分離抵抗性ラットの動脈のビデオ顕微鏡を用いた血管制御機構の評価

The overall goal of this procedure is to utilize isolated resistance arteries to test vessel reactivity to vasoactive stimuli. It will also show how to assess changes in smooth muscle control mechanisms and passive mechanical properties. This method can help answer key questions in the field of vascular biology regarding endothelial and vascular smooth muscle control mechanisms in small-resistance arteries during various conditions.

The main advantage of this technique is that small-resistance arteries can be isolated from the parenchymal cell environment and maintained at their normal pressure while responses to vasoactive stimuli are tested. The implications of this technique extend toward therapy and diagnosis of vascular dysfunction. This type of manipulation is not possible in vivo.

Though this method can provide insight into peripheral vascular disease, it can also be applied to coronary artery disease. Visual demonstration of this method is critical as the microcannulation steps are difficult to learn because of the delicacy of the procedure and the need to avoid damaging the artery. On the day of the experiment, prepare two liters of a physiological salt solution in a two-liter Erlenmeyer flask.

While adding the 20X buffer stock, ensure that the solution is continuously equilibrating with the gas mixture and stirring it with a magnetic stirring bar. Next, watch the pH while adding 0.28 grams of monosodium phosphate to the mixture. As necessary, adust the pH by adding drops of either six normal hydrochloric acid or 6.5 normal sodium hydroxide solution from a Pasteur pipette so that the pH stays at 7.4.

Then add 1.98 grams of glucose to the physiological salt solution. Next, prepare 500 milliliters of a calcium-free physiological salt solution by diluting the 20X stock solution in deionized water. Then, add 25 milliliters of the 20X buffer stock in an Erlenmeyer flask and add 0.07 grams of monosodium phosphate to the solution while monitoring and adjusting the pH of the solution.

Use tetrafluoroethylene tubing to connect a gas tank containing the appropriate gas mixture to the organ bath. Continuously bubble the solution at a rate sufficient to ensure continuous, but not turbulent bubbling of the solution flowing into the vessel chamber. Also, place a small air stone connected to the equilibration gas mixture in the vessel chamber to help maintain the physiological salt solution's gas composition.

Place the physiological salt solution in a two-liter Mariotte bottle. Add a central glass tube to a stopper to serve as a reservoir which will continuously deliver the physiological salt solution into the pharmacological organ bath that supplies the solution to the vessel chamber. Place the opening of the central glass tube in the Mariotte bottle at the same level as the top of the physiological salt solution in the organ bath.

This will ensure constant hydrostatic pressure head for delivery of the physiological salt solution into the organ bath. Use a polyethylene tube connected to a J-shaped glass tube to deliver the physiological salt solution from the Mariotte bottle and into the organ bath. For lumenal profusion, use polyethylene tubing to connect the inflow pipette to a physiological salt solution reservoir composed of a 66-CC plastic syringe elevated to a position that maintains the desired inflow pressure.

Monitor the pressure by connecting a pressure transducer to the system via a stopcock. Then, connect the outflow pipette to a polyethylene tube to a reservoir similar to the inflow reservoir in order to allow the physiological salt solution to flow through the vessel in response to a pressure gradient. Also, use a similar stopcock and pressure transducer connection to monitor the outflow pressure.

Remove the brain of a Sprague Dawley rat and place it in the supine position in a glass Petri dish filled with ice-cold physiological salt solution. Then, place the dish under a stereoscope. Use a pair of Vannas scissors and a Dumont number five fine-tip forceps to excise the middle cerebral arteries from the brain.

Then, clean any residual brain tissue from the middle cerebral artery using the forceps. Transfer the artery to the vessel chamber by gently holding it by the end of the excised segment and carefully place it into the chamber. Next thread the cerebral artery onto the inflow micropipette by pulling it toward the pipette base until the tip advances into the lumen of the artery.

Secure the artery on the inflow pipette by tying a loop prepared from a single-strand fiber previously teased from 10-0 sutures around the artery. Then, secure the opposite end of the middle cerebral artery to the outflow pipette by tightening a second suture loop around the vessel and use the micrometer connected to the inflow pipette holder to stretch the artery to its in situ length. Tie off all the side branches using single strands teased from 10-0 sutures in order to maintain a constant pressure in the artery.

Next, set up a video microscope consisting of a video camera attached to a dissecting microscope that is connected to a video micrometer and a television monitor. Use this setup to measure the internal diameter of the artery. Assess the myogenic tone and vessel responses to vasodilator stimuli by making sure that the artery exhibits a suitable level of active tone prior to the experiment.

Discard any arteries lacking active tone at rest with the exception of vessels such as small mesenteric arteries that do not normally exhibit active resting tone. Next, add increasing concentrations of acetylcholine to the vessel chamber and measure the vessel diameters to test the endothelium-dependent reactivity of the cannulated resistance artery. In this example, the artery exhibits endothelial dysfunction as demonstrated by its failure to dilate in response to acetylcholine.

At the end of the experiment, determine the maximum diameter of the artery by adding calcium-free physiological salt solution to the perfusate and superfusate. Use this measurement to calculate active resting tone percent. Demonstration of a maximum dilation in calcium-free physiological salt solution verifies that the failure of the artery to dilate in response to acetylcholine was due to endothelial dysfunction and not due to an inability of the artery to relax due to structural remodeling of the vessel.

This figure illustrates the responses to the endothelium-dependent vasodilator acetylcholine in isolated middle cerebral arteries from male Dahl Salt Sensitive rats in the three narrowed congenic strains. This shows that endothelium-dependent dilation to acetylcholine was absent in the salt-sensitive parental strain and in both the congenic strains retaining the salt-sensitive Renan allele. Normal dilation was restored in the congenic strain carrying the brown Norway Renan allele in the salt-sensitive genetic background supporting the hypothesis that endothelial dysfunction in SS rats is due to an impaired regulation of the Renan gene.

In this experiment, the failure of the high-salt diet to eliminate the endothelium-dependent dilation to acetylcholine in one particular strain of rat, the findings regarding contributors to vascular oxidant stress and endothelial dysfunction during elevations in dietary salt intake. Once mastered, this technique can be done in three to 3 1/2 hours if it's performed properly. While attempting this procedure, it's important to remember to handle the isolated artery very carefully to avoid damage to the smooth muscle and especially the endothelium.

Following this procedure, other methods like the addition of vasoconstrictor and vasodilator agonists or receptor antagonists can be performed in order to answer questions including mechanisms affecting vascular reactivity to different substances and mechanisms of drug action on blood vessels. After its development, this technique paved the way for researchers in the fields of vascular biology and microcirculatory physiology to explore smooth muscle and endothelial function in resistance arteries from different vascular beds of experimental animals and humans. After watching this video, you should have a good understanding of how to use this method to evaluate endothelial and vascular smooth muscle control mechanisms in small-resistance arteries from different vascular beds.