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 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.
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'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.
All experimental procedures followed the guidelines of the Canadian Council on Animal Care.
1. Sample collection and preparation
2. Sample staining
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. Hallgrímsson'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.
4. Image processing
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 were digitized. As can be observed in Figure 2, points are placed all along the border of the brain at the chosen planes. Once digitization is finished, the coordinates of the points can be read in a .txt file (Figure 3). The purpose of point digitization was to obtain raw coordinates that can be further used in geometric morphometric analyses. The presented protocol is focused on a single specimen, and morphometric analyses are suitable for larger samples, but the results obtained using the same set of points are presented elsewhere14.
Finally, the result of manual segmentation of the entire brain is a 3D model of the brain (Figure 4). In these volumes, diverse approaches can be taken, from simply estimating their area and volume to extracting surface points, to performing analyses that are similar to the one presented here for curve points.
Figure 1: Representative slices of a micro-CT image of a neonate's head. (A–C) Axial, (D) coronal, and (E) parasagittal planes are presented. Regions and structures that can be distinguished in the parasagittal plane: (a) olfactory bulbs, (b) cortex, (c) midbrain, (d) cerebellum, (e) hindbrain. Please click here to view a larger version of this figure.
Figure 2: Landmarks and semilandmarks digitized in the proposed protocol. (A) Axial and (B) parasagittal planes are presented. Numbers indicate landmarks. Please click here to view a larger version of this figure.
Figure 3: Example of a file containing point coordinates as exported from the software. The main items are indicated. Please click here to view a larger version of this figure.
Figure 4: 3D reconstruction of a neonate's brain after manual segmentation. An example of an entire reconstructed brain is presented in two views. Please click here to view a larger version of this figure.
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 coordinates of points and applying geometric morphometric techniques8,14. The digitization of coordinates is also useful for obtaining linear measurements, using a simple Euclidean distance estimation. This, together with the volumes derived from brain and ROI segmentations, are valuable inputs for traditional morphometric assessment.
Although the protocol presented here is easily applied to ex vivo small samples, such as the ones we used, it is not applicable for in vivo studies, as fixation, and especially Lugol’s solution as a contrast agent, is not viable. Micro-CT imaging is used in vivo, usually for hard tissue examination19, but the administration of contrast agents that reach the brain crossing the hematoencephalic barrier in living neonates is not simple. Thus, the technique presented here is still a promising tool to answer different questions of morphological variation.
Since pioneering studies showed that iodine is a good choice for vertebrate embryos20, its application has expanded rapidly, and it became evident that one of the main drawbacks of this technique is the shrinking produced by the contrast agent12. It is a ubiquitous problem in studies using this approach, and users should consider it when reproducing the protocol presented here, since some amount of shrinking is expected. Diverse strategies have been proposed to reduce this effect; Vickerton et al.21 examined different concentrations of iodine and concluded that shrinking depends directly on concentration, and reducing it largely helps to restrain deformation due to this effect. Another point to adjust is the proper time of immersion, which depends on the size and the hardness of the structures. After some tests, we found that 15-20 h is a sufficient period to stain the brain tissues of mouse neonates and preserve the main morphological properties. Some authors have suggested the use of some solutions before staining, such as agarose7 and hydrogel11,13, or the application of a buffered Lugol’s solution that prevents acidification and, consequently, shrinkage22. All these procedures could be added to the protocol presented here, which is a general and basic approach to micro-CT imaging with iodine contrast.
The use of micro-CT scanners for imaging soft tissues in larger samples, such as adult mice, has some drawbacks. An immersion procedure for staining is not recommendable because the distribution of the contrast agent will probably be heterogeneous. It will be important to explore some alternative options, like administering Lugol’s solution via cardiac perfusion.
In conclusion, micro-CT with the addition of Lugol’s solution is a suitable alternative for imaging perinatal brains of small animals, in particular mice. It is a simple way to obtain high-resolution images that, complemented with histology, can provide a consistent characterization of brain morphological variation.
The authors have nothing to disclose.
We thank Wei Liu for his technical assistance. This work is funded by ANPCyT PICT 2017-2497 and PICT 2018-4113.
µCT 35 | Scanco Medical AG | Note that Scanco does not offer the µCT 35 anymore. Their smallest scanner is now the µCT 45 | |
Avizo | Visualization Sciences Group, VSG | ||
C57BL/6 Mice | Bioterio Facultad de Ciencias Veterinarias Universidad Nacional de La Plata | ||
Conical tubes | Daigger | CH-CI4610-1856 | |
Flux cabinet | Esco | AC2-458 | |
Glass beaker | Glassco | GL-229.202.10 | |
Glass bottle | Simax | CFB017 | |
Glass funnel | HDA | VI1108 | |
HCl | Carlo Erba | 403872 | Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves) |
I2 | Cicarelli | 804211 | When preparing I2KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves) |
KI | Cicarelli | PA131542.1210 | When preparing I2KI, manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves) |
Magnetic stirring | Arcano | 4925 | |
NaOH | Cicarelli | 1580110 | Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves) |
Orbital shaker | Biomint | BM021 | |
Paraformaldehyde | Biopack | 2000959400 | Manipulate under a flux cabinet and use personal protective equipment (mask, glass and gloves) |
Paton spatula | Glassco | GL-377.303.01 | |
PBS | Biopack | 2000988800 | |
Plastic Pasteur pipette | Daigger | 9153 | |
R | R Project | The package geomorph for R was used in the protocol (https://cran.r-project.org/web/packages/geomorph/index.html) | |
Scissors | Belmed | ||
Sodium azide | Biopack | 2000163500 | |
Thermometer | Daigger | 7650 |