Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain’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 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.
An ependymal cell monolayer lines the ventricular system of the brain providing bi-directional barrier and transport functions between the cerebral spinal fluid (CSF) and interstitial fluid (ISF) 1-3. These functions help to keep the brain toxicant-free and in physiological balance 2,3. In humans loss of portions of this lining through injury or disease does not appear to result in regenerative replacement as found in other epithelial linings; rather loss of ependymal cell coverage appears to result in periventricular astrogliosis with a meshwork of astrocytes covering regions denuded of ependymal cells at the ventricle surface. Serious repercussions to important CSF/ISF exchange and clearance mechanisms would be predicted to result from loss of this epithelial layer 1,2,4-7.
A common feature of human aging is enlarged lateral ventricles (ventriculomegaly) and associated periventricular edema as observed by MRI and fluid-attenuated inversion recovery MRI (MRI/FLAIR) 8-14. To investigate the relationship between ventriculomegaly and the cellular organization of the ventricle lining, postmortem human MRI sequences were matched with histological preparations of lateral ventricle periventricular tissue. In cases of ventriculomegaly, substantial areas of gliosis had replaced ependymal cell coverage along the lateral ventricle wall. When ventricle expansion was not detected by MRI-based volume analysis, the ependymal cell lining was intact and gliosis was not detected along the ventricle lining 6. This combinatorial approach represents the first comprehensive documentation detailing changes in cellular integrity of the lateral ventricle lining using wholemount preparations of portions or the entire lateral ventricle wall and 3D modeling of ventricle volumes 6. Several diseases (Alzheimer’s disease, schizophrenia) and injuries (traumatic brain injury) show ventriculomegaly as an early neuropathological feature. Denudation of areas of the ependymal cell lining thereby would be predicted to interfere with normal ependymal cell function and compromise the homeostatic balance between CSF/ISF fluid and solute exchange. Thus, a more thorough examination of changes to the ventricular system, its cellular composition, and the consequence to underlying or neighboring brain structures will ultimately begin to reveal more about the neuropathology associated with ventricle enlargement.
The lack of multimodal imaging data, and in particular longitudinal data sequences, together with limited access to corresponding histological tissue samples makes analysis of human brain pathologies difficult. Modeling phenotypes found in human aging or disease can often be achieved with mouse models and animal models become one of our best means to explore questions about human disease initiation and progression. Several studies in healthy young mice have described the cytoarchitecture of the lateral ventricle walls and the underlying stem cell niche 4,7-15. These studies have been extended to include 3D modeling and cellular analysis of the ventricle walls through aging 6,15. Neither periventricular gliosis nor ventriculomegaly are observed in aged mice, rather mice display a relatively robust subventicular zone (SVZ) stem cell niche subjacent to an intact ependymal cell lining 6,15. Thus, striking species-specific differences exist in both the general maintenance and integrity of the lateral ventricle lining during the process of aging 6,15. Therefore, to best use mice to interrogate conditions found in humans, differences between the two species need to be characterized and appropriately considered in any modeling paradigm. Here, we present procedures to evaluate longitudinal changes to the lateral ventricles and associated periventricular tissue in both humans and mouse. Our procedures include 3D rendering and volumetry of both mouse and human ventricles, and use of immunohistochemical analysis of whole mount preparations of periventricular tissue to characterize both cellular organization and structure. Together these procedures provide a means to characterize changes in the ventricular system and associated periventricular tissue.
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
1.2) Immunohistochemistry for Lateral Ventricle Analysis
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.
1.4) Lateral Ventricle 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.
2.2) Human Periventricular Tissue Preparation and Analysis
Contour tracing of the mouse lateral ventricles based on immunostained 50 µm coronal sections and 3D reconstructions (Figure 3) allows volume data to be collected in different experimental paradigms using mouse as a model system for disease or injury. Critical to this procedure is the exclusion of regions where the lateral ventricle walls adhere to each other. By subsegmenting regions of the ventricles and designating a different color for each region (Figure 3C), contiguous sections can be complied and regional and total volume can be calculated from compiled subsegments.
Similar studies can be performed using MRI scans of the brain together with semi-automated segmentation (ITK-SNAP) of the lateral ventricles for 3D renderings. For direct pairing of ventricle volumes and periventricular tissue analysis, pre- or post-mortem MRI scans are segmented and aligned to create 3D reconstructions (Figure 4). If longitudinal MRI scans are available, 3D reconstructions can be aligned and overlaid for analysis of expansion over time (Figure 5). Longitudinal analysis provides information about regions of particular interest for future immunohistochemical analysis. Corresponding tissue is analyzed immunohistochemically to reveal regions of astrogliosis versus intact ependymal cell monolayer at the ventricle surface (Figure 6A). Tissue images are captured and then montaged to cover large areas of ventricle surface (Figure 6B). Compiled montages are converted to cartoon representations and then mapped onto an MRI-based 2D model to show regional alterations in periventricular cellular integrity (Figure 6C). Whole mount preparation of the ventricle wall allows for panoramic view of large expanses of the ventricle surface, or as we have demonstrated the entire lateral ventricle surface 6. Increased levels of Aquaporin-4 expression in areas of surface gliosis suggesting edema can be used to assess ventricle lining integrity 6.
Figure 1: Performing lateral ventricle segmentation using the ITK/Snap Snake ROI Tool (A) ‘Bubbles’ are added to lateral ventricles. (B) ‘Bubbles’ expand during active contour evolution. (C) Segmentation is complete when entire lateral ventricle is filled. (Care is taken to avoid the 3rd ventricle.)
Figure 2: Human lateral ventricle wall dissection (A-D) Intact hemisphere is cut into 1.5 cm thick coronal slabs. Isolated 2D representation of ventricle is shown in (D). (E) A section of ventricle wall is dissected out and processed for IHC. Subdivision of tissue may be required depending on size and curvature. To maintain orientation, tissue is notched at top on opposite side of ventricle surface (green). Pins (black dots) are used to secure tissue and guide final dissection (red dashed line). Final whole mount preparation is mounted on slide with ventricle surface facing up (green).
Figure 3: Tracing and 3D reconstruction of mouse lateral ventricles (A Coronal mouse brain section including lateral ventricles, outlined by S100β-immunoreactive ependymal cells. (*, Lateral Ventricles; bracket, region of adhesion; scale bar, 500 µm). (B) Mouse lateral ventricles are traced as ‘contours’ and arranged as sub-segmented sections, excluding regions of intraventricular adhesion from interfering with volumetric analysis. (C)3D reconstruction of lateral ventricle contours. Yellow, contour of whole brain volume spanning the region of interest containing the lateral ventricles.
Figure 4: MRI-based lateral ventricle segmentation (A) MR Images are assembled. (B) Lateral ventricle is defined as ROI (red). (C) 3D image reconstruction allows for volumetric quantification and qualitative visualization of lateral ventricle.
Figure 5: Assessment of longitudinal ventricle expansion Longitudinal MRIs from multiple points can be aligned and overlaid to visualize and quantify ventricle volume expansion within subjects over time.
Figure 6: Immunohistochemical evaluation and regional mapping of human lateral ventricle surface (A) Areas of intact ependyma cells, outlined by β-catenin staining, show a cobblestone appearance (asterisk) and are demarcated by a dotted line from regions of astrogliosis on ventricle surface (GFAP+ staining). (B) Serial confocal images are overlaid to generate a regional montage and traced using Adobe Photoshop for a cartoon representation of cellular organization at the ventricle surface. (C) Cartoon of image montage is mapped to ventricle surface on corresponding MRI based 3D ventricle reconstruction. (Scale bar (A), 40 µm; Scale bar (B), 1 mm)
We present tools and protocols that can be used to evaluate the integrity of the brain’s ventricular system in mice and in humans. These tools, however, can also be applied to other brain structures or organ systems that undergo changes due to injury, disease, or during the process of aging 14,21,22. The strategies presented take advantage of software that allows the alignment of cross-sectional and longitudinal MRI sequences to generate 3D volume representations of specific regions or structures of interest. Longitudinal MRI sequences allow compilation of 3D volume changes that occur over time and can be extended to include total brain volume, for lateral ventricle to total brain volume ratios, and/or other brain structures (e.g., subthalamic nucleus 23 or corpus callosum white matter tracts using diffusion-weighted tensor imaging). Together, a comprehensive analysis of brain structural changes can be performed. Ultimately, to evaluate age-related and disease-related changes to brain structures a collection of multimodal imaging techniques are needed to provide the most accurate picture of brain health status 24. Documentation and compilation of critical structural data, together with subject-subject variability and the range of variability across subjects, could ideally be used to generate ultra-high field MRI atlases to best guide clinical diagnosis of tissue in health or disease.
Several critical steps are important to note in order to decrease acquisition and processing variability between and within subject data sets. Ideally to obtain matched data sequences, the same MRI scanner and sequence type should be used throughout the study. Postmortem brain tissue is often of varying quality; critical factors such as the cause of death, the postmortem interval, quality of the perfusion, and length of time in fixative all may affect staining efficacy and the need for an antigen retrieval protocol. In addition, the size of the human brain requires multiple slice sections with each slice requiring further manipulation to obtain tissue containing the regions of interest. Therefore, it is critical that the location and orientation of each section is clearly marked and recorded. Ultimately, the primary problem with postmortem tissue analysis is precisely that – it is postmortem – and therefore provides only a snapshot view of the tissue at the end of life. Improved multimodal imaging techniques is required for real-time analysis of changes to brain structures and morpho-functional information with cellular resolution.
Mouse studies allow interrogation of the human condition variable by variable and do not present many of the difficulties found when working with human tissue. Mouse studies used to model human disease or injury through analysis of causal structural dynamics may offer improvements for disease-model validation. However, species-specific differences should be noted. In our analysis of the lateral ventricles, it is important to note that mice retain an active stem cell niche along the lateral wall of the lateral ventricles and stem cell-mediated regenerative repair of the ventricle lining occurs in cases of modest ependymal cell loss, as found in aged mice or when limited ependymal cell denudation occurs through exposure of the ependyma to neuraminidase 19. In contrast, humans do not maintain a robust stem cell niche along the lateral ventricle walls 25-27 and little, if any, regeneration is likely. In contrast to aged mice, aged humans show extensive astrogliosis at the ventricle surface associated with ventricle expansion6. This level of denudation and ‘scarring’ of the ventricle lining can be modeled in mouse using neuraminidase to denude the ventricle lining of ependymal cells 6,28. In addition, it is important to recognize that vivarium-maintained mice do not experience infections, disease or trauma, presenting a much different medical history from most human subjects. Furthermore, mice typically do not show age-associated neurodegenerative disease, ventriculomegaly or periventricular gliosis. Therefore, to observe these phenotypes they have to be modeled. In the end, no animal model can fully recapitulate the ontogeny, pathophysiology, and symptomatic complexity of human disease and these limitations need to be acknowledged and properly communicated.
In summary, we present techniques and protocols to assess lateral ventricle volumes in mouse and human. Ventricles can be rendered in 3D and longitudinal analysis allows compilation of spatiotemporal volume changes. In addition, using paired brain tissue we demonstrate how cellular features can be linked to changes in ventricle volumes. Recent results demonstrating increased levels of Aquaporin-4 expression in areas of periventricular gliosis suggest edema along the ventricle surface. Therefore future investigations pairing FLAIR-MRI with tissue histology will improve our understanding of CSF and interstitial fluid exchange, and provide critical information about ventricle system health and how its deterioration affects various brain functions.
The authors have nothing to disclose.
An NINDS Grant NS05033 (JCC) supported this work. The University of Connecticut RAC, SURF and OUR programs provided additional support.
Name of the Materal/Equipment | Company | Catalog Number | Comments/Description |
Phosphate buffered saline (PBS) | Life Technologies | 21600-069 | |
Paraformaldehyde (PFA) | Electron Microscopy Sciences | 19210 | Use at 4% in PBS, 4 °C |
Normal Horse Serum | Life Technologies | 16050 | 10% in PBS-TX (v/v) |
Normal Goat Serum | Life Technologies | 16210 | 10% in PBS-TX (v/v) |
Triton X-100 (TX) | Sigma-Aldrich | T8787 | 0.1% in PBS (v/v) |
Vibratome | Leica | VT1000S | |
Fluorescence Microscope | Zeiss | Imager.M2 | |
Camera | Hamamatsu | ORCA R2 | |
Microscope Stage Controller | Ludl Electronic Products | MAC 6000 | |
Stereology software | MBF Bioscience | Stereo Investigator 11 | |
Stereology software | ImageJ/NIH | NIH freeware | |
3D Reconstruction software | MBF Bioscience | Neurolucida Explorer | |
Confocal Microscope | Leica | TCS SP2 | |
MRI Software | |||
Freesurfer | https://surfer.nmr.mgh.harvard.edu/fswiki/DownloadAndInstall | Segmentation and Volume | |
ITK-Snap | http://www.itksnap.org/pmwiki/pmwiki.php | Segmentation and Volume | |
Multi-image Analysis GUI (Mango) | http://ric.uthscsa.edu/mango/ | Longitudinal overlay | |
Whole Mount Equipment | |||
22.5° microsurgical straight stab knife | Fisher Scientific | NC9854830 | |
parafilm | |||
wax bottom dissecting dish | |||
pins | |||
fine forceps | |||
aquapolymount | |||
Dissecting Microscope | Leica | MZ95 | |
Whole Mount Antibodies | |||
mouse anti-b-catenin | BD Bioschiences, San Jose, CA, USA | 1:250 | |
goat anti-GFAP | Santa Cruz Biotechnology | 1:250 | |
rabbit anti-AQP4 (aquaporin-4) | Sigma-Aldrich | 1:400 | |
Coronal Antibodies | |||
Anti-S100β antibody | Sigma-Aldrich | 1:500 | |
4’,6-diamidino-2-phenylindole (DAPI) | Life Technologies | D-1306 | 10 µg/mL in PBS |