Murine Model of Thoracic Aortic Dissection Induced by Oral β-Aminopropionitrile and Subcutaneous Angiotensin II Infusion

This protocol provides a detailed, step-by-step procedure for the induction of thoracic aortic dissection in mice. Specifically, it includes the precise calculation of the required doses of β-aminopropionitrile and angiotensin II, the procedure for osmotic pump filling, and the implantation technique of the osmotic pump.

Thoracic aortic dissection (TAD) is a highly fatal cardiovascular disease that lacks efficient medical treatment. Replication of animal models of TAD pathophysiology is essential for studying the intrinsic mechanisms of TAD. The widely used TAD model induced by β-aminopropionitrile (BAPN, an irreversible and orally active lysyl oxidase inhibitor) in mice has the limitation of an inconsistent success rate. This protocol describes in detail a reported modified murine model of TAD induced by oral BAPN combined with subcutaneous angiotensin II (Ang II) infusion. After four weeks of BAPN administration followed by 24 h of Ang II infusion, a murine model with characteristics similar to human TAD was reliably induced, and the success rate of TAD model construction was significantly improved. Oral BAPN inhibits the cross-linking of elastin and collagen, resulting in the destruction of the aortic wall structure and inducing aortic dilation and dissection formation to a certain extent. The subsequent induction of Ang II further exacerbates the degeneration of the aortic wall, thereby promoting the occurrence of TAD. Consequently, the combination of BAPN and Ang II represents a refined approach to constructing a murine TAD model, offering a valuable tool to explore the pathogenesis and potential therapeutic approaches for TAD.

Thoracic aortic dissection (TAD) is a serious aortic disease caused by an intimal tear due to bleeding within the wall of the thoracic aorta, resulting in separation of the aortic wall layers, blood entering the media of the aortic wall, forming a false lumen, and causing pressure on the true lumen^1,2,3. Epidemiologic studies suggest that the incidence of TAD is between 7 and 9 cases per 100,000 people per year⁴. At present, it is believed that the pathogenesis of TAD is caused by the abnormal structure and hemodynamics of the aortic media, and factors such as hypertension, dyslipidemia, and hereditary vascular disease increase the risk of TAD⁵. Surgical intervention remains the primary treatment option for TAD. However, due to the high perioperative risks, exploring the pathogenesis of TAD and early intervention methods to delay its progression is of significant importance for improving the prognosis of TAD. As it is very difficult to obtain human samples and perform experiments directly in humans, it is necessary to establish animal models of TAD that mimic the characteristics of human TAD.

Over the past few decades, many animal models of aortic aneurysm (AA) have been widely reported. However, there are still few studies on the establishment of TAD models; some researchers have even considered TAD to be a byproduct of the AA animal model⁶. In fact, given that TAD results from an initial intimal tear of the thoracic aorta followed by rapid expansion of the false lumen, this significant difference in mechanism distinguishes TAD from aortic aneurysm⁷. To date, β-aminopropionitrile (BAPN)-induced rodent aortic dissection is the most used model of TAD. BAPN, a specific and irreversible inhibitor of lysyl oxidase, inhibits the cross-linking of elastic fibers and collagen fibers in the aortic wall, and is widely used in animal models of aortic dissection^8,9,10. In most cases, BAPN has been added to the drinking water of mice to construct TAD models, and a combination of BAPN and angiotensin II (Ang II) via osmotic pump has been reported to construct TAD models^11,12. However, these methods for building TAD models are not described in detail. Because of differences in mouse strains, BAPN administration, and the concentration and duration of Ang II, the incidence and extent of TAD lesions have been unstable across different experiments. Therefore, there is an urgent need for a stable method to construct mouse TAD models.

Here, this protocol describes in detail, step by step, a simple and highly successful method using a combination of BAPN-supplemented water and Ang II osmotic pump for constructing a mouse TAD model. This protocol is applicable to most labs and is easy to learn, allowing even researchers with no experience in mouse model construction to perform it consistently.

Animal protocols were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (Approval Number TMUaMEC 2022036). Three-week-old C57BL/6J male mice were used in this study. Details of the reagents and equipment used are listed in the Table of Materials.

1. Animal maintenance and grouping

1. Raise the mice on standard maintenance chow. Use three-week-old mice for this study.
2. Randomly assign the mice to the control group (Control), the oral BAPN group (BAPN), the oral BAPN and saline infusion group (BAPN + Saline), and the oral BAPN and Ang II infusion group (BAPN + Ang II) (Figure 1). Provide the Control group mice with normal drinking water.
3. Provide the BAPN group mice with drinking water supplemented with BAPN at a dose of 1 mg/g/day for 4 weeks. Infuse the mice in the BAPN + Ang II group with Ang II (1 ng/g/min) and the mice in the BAPN + Saline group with an equivalent amount of saline for 24 h after 4 weeks of oral BAPN administration.

2. Preparation for BAPN-supplemented drinking water

1. Provide the mice with BAPN-supplemented drinking water for 4 consecutive weeks after 3 days of adaptive feeding, setting 1 day as an induction cycle.
2. Weigh the mice in each cage to calculate the amount of BAPN required in the drinking water.
3. Record the water volume (vol) of each cage over the past day.
4. To ensure adequate water intake, use 1.3 times the daily water volume (1.3 x vol) as the amount of water for an induction cycle. Accordingly, dissolve 1.3 times the required weight of BAPN in the calculated volume of drinking water. Detailed calculation method and an example are shown in Table 1.
   NOTE: Mice at this age are still in a period of rapid growth. It is recommended that the water intake volume of the mice be recorded daily throughout the model induction period and that the BAPN-supplemented drinking water in the cage be changed every day. To prevent the decomposition of BAPN, wrap the bottle in aluminum foil to protect it from light.

3. Calculation of Ang II mass

1. Weigh each mouse and calculate the Ang II mass required for the experiment based on the maximum body weight.
2. Use the calculation template (Table 2) to calculate the Ang II mass needed for the experiment.
3. Calculate the mass of Ang II required for 130 µL of Ang II solution per mouse, since each pump requires approximately 100 µL.
4. Use the calculation template (Table 3) to calculate the filling volume of Ang II solution and saline needed for the experiment.

4. Ang II dissolution

1. Store lyophilized Ang II powder in a sealed vial at -80 °C. To prevent moisture condensation, allow the Ang II to equilibrate to room temperature and then centrifuge it before opening.
2. Weigh the calculated amount of Ang II in a sterile microtube using an analytical balance.
3. Add the calculated volume of normal saline into the microtube containing the lyophilized Ang II and vortex thoroughly until fully dissolved.
4. Prepare the required Ang II solution separately for each mouse on a clean bench based on body weight. Turn the microtube upside down to ensure the solution is well-mixed.

5. Osmotic pump filling

1. Obtain the two parts of the osmotic pump from the package and prepare the pump using sterile instruments to avoid the risk of infection.
2. Weigh each pump, including the pump body and flow moderator, with an analytical balance. Record the data and use it to calculate the filling ratio.
3. Attach the filling tube to a freshly opened 1 mL sterile syringe and carefully aspirate the prepared Ang II solution described above, taking care not to aspirate air bubbles.
4. Hold the filling tube in the upward position and carefully remove any air bubbles from the syringe. Maintain this position to prevent air bubbles from entering the filling tube.
5. Gently insert the end of the filling tube into the opening at the top of the osmotic pump until it cannot be inserted any further.
6. Hold the osmotic pump upright and slowly squeeze the syringe plunger. Once the Ang II solution appears at the outlet, stop immediately and carefully remove the filling tube.
7. Carefully and slowly insert the flow moderator into the opening of the osmotic pump until no gap is visible between the flow moderator and the top of the pump. Wipe off any excess Ang II solution with sterile absorbent paper.
8. Weigh the loaded pump on an analytical balance and record it. The difference in pump weight before and after infusion is the mass of the loaded Ang II solution.
9. Calculate the filling rate using the following formula:
   Filling rate = (mass of filled pump - mass of empty pump) / (standard volume) x 100%
   NOTE: The fill volume should be more than 90% of the standard volume indicated on the instruction sheet. If so, the fill is successful. If not, the pump should be drained and refilled.
10. Place the filled pumps in sterile saline at 37 °C with the moderator head up for at least 6 h until implantation.

6. Surgical procedure for pump implantation

1. Autoclave all surgical instruments, including scissors, hemostats, tweezers, needle forceps, and suture needles, 24 h prior to pump implantation.
2. Place the mouse in an anesthetic induction chamber with 1.5%-2% isoflurane at a flow rate of 2 L/min and stabilize the mouse for 2 min. Shave the mouse hair in an area of about 2 cm × 1 cm on the mid-scapular skin.
   NOTE: Because the mouse's response to isoflurane is variable, the concentration may need to be adjusted to maintain stable anesthesia.
3. Place the mouse in a prone position with the nose in the nose cone connected to the isoflurane anesthetic machine. Apply veterinary ointment to the eyes to prevent dryness while under anesthesia. Ensure that the mouse does not respond to painful stimuli before and during pump implantation.
4. Disinfect the dorsal skin three times with medical iodophor.
5. Carefully make a transverse incision of about 1 cm on the skin using a scalpel.
6. Gently grasp the incisal margin with curved forceps and bluntly dissect the subcutaneous tissue with another pair of curved forceps to create a pocket for the pump. Ensure the pocket is large enough to allow the pump to move freely.
7. Insert the filled pump into the subcutaneous pocket with the flow moderator head positioned toward the caudal end of the mouse. Leave sufficient space to close the incision and avoid overstretching the skin.
8. Once the pump has been implanted, neatly align the incisal margin and close the skin with 6-0 non-absorbable sutures.
   NOTE: Carefully inspect the incision site to ensure that the wound is completely closed and that the pump is not pressing directly against the incision.
9. Clean the incision again with iodophor and apply 5% lidocaine anesthetic gel topically using a sterile cotton swab. Turn the isoflurane off (0%).

7. Postoperative animal care

1. Monitor mice closely after pump implantation and place them in a recovery cage along with an electric heated blanket.
2. Allow the mice to recover alone in a warm cage for at least 20 min until they wake up, and then return them to their original housing cage.
3. Observe the mice hourly for the first 6 h after surgery. For the next 18 h, observe the mice at 6-h intervals. Collect samples immediately if mice are in a moribund state or die during Ang II administration.

8. Harvesting, fixing, cleaning, and imaging of aortas

1. Anesthetize mice from all experimental groups with isoflurane and then sacrifice them by cervical dislocation at the end of the study.
2. Place the mice in a supine position and secure them on a mouse plate. Make a lower abdominal incision and extend it across the chest wall until the thoracic and abdominal cavities are fully exposed.
   1. Immediately make a small incision in the right atrium, insert a sterile syringe into the left ventricle, and slowly inject approximately 10 mL of ice-cold phosphate-buffered saline (PBS) through the left ventricle until the lungs and liver turn white.
3. Subsequently, perform resection of the lungs, liver, spleen, and intestines. Fully expose the entire aorta and measure the maximum diameter of the thoracic aorta using a digital caliper. Dissect the aorta from the heart and transect all arterial branches and common iliac arteries to harvest the entire aorta.
4. Image the aorta with a digital camera, preserve it in 4% paraformaldehyde for 24-48 h, and embed it in paraffin for sectioning.
   NOTE: Take care to avoid injury to the intestinal tract, as it may interfere with tissue analysis.
5. Cut aortic paraffin sections at a thickness of 3-5 µm using a microtome, then perform dewaxing and hydration. Stain the sections with hematoxylin and eosin (H&E) and Elastic-Van Gieson (EVG) using the respective staining kits according to the manufacturer's instructions.

A total of 70 male C57BL/6J mice, aged 3 weeks, were included in this study and randomly assigned to four groups: Control (n = 10), BAPN (n = 20), BAPN + Saline (n = 20), and BAPN + Ang II (n = 20). In the BAPN group, 11 out of 20 mice developed thoracic aortic dissection (TAD) 28 days after BAPN administration, with 4 mice dying from aortic rupture. In the BAPN + Saline group, 12 out of 20 mice developed TAD, with 4 deaths due to rupture. Notably, in the BAPN + Ang II group, all 20 mice ...

Due to the limited understanding of life-threatening thoracic aortic dissection (TAD), the establishment of stable animal models is essential for exploring the molecular mechanisms underlying TAD onset and progression. β-Aminopropionitrile (BAPN), a lysyl oxidase inhibitor, is widely used in rodent models of TAD because it disrupts the cross-linking of collagen and elastin, thereby weakening the aortic wall and increasing its susceptibility to mechanical stress¹³. However, BAPN administration...

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

This work was supported by a grant from the National Natural Science Foundation of China (82370299) and the Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-060B).