The authors thank Voltaire Gungab for assistance with manuscript preparation. This work was supported in part by grants from: the National Institutes of Health (T32 GM008440 to E.J. Walker, R01 NS27713 to W.L. Young, P01 NS44155 to W.L. Young and H. Su), and R21 NS070153 to H. Su; and the American Heart Association (AHA 10GRNT3130004 to H. Su).

1. Clearing Blood from Vasculature
<ol>
	<li>Anesthetize mouse by intraperitoneal injection of 100 mg/kg ketamine with 10 mg/kg xylazine diluted in 0.25 ml saline.</li>
	<li>Once anesthesia has been confirmed by no response to a paw pinch, use scissors to make an incision across the chest with scissors just below the xyphoid process, cut through diaphragm, and cut up between dorsal and ventral segment of ribcage to expose thoracic cavity. Expose heart by folding up sternum and adjacent chest wall and secure with hemostat. Open the pericardial sac with tweezers to expose the heart.</li>
	<li>Turn on perfusion pump (100mmHg pressure) connected to warm PBS plus heparin (5 unit/ml) solution in hot water bath at 37&deg;C and tubing with a blunt 22 gauge needle on the outflow.</li>
	<li>Insert the needle into the left ventricle, near the apex of the heart. Immediately cut open the right atrium to enable systemic blood outflow and continue flushing until blood is removed from circulation.</li>
</ol>
2. Perfusion of Casting Agent into the Vascular System
<ol>
	<li>Prepare Microfil (Flow Tech, Inc. Carver, MA) casting solution per manufacturer's protocol: Mix 5 ml of MV diluent with 4 ml of filtered MV compound. Add 450&mu;l (5%) of catalyst (MV curing agent). Solution working time is 20 minutes, but mix immediately before use to ensure proper viscosity.</li>
	<li>Withdraw casting agent solution into a 10ml syringe, attach a blunt 20 gauge needle to the syringe and fill needle with solution.</li>
	<li>Insert needle into the left ventricle through the same hole used for flushing the blood. Secure needle into place with hemostat clamp on the heart.</li>
	<li>Inject casting agent solution slowly into the left ventricle at approximately 3 ml/min. Excess pressure may lead to incorrect flow of casting agent or potential vessel rupture.</li>
	<li>Look for signs of successful perfusion, including: visualization of casting agent in intestine and liver vasculature, distal limb discoloration, and nose and tongue discoloration. Readjust needle if necessary to achieve proper perfusion.</li>
</ol>
3. Collection and Processing of Tissue
<ol>
	<li>Remove brain and place in 4% paraformaldehyde overnight at 4&deg;C.</li>
	<li>Dehydrate brain by placing from paraformaldehyde directly into increasingly concentrated EtOH at room temperature: 25% EtOH, 1 day; 50% EtOH, 1 day; 75% EtOH, 1 day; 95% EtOH, 2 days; and 100% EtOH, 2 days.</li>
	<li>Clarify brain tissue around blood vessels by immersing the perfused brain in methyl salicylate for 2 days at room temperature.</li>
	<li>Vasculature of the whole unsectioned clarified brain can be imaged with brain immersed in methyl salicylate under a dissecting microscope (Fig. 1) (Leica MZFL III microscope, Leica Microsystems, Bannockburn, IL) or brightfield microscope (Fig. 2) (Leica DM/LS, Leica Microsystems).</li>
	<li>Reclarification can be done if tissue is not completely cleared. From methyl salicylate, transfer brain to 95% EtOH for 2 days, 100% EtOH for 2 days, and methyl salicylate for 2 days.</li>
</ol>
4. Micro-CT Imaging or Histological Application- Tissue Preparation
<ol>
	<li>The brain can be imaged using micro-CT directly after clarification or after re-hydration (Fig. 4).</li>
	<li>To prepare brain tissue for histological analysis, rehydrate the brain through serial diluted EtOH: 100% EtOH, 1 day; 95% EtOH, 1 day; 75% EtOH, 1 day; 50% EtOH, 2 days; 25% EtOH, 2 days; and store in PBS. The brain can then be embedded in paraffin using a standard procedure.</li>
</ol>
5. Representative Results
Following vascular casting, the cerebrovasculature can be seen macroscopically on the brain surface (Fig.1A). The vascular tree inside the parenchyma can be visualized following clarification (Fig. 1B). The detailed vascular structure can be imaged and analyzed under a brightfield microscope (Fig. 2), dissecting scope (Fig. 3), and with micro-CT scanning (Fig. 4). Using brightfield microscopy, the vasculature can be viewed at different focal planes without sectioning the brain (Fig. 2A) as well as individual microvessels at higher magnification (Fig. 2B). Under a dissecting scope, one can see the entire vascular structure in one focal plane, allowing better visualization of the three dimensional structure of the cerebrovasculature (Fig. 3). Images taken under a dissecting microscope can show large vessel morphology and whole brain vasculature, but lack the details shown in pictures taken with brightfield microscopy (Fig. 2B). This technique has allowed us to detect irregular vessels in our experimental BAVM adult mouse model2. Figure 5 shows an example of the same irregular vessels detected by micro-CT scanning (Fig. 5A) and brightfield microscope (Fig. 5B). Together, this data shows that we can analyze the same sample with multiple imaging tools after vascular casting.
<img alt="Figure 1" src="/files/ftp_upload/2958/2958fig1.jpg" /> 
Figure 1. Whole mouse brain following vascular casting. Before (A) and after (B) clarification by methyl salicylate solvent. Vessels can be clearly visualized on the brain surface (A) and in parenchyma following clarification procedure (B).
<img alt="Figure 2" src="/files/ftp_upload/2958/2958fig2.jpg" /> 
Figure 2. Brightfield microscope images of clarified vascular casted brain. Brain can be imaged at various planes and at different magnification levels. Scale Bar 200&mu;m (A) and 50&mu;m (B).
<img alt="Figure 3" src="/files/ftp_upload/2958/2958fig3.jpg" /> 
Figure 3. Dissecting microscope image at 1x showing whole adult mouse brain cerebrovasculature from a dorsal view.
<img alt="Figure 4" src="/files/ftp_upload/2958/2958fig4.jpg" /> 
Figure 4. Micro-CT scanning image of the same whole adult mouse brain shown in Figure 3 from a dorsal view.
<img alt="Figure 5" src="/files/ftp_upload/2958/2958fig5.jpg" /> 
Figure 5. Application of vascular casting method to detect a brain arteriovenous malformation. A) Micro-CT scan of vascular casted brain shows region of enlarged, irregular AVM-like vessels in right hemisphere from a ventral view. Enlarged image of region (white box) is shown on the right. B) Same irregular vessels visualized under brightfield microscope at 50x magnification. Scale bar 50 &mu;m.

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Experimental procedures for using laboratory animals were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (UCSF).

We report here a method for casting of adult mouse cerebrovasculature that may be imaged with various modalities. Application of this method to the disease model BAVM enables 3D analysis of irregular vessels. Potential future studies include quantification of micro-CT images, histological analysis of tissue, and diagnosis of vascular disease progression.
Common Problems Encountered and Suggestions
Complete perfusion of the brain vasculature is not always achieved. Having enough pressure to reach the distal blood vessels without rupturing the left atrium or aorta is a challenge. To overcome this, apply consistent pressure of approximately 100mmHg. Also, adjust needle position to ensure appropriate outflow of casting agent into the aorta. It is important that the carotid arteries are not restricted so the casting agent can reach the brain vessels. Critical procedures that correlate with improved small vessel perfusion are: (1) proper clearing of the blood from the vasculature; (2) filtration of the casting agent; and (3) needle placement during casting agent injection. Care should be taken to avoid injection of casting agent into the left atrium and lung. Other investigators have successfully perfused mouse3 and rat4 cerebrovasculature without using additional procedures. Manual analysis of Microfil perfused vessels counterstained with hemotoxylin showed 93% vessel lumen perfusion5. Always discard or completely clean any tool that comes in contact with the casting agent as it will polymerize quickly, and tools cannot be used for multiple subjects. Clarification of the surrounding mouse brain tissue can be challenging. Following immersion in methyl salicylate, the tissue may still appear opaque, making vessel imaging difficult. Ensuring proper dehydration and clarification should solve this problem. Placing brain tissue on a rocker during alcohol and methyl salicylate treatment can improve tissue clarification. 
Drawbacks of the Technique
The limitations of this technique include: (1) it is necessary to inject the casting agent solution immediately after mixing it as it will thicken quickly after the catalyst is added; (2) the casting agent fills large conductance vessels easily, but it does not reliably fill smaller vessels, such as arterioles (resistance vessels) and capillaries (nutritive vessels); and (3) it is not the best radiopaque contrast for micro-CT imaging, although some groups prefer Microfil for micro-CT imaging1,6. However, following the suggestions proposed above, including proper needle placement, vessel position and perfusion pressure, complete casting of smaller vessels can be achieved. The adaptability to various analysis tools makes this agent a valuable tool for analyzing cerebrovascular structure, including arterial and venous circulation, with various perspectives.
Applications of the Technique
The benefit of this method is its broad application potential. The cast fills all vessels, including arteries, veins, and capillaries of all tissue within the body, so vascular structures in multiple organs of various species can be analyzed. Previously, other groups have used this perfusion for large7 and small8 vessels in human, monkey9, sheep10, rat4,11,12, and mouse3,13. We now show evidence of using this technique in analyzing the structure of microvessels in the mouse brain, in both healthy and diseased states2. In addition, there are Microfil solutions with various viscosities available to perfuse vessels of different sizes and different colors to improve the visualization of the vessels in various organs. For example, red can be easily visualized in the pale brain parenchyma, and yellow contrasts well with dark red myocardium. Moreover, as the material is radiopaque, vasculature can be imaged with micro-CT scanning to obtain a 3D rotatable image. The tissue can also be used for paraffin sections and histological analysis after being imaged.
Conclusion
We demonstrate here a protocol to make a vascular cast of the blood vessels of the adult mouse brain, which can be adapted using a variety of analysis techniques to study the morphological structure of the cerebrovasculature. We have also provided evidence for the application of this method to a model of the BAVM disease state, represented by enlarged, irregularly shaped vessels that shunt blood from arterial to venous circulations. There are a variety of vascular casting methods available. The simple protocol we provide here for a vascular cast may be used as a tool to examine the vasculature of the adult mouse brain using various imaging modalities.

<table><tbody><tr><td>Microfil</td><td>FlowTech</td><td>MV130</td><td>4 month shelf life</td></tr><tr><td>Methyl Salicylate</td><td>Fisher Scientific</td><td>O3695-500</td><td>to clarify tissue</td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

Research

JoVE Journal

Neuroscience

21.5K Views.  University of California, San Francisco. In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Video: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Developmental Biology

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 ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Medicine

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Biology

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the context of tissue regeneration, tumor development, and therapeutic responses. Particularly, the calvarial bone marrow is a highly dynamic tissue, where the hematopoietic fate is interconnected with the surrounding microenvironment, with specialized vessels responding to normal and pathologic hematopoiesis. Traditional imaging of fixed tissues offers static information, often limiting a comprehensive understanding of these processes. 
The integration of transgenic animals expressing cell-specific markers, live cell tracers, advancements in imaging equipment, and the use of specialized chambers has elevated intravital microscopy to a pivotal tool for gaining insights into dynamic biological phenomena. One application of intravital imaging is the investigation of tumor vessel behavior and therapeutic effects. A newly designed 3D-printed titanium head fixation implant can be stably connected to the mouse skull and is suitable for longitudinal imaging during multiple sessions. 
The proposed protocol allows for the spatial and temporal examination of vascular dynamics in the calvarial bone marrow, including visualization and quantification of vascular heterogeneity, interaction with stromal and hematopoietic cells, and measurement of vascular functional parameters. Additionally, the technique enables the visualization of established vascular beds and the monitoring of therapeutic effects, stem cell mobilization, and the localization of chemotherapeutic compounds over time using two-photon microscopy. Overall, this intravital longitudinal imaging protocol provides a comprehensive platform for investigating both tumor vessel behavior and hematopoietic cell dynamics, offering valuable insights into the intricate processes governing these biological phenomena.

Intravital microscopy allows the study of dynamic biological processes such as tissue regeneration and tumor development. The calvarial bone marrow, a highly dynamic tissue, offers insights into hematopoiesis and vascular function. Using a biocompatible 3D-printed head fixation implant allows for repetitive longitudinal imaging, enhancing our understanding of tissue dynamics and the tumor microenvironment.

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

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Chapters in this video

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

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

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow