The overall goal of this videomicroscopy technique is to examine the functional properties of isolated microvessels in response to pharmacological and physiological stimuli, providing insight into the pathophysiology and molecular mechanisms that contribute to vascular dysfunction in humans. This method is useful for understanding the molecular mechanisms that contribute to dysfunction of the local microvasculature within fat, which has been linked to systemic diseases. The main advantages of this technique are that blood vessels remain functional after removal from the human body for a period of time and are easily examined for their physiologic properties.
Clinical relevance of this experimental approach is that it allows us to identify pathways that are differentially altered in disease conditions and to potentially discover novel therapeutic targets. Under a tissue dissecting microscope, use microscissors and microforceps to carefully remove the surrounding fat and connective tissue from the small arterials within the adipose sample. It is essential to distinguish the arterials from the venules.
Arterials are typically smaller in diameter, demonstrate a greater tone, and respond more robustly than do venules. When the arterial has been isolated, use nylon or silk sutures to tie off any branches. Next, use a 10 milliliter syringe to slowly fill the tubing with fresh Krebs solution.
Then, attach rubber tubes to the pressure reservoirs and glass capillary pipettes within the chamber. Next, move the dissected arterial to the organ chamber and cannulate the vessel onto the glass capillary pipettes. Carefully secure both ends of the arterial on the pipettes with nylon sutures.
Once the vessel is secure, slowly remove the Krebs buffer from the chamber and add two milliliters of fresh Krebs solution to the chamber. Next, attach the organ chamber to the stage of an inverted microscope equipped with a video camera. Turn on the edge detection software at a sampling rate of one kilohertz.
Connect the remaining pressurized tubing to the second Krebs solution-filled pressure reservoir and then connect the pressure reservoirs to the pressure transducer. When all of the tubing has been connected, set the heating block to 37 degrees celsius. Next, use the pressure control unit to gradually increase the intraluminal pressure to five milliliters of mercury every five minutes to achieve the appropriate experimental pressure within the lumen of the isolated blood vessel.
Allow the vessel to equilibrate for 20 to 30 minutes once the pressure reaches 60 milliliters of mercury and record the diameter of the adipose arterial at rest. At the end of the equilibration period, pre-constrict the blood vessel to approximately 55%of the resting baseline diameter by adding one microliter of endothelin 1 directly to the bath every five minutes until vessel diameter has been appropriately constricted. For flow-induced endothelial dependent vasodilation, induce continuous flow into the intraluminal space of the arterial in equal and opposite directions so that a pressure difference can be developed across the vessel without altering the mean intraluminal pressure of 60 millimeters mercury.
After three to five minutes, measure the flow-mediated dilation. Increase each increment of pressure gradient by a 10 centimeter of water change every five to six minutes up to a maximum of 100 centimeters of water. After assessing the flow-mediated vessel dilation, return the pressure reservoirs to the same height to stop the induction of flow.
Then immediately, but carefully replace the chamber solution with fresh Krebs solution without disturbing the suspended arterial and allow the vessel to begin to return to the baseline diameter for 20 to 30 minutes. Once the arterial has returned to the resting baseline diameter, a subtle choline-mediated endothelial dependent vasodilation can be assessed. This begins by pre-constricting the vessel to approximately 55%of its resting diameter with endothelin 1, as previously demonstrated.
Once pre-constriction is achieved, add two microliters of increasing doses of acetylcholine directly to the bath. Record the change in arterial diameter five minutes following administration of each dose. Upon completion of the acetylcholine dose response, assess the endothelium independent vasodilation and vessel viability by the sequential administration of papaverine and endothelium independent vasodilator directly to the bath.
Endothelium dependent vasodilation responses to increased flow and sheer stress or acetylcholine are significantly blunted in visceral versus subcutaneous adipose arterials in human obesity. Endothelium independent vasodilation in response to papaverine, however, is not differentially altered between the two dipose, suggesting that vascular dysfunction in visceral domains is largely a result of dysfunction at the level of the endothelium, at least in the early stages of the disease. Once mastered, this technique can be completed in three to five hours, if performed properly.
While attempting this procedure, it's important to remember to take your time and be diligent, as even minor damages to the blood vessels may skew the results. Following this procedure, other methods like siRNA transfection of the blood vessels can be performed to answer additional questions about the impact of specific genes on vasomotor function in human disease. After it's development, this technique paved the way for researchers in the field of adipose tissue biology and microcirculation to explore the impact of the adipose tissue microenvironment on local vascular health in human obesity.
After watching this video, you should have a good understanding of how to directly probe the pathophysiology of whole intact segments of human blood vessels, which are removed from living subjects, a process that cannot be replicated by non-invasive imaging.
