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...

Watch this Scientific Journal Video about Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus at JoVE.com

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.

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Research

JoVE Journal

Neuroscience

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...