The authors acknowledge the following funding sources, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Dorothy Pitts Research Fund (NG), and the Nicky and Thor Eaton Research Fund. The authors acknowledge Kevin DeSimone, and Aman Goyal and for their knowledge in MRI acquisition and analysis expertise.

1. Participant and Postmortem Brain Set-Up
NOTE: All images were acquired using a 3 T MRI scanner with a 32-channel head coil and all MRI scanning was performed at RT, approximately 20 &#176;C. All participants were right handed and gave written informed consent. Each participant was in good health with no history of neurological disorders. The experimental protocol was approved and follows the guidelines of York University Human Participants Review Committee.
<ol>
	<li>Ask each participant fill out and sign a patient consent form that details MRI safety guidelines and the neuro-imaging protocol.</li>
	<li>For each participant, place earplugs in each ear and secure their head with pads to minimize head motion.</li>
	<li>For post-mortem brain imaging, make sure the brain is fixed prior to neuroimaging and is contained within a bag or container that fits within the MRI head-coil. Place the postmortem brain in the head-coil with its z-axis (superior to inferior) aligned with the bore of the scanner. The brainstem (posterior) should face towards the foot of the scanner bed.</li>
	<li>Place vacuum cushion hands around the post-mortem brain for additional support.</li>
</ol>
2. Localizing and Prescribing the Subcortex
NOTE: The thalamus is a dual lobed structure located near the center of the brain situated between the midbrain and the cerebral cortex. Located within the dorsal thalamus, the human LGN is a small subcortical structure that extends a maximum of ~10 mm.
<ol>
	<li>To register a new participant, open the MRI imaging software and click on the Patient tab in the upper left hand corner. Then click on Register.</li>
	<li>Fill in the appropriate patient information, and then click on the Exam tab.</li>
	<li>To obtain a localizer scan, click on the Exam Explorer tab to create a new protocol. Observe the set-up window on the screen, click the Routine tab, and enter the following parameters: acquisition time 28 sec, acquisition matrix 160 &#215; 160, 1 slice, 1.6 mm thick isotropic voxel size, FOV = 260 mm, FoV phase = 100%, slice resolution = 69%, phase and slice partial phase Fourier = 6/8, TR = 3.15 ms, TE = 1.37 msec, Flip Angle = 8&#176;.</li>
	<li>Overlay the slice selection box used for acquiring the PD images over the localizer covering the subcortical nuclei within the thalamus as well as surrounding structures (Figure 4).</li>
</ol>
3. High-resolution Structural Parameters
<ol>
	<li>Create a new protocol for obtaining high-resolution PD-weighted scans. In the set-up window on the screen, click the Routine tab, and enter the following parameters in the coronal orientation: acquisition time 179 sec, acquisition matrix 512 &#215; 512, 0.3 &#215; 0.3 &#215; 1 mm3 voxel size, TR = 3.25 sec, TE = 32 msec, flip angle = 120&#176;, interleaved slice acquisition, FoV read = 160 mm, FoV phase = 100%, parallel imaging (GRAPPA) with an acceleration factor of 2.
	<ol>
		<li>Use a Turbo Spin Echo sequence, with an Echo Train Length of 5. The first echo at 32 msec is the effective echo for this sequence. Reduce the bandwidth (BW) to the minimum possible, 40 Hz/pixel, to maximize the SNR. To reduce scan duration, choose 18 slices, each 1 mm thick, with an FOV = 160 mm. This slab provides enough coverage of subcortical regions of interest. 
		NOTE: For reliable identification of subcortical structures, acquire 5 runs with the above parameters. The total scan duration is only ~15 min (Figure 5). Fat-saturation was not employed.</li>
	</ol>
	</li>
	<li>In post-mortem brain imaging, reliable identification of subcortical structures can be observed in just one scan with the total duration of only ~3 min following the same scanning protocol as in 3.1 (Figure 6).</li>
</ol>
4. Image Analysis
NOTE: To analyze the MRI data, use the freely available FMRIB&#39;s Software Library (FSL) package available for download at (<a href="https://www.fmrib.ox.ac.uk/fsl/">https://www.fmrib.ox.ac.uk/fsl/</a>).
<ol>
	<li>Open a terminal window, and convert the raw DICOM files from the scanner for each PD volume to a NIfTI format with a DICOM to NIfTI converter. A number of which are freely available for download (e.g.,&#160;<a href="https://www.nitrc.org/projects/mricron">https://www.nitrc.org/projects/mricron</a>). In the command line, type dcm2nii followed by the directory of each PD weighted image run.</li>
	<li>In a terminal window obtain the parameters of the original PD scan. Type fslinfo in the command line followed by the PD scan in NIfTI format.</li>
	<li>Create a high-resolution blank image target volume that has twice the resolution and half the voxel size given by the parameters from fslinfo from the original PD scan. The order of data inputs for this command are as follows: 
	fslcreatehd &#60;xsize&#62; &#60;ysize&#62; &#60;zsize&#62; &#60;tsize&#62; &#60;xvoxsize&#62; &#60;yvoxsize&#62; &#60;zvoxsize&#62; &#60;tr&#62; &#60;xorigin&#62; &#60;yorigin&#62; &#60;zorigin&#62; &#60;datatype&#62; &#60;headername&#62; 
	NOTE: For example, if the original PD scan with the following parameters as described in 3.1 are collected (i.e., 512 &#215; 512 matrix, 18 slice, 0.3 &#215; 0.3 &#215; 1 mm3 voxel size, TR = 3.25 s), type the following into the command window: 
	fslcreatehd 1024 1024 36 1 0.15 0.15 0.5 3.25 0 0 0 4 blankhr.nii.gz</li>
	<li>Define the transformation using an identity matrix. Type in any text editor program a text file saved as &#39;identity.mat&#39; that looks like this: 
	0 0 0 
	1 0 0 
	0 1 0 
	0 0 1</li>
	<li>Use the flirt command to apply the transformation, upsampling each original PD weighted run to double the total resolution from a 512 to a 1024 matrix, and halve the voxel size in each dimension resulting in a resolution of 0.15 &#215; 0.15 &#215; 0.5 mm3. In a terminal window for each PD volume, type the following flirt command changing the original and output names per run: 
	flirt -interp sinc -in originalPD.nii.gz -ref blankhr.nii.gz -applyxfm -init identity.mat -out highresPD.nii.gz 
	NOTE: Where originalPD.nii.gz is the source volume, blankhr.nii.gz is the desired output resolution, and highresPD.nii.gz is the name of the output volume.</li>
	<li>Move all the high-resolution images to a new folder, and navigate to it in a terminal window.</li>
	<li>For each participant, concatenate all the upsampled PD images into a single 4D file using fslmerge. In a terminal window type: 
	fslmerge -t concat_highresPD *.nii.gz 
	NOTE: This creates a 4D file called concat_highresPD.nii.gz.</li>
	<li>Motion correct the concatenated file using mcflirt 10 . This tool allows for an automated robust registration for linear (affine) inter and inter-modal brain images. Select a 4-stage correction, which utilizes sinc interpolation (internally) as a further optimization pass for greater accuracy. In a terminal window type: 
	mcflirt -in concat_highresPD -out mcf_concat_highresPD.nii.gz -stages 4 -plots 
	NOTE: This creates a 4D file called mcf_concat_highresPD.nii.gz.</li>
	<li>Finally, create the 3D mean image using fslmaths. In a terminal window type: 
	fslmaths mcf_concat_highresPD.nii.gz -Tmean mean_highresPD.nii.gz 
	NOTE: This creates a 3D file called mean_highresPD.nii.gz that is of high quality</li>
	<li>Visualize the final outcome 3D high-resolution image using the fslview command. In the directory of where your image is, type the following in a terminal window: 
	fslview mean_highresPD.nii.gz.&#34;</li>
	<li>Inspect intensity profiles of ROIs in question. Create an ROI using fslview (this can be a vertical line drawn across a region of the LGN for example). In fslview load the high-resolution PD image. Click on the tools tab, then click on the single image tab to enlarge the image for drawing ROIs. Then, click the File tab followed by the Create Mask tab. Draw a line in the ROI of interest. Save the ROI by clicking File, then the Save As. Repeat the line masks for multiple areas within the ROI for intensity comparisons and other ROIs in question.</li>
	<li>Use AFNI&#39;s 3dmaskdump command to analyze the resulting intensity of the image. In the directory of where the images are, use the following command in a terminal window to extract the image intensities and location (given as result_mask.txt) of your ROI mask: 
	3dmaskdump -o result_mask.txt -noijk -xyz -mask ROI_linemask.nii.gz PDaverage_image.nii.gz</li>
</ol>

<ol>
	<li>Devlin, J. T., 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=Reliable+identification+of+the+auditory+thalamus+using+multi-modal+structural+analyses.">Reliable identification of the auditory thalamus using multi-modal structural analyses.</a> NeuroImage. 30 (4), 1112-1120 (2006).</li><li>Fellner, F., 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=True+proton+density+and+T2-weighted+turbo+spin-echo+sequences+for+routine+MRI+of+the+brain.">True proton density and T2-weighted turbo spin-echo sequences for routine MRI of the brain.</a> Neuroradiology. 36 (8), 591-597 (1994).</li><li>Schumann, C. M., Buonocore, M. H., Amaral, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+the+post-mortem+autistic+brain.">Magnetic resonance imaging of the post-mortem autistic brain.</a> J Autism Dev Disord. 31 (6), 561-568 (2001).</li><li>Tovi, M., Ericsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+T1+and+T2+over+time+in+formalin-fixed+human+whole-brain+specimens.">Measurements of T1 and T2 over time in formalin-fixed human whole-brain specimens.</a> Acta Radiol. 33 (5), 400-404 (1992).</li><li>Fujita, N., 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=Lateral+geniculate+nucleus:+anatomic+and+functional+identification+by+use+of+MR+imaging.">Lateral geniculate nucleus: anatomic and functional identification by use of MR imaging.</a> Am J Neuroradiol. 22 (9), 1719-1726 (2001).</li><li>Bridge, H., Thomas, O., Jbabdi, S., Cowey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+connectivity+after+visual+cortical+brain+damage+underlie+altered+visual+function.">Changes in connectivity after visual cortical brain damage underlie altered visual function.</a> Brain. 131 (6), 1433-1444 (2008).</li><li>Gupta, N., 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=Atrophy+of+the+lateral+geniculate+nucleus+in+human+glaucoma+detected+by+magnetic+resonance+imaging.">Atrophy of the lateral geniculate nucleus in human glaucoma detected by magnetic resonance imaging.</a> Br J Opthalmol. 93 (1), 56-60 (2009).</li><li>Dai, 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=Assessment+of+lateral+geniculate+nucleus+atrophy+with+3T+MR+imaging+and+correlation+with+clinical+stage+of+glaucoma.">Assessment of lateral geniculate nucleus atrophy with 3T MR imaging and correlation with clinical stage of glaucoma.</a> Am J Neuroradiol. 32 (7), 1347-1353 (2011).</li><li>McKetton, L., Kelly, K. R., Schneider, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+lateral+geniculate+nucleus+and+optic+chiasm+in+human+albinism.">Abnormal lateral geniculate nucleus and optic chiasm in human albinism.</a> J Comp Neurol. 522 (11), 2680-2687 (2014).</li><li>Jenkinson, M., Bannister, P., 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=Improved+optimization+for+the+robust+and+accurate+linear+registration+and+motion+correction+of+brain+images.">Improved optimization for the robust and accurate linear registration and motion correction of brain images.</a> NeuroImage. 17 (2), 825-841 (2002).</li><li>Dietrich, O., Raya, J. G., Reeder, S. B., Reiser, M. F., Schoenberg, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+signal-to-noise+ratios+in+MR+images:+influence+of+multichannel+coils,+parallel+imaging,+and+reconstruction+filters.">Measurement of signal-to-noise ratios in MR images: influence of multichannel coils, parallel imaging, and reconstruction filters.</a> J Magn Reson Imaging. 26 (2), 375-385 (2007).</li><li>Andrews, T. J., Halpern, S. D., Purves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+size+variations+in+human+visual+cortex,+lateral+geniculate+nucleus,+and+optic+tract.">Correlated size variations in human visual cortex, lateral geniculate nucleus, and optic tract.</a> J Neurosci. 17 (8), 2859-2868 (1997).</li><li>Pfefferbaum, A., Sullivan, E. V., Adalsteinsson, E., Garrick, T., Harper, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+MR+imaging+of+formalin-fixed+human+brain.">Postmortem MR imaging of formalin-fixed human brain.</a> NeuroImage. 21 (4), 1585-1595 (2004).</li></ol>

The authors have nothing to disclose.

This study describes an optimized protocol in acquisition and analysis technique in order to obtain high-resolution PD weighted images of subcortical regions. A number of scanning parameters were tested and modified with the most significant ones pertaining to matrix size, voxel size, and bandwidth to increase the SNR and decrease the number of acquisitions, a critical step in being able to determine high-resolution subcortical structures. In conjunction with finding the optimal parameters within living humans, this rese...

The purpose of this research was to test the resolution limits of structural MRI in the absence of physiological noise. Proton density (PD) weighted images were acquired in a postmortem brain over a long duration (two ~24 hr sessions) to determine the minimum number of images that needed to be registered and averaged to resolve the subcortical structures. For comparison, PD weighted images were also acquired in living humans over a number of sessions. In particular, the objective was to ascertain whether it would be possible in a best-case scenario to resolve all six individual layers of the human LGN, which are approximately 1 mm thick (Figure 1).



<img alt="Figure 1" src="/files/ftp_upload/53309/53309fig1.jpg" /> 
Figure 1.&#160;Human Lateral Geniculate Nucleus layers. Schematic of the laminar structure of the LGN. Magnocellular (M) layers are comprised of larger neuronal cell size and smaller cell density that are responsible for resolving motion and course outlines (layers 1-2, depicted as dark grey). Parvocellular layers (P) are comprised of smaller neuronal cell size and larger cell density that are responsible for resolving fine-form and colour (layers 4-6, depicted as light grey). Scale bar 1 mm. Figure based on stained human LGN 12.
Spatial resolution in MRI is improved when the matrix size is increased, and when field-of view (FOV) and slice thickness are decreased. However, increased resolution decreases the signal to noise ratio (SNR), which is proportional to the voxel volume. SNR is also proportional to the square root of the number of measurements. In living humans, although multiple images can be acquired over a number of separate imaging sessions, the ultimate resolution is limited by physiological noise, such as respiration, circulatory pulsations and head movement.
High-resolution (0.35 mm in-plane voxels) PD weighted scans were acquired. PD scans enhance grey and white contrast in the thalamus1, and result in images that minimize T1 and T2 effects. Its image is dependent on the density of protons in the form of water and macromolecules such as proteins and fat in the imaging volume. The increased numbers of protons in a tissue results in a brighter signal on the image due to the higher longitudinal component of magnetization2.
PD-weighted scans were collected since they provide a higher contrast of subcortical structures with surrounding tissue. Other contrasts, such as T1- and T2-weighted images result in difficulty in delineating subcortical structures like the LGN due to smaller contrast-to-noise ratios, as determined &#402; 1,3.
Likewise, earlier studies found that PD-weighted images of formalin fixed post-mortem brains resulted in higher contrast differences between gray and white matter as compared to T1- and T2-weighted images that had similar grey and white matter image intensities 3,4. The underlying biophysical determinants can explain these differences. T1 (longitudinal) and T2 (transverse) relaxation times of hydrogen protons depend on how water moves within the tissue. Fixatives such as formalin work by cross-linking proteins. The differences between water mobility are reduced between different tissue types when fixatives are used. Reduced T1 tissue contrast has been observed after fixation, whereas the differences in the relative density of protons within brain tissues increased with fixation, providing better contrast differentiation 3, 4.
Previous studies have identified the LGN in PD-weighted scans using a 1.5 T 5,6,7, and at 3 T scanner 8,9. It is critical to obtain these scans to be able to precisely outline the extent of the LGN. To maintain full coverage of the subcortical nuclei, 18 PD-weighted slices were obtained within the thalamus. Each volume was re-sampled to twice the resolution 1024 matrix, (0.15 mm in-plane voxel size), concatenated, motion corrected and averaged to produce a high-resolution 3D image of subcortical structures. The optimum number of PD images required for the following slice prescription was 5, reducing scan time to less than 15 min&#160;in living humans. Only 1 PD image was required to clearly demarcate subcortical regions in the postmortem brain, reducing scan time to less than 3 min&#160;(Figure 2 and 3).
A whole formalin-fixed postmortem brain specimen was scanned from a woman who had died of cardiopulmonary arrest at age 82 years. Review of medical records revealed that she had: chronic obstructive pulmonary disease, angina, triple bypass surgery 8 years prior to death, uterine cancer treated with hysterectomy 7 years prior to death, hyperlipidemia, glaucoma, and cataract surgery. The postmortem brain specimen was immersion-fixed in 10% neutral buffered formalin for at least 3 weeks at 4 &#176;C.The postmortem brain was scanned with the same imaging protocol as well as with other parameters over the course of many hours for image quality comparisons. Only the optimized parameters will be described for the protocol.

<table><tbody><tr><td>Magnetom Trio 3T&amp;nbsp; MRI</td><td>Siemens (Erlangen, Germany).</td><td></td><td></td></tr><tr><td>Vacuum cushion hand</td><td>Siemens</td><td>Mat No: 4765454</td><td>Manufactured by: Johannes-Stark-Stk. 8 D-92224 Amberg</td></tr></tbody></table>

high-resolution structural magnetic resonance imaging of the human subcortex in vivo and postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

Once the subcortex is prescribed within the thalamus, PD weighted images are collected within the slice selection box (Figure 4). The SNR improved by increasing the number of averages in both postmortem and in vivo scans. To determine image quality, the SNR from different scan averages was compared by dividing the signal of the mean brain region by the standard deviation in some area outside the brain. The SNR was calculated as SNR = 0.655 * &#181;tissue/&#963;a...

Watch this Scientific Journal Video about High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem at JoVE.com

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

high-resolution-structural-magnetic-resonance-imaging-human-subcortex

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.3K Views.  York University. Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise. 

Video: High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Biology

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 ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes ...

Medicine

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 ...

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 ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

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

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

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

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

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis