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

<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>

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.

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			<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>
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		<tr>
			<td>Avizo</td>
			<td>Visualization Sciences Group, VSG</td>
			<td></td>
			<td></td>
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			<td>C57BL/6 Mice</td>
			<td>Bioterio Facultad de Ciencias Veterinarias Universidad Nacional de La Plata</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Conical tubes</td>
			<td>Daigger</td>
			<td>CH-CI4610-1856</td>
			<td></td>
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			<td>Flux cabinet</td>
			<td>Esco</td>
			<td>AC2-458&#160;</td>
			<td></td>
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			<td>Glass beaker&#160;</td>
			<td>Glassco</td>
			<td>GL-229.202.10</td>
			<td></td>
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			<td>Glass bottle</td>
			<td>Simax</td>
			<td>CFB017</td>
			<td></td>
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			<td>Glass funnel</td>
			<td>HDA</td>
			<td>VI1108</td>
			<td></td>
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			<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>
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			<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>
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		<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>
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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.

1.0K 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.

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Summary