A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction
Summary
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.
Abstract
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.
Introduction
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 (Aβ) 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 Aβ 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.
Protocol
Representative Results
Discussion
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 …
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 23 G catheter | University Medstore | 2546-CABD305145 | Needed for perfusion (https://www.uoftmedstore.com/index.sz) |
| Absolute ethanol | University Medstore | https://www.uoftmedstore.com/index.sz | For removing the microfil |
| Betadine scrub | # | https://www.pittsborofeed.com/products/betadine-surgical-scrub | Sterilization |
| Betadine solution | Amazon | https://www.amazon.ca/Povidone-Iodine-10-Topical-Solution-100ml/dp/B09DTKJGHW | Sterilization |
| Bupivacaine | Provided by animal facility | N/A | Analgesic |
| Clippers | BrainTree Scientific Inc | CLP-41590 | Shave fur |
| Cotton Q-tip | University Medstore | 1962 | For surgery (https://www.uoftmedstore.com/index.sz) |
| Elastase | Sigma-aldrich | E7885 | Used for the dilatation of blood vessel |
| Ethanol | University Medstore | 39752-P016-EAAN | Sterilization (https://www.uoftmedstore.com/index.sz) |
| Goat anti-mouse 568 | Invitrogen | A11004 | For staining mature neurons |
| Graphpad prism 10 | Graphpad prism 10 | https://www.graphpad.com/ | Statistical analysis software |
| Hamilton syringe | Sigma-aldrich | 28614-U | Injection elastase |
| Heat pad | Amazon | https://www.amazon.ca/iPower-Temperature-Controller-Terrarium-Amphibians/dp/B08L4DBFFZ | Maintain body temperature |
| ImageJ software | Fiji Imagej software | imagej.net (USA) | Image analysis software |
| Induction chamber | Provided by animal facility | N/A | Anesthesia induction |
| Metacam | Provided by animal facility | N/A | Analgesic |
| Methyl salicylate | Sigma-aldrich | M6752 | For removing the microfil |
| Microfil | Flow Tech, Carver, Massachusetts | https://www.flowtech-inc.com/order/ | Dye (yellow) |
| Mouse monoclonal anti-NeuN | Millipore Sigma | MAB377 | For staining mature neurons |
| Olympus VS200 slide scanner and VSI software. | Olympus Life Science | https://www.olympus-lifescience.com/en/downloads/detail-iframe/?0[downloads][id]=847254104 | Imaging software |
| Paraformaldehyde | University Medstore | PAR070.1 | For protein fixation (https://www.uoftmedstore.com/index.sz) |
| Perfusion pump | VWR International | https://pr.vwr.com/store/product/4787969/vwr-variable-speed-peristaltic-pumps | Needed for perfusion |
| Scalpel | University Medstore | 2580-M90-10 | For surgery (https://www.uoftmedstore.com/index.sz) |
| Stereotaxic | Provided by animal facility | N/A | So secure the animal for surgery |
| Surgical scissor | University Medstore | 22751-A9-240 | For surgery (https://www.uoftmedstore.com/index.sz) |
| Surgical tape | University Medstore | https://www.amazon.ca/3M-Micropore-Tape-1530-2-Rolls/dp/B0082A9GS2 | Secure the animal on the diaper |
| Sutures | University Medstore | 2297-VS881 | For surgery (https://www.uoftmedstore.com/index.sz) |
| X2 tweezers | University Medstore | 7731-A10-612 | For surgery (https://www.uoftmedstore.com/index.sz) |
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