Analysis of Calcium Dynamics and Membrane Potential Changes in a Mouse Arteriolar Endothelium

Protokół

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

 

1. Materials and equipment

NOTE: See the Table of Materials for all reagents and materials required for this protocol. In addition, manuals and websites associated with the respective vendors can also be consulted as needed.

1. Flow chamber
   1. Fasten a superfusion chamber with a glass coverslip onto a platform composed of anodized aluminum. Secure the platform with the chamber onto an aluminum microscope stage.
   2. Set a micromanipulator holding a pinning pipette at each end of the platform on the aluminum microscope stage.
      NOTE: If necessary, use a transferable stage apparatus with a flow chamber unit to move a secured, isolated endothelial tube from one microscope apparatus to another for experimentation.
2. Microscopes
   1. Set up the experimental apparatus by arranging an inverted microscope (objectives: 4x, 10x, 20x, 40x, and 60x) and a manual aluminum stage on a vibration isolation table.
3. Intracellular V_m recording equipment
   1. Connect the electrometer to a compatible headstage. Use accessories, such as a function generator and stimulator, for protocols requiring current injection.
   2. Connect amplifier outputs to a data digitizer system, oscilloscope, and audible baseline monitors. Secure the reference bath electrode (Ag/AgCl pellet) near the flow chamber exit.
   3. Assemble a photometric system with integrated components of a fluorescence system interface, high-intensity arc lamp and power supply, hyperswitch, photomultiplier tube (or PMT), and camera to measure [Ca²⁺]_i in endothelial cells.
   4. Assemble a temperature controller equipped with an inline heater to raise and maintain a physiological temperature (37 °C) throughout the experiment.
   5. Assemble a multichannel platform connected to a valve controller with an inline flow control valve to control the delivery of solutions to endothelial tubes secured in the chamber.
4. Micropipettes and sharp electrodes
   ​NOTE: The experimenter will need an electronic glass puller and a microforge to prepare pinning pipettes.
   1. To secure the endothelial tube in the superfusion chamber, prepare heat-polished pinning pipettes with a blunted, spherical end (outer diameter: 50-70 µm) prepared from thin-wall borosilicate glass capillaries.
   2. To record the V_m of an endothelial cell, prepare sharp electrodes with a tip resistance of ~150 ± 30 MΩ from glass capillaries using the glass puller only.

2. Solutions and drugs

1. Physiological salt solution (PSS)
   1. Prepare a minimum of 1 L of PSS using 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 mM glucose.
   2. Prepare necessary solutions lacking CaCl₂ (zero Ca²⁺ PSS) for dissection of cerebral arterioles and isolation of endothelial tubes.
      NOTE: Prepare all solutions in ultrapure deionized H₂O, followed by filtration (0.22 µm). Ensure that the final product contains a pH 7.4 with osmolality between 290 and 300 mOsm.
2. Fura-2 and pharmacological agents
   1. Prepare Fura-2 AM stock in dimethyl sulfoxide (DMSO; 1 mM). Prepare 500 µL of working concentration (10 µM) by adding 5 µL of the stock to 495 µL of PSS for loading.
   2. Prepare at least 50 mL of working concentrations of pharmacological agents in PSS or DMSO as appropriate.
3. Conducting solution
   1. Prepare 2 M KCl by dissolving KCl in deionized H₂O (7.455 g of KCl in 50 mL of H₂O). Pass the solution through a syringe with a 0.22 µm filter prior to backfilling the sharp electrodes.

3. Utilization of arteriolar endothelial tubes for the examination of cellular physiology

NOTE: Isolated and secured arteriolar endothelial tubes can be used for intracellular recordings of [Ca²⁺]_i dynamics and V_m using photometry and sharp electrode electrophysiology, respectively, as previously illustrated (Figure 1). [Ca²⁺]_i and V_m can be measured as separate or combined experimental variables (Figure 1). However, arteriolar endothelial tubes are more delicate than arterial endothelium, and experimentation time should not exceed 1 h.

1. Measurement of [Ca²⁺]_i
   1. Turn on the equipment and software for [Ca²⁺]_i recordings while maintaining continuous superfusion at a flow rate of 5-7 mL/min.
   2. Load the endothelial tube with the Ca²⁺ dye Fura-2 AM for 30 min at room temperature. Wash the cells with superfusion solution for another 20-30 min while gradually raising the bath temperature to 37 °C. Maintain the temperature at 37 °C throughout the experiment.
   3. Manually adjust the imaging window using photometry software to focus on ~20 endothelial cells (Figure 1A). In the absence of light, turn the PMT on the fluorescence interface and begin acquisition of [Ca²⁺]_i by exciting Fura-2 alternately (≥10 Hz) at 340 nm and 380 nm while collecting fluorescence emission at 510 nm. Once a stable baseline recording of [Ca²⁺]_i is established, apply pharmacological agents (e.g., purinergic receptor agonists) per the experimental objective (Figure 1B).
2. Measurement of V_m​
   1. Turn on the equipment and software for V_m recordings and set the data acquisition rate (≥10 Hz) while maintaining continuous superfusion at a flow rate of 5-7 mL/min. Gradually raise the bath temperature to 37 °C and maintain it until the end of the experiment.
   2. Pull a sharp electrode using a borosilicate glass capillary, backfill with 2 M KCl, and secure it over a silver wire coated with chloride in the pipette holder attached to an electrometer that, in turn, is held by a micromanipulator.
   3. While viewing through the 4x objective, use a micromanipulator to carefully position the sharp electrode tip just over a cell of the arteriolar endothelial tube into the flowing PSS in the chamber.
   4. Gradually increase magnification to 400x and reposition the electrode tip as needed.
   5. Using the micromanipulator, gently insert the tip of a sharp electrode into one of the cells of the endothelial tube and start recording V_m using an electrometer (Figure 1A).

Once the endothelial resting V_m is stable (−30 to −40 mV), apply the desired pharmacological agents per experimental objective (Figure 1C).

Wyniki

Figure 1: Application of endothelial tube for physiological examination of pathways related to blood flow regulation. (A) A sharp electrode (purple arrow) is positioned in a cell of an endothelial tube focused in the data acquisition window for photometry. (B) Representative recording of intracellular Ca²⁺ using Fura-2 photometry in response to MT...

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