Name: microdissection and whole mount scanning electron microscopy visualization of mouse choroid plexus
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This work was supported by the Belgian Foundation of Alzheimer&#39;s Research (SAO; project number: 20200032), the Research Foundation Flanders (FWO Vlaanderen; project numbers: 1268823N, 11D0520N, 1195021N) and the Baillet Latour Fund. We thank the VIB BioImaging Core for training, support, and access to the instrument park.

All animal experiments described in this study were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European legislation (EU Directives 2010/63/EU, 86/609/EEC). All experiments on mice and animal protocols were approved by the ethics committee of Ghent University (permit numbers LA1400091 and EC 2017-026).
1. Preparation
<ol>
	<li>Anesthetics: Prepare a terminal anesthetic. For instance, a sodium pentobarbital ...

<ol>
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Here, a method to isolate the choroid plexus (CP) out of the lateral ventricle and the fourth ventricle of a mouse brain is described. This whole mounting method of the CP facilitates further analysis using a repertoire of techniques to get a complete view of the CP morphology, cellular composition, transcriptome, proteome, and secretome. Such analyses are crucial to gain a better understanding of this remarkable structure protruding from the ventricles of the brain. This knowledge is of immense research interest, as it ...

Tight barriers separate the central nervous system (CNS) from the periphery, including the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. These barriers protect the CNS against external insults and ensure a balanced and controlled microenvironment1,2,3. While the BBB has been extensively studied over time, the blood-CSF barrier located at the choroid plexus (CP) has only gained increasing research interest during the last decade. This latter barrier can be found in the four ventricles of the brain (Figure 1A, B)&#160;and is characterized by a single layer of choroid plexus epithelial (CPE) cells surrounding a central stroma, leaky capillaries, fibroblasts, and a lymphoid and myeloid cell population (Figure 1C)4,5,6. The CPE cells are firmly interconnected by tight junctions, thus preventing leakage from the underlying fenestrated blood capillaries into the CSF and brain. Additionally, transport across the CPE cells is regulated by a number of inward and outward transport systems that manage the influx of beneficial compounds (e.g., nutrients and hormones) from the blood to the CSF and the efflux of harmful molecules (e.g., metabolic waste, excess neurotransmitters) in the other direction1,6. To be able to exert their active transport function, the CPE cells contain numerous mitochondria in their cytoplasm7. Moreover, the CP is the main source of CSF and acts as the gatekeeper of the brain by the presence of resident inflammatory cells1. Due to its unique location between the blood and the brain, the CP is also perfectly positioned to carry out immune surveillance8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64733/64733fig01.jpg" /> 
Figure 1: Schematic overview of the location and composition of the choroid plexus (CP). (A,B)&#160;CP tissue is found within the two lateral, the third, and the fourth ventricles of (A) human and (B) mouse brains. (C) The CP tissue consists of a single layer of tightly connected cuboidal CP epithelium (CPE) cells surrounding fenestrated capillaries, loose connective tissue, and lymphoid and myeloid cells, and forms the blood-cerebrospinal fluid barrier (adapted and modified from reference23). Figure created with Biorender.com. <a href="https://www.jove.com/files/ftp_upload/64733/64733fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Over the past decade, increasing evidence, including several reports from our research group, have revealed that the CP plays a central role in health and disease9,10,11,12,13,14,15,16,17,18. For example, it is known that the aging blood-CSF barrier displays morphological alterations in, among others, the nuclei, microvilli, and the basement membrane1,19. Additionally, in the context of Alzheimer&#39;s disease, the overall barrier integrity is compromised and all of these age-related changes appear to be even more pronounced1,8,20. In addition to morphological changes, the transcriptome, proteome, and secretome of the CP are altered during disease12,21,22,23. Thus, advanced knowledge of the CP is essential to better understand its role in neurological diseases and potentially develop new therapeutic strategies.
An efficient method for accurate microdissection of the CP out of the brain ventricles is the first invaluable step to allow proper investigation of this tiny brain structure. On account of its highly vascularized nature (Figure 2B), the CP floating inside the ventricular cavities of the brain can be identified using a binocular microscope. However, transcardial perfusion is often required for downstream analysis, complicating the proper identification and isolation of the CP tissue (Figure 2C). If the further processing steps allow (e.g., in the case of RNA and protein analysis), the CP can be visualized via transcardial perfusion with bromophenol blue (Figure 2A). Several publications already describe the isolation of the CP from rat24 and mouse pup brains25. Here, a microdissection isolation technique is described to isolate the CP from adult mice. Importantly, this isolation technique preserves the viability, function, and structure of the cells within the CP. The isolation of the CP floating in the fourth and lateral ventricles is described here. In short, the mice are terminally anesthetized&#160;and, if necessary, transcardially perfused. However, it should be noted that perfusion can damage the structure of the cells within the CP. Consequently, if the sample is to be analyzed using transmission electron microscopy (TEM), serial block face scanning electron microscopy (SBF-SEM), or focused ion beam SEM (FIB-SEM), perfusion should not be performed. Next, the whole brain is isolated, and forceps are used to sagittally hemisect the brain. From here, the CPs floating in the lateral ventricles can be identified and dissected, while the CP from the fourth ventricle can be isolated from the cerebellar side of the brain.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64733/64733fig02.jpg" /> 
Figure 2: Visualization of the (A-C) fourth and (D-F) lateral ventricle choroid plexus (CP) after (A,D) bromophenol blue perfusion, (B,E) no perfusion, and (C,F) perfusion with PBS/heparin. The images are taken with a stereo microscope (8x-32x magnification). <a href="https://www.jove.com/files/ftp_upload/64733/64733fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Once the CP is properly dissected out of the brain ventricles, a whole repertoire of techniques can be applied to gain further understanding on the function of this structure. For example, flow cytometry or single cell RNA sequencing can be performed to quantify and phenotypically analyze the infiltrating inflammatory cells under certain disease conditions26,27. In addition to the cellular composition, the molecular composition of the CP can be analyzed to assess the presence of cytokines and chemokines via enzyme-linked immunosorbent assay (ELISA), immunoblot, or through simultaneous analysis of multiple cytokines using the cytokine bead array28. Moreover, transcriptome, vascular, immune cell histology, and secretome analyses can be performed on the microdissected CP explants29. Here, scanning electron microscopy (SEM) on whole mount CP is used to obtain an overall view of the CP structure. SEM uses a beam of focused electrons to scan over the surface and create an image of the surface&#39;s topography and composition. Since the wavelength of electrons is much smaller than that of light, the resolution of SEM is in the nanometer range and superior to that of a light microscope. Consequently, morphological studies on the subcellular level can be performed via SEM. Briefly, the dissected CP is immediately transferred into a glutaraldehyde-containing fixative for an overnight fixation, followed by osmication and uranyl acetate staining. The samples are then treated with lead aspartate stain, dehydrated, and ultimately embedded for imaging.
Thus, this protocol facilitates the efficient isolation of the CP from the mouse brain ventricles, which can be further analyzed using a variety of downstream techniques to investigate its structure and function.

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			<td>26G x 1/2 needle</td>
			<td>Henke Sass Wolf</td>
			<td>4710004512</td>
			<td></td>
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			<td>Aluminium specimen mounts</td>
			<td>EM Sciences</td>
			<td>75220</td>
			<td></td>
		</tr>
		<tr>
			<td>Cacodylate buffer</td>
			<td>EM Sciences</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon steel surgial blades</td>
			<td>Swann-Morton</td>
			<td>0210</td>
			<td>size: 0.45 mm x 12 mm</td>
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			<td>Carbon adhesive tabs -12 mm</td>
			<td>EM Sciences</td>
			<td>77825-12</td>
			<td></td>
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			<td>Critical point dryer&#160;</td>
			<td>Bal-Tec&#160;</td>
			<td>CPD030</td>
			<td></td>
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		<tr>
			<td>Crossbeam 540</td>
			<td>Zeiss</td>
			<td></td>
			<td>SEM system</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools GmbH&#160;</td>
			<td>91197-00</td>
			<td></td>
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			<td>Glutaraldehyde</td>
			<td>EM Sciences</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H-3125</td>
			<td></td>
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		<tr>
			<td>Ismatec Reglo ICC Digital Peristaltic pump 2-channel</td>
			<td>Metrohm Belgium N.V</td>
			<td>CPA-7800160</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide&#160;</td>
			<td>EM Sciences</td>
			<td>19170</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
			<td></td>
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		<tr>
			<td>Platinum&#160;</td>
			<td>Quorum&#160;</td>
			<td>Q150T ES</td>
			<td>PBS without Ca++ Mg++ or phenol red; sterile filtered</td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Kela NV</td>
			<td>514</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Basket Stainless Steel</td>
			<td>EM Sciences</td>
			<td>70190-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi DV4 Stereo microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools GmbH&#160;</td>
			<td>91460-11</td>
			<td></td>
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microdissection and whole mount scanning electron microscopy visualization of mouse choroid plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The described protocol facilitates the efficient isolation of the CP from the mouse brain lateral (Figure 2A-C) and fourth (Figure 2D-F)&#160;ventricles. After isolating the whole brain, forceps are used to sagittally hemisect the brain and identify the CPs floating in the lateral ventricles. The CP from the fourth ventricle can be isolated from the cerebellar side of the brain. Perfusion with b...

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

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

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.

Research

JoVE Journal

Neuroscience

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