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

The overall goal of the following procedures is to provide a means to allow investigations into the cause and effect of ventricular magaly and to highlight techniques that allow study of ventricular system health and its important barrier and filtration functions within the brain. This is achieved by collecting either coronal sections of the mouse brain or MRI scans of the human brain to generate 3D reconstructions of the ventricles and to calculate ventricle volumes as a second step postmortem tissue that includes en foss preparations of the apical surface of the ventricles is stained with cell specific antibodies to reveal the cellular integrity of the ventricle lining. Next, a montage of the cellular organization at the entire ventricle surface is prepared and rendered over the 3D model of the ventricle to demonstrate regions of gliosis versus intact append cells.

The results show regions of ventricle expansion based on longitudinal 3D modeling of the ventricles and areas where the append themal cell layer of the ventricle lining is no longer intact, but replaced by glial scars. By combining the 3D modeling data with the immunohistochemical data, a 3D region specific map of the cellular composition of the ventricle lining can be generated. The implications of this technique extend toward therapy or diagnosis of brain injury, neurodegenerative diseases such as Alzheimer's disease and normal aging, all of which typically show some level of ventricle expansion and thereby likely ventricle surface scoliosis.

Demonstrating the procedures in mouse will be Richard Wolffer and undergraduate student from our laboratory procedures performed using human MRI data and postmortem human tissue will be demonstrated by yeun. Currently a medical student and Rebecca Chu, a grad student in the lab. Prepare the slides to be imaged by fixing sectioning immuno staining and mounting the mouse brain as outlined in the written portion of the protocol To begin the procedure, to trace the mouse lateral ventricles turn on the upright fluorescence microscope and computer, the microscope should be equipped with an automated stage and a digital CCD camera.

Launch the mapping software in fluorescence mode, open display options to load the toolbars and docking pool panels needed for tracing. Next click options, display options accessories, and select the main and marker toolbars in the same menu. Under docking pool panels, select camera histogram, multi-channel control, camera settings, and image acquisition.

Identify the first piece of tissue containing the lateral ventricles based on strong S 100 beta fluorescence that labels the append cells. Lining the lateral ventricle. Orient the left and right hemispheres by identifying the small incision made during sectioning.

Now create a new data file and designate a reference point for stage movement by clicking in the image window above the left lateral ventricle. Keep this reference point in a consistent location relative to the lateral ventricles across tracing files. To help when importing contours for serial reconstruction, select the appropriate preset contour type from the contour dropdown menu.

Use a unique color for each of the left and right lateral ventricle tracings. Now click along the whole apical surface of the append lining to trace the contour of the ventricle. Move the tracing window using the arrow keys.

After tracing the contour right click and select close contour. Repeat the tracing for the right ventricle using a different color. Next, select the appropriate marker from the markers toolbar on the left, and click to drop a marker on the tracing screen along the midline to assist in aligning serial contours for 3D reconstruction.

Repeat this procedure to make a second mark. Save the tissue traces by selecting file Save data file as and create a new folder and file name number the tracings according to the convention slide number dash tissue number for easy identification of traces from serial sections. Then repeat the procedure just shown for all sections containing a ventricle.

To perform the lateral ventricle 3D reconstruction. Open the 3D reconstruction program. Then click file Open and select the contour file of the first trace serial section.

Import all of the contour tracings of the left and right ventricle, as well as any added markers. Align the contours according to the instructions in the written portion of the protocol. Then select all contours in the left hand panel, and then click the 3D visualization button to display the final 3D reconstruction.

This image shows coronal mouse brain section including lateral ventricles outlined by S 100 beta immunoreactive append cells. The asterisk indicates the lateral ventricles. The brackets delineate the adhesion and the scale bar represents 500 microns.

The mouse lateral ventricles are traced as contours and arranged as subsegment sections. Regions of intraventricular are eliminated from interfering with volumetric analysis. This image shows the final 3D reconstruction of lateral ventricle contours.

The yellow color represents the contour of whole brain volumes spanning the region of interest containing the lateral ventricles. You should have a good understanding of how to create 3D ventricle reconstructions of Corona sliced brain tissue Protocols are listed to create 3D image reconstruction and volumetric quantification of lateral ventricles and to assess volumetric changes over time using longitudinal overlay analysis, it's important to note that consistency in MR.R data collection and post-acquisition processing are extremely important criteria for inclusion of datasets To perform ventricular segmentation, ITK SNAP is used to segment the ventricles from high resolution T one weighted MR images. After starting the program, open grayscale image, select the desired file and open it as a nifty file.

Then scroll through each anatomical plane and note ventricular abnormalities such as nose regions, large or small temporal horns or similar. Next in the main toolbox, click snake, ROI tool. Then segment 3D and select the intensity region option.

Now click pre-process image and select above. Record the maximum intensity using the sliding bar. Then set the threshold to 15%of maximum intensity and record this value.

This should highlight the ROI in white. Set the smoothness parameter to 10. Click okay.

Then next. Next, add 10 to 12 small size two 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.

Next, select parameters and set curvature force to 0.4. Then click the play symbol to begin active contour evolution. Now click update mesh to visualize the surface.

Then check auto update. Stop the segmentation process when all areas of the lateral ventricle are included in the region of interest. Avoid inclusion of the third ventricle.

Finally, click finish and save the segmentation results as a nifty file format. To obtain total ventricle volume, select segmentation, volume and statistics obtain the total volume of the ventricle using set color label. In this segment, longitudinal ROIs are overlaid using mango software to qualitatively and quantitatively demonstrate changes in ventricle volume within subjects over time.

After opening the software, click open, open file load previously saved nifty file. After the image file is loaded, click file load ROI and load the ROI of the baseline ROI time point as green. Click file load ROI and load the second ROI time point as red.

Save the longitudinal overlay by selecting file save ROI assign each image a file name according to the convention now shown on screen. Next, open ITK snap. Then reopen the combined ROI as a gray scale image load combined ROI by selecting segmentation load from image combined ROI file to obtain longitudinal volume data, run the following segmentation volume and statistics.

Label one red equals expansion volume label two green equals stenosis volume label three blue equals base volume, beginning with an intact hemisphere that has been fixed in 10%Formin for a maximum of six weeks and rinsed thoroughly with 0.1 molar PBS slice, 1.5 centimeter thick coronal sections through the entire hemisphere using a large knife using a microsurgical scalpel. Dissect the ventricle wall one centimeter deep, maintaining one continuous section for the entire lateral wall, subdivide the wall as needed to create appropriately sized sections for staining. Well notch the section on the opposite side of the ventricle wall.

To identify the superior inferior orientation, perform immunohisto chemistry and mount the sections according to the written protocol. Under a dissecting scope, secure tissue using pins with a microsurgical stab knife, create a uniformly thin section of lateral ventricle wall and mount a penem aside up onto a slide When the slides are fully dry, examine the sections by immunofluorescence confocal microscopy. Set the imaging focal plane at the ventricle surface as determined by the use of beta-catenin, A marker for apical adherence.

Junction proteins of append cells. Then delineate regions of append cell coverage and surface astro gliosis according to GFAP staining at the ventricle surface to document and montage the entire ventricle surface. Use overlapping images of the entire surface to create a montage of serial images.

Open Adobe Photoshop and use file automate auto emerge interactive layout to select images to overlay, making manual adjustments if necessary. Create a new layer to trace montage to generate a cartoon representation of intact. Depend cell coverage or ventricle surface G scoliosis, compile all sections to reconstruct the map of ventricle wall and link to the corresponding region on the MRI reconstruction.

This is the assembly of human MR images here. The lateral ventricle has been defined as the region of interest in red. The 3D image reconstruction as shown here, allows for volumetric quantification and qualitative visualization of the lateral ventricle.

This image shows the assessment of longitudinal ventricle expansion in an elderly patient. Longitudinal MRIs from multiple points can be aligned and overlaid to visualize and quantify ventricle volume expansion within subjects over time. These last three images show immunohistochemical evaluation and regional mapping of the human lateral ventricle surface.Here.

The areas of intact append cells outlined by beta catine and staining show a cobblestone appearance as indicated by the asterisk and are demarcated by a dotted line from regions of astro gliosis on the ventricle surface outlined by GFAP staining. Serial confocal images are overlaid to generate a regional montage and traced using Adobe Photoshop or a cartoon representation of cellular organization at the ventricle surface. This cartoon image of the montage is mapped to the ventricle surface on the corresponding MRI based 3D ventricle reconstruction.

As shown the green color indicates intact append and the red areas of gliosis. While performing this procedure, it's important to maintain careful documentation of each tissue sample in order to perform serial reconstruction and mapping of cellular integrity. Following this procedure, other methods like subsegmentation of the lateral ventricles can be performed in order to answer additional questions concerning region specific expansion over time.