Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound
December 2nd, 2014
We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.
The overall goal of this procedure is to measure the aortic pressure diameter relationship in vivo to determine aortic stiffness. This is accomplished by first introducing a catheter into the tail vein for intravenous administration of vasoactive drugs. The second step is to insert a pressure catheter into the aorta via the femoral artery for invasive blood pressure measurement.
Next, the ascending aorta is visualized by ultrasound and its diameter is tracked by M mode. The final step is to infuse vasoactive drugs to raise and lower blood pressure and record the corresponding changes in aortic diameter. Ultimately, the aortic pressure diameter data collected can be used to calculate aortic compliance to show the pressure dependency of aortic stiffness.
The main advantage of this technique of our existing methods like pulse wave velocity, is that local aortic stiffness can be be measured. To begin, prepare solutions of phenylephrine and sodium nitropress side in 0.9%saline. Next, place the pressure catheter into a 30 milliliter syringe filled with distilled water and connect it to the pressure control unit.
Soak the catheter plugged in for 30 to 45 minutes. During the setup and surgery procedures, the catheter used for intravenous drug infusion is made from two 30 gauge needles and PE 10 polyethylene tubing. Insert one needle into one end of the tubing.
Next, remove the needle portion of the other needle and insert the blunt end into the other end of the tubing. Attach the catheter to a one milliliter syringe and fill with heparin saline solution. After confirming the absence of a hind limb reflex, place the mouse on a heated electrocardiogram pad.
Maintain anesthesia with 2%isof fluorine. After placing ointment on the eyes, place the animal on its side to gain better access to the tail veins. Secure the mouse onto the ECG pad with tape and make sure the animal is kept warm to promote vasodilation.
Next, use a piece of elastic tubing to tie a tourniquet around the base of the tail. Tight enough to collapse the veins, but not enough to cut off the arterial circulation. After two to three minutes, the vein should bulge out and become more visible.
Gently pull the tail taut and bend it at an angle with one hand while holding the needle parallel to the tail with the other pierce the tail at the bend through the skin and into the vein. Blood will push back into the catheter. If the needle is inserted correctly.
Place one drop of tissue glue where the needle is inserted. To secure the catheter, remove the tourniquet and confirm patency by injecting saline with little resistance. To insert the blood pressure catheter, place the animal in a supine position and tape its paws onto the ECG pad.
After removing the hair on the chest and leg with depilatory cream, use fine scissors to make an incision in the skin above the location of the femoral artery. Dissect through the subcutaneous fat tissue and use hemostats to move the abdomen aside. To visualize the artery using fine forceps, separate the nerve away from the artery vein bundle.
Gently pierce through the sheath around the artery vein bundle to separate the artery from the vein. Pass one suture around the artery at the proximal end, and place two sutures at the distal end securely, not the most distal suture to stop distal blood flow. Use hemostats to pull the proximal suture to temporarily stop blood flow into the femoral artery.
Next, use micro scissors to make a small incision into the femoral artery near the distal knot. Calibrate the blood pressure catheter just before inserting it into the femoral artery. When ready, open the incision with fine forceps with one hand and insert the catheter head into the artery with the other, not the middle.
Suture around the catheter wire to secure the catheter into place. Relax the proximal suture and advance the catheter forward into the abdominal aorta, not the proximal suture. To further secure the catheter and to prevent bleeding carefully move the ECG pad with mouse pressure catheter and saline syringe to the ultrasound imaging stage.
Connect the blood pressure catheter to the pressure control unit. Place the saline syringe in the syringe pump. Allow the animal and the catheter to equilibrate for 20 minutes.
First, reduce the isoflurane to 1.5%Mount the transducer onto the rail system so that the same view is maintained for the duration of the experiment. Visualize the ascending aorta longitudinally on B mode using a long axis view on the ultrasound mainframe. Place the M mode cursor over the section of aorta to be tracked.
Track the aortic diameter change over the cardiac cycle using M mode. Next, replace the saline syringe in the syringe pump with one containing PE solution. Record M mode at baseline aortic pressure.
Begin infusion at 360 micrograms per kilogram per minute and infuse for one minute until aortic pressure reaches a plateau. At this point, stop the infusion and wait two minutes for blood pressure to return to baseline. Next, lower the infusion rate to 240 micrograms per kilogram per minute.
Infuse for one minute until the blood pressure plateaus and record M mode. Stop the infusion and wait two minutes for blood pressure to return to baseline. Repeat these steps for 120 micrograms per kilogram per minute pe after returning to baseline, replace the PE with saline and infuse at a rate of 30 microliters per minute.
Infuse for two to three minutes until further infusion does not produce an increase in aortic pressure, and pressure is returning to baseline. Wait five minutes for the blood pressure to stabilize at baseline. Next, replace the saline with SNP solution.
Record M mode at baseline. Aortic pressure infuse for one minute at 240 micrograms per kilogram per minute, and when aortic pressure reaches a plateau, record the M mode. Stop the infusion and wait two minutes for blood pressure to return to baseline line.
Next, lower the infusion rate to 120 micrograms per kilogram per minute. Infuse for one minute until the blood pressure plateaus while recording m mode, stop the infusion and wait two minutes for the blood pressure to return to baseline. Lastly, repeat these steps With 60 micrograms per kilogram per minute.
SNP aortic pressure is increased with infusion of the vasoconstrictor phenyl rine and decreased with infusion of the vasodilator sodium nitropress side. Aortic pressure plateaus one minute after the start of the drug infusion. This graph demonstrates that aortic pressure is changed incrementally by the dose of drug infused.
Aortic diameter can be plotted against its corresponding aortic pressure to show the pressure diameter relationship. In addition, compliance can be calculated for each pressure increment and plotted against the mean aortic pressure to show the pressure dependency of aortic stiffness. After watching this video, you should have a good understanding of how to insert tail vein catheters and how to obtain images of the aorta by ultrasound.
The pressure diameter data collected by this procedure can be used to calculate in vivo aortic compliance.
Overview
This article describes a technique for measuring aortic stiffness in vivo in mice by assessing the pressure-diameter relationship of the aorta. The method involves using ultrasound to record aortic diameter and an invasive solid-state pressure catheter to measure aortic pressure.
Key Study Components
Area of Science
- Cardiovascular physiology
- In vivo imaging techniques
- Aortic compliance measurement
Background
- Aortic stiffness is an important indicator of cardiovascular health.
- Traditional methods like pulse wave velocity do not measure local aortic stiffness.
- This technique allows for direct measurement of aortic diameter changes in response to pressure variations.
- Vasoactive drugs are used to manipulate blood pressure during measurements.
Purpose of Study
- To develop a reliable method for measuring aortic stiffness in live mice.
- To assess the pressure dependency of aortic stiffness through direct measurement.
- To improve understanding of cardiovascular dynamics in experimental models.
Methods Used
- Intravenous administration of vasoactive drugs via a catheter.
- Invasive blood pressure measurement using a pressure catheter.
- Ultrasound imaging to visualize and track aortic diameter.
- Incremental changes in blood pressure to record corresponding aortic diameter changes.
Main Results
- Successful measurement of aortic diameter changes in response to varying pressures.
- Data collected can be used to calculate aortic compliance.
- Demonstrated the effectiveness of the technique in measuring local aortic stiffness.
- Provided insights into the pressure dependency of aortic stiffness.
Conclusions
- This technique offers a novel approach to assess aortic stiffness in vivo.
- It enhances the understanding of cardiovascular mechanics in experimental settings.
- The method could be beneficial for future research in cardiovascular diseases.
Frequently Asked Questions
Executive Industry Relevance
Quantitative measurement of ascending aortic stiffness in vivo using ultrasound and invasive pressure monitoring enables precise evaluation of vascular compliance in preclinical models. This approach supports mechanistic de-risking and target validation for cardiovascular drug discovery by providing physiologically relevant data on vessel biomechanics. The method enhances predictive confidence at the early discovery and translational inflection points for cardiovascular portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of vascular stiffness mechanisms in disease-relevant mouse models.
- Supports functional target validation by quantifying pressure-diameter relationships under controlled hemodynamic conditions.
- Facilitates biological de-risking by distinguishing local aortic stiffness from systemic vascular changes.
Screening & Assay Development
- Provides validated, quantitative readouts of aortic compliance for downstream compound evaluation.
- Standardizes measurement of biomechanical endpoints, improving reproducibility across studies.
- Establishes a platform for scalable assessment of vascular-targeted interventions in vivo.
Translational & Preclinical Research
- Aligns preclinical vascular stiffness measurements with translational biomarker strategies for cardiovascular disease.
- Enables continuity from mechanistic discovery to preclinical validation of therapeutic hypotheses.
- Supports risk-adjusted advancement decisions by providing physiologically relevant endpoints.
Pipeline & Workflow Integration
This method integrates into the discovery-to-preclinical continuum by enabling hypothesis testing, target validation, and quantitative assessment of vascular function in live animal models.
- Discovery Biology: Supports mechanistic hypothesis testing and pathway clarification for vascular stiffness.
- Screening: Delivers reproducible, quantitative compliance data for compound triage.
- Analytics: Generates pressure-diameter curves and compliance indices for robust statistical comparison.
- Translational Research: Bridges discovery findings to preclinical biomarker development in cardiovascular research.
- Enterprise Reuse: Provides a reusable platform for evaluating diverse vascular-targeted modalities.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in vascular target validation.
- Operational Value: Enhances standardization, reproducibility, and scalability of in vivo vascular measurements.
- Strategic Value: Improves go/no-go decision-making and capital efficiency in cardiovascular portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of vascular-targeted assets.
Implementation Considerations
- Requires expertise in small animal surgery, ultrasound imaging, and invasive pressure monitoring.
- Demands access to high-resolution ultrasound and solid-state pressure catheter systems.
- Necessitates cross-team standardization of measurement protocols and data analysis.
- Adaptation may be needed for different mouse strains or vascular regions.
- Measurement error from aortic motion must be mitigated by probe adjustment and increased sample size.
