Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

This method can help answer key questions in the field of cerebral vascular physiology as it relates to cerebral blood flow regulation during aging and development of chronic diseases. The primary advantage of this technique is that it allows simultaneous measurement of intracellular calcium and membrane potential of cerebral arterial endothelium in it's native form under physiological conditions. To isolate the mouse brain, under the microscope remove the skin and hair over the skull.

And remove excessive blood with cold calcium-free PSS. Next, make an incision using only the tips of standard dissection scissors. Starting from the occipital bone and extending up through the nasal bone of the skull.

Then open she skull carefully along the incision, using coarse-tipped forceps and separate the connective tissue to expose the brain. Gently wash the isolated brain with cold calcium-free PSS in a beaker to remove the blood. Place the brain ventral side up in a chamber containing cold dissection solution for the isolation of cerebral arteries.

To isolate the cerebral arteries, secure the isolated brain in cold dissection solution by inserting two stainless steel pins through it, into a charcoal-infused silicon polymer coating at the bottom of a glass Petri dish. Subsequently, surgically isolate the posterior cerebral arteries about 0.3 to 0.5 centimeter segments from the posterior communicating and basilar arteries. Use stainless steel pins to secure both isolated posterior cerebral arteries in the dissection solution in the Petri dish.

Then clean the isolated posterior cerebral arteries carefully by removing connective tissue using sharpened fine-tipped forceps. Cut the intact arteries into one to two millimeter segments for enzymatic digestion. In this procedure, prepare the trituration apparatus using a microscope, a camera, and an aluminum stage holding a chamber and micromanipulators.

Secure a microsyringe with a pump controller adjacent to the stage and the specimen. Next completely backfill a titration pipette with mineral oil and secure it over the microsyringe piston. Then using the microsyringe with the pump controller withdraw about 130 nanoliters of dissociation solution into the pipette while ensuring the absence of air bubbles.

Subsequently, place intact arterial segments into one milliliter of dissociation solution with required concentrations of enzymes in a 10 milliliter glass tube. Incubate at 34 degrees Celsius for 10 to 12 minutes for partial digestion. Following digestion, replace the enzyme solution with five milliliters of fresh dissociation solution.

Using a one milliliter pipette, transfer one segment into the chamber containing dissociation solution at room temperature. Next place the pipette into the dissociation solution in the chamber and position it close to one end of the digested vessel. Set a rate within the range of two to five nanoliters per second on the pump controller for gentle trituration.

While viewing through 100 times to 200 times magnification withdraw and eject the arterial segment to dissociate smooth muscle cells while producing an endothelial tube. If necessary, carefully use fine-tipped forceps to separate dissociated adventitia and internal elastic lamina from the endothelial tube. Confirm that all smooth muscle cells are dissociated and that only endothelial cells remain as intact tube.

Using micromanipulators, secure each end of the endothelial tube on the glass cover slip of the superfusion chamber using borosilicate glass pinning pipettes. Wash out the dissociated adventitia and smooth muscle cells from the chamber. And replace the dissociation solution with two molar more calcium chloride PSS.

Transfer the mobile platform with secured endothelial tube onto the microscope of superfusion and experimental rig. Then use six clean 50 milliliter reservoirs for continuous delivery of PSS and respective drug solutions during the experiment. Use the in-line flow control valve to manually set the flow rate as consistent as laminer flow while matching flow feed to vacuum suction.

Deliver PSS to the chamber for the superfusion of the endothelial tube for at least five minutes before recording the background data and dye loading. To measure the membrane potential simultaneously with intracellular calcium concentration, pull a sharp electrode and backfill it with two molar potassium chloride to record from cells loaded with fura-2 dye. To study the intracellular coupling via dye transfer, backfill the microelectrode with 0.1%propidium iodide dissolved in two molar potassium chloride.

Next place the electrode over a silver wire coated with chloride in the pipette holder attached to an electrometer head stage secured with a micromanipulator. Use the micromanipulator to briefly position the tip of the electrode into the flowing PSS in the chamber, while viewing through the four times objective. Subsequently, set the resting membrane potential to zero as consistent with grounded bath potential.

If desired, use audible baseline monitors linked to electrometers to associate sound pitch with potential recordings. Afterward, increase magnification to 400 times using a 40 times objective and position the tip of electrode just over the cell of the endothelial tube. Adjust the photometric window using the photometry software to focus on about 50 to 80 endothelial cells.

Then gently place the electrode into one of the cells of the endothelial tube using the micromanipulator and wait at least two minutes for the resting membrane potential to stabilize. Once resting membrane potential is stable at its expected value, turn on the photomultiplier tube on the fluorescence interface in the absence of light and begin acquisition of the intracellular calcium concentration by exciting Fura-2 alternately at 340 and 380 nanometers while collecting florescence emission at 510 nanometers. Once simultaneous measurements of membrane potential and intracellular calcium concentration are established, allow about five minutes for the superfusion of endothelial tube with PSS at a constant laminar flow rate before the application of drugs.

Apply the drug prepared in PSS to the superfusion chamber at a constant flow rate. During drug treatment measure the membrane potential in the F340/F380 ratios simultaneously. Once the experiment is done, withdraw the electrode from the cell.

Then stop respective recordings of membrane potential and intracellular calcium concentration and save the files for data analysis. This is a differential interference contrast image of a mouse cerebral arterial endothelial tube, adjusted for experiment in photometric window using a 40 times objective. A sharp electrode is placed into a cell, as shown at the top of the image.

Here are the examples of raw traces for simultaneous measurement of F340/F380 ratio and membrane potential in response to 100 micromolar ATP. Following this procedure, other methods like confocal fluorescent microscopy, can be performed in order to answer additional questions regarding microdomain signaling of endothelial calcium, and reactive oxygen species, as examples. In general this technique will continue to pave the way for researches in cellular physiology to explore vascular function and aging in the blood and lymphatic circulation.