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 th...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

<ol>
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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.

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

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

In experimental neuroscience, specifically for those studies addressing questions about development, it is relevant to count on quantitative characterization of brains of embryonic and prenatal specimens. Micro computed tomography in combination with a common contrast agent is capable of producing high solution images suitable for further morphometric analysis. Micro computed tomography has been applied to imaging hard tissue such as bone because of x-ray absorption in mineralized tissues.However, in the last decades, the use of Lugol's solution as a contrast agent has upgraded the application of using machine technique for soft tissues. This protocol combines procedures and tools using the field to demonstrate the great potential of micro computed tomography for studying the prenatal brains of small animals. The strength of our proposal, precisely depicting the process from the sample preparation to morphometric data processing.Images derived from micro computed tomography preserve a special information, which is difficult with standard histological techniques. Additionally, micro computed tomography with the use of a contrast agent has two advantages when compared with magnetic resonance. Scanners are less expensive and easy to operate and allow a higher spatial resolution.The protocol presented here could be applied with subtle modifications to fix samples of different organs derived from diverse species. Then a variety of scientific questions could be approached. Since the contrast agent is administered by emersion, the main limitations should be related to the sample size, but the potential applications are enormous.

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

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 ...

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

Research

JoVE Journal

Neuroscience

Murine Neonatal Brain Sample Fixation and Staining for Micro-CT Scanning

To begin, fill the 50 milliliter conical tube with 40 milliliters of 4%paraformaldehyde. And immerse freshly decapitated neonatal heads into it using a paton spatula or a spoon. Store the tubes at minus four degrees celsius for 48 hours.Then transfer the heads to 40 milliliters of PBS containing 0.1%sodium azide, and continue to keep the heads refrigerated. For staining, immerse each head in a 50 milliliter conical tube containing 40 milliliters of Logul's solution, and mix it for 15 to 20 hours on an orbital shaker at a constant stir. Before scanning, place the stained sample in a 15 milliliter tube containing 10 milliliters of 1%agros for 30 minutes.

Processing Micro-CT Scanned Image of Stained Murine Neonatal Brain Sample

To begin, launch the image processing software, select file, then open data and choose the previously generated micro CT scanned images in dot BMP format. When the window of the image read parameters opens, confirm the parameters and accept if correct. In the main panel, click on project view to locate a new object created with the name of the dot BMP file.Right click on the created object and select the option ortho slice, then click create. Under properties, select the orientation plane and the slice number to see. To obtain 3D volumes of the complete head in the main panel, go to project view, right click the image object, select the interactive thresholding option, and click create.Under properties, change the values for minimum threshold RGB and maximum threshold RGB until the part of the image corresponding to the head of the animal is selected. Then select apply. Right click on the threshold object created under the project view.In properties select isosurface, choose a threshold to visualize the surface, then click apply. To digitize the landmarks, right click on project view, select create object, then select points and lines followed by landmarks, then click on create. Then right click the new object landmarks.Select landmark view and click on create. Under size, modify the point size. To add landmarks in properties, in the edit mode, select add.Digitize five equally distributed points around the curves until the next landmark at the midbrain cortex intersection, then digitize eight semi landmarks around the cortex. To save the digitized points right click on object landmarks and choose save data. To segment the brain, right click on project view, followed by create object, select images and fields, and then label field.Click on create. Select the label field object, and press the segmentation editor option in properties. Under materials, add a new material and rename it as brain.Under selection, choose the current slice to segment the tissue corresponding to the brain in each slice. In tools, use the magic wand and brush to select the brain tissue in each slice. Then under selection, press add.Now return to project view, right click on label field and select generate surface, then click apply. Once the surface is obtained, apply extract surface to export it. With micro CT scanning, despite the small size of neonatal mouse brains, the anatomical structures such as the olfactory bulbs, cortex, midbrain, cerebellum, and hind brain can be distinguished.The manual segmentation of the entire brain resulted in the 3D model reconstruction of the neonates brain.