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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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. T....

Protocol

All experimental procedures followed the guidelines of the Canadian Council on Animal Care.

1. Sample collection and preparation

  1. Prepare 500 mL of 4% paraformaldehyde (PFA).
    1. 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.
    2. Stir while heating. With a thermometer, constantly check the temperature of th.......

Representative Results

Here, a basic protocol to obtain high-resolution images of neonatal mouse brains is presented. Heads were scanned after immersion in Lugol'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.......

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
 µCT 35Scanco Medical AGNote that Scanco does not offer the  µCT 35 anymore. Their smallest scanner is now the  µCT 45 
AvizoVisualization Sciences Group, VSG
C57BL/6 MiceBioterio Facultad de Ciencias Veterinarias Universidad Nacional de La Plata
Conical tubesDaiggerCH-CI4610-1856
Flux cabinetEscoAC2-458 
Glass beaker GlasscoGL-229.202.10
Glass bottleSimaxCFB017
Glass funnelHDAVI1108
HClCarlo Erba403872Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)
I2Cicarelli804211When preparing I2KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)
KICicarelliPA131542.1210When preparing I2KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)
Magnetic stirringArcano4925
NaOHCicarelli1580110Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)
Orbital shakerBiomintBM021
Paraformaldehyde Biopack2000959400Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves)
Paton spatulaGlasscoGL-377.303.01
PBSBiopack2000988800
Plastic Pasteur pipetteDaigger9153
RR ProjectThe package geomorph for R was used in the protocol (https://cran.r-project.org/web/packages/geomorph/index.html)
Scissors Belmed
Sodium azideBiopack2000163500
ThermometerDaigger7650

References

  1. Altman, A. R., et al. Quantification of skeletal growth, modeling, and remodeling by in vivo micro-computed tomography. Bone. 81, 370-379 (2015).
  2. Wehrle, E., et al.

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Micro CTMicro computed TomographyNeuroimagingBrain MorphologyMouse Neonatal BrainContrast AgentLugol s SolutionMorphometric Analysis3D ImagingMRISoft Tissue ImagingSample PreparationImage SegmentationRegion Of Interest Analysis

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