The overall goal of this procedure is to quantify the passive arterial mechanical properties and mice of different ages. This is accomplished by first dissecting out the carotid arteries with careful measurement of the in vivo and ex vivo lengths. Next bi axial mechanical tests are performed on the carotid arteries.
After this, the arteries unloaded dimensions and circumferential residual strain are measured. The final step is to calculate parameters of interest such as stiffness, stressor, strain, or to fit the data to constitutive equations. The complete set of data can be used to fully characterize the passive mechanical behavior of the arterial wall through bi axial measurements of stress, strain relationships, and residual strain.
The main advantage of this technique over existing methods is that we carefully measure in vivo and exvivo lengths and dimensions and perform multiple biaxial testing protocols. This method introduces a comprehensive set of procedures for arterial mechanical testing to make future results by scientists in the field. More consistent and comparable.
Though this method can provide very good insight into the mechanical properties of mouse arteries. It can also be applied to the mechanical testing of humans and other animals arteries with some modifications Before beginning the dissection, tape down the limbs of the sacrificed mouse to a dissecting board under a dissecting microscope. Use dissecting scissors and curved forceps to open the chest and expose the right and left carotid arteries.
Both arteries can be used for mechanical testing to increase numbers or to provide an internal control for different treatment protocols. Using curved forceps and fine tweezers. Carefully clear fat and nearby tissue until the full length of both carotids from the aorta to the common carotid bifurcation can be visualized.
Make sure to keep the arteries moist with PSS during the dissection procedure. Cut a piece of seven oh suture to the desired length. Using the digital calipers and dissection scissors, lay the suture on the vessel and use an 18 gauge needle dipped in activated charcoal.
To mark the ends of the suture on the vessel. Make sure that the complete length of the artery is lying in a single horizontal plane. For an accurate measurement, the head of the mouse may have to be tilted up to accomplish this.
Then document the in vivo length of the carotid with a photo. Remove the carotids by cutting them free at the charcoal marks with micro scissors. Normally the blood is flushed from the arteries prior to dissection.
However, for video purposes, the blood was left in the vessel. To improve visualization, place the cut carotids in a Petri dish with a small amount of PSS. Remove any fat from the outside of the arteries and let them equilibrate for two to three minutes.
Document the ex vivo length of each carotid with a photo. Place the carotids in a fuge tube filled with PSS until ready for testing. They can be stored in the refrigerator for up to three days with no changes in passive mechanical behavior.
Place the heated PSS filled bath of the Danish Myo technology system under the dissection microscope. For details on preparing the system. Consult the text.
Next, use a micrometer to just touch the cannula tips together. Record this value as the zero length of the micrometer. Now place a small loop of suture tied with a loose overhand knot on the end of each cannula.
Then with fine tweezers, carefully grasp the end of an artery brought up to room temperature and place it in the bath. Do not tweeze any other parts of the artery. Using another tweezer slip the end of the artery over one cannula for a mice of 21 days or older.
Tighten the suture knot and repeat the process for the other side. If it is difficult to mount the second side, apply a slight pressure to open up the artery When mounting the vessel. Be sure not to apply excessive force or torque to the force transducer connected to the mounting rod For mice younger than 21 days.
Slide the vessel completely onto one cannula. Touch the two cannula together and slide the vessel back over the second cannula. Then separate the cannula and tighten the suture knot.
Do not twist the artery. Use the micrometer to position the artery at its ex vivo length. The artery lengths beyond the suture ties must be subtracted from that measurement.
The artery should look unstretched. The axial force readings should be close to zero. Double check the ex vivo length.
Using the digital calipers, We have found that setting the artery at the measured ex vivo length is easier than trying to estimate the unloaded length from zero force measurements or visualization alone. Then using the in vivo stretch ratio, calculate and set the artery length between the suture ties to the in vivo testing length. The artery is now ready for testing to perform diameter tracking of a mounted artery under an inverted microscope focus on the edges of the artery wall.
The outer diameter can be measured and the inner diameter must be calculated from those values. Start the diameter tracking protocol in myo view and check tracking of the outer diameter. Tracking problems may be remedied by ensuring no fat is on the artery wall by adjusting the contrast in brightness of the image and by eliminating all the bubbles from the fluid inside and outside the tubing.
A small bubble in the artery will not affect the mechanical testing results, but it can interfere with the automated diameter tracking. For further testing, the tissue must be preconditioned to obtain a repeatable mechanical loading curve from the in vivo testing length, manually increased and decrease the pressure three times between zero and the maximum pressure for the specimen age pressure is applied from fluid loading through the cannula. When the artery is at the in vivo stretch ratio, the axial forces should decrease slightly as the pressure is increased to precondition a mounted artery with axial stretching begin by manually setting the pressure to one third of the maximum pressure and stretch the artery axially by manually turning the micrometer from the in vivo stretch ratio to the maximum axial stretch In preliminary tests.
This was a approximate maximum stretch that could be applied to an artery without causing any permanent creep. After preconditioning three pressure and three axial stretch protocols are performed on each artery. For pressure protocols, put the vessel at the in vivo stretch ratio and run the appropriate inflation protocol for the specimen age.
This will increase pressure from zero to the maximum pressure three times, stretch the artery to half of the maximum axial stretch and repeat the pressure protocol at this length. The axial force should remain approximately constant as the pressure increases. Finally, stretch the artery to the maximum axial stretch and repeat the protocol at this length.
The axial force should increase as the pressure increases. Axial stretch protocols must be done manually with this test system. First, set the pressure to one third of the maximum pressure.
Second, gradually stretch the artery to the maximum axial stretch at an approximate rate of 10 microns per second by turning the micrometer at the maximum stretch, return the artery to the in vivo length. Repeat this step three times. Repeat the protocol at two thirds of the maximum pressure and at maximum pressure.
Take care to apply the correct stretch distance and a constant stretch rate. During this protocol, remove the artery from the bath and place in a Petri dish with PSS under a dissection microscope, hold one end of the artery with fine tweezers and use a scalpel to cut three to five rings 0.5 millimeters thick. Then with tweezers, gently open up each ring into a circle and photo.
Document the most circular rings for unloaded dimension. Next, use a scalpel to cut each ring at one location and allow it to open into an arc. Allow the arcs to equilibrate for 10 minutes and then photo document them for opening angle measurements.
The remaining length of the carotid can be fixed for histology. Measurements were taken from carotid arteries from a three day old C 57 black six J mouse. Closed rings and open rings were used to calculate unloaded dimensions and opening angles.
The raw pressure, diameter, and force data versus time were collected Single loading cycles with no artifacts were isolated from this data. For each protocol, the data was next used for further calculations, analyses, and modeling to determine differences in the mechanical behavior between arteries. Once mastered, eight to 10 arteries can be characterized in a single day, especially if one person performs the dissection and ring cutting and another person performs the mechanical tests.
This technique paves the way for researchers in the field of vascular mechanics to explore vascular pathologies in mouse models of human disease. After watching this video, you should have a good understanding how to prepare mouse arteries for mechanical testing, perform the tests, and make accurate measurements on exci arteries.
