The overall goal of the following experiment is to assess vascular function In patients with chronic kidney disease, this is achieved by first collecting vascular endothelial cells from patients. Aortic pulse wave velocity is then assessed to measure large elastic artery stiffness. Next brachial artery flow mediated dilation is measured in order to assess vascular endothelial function.
Ultimately, results can be obtained that characterize vascular endothelial function. This method can help answer key questions in the vascular field, such as what happens at the cellular level to the expression of various proteins. Generally, individuals new to this method will struggle because extensive practice and training is required to master ultrasound imaging.
Demonstrating the procedure will be Lonnie Perino, a research assistant from my laboratory. To begin prepare the site with topical antiseptic. Next place sterile fenestrated drapes over the site.
A trained nurse or physician should place an IV and attach a hep lock adapter. Place two J wires on the drapes to uncoil the J shape. Pull the arc of the J on both wires.
Once uncoiled, uncap the hep lock and feed the JWI into the vein approximately eight centimeters. Push the wire back and forth several times before removing to avoid accumulating too much blood on the wire. Next, use wire cutters to snip the wires so that they fit in a 50 milliliter conical tube containing about 30 milliliters of dissociation buffer.
Repeat these steps for the second wire when sample collection is complete. Return to the wet laboratory. Clasp the wires with a pair of forceps and hold them inside of the tube but above the solution for 10 minutes.
Use a motorized pipetter to rinse the wires with dissociation buffer from the tube. Release the buffer so that it runs down the length of the wires and shake off excess fluid into the tube. Remove the wires and centrifuge the sample to pellet.
Retrieve the samples and resuspend the pellet in PBS before centrifuging. For an additional five minutes afterwards, use a pipette tip on the end of a suction hose to leave about two milliliters in the tube. Vacuum off the rest without disturbing the pellet.
Resuspend the pellet and pipette the sample evenly into the oval areas of eight previously labeled slides. Place the slides in the incubator at 37 degrees Celsius for five hours before storing at minus 80 degrees Celsius until ready for analysis to begin. The subject should change into disposable shorts and lie supine in a quiet, dim, climate controlled room.
When ready to begin position the electrodes for the ultrasound and our arterial stiffness device. Next, place the blood pressure cuff on the subject. Begin the blood pressure readings after 20 minutes, repeat the readings until the measurements are within five millimeters of mercury.
Resting two minutes between each reading and performing at least three. Next palpate for the brachial artery pulse and place the sonometer to record brachial wave forms. Repeat for the radial, femoral, and carotid arteries.
Measure the distance to each of these sites from the super sternal notch using a tape measure for the brachial, radial, and carotid arteries and custom ruler for the femoral. Next, calculate the carotid brachial carotid radial and carotid femoral pulse wave velocity using specialized software. Place the forearm blood pressure cuff just distal to the reon process.
Set the vascular software to trigger mode and record at least 10 cardiac cycles of baseline brachial artery, ultrasound images and blood flow velocity measurements. A mechanical arm can be used to steady the ultrasound probe if desired. Next, inflate the forearm blood pressure cuff to 250 millimeters of mercury.
And begin a timer. Instruct the participant to remain very still. Begin recording velocities when the timer reads four minutes, 45 seconds trigger.
Release the cuff at five minutes and change the ultrasound to record B mode images. When the clock reads five minutes, 10 seconds. Continue recording until the clock reads seven minutes.
Next, take the subject's blood pressure. If systolic blood pressure is greater than or equal to 100 millimeters of mercury, place 0.4 milligrams of sublingual nitroglycerin under the subject's tongue and begin the timer. Begin recording B mode when the clock reads three minutes and stop recording.
When the clock reads eight minutes. To analyze the stained endothelial cells, a single blinded experimenter should scan the slides Systematically, endothelial cells are identified by positive staining for VE cadherin and nuclear integrity is confirmed by positive staining for dapi. In addition, the cells should be stained for the protein of interest.
A total of 30 cells per slide should be imaged for later analysis. Repeat this process for each slide in the stained batch, including the VE control slide. Qualitative software is used to analyze the intensity of the staining for the primary antibody of interest.
This image shows the baseline brachial artery diameter obtained during assessment of a patient with chronic kidney disease after cuff release for two minutes. This recording shows an R wave gated change in diameter. These wave forms were obtained at each of the carotid and femoral arteries.
The difference in time and distance was used to calculate a report of aortic pulse wave velocity. Here, control and patient cells were analyzed for nuclear integrity with DPI endothelial cell expression with VE cadherin and for the oxidant enzyme N-A-D-P-H oxidase. While attempting this procedure, it's important to remember to obtain the highest quality ultrasound images, waveforms and endothelial cell images possible for accurate and quantitative assessment.
Don't forget that working with human samples can be extremely hazardous, and precautions such as wearing appropriate personal protective equipment should always be taken while performing this procedure.
