This investigation was supported by the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH) under 1R01HL149404-01A1 (BL), and the Ruth L. Kirschstein National Research Service Award T32 HL 007936 to the University of Wisconsin-Madison Cardiovascular Research Center (JB). Figures were created or edited with Biorender.com. Statistical analysis was performed using GraphPad Prism 10 software.

Animal protocols are approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (M005792).
1. Animal maintenance
<ol>
	<li>Raise mice on standard maintenance chow. Use adult mice or young adult mice (8-12 weeks old). 
	NOTE: Using adults ensures that the animals have reached full maturity and limits any chance that changes in aortic diameter could be related to animal growth. For this study, we utilized male and female C57BL/6J mice aged 22-24 weeks at the time of surgery. Lu and colleagues did not observe significant differences in the aneurysmal response between younger and older mice8. Additionally, while most aneurysm modeling is performed in male mice, this model successfully induces AAAs in both male and female mice9.</li>
	<li>Determine the study duration and assign animals to treatment or sham (control) groups. Administer 0.2% BAPN drinking water to treatment group mice and subject them to surgery with topical application of active elastase to the infrarenal aorta. Administer untreated water to control animals and subject them to surgery with the application of denatured elastase to the infrarenal aorta.</li>
</ol>
2. Initiation of B-aminopropionitrile (BAPN)-supplemented drinking water
<ol>
	<li>Two days before surgery, start treatment mice on 0.2% BAPN drinking water. Prepare BAPN water in larger volumes and store it in the dark at 4 &#176;C for up to 28 days. Be sure the BAPN water reaches room temperature before giving it to the mice. 
	NOTE: We recommend BAPN water be replaced in cages every 7 days throughout the duration of the study.</li>
</ol>
3. Day of surgery material preparation
<ol>
	<li>Cut surgical gloves into 5 mm x 10 mm strips, which will be used later to help isolate the aorta before treatment with elastase. Prepare a surgical drape by cutting a ~ 1.5 cm x 3 cm sized oval in the center of a surgical drape. Unfold the 2 in x 2 in gauze and cut in half to create strips of gauze roughly 2.5 cm x 10 cm to be used later for retraction of abdominal contents. Autoclave all surgical instruments (see the Table of Materials) and set up a sterile surgical field as shown by the example in Figure 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66812/66812fig01.jpg" /> 
Figure 1: Example of the sterile surgery setup in preparation for the elastase/BAPN murine model of AAA. Abbreviations: BAPN = &#223;-aminopropionitrile; AAA = abdominal aortic aneurysm. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Prepare a postoperative recovery cage by placing a clean cage under a heat lamp and place saline near the lamp to warm to body temperature (37 &#176;C). Ensure the heat lamp is safely positioned so that the recovery cage and saline are warm but are not beyond 37 &#176;C. Turn on the water pump to begin circulating warm water through the heating pad.</li>
</ol>
4. Animal preparation for surgery
<ol>
	<li>Place the mice in an induction chamber and anesthetize them with 5% isoflurane at 200 mL/min using a low-flow electronic vaporizer. While anesthetized, weigh each mouse and deliver 0.6 mg/kg of buprenorphine ER and 20 mg/kg of carprofen subcutaneously for analgesia. Use electric hair trimmers to clip the fur on the abdomen, from the lower abdomen to inferior of the xyphoid process. Use gauze or a laboratory wipe to brush away excess hair. Return mice to their cage and wait at least 20 min for analgesia to take effect before proceeding with surgery.</li>
	<li>After at least 20 min since analgesia was administered, place the mouse in an anesthesia induction chamber and again deliver 5% isoflurane at 200 mL/min using a low-flow electronic vaporizer until the mouse is sedated.</li>
	<li>Remove the sedated mouse from the induction chamber and place it supine on the surgical field. Apply eye gel and secure the nose cone with surgical tape. Reduce the delivered inhaled isoflurane to a maintenance rate of 1-2% at 50 mL/min. Secure the mouse&#39;s front and hind paws with surgical tape.</li>
	<li>Examine the mouse&#39;s lower abdomen for the bladder. Gently apply external pressure to the bladder between the thumb, index, and middle fingers to induce voiding; meanwhile, use a piece of gauze to wick away the urine. 
	NOTE: Be careful to not contaminate the surgical field.</li>
	<li>Begin disinfecting the abdomen by applying an iodine-based or chlorhexidine-based scrub and 70% alcohol with cotton-tipped swabs. Start in the center of the abdomen and work outward in a circular motion 3x. Allow the area to briefly dry between applications.</li>
	<li>Check for a lack of a toe-pinch response to ensure anesthesia is adequate. Ensure the nose cone and limbs are secured. Place a surgical drape over the mouse, with the opening directly over the surgically prepped abdomen. 
	NOTE: Do not drag the drape across the mouse to avoid potential contamination.</li>
</ol>
5. Surgical induction of AAA
<ol>
	<li>Entering the abdominal cavity:
	<ol>
		<li>Wash hands and wear clean nitrile or sterile surgical gloves. Before contacting the surgical field, always spray gloves with 70% EtOH and rub gloved hands together until dry.</li>
		<li>Use blunt forceps to tent the skin in the midline abdomen. Use a scalpel to make a midline longitudinal abdominal incision, roughly 2-3 cm in length.</li>
		<li>Use forceps to lift the rectus muscles to identify the translucent linea alba. Use scissors to enter the abdominal cavity through the linea alba, then extend along the linea alba proximally and distally.</li>
	</ol>
	</li>
	<li>Exposing the abdominal aorta:
	<ol>
		<li>Dampen a strip of gauze and two cotton-tipped swabs with warmed saline. Create an abdominal roll by tightly rolling one end of the gauze halfway, leaving a generous tail.</li>
		<li>Use a skin retractor to retract the right abdominal wall.</li>
		<li>Using dampened cotton-tipped swabs, perform a right-medial visceral rotation by gently sweeping the small and large intestines to the left upper quadrant and visualize the aorta and inferior vena cava (IVC). Use an abdominal roll to retract the bowel out of view &#8211; tuck the rolled end of the gauze underneath the bowel, and then bring the tail end around and out of the body to gently swaddle the bowel. Apply gentle tension to the tail of the gauze to hold the bowel out of the field of view. Adjust the abdominal roll and skin retractor to gain an optimal view of the retroperitoneal organs, as shown in Figure 2A. 
		NOTE: The abdominal roll helps to keep the bowel moist and protect it from being inadvertently damaged by surgical instruments. Be sure the gauze remains damp during the procedure to prevent the bowel from drying out. Be careful to not forcefully retract the bowel as this can cause kinking of the superior mesenteric artery and bowel vasculature, which can potentially cause ischemic damage. Moreover, when initially sweeping the small intestine, be cautious of a thin translucent attachment between the large bowel and the inferior liver (hepatocolic ligament), which if not careful, can easily tear off the liver capsule and cause bleeding. If there is tension on this ligament during retraction, divide it sharply with scissors.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66812/66812fig02.jpg" /> 
Figure 2: Representation of abdominal retraction and the optimal surgical view for exposure of the mouse infrarenal aorta. (A) Placement of a gauze abdominal roll helps to retract the intraabdominal organs, while an opposing retractor assists in providing visualization of the retroperitoneum. A sterile surgical drape (transparent to show animal orientation) is placed over the anesthetized animal to help maintain sterility. (B) The retroperitoneal fascia (green box) overlies the aorta anteriorly. (C) Example of the infrarenal aorta following dissection of the retroperitoneal fascia. Isolation of the aorta from the IVC can be achieved by starting at&#160;a potential space between the aorta and IVC located just distal to the left renal vein as it crosses anteriorly (yellow circle). Abbreviation: IVC = inferior vena cava. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Circumferential dissection and isolation of the infrarenal aorta:
	<ol>
		<li>Confirm that the IVC and infrarenal aorta are in full view. Begin exposing the aorta by first entering and dividing the retroperitoneal (RP) fascia (Figure 2B). Identify the gonadal (testicular or ovarian) arteries that run parallel along the anterior infrarenal aorta (Figure 2B and Figure 3). Use forceps to bluntly divide the fascia between the gonadal arteries and continue longitudinally to expose the aorta anteriorly (Figure 2C). 
		NOTE: The RP fascia is a thin, translucent layer of connective tissue that contains lymphatics and the splanchnic plexus. It is necessary to dissect through the RP fascia to expose the aorta. However, do not dissect through the connective tissue of the aortic adventitia. A tear in the adventitia (white connective tissue) will expose the media (appears bright red), and the aorta will likely rupture at this site once elastase is applied.</li>
		<li>Next, start to isolate the abdominal aorta off the IVC. Begin this dissection at a small gap between the IVC and aorta, located just below the inferior edge of the left renal vein as it crosses the aorta (Figure 2C). Use the tips of forceps to gently spread apart the connective tissue fibers between the aorta and IVC and continue working circumferentially around the aorta at this level. 
		NOTE: The IVC is very thin-walled and closely adheres to the aorta by a fine layer of fibrous connective tissue. Be careful to avoid handling the IVC or cleaning off the IVC as much as possible. Dissecting the right side of aorta off the IVC first (before dissecting the left side of the aorta from the surrounding musculature) will help the aorta to &#34;fall away&#34; from the IVC.</li>
		<li>Continue bluntly dissecting the plane between the aorta and IVC, working caudally toward the aortic bifurcation. Stop further distal dissection once reaching the aortic bifurcation. 
		NOTE: Take extra precaution when dissecting around the inferior mesenteric artery (IMA), which is typically located near the mid-section of the infrarenal aorta and travels laterally across the IVC.</li>
		<li>Once the right edge of the aorta is separated from the IVC, return proximally to the level of the left renal vein. Dissect the RP fascia off the lateral left edge of the aorta, working circumferentially around until the aorta is fully isolated. See Figure 3 for relevant anatomy of the retroperitoneal dissection. 
		NOTE: Be careful dissecting behind the aorta as there is high variability in the location and number of lumbar veins and arteries. See Figure 4 for reference of areas at high risk for bleeding with this dissection.</li>
		<li>Carefully inspect that the aorta is circumferentially isolated from the IVC and the surrounding musculature as much as possible, with careful dissection around the aortic segments tethered by the IMA and lumbar arteries.</li>
		<li>Place a strip of glove along the right and left edges of the aorta, as demonstrated in Figure 5A. Attempt to cover as much of the IVC as possible.</li>
		<li>Use handheld calipers to measure the widest aortic diameter and record three measurements. Spray the tips of the calipers with 70% EtOH before and after measurements. Avoid directly contacting the aorta with the caliper tips to prevent contamination. 
		NOTE: Photos using a calibrated camera-capable microscope can also be utilized.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66812/66812fig03.jpg" /> 
Figure 3: Anatomy of the blood supply to the lower abdomen, pelvis, and retroperitoneum of the mouse. Abbreviations: R = right; L = left. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66812/66812fig04v2.jpg" /> 
Figure 4: Sites of high risk for injury and hemorrhage during the retroperitoneal dissection and circumferential isolation of the infrarenal aorta. Abbreviations: L = left; IMA = inferior mesenteric artery. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66812/66812fig05.jpg" /> 
Figure 5: Intraoperative responses to elastase application or sham during the elastase/BAPN murine AAA model. (A) Segments of glove are placed along the length of the aorta prior to elastase application to help protect the IVC and bowel from exposure to elastase while keeping the aorta soaked in elastase (B) Application of denatured elastase does not cause dilation of the aorta (blue box). The maximal aortic diameter measured 0.627 mm at baseline, then 0.607 mm after 5 min of topical denatured elastase. (C) Application of elastase causes aortic dilation after 5 min of treatment. In this example, the aorta (green) dilated to 0.953 mm from 0.607 mm, a 57% increase in diameter. Abbreviations: BAPN = &#223;-aminopropionitrile; AAA = abdominal aortic aneurysm. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Elastase application:
	<ol>
		<li>Use a cotton-tipped swab to dab any extra blood or fluid off the aorta.</li>
		<li>Next, place a 10 mm x 2 mm piece of dry gauze on top of the aorta. Use a pipette to dispense 5 &#181;L of elastase (or control denatured elastase) to saturate the gauze and aorta. Gently fold the pieces of the glove around the aorta. 
		NOTE: To prepare denatured elastase for use in sham or control groups, boil elastase at 100 &#176;C for 30 min.</li>
		<li>Allow 5 min for elastase to act on the aorta. During this incubation period, if required, release some of the tension placed by the abdominal roll and the skin retractor. 
		NOTE: Due to the batch effect with elastase, we encourage investigators to use the same bottle of elastase for all experiments within a given study. With each new bottle of elastase, we recommend performing a dose-response to ensure there is not an overwhelming number of early ruptures (prior to 4 weeks). The duration of elastase application can also be adjusted between 4 and 6 min depending on the response to elastase.</li>
		<li>After 5 min, reset bowel retraction and unfold the pieces of the glove. Irrigate the abdominal cavity with 1 mL of warm 0.9% sterile normal saline, while carefully removing the gauze and pieces of glove off the aorta. Absorb the saline in the abdomen with 10 mm x 10 mm gauze. Repeat irrigating the abdomen for a total of 3 x 1 mL.</li>
		<li>Use handheld calipers to remeasure the widest aortic diameter post elastase application and record 3x. See Figure 5B,C for examples of the aortic dilation to treatment with sham and active elastase. 
		NOTE: The averages of the three pre- and post- elastase measurements can be used to calculate the percent change in aortic diameter with treatment. Typically, there is a notable dilation ~30-50% immediately after elastase treatment, which can help to ensure the elastase is functional and the aorta was adequately treated. The diameter of the aorta should not change with the application of denatured elastase or may measure slightly smaller (likely from spasm).</li>
	</ol>
	</li>
	<li>Closure of the abdominal cavity:
	<ol>
		<li>Carefully remove the abdominal roll from underneath the bowel and out of the body.&#160;If needed, apply additional saline to the bowel to prevent sticking to the abdominal roll during removal. Check to make sure the bowel appears pink and adequately perfused. 
		NOTE: It is not necessary to try and reposition the bowel back into its original location; attempting so can risk twisting of the bowel or internal hernias.</li>
		<li>Reapproximate the abdominal fascia using a running 5-0 non-absorbable monofilament suture. Close the skin with 3-4 skin staples.</li>
	</ol>
	</li>
</ol>
6. Postoperative animal care
<ol>
	<li>Place the mouse in the recovery cage with a heat lamp. Ensure the temperature of the cage is warm, not hot.</li>
	<li>Administer a 0.5-1 mL subcutaneous fluid bolus of 0.9% normal saline.</li>
	<li>Allow the mouse to recover by itself in the warmed cage for ~20 min until active as per institutional protocol, then return to a housing cage.</li>
	<li>Per institutional protocol, administer carprofen 20 mg/kg at 24 h after surgery on postoperative day 1, and continue daily for 3 days.</li>
</ol>
7. Aortic measurement and tissue harvest
<ol>
	<li>Following euthanasia with isoflurane and cervical dislocation, reopen the abdominal cavity. Extend the incision through the sternum to access the thorax. Excise the right atria and perfuse the left ventricle with 10 mL of cold 1% Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) solution over 2 min. Resect the lungs, liver, and spleen. 
	NOTE: Be careful to not injure the bowel; spillage of enteric contents can impact tissue analysis.</li>
	<li>Expose the abdominal aorta, and measure the maximal infrarenal aorta diameter, as described above. Continue to dissect the entire aorta and heart. Once the heart and aorta are isolated, cut all arterial branches and common iliac arteries leaving short segments intact on the aorta. Place the heart and aorta on a contrasted background next to a ruler and image.</li>
</ol>
8. Data analysis and reporting
<ol>
	<li>To help account for human error, measure aortic diameters at least 3x each when using handheld calipers, then report the diameter as the averaged value.</li>
	<li>Define AAA as a 50% increase in the healthy aorta diameter. Be sure to include both the gross aortic diameters and the percent change in diameter in the study results.</li>
</ol>

Abdominal Aortic Aneurysm Development in Mice Using the Elastase and BAPN Method

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The authors of this manuscript have no conflicts of interest to declare.

Understanding the complex pathophysiology of AAA is critical for improving the management of aortic aneurysm disease. While newer strategies are actively developed to improve surgical outcomes, AAAs remain prevalent in our aging society and aneurysm rupture remains a leading cause of death in the United States10. Therefore, the unmet needs in AAA detection, prevention, and treatment strategies warrant further foundational aneurysm research11.
Animal models that accurately and efficiently recapitulate the features and behaviors of human AAAs are essential for mechanistic studies of aneurysm pathophysiology and identifying potential therapeutic targets. While current animal models can mimic the major aspects of the aneurysmal changes that occur in human disease, no single model fully represents the true complexity of human AAAs. Currently, mice are the most widely accepted species for animal AAA modeling. Researchers should consider the various strengths and weaknesses of each murine model for their particular aneurysm study, such as those expertly described in reviews by Daugherty et al. and Busch et al.12,13.
The use of elastase to induce AAA in rodents was first described by Anidjar et al. in 199014. Perfusing the aorta with porcine pancreatic elastase using a syringe pump creates an initial dilation roughly between 50% and 70%, and the dilated segments favorably demonstrate similar pathologic features of human AAAs, such as medial degeneration and adventitial inflammation. The classic perfusion model, however, is arguably the most technically challenging aneurysm model, and the aneurysms that are typically formed by the second week begin to gradually resolve thereafter. Bhamidipati et al. in 2012 then demonstrated that adventitial application of elastase could also successfully induce similar aneurysms that are more reproducible in size15. A far less challenging model, the topical elastase model became widely adopted in aneurysm research. Additional methodology and advantages of the topical elastase model are discussed in the methods paper by Xue and colleagues16.
The elastase/BAPN model of murine AAA was developed by Lu and colleagues in 20178. Introducing 0.2% BAPN drinking water improved upon many of the critiques of the classic topical elastase model, now producing aneurysms that continually expand to the point of AAA rupture. In their 2017 study, they demonstrated mice in the elastase/BAPN-treated group had significantly higher AAA formation rates compared to the elastase group (93% vs 65%, P &#60; 0.01), which were also more advanced-staged AAAs. Over a 100-day study period, AAAs in the elastase/BAPN group continued to dilate to &#62;800% baseline diameter and formed intraluminal thrombus (53.8%), and 46.2% spontaneously ruptured before the end of the experiment. This model has allowed researchers to investigate factors that may impact aneurysm progression and stability over time.
Berman et al. further explored the elastase/BAPN model by varying the concentration of topical elastase, study duration, timing of BAPN administration, and the impact of animal sex9. Treatment with 5 &#181;L of higher concentrated elastase (5 mg/mL or 10 mg/mL) produced larger aneurysms than 2.5 mg/mL over 56 days. The prevalence of intraluminal thrombus formation also depended on the elastase concentration, which occurred in 28.6% of the 5 mg/mL-treated mice, and 62.5% of the 10 mg/mL-treated mice. They also demonstrated the elastase/BAPN model could induce aneurysms in female mice. Although only a few female mice were studied (n=5), they found the aneurysms in females were more prone to rupture (2 of 5 mice) and were significantly larger than male AAAs at 56 days.
In this paper, we aim to provide a method to address one of the largest limitations of surgical modeling, which is the variation in the surgical procedure. Without a clear consensus on the degree of dissection and the area of the aorta treated with elastase, the results of this model could vary dramatically between animals, investigators, and institutions. We have observed numerous anatomical variations between mice, including the number and size of lumbar arteries and veins, and the location of the IMA, takeoff of the left gonadal vein, among others, which can be limiting when attempting to treat only a portion or specific segment of the infrarenal aorta. Here, we demonstrate that circumferentially dissecting the entire length of the infrarenal aorta from the left renal artery proximally to the aortic bifurcation distally helps to provide reproducible degrees of aortic exposure despite anatomical differences while increasing the success of aneurysm induction and providing clear boundaries for the operator. Additionally, the size and more anterior position of the IVC tends to cover a majority of the aorta, which can affect the amount of aorta treated if not isolated from the IVC. While it is necessary to remove the retroperitoneal fascia to expose the aorta, it is important to not fully dissect the connective tissue of the adventitia off the aorta and expose any of the media layers, as this typically results in rupture during the 5 min elastase incubation period. This could serve as an additional internal control to the degree of the dissection with this model yet can be a frustrating learning curve when adopting this model. Operators will additionally learn higher risk areas (Figure 4) that can be easily injured during surgery and lead to uncontrollable hemorrhage.
While it is important that the procedural steps of this model are consistent, the duration of the study and timing of interval ultrasonography can vary depending on the goal of the research. Aortic dilation begins immediately with elastase application, yet studies using this model commonly follow mice for 28 days post surgery7, as in this example experiment. Extending the study duration should be considered when studying advanced AAAs, long-term growth, intraluminal thrombus formation, or rupture.
Additional perioperative measures, such as maintaining animal body temperature and hydration status can help to improve animal survival of this invasive procedure. The use of a heating pad during surgery and placement in a warm recovery cage can help avoid hypothermia. Saline should be warmed before it is used to irrigate the abdominal cavity. A subcutaneous fluid bolus directly following surgery can account for insensible fluid losses during the operation, and help the animal maintain adequate hydration during the immediate recovery phase. With careful tissue handling and a consistent methodical approach, the elastase/BAPN model can be performed by an experienced operator between 30 min and 45 min per mouse and reliably produce AAA with very low perioperative complications.
Our results demonstrate that the combination of BAPN in addition to circumferential dissection of the infrarenal aorta prior to elastase application produces large, continually expanding AAAs, with larger diameters and rupture incidence at shorter periods. In this experiment, AAAs were successfully induced in all male (6 of 6) and female (6 of 6) mice treated with active elastase. Elastase exposure for 5 min resulted in an immediate increase in aortic diameter by roughly 30-40%, which is helpful in confirming successful and consistent elastase application among treatment groups. Similar to Berman et al., we have shown that this model can induce AAAs in female mice, which also have a greater rupture response than males. Half of the female mice (3 of 6) ruptured within 28 days, compared to 0 of 6 of the males, however, female mice weigh less than males. Male mice demonstrated an increase in AAA diameter by 257% compared to -4% of male controls, while the surviving females showed a 417% diameter increase, compared to -16% of female controls. Aortic diameters were not significantly different between the surviving male and female treated mice at 28 days due to the higher number of ruptures in the female group. We speculate the sham mice exhibit smaller aortic diameters by the end of the study as the aorta tends to dilate slightly during the initial dissection and then, forms scar tissue by 28 days.
The elastase/BAPN model possesses certain limitations. Circumferential dissection of the aorta requires fine surgical skills yet helps improve replicability and the degree of aneurysm induction. Similar to the topical elastase model, there is also a batch effect in elastase enzyme activity, which as mentioned earlier, is therefore important to utilize the same bottle of elastase for all animals in a given experiment. While the incidence of AAA intraluminal thrombus and rupture increases with time and aneurysm severity, these are not guaranteed nor fully predictable in this model.
In summary, the elastase/BAPN model produces large, true infrarenal AAAs in both male and female mice, which progressively expand over time, form intraluminal thrombus, and are capable of rupture. These strengths of this murine model help to better recapitulate some of the behaviors and characteristics of aneurysms in humans. Although technically difficult, careful and thorough dissection of the aorta can augment the aneurysmal response. Currently, the elastase/BAPN method is an advanced model for studying infrarenal abdominal aortic aneurysms.

An aneurysm is defined as a pathologic dilation of a blood vessel greater than 50% of the healthy vessel diameter1. Despite abdominal aortic aneurysms (AAA) being a commonly encountered condition in the aging population, with an incidence of roughly &#62;5% of males &#62; 65 years of age, there are no directed therapeutic strategies for treating AAA1. Current management of AAA is limited to risk factor reduction and surgical repair with either open or endovascular surgery based on aortic diameter or growth rate2. The greatest danger of AAA is aneurysm rupture, which is fatal if untreated, and repair in this emergent setting can carry mortality risks upwards of 90%1.
The pathophysiology of AAA is complicated, multifactorial, and not fully understood3. Features of human AAA include true aneurysmal dilation of the aortic wall with an infiltration of inflammatory cells, the presence of intraluminal thrombus, and progressive dilation that leads to eventual rupture3,4. Additionally, AAAs are associated with advanced age, have a 9:1 male:female sex predominance, and most commonly occur in the infrarenal aorta5. Modeling all features and behaviors of human AAAs in animals remains an ongoing challenge6.
Current AAA modeling is primarily conducted in mice, and aneurysms are commonly induced using one of three methods-by angiotensin II (AngII) infusion via a subcutaneously implanted osmotic pump, and by direct application of calcium chloride (CaCl2) or elastase to the aorta7. In the latter method, porcine pancreatic elastase (PPE) is applied to a segment of the infrarenal aorta and causes enzymatic degradation of elastin fibers within the elastic lamella of the tunica media. This structural damage results in the weakening of the aortic wall and outward aneurysmal dilation. The use of topical elastase alone, however, produces relatively small infrarenal aneurysms, which fail to progressively enlarge or rupture over time. More recently, Lu et al. improved upon this model by additionally administering &#946;-aminopropionitrile (BAPN), an irreversible inhibitor of lysyl oxidase, to their elastase-treated mice8. By preventing the crosslinking of elastin and collagen fibers, BAPN supplementation causes elastase-damaged aortas to progressively dilate to the point of rupture. The elastase/BAPN model additionally has a higher incidence rate of AAA than the topical elastase model, and the aneurysms produced are also larger and contain intraluminal thrombus8.
In the elastase/BAPN model, the degree of surgical dissection and exposure of the aorta to elastase can impact the success and replicability of this model. In this manuscript, we describe that co-administration of BAPN drinking water and topical elastase application to the aorta following circumferential isolation of the entire infrarenal aortic segment improves replicability, accounts for anatomical differences between animals, and results in a greater AAA induction rate, aneurysm sizes and rupture incidence. In this article, we will describe a standardized approach to reliably inducing advanced abdominal aortic aneurysms in mice using a combination of topical elastase- and BAPN-supplemented water.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 L induction chamber</td>
			<td>Kent Scientific Corporation</td>
			<td>SOMNO-0530XXS</td>
			<td>anesthesia induction chamber</td>
		</tr>
		<tr>
			<td>0.9% sodicum chloride injection, USP, 20 mL</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-03</td>
			<td>normal saline</td>
		</tr>
		<tr>
			<td>3 mL syringe Luer-Lok Tip with BD PrecisionGlide Needle 22 G x 3/4</td>
			<td>BD</td>
			<td>REF 309569</td>
			<td>syringe, 22 G needle</td>
		</tr>
		<tr>
			<td>3-Aminopropionitrile Fumarate</td>
			<td>TCI</td>
			<td>A0796</td>
			<td>BAPN</td>
		</tr>
		<tr>
			<td>3-Aminopropionitrile Fumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>A3134-25G</td>
			<td>BAPN</td>
		</tr>
		<tr>
			<td>Avant Delux gauze sponges, 2&#34; x 2&#34; 4-Ply</td>
			<td>Medline</td>
			<td>NON26224</td>
			<td>gauze sponges</td>
		</tr>
		<tr>
			<td>Balding clipper</td>
			<td>Whal Clipper Corporation</td>
			<td>8110</td>
			<td>hair clippers</td>
		</tr>
		<tr>
			<td>betadine surgical scrub (povidone-iodine, 7.5%)</td>
			<td>Avrio</td>
			<td>NCD 67618-154-16</td>
			<td>betadine surgical scrub</td>
		</tr>
		<tr>
			<td>blunt forceps</td>
			<td>ROBOZ</td>
			<td>RS-5130</td>
			<td>blunt forceps</td>
		</tr>
		<tr>
			<td>Buprenorphine ER-lab</td>
			<td>ZooPharm</td>
			<td>BERLAB0.5</td>
			<td>buprenorphine</td>
		</tr>
		<tr>
			<td>carprofen</td>
			<td>Norbrook</td>
			<td>NDC 55529-131-11</td>
			<td>carprofen</td>
		</tr>
		<tr>
			<td>CASTROVIEJO 5.75&#34; straight with lock</td>
			<td>ROBOZ</td>
			<td>RS-6412</td>
			<td>Castroviejo needle driver</td>
		</tr>
		<tr>
			<td>cotton tipped wood applicators, 6&#34;</td>
			<td>Dynarex</td>
			<td>No. 4302</td>
			<td>cotton tipped wood applicators</td>
		</tr>
		<tr>
			<td>DESMARRES 5.5&#39; rectractor&#160;</td>
			<td>ROBOZ</td>
			<td>RS-6672</td>
			<td>skin rectractor&#160;</td>
		</tr>
		<tr>
			<td>digital caliper, 0-150 mm</td>
			<td>World Precision Instruments</td>
			<td>501601</td>
			<td>digital caliper</td>
		</tr>
		<tr>
			<td>DPBS (1x)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td>Elastase from porcine pancrease Type I</td>
			<td>Sigma-Aldrich</td>
			<td>E1250-10MG</td>
			<td>elastase &#62;4.0 units/mg protein</td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td>ethanol diluted to 70%</td>
		</tr>
		<tr>
			<td>eye lube</td>
			<td>Optixcare</td>
			<td>14716</td>
			<td>eye lube</td>
		</tr>
		<tr>
			<td>Germinator 500 dry sterilizer</td>
			<td>CellPoint Scientific, Inc</td>
			<td>5-1450</td>
			<td>dry bead sterilizer</td>
		</tr>
		<tr>
			<td>heat therapy mat</td>
			<td>Adroit Medical Systems</td>
			<td>V016</td>
			<td>heat therapy mat</td>
		</tr>
		<tr>
			<td>heat therapy pump</td>
			<td>Adroit Medical Systems</td>
			<td>HTP-1500</td>
			<td>heat therapy pump</td>
		</tr>
		<tr>
			<td>isoflurane, USP</td>
			<td>Akorn Animal Health</td>
			<td>NCD 59399-106-01</td>
			<td>isoflurane</td>
		</tr>
		<tr>
			<td>L-10 pipette</td>
			<td>Rainin</td>
			<td>LTS 0.5-10 uL</td>
			<td>pipette</td>
		</tr>
		<tr>
			<td>Low profile anesthesia mask, small</td>
			<td>Kent Scientific Corporation</td>
			<td>SOMNO-0801</td>
			<td>anesthesia nose cone</td>
		</tr>
		<tr>
			<td>micro dissector scissors</td>
			<td>ROBOZ</td>
			<td>RS-5619</td>
			<td>micro dissector scissors</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>microscope</td>
		</tr>
		<tr>
			<td>Nii-LED high intensity LED illuminatorLED exertnal light</td>
			<td>Nikon Instruments, Inc</td>
			<td>83359 NII-LED</td>
			<td>external dissection light</td>
		</tr>
		<tr>
			<td>nylon 5-0 monofilament, black non-absorbable suture</td>
			<td>Oasis</td>
			<td>MV-661-V</td>
			<td>5-0 nylon suture</td>
		</tr>
		<tr>
			<td>polyisoprene surgical gloves, GAMMEX Non-Latex PI Micro, size 7.5</td>
			<td>Ansell</td>
			<td>20685975</td>
			<td>non-latex surgical gloves</td>
		</tr>
		<tr>
			<td>Reflex 7 mm stainless steel wound clips</td>
			<td>CellPoint Scientific, Inc</td>
			<td>203-1000</td>
			<td>wound clips</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>Ohaus</td>
			<td>Compass CR2200</td>
			<td>scale</td>
		</tr>
		<tr>
			<td>SomnofFlo Accessory Kit</td>
			<td>Kent Scientific Corporation</td>
			<td>10-8000-71</td>
			<td>tubing for electronic vaporizer&#160;</td>
		</tr>
		<tr>
			<td>SomnoFlo electronic vaporizer</td>
			<td>Kent Scientific Corporation</td>
			<td>SF2992</td>
			<td>low-flow electronic vaporizer</td>
		</tr>
		<tr>
			<td>SomnoPath Flow Diverter</td>
			<td>Kent Scientific Corporation</td>
			<td>SP1016</td>
			<td>flow diverter for electronic vaporizer</td>
		</tr>
		<tr>
			<td>SS/45 sharp forceps</td>
			<td>ROBOZ</td>
			<td>RS-4941</td>
			<td>sharp forceps</td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>ROBOZ</td>
			<td>RS-6010SC</td>
			<td>surgical scissors</td>
		</tr>
		<tr>
			<td>vessel forceps</td>
			<td>Dumont</td>
			<td>VES 0.35</td>
			<td>vessel forceps</td>
		</tr>
	</tbody>
</table>

advanced abdominal aortic aneurysm modeling in mice by combination of topical elastase and oral &#223;-aminopropionitrile

The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with &#223;-aminopropionitrile (BAPN)-supplemented drinking water to reliably produce true infrarenal aneurysms with behaviors that mimic human AAAs. Topically applying elastase to the adventitia of the infrarenal aorta causes structural damage to the elastic layers of the aortic wall and initiates aneurysmal dilation. Co-administering BAPN, a lysyl oxidase inhibitor, promotes sustained wall degeneration by reducing collagen and elastin crosslinking. This combination results in large AAAs that progressively expand, form intraluminal thrombus, and are capable of rupture. Refining surgical techniques, such as circumferentially isolating the entire infrarenal aortic segment, can help standardize the procedure for a consistent and thorough application of porcine pancreatic elastase despite different operators and anatomic variations between mice. Therefore, the elastase/BAPN model is a refined approach to surgically inducing AAA in mice, which may better recapitulate human aneurysms and provide additional opportunities to study aneurysm growth and rupture risk.

Male and female C57BL/6J mice ages 22-24 weeks were used in this study. Infrarenal aortas were treated with 5 &#181;L of elastase enzyme (6.9 mg protein/mL, 6 units/mg protein) or denatured elastase for 5 min. Elastase-treated male mice demonstrated a 43.4% increase in aortic diameter after 5 min exposure to elastase compared to untreated baseline aortic diameters, while treated female aortas increased by 33.6% (P = 0.0342). Aortic diameters of shams exhibited relatively no change over 5 min exposure to denatured elastase (males 0.5%; females -2.8%). There were no surgically related deaths among the 12 treated and 6 sham mice. Data for the 28-day study is demonstrated in Table 1. Of the female treated mice, 3 of 6 died from AAA rupture; one on postoperative day 20 and two on day 25 (Figure 6). There were no AAA ruptures among treated males. AAAs (defined as an increase &#62;50% of the baseline aorta diameter or death by AAA rupture) were successfully induced in all treated mice (12 of 12). At 28 days, the average AAA diameter of treated males was 2.86 &#177; 0.31 mm, with an average change percent change of 257 &#177; 54%, while AAA diameters of the surviving treated female mice were 3.60 &#177; 1.87 mm, with an average percent change of 417 &#177; 286% (Figure 7). Sham mice exhibited relatively no change in aortic diameters.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66812/66812fig06.jpg" /> 
Figure 6: Survival of male and female B6 mice during a 28-day elastase/BAPN model of AAA. (A) AAA rupture occurred in 3 of the 6 treated female mice, (one mouse at 20 days, then two mice at 25 days) while there were no ruptures among the 6 treated male mice at 28 days. (B) Representative images at necropsy of a female mouse that died from AAA rupture. AAA rupture is demonstrated by a large retroperitoneal hematoma (left) and presence of an infrarenal AAA with a wall defect (right). Abbreviations: BAPN = &#223;-aminopropionitrile; AAA = abdominal aortic aneurysm. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66812/66812fig07.jpg" /> 
Figure 7: Maximal aortic diameters of elastase/BAPN and sham male and female B6 mice at 28 days. (A) Treated mice exhibit significantly larger infrarenal diameters at 28 days compared to shams. (B) The combination of elastase and BAPN successfully produces large infrarenal AAAs in both male and female B6 mice. Abbreviations: BAPN = &#223;-aminopropionitrile; AAA = abdominal aortic aneurysm. <a href="https://www.jove.com/files/ftp_upload/66812/66812fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3"></td>
			<td>B6 male Sham</td>
			<td>B6 female Sham</td>
			<td>B6 male Elastase/BAPN</td>
			<td>B6 female Elastase/BAPN</td>
		</tr>
		<tr>
			<td colspan="3">Number of mice</td>
			<td>3</td>
			<td>3</td>
			<td>6</td>
			<td>6</td>
		</tr>
		<tr>
			<td colspan="3">Age (weeks)</td>
			<td>22.3 &#177; 0.0</td>
			<td>22.7 &#177; 0.7</td>
			<td>23.1 &#177; 0.2</td>
			<td>23.2 &#177; 0.2</td>
		</tr>
		<tr>
			<td colspan="3">Weight (g; at surgery)</td>
			<td>36.3 &#177; 2.5</td>
			<td>23.7 &#177; 1.2</td>
			<td>32.8 &#177; 1.7*</td>
			<td>23.7 &#177; 0.8</td>
		</tr>
		<tr>
			<td colspan="3">Pre-treatment aortic diameter (mm)</td>
			<td>0.89 &#177; 0.02</td>
			<td>0.75 &#177; 0.04</td>
			<td>0.81 &#177; 0.07</td>
			<td>0.73 &#177; 0.09</td>
		</tr>
		<tr>
			<td colspan="3">Post-treatment aortic diameter (mm)</td>
			<td>0.90 &#177; 0.03</td>
			<td>0.73 &#177; 0.01</td>
			<td>1.15 &#177; 0.03**</td>
			<td>0.98 &#177; 0.12**</td>
		</tr>
		<tr>
			<td colspan="3">Percent change post 5 min treatment (%)</td>
			<td>0.5 &#177; 4.4</td>
			<td>-2.8 &#177; 5.3</td>
			<td>43.4 &#177; 10.2***</td>
			<td>33.6 &#177; 4.5***</td>
		</tr>
		<tr>
			<td colspan="3">AAA incidence (%)</td>
			<td>0 / 3</td>
			<td>0 / 3</td>
			<td>6 / 6</td>
			<td>6 / 6</td>
		</tr>
		<tr>
			<td colspan="3">AAA ruptures by 28 days</td>
			<td>0 / 3</td>
			<td>0 / 3</td>
			<td>0 / 6</td>
			<td>3 / 6</td>
		</tr>
		<tr>
			<td colspan="3">Survival to 28 days</td>
			<td>3 / 3</td>
			<td>3 / 3</td>
			<td>6 / 6</td>
			<td>3 / 6</td>
		</tr>
		<tr>
			<td colspan="3">Maximal aortic diameter at 28 days (mm)</td>
			<td>0.85 &#177; 0.01</td>
			<td>0.64 &#177; 0.01</td>
			<td>2.86 &#177; 0.31*</td>
			<td>3.60 &#177; 1.87**</td>
		</tr>
		<tr>
			<td colspan="3">Percent change aortic diameter at 28 days (%)</td>
			<td>-4 &#177; 2</td>
			<td>-16 &#177; 2</td>
			<td>257 &#177; 54*</td>
			<td>417 &#177; 286**</td>
		</tr>
	</tbody>
</table>
Table 1: Results of a 28-day model of the elastase/BAPN murine model of AAA. Data are mean &#177; SD. *P&#60;0.05, **P&#60;0.005, ***P&#60;0.0001 compared to sham of the same sex via one-way ANOVA Fischer&#39;s test. Abbreviations: BAPN = &#223;-aminopropionitrile; AAA = abdominal aortic aneurysm.

Author Spotlight: Enhanced Murine AAA Model Using Elastase to Mimic Human Aneurysms

This protocol describes a methodical surgical approach to modeling advanced abdominal aortic aneurysms in mice by a combination of directly applying elastase to the infrarenal aorta and administering &#223;-aminopropionitrile through drinking water.

Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral &#223;-aminopropionitrile

The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with &#223;-aminopropionitrile (BAPN)-supplemented drinking ...

advanced-abdominal-aortic-aneurysm-modeling-mice-combination-topical

Research

JoVE Journal

Biology

1.2K Views.  University of Wisconsin-Madison School of Medicine and Public Health. Ons onderzoek heeft tot doel de pathofysiologie van abdominaal aorta-aneurysma te begrijpen. In het bijzonder willen we graag op moleculair gebied weten hoe een aneurysma scheurt. Wij geloven dat het modelleren van aorta-aneurysma bij muizen, mannen en vrouwen, ons zal helpen een nieuwe behandeling te ontwikkelen voor deze zeer dodelijke ziekte die dringend farmacologische behandeling nodig heeft.De pathofysiologie van abdominaal aorta-aneurysma is mul...

Video: Auteur in de schijnwerpers: Verbeterd AAA-model voor muizen met behulp van elastase om menselijke aneurysma's na te bootsen

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral ß-aminopropionitrile

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