Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

To get a better insight in the composition and structure of the choroid plexus an efficient method to isolate the choroid plexus out of the brain ventricles is a first invaluable step. With this microdissection technique it is possible to dissect the choroid plexus out of the brain ventricles while containing its function, viability, and structure without contaminating the choroid plexus with surrounding tissue. A significant hurdle in choroid plexus isolation is identifying the structure as it still floats in the brain ventricles.

Staining the choroid plexus with bromophenol blue facilitates the isolation. Demonstrating the procedure will be Elien Van Wonterghem, our lab manager. Begin with preparing a terminal anesthetic, transcardial perfusion solution, and a solution required for scanning electron microscopy, or SEM, and other solutions as mentioned in the text.

To isolate the mouse brain place the terminally anesthetized animal in the dorsal decubitus position, and fix the mouse by pinning the limbs on a plate. Disinfect the chest by spraying 70%ethanol before making an incision of approximately four centimeters just under the diaphragm using a carbon steel surgical blade. Open the skin and expose the chest using surgical scissors.

Cut the diaphragm completely open and separate the thorax to expose the lungs and the beating heart. Using a perfusion pump transcardially perfuse the mouse with 10 milliliters of the perfusion solution at a rate of 4.5 milliliters per minute. Insert a 26-gauge needle in the left ventricle to pump the solution into the systemic circuit.

Using surgical scissors, cut the right atrium for the blood to get out of circulation. After decapitation of the mouse cut open the scalp by making an incision starting between the ears to the superior to the eyes. Pull the skin laterally to expose the skull.

Then cut open the skull by following the squamosal sutures towards the nasal part. Once done, place the brain in an ice cold Petri dish, and add one milliliter of ice cold PBS to the brain tissue. To isolate the choroid plexus floating in the fourth ventricle gently cut off the cerebellum from the cerebrum using a scalpel.

Remove the remaining brainstem tissue parts from the cerebellum. Rotate the brain so the cutting line is facing upward, ensuring that the fourth ventricle cavity is visible in the middle of the section with the choroid plexus lying at the dorsal site. If necessary, open the ventricle by pulling the connective tissue with sharp forceps.

Gently tear the choroid plexus out of the ventricle wall using tiny sharp forceps. To isolate the choroid plexus from the lateral ventricle, sagittally cut the brain into two hemispheres using tiny sharp forceps. Rotate one brain hemisphere so the cutting line is upward.

To reveal the lateral ventricle gently pull away the cortex from the thalamus. Retract the hippocampus to the mid-sagittal cutting line. The lateral ventricle is now visible with the choroid plexus lying at the bottom of the ventricle.

If necessary, open the ventricle a bit by pulling open the connective tissue with sharp forceps. Use tiny sharp forceps to gently tear the choroid plexus out of the ventricle wall. To perform the sample preparation for SEM transfer the freshly isolated choroid plexus into freshly made fixation solution and incubate overnight at four degrees Celsius.

Once done, wash the sample three times using three to five milliliters of 0.1 molar sodium cacodylate buffer for five minutes each. Postfix the samples in three to five milliliters of 2%osmium tetroxide in 0.1 molar sodium cacodylate buffer for 30 minutes. Then wash the samples three times for five minutes each using three to five milliliters of ultrapure water.

Next, dehydrate the samples in an increasing series of ice cold ethyl alcohol concentrations with 15 minutes per ethyl alcohol solution. Use a critical point dryer to dry the sample properly. Position the sample carefully on a specimen mount with a carbon sticker and visualize the choroid plexus samples using SEM.

The present protocol facilitated the efficient isolation of the choroid plexus from the mouse brain lateral and fourth ventricles. Perfusion with bromophenol blue resulted in the visualization of the choroid plexus. When bromophenol blue was not allowed in the further processing steps perfusion with PBS-heparin or no perfusion was performed for processing.

The SEM morphological analysis of the surface of choroid plexus epithelial, or CPE, cells clearly show the two arm structure of the fourth ventricle and the typical C-shaped form of the lateral choroid plexus isolated from the lateral ventricle. A higher SEM magnification revealed vesicle-like structures on the apical side of CPE cells and microvilli, indicating that morphological alterations of the CPE cells in disease conditions can be investigated using SEM. It is important to only touch and take out the choroid plexus tissue in order not to contaminate the sample with surrounding tissue.

Use necessary caution to preserve the structure of the choroid plexus. A variety of downstream techniques can be applied on the isolated choroid plexus to investigate its structure and function, going from microscope analysis over transcriptome analysis to flow cytometry. Microdissecting the choroid plexus out of the brain allows us to get a better insight into the structure and function of this tiny brain tissue.

This knowledge is essential to better understand its role in neurological diseases.