We thank Wei Liu for his technical assistance. This work is funded by ANPCyT PICT 2017-2497 and PICT 2018-4113.

All experimental procedures followed the guidelines of the Canadian Council on Animal Care.
1. Sample collection and preparation
<ol>
	<li>Prepare 500 mL of 4% paraformaldehyde (PFA).
	<ol>
		<li>Under an extraction flux in a cabinet, add 20 g of PFA powder to 250 mL of 1x phosphate-buffered saline (PBS) in a 1 L glass beaker. Place the beaker with a magnet on a magnetic stirring plate.</li>
		<li>Stir while heating. With a thermometer, constantly check the temperature of the solution to keep it under 60 &#176;C.</li>
		<li>With a plastic Pasteur pipette, add drops of 1 M NaOH to the solution to dissolve the PFA. Once the PFA powder has completely dissolved, cool the solution at room temperature (RT).</li>
		<li>Adjust the volume to 500 mL with 1x PBS. Adjust the pH to 7.2-7.3 by adding drops of 1 M HCl with a plastic Pasteur pipette.</li>
		<li>Using a paper filter and a glass funnel, filter the solution in a 1 L glass bottle.</li>
		<li>Close the glass bottle, remove it from the flux cabinet, and store in a refrigerator at -4 &#176;C.</li>
	</ol>
	</li>
	<li>Collect brain samples.
	<ol>
		<li>To obtain samples, sacrifice mouse neonates the morning following their birth, at 0.5 days of age (P 0.5), using surgical scissors to decapitate them.</li>
		<li>Place the scissors in the neck of the neonate while holding the body, and perform a single and clean cut.</li>
	</ol>
	</li>
	<li>Fixate the samples in PFA.
	<ol>
		<li>Fill a 50 mL conical tube with 40 mL of 4% PFA.</li>
		<li>Immerse each head in the tube containing PFA as a fixation agent. For this action, use a Paton spatula or a spoon.</li>
		<li>Store the conical tubes in the refrigerator at -4 &#176;C for 48 h, then change to 40 mL of 1x PBS with 0.1% sodium azide as a preservative. Maintain refrigerated at -4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Sample staining
<ol>
	<li>Prepare Lugol&#39;s solution (3.75% w/v).
	<ol>
		<li>Under an extraction flux in a cabinet, in a 1 L glass beaker, dissolve 10 g of Kl and 5 g of I2 in 400 mL of distilled water, with constant mixing using a magnetic stirrer.</li>
	</ol>
	</li>
	<li>Immerse each head in a 50 mL conical tube containing 40 mL of Lugol&#39;s solution.
	<ol>
		<li>For neonates of an approximate body size of 1.3 g, leave the samples immersed for 15-20 h with constant mixing using an orbital shaker.</li>
		<li>Place the samples in a 15 mL conical tube containing 10 mL of 1% agarose for 30 min before scanning.</li>
	</ol>
	</li>
</ol>
3. Micro-CT scanning
NOTE: Micro-CT scanning requires specific equipment. There are different options for this kind of scanner, and details on acquisition depend on the characteristics of the used equipment. Dr. Hallgr&#237;msson&#39;s lab counts on some alternatives of micro-CT scanners. Here, the protocol is based on the use of a basic desktop scanner, which is among the more accessible small animal imaging machines used by labs around the world. If the scanning objective is the skull, skip the steps in section 2 of the protocol and proceed to section 3. After scanning the skull, the staining process and the scanning of the brain could be performed to obtain images from the same specimens of both the brain and the skull.
<ol>
	<li>Place the sample in the micro-CT holder.
	<ol>
		<li>Place the sample in a 50 mL conical tube. Fix the sample to avoid movements during the scanner session. For this purpose, some polyurethane material can be used to fill the empty parts of the tube.</li>
		<li>Open the equipment and place the tube with the sample in the micro-CT holder.</li>
		<li>Log into the micro-CT control panel and register the sample to be scanned. This will automatically generate a sample number. In addition to the number, fill out the &#34;Name&#34; field. Both the sample name and number should be recorded elsewhere (e.g., in a spreadsheet) in case the raw data needs to be retrieved again.</li>
		<li>Start the scanning program. Enter the sample name or number and select a Control file, which should contain the scanning parameters.</li>
	</ol>
	</li>
	<li>Scan the sample.
	<ol>
		<li>In the screen of the micro-CT software, set the following parameters: isotropic voxel size of 0.012 mm, 45 kVp, 177 lA, 800,000 ms integration time, and 500 projections per 180&#176;.</li>
		<li>Set the scanning region by selecting Scout View. Once the reference image appears, press Reference Line to set the region. Move the solid green line to the edge of the sample, then hold Shift while dragging the cursor to extend the scan region to the other edge of the sample.</li>
		<li>Select OK to begin the scan.</li>
	</ol>
	</li>
	<li>Evaluate and export the scan.
	<ol>
		<li>Open up the micro-CT scanner Evaluation Program and click on Select Sample and Measurement. In the Filter field, type the sample name, select the sample, and select the measurement file.</li>
		<li>The file will load up as slices, the number of which depends on the initial scanning window. Click on the Load All button, which will load every slice and facilitate movement between slices later on.</li>
		<li>Select Tasks &#62; Evaluation 3D to initialize a cropping box around the slices. A 3D-Evaluation prompt will appear with a number of fields, including Task/Evaluation, VOI (Volume of Interest), Start X, Y, Z, and Dimension X, Y, Z.</li>
		<li>Set the Task &#62; Evaluation 3D field to the appropriate script, as a variety of evaluation tasks can be performed, such as reconstruction, segmentation, morphometry (e.g., bone mineral density), and file conversions or transfers. The Default Evaluation script can be used as a guide.</li>
		<li>After selecting the script, adjust the VOI box on the main image. Start on the first slice. Ensure that the box contains the anatomy to be evaluated. Move the VOI by right-clicking on one of the small white squares toward the box edge and resizing the VOI using the middle mouse button. Alternatively, adjust the Dimension fields numerically.</li>
		<li>Pass through the sample using the scroll bar underneath the image to ensure that all of the anatomies have been captured in the VOI box. Click on XY, XZ, or YZ near the top left of the software to change the orientation.</li>
		<li>Select Start Evaluation. Depending on the Task Evaluation script, this will generate a 3D .aim image file with a corresponding .txt header file. Additionally, a path can be specified in the script to File Transfer Protocol (FTP) the image to a particular location.</li>
	</ol>
	</li>
</ol>
4. Image processing
<ol>
	<li>Open the images in the image processing software. 
	NOTE: Image processing can be carried out using various software. This protocol includes the basic steps in a commercial software, which is widely accepted in the area and has versatile tools for basic and advanced processing. In addition, there are different acceptable formats for reconstructed micro-CT images. This aspect usually depends on the equipment used for acquisition and its software. Here, image processing is carried out with .bmp images. The same procedure can be applied for .tiff images and other types. Since the acquisition is carried out in a scanner with .aim as the default format, a conversion from .aim to .bmp is first presented.
	<ol>
		<li>To open .aim files, select File &#62; Open Data and choose the file. After that, a window with the title File Format Selection will open. Select Raw Data.</li>
		<li>A window with the title Raw Data Parameters will open. On this window, fill in the parameters following the information on the header .txt file that is generated for each .aim file. For Data Type, choose 16-bit for header complete with the number after Image data starts at byte offset. Also, set the dimensions and voxel size using the information on the header .txt file. Once the parameters are complete, press OK.</li>
		<li>If conversion from .aim to .bmp is needed, in Main Panel &#62; Project View, right-click on the .aim object and select the option Convert &#62; Convert Image Type. On Properties, select 8-bit as the Output Type. Select Apply.</li>
		<li>Right-click on the new 8-bit object. Select Save as. Choose .bmp and save.</li>
		<li>To open the .bmp file, select File &#62; Open Data and choose the file. After that, a window with the parameters of the image opens. Confirm the parameters and accept if correct.</li>
		<li>In Main Panel &#62; Project View, a new object is created with the name of the .bmp files. Right-click on this object and select the option Ortho Slice. In Properties, select the slice number and the orientation plane to see.</li>
	</ol>
	</li>
	<li>Obtain 3D volumes of the complete head.
	<ol>
		<li>In Main Panel &#62; Project View, right-click on the image object. Select Interactive Thresholding option &#62; Create. In Properties, change the values for Minimum RGB and Maximum RGB until the part of the image corresponding to the head of the animal is selected. Select Apply.</li>
		<li>In Main Panel &#62; Project View, a thresholded object is created. Right-click on this object and, in Properties, select Isosurface. In Properties, choose a threshold to visualize the surface and click Apply. This threshold needs to be chosen empirically, then different values might need to be examined.</li>
	</ol>
	</li>
	<li>Digitizing point coordinates
	<ol>
		<li>3D point coordinates can be digitized both based on slices or extracted surfaces. For the first option, right-click in the image option and select Slice. In Translate, choose the desired slice in the axial plane. If the plane is not correctly placed, click Options &#62; Rotate in Properties to establish the right orientation.</li>
		<li>To digitize landmarks, right-click in Main Panel &#62; Project View and select Create Object &#62; Points and Lines &#62; Landmarks. Right-click the new Object Landmarks and select Landmark View. In Landmark View, modify the size of the point in Size. To add landmarks in Properties, use the option Add in Edit Mode. 
		NOTE: For neonates, a set of landmarks and semilandmarks around the axial and sagittal curves of the brain was previously published14.</li>
		<li>First, choose the axial plane that crosses the most rostral point of the olfactory bulbs and the most caudal point of the cortex.</li>
		<li>Select the Object Landmarks and, in Properties, select Landmark Editor. Using the arrow, click on the place the point is to be digitized.</li>
		<li>In the proposed set of landmarks, 24 points are digitized in the selected axial plane. Digitize the first in the most caudal point of the brain at the midline.</li>
		<li>Digitize five points equally distributed around the curve (semilandmarks) until the next landmark at the intersection between the midbrain and the cortex.</li>
		<li>Then, digitize eight points (semilandmarks) around the cortex.</li>
		<li>Digitize a new landmark at the intersection between the cortex and the olfactory bulbs. Then, digitize four points (semilandmarks) around the olfactory bulbs.</li>
		<li>Digitize a new landmark at the most rostral point of the olfactory bulbs at the midline. Then, digitize two points (semilandmarks) in between both olfactory bulbs.</li>
		<li>Digitize a new landmark at the most caudal point of the olfactory bulbs at the midline.</li>
		<li>For sagittal points, establish a slice in the right side of the brain at a parasagittal plane where the brain is most caudally prominent.</li>
		<li>In the proposed set of landmarks, 33 points are digitized in the selected parasagittal plane. Digitize the first in the intersection between the diencephalon and the hindbrain. Then, digitize nine points (semilandmarks) in the ventral limit of the brain.</li>
		<li>Digitize a new landmark at the most rostral point of the olfactory bulb. Then, digitize three points (semilandmarks) around the olfactory bulbs.</li>
		<li>Digitize a new landmark in the intersection between the olfactory bulb and the cortex. Then, digitize three points (semilandmarks) around the olfactory bulbs.</li>
		<li>Digitize a new landmark in the intersection between the olfactory bulb and the cortex. Then, digitize seven points (semilandmarks) around the cortex.</li>
		<li>Digitize a new landmark in the intersection between the cortex and the midbrain. Then, digitize seven points (semilandmarks) around the cortex.</li>
		<li>Digitize a new landmark in the intersection between the cortex and the midbrain. Then, digitize six points (semilandmarks) around the midbrain.</li>
		<li>Digitize a new landmark in the intersection between the midbrain and the cerebellum. Then, digitize two points (semilandmarks) around the cerebellum.</li>
		<li>Digitize a new landmark in the intersection between the cerebellum and the hindbrain.</li>
		<li>Save the digitized points. Right-click on the Object Landmarks and choose the option Save Data.</li>
	</ol>
	</li>
	<li>Analyze the point coordinates. 
	NOTE: Once the point coordinates are digitized for a set of specimens, basic analyses can be performed using geometric morphometrics tools to obtain size (centroid size) and shape variables (shape or Procrustes coordinates). Analyses are carried out in a free open software environment, which is especially suitable for statistical analyses.
	<ol>
		<li>Using a notepad, build a file containing the coordinates of all the digitized specimens. For this purpose, follow the TPS format, as described in&#160;https://morphometrics.uk/MorphoJ_guide/frameset.htm?index.htm.</li>
		<li>Open the statistical software. Select File &#62; Change Dir to choose the directory where the .tps file is saved.</li>
		<li>Select Packages &#62; Install packages. Choose one Cran Mirror. Look for geomorph and install it.</li>
		<li>Load packages by typing in the console: library(geomorph) and library(Morpho).</li>
		<li>Load the dataset by typing in the console: dataset &#60;- readland.tps(file=&#34;NAME_OF_FILE.tps&#34;, specID=&#34;ID&#34;).</li>
		<li>Perform generalized Procrustes analysis by typing in the console: GPA&#60;- gpagen(dataset).</li>
		<li>Obtain the centroid size by typing in the console: CS&#60;-GPA$Csize.</li>
		<li>Obtain Procrustes coordinates by typing in the console: ProcCoord&#60;- GPA$coords.</li>
		<li>Plot the Procrustes coordinates and the mean shape by typing in the console: plotAllSpecimens(ProcCoord).</li>
	</ol>
	</li>
	<li>Segmentation of ROIs 
	NOTE: To segment the brain and obtain a 3D reconstruction, manually identify the brain tissues in each slice. The same procedure can be applied to specific ROIs, such as the olfactory bulbs, cortex, etc.
	<ol>
		<li>Right-click on Main Panel &#62; Project View and select Create Object &#62; Label Field.</li>
		<li>Selecting the Label Field object, press the Segmentation Editor option in Properties.</li>
		<li>In Materials, add a new material and, if desired, rename it as Brain.</li>
		<li>In Selection, choose the option Current Slice to segment the tissue corresponding to the brain in each slice.</li>
		<li>Using the options present in Tools, select the brain tissue in each slice. For this case, the Magic Wand and the Brush are the most suitable.</li>
		<li>Once the selection is made in each slice, press Add in Selection.</li>
		<li>When selection in all slices is finished, return to Project View in Main Panel and right-click on Label Field to select Generate Surface, then click Apply.</li>
		<li>Once the surface is obtained, export it by applying Extract Surface.</li>
		<li>To obtain the volume of the segmented structures, in Main Panel &#62; Project View, select the object that contains the label, right-click, and select Measure and Analyze &#62; Volume Fraction. Then, click Apply.</li>
	</ol>
	</li>
</ol>

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

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The authors declare that they have no competing interests.

In this work, a concise protocol to scan neonatal brain tissues of mice using micro-CT with a contrast agent is introduced. In addition, it includes simple procedures to obtain quantitative and qualitative outputs. Building on these methods, further alternative or complementary analyses can be performed.
As shown in the protocol, micro-CT images can be analyzed in different ways. In previous studies, our group estimated the size and shape variation in perinatal brains of mice by digitizing coo...

Micro-computed tomography (micro-CT) imaging is a valuable tool for different fields of research. In biology, it is especially suitable for bone research because of X-ray absorption in mineralized tissues. Due to this feature, diverse questions regarding bone development1, metabolism2, and evolution3,4, among other topics, have been approached with the assistance of micro-CT. In 2008, de Crespigny et al.5 showed that micro-CT images of adult mouse and rabbit brains could be obtained using iodine as a contrast agent. This work opened a new application for this imaging technique, since iodine allowed the acquisition of images from soft tissues which otherwise would be insensitive to X-rays. Thus, the general goal of combining micro-CT and an iodine-based contrast agent is to obtain high-resolution images, in which soft tissues can be distinguished and identified at a meso or macro anatomical level.
This technique has notable potential for studies that require detailed ex vivo phenotypic characterization of small specimens, such as mouse embryos, which are widely used in experimental designs6. Iodine contrast in combination with micro-CT imaging has been used to obtain volumetric quantifications of organs7 and landmark three-dimensional (3D) structures8,9. In recent years, micro-CT scanning of stained samples has been applied to describe brain phenotypic features of rodents10, and different improvements to the technique have been proposed. For adult brains, a protocol of 48 h of immersion in iodine, with a previous step of perfusion with a hydrogel, was found to produce images of high quality11. Gignac et al.12 expanded the limits of this technique by showing that rat brains stained with iodine could be processed to perform routine histological techniques. Similarly, these procedures demonstrate promising results for embryonic and pre-weaning rodent brains8,13,14,15.
Although neuroscience has largely applied microscope-based techniques to assess different structural and functional aspects of brain development, such studies are more suitable for characterizing specific cell populations or spatially limited structures. Conversely, micro-CT imaging allows the description of whole structures and the acquisition of 3D models that preserve relevant spatial information, which is complementary to microscopic techniques. Magnetic resonance imaging (MRI) is also a standard technique applied to explore the structural features of small animals16,17,18. However, micro-CT, with the use of a contrast agent, has two main advantages for ex vivo fixed samples: micro-CT scanners are largely less expensive and easy to operate, and allow a higher spatial resolution than MRI12.
This work aims to describe the procedure to obtain high-resolution images from neonatal mouse brains using micro-CT scanning after staining with Lugol&#39;s solution, an iodine-based contrast agent. A comprehensive protocol is presented, which starts with preliminary stages such as sample collection and fixation of tissues, and goes through staining, micro-CT image acquisition, and standard processing. Image processing includes the segmentation of a 3D volume of the complete head, as well as of the brain, and the selection of specific anatomical planes to digitize point coordinates that could be then used in morphometric analyses. Although the focus here is the neonatal mouse brain, similar strategies can be applied to other soft tissues. Thus, the protocol presented here is flexible enough to be applied, with subtle modifications, to other types of samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;&#181;CT 35</td>
			<td>Scanco Medical AG</td>
			<td></td>
			<td>Note that Scanco does not offer the&#160; &#181;CT 35 anymore. Their smallest scanner is now the&#160; &#181;CT 45&#160;</td>
		</tr>
		<tr>
			<td>Avizo</td>
			<td>Visualization Sciences Group, VSG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Bioterio Facultad de Ciencias Veterinarias Universidad Nacional de La Plata</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes</td>
			<td>Daigger</td>
			<td>CH-CI4610-1856</td>
			<td></td>
		</tr>
		<tr>
			<td>Flux cabinet</td>
			<td>Esco</td>
			<td>AC2-458&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beaker&#160;</td>
			<td>Glassco</td>
			<td>GL-229.202.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle</td>
			<td>Simax</td>
			<td>CFB017</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass funnel</td>
			<td>HDA</td>
			<td>VI1108</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carlo Erba</td>
			<td>403872</td>
			<td>Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td>
		</tr>
		<tr>
			<td>I2</td>
			<td>Cicarelli</td>
			<td>804211</td>
			<td>When preparing I2KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td>
		</tr>
		<tr>
			<td>KI</td>
			<td>Cicarelli</td>
			<td>PA131542.1210</td>
			<td>When preparing I2KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td>
		</tr>
		<tr>
			<td>Magnetic stirring</td>
			<td>Arcano</td>
			<td>4925</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Cicarelli</td>
			<td>1580110</td>
			<td>Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Biomint</td>
			<td>BM021</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Biopack</td>
			<td>2000959400</td>
			<td>Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)</td>
		</tr>
		<tr>
			<td>Paton spatula</td>
			<td>Glassco</td>
			<td>GL-377.303.01</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biopack</td>
			<td>2000988800</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td>Daigger</td>
			<td>9153</td>
			<td></td>
		</tr>
		<tr>
			<td>R</td>
			<td>R Project</td>
			<td></td>
			<td>The package geomorph for R was used in the protocol (https://cran.r-project.org/web/packages/geomorph/index.html)</td>
		</tr>
		<tr>
			<td>Scissors&#160;</td>
			<td>Belmed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Biopack</td>
			<td>2000163500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Daigger</td>
			<td>7650</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Here, a basic protocol to obtain high-resolution images of neonatal mouse brains is presented. Heads were scanned after immersion in Lugol&#39;s solution. Despite their small size, the main brain anatomical structures, such as the olfactory bulbs, cortex, midbrain, cerebellum, and hindbrain, can be distinguished (Figure 1).
Different analyses can be carried out using these images as inputs. A set of landmarks and semilandmarks in two different anatomical planes we...

Watch this Scientific Journal Video about Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains at JoVE.com

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.

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 ...

micro-ct-imaging-and-morphometric-analysis-of-mouse-neonatal-brains

Research

JoVE Journal

Neuroscience

1.3K Views.  Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos (ENyS, CONICET-UNAJ-HEC). 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.

Video: Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains – A Micro-CT Protocol with Lugol's Solution Contrast Agent

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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.

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). ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

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 ...

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

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Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

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

Morphometric Analyses of Retinal Sections

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

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