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JoVE Journal Neuroscience

Neuroscience

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

Published: May 17, 2024 doi: 10.3791/66792
1,2, 1, 1,2, 3, 2
1Brain Health Imaging Centre, Centre for Addiction and Mental Health, 2Department of Pharmacology and Toxicology, University of Toronto, 3Department of Neurology, Columbia University

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.

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Protocol

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

  1. Surgical procedure
    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. Place the nose bar from the stereotaxic frame on top of the mouse to provide stability and ensure the upper teeth are fastened.
    6. Position the animal head at a 120° angle from the body to elevate and distend the nape to expose the surgical area of interest.
    7. Clean the surgical site with Betadine scrub 3x, 70% ethanol 3x, and betadine solution 1x with sterile 2 inch x 2 inch gauze.
    8. Fold the skin gently with the help of tweezers and make a small incision (1 cm) using surgical scissors.
      NOTE: This should reveal the midline of the nape of the neck.
    9. Carefully run the scalpel along the midline to cut through the muscles. Then, using tweezers 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).
  2. 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 µL. The amount of elastase that is supposed to be in the 2.5 µL should be 15 milliUnits mU.
    1. First calculate the concentration of the solution to be injected.
      Concentration = mass/ volume
      = 15 mU/2.5 µL
      = 6 mU/µL
      Therefore, the concentration that is required is 6 mU/µL.
    2. The quantity of the elastase is 250 U and the study works with milliUnits, so convert the units to milliUnits which gives 250000 mU.
    3. 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.
    4. Using the same formula above, rearrange to solve for volume:
      Volume = mass/Conc.
      = 250000 mU/ 6 mU/µL
      ​= 41666.67 µL
    5. Since it is hard to pipette 41666.67 µL, simply convert it to milliliters by dividing it by 1000 to get 41.66667 mL to obtain a 1x solution.
      1. To make the solution more concentrated,increase it by 10x or 100x.
      2. Create a 10x solution since it is simpler to work with (see below).
        ​10x solution ---------- 4.166667 mL (conc.)
    6. 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.
    7. Pipette 10 µL of stock solution and place in 100 aliquot tubes giving 1000 µL of the 4.167 mL stock solution. Store the tubes at -20 °C.
    8. Store the remainder of stock solution in aliquot tubes at 100 µL per tube and store in freezer at -20 °C.
      NOTE: The solutions are stable at -20 °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.
    9. After thawing one of the aliquot tubes that contains 10 µL of a 10x solution, add 90 µL of sterile PBS to get of a 100 µL 1x solution.
    10. If the aliquot tube that contains 100 µL of a 10x solution was thawed instead, simply add 900 µL of PBS to get 1000 µL, which is a 1x solution. Store either solution at 4 °C for 1 week.
  3. 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.
    1. Load the Hamilton syringe with 2.5 µL of elastase (concentration of 15 mU) and ensure there are no air bubbles.
    2. 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.
    3. 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.
    4. 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.
    5. After removing the bevel, close the incision site with a non-absorbable surgical suture (#4-0 with 3/8" needle), and turn off the anesthetic.
    6. Remove the animal from the stereotaxic frame and place on a warm heat pad set at 37 °C until the subject recovers.
      NOTE: Another dose of Metacam is recommended after surgery to help with pain during post-surgical recovery.

2. Silicone rubber compound injection and tissue harvest

  1. Anesthetize the animal with an overdose of avertin (1 mL, 125-250 mg/kg).
    NOTE: Pinch the toe to ensure reflexes cease.
  2. Place the animal on a diaper and secure all limbs with surgical tape with the chest cavity facing upward
  3. Open the chest cavity using tweezers and scissors to expose the heart.
  4. 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.
  5. Replace the buffer bottle with one containing 4% paraformaldehyde (PFA)(made up in PBS) and perfuse for another 4 min.
  6. 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.
  7. 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.
  8. Remove the mouse's head after injection and store at 4 °C overnight allowing the silicone rubber compound to cure in the vessels.
  9. Next day, gently remove the skull of the mouse (preferably using fine tweezers), extract the brain, and incubate in 4% PFA overnight at room temperature (RT).
  10. After a 24 h incubation in PFA, wash the brain in PBS 3x, place in 30% sucrose, and store at 4 °C.

3. Quantitative analysis

  1. To capture high-quality images of the Circle of Willis in the entire brain (Figure 3):
    1. Remove the brain from the sucrose solution. Thoroughly dry the brain using a paper towel to wick the surface moisture.
    2. Position the brain in a brain slicer. Place the brain slicer under a light microscope equipped with a camera.
    3. Focus the microscope to a clear, high-resolution resolution of the Circle of Willis.
  2. 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's thickness horizontally to find its diameter.
  3. 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.
  4. Use a 2-way analysis of variance (ANOVA) to compare the average change in diameter between the elastase and control group.
    1. Calculate the percentage change in diameter using the formula below 21:
      Equation 1
      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.

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.

  1. Wash the brains 3x with PBS to remove the sucrose.
  2. Place the brain in 25%, 50%, 75%, 95%, and absolute ethanol for 24 h at each concentration, for a total time of 5 days.
  3. After dehydration of tissue, place the tissue in methyl salicylate (conc. ≤100%) for 12-24 h. The ethanol and methyl salicylate will allow for the removal of excess silicone in the tissue.
  4. Wash the brain in PBS 3x to remove residual chemicals and leave overnight on a rocker at 4 °C in fresh PBS.
  5. Place the brain in sucrose and store at 4 °C prior to sectioning and immunostaining.

5. Immunohistochemical staining

  1. Sectioning of brains
    1. Prior to immunosectioning, allow the brain to settle at the bottom of the tube filled with sucrose.
    2. Freeze the brain on the microtome platform using optimal cutting temperature (OCT) compound surrounded by dry ice. Slice the brain into 40 µm sections using the microtome blade.
    3. Transfer each section into a 96-well plate containing cryoprotectant to prevent freezing using a fine tip paintbrush.
    4. Securely seal the 96-well plate with plastic wrap. Store the sealed plate in a -20 °C freezer.
  2. Immunofluorescence staining (using NeuN as an example)
    1. Place the sections in a 24-well plate. Perform three washes with PBS to eliminate the cryoprotectant.
    2. 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.
    3. 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.
    4. Dilute the monoclonal mouse anti-NeuN in the blocking solution mentioned above.
    5. Add the diluted antibody to each well. Incubate overnight at 4 °C, shielded from light by foil.
    6. After the primary incubation, wash the sections 3x with PBS. Dilute the polyclonal goat anti-mouse 568 in the same blocking solution.
    7. Add the diluted antibody to each well. Incubate for 2 h at room temperature, shielded from light by foil.
    8. Wash the sections 3x with PBS after the secondary incubation.
    9. Prepare for mounting by slowly transferring sections onto slides using fine paintbrushes. Allow all sections to dry.
    10. Apply 125-150 µL of anti-fade mounting media once sections are dried. Gently place coverslips.
    11. Allow slides to dry for 24 h at RT in the dark, sealing edges with nail polish. Capture fluorescent images using imaging software.

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Representative Results

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.

After the elastase injection, the animals were perfused with PBS and 4% PFA at three distinct time intervals: 2 weeks, 1 month, and 3 months. Subsequently, their blood vessels were fixed with a silicone rubber compound to improve the visualization of blood vessels, as depicted in Figure 2. Qualitatively, the elastase-induced dilatation reveals an increased visibility of the small blood vessels around the Circle of Willis and an enlarged and tortuous basilar artery, compared to the control group that received a vehicle injection (Figure 3).

Quantitative analysis revealed a significant difference between the elastase and control groups, with enlargement of blood vessels in the elastase group seen at 2 weeks (32% increase; p-value= 0.019), 1 month (19% increase; p-value= 0.020), and 3 months (20% increase; p-value= 0.020) after injection (Figure 4). Subsequent dehydration of the brains through a gradual increase in ethanol concentration, followed by immersion in methyl salicylate, facilitated the removal of excess silicone rubber compound and contributed to an enhanced quality of histological staining (Figure 5).

Figure 1
Figure 1: Location of the Cisterna magna. Enhanced visualization reveals the Cisterna magna isolated from adjacent neck muscles, showcasing its position in relation to the brain and cranial structure, particularly the occipital bone. Highlighted by a yellow triangle, the Cisterna magna resembles an inverted triangle, offering clarity in anatomical depiction. Scale bars: 5 mm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Injection of yellow dye. Transcardial perfusion was utilized to prepare the tissue for histological staining. (A) The perfusion setup for mice involved pumping PBS into the body for 4 minutes to remove the blood, followed by PFA for another 4 min for protein fixation. (B) The injection of dye into the left ventricle of a mouse after perfusion enhances the visibility of blood vessels in the brain, facilitating easy observation with the naked eye. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Pictograph of brains injected with elastase and PBS. The visualization illustrates the enlargement of the BA and increased tortuosity around the CW following elastase injection, in comparison with the control group (PBS). CW, circle of willis; BA, basilar artery. Scale bars: 3 mm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Elastase-induced and sustained dilatation of blood vessels over 3 months. Compared to the control group treated with PBS, we observed significant blood vessel dilation following a single elastase injection into the Cisterna magna of 3-month-old mice, which sustained for 3 months. The basilar artery was measured at 2 weeks, 1 month, and 3 months after elastase injection. Data are mean ± SD; 2- way Anova, Sidak (post hoc testing); *p < 0.019 (2 weeks), *p < 0.020 (1 month), and *p < 0.020 (3 months). Please click here to view a larger version of this figure.

Figure 5
Figure 5: NeuN staining of dehydrated brain after the removal of excessive yellow dye. After placing the brain in increasing concentrations of ethanol and methyl salicylate for 24 h each, the brain was sectioned using a microtome and stained for the neuronal marker NeuN. In the diagram, it is evident that the (A) non-dehydrated brain exhibits greater visible blood vessels compared to the (B) dehydrated brain, as indicated by the white arrow. This facilitates a more accurate analysis of these sections, as it reduces the background interference related to blood vessels. Scale bars: 3 µm. Please click here to view a larger version of this figure.

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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 blood vessels for at least 3 months without the need for repeatable injection or implanting a cannula. The approach of administering elastase into an AD mouse model is another notable advantage, particularly for accelerating the deposition of amyloid plaques in the brain. Typically, AD models such as AppNL-F mice (utilized in this study) showed plaque formation at 6 months of age28. This model was selected because these mice naturally exhibit human-like amyloid deposition under normal aging conditions. Injecting elastase into the Cisterna magna at 3 months of age could exacerbate AD pathology by reducing cerebral blood flow16 and increasing blood-brain barrier disruptions12,13.

It is important to note that injecting substances into the Cisterna magna can be impeded by CSF pressure. We addressed this limitation by developing a method aimed at minimizing its impact, recognizing that we cannot eliminate the potential interference of cerebrospinal fluid (CSF) on elastase delivery to the brain. Elastase's ability to digest elastin results in a reduction in arterial stiffness, thereby weakening the arteries and causing blood vessels to elongate and become tortuous29,30, making it a suitable intervention for causing dolichoectasia.

Typically, it takes around 20 min to complete the surgical injection into the Cisterna magna per mouse. During the process, the mice are anesthetized with 1.5%-2% isoflurane used as general anesthesia. It is recommended to deploy the use of a stereotaxic frame with a tooth bar and ear bars to ensure stability and firmness of the mouse head during the operation, which is a delicate and crucial step. Incorrect positioning on the stereotaxic frame may result in a tilted head and imprecise delivery, as well as the potential to hit a blood vessel. Instead of cutting the skin with a scalpel, which can be messy, we pinch the skin with a fine tweezer and make a clean cut with a surgical scissorss to reveal the midline of the nape of the neck. The scalpel then ran along the midline to make a neat cut, and fine tweezers were used to separate muscles. Overall, this minimizes bleeding, which helps the mouse recover much faster.

We opted for a 15 mU dosage of elastase based on the findings of previous work20, which demonstrated that there was no difference between 15 mU and 25 mU dosages. Furthermore, they noted that higher concentrations led to increased mortality rates. While a 28% mortality rate was reported with the 15 mU dosage20, the method used in this study yielded no fatalities among the cohort. We had zero mortalities during the entire surgical process of all mice and a 5-10% mortality post-surgery up to 3 months afterward. However, the primary focus of this study lies in understanding dolichoectasia, the dilation of large blood vessels. This emphasis led us to concentrate on the basilar artery as the primary reference for measuring dilatation. Additionally, injecting elastase into the Cisterna magna of mice induces a broad effect, dilating both large and small blood vessels.

When injecting into the cistern magna of any animal, it is crucial to consider the distance between brain tissue and the Cisterna magna space to avoid damaging the brain with the needle. Due to mice having a relatively small distance, we only insert the bevel of the syringe into the Cisterna magna. To minimize the risk of infection, the procedure must be performed with sterile surgical equipment, with the mouse anesthetized. Administering bupivacaine and Metacam before the procedure can help reduce post-operative discomfort and promote faster recovery for the animals. The Cisterna magna, located at the base of the skull, is made visible by thoroughly drying the surgical site with a sterile cotton swab. For the current investigation, the surgical region is around 1 cm in size, which is the maximum area that can be used to obtain a clear view of the Cisterna magna. Additionally, the small incision site helps the animal to heal more quickly.

It is crucial to maximize drug retention within the Cisterna magna space. Previous research attempted to prevent drug leakage by placing a cotton swab at the injection site following elastase administration21. However, we have concerns about the efficacy of this method, as the absorbent nature of cotton at the injection site could potentially compromise the effectiveness of elastase by reabsorbing it after injection. Likewise, in another study20, the needle was quickly removed after injecting elastase into the Cisterna magna, posing another potential challenge in ensuring optimal drug enters the Cisterna magna space, potentially leading to elastase leakage into the cerebrospinal fluid. Therefore, the proposed method here begins with puncturing the Cisterna magna to release cerebrospinal fluid (CSF), followed by placing a sterile cotton swab at the puncture site to absorb excess CSF and alleviate pressure within the space. Subsequently, the cotton swab is removed, and the needle is reinserted into the same spot for elastase injection into the cisterna space without reapplying the cotton swab. Furthermore, we opted to keep the needle in place for 1-2 min post-injection, allowing the elastase to diffuse adequately throughout the Cisterna magna before gently withdrawing it. However, it should be noted that this technique requires performing the injection-free hand and steady anchoring of the needle while puncturing the Cisterna magna and injecting the elastase slowly. In addition, it is important to avoid damaging the large blood vessels across the Cisterna magna when injecting since it can result in complications like circling after surgery, which is an endpoint and requires euthanization. Overall, the method demonstrates dilatation lasting up to 3 months from a single elastase injection into the Cisterna magna, surpassing the 2-week dilatation observed in previous methods20,21. In conclusion, this approach extends the duration of dilatation but also consistently delivers substances, such as elastase, directly to the brain. This method is preferred for its minimally invasive approach to transporting substances across the blood-brain barrier, offering an additional advantage as it can further expanded for drug delivery to the central nervous system.

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Disclosures

There is no conflict of interest.

Acknowledgments

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

Name Company Catalog Number Comments
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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References

  1. Kadry, H., Noorani, B., Cucullo, L. A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids Barriers CNS. 17 (1), 69 (2020).
  2. Sethi, B., Kumar, V., Mahato, K., Coulter, D. W., Mahato, R. I. Recent advances in drug delivery and targeting to the brain. J Control Release. 350, 668-687 (2022).
  3. Vagnucci, A. H. Jr, Li, W. W. Alzheimer's disease and angiogenesis. Lancet. 361 (9357), 605-608 (2003).
  4. Banks, W. A., et al. Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit. J Neuroinflamm. 12, 223 (2015).
  5. Archie, S. R., Shoyaib, A. A., Cucullo, L. Blood-brain barrier dysfunction in CNS disorders and putative therapeutic targets: an overview. Pharmaceutics. 13 (11), 1779 (2021).
  6. Jefferies, W. A., Price, K. A., Biron, K. E., Fenninger, F., Pfeifer, C. G., Dickstein, D. L. Adjusting the compass: new insights into the role of angiogenesis in Alzheimer's disease. Alzheimers Res Ther. 5 (6), 64 (2013).
  7. Kook, S. Y., Seok-Hong, H., Moon, M., Mook-Jung, I. Disruption of blood-brain barrier in Alzheimer disease pathogenesis. Tissue Barriers. 1 (2), e23993 (2013).
  8. Kempermann, G. Activity dependency and aging in the regulation of adult neurogenesis. Cold Spring Harb Perspect Biol. 7 (11), a018929 (2015).
  9. Del Brutto, V. J., Ortiz, J. G., Biller, J. Intracranial arterial dolichoectasia. Front Neurol. 8, 344 (2017).
  10. de la Torre, J. C., Mussivand, T. Can disturbed brain microcirculation cause Alzheimer's disease. Neurol Res. 15 (3), 146-153 (1993).
  11. Gutierrez, J., Sacco, R. L., Wright, C. B. Dolichoectasia-an evolving arterial disease. Nat Rev Neurol. 7 (1), 41-50 (2011).
  12. Desai, B. S., Schneider, J. A., Li, J. L., Carvey, P. M., Hendey, B. Evidence of angiogenic vessels in Alzheimer's disease. J Neural Transm. 116 (5), 587-597 (2009).
  13. Biron, K. E., Dickstein, D. L., Gopaul, R., Jefferies, W. A. Amyloid triggers extensive cerebral angiogenesis causing blood brain barrier permeability and hypervascularity in Alzheimer's disease. PLoS One. 6 (8), e23789 (2011).
  14. Zenaro, E., Piacentino, G., Constantin, G. The blood-brain barrier in Alzheimer's disease. Neurobiol Dis. 107, 41-56 (2017).
  15. Brandl, S., Reindl, M. Blood-Brain barrier breakdown in neuroinflammation: current in vitro models. Int J Mol Sci. 24 (16), 12699 (2023).
  16. Austin, B. P., et al. Effects of hypoperfusion in Alzheimer's disease. J Alzheimers Dis. 26 (Suppl 3), 123-133 (2011).
  17. Jagust, W. J., Budinger, T. F., Reed, B. R. The diagnosis of dementia with single photon emission computed tomography. Arch Neurol. 44 (3), 258-262 (1987).
  18. Schuff, N., et al. Cerebral blood flow in ischemic vascular dementia and Alzheimer's disease, measured by arterial spin-labeling magnetic resonance imaging. Alzheimers Dement. 5 (6), 454-462 (2009).
  19. Singh, C., Pfeifer, C. G., Jefferies, W. A. Pathogenic Angiogenic Mechanisms in Alzheimer's Disease. Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy. , InTech (2017).
  20. Dai, D., Kadirvel, R., Rezek, I., Ding, Y. H., Lingineni, R., Kallmes, D. Elastase-induced intracranial dolichoectasia model in mice. Neurosurgery. 76 (3), discussion 343 337-343 (2015).
  21. Liu, F. X., et al. Modified protocol for establishment of intracranial arterial Dolichoectasia model by injection of elastase into cerebellomedullary cistern in mice. Front Neurol. 13, 860541 (2022).
  22. Lee, A. Y., Han, B., Lamm, S. D., Fierro, C. A., Han, H. Effects of elastin degradation and surrounding matrix support on artery stability. Am J Physiol Heart Circ Physiol. 302 (4), H873-H884 (2012).
  23. Li, Y., et al. The role of elastase in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins. Invest Ophthalmol Vis Sci. 62 (9), 7 (2021).
  24. Temesvári, P., Ábrahám, C. S., Gellén, J. Jr, Speer, C. P., Kovács, J., Megyeri, P. Elastase given intracisternally opens blood-brain barrier in newborn piglets. Biol Neonatol. 67 (1), 59-63 (1995).
  25. Takata, F., et al. Elevated permeability of the blood-brain barrier in mice intratracheally administered porcine pancreatic elastase. J Pharmacol Sci. 129 (1), 78-81 (2015).
  26. Hanyu, H. Diagnosis and treatment of mixed dementia. Brain Nerve. 64 (9), 1047-1055 (2012).
  27. Chui, H. C., Ramirez-Gomez, L. Clinical and imaging features of mixed alzheimer and vascular pathologies. Alzheimers Res Ther. 7 (1), 21 (2015).
  28. Saito, T., et al. Single app knock-in mouse models of alzheimer's disease. Nat Neurosci. 17 (5), 661-663 (2014).
  29. Dobrin, P. B., Canfield, T. R. Elastase, collagenase, and the biaxial elastic properties of dog carotid artery. Am J Physiol. 247 (1 Pt 2), H124-H131 (1984).
  30. Wagenseil, J. E., Ciliberto, C. H., Knutsen, R. H., Levy, M. A., Kovacs, A., Mecham, R. P. Reduced vessel elasticity alters cardiovascular structure and function in newborn mice. Circ Res. 104 (10), 1217-1224 (2009).
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Simpson, D., Morrone, C. D., Wear,… More

Simpson, D., Morrone, C. D., Wear, D., Gutierrez, J., Yu, W. H. A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction. J. Vis. Exp. (207), e66792, doi:10.3791/66792 (2024).

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