The authors would like to acknowledge the Ligue Francaise contre l&#39;&#233;pilepsie (to M.A-L), Institut National de la Sant&#233; et de la Recherche M&#233;dicale Grant AVENIR R12087FS (to F.J), grant from the university of Montpellier (to F.J) and grant from Federation pour la Recherche sur le Cerveau (to N.M). We acknowledge the technical assistance of Chrystel Lafont at the IPAM in vivo imaging core platform facility of Montpellier. We also thank Mary Vernov (Weill Cornell Medical College) for proofreading the manuscript.

Mice are allowed ad libitum access to food, water, and maintained on a 12-hr light-dark cycle. All procedures involving laboratory animals conformed to National and European laws and were approved by the French Ministry for Education and Scientific Research (CEEA-LR-00651-01). A total of 6 transgenic 5xFAD and 4 littermate wildtype (WT) control mice were used for this procedure.
1. Pre-operative Preparation
<ol>
	<li>Intraperitoneally (IP) inject Methoxy-X04 (10 mg/kg) 48 hr before surgery to label A&#9...

<ol>
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Authors have no competing financial interests.

The open-skull technique for in vivo two-photon microscopy offers the advantage of unlimited imaging sessions of large imaging fields13,14. However, this technique also produces inflammation in the region of interest14, often incompatible or impacting neuro-vascular read-outs15. On the contrary, the thinned skull transcranial technique does not result in neuro-inflammation, enabling reliable imaging of the cerebrovascular structures and plaque accumulation10,14. A seco...

The brain vasculature is a multi-cellular structure, which is anatomically and functionally coupled to neurons. A dynamic remodeling of vessels occurs throughout brain development and during the progression of pathologies of the central nervous system (CNS) 1,2. It is widely accepted that cerebrovascular damage is a hallmark of several CNS diseases, including epilepsy, Alzheimer&#39;s disease (AD), traumatic brain injury and encephalitis 3,4. Therefore, tracking cerebrovascular changes in vivo becomes significant when modeling CNS diseases, from onset and into chronic phases. As cerebrovascular modifications often occur concomitantly with neuronal damage or plasticity, imaging of the neuro-vasculature represents a key entry point to decipher CNS disease pathophysiology.
This protocol describes a longitudinal two-photon based procedure to track the remodeling of the cerebrovasculature in a mouse model of AD, a progressive pathology marked by cerebrovascular defects on large and small caliber vessels due to amyloidogenic plaque deposition 5-7. This procedure allows for the visualization of amyloid deposits and tracking of their position and growth with respect to neurovascular remodeling throughout the course of the disease. Vital fluorescent dyes are injected before each imaging session for the visualization of the cerebrovasculature and amyloid plaques in transgenic AD mice8. Repeated imaging sessions of a ROI through a thinned skull transcranial window is non-invasive and the method of choice to assess neurovascular remodeling in the living mouse brain 2,5,9,10.
The procedure below outlines the surgical protocol, image acquisition and processing. The early progression of cerebral amyloid angiopathy (CAA) mostly at large leptomeningeal and penetrating arterioles is characterized.

<table border="0" cellpadding="0" cellspacing="0" width="627">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">methoxy-X04</td>
			<td style="width:125px;">tocris</td>
			<td align="right" style="width:119px;">4920</td>
			<td style="width:167px;">use 10 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-Dextran 70Kda</td>
			<td style="width:125px;">sigma</td>
			<td align="right" style="width:119px;">46945</td>
			<td>use 100 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gelfoam/Bloxang</td>
			<td style="width:125px;">Bausch and Lomb</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micorsurgical blade</td>
			<td style="width:125px;">surgistar</td>
			<td align="right" style="width:119px;">6900</td>
			<td>must be sharp and not dented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">povidone-iodine</td>
			<td style="width:125px;">betadine</td>
			<td style="width:119px;"></td>
			<td>antisceptic solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">binocular stereomicroscope</td>
			<td style="width:125px;">olympus</td>
			<td style="width:119px;">SX10</td>
			<td>optimal image contrast is crucial for this procedure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-photon microscope</td>
			<td style="width:125px;">zeiss</td>
			<td style="width:119px;"></td>
			<td>Zeiss LSM 710mp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine scissors-toughcut</td>
			<td style="width:125px;">Fine science tools</td>
			<td>14058-09</td>
			<td>this scissors are optimized for cutting skin and soft tissue</td>
		</tr>
	</tbody>
</table>

longitudinal in vivo imaging of the cerebrovasculature: relevance to cns diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

This protocol describes a method for visualizing the cerebrovasculature and amyloid deposits overtime. Fluorescent dyes were injected to label amyloid depositions (methoxy-XO4) 11 and to fill the cerebrovascular lumen (FITC-Dextran)1. 3D image analysis software modules were used to create 3D images of a constant field of view captured at consecutive time points. Representative images obtained in the somatosensory cortex of 5XFAD mice5 (genetic model of AD)...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases at JoVE.com

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

The overall goal of this procedure is to track the remodeling of the vasculature in the mouse brain during the progression of a chronic disease over extended periods of time. This method can help answer key questions in the field of neuroscience, such as the role of cerebral vasculature remodeling in diseases of the central nervous system. The main advantage of this technique is that it is minimally invasive and without neural inflammation, making it ideal to study pathologies where inflammation is actually implicated.This technique allows the visualization of pial and and penetrating cortical vessels, complementing histological techniques direct at the visualization of microcapillaries in the parenchymal of the blood-brain barrier. Demonstrating the procedure will be Margarita Arango-Lievano. She's a post-doc from my laboratory.To begin this procedure, clean the surgical bench and microscope plate with 70%ethanol and cover them with a clean, absorbent cloth. Next, check the appropriate level of anesthesia with paw or tail pinches. Then apply sterile ophthalmic ointment to the eyes and shave the fur from the nose to the ears with a razor blade.Afterward, sterilize the skin with povidone iodine antiseptic solution. Place the mouse under the binocular microscope and make a skin incision from the ears to the nose. Pull the skin sideways to expose the skull.Then tear the periosteum and repeatedly rinse the skull with sterile ACSF to stop possible bleeding. In this procedure, remove the eye ointment with a cotton tip. Then apply a drop of topical ophthalmic anesthetic.Gently pressure the skin around the eye socket to achieve protrusion. After that, inject FITC conjugated with dextran in the retro-orbital sinus. Subsequently, apply sterile ophthalmic ointment to the eyes.Ensure that the skin is retracted and the skull is clean and dry around the selected imaging area. Then apply a small amount of cyanoacrylic glue around the window of the head mount device and glue the head mount device around the target region. Subsequently, apply gentle pressure to the head mount for several seconds.Pull the ends of the skin to the edge of the window of the head mount device, making sure the area is dry. Secure the skin and the edge of the window with a small amount of cyanoacrylic glue and wait until dry. Afterward, secure the animal at the stage using the head mount device.Rinse the bone in the target area with sterile ACSF and ensure that the well between the skin and the head mount device is perfectly sealed. Following that, wrap the mouse in a survival blanket to maintain normothermia. Now perform thinning of the skull in ACSF solution to attenuate vibrations created by drilling.And keep in mind to change the ACSF regularly to clear the bone debris and to avoid tissue overheating. Using a dental drill, start thinning the skull surface in an area with the diameter of one millimeter using regular vertical motion parallel to the skull, and remove most of the spongy bone. Note that the bone near the sutures is highly vascularized.If bleeding occurs, apply ACSF pre-soaked gel foam to stop it. So we keep the bone to limit inflammation, but excessive pressure or heating during the thinning procedure can also result in inflammation. Use a sharp, disposable, ophthalmic microsurgical blade to continue with the thinning of the skull to result in a final region of about 0.5 millimeters in diameter with a thickness of 20 to 35 micrometers, being careful not apply excessive pressure.Due to bone regrowth and scarring, it is necessary to further thin the window at each imaging session. In this step, transfer the mouse with the head mount stage under the two-photon laser microscope. Using a 20X water immersion objective with a numerical aperture of 1.0, locate the thin cranial window at the center of the optical field using the epifluorescent lamp.Ensure that the objective is always immersed in ACSF. Next, start laser scanning with a mode-locked pulse laser. Set the excitation wavelength at 750 nanometers to detect the emitted fluorescence of the methoxy-X04 in the blue channel and FITC-dextran in the green channel.Acquire a low magnification stack at 0.7X numerical zoom in order to create a 3D map for the precise re-localization of the ROI at later time points. Then acquire a mosaic of four high-digital magnification images with a two X numerical zoom. Using the imaging software, move the window in 200-micrometer steps to capture all regions.The depth of the stacks is typically 250 micrometers starting from the pial surface in each image of the mosaic. Before removing the mouse from the microscope, take a picture or make a hand-drawn 2D map of the pial vasculature for future re-localization of the imaging field. Shown here is the comparison of two reconstructed images of the microvasculature and amyloid depositions on a five X FAD mouse at four and five months of age.Amyloid plaques are indicated with white asterisks, and new plaques appearing at five months are indicated by yellow asterisks. A rare instance of plaque reduction is indicated with an orange asterisk. Newly formed microvessels are indicated with yellow arrows, while vascular occlusion is indicated with a red arrow.This figure shows that the amyloid vascular deposits on large-caliber vessels. At the same time, amyloid plaques can be also observed on small-caliber vessels. Red arrows indicate occluded vessels, whereas white asterisks indicate growing amyloid plaques.So once mastered, this technique can be performed in 1 1/2 hours, including the surgery and imaging steps. While attempting this procedure, it is important to plan the number of imaging session in order to limit excessive thinning in the first session. Overall, these procedures allow tracking changes in cerebral vasculature during disease progression.

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7.0K 조회수.  Institute of Functional Genomics. The overall goal of this procedure is to track the remodeling of the vasculature in the mouse brain during the progression of a chronic disease over extended periods of time. This method can help answer key questions in the field of neuroscience, such as the role of cerebral vasculature remodeling in diseases of the central nervous system. The main advantage of this technique is that it is minimally invasive and without neural inflammation, making it ideal to study pathologies where inflammat...