The overall goal of this procedure is to determine the biological response of aortic heart valve tissue, or cells to cyclic pressure. This is accomplished by first isolating valve tissue from poor sign hearts and incubating them in tissue culture medium overnight. The second step of the procedure is to ensure that the pressure system is calibrated.Next.
The valve tissue or cells are placed into the pressure system. The chamber is placed in a 37 degrees Celsius incubator and the cyclic pressure regime is started for the desired period of time at the desired magnitude and frequency. Finally, the tissue is removed from the system for biological analysis.
Ultimately, results can be obtained that show how elevated pressure affects gene and protein expression through real-time PCR, Western blotting or microscopy. The main advantage of this technique is that we're able to separate the effects of mechanical pressure from other mechanical forces and biochemical stimulants. This is an advantage over other techniques such as animal models, though this can be used to help us understand the way in which mechanical forces impacted biology and the pathogenesis of a heart valves.
It's also can be used for developing tissue engineered constructs. Demonstrating this procedure for us now is toy fate. A graduate student in my laboratory To prepare tissue for pressure.
Studies collect aortic valves from adult female pigs weighing no more than 120 pounds immediately after death. Wash the valves twice with sterile PBS, then place on ice and transport to the laboratory. All subsequent steps should be performed under sterile conditions.
Ensure that the leaflets do not show any sign of degeneration, tearing, or calcification. Remove leaflets from the aortic root by cutting one third of the distance from the annulus. Place the leaflets in individual wells of a six well plate and incubate with three milliliters of DMEM supplemented with 1%antibiotic antimycotic solution, and 10%fetal bovine serum overnight at 37 degrees Celsius and 5%carbon dioxide.
This pressure system is custom made and has been designed to study the mechano biological effects of cyclic pressure on aortic valve tissue. To use the system, log onto the computer and open the lab view program. Check that the compressed air is connected to the system and open the air supply to full velocity.
Turn on the signal amplifier. Make sure the voltage reading is 0.00. Adjust as necessary.
The lab view interface has a switch marked test record with the switch set to test. Click the button marked air supply to open the inlet solenoid valve. Using the gas pressure regulator pressurize the chamber with compressed air at one PS.I read the pressure in the chamber using the digital pressure gauge located on the rear end plate of the chamber.
Once the pressure has equilibrated, record the voltage reading from the signal amplifier. Repeat for 2, 3, 4, and five PSI. Next construct a calibration curve of pressure versus voltage.
Convert the pressure from PSI to millimeters of mercury. Place the equation from the graph into the code of the lab view program. Remove the aluminum front plate from the pressure chamber and spray the chamber with 70%ethanol.
Place the six well plate containing the leaflet samples into the chamber and replace the front end plate. Ensure that the seal is airtight by tightening the nuts located on the four threaded rods by hand. Place the pressure chamber in the 37 degrees Celsius incubator.
Enter the amount of time the system cycles between compressed air input and output. These should be set to 0.6 seconds and 0.4 seconds to mimic diastolic and systolic conditions respectively at a frequency of one hertz. Then enter a data file path in lab view.
Click run and switch the test record toggle to record. Check that the pressure is at the desired level using the graph on the lab view interface. If necessary, adjust the pressure using the gas pressure regulator.
Once the experiment is complete, click stop in lab view and turn off the air supply and open the exhaust valve on the pressure chamber. Finally retrieve the pressure system from the incubator. Then remove the front end plate from the chamber and retrieve the six well plate.
The samples can now be analyzed for gene expression, protein expression, histology and mechanical properties represent pressure wave forms of normal and elevated pressure. Conditions obtained from the system are shown here under in vivo conditions. There is a difference in pressure between the left ventricle and the ascending aorta during diastole that enables the aortic valve to remain closed under normal physiological conditions.
This trans valvular pressure is 80 millimeters of mercury. However, under stage one and stage two hypertension, the trans valvular pressure can increase to 90 and 100 millimeters of mercury respectively. The pressure system is capable of simulating the maximum trans valvular pressures observed under norm 10, stage one and stage two hypertensive conditions.
Representative pressure wave forms of normal and elevated pressure conditions obtained from the system are shown here. So following this procedure, we're able to do other methods such as PCR and Western blotting and microscopy on the on the leaflets. And this helps us to understand things like changes in gene expression and protein synthesis, and helps us to understand mechano transduction in the heart valves.
This technique has been really useful in helping us understand mechanical biology and the mechanisms of disease, and it has been able to, or it can help us in the future to understand changes that occur in other tissues that may undergo changes in pressure in vivo.
