murine neonatal brain sample fixation and staining for micro-ct scanning

Micro-TC y procesamiento de imágenes del cerebro neonatal del ratón

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.

Autor destacado: Imágenes de alta resolución de cerebros neonatos de ratón: un protocolo de micro-TC con el agente de contraste en solución de Lugol

Imágenes de micro-TC y análisis morfométrico de cerebros neonatales de ratones

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.

Fijación y tinción de muestras cerebrales neonatales murinas para microtomografía computarizada

Para comenzar, llene el tubo cónico de 50 mililitros con 40 mililitros de paraformaldehído al 4%. Y sumerja en él cabezas neonatales recién decapitadas con una espátula paton o una cuchara. Guarde los tubos a menos cuatro grados centígrados durante 48 horas.Luego transfiera las cabezas a 40 mililitros de PBS que contengan 0.1% de azida de sodio y continúe manteniendo las cabezas refrigeradas. Para teñir, sumerja cada cabezal en un tubo cónico de 50 mililitros que contenga 40 mililitros de solución de Logul y mézclelo durante 15 a 20 horas en un agitador orbital a una agitación constante. Antes de escanear, coloque la muestra teñida en un tubo de 15 mililitros que contenga 10 mililitros de 1% de agros durante 30 minutos.

Research

JoVE Journal

Neuroscience

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

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 ...

Investigación

Neurociencias

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

Procesamiento de una imagen escaneada por microtomografía computarizada de una muestra de cerebro neonatal murino teñida