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

NOTE: Animal procedures were approved by the University of Connecticut IACUC and conform to NIH guidelines. Human tissue and data analysis and procedures were in compliance with and approved by the University of Connecticut IRB and conform to NIH guidelines.

1. Mouse: Analysis of Periventricular Cellular Integrity and 3D Modeling of the Lateral Ventricle

1.1) Preparation of Mouse Lateral Ventricle Wall Whole Mounts

1. Prepare mouse lateral ventricle whole mounts for immunohistochemistry (IHC) as previously described ^16,17.

1.2) Immunohistochemistry for Lateral Ventricle Analysis

1. Fix and section mouse brain tissue as previously described ^18,19.
   1. Briefly, flush mice transcardially with normal, room temperature saline, until organs have cleared (indicated by a pale coloration), followed by room temperature 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS) until tissue has stiffened.
   2. Remove brain from the skull. Use iris dissection scissors to cut from base of skull along the sagittal suture.
   3. Expose skull and remove brain with forceps. Immerse the brain in 4% PFA fixative overnight at 4 °C. Then wash 3 times for 20 min with PBS.
2. Prepare serial sections for immunohistochemistry and volume analysis.
   1. Using a microsurgical scalpel, make a small incision longitudinally along the cortex of the left hemisphere to allow the identification of the left hemisphere from the right hemisphere when floating sections are immunostained.
   2. Encase fixed brains in 3% agarose for added structural support during vibratome sectioning. Warm 3% agarose in distilled water (w/v) until completely dissolved using either a microwave or hot plate.
   3. With a razor blade, remove the caudal section of the brain at the cerebellum with a coronal cut, leaving a flat and even surface. Apply super glue to the tissue stage and position the brain on the newly cut surface with the olfactory bulbs facing upward.
   4. When liquid agarose solution has cooled to a temperature just warm to touch, apply evenly over the brain to completely encase the entire brain. Allow the agarose to solidify.
   5. Section agarose encased brains coronally on a vibratome into 50 µm sections, with sections placed serially into a 24-well plate containing PBS in order to preserve sequential order.
      NOTE: Generally 12 wells are used for one brain, with collection of tissue beginning 5 sections prior to seeing the emergence of the lateral ventricles beginning with well A1, moving numerically through to B6 and cycling back to A1. This allows multiple distinguishable sections from the same brain to be processed within the same well. For lateral ventricle volume analysis, tissue collection ceased when the lateral ventricles and third ventricle merge, resulting in approximately 3 sections per well or 36 sections total.
   6. Aspirate the PBS using a transfer pipette affixed with a 20-200 µl micropipette tip to ensure that floating sections are not accidently aspirated. Block and permeabilize floating sections in 10% serum, 0.1% Triton X-100 (TX) in PBS (PBS-TX) for 1 hr at room temperature. Use 300 µl of solution per well to cover free-floating tissue sections in a 24-well plate and perform all incubations of sections on a rocking plate.
   7. Aspirate the blocking solution, and incubate sections with primary antibodies in PBS-TX overnight (at least 12 hr) at 4 °C. For ventricle volume traces using coronal sections, use anti-S100β antibody to label ependymal cells.
   8. Aspirate primary antibody solution, and wash sections 3 times for 10 min each with PBS-TX.
   9. Incubate sections in dark for 1 hr at room temperature with fluorescent secondary antibodies, at concentrations of 1:1,000 in 10% serum, PBS-TX. Keep sections in the dark for all remaining incubation steps.
   10. Aspirate secondary antibody solution and incubate sections with 4’,6-diamidino-2-phenylindole in PBS (DAPI, 10 µg/ml) for 5 min to counterstain cell nuclei. Aspirate the DAPI solution and rinse sections 3 times for 10 min each with PBS.
   11. Mount sections serially onto slides using the cortical mark to preserve left versus right hemisphere orientation. Air dry sections in the dark and then coverslip by applying a thin layer of mounting media to the slide and gently place a glass coverslip on top. Allow coverslipped slides to dry overnight at room temperature.

1.3) Lateral Ventricle Segmentation for 3D Reconstructions

NOTE: Perform tracing of lateral ventricles using mapping software on an upright epifluorescence microscope with an automated stage and a digital CCD camera for fluorescence detection.

1. Turn on the fluorescence microscope equipment and launch mapping software in fluorescence mode. Open ‘Display Options’ to load the proper toolbars and docking tool panels needed for tracing. Options>Display Options>Accessories, and select the ‘Main’ and ‘Marker’ toolbars. In the same menu, select ‘Camera Histogram’, ‘Multichannel Control’, ‘Camera Settings’, and ‘Image Acquisition’ under ‘Docking Tool Panels.’
2. Observe beginnings of the ventricles based on strong S-100β fluorescence that labels ependymal cells that line the lateral ventricle. Identify the first piece of tissue containing the lateral ventricles.
3. Create a new data file and designate a reference point for stage movement by clicking in the image window above the left lateral ventricle. Look for the small incision to orient left from right hemisphere. Keep the reference point in a consistent location relative to the lateral ventricles across tracing files to help when importing contours for serial reconstruction.
4. Select appropriate preset contour type from the contour drop down menu, e.g., ‘Left Lateral Ventricle.’ Use a unique color for each of the left and right lateral ventricle tracings. If changes in the contour name and color are required, go to Options>Display Preferences>Contours, then modify the names of the contour colors or select ‘Add Contour Type’.
5. With the ‘Left Lateral Ventricle’ contour selected, click along the apical surface of the ependymal lining to begin the trace contour. Trace the ventricle by continuing to click successively along the apical surface.
   1. For irregular areas requiring more finesse, click and hold the left mouse button in order to trace free hand. Press Ctrl+Z, or go to Options>Undo to undo the previous contour point. Move the tracing window using the arrow keys or by making a contour point off screen and allowing the window to automatically center itself. To finish the ventricle tracing right click and select ‘Close Contour’.
6. Select a new contour color (contour type) for the right lateral ventricle using the drop down menu. Trace the right ventricle as in Step 1.3.4 for the left.
7. If changes in the contour name and color are required, right click on the contour and select Change Contour Type to select the appropriate color.
8. Add markers along the midline to assist in aligning serial contours for 3D reconstruction. To add markers, select the appropriate marker (use the ‘X’) from the Markers toolbar on the left and click to drop them on the tracing screen. Import markers along with the contours for each tissue section trace.
9. To save the tissue traces, select File>Save Data File As and create a new folder and file name. Number the tracings [slide #]-[tissue #] for easy identification of traces from serial sections. For example, the third tissue section on the second slide has the file name ‘2-3’ within the directory for each brain.
10. Repeat Steps 1.3.4 to 1.3.9 for each serial section of brain tissue to trace the ventricles through the entire brain, excluding regions of ventricle wall adhesion.
11. If the ventricle has a region of adhesion, sub-segment the anterior region from those dorsal and ventral to the adhesion site. Create two new contour types for each ventricle (e.g., ‘Dorsal Left Ventricle’ and ‘Ventral Left Ventricle’). On the first tissue slice with adhesion, trace the lateral ventricle with both the original lateral ventricle contour and the new dorsal and ventral contours to ensure a complete ventricle reconstruction can be created.
12. In sections posterior to a ventricle wall adhesion, create a set of new contour colors for the posterior portion of each ventricle (e.g., ‘Posterior Left Ventricle’). Double-trace this first re-joined section with both the current adhesion and new posterior contour types.

1.4) Lateral Ventricle 3D Reconstruction

1. Align and compile serial contour tracings of the mouse lateral ventricles to generate 3D reconstructions and volumetric data.
2. Open 3D reconstruction program. Click File>Open and select the contour file of the first traced serial section. Import the contour tracings of the left and right ventricle as well as any added markers.
3. To select contours, click the ‘Select Objects’ button (cursor icon) and select the two ventricles and markers by holding ‘Ctrl’. To establish the Z position of the contours, right click and select ‘Modify Z Position’. Set the first tissue slice to 0 µm.
4. To save the reconstruction, select File>Save Data File As. Save after every file import, so if a mistake is made recovery is as easy as re-loading the previous file.
5. To add the next tissue slice to the reconstruction, click File>Append to Display. Select the .DAT file to be appended, and check the ‘Merge’ box.
6. Follow Step 1.4.3 to again adjust the Z position of the newly imported trace file. Select only the newly added left and right contours in the main window to avoid moving the other traces. Set each successive trace file to 50 µm from the Z position of the previous contour for 50 µm sections. (First contour: z = 0, second contour: z = 50, third contour: z = 100, etc.)
7. To adjust the X, Y position of selected contours and markers, click the ‘Enable Translation with Mouse’ button and drag the contours to align with the previous tissue contours. Keep both the left and right contours of the current trace selected to allow synchronous movement.
8. To rotate the traces clockwise or counter clockwise to line up the midline markers, select the ‘Z-axis Rotation’ button. To flip contours across the Y-axis (if the left contour is on the right) select both imported contours and markers, right click, select ‘Set Rotation Angle’, and enter ‘180’ into the ‘Y rotation’.
9. Ensure that the ventricles and midline markers are best aligned before saving the reconstruction file, as in Step 1.4.4.
10. Repeat Steps 1.4.5 to 1.4.9 for each subsequent tissue slice until all have been imported and properly aligned.
11. To view volume data of the serial reconstruction, click Analysis>‘Markers and Region Analysis’ and select ‘3D Contour Summary’. Select all contours in the left hand panel and then click the ‘3D Visualization’ button to display the final 3D reconstruction.

2. Human: Analysis of Periventricular Cellular Integrity and 3D Modeling of the Lateral Ventricle

2.1) Human MRI Data Analysis

NOTE: Protocols are listed to create 3D image reconstructions and volumetric quantification of the lateral ventricles and assess volumetric changes over time using longitudinal overlay analysis. It is important to note that consistency in MR data collection (e.g., machine and magnet strength, section thickness, orientation and resolution) and post-acquisition processing are extremely important criteria for inclusion of data sets ²⁰.

1. Ventricular Segmentation
   NOTE: ITK/Snap is used to segment the ventricles from high-resolution T1-weighted MR images. Alternatively, other freeware software such as Freesurfer can be used.
   1. Open ITK/Snap, File > Open Grayscale Image. Select desired file and open as a .nii file (NITFI file).
   2. Scroll through each anatomical plane, noting ventricular abnormalities (stenosed regions, large or small temporal horns, etc.). In the Main Toolbox click ‘Snake ROI Tool’. Click ‘Segment 3D’. Select the ‘Intensity Region’ option.
   3. Click ‘Preprocess Image’. Select ‘Above’, to include regions below a certain intensity threshold in order to highlight ROI in white. Record maximum intensity using sliding bar. Set the threshold to 15% of maximum intensity (record this value). Set smoothness parameter to 10. Click ‘OK’, then ‘Next’.
   4. Add 10-12 small (size 2) ‘bubbles’ to each ventricle: two in the anterior frontal horn, seven evenly-spaced along the superior surface of the lateral ventricle body, one in the occipital horn and one in the temporal horn of the lateral ventricle. ‘Select Parameters’ and set curvature force to 0.4. Click the Play Symbol to begin active contour evolution.
   5. Click ‘Update Mesh’ to visualize surface; check ‘Auto-Update’. Stop segmentation when all areas of the lateral ventricle are included in the region of interest (ROI). Avoid inclusion of the third ventricle. Click ‘Finish’.
   6. Manually edit image if necessary to remove undesired regions by the following methods (typically need to erase third ventricle leakage). Manually use the ‘3D Scalpel’ tool to remove excess regions or manually use the ‘Paintbrush’ tool with ‘Clear Label’ as the active drawing label to erase any inclusion beyond ROI.
   7. Save segmentation results as a .nii image (‘NIFTI’ file format).
   8. To obtain total ventricle volume, select ‘Segmentation>Volume’ and Statistics; obtain the total volume of the ventricle using set color label.
2. Longitudinal Representation Of Lateral Ventricles From MRIScans
   NOTE: Using Mango software, longitudinal ROIs are overlaid to qualitatively and quantitatively demonstrate changes in ventricle volume within subjects over time.
   1. Open Mango. Click File>Load ROI of the first ROI time point (baseline) as GREEN. File>Load ROI of the second ROI time point as RED. Save longitudinal overlay by selecting File>Save as; assign each image as ‘file name_[first time point age]-[second time point age].nii’.
   2. Open ITK-SNAP. Reopen the combined ROI as ‘greyscale image’, by selecting Segmentation>Load From Image>combined ROI file. To obtain longitudinal volume data run the following: Segmentation>Volume and Statistics>Label 1 (red) = expansion volume; Label 2 (green) = stenosis volume; Label 3 (blue) = base volume.

2.2) Human Periventricular Tissue Preparation and Analysis

1. Safety instructions for working with human tissue
   1. Obtain appropriate safety training (e.g., Blood-borne Pathogens course).
   2. Observe standard (universal) safety precautions. Wear gloves. Change gloves before touching any common equipment or surfaces that are not disposable. Label any items routinely handled when working with human tissue with ‘human tissue use’ designations.
   3. Follow decontamination practices for all items contacting human tissue. Bleach all contaminated liquids for a minimum of 24 hr prior to disposal, treat contaminated surfaces with a bleach soaked towel (e.g., bench top) or by placing object directly in bleach (e.g. forceps).
   4. Prepare contingency plan for contact with human tissue directly on skin, which may include soaking a paper towel in bleach and holding on the affected area (5-10 min).
   5. Use appropriate waste disposal for all items coming in contact with human tissue.
2. Lateral Ventricle Whole Mount Preparation
   1. Beginning with an intact hemisphere (fixed in 10% formalin and rinsed thoroughly with 0.1 M PBS), slice 1.5 cm thick coronal sections through entire hemisphere using large knife.
      NOTE: All fixation protocols require prior antibody verification.
   2. Label sections front to back starting with first section containing the lateral ventricle. Use an alpha-numeric labeling system, labeling the slices with letters (anterior to posterior) and subsection with numbers (top down). Avoid disrupting ependymal surface.
   3. Using microsurgical scalpel, dissect ventricle wall 1 cm deep, maintaining one continuous section for entire lateral wall. Subdivide wall as needed to create appropriately sized sections for staining well. Notch section on opposite side of ventricle wall to identify superior/inferior orientation.
   4. Perform antigen retrieval by incubating tissue sections in 10 mM sodium citrate buffer (pH 6.0) at 100 °C for 10-20 min using a double boiler (optimal incubation time is determined empirically). Rinse 3 times in PBS/0.1%Triton X-100 (PBS-TX).
   5. Block in 10% horse serum using PBS/ PBS-TX for 1 hr at room temperature.
   6. Incubate with primary antibodies for 48 hr at 4 °C. To visualize ventricle wall lining, immunostain whole mounts with the following antibody combinations: mouse anti-β-catenin (1:250); goat anti-GFAP (1:250); rabbit anti-AQP4 (1:400).
   7. Perform all subsequent steps in the dark. Rinse tissue 3 times in PBS, incubate with secondary antibodies (1:500) for 24 hr at 4 °C or 2 hr at room temperature, and rinse another 3 times in PBS. Incubate tissue in DAPI for 7 min at room temperature followed with another 3 rinses in PBS.
   8. Prepare tissue for final dissection by covering a wax bottom dish with parafilm. Fill dish with PBS, place tissue in dish using fine forceps, avoiding making contact with ependymal wall.
   9. Under the dissecting microscope, firmly secure tissue on its side using pins. With a 22.5° microsurgical stab knife, create uniform thin sections (approximately 300 µm thick) of lateral ventricle wall. For large sections or sections with difficult curvature, cut into smaller cubes before cutting into final sections for mounting and note location.
   10. Carefully place dissected section ependyma side up onto slide using fine forceps, taking note of regional location and orientation on the slide. Cover tissue with thin layer of aquapolymount, taking care to avoid bubbles. Place coverslip over tissue, add additional aquapolymount as necessary to fill any gaps under coverslip.
   11. Place mounted sections in dark and dry for 2-3 days at room temperature. Store in slidebox at 4 °C.
3. Immunohistochemistry and Histological Analysis
   1. Using confocal microscopy to view fluorescent immunohistochemistry, set the imaging focal plane at the ventricle surface, as determined by the use of β-catenin (marker for apical adherens junction proteins of ependymal cells). Delineate regions of ependymal cell coverage and surface astrogliosis (GFAP staining at ventricle surface).
   2. To document and montage the entire ventricle surface, use overlapping images to cover entire surface. To create montage of serial images, open Adobe Photoshop. Use File>Automate>Photomerge>Interactive layout to select images to overlay, making manual adjustments if necessary.
   3. Create new layer to trace montage to generate cartoon representation of intact ependyma cell coverage or ventricle surface gliosis. Repeat for each slide or ROI. Compile all sections to reconstruct map of ventricle wall and link to corresponding region on MRI reconstruction.