分离及脑实质小动脉插管

The overall goal of this procedure is to isolate and mount cerebral arterioles for studies of vascular function and structure using pressure myography. This method can be used to answer key questions in the cerebrovascular field, such as, how did cerebral microvasculature quickly adapts to changes in intraluminal pressure or nutrient demand or during pathological conditions. The main advantage of this technique is that it allows for pressurization of the arteriole in addition of intraluminal pressure, which are important physiological determinants of vascular function.

To begin, using a micropipette puller with the appropriate settings, such as those listed here, pull capillaries to generate cannulas. After inserting a cannula into the holder of a myograph chamber and aligning it, under a dissecting microscope, use forceps to carefully break the tip to the desired diameter, such as 10 micrometers as shown here. Fill both cannulas with cerebrospinal fluid or aCSF containing 1.8 millimolar calcium, and use five to 20 milliliters of calcium free aCSF supplemented with 1%BSA and 10 micromolar diltiazem to fill the chamber.

Store the chamber at four degrees Celsius until just before the cannulation. After isolating a mouse brain according to the text protocol, under the dissecting scope, locate the circle of Willis and the middle cerebral artery, or MCA, branching from it. Using sharp and aligned Vannas spring scissors, cut a small rectangle around the MCA.

The top part of the rectangle should be past a branching point from the circle of Willis. Then, with the Vannas spring scissors, perform an undercut into the depth of the tissue for approximately two to three millimeters. Using insect pins, pin down the most distal end of the brain slice into the dissecting dish, with the MCA facing upwards.

Make a shallow incision to cut the pia near the pin, being careful not to cut too deep into the underlying cerebral cortex. Next, with small forceps, carefully grab each side of the pia and start peeling the pia from the cortex. If necessary, hold onto the MCA ensuring that the surrounding pial membrane is not damaged.

Close to the circle of Willis, resistance against peeling will increase. Take extra care at this point, because the longer parenchymal arterioles will be in this region. Continue to peel until the pia containing the MCA is free from the cortex.

Care and patience should be taken during this step, as the arterioles are very fragile and sudden movements or pulling with too much force will damage them. Once the pia is free, place it carefully on top of the pad on the dissecting dish, with the surface opposite the parenchymal arterioles facing down. With Vannas spring scissors, free the dissected arterioles from the MCA by bluntly cutting the ends of the vessels.

After preparing sutures and securing two onto each cannula, use 10%BSA to coat a glass micropipette, and with calcium free aCSF supplemented with 1%BSA, rinse it off. Move 50 microliters of calcium free aCSF supplemented with 1%BSA into the glass micropipette. Then, pull up a parenchymal arteriole, or PA, and transfer to the pressure myography chamber.

Push the plunger in one fluid motion to dispense the parenchymal arteriole into the chamber to prevent it from sticking to the internal chamber of the glass micropipette. With two super fine forceps, open the lumen of the PA, taking care to only touch the very end of the vessel. Then, using the forceps to hold the edges of the PA, carefully pull the vessel onto the cannula.

Next, loosen the two sutures on the cannula, and after spacing them slightly apart on the vessel, pull them toward the operator while tightening them on the vessel. Make sure that any branches are tied off before closing the opposite end of the PA.These are the most difficult steps. Opening up and sliding the arteriole onto the cannula requires practice.

When tying up the knot, hold both sides evenly as to not break the tip of the cannula. Then, turn the chamber around and close the opposite end of the PA as a blind-sac by opening the ties on the opposite cannula and passing it onto the arteriole. Slowly close the knot in such a way that the PA will be tied to the side of the cannula.

Avoid a longitudinal stretch of the arteriole. Carefully transfer the chamber to the stage of the microscope to be used for recording vessel diameter. Attach the chamber onto the microscope's stage, connecting a temperature probe and inlet and outlets for perfusion to the correct tubes.

Pressurize the arteriole to 40 millimeters mercury intraluminal pressure for mouse parenchymal arterioles, or 50 millimeters mercury for rat arterioles. To carry out an agonist induced constriction experiment, after preparing the agonist solution and adding it to the PA chamber according to the text protocol, incubate the vessel in the solution for 10 minutes to allow the luminal diameter to reach a steady state. Then, record the diameter of the arteriole.

After washing out the agonist and adding a drug to induce maximum dilation, record the passive diameter of the arteriole. This figure shows a representative constriction of mouse PAs to 60 millimolar potassium chloride in aCSF to evaluate the integrity of the preparation. PA should constrict between 15 to 30 percent in the presence of 60 millimolar potassium chloride.

Illustrated here is the PA constriction in response to incubation with increasing concentrations of the Thromboxane A2 analog, U-46619 into the superfusing bath. The data can be presented as a change in diameter or as a percent vasoconstriction to potassium chloride, which normalizes the change in diameter by the maximum constriction to 60 millimolar potassium chloride. In this figure, a representative tracing of the lumen diameter of a PA in a myogenic reactivity experiment is shown.

Stepwise increases in intraluminal pressure cause a graded constriction of PAs in the presence of 1.8 millimolar extracellular calcium. Incubation of the same PA with aCSF without calcium and supplemented with 10 micromolar diltiazem plus two millimolar EGTA abolishes myogenic tone generation, and the lumen diameter of the PA increases according to intraluminal pressure, which is the same as the passive lumen diameter of the arteriole. While attempting this technique, it's important to remember to keep your shoulders relaxed as to prevent pain.

And once this technique is mastered, it should take no longer than 30 minutes from the isolation up to the cannulation of the arteriole. Following this method, other techniques like imaging of intracellular calcium can be used to answer additional questions, such as the role of intracellular calcium in control of microvascular tone. After its development, this technique has paved the way for researchers in the field of vascular biology to explore the physiological control and pathological changes of the intercerebral vasculature in many organ systems, including humans and rodents.

After watching this video, you should have a good understanding of how to isolate and cannulate a cerebral parenchymal arteriole.