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>
	<li>Altman, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+skeletal+growth,+modeling,+and+remodeling+by+in+vivo+micro-computed+tomography.">Quantification of skeletal growth, modeling, and remodeling by in vivo micro-computed tomography.</a> Bone. 81, 370-379 (2015).</li><li>Wehrle, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+characterization+of+fracture+healing+patterns+and+assessment+of+biomaterials+by+time-lapsed+in+vivo+micro-computed+tomography.">Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography.</a> Scientific Reports. 11 (1), 8660 (2021).</li><li>Ar&#237;stide, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+shape+convergence+in+the+adaptive+radiation+of+New+World+monkeys.">Brain shape convergence in the adaptive radiation of New World monkeys.</a> Proceedings of the National Academy of Sciences. 113 (8), 2158-2163 (2016).</li><li>Paluh, D. J., Stanley, E. L., Blackburn, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+hyperossification+expands+skull+diversity+in+frogs.">Evolution of hyperossification expands skull diversity in frogs.</a> Proceedings of the National Academy of Sciences. 117 (15), 8554-8562 (2020).</li><li>de Crespigny, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+micro-CT+imaging+of+the+postmortem+brain.">3D micro-CT imaging of the postmortem brain.</a> Journal of Neuroscience Methods. 171 (2), 207-213 (2008).</li><li>Gignac, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusible+iodine-based+contrast-enhanced+computed+tomography+(diceCT):+an+emerging+tool+for+rapid,+high-resolution,+3-D+imaging+of+metazoan+soft+tissues.">Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues.</a> Journal of Anatomy. 228 (6), 889-909 (2016).</li><li>Wong, M. D., Dorr, A. E., Walls, J. R., Lerch, J. P., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+mouse+embryo+atlas+based+on+micro-CT.">A novel 3D mouse embryo atlas based on micro-CT.</a> Development. 139 (17), 3248-3256 (2012).</li><li>Gonzalez, P. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+protein+restriction+in+mice+impacts+placental+function+and+maternal+body+weight+before+fetal+growth.">Chronic protein restriction in mice impacts placental function and maternal body weight before fetal growth.</a> PLoS One. 11 (3), 0152227 (2016).</li><li>Watanabe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+endocasts+good+proxies+for+brain+size+and+shape+in+archosaurs+throughout+ontogeny.">Are endocasts good proxies for brain size and shape in archosaurs throughout ontogeny.</a> Journal of Anatomy. 234 (3), 291-305 (2019).</li><li>Gignac, P. M., Kley, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+diceCT+imaging+for+high-throughput+comparative+neuroanatomical+studies.">The utility of diceCT imaging for high-throughput comparative neuroanatomical studies.</a> Brain, Behavior and Evolution. 91 (3), 180-190 (2018).</li><li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+microCT+using+iodine-based+contrast.">A novel procedure for rapid imaging of adult mouse brains with microCT using iodine-based contrast.</a> PLoS One. 10 (11), e0142974 (2015).</li><li>Gignac, P. M., O'Brien, H. D., Sanchez, J., Vazquez-Sanroman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging+of+the+rat+brain+using+an+integrated+diceCT+and+histology+workflow.">Multiscale imaging of the rat brain using an integrated diceCT and histology workflow.</a> Brain Structure &amp; Function. 226 (7), 2153-2168 (2021).</li><li>Wong, M. D., Spring, S., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+stabilization+of+tissue+for+embryo+phenotyping+using+micro-CT+with+iodine+staining.">Structural stabilization of tissue for embryo phenotyping using micro-CT with iodine staining.</a> PLoS One. 8 (12), e84321 (2013).</li><li>Barbeito-Andr&#233;s, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+Zika+syndrome+is+associated+with+maternal+protein+malnutrition.">Congenital Zika syndrome is associated with maternal protein malnutrition.</a> Science Advances. 6 (2), (2020).</li><li>Handschuh, S., Gl&#246;smann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryo+phenotyping+using+X-ray+microCT.">Mouse embryo phenotyping using X-ray microCT.</a> Frontiers in Cell and Developmental Biology. 10, 949184 (2022).</li><li>Turnbull, D. H., Mori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+in+mouse+developmental+biology.">MRI in mouse developmental biology.</a> NMR in Biomedicine. 20 (3), 265-274 (2007).</li><li>Qiu, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+MRI+shows+brain+areas+relatively+larger+in+males+emerge+before+those+larger+in+females.">Mouse MRI shows brain areas relatively larger in males emerge before those larger in females.</a> Nature Communications. 9, 2615 (2018).</li><li>Lerch, J. P., Sled, J. G., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+phenotyping+of+genetically+altered+mice.">MRI phenotyping of genetically altered mice.</a> Methods in Molecular Biology. 711, 349-361 (2011).</li><li>Gonzalez, P. N., Kristensen, E., Morck, D. W., Boyd, S., Hallgr&#237;msson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+growth+hormone+on+the+ontogenetic+allometry+of+craniofacial+bones.">Effects of growth hormone on the ontogenetic allometry of craniofacial bones.</a> Evolution &amp; Development. 15 (2), 133-145 (2016).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+developmental+biology:+a+versatile+tool+for+high-contrast+3D+imaging+at+histological+resolutions.">MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions.</a> Developmental Dynamics. 238 (3), 632-640 (2009).</li><li>Vickerton, P., Jarvis, J., Jeffery, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration-dependent+specimen+shrinkage+in+iodine-enhanced+microCT.">Concentration-dependent specimen shrinkage in iodine-enhanced microCT.</a> Journal of Anatomy. 223 (2), 185-193 (2013).</li><li>Dawood, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+soft-tissue+shrinkage+artefacts+caused+by+staining+with+Lugol's+solution.">Reducing soft-tissue shrinkage artefacts caused by staining with Lugol's solution.</a> Scientific Reports. 11, 19781 (2021).</li></ol>

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