultaneously from brain regions deeper than ~1.5 mm and in high temporal resolution (&#60;1 ms). Second, when we hope to know the internal architecture of a local neuronal circuit, we need to stop all inputs coming from external brain regions to eliminate confounding factors. In order to identify the directions and positions of produced brain slices, it will be further necessary to integrate the spatial positions of these produced brain slices using coordinates. There are, however, a few systematic and reliable ways to make brain slices in an organized way10,11. Here, a new coregistration protocol is introduced, using 3D scanning technology for neuroscientific research in order to provide an integrative protocol. This protocol acts to coordinate micro- and macroscales and embed multielectrode array (MEA) microdata12,13 and staining data onto a macroscopic MRI space through 3D scan surfaces of extracted brains, as well as of noninvasively recorded brains. Surprisingly, this showed a distance error of only ~50 &#956;m as the mode value of the histogram. As a result, the mode values of minimum distances between two surfaces between the MRI surface and the scanned 3D surface were nearly 50 &#181;m for all six mice, which is a suitable number when checking for commonality among individuals. The typical slice width had a recorded spike activity of around 300 &#181;m.

<table><tbody><tr><td>Air compressor</td><td>Kimura Medical</td><td>KA-100</td><td>Animal preparation for MRI</td></tr><tr><td>All-in-one fluorescence microscope</td><td>KEYENCE</td><td>BZ-X710</td><td></td></tr><tr><td>Anesthesia box</td><td>Bio Research Center</td><td>RIC-01</td><td>Animal preparation for MRI</td></tr><tr><td>Anesthesia system</td><td>ACOMA Medical Industry</td><td>NS-5000A</td><td>Animal preparation for MRI</td></tr><tr><td>Anti-GAD67, clone 1G10.2</td><td>Merk Millipore</td><td>MAB5406</td><td>For immunostaining</td></tr><tr><td>Calcium Chrolide</td><td>nacalai tesque</td><td>06729-55</td><td>aCSF</td></tr><tr><td>Choline Chloride</td><td>nacalai tesque</td><td>08809-45</td><td>aCSF</td></tr><tr><td>Curved blunt forceps</td><td></td><td></td><td></td></tr><tr><td>Disposal scalpel</td><td>Kai</td><td>10</td><td></td></tr><tr><td>D-PBS(-) without Ca and Mg, liquid (10x)</td><td>nacalai tesque</td><td></td><td>For immunostaining</td></tr><tr><td>D(+)-Glucose</td><td>Wako</td><td>049-31165</td><td>aCSF</td></tr><tr><td>Gelatin</td><td>nacalai tesque</td><td>16605-42</td><td>re-secctioning</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td><td>Invitrogen</td><td>A32723</td><td>For immunostaining</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td><td>Invitrogen</td><td>A32732</td><td>For immunostaining</td></tr><tr><td>Heater mat</td><td>Bio Research Center</td><td>HM-10</td><td>Animal preparation for MRI</td></tr><tr><td>Heater mat controller</td><td>Bio Research Center</td><td>BWT-100A</td><td>Animal preparation for MRI</td></tr><tr><td>Heater system</td><td>SA Instruments</td><td>MR-compatible Small Animal Heating System</td><td>Animal preparation for MRI</td></tr><tr><td>Isoflurane</td><td>AbbVie</td><td></td><td>Animal preparation for MRI</td></tr><tr><td>Isoflurane vaporizer</td><td>ACOMA Medical Industry</td><td>MKIIIai</td><td>Animal preparation for MRI</td></tr><tr><td>Linear Slicer</td><td>DOSAKA</td><td>Neo Linear Slicer MT</td><td></td></tr><tr><td>L(+)-Ascorbic Acid Sodium Salt</td><td>Wako</td><td>196-01252</td><td>aCSF</td></tr><tr><td>Magnesium Chrolide Hexahydrate</td><td>Wako</td><td>135-00165</td><td>aCSF</td></tr><tr><td>MaxOne Single-Well MEA</td><td>MaxWell Biosystems</td><td></td><td></td></tr><tr><td>Metal Spatula</td><td></td><td></td><td></td></tr><tr><td>Monitoring system</td><td>SA Instruments</td><td>Model 1025</td><td>Animal preparation for MRI</td></tr><tr><td>Monitoring software</td><td>SA Instruments</td><td>PC-SAM V.5.12</td><td>Animal preparation for MRI</td></tr><tr><td>MRI compatible cradle</td><td>Bruker BioSpin</td><td>T12812</td><td>Animal preparation for MRI</td></tr><tr><td>MRI coil</td><td>Bruker BioSpin</td><td>T9988</td><td>For MRI</td></tr><tr><td>MRI operation software</td><td>Bruker BioSpin</td><td>ParaVision 5.1</td><td>For MRI</td></tr><tr><td>Neo LinearSlicer MT</td><td>D.S.K.</td><td>NLS-MT</td><td></td></tr><tr><td>NeuN (D4G40) XP Rabbit mAb</td><td>Cell Signaling</td><td>24307</td><td>For immunostaining</td></tr><tr><td>Normal Goat Serum</td><td>Wako</td><td>143-06561</td><td>For immunostaining</td></tr><tr><td>Potassium Chloride</td><td>Wako</td><td>163-03545</td><td>aCSF</td></tr><tr><td>Polyethylene Glycol Mono-p-isooctylphenyl Ether</td><td>nacalai tesque</td><td>12967-45</td><td>For immunostaining</td></tr><tr><td>Pressure-sensitive respiration sensor</td><td>SA Instruments</td><td>RS-301</td><td>Animal preparation for MRI</td></tr><tr><td>Preclinical MRI scanner</td><td>Bruker BioSpin</td><td>BioSpec 70/20 USR</td><td>For MRI</td></tr><tr><td>Pyruvic Acid Sodium Salt</td><td>nacalai tesque</td><td>29806-54</td><td>aCSF</td></tr><tr><td>SCAN in a BOX</td><td>Open Technologies srl</td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Sieve bottle</td><td>TIGERCROWN</td><td>81</td><td>For 3D scan</td></tr><tr><td>SlowFade Gold Antifade Mountant</td><td>Invitrogen</td><td>S36937</td><td>For immunostaining</td></tr><tr><td>Sodium Chloride</td><td>Wako</td><td>191-01665</td><td>aCSF</td></tr><tr><td>Sodium Dihydrogenphosphate</td><td>Wako</td><td>197-09705</td><td>aCSF</td></tr><tr><td>Sodium Hydrogen Carbonate</td><td>Wako</td><td>191-01305</td><td>aCSF</td></tr><tr><td>Sodium Hydrogensulfite</td><td>nacalai tesque</td><td>31220-15</td><td>For immunostaining</td></tr><tr><td>Thermistor temperature probe</td><td>SA Instruments</td><td>RTP-101-B, PLTPC-300</td><td>Animal preparation for MRI</td></tr><tr><td>Tooth bar</td><td>Bruker BioSpin</td><td>T10146</td><td>Animal preparation for MRI</td></tr><tr><td>Winged intravenous needle</td><td>TERUMO</td><td>SV-23CLK</td><td>For perfusion</td></tr><tr><td>1 mol/l-Tris-HCl Buffer Solution</td><td>nacalai tesque</td><td>35436-01</td><td>For immunostaining</td></tr><tr><td>1 mol/l-Hydrochloric Acid</td><td>nacalai tesque</td><td>37314-15</td><td>For pH adjustment of solution</td></tr><tr><td>16%-Paraformaldehyde Aqueous Solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td>For immunostaining</td></tr></tbody></table>

3d scanning technology bridging microcircuits and macroscale brain images in 3d novel embedding overlapping protocol

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and microscopic neuronal spikes, propagating along axons and dendrites. Both scales complement different aspects of human cognitive and behavioral functions. At the macroscopic level, MRI has been the current standard imaging technology, in which the smallest spatial resolution, voxel size, is 0.1&#8211;1 mm3. Also, at the microscopic level, previous physiological studies were aware of nonuniform neuronal architectures within such voxels. This study develops a powerful way to accurately embed microscopic data into a macroscopic map by interfacing biological scientific research with technological advancements in 3D scanning technology. Since 3D scanning technology has mostly been used for engineering and industrial design until now, it is repurposed for the first time to embed microconnectomes into the whole brain while preserving natural spiking in living brain cells. In order to achieve this purpose, first, we constructed a scanning protocol to obtain accurate 3D images from living bio-organisms inherently challenging to image due to moist and reflective surfaces. Second, we trained to keep speed to prevent the degradation of living brain tissue, which is a key factor in retaining better conditions and recording more natural neuronal spikes from active neurons in the brain tissue. Two cortical surface images, independently extracted from two different imaging modules, namely MRI and 3D scanner surface images, surprisingly show a distance error of only 50 &#956;m as mode value of the histogram. This accuracy is comparable in scale to the microscopic resolution of intercellular distances; also, it is stable among different individual mice. This new protocol, the 3D novel embedding overlapping (3D-NEO) protocol, bridges macroscopic and microscopic levels derived by this integrative protocol and accelerates new scientific findings to study comprehensive connectivity architectures (i.e., microconnectome).

We evaluated distances between cortical surfaces, produced by stripping MRI volume, and surfaces obtained from 3D scans of extracted brains. The mode values of the histogram of the distances are only 55 &#956;m (Figure 3a). Additionally, when accumulating the histogram from the point where the distance equals zero, the accumulated value reaches 90% of total sample numbers at ~300 &#956;m (Figure 3b). The final histogram of the di...

Watch this Scientific Journal Video about 3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol at JoVE.com

3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol

This article introduces an experimental protocol using 3D scanning technology bridging two spatial scales: the macroscopic spatial scale of whole-brain anatomy imaged by MRI at &#62;100 &#956;m and the microscopic spatial scale of neuronal distributions using immunohistochemistry staining and a multielectrode array system and other methods (~10 &#956;m).

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and ...

3d-scanning-technology-bridging-microcircuits-macroscale-brain-images

Research

JoVE Journal

Neuroscience

7.2K Views.  Kyoto University. Dit protocol is de eerste die 3D-scantechnologie introduceert in het anatomische, met name neuroanatomisch onderzoeksveld, en heeft zeer nauwkeurige overlappende prestaties bereikt. De 3D neuro inbedding overlappen, of 3D-neuro protocol, integreert micro-schaal steriele sockets in macro-schaal hersenbeelden, en bruggen deze verschillende ruimtelijke schalen naadloos. We moeten ook het 3D neuro protocol toepassen op menselijke MRI's en postmortem hersenen.De overlappende stelt ons in st...

Video: 3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol

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