Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

Our method allows the study of blood vessels located in structures that would otherwise be difficult to image in Vivo, such as the hippocampus. Because the blood vessels are removed from the brain tissue, the signaling between the capillaries and the arteriole can be studied without off-target effects on the surrounding tissue. To isolate the hippocampus, use small dissection scissors to cut the skin along the midline at the top of the head of the mouse and move the skin to the sides.

Starting at the caudal side of the skull, cut the skull along the midline until the olfactory bulbs are reached and remove portions of the skull until the cerebrum is exposed. Starting near the nose of the mouse, slowly remove the brain, using the scissors to cut through the olfactory bulbs, cranial nerves, and spinal cord. Submerge the brain, ventral side down in MOPS Buffered Saline, in the center of a dissection plate, and place the plate under a dissection microscope.

Holding the sharp edge parallel to the bottom of the dish, use razor blade to cut the brain in half along the longitudinal fissure in one stroke. Place one hemisphere in the center of the plate with the midline facing down and push the razor blade straight through the tissue along the transverse fissure to remove the cerebellum and brain stem. Rotate the hemisphere so that the medial side is facing up and use a spatula to hold the brain in place.

Insert the tip of a second spatula below the corpus callosum, scooping under the tissue to remove the thalamus, septum, and hypothalamus from the hippocampus. The hippocampus should now be visible as a curved structure near the posterior side of the cerebrum. Using one spatula to hold the cerebrum in place, use the second spatula to scoop the hippocampus out of the cerebrum.

Then transfer the hippocampi to a new dissection plate, filled about halfway with fresh MOP solution. For hippocampal arteriole isolation, place small pins at each end of one hippocampus to secure the sample hippocampal artery side up. Using very sharp forceps, gently stretch small sections of the hippocampi to loosen the tissue surrounding the arterioles.

And search through the dorsal hippocampal tissues to identify the external transverse arteries. When the artery has been located, grab the external transverse artery from the hippocampus and slowly pull it away from the tissue to collect the arterioles and capillaries. The artery from each hippocampus is removed separately.

When there are no more vessels to be removed, place the samples on ice and discard the remaining hippocampal tissue. For cannulation of the arterioles, locate and arteriole with a branch that ends with capillaries and place the arteriole into an organ chamber. Carefully push the cannula tip through the arteriole wall below the target area to mount the blood vessel.

Then, carefully slide the vessel onto the cannula until there is enough tissue on which to place the tie. Use 12-0 nylon sutures to make a loose knot that will fit over the blood vessel and cannula, and use a half hitch knot to secure the ties, pull the ends to tighten the knot to secure the arteriole to the cannula. Secure another tie on the other end of the arteriole to seal it.

On the opposite side of the chamber, lower a second cannula until the point of the cannula pins down the tie on the end of the arteriole. Then use a third cannula to pin the capillary branch to the coverslip, placing the tip close to the end of the branch, while leaving the ends of the capillaries exposed. As the brain microcirculation is exquisitely fragile, be sure to minimize the stretching and handling of the vessels during the cannulation procedure to ensure survival of the arterioles.

For pressure myography analysis, transfer the chamber to a light microscope stage with recording software and connect the inflow and outflow tubing to the chamber for profusion. Start the profusion with 37 degrees Celsius Artificial cerebrospinal fluid at a four milliliter per minute flow rate, and attach the pressuring cannula to peristaltic pump paired with a pressure transducer. Bring the internal pressure to 20 millimeters of Mercury and start the recording software.

Adjust the microscope and the image settings to achieve the clearest image possible. And use the Edge Detection software to check that the arteriole is about 15 to 30 micrometers in diameter when it is fully dilated. Once the settings have been optimized, begin the recording and increase the intravascular pressure in the vessel up to 40 millimeters of Mercury, while recording the arteriole diameter with the Edge Detection software.

Then profuse the chamber for 15 to 20 minutes to wash out the MOP solution. To test the viability of the vessel, apply one micromolar NS309 solution to the bath profusion, the arteriolar segment should dilate, demonstrating a 30 to 40%myogenic tone. Once the baseline tone for the arteriole has been established, use a glass puller to make cannulas with a fine point at one end, and use forceps to break the tip of each cannula, so that the drug of interest can flow smoothly through the tip at five pounds of pressure per square inch.

Fill the cannula with the drug of interest. And load the cannula onto a three-axis micromanipulator attached to the microscope. Connect the tubing from the pressure ejection system to the cannula, and slowly lower the cannula into the bath near the capillaries, taking care not to hit any part of the vessel or the chamber.

To stimulate the capillaries, lower the cannula to the coverslip just next to the capillaries and activate the pressure ejection system with the desired ejection time. At the end of the stimulation, raise the cannula slightly to avoid further stimulation. To confirm that only the capillaries were being stimulated, fill the new cannula with one micromolar of NS309 solution and stimulate the capillaries as demonstrated.

Then obtain the maximal vasodilation by bathing the preparation with the Calcium-free solution. Bath application of a one micromolar NS309 causes a near maximal dilation of the arteriole due to the Calcium-sensitive Potassium channels in the endothelium. Capillary endothelial cells however, lack intermediate and small conductance channels and do not hyperpolarize in response to the agonist, as a result, stimulating capillary ends with NS309, does not cause upstream arteriolar dilation, this indicates that NS309 did not reach the arteriole, and can be used as a control to access the spacial restriction of the compound applied onto capillaries by pressure ejection.

Using the hippocampal capillary parenchymal arteriole preparation as demonstrated, the application of a Artificial cerebrospinal fluid supplemented with 10 millimolar Potassium to the capillary ends, results in an upstream arteriolar dilation that does not differ between preparations from male and female mice. Further, the addition of the Kir2 inhibitor ML 133, virtually abolishes the capillary induced arteriolar dilation, in response to 10 millimolar Potassium in preparations from both male and female mice. When mounting the blood vesssel, limit direct interactions with the blood vessel to minimize the damage to, and increase the viability of the vessel.

Once the blood vessel has been isolated, different drug compounds can be tested or the blood vessel can be further processed for olecular biology, immunochemistry, or electrophysiology studies.