This study was made possible by the invaluable contributions of Stephanie Tam who provided support in assisting with the surgeries. We extend our sincere gratitude for her help. The National Institutes of Health (AG066162) for support of this research.

AppNL-Fmice (3 months old) that express human amyloid plaque at a physiological level were used for this study though this system can be used with any rodent model. All animal procedures were approved by the Animal Care Committee of CAMH (Protocol #843) and were in accordance with the ethical standards of the Canadian Council on Animal Care guidelines. Mice were bred in-house and kept on a 12-h light-dark cycle with ad libitum access to chow and water.
1. Procedure for Cisterna magna (CM) injection
<ol>
	<li>Surgical procedure
	<ol>
		<li>Place the mouse (APPNL-F, both sexes, 3 months) in an anesthesia induction chamber for 1 min. 
		NOTE: Flush the chamber with 5% isoflurane mixed with 1% oxygen for at least 1 min.</li>
		<li>After the mouse is anesthetized, remove the animal from the chamber and place it on a fresh surgical drape and maintain anesthesia (isoflurane, 2.5%-3%) by placing a nasal cone over the nose. 
		NOTE: Check depth of anesthesia by pinching the toe to ensure no reflexes.</li>
		<li>Apply ophthalmic ointment to prevent dryness. Then, subcutaneously inject 0.1 mL of bupivacaine (local anesthetic; 1-2 mg/kg 0.125%, with a dilution ratio of 1:2) to the incision site and Metacam (analgesic; 5 mg/kg, with a dilution ratio of 1:10). Keep the animal hydrated by injecting 0.5 mL of saline subcutaneously before doing the procedure to compensate for any potential blood loss during surgery.</li>
		<li>For a clear incision site, shave the neck at the base of the occipital bone, wipe down the surface with sterile PBS and transfer the subject to the stereotaxic frame in the prone position. 
		NOTE: Ensure that anesthesia is maintained through the stereotaxic nose cone.</li>
		<li>Place the nose bar from the stereotaxic frame on top of the mouse to provide stability and ensure the upper teeth are fastened.</li>
		<li>Position the animal head at a 120&#176; angle from the body to elevate and distend the nape to expose the surgical area of interest.</li>
		<li>Clean the surgical site with Betadine scrub 3x, 70% ethanol 3x, and betadine solution 1x with sterile 2 inch x 2 inch gauze. 
		NOTE: Gloves and instruments should be sterile before the procedure. A sterile drape should also be placed over the incision site.</li>
		<li>Make a small incision (1 cm) using a scalpel. 
		NOTE: This should reveal the midline of the nape of the neck.</li>
		<li>Carefully run the scalpel along the midline to cut through the muscles. Then, using forceps gently separate the muscles by pulling them apart to the left and right. 
		NOTE: This should reveal the Cisterna magna (inverted triangle) which is located beneath the base of the skull (Figure 1).</li>
	</ol>
	</li>
	<li>Preparation of elastase 
	NOTE: The package of elastase (powder form) that was ordered contained 250 Units (U). The amount of the elastase to be injected in each animal is 2.5 &#181;L. The amount of elastase that is supposed to be in the 2.5 &#181;L should be 15 milliUnits mU.
	<ol>
		<li>First calculate the concentration of the solution to be injected. 
		Concentration = mass/ volume 
		=&#160;15 mU/2.5 &#181;L 
		=&#160;6 mU/&#181;L 
		Therefore, the concentration that is required is 6 mU/&#181;L.</li>
		<li>The quantity of the elastase is 250 U and the study works with milliUnits, so convert the units to milliUnits which gives 250000 mU.</li>
		<li>After having a mass of 250000 mU and concentration of 6 mU/uL, figure out the volume (PBS) that would require diluting the elastase and maintaining a concentration of 6 mU/uL.</li>
		<li>Using the same formula above, rearrange to solve for volume: 
		Volume = mass/Conc. 
		= 250000&#160;mU/ 6 mU/&#181;L 
		&#8203;=&#160;41666.67 &#181;L</li>
		<li>Since it is hard to pipette 41666.67 &#181;L, simply convert it to milliliters by dividing it by 1000 to get 41.66667 mL to obtain a 1x solution.
		<ol>
			<li>To make the solution more concentrated,increase it by 10x or 100x.</li>
			<li>Create a 10x solution since it is simpler to work with (see below). 
			&#8203;10x solution ---------- 4.166667 mL (conc.)</li>
		</ol>
		</li>
		<li>Place the 250 U in a 5 mL aliquot tube and add 4.167 mL of sterile PBS into the aliquot tube to make the 10x stock solution. 
		NOTE: Prepare the elastase inside a sterile biosafety cabinet to maintain sterility.</li>
		<li>Pipette 10 &#181;L of stock solution and place in 100 aliquot tubes giving 1000 &#181;L of the 4.167 mL stock solution. Store the tubes at -20 &#176;C.</li>
		<li>Store the remainder of stock solution in aliquot tubes at 100 &#181;L per tube and store in freezer at -20 &#176;C. 
		NOTE: The solutions are stable at -20 &#176;C for up to 6 months with no significant loss of activity. In addition, it is important to note that excessive freezing and thawing of the elastase can reduce stability. Therefore, to prevent that from happening, the stock solution can be divided before freezing such that only one sample needs to be thawed at a time for injection.</li>
		<li>After thawing one of the aliquot tubes that contains 10 &#181;L of a 10x solution, add 90 &#181;L of sterile PBS to get of a 100 &#181;L 1x solution.</li>
		<li>If the aliquot tube that contains 100 &#181;L of a 10x solution was thawed instead, simply add 900 &#181;L of PBS to get 1000 &#181;L, which is a 1x solution. Store either solution at 4 &#176;C for 1 week.</li>
	</ol>
	</li>
	<li>Injecting into the Cisterna magna 
	NOTE: Clean the Hamilton syringe before loading with elastase by loading and aspirating with distilled water 5x, then ethanol 5x, and finally distilled water 5x.
	<ol>
		<li>Load the Hamilton syringe with 2.5 &#181;L of elastase (concentration of 15 mU) and ensure there are no air bubbles.</li>
		<li>Slowly insert the syringe with the bevel facing upward into the center of the Cisterna magna. 
		NOTE: Avoid puncturing any blood vessels that run across the Cisterna magna.</li>
		<li>After the puncture, remove the bevel. This will facilitate the release of a small amount of cerebrospinal fluid (CSF). Use a sterile cotton swab to absorb the excess CSF.</li>
		<li>Reinsert the bevel into the puncture site, slowly inject elastase or vehicle over 1 min into the Cisterna magna and leave the needle in place for 1 min to prevent any leakage of elastase.</li>
		<li>After removing the bevel, close the incision site with a non-absorbable surgical suture (#4-0 with 3/8&#34; needle), and turn off the anesthetic.</li>
		<li>Remove the animal from the stereotaxic frame and place on a warm heat pad set at 37 &#176;C until the subject recovers. 
		NOTE: Another dose of Metacam is recommended after surgery to help with pain during post-surgical recovery.</li>
	</ol>
	</li>
</ol>
2. Silicone rubber compound injection and tissue harvest
<ol>
	<li>Anesthetize the animal with an overdose of avertin (1 mL, 125-250 mg/kg). 
	NOTE: Pinch the toe to ensure reflexes cease.</li>
	<li>Place the animal on a diaper and secure all limbs with surgical tape with the chest cavity facing upward</li>
	<li>Open the chest cavity using forceps and scissors to expose the heart.</li>
	<li>Insert a 23 G blunt needle into the left ventricle of the heart and perfuse with PBS infused with heparin (100 U/mL) for 4 min to clear the blood vessels using a micro-perfusion pump.</li>
	<li>Replace the buffer bottle with one containing 4% paraformaldehyde (PFA)(made up in PBS) and perfuse for another 4 min.</li>
	<li>Prepare the silicone rubber compound (yellow) solution by combining 5 mL of compound with 5 mL of the diluent in a 50 mL tube. Mix thoroughly before use.</li>
	<li>Fill a 10 mL syringe with 10 mL of silicone rubber compo und. Attach the syringe to the hose connected to a 23 G blunt needle, and carefully inject the solution manually into the heart. 
	NOTE: Due to the high viscosity of the silicone rubber compound, it is important to use a larger barrel syringe or the pressure will be too great to push the plunger.</li>
	<li>Remove the mouse&#39;s head after injection and store at 4 &#176;C overnight allowing the silicone rubber compound to cure in the vessels.</li>
	<li>Next day, gently remove the skull of the mouse (preferably using fine forceps), extract the brain, and incubate in 4% PFA overnight at room temperature (RT).</li>
	<li>After a 24 h incubation in PFA, wash the brain in PBS 3x, place in 30% sucrose, and store at 4 &#176;C.</li>
</ol>
3. Quantitative analysis
<ol>
	<li>To capture high-quality images of the Circle of Willis in the entire brain (Figure 3):
	<ol>
		<li>Remove the brain from the sucrose solution. Thoroughly dry the brain using a paper towel to wick the surface moisture.</li>
		<li>Position the brain in a brain slicer. Place the brain slicer under a light microscope equipped with a camera.</li>
		<li>Focus the microscope to a clear, high-resolution resolution of the Circle of Willis.</li>
	</ol>
	</li>
	<li>Use image analysis software with a 10 cm ruler reference to measure approximately 1 cm and note the pixel count. Set the 1 cm scale based on this measurement, then measure the basilar artery&#39;s thickness horizontally to find its diameter.</li>
	<li>Use a standard pixel densitometry software to analyze the image. Measure (in centimeters) five separate readings of the basilar artery using an image analysis software and calculate the average to get an accurate representation of the basilar artery diameter.</li>
	<li>Use a 2-way analysis of variance (ANOVA) to compare the average change in diameter between the elastase and control group.
	<ol>
		<li>Calculate the percentage change in diameter using the formula below 21: 
		<img alt="Equation 1" src="/files/ftp_upload/66792/66792eq01.jpg" style="margin: auto;" /> 
		C = relative change 
		X1 = Initial value (Vehicle) 
		X2 = Final value (Elastase) 
		Consider P values less than 0.05 statistically significant. Perform all statistical analyses using a statistical analysis software.</li>
	</ol>
	</li>
</ol>
4. Removal of silicone rubber compound using alcohol dehydration
NOTE: It is important to dehydrate the brain to help remove the excess silicone rubber compound from the blood vessel which can potentially improve the quality of immunostaining.
<ol>
	<li>Wash the brains 3x with PBS to remove the sucrose.</li>
	<li>Place the brain in 25%, 50%, 75%, 95%, and absolute ethanol for 24 h at each concentration, for a total time of 5 days.</li>
	<li>After dehydration of tissue, place the tissue in methyl salicylate (conc. &#8804;100%) for 12-24 h. The ethanol and methyl salicylate will allow for the removal of excess silicone in the tissue.</li>
	<li>Wash the brain in PBS 3x to remove residual chemicals and leave overnight on a rocker at 4&#160;&#176;C in fresh PBS.</li>
	<li>Place the brain in sucrose and store at 4&#160;&#176;C prior to sectioning and immunostaining.</li>
</ol>
5. Immunohistochemical staining
<ol>
	<li>Sectioning of brains
	<ol>
		<li>Prior to immunosectioning, allow the brain to settle at the bottom of the tube filled with sucrose.</li>
		<li>Freeze the brain on the microtome platform using optimal cutting temperature (OCT) compound surrounded by dry ice. Slice the brain into 40 &#181;m sections using the microtome blade.</li>
		<li>Transfer each section into a 96-well plate containing cryoprotectant to prevent freezing using a fine tip paintbrush.</li>
		<li>Securely seal the 96-well plate with plastic wrap. Store the sealed plate in a -20 &#176;C freezer.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining (using NeuN as an example)
	<ol>
		<li>Place the sections in a 24-well plate. Perform three washes with PBS to eliminate the cryoprotectant.</li>
		<li>To prevent non-specific binding, incubate for 1 h at RT using a blocking solution containing 2% goat serum, 0.1% Triton-X 100, and 1% BSA.</li>
		<li>Prepare all components in PBS. 
		NOTE: The primary antibody used included monoclonal mouse anti-NeuN with a 1: 500 dilution. The secondary antibody used included polyclonal goat anti-mouse 568 with a 1:200 dilution.</li>
		<li>Dilute the monoclonal mouse anti-NeuN in the blocking solution mentioned above.</li>
		<li>Add the diluted antibody to each well. Incubate overnight at 4 &#176;C, shielded from light by foil.</li>
		<li>After the primary incubation, wash the sections 3x with PBS. Dilute the polyclonal goat anti-mouse 568 in the same blocking solution.</li>
		<li>Add the diluted antibody to each well. Incubate for 2 h at room temperature, shielded from light by foil.</li>
		<li>Wash the sections 3x with PBS after the secondary incubation.</li>
		<li>Prepare for mounting by slowly transferring sections onto slides using fine paintbrushes. Allow all sections to dry.</li>
		<li>Apply 125-150 &#181;L of anti-fade mounting media once sections are dried. Gently place coverslips.</li>
		<li>Allow slides to dry for 24 h at RT in the dark, sealing edges with nail polish. Capture fluorescent images using imaging software.</li>
	</ol>
	</li>
</ol>

Dolichoectasia: Vascular Dementia and Alzheimer&#39;s Disease Models

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There is no conflict of interest.

This article demonstrates an improved protocol for cerebrovascular dilatation, providing a precise and straightforward approach for elastase injection into the Cisterna magna of mice. This anatomical point serves as a direct gateway to the cerebrospinal fluid, offering a valuable avenue for the investigation of different neurological diseases. One of the main advantages of this modified technique is that injecting a single dose of elastase into the Cisterna magna of mice was able to cause and sustain dilatation of large ...

Microvascular endothelial cells that line the cerebral capillaries are the main components for forming the blood-brain barrier (BBB)1 which plays a crucial role in regulating what enters or leaves the brain circulation with the periphery. Essential nutrients needed for nervous tissue are permitted to enter the BBB, while some essential amino acids like glutamate are expelled from the brain, as high concentrations can cause permanent neuroexcitatory damage to brain tissue2. Under normal physiological conditions, the BBB limits the amount of plasma proteins like albumin3,4 and prothrombin from entering the brain since those can have detrimental effects5,6,7. Finally, the BBB protects the brain from neurotoxins that are circulating in the periphery, such as xenobiotics from food or the environment1. Overall, damage to brain tissues is irreversible, and aging that correlates with low levels of neurogenesis8 highlights the importance of the BBB in protecting and preventing any factors from accelerating the neurodegenerative process.
In dolichoectasia (or large blood vessel dilatation), a decrease in vessel elasticity is observed, which results in vessels undergoing morphological changes, thus rendering them dysfunctional9 and leading to reduced blood flow in the brain. This reduction in blood flow subsequently diminishes oxygen and glucose supply, ultimately triggering damage to the BBB through the activation of reactive astrocytes10. When the internal elastin lamina of vessels is damaged from dolichoectasia11, repeated stimulation of the vascular endothelial growth factor (VEGF) is necessary for angiogenesis. This can lead to the formation of leaky vessels and ultimately result in pathological angiogenesis, characterized by the development of defective vessels12. During pathological angiogenesis, when blood vessels become defective, a compensatory mechanism appears to restore vessel integrity by upregulating tight junction proteins. However, this process can inadvertently disrupt the BBB when the structural integrity of a blood vessel is lost13. This may occur through further disrupting the BBB and promoting the production of amyloid plaque14. Additionally, leakage from the periphery can cause neuroinflammation15, which results in neuronal degeneration and subsequent memory loss.
Structurally the protection that the BBB provides is through the tight junctions that prevent xenobiotic agents from the blood entering the brain. When permitting certain substances to enter the brain, the BBB mainly does it through two major processes, passive diffusion, or specific channels (like ion channels and transporters)1. In AD, research has demonstrated that a dysfunctional vascular system plays a significant role in the progression of the condition12,13. The formation of amyloid-beta (A&#946;) plaques and neurodegeneration can result from the breakdown of the BBB12,13 and disturbances in cerebral blood flow16. A reduction in cerebral blood flow can be seen in elderly individuals diagnosed with vascular dementia and AD17,18. Damage to the blood-brain barrier (BBB) along with a dysfunctional cerebral blood flow (CBF) can contribute to the increased production of A&#946; concentration in the brain, accompanied by the infiltration of foreign materials from the peripheral circulation19.
To investigate the pathogenesis of neurological diseases like AD and vascular dementia (VaD), models are developed to replicate the disease. In vitro models are extensively used but lack the biological environment for extensive disease modeling like mixed cell population, thus necessitating the importance of in vivo models. Mice are commonly used due to their ease of genetic manipulation in generating human-like properties (e.g., pathology) in disease. With the progression that has been made so far, there is still a continued need for improved models to emulate disease phenotypes like large vessel dilatation and their role in AD. To this end, we saw an opportunity and modified a technique that involved the injection of elastase into the Cisterna magna of mice20,21. Elastase is an enzyme that has been shown to break down elastin in connective tissue22 and in surrounding tight junctions23. The Cisterna magna was chosen as the point of injection due to it being located directly above the circle of Willis, the largest blood vessel in the brain. By injecting elastase into the Cisterna magna, we can compromise the BBB and blood vessels by breaking down the tight junctions and inducing dilatation of blood vessels (circle of Willis)24,25. Combining this technique with the use of an AD mouse model of pathology, for improved understanding of the pathogenesis for the vascular component of AD, can provide valuable insights into the complex interactions and influences between these two distinct pathologies.
Previous studies have demonstrated instances where patients display both the pathological features of AD and VaD, a condition typically referred to as mixed dementia26,27. Thus, understanding the interconnected mechanisms between both conditions can offer a more comprehensive perspective on the progression and manifestation of these neurodegenerative disorders, enhances our comprehension of the underlying mechanisms and potential therapeutic strategies. To this end, we demonstrate the application of elastase in an AD pathology mouse model (AppNL-F) to identify vascular changes.

<table><tbody><tr><td>23 G catheter</td><td>University Medstore</td><td>2546-CABD305145</td><td>Needed for perfusion&amp;nbsp; (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>Absolute ethanol</td><td>University Medstore</td><td>https://www.uoftmedstore.com/index.sz</td><td>For removing the microfil</td></tr><tr><td>Betadine scrub</td><td>#</td><td>https://www.pittsborofeed.com/products/betadine-surgical-scrub</td><td>Sterilization</td></tr><tr><td>Betadine solution</td><td>Amazon</td><td>https://www.amazon.ca/Povidone-Iodine-10-Topical-Solution-100ml/dp/B09DTKJGHW</td><td>Sterilization</td></tr><tr><td>Bupivacaine</td><td>Provided by animal facility</td><td>N/A</td><td>Analgesic</td></tr><tr><td>Clippers</td><td>BrainTree Scientific Inc</td><td>CLP-41590</td><td>Shave fur</td></tr><tr><td>Cotton Q-tip</td><td>University Medstore</td><td>1962</td><td>For surgery (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>Elastase</td><td>Sigma-aldrich</td><td>&amp;nbsp;E7885</td><td>Used for the dilatation of blood vessel</td></tr><tr><td>Ethanol</td><td>University Medstore</td><td>39752-P016-EAAN</td><td>Sterilization (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>Goat anti-mouse 568</td><td>Invitrogen</td><td>A11004</td><td>For staining mature neurons</td></tr><tr><td>Graphpad prism 10</td><td>Graphpad prism 10</td><td>https://www.graphpad.com/</td><td>Statistical analysis software</td></tr><tr><td>Hamilton syringe</td><td>Sigma-aldrich</td><td>28614-U</td><td>Injection elastase</td></tr><tr><td>Heat pad</td><td>Amazon</td><td>https://www.amazon.ca/iPower-Temperature-Controller-Terrarium-Amphibians/dp/B08L4DBFFZ</td><td>Maintain body temperature</td></tr><tr><td>ImageJ software</td><td>Fiji Imagej software</td><td>imagej.net (USA)</td><td>Image analysis software</td></tr><tr><td>Induction chamber</td><td>Provided by animal facility</td><td>N/A</td><td>Anesthesia induction</td></tr><tr><td>Metacam</td><td>Provided by animal facility</td><td>N/A</td><td>Analgesic</td></tr><tr><td>Methyl salicylate</td><td>Sigma-aldrich</td><td>M6752</td><td>For removing the microfil</td></tr><tr><td>Microfil</td><td>Flow Tech, Carver, Massachusetts</td><td>https://www.flowtech-inc.com/order/</td><td>&amp;nbsp;Dye (yellow)</td></tr><tr><td>Mouse monoclonal anti-NeuN</td><td>Millipore Sigma</td><td>MAB377</td><td>For staining mature neurons</td></tr><tr><td>Olympus VS200 slide scanner and VSI software.</td><td>Olympus Life Science</td><td>https://www.olympus-lifescience.com/en/downloads/detail-iframe/?0[downloads][id]=847254104</td><td>Imaging software</td></tr><tr><td>Paraformaldehyde</td><td>University Medstore</td><td>PAR070.1</td><td>For protein fixation&amp;nbsp; (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>Perfusion pump</td><td>VWR International</td><td>https://pr.vwr.com/store/product/4787969/vwr-variable-speed-peristaltic-pumps</td><td>Needed for perfusion</td></tr><tr><td>Scalpel</td><td>University Medstore</td><td>2580-M90-10</td><td>For surgery (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>Stereotaxic</td><td>Provided by animal facility</td><td>N/A</td><td>So secure the animal for surgery</td></tr><tr><td>Surgical scissor</td><td>University Medstore</td><td>22751-A9-240</td><td>For surgery (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>Surgical tape</td><td>University Medstore</td><td>https://www.amazon.ca/3M-Micropore-Tape-1530-2-Rolls/dp/B0082A9GS2</td><td>Secure the animal on the diaper</td></tr><tr><td>Sutures</td><td>University Medstore</td><td>2297-VS881</td><td>For surgery (https://www.uoftmedstore.com/index.sz)</td></tr><tr><td>X2 tweezers</td><td>University Medstore</td><td>7731-A10-612</td><td>For surgery (https://www.uoftmedstore.com/index.sz)</td></tr></tbody></table>

a visual approach for inducing dolichoectasia in mice to model large vessel-mediated cerebrovascular dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

We were successful in locating the Cisterna magna beneath the occipital region of the skull after carefully positioning the mouse on the stereotaxic frame and dissecting the muscles. This anatomical structure, resembling an inverted triangle and highlighted in yellow, is situated beneath the base of the skull (Figure 1). To ensure precision and prevent any damage to the brain tissue, 1-2 mm of the Hamilton syringe bevel was gently inserted into the Cisterna magna.
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Read this Scientific Article Protocol about A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction at JoVE.com

Author Spotlight: Modeling Vascular Contributions to Alzheimer's Disease in Transgenic Mice

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

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JoVE Journal

Neuroscience

541 Views.  Centre for Addiction and Mental Health. De reikwijdte van ons onderzoek is het model dolichoectasie in een transgeen muismodel om de maximale impact te zien die vasculaire veranderingen hebben op de pathologie van de ziekte van Alzheimer. We zijn in staat om aan te tonen dat een enkele dosis elastase injectie in de Cisterna magna van muizen in staat is om dilatatie te veroorzaken tot de drie maanden. Er is niet veel bekend over personen die lijden aan zowel vasculaire dementie als de ziekte van Alz...

Video: Auteur in de schijnwerpers: modellering van vasculaire bijdragen aan de ziekte van Alzheimer bij transgene muizen

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Medicine

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

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.

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.

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

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

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