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

Abstract

Source: Hakim, M. A. et al., Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma. J. Vis. Exp. (2022).

This video demonstrates the functional analysis of an arteriolar endothelial tube isolated from a mouse brain. It outlines the steps for measuring intracellular calcium levels and membrane potential, both at baseline and in response to pharmacological stimulation.

Protocol

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).

Results

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 f...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amplifiers	Molecular Devices, Sunnyvale, CA, USA	Axoclamp 2B & Axoclamp 900A
Audible baseline monitors	Ampol US LLC, Sarasota, FL, USA	BM-A-TM
Bath Chiller (Isotemp 500LCU)	ThermoFisher Scientific	13874647
Borosilicate glass capillaries (Pinning)	Warner Instruments	G150T-6
Borosilicate glass capillaries (Sharp Electrodes)	Warner Instruments	GC100F-10
BSA: Bovine Serum Albumin	Sigma	A7906
CaCl2: Calcium Chloride	Sigma	223506
DMSO: Dimethyl Sulfoxide	Sigma	D8418
Flow Control Valve	Warner Instruments	FR-50
Fluorescence system interface, ARC lamp & power supply, hyperswitch and PMT	Molecular Devices, Sunnyvale, CA, USA	IonOptix Systems
Function Generator	EZ Digital, Seoul, South Korea	FG-8002
Fura-2 AM dye	Invitrogen, Carlsbad, CA, USA	F14185
Glucose	Sigma-Aldrich (St. Louis, MO, USA)	G7021
HCl: Hydrochloric Acid	ThermoFisher Scientific (Pittsburgh, PA, USA)	A466250
Headstages	Molecular Devices	HS-2A & HS-9A
HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma	H4034
Inline Solution Heater	Warner Instruments	SH-27B
KCl: Potassium Chloride	Sigma	P9541
MgCl2: Magnesium Chloride	Sigma	M2670
Microforge	Narishige, East Meadow, NY, USA	MF-900
Micromanipulator	Siskiyou	MX10
Micropipette puller (digital)	Sutter Instruments, Novato, CA, USA	P-97 or P-1000
Microscope (Nikon-inverted)	Nikon Instruments Inc, Melville, NY, USA	Ts2
Microscope (Nikon-inverted)	Nikon Instruments Inc	Eclipse TS100
Microscope objectives	Nikon Instruments Inc	20X (S-Fluor) and 40X (Plan Fluor)
Microscope platform (anodized aluminum; diameter, 7.8 cm)	Warner Instruments	PM6 or PH6
Microscope Stage (Aluminum)	Siskiyou, Grants Pass, OR, USA	8090P
MTA: 2-Methylthioadenosine diphosphate trisodium salt	Tocris	1624
NaCl: Sodium Chloride	Sigma	S7653
NaOH: Sodium Hydroxide	Sigma	S8045
Oscilloscope	Tektronix, Beaverton, Oregon, USA	TDS 2024B
Phase contrast objectives	Nikon Instruments Inc	(Ph1 DL; 10X & 20X)
Plexiglas superfusion chamber	Warner Instruments, Camden, CT, USA	RC-27
Stereomicroscopes	Zeiss, NY, USA	Stemi 2000 & 2000-C
Syringe filter (0.22 µm)	ThermoFisher Scientific	722-2520
Temperature Controller (Dual Channel)	Warner Instruments	TC-344B or C
Valve Control System	Warner Instruments	VC-6
Vibration Isolation Table	Technical Manufacturing, Peabody, MA, USA	Micro-g