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 perinatal specimens. Micro computed tomography in combination with a common contrast agent is capable of producing high resolution images suitable for further morphometric analysis. Micro computed tomography has been applied to immature heart tissue such as bone because of x-ray absorption in mineralized tissues.

However, in the last decades, the use of lugol solution as a contrast agent has upgraded the application of this emerging technique for soft tissues. This protocol combines procedures and tools used in the field to demonstrate the great potential of micro computed tomography for studying the prenatal brains of small animals. The strength of our proposal precise in depicting the process from the sample preparation to morphometric data processing.

Images derived from microcomputer tomography preserve special information, which is difficult with a standard histological techniques. Additionally, microcomputer 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 immersion, the main limitations should be related to the sample size, but the potential applications are enormous.

To begin, fill the 50 milliliter conical tube but with 40 milliliters of 4%paraformaldehyde and immerse freshly decapitated neonatal heads into it using a baton 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 lugol 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%agarose for 30 minutes. To begin, launch the image processing software, select file, then open data and choose the previously generated micro CT scanned images in 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 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 iso surface. 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 hindbrain can be distinguished. The manual segmentation of the entire brain resulted in the 3D model reconstruction of the neonate's brain.