Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

Summary

Intradermal microdialysis is a minimally invasive technique used to investigate microvascular function in health and disease. Both dose-response and local heating protocols can be utilized for this technique to explore mechanisms of vasodilation and vasoconstriction in the cutaneous circulation.

Abstract

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally invasive technique used to investigate mechanisms of vascular smooth muscle and endothelial function in the cutaneous circulation. This technique allows for the pharmacological dissection of the pathophysiology of microvascular endothelial dysfunction as indexed by decreased nitric oxide-mediated vasodilation, an indicator of cardiovascular disease development risk. In this technique, a microdialysis probe is placed in the dermal layer of the skin, and a local heating unit with a laser Doppler flowmetry probe is placed over the probe to measure the red blood cell flux. The local skin temperature is clamped or stimulated with direct heat application, and pharmacological agents are perfused through the probe to stimulate or inhibit intracellular signaling pathways in order to induce vasodilation or vasoconstriction or to interrogate mechanisms of interest (co-factors, antioxidants, etc.). The cutaneous vascular conductance is quantified, and mechanisms of endothelial dysfunction in disease states can be delineated.

Introduction

Cardiovascular disease (CVD) is the leading cause of death in the United States¹. Hypertension (HTN) is an independent risk factor for stroke, coronary heart disease, and heart failure and is estimated to affect upward of ~50% of the United States population². HTN can develop as an independent CVD (primary HTN) or as a result of another condition, such as polycystic kidney disease and/or endocrine disorders (secondary HTN). The breadth of etiologies of HTN complicates investigations into the underlying mechanisms and end-organ damage observed with HTN. Diverse and novel research approaches into the pathophysiology of the end-organ damage associated with HTN are needed.

One of the earliest pathological signs of CVD is endothelial dysfunction, as characterized by impaired nitric oxide (NO)-mediated vasodilation^3,4,5. Flow-mediated dilation is a common approach used to quantify the endothelial dysfunction associated with CVD, but endothelial dysfunction in microvascular beds can be both independent of and precursory to that of large conduit arteries^6,7,8. Furthermore, resistance arterioles are more directly acted on by local tissue than conduit arteries and have more immediate control over the delivery of oxygen-rich blood. Microvascular function is predictive of adverse cardiovascular event-free survival^9,10,11. The cutaneous microvasculature is an accessible vascular bed that can be used to examine responses to physiological and pharmacological vasoconstrictive or vasodilatory stimuli. Intradermal microdialysis is a minimally invasive technique, the goal of which is to investigate the mechanisms of both vascular smooth muscle and endothelial function in the cutaneous microvasculature with targeted pharmacological dissection. This method contrasts with other techniques, such as post-occlusive reactive hyperemia, which does not allow for pharmacological dissection, and iontophoresis, which allows for pharmacological delivery but is less precise in its mechanism of action (reviewed thoroughly elsewhere¹²).

The rationale behind the development and use of this technique is extensively reviewed elsewhere¹³. This approach was originally developed for use in neurological research in rodents and then was first applied to humans to investigate the mechanisms underlying active vasodilation from a thermoregulatory standpoint. In the late 1990s, this method was used to examine both neural and endothelial mechanisms with regard to local heating of the skin. Since that time, the technique has been utilized to investigate a number of neurovascular signaling mechanisms in the skin.

Using this technique, our group and others have interrogated the mechanisms of endothelial dysfunction in the microvasculature of several clinical populations, including, but not limited to, dyslipidemia, primary aging, diabetes, chronic kidney disease, polycystic ovary syndrome, preeclampsia, major depressive disorder^{14,15,16,17,18,19}, and hypertension^{20,21,22,23,24}. For example, a previous study found that normotensive women with a history of preeclampsia, who are at an increased risk for CVD, had reduced NO-mediated vasodilation in the cutaneous circulation compared with women with a history of normotensive pregnancy²⁰. In another study, adults diagnosed with primary HTN demonstrated increased angiotensin II sensitivity in the microvasculature compared with healthy controls²¹, and chronic sulfhydryl-donating antihypertensive pharmacotherapy in primary HTN patients has been shown to decrease blood pressure and improve both hydrogen sulfide- and NO-mediated vasodilation²². Wong et al.²³ found impaired sensory-mediated and NO-mediated vasodilation in prehypertensive adults, coinciding with our finding of a progression of endothelial dysfunction with increasing HTN stages, as categorized by the 2017 American Heart Association and American College of Cardiology guidelines²⁴.

The intradermal microdialysis technique allows for tightly controlled mechanistic investigations into microvascular function in health and disease states. Therefore, this paper aims to describe the intradermal microdialysis technique as applied by our group and others. We detail the procedures for both pharmacological stimulation of the endothelium with acetylcholine (ACh) to examine the dose-response relationship and physiological stimulation of endogenous NO production with either a 39 °C or 42 °C local heating stimulus protocol. We present representative results for each approach and discuss the clinical implications of the findings that have arisen from this technique.

Protocol

All procedures are approved by the Institutional Review Board of the Pennsylvania State University prior to participant recruitment.

1. Equipment setup

1. Turn on the local heating unit and the laser Doppler flowmeter.
   NOTE: Both should be calibrated prior to data collection according to the manufacturer's instructions. The laser Doppler flowmeter should be connected to data acquisition hardware with sampling at 100 Hz (100 samples/min) and continuous recording in a data acquisition software. While other data acquisition hardware and software can be used, for simplicity, the remaining instructions reflect the PowerLab hardware and LabChart software capabilities.
2. Open a LabChart software file.
   NOTE: A reference file with the desired data input and continuous data collection capabilities should be made in advance. There should be one panel for each laser Doppler and local heater that corresponds to each microdialysis site, and the panels should correspond to the appropriate channel inputs in the data acquisition hardware unit.

2. Microdialysis fiber placement

1. Identify the large, visible blood vessels of the skin in the ventral aspect of the forearm, and indicate them with a permanent marker (utilize a tourniquet to visualize the vessels if necessary; identifying vessels in darkly pigmented skin may require greater reliance on palpation).
2. Swab the area encompassing the marks and a generous portion of the surrounding area using betadine swabs. Wipe away the betadine with alcohol swabs. Cover the sterilized area of skin with a sterile drape, and apply ice for ~5 min to numb the area.
3. Remove the ice, and insert an introducer needle (23 G, 25 mm length), bevel facing upwards, into the dermal layer of the skin at a depth of 2-3 mm (depending on the individual skin thickness). Advance the needle, being careful to remain in the dermal layer, and exit the skin ~20 mm from the point of insertion.
   NOTE: To confirm the proper depth of placement in the skin, the shape of the needle should be visible and easily palpable, but the color of the needle should be concealed. If more than one microdialysis probe is required for the experiment, any two introducer needles will need to be placed ≥2.5 cm apart and positioned prior to microdialysis probe insertion. Probes should not be placed along the same major vessel.
4. Leaving the needle in place, connect the probe (via the Luer lock) to a syringe containing lactated Ringer's solution. Feed the opposite end of the probe through the introducer needle until the semipermeable membrane of the probe is near but still outside of the opening of the introducer needle. Slowly perfuse a small amount of Ringer's solution through the fiber until the solution is visibly perfused through the pores of the membrane to confirm the integrity of the membrane.
5. If using a Harvard Bioscience microdialysis probe and introducer needle, follow steps 2.5.1-2.5.2.
   1. Upon confirmation of the probe function, further feed the probe through the introducer needle until the membrane is completely contained in the dermal layer of the skin within the introducer needle.
   2. Using a finger, secure the probe in place proximal to the needle, and withdraw the needle in the opposite direction from insertion. Tape the external portion of the fiber in place on the skin to prevent displacement of the semipermeable membrane during the experiment.
6. If using a Bioanalytical Systems microdialysis probe and introducer needle, follow steps 2.6.1-2.6.2.
   1. Upon confirmation of the probe function, take hold of the hub of the introducer needle and the distal portion of the microdialysis probe in one hand, and simultaneously withdraw the needle opposite to the direction of insertion, moving the microdialysis probe into place.
   2. Adjust the probe as needed to ensure the semipermeable membrane is buried in the skin completely. Tape the external fiber in place on the skin to prevent displacement of the semipermeable membrane during the experiment.

3. Hyperemia

1. While waiting for the hyperemic response to needle insertion to subside (~60-90 min), place the single-use syringe in the syringe-holder tray on the microinfusion pumps. Perfuse lactated Ringer's solution, saline, or vehicle solution (the solution in which the experimental pharmacological agent is dissolved; 2 µL/min) during the hyperemia phase.
   NOTE: While the microdialysis probes cannot be removed during this ~60-90 min phase, the participant can adjust their body position or move their hand, or the Luer lock of the probe can be removed from the syringe and secured with tape to the participant's arm to allow them free range of motion to briefly stand. Once instrumented with local heaters and laser Doppler flowmetry (LDF) probes and once data collection has begun, the LDF probes cannot be moved.
2. When the skin redness, which is an indicator of the hyperemic response to needle trauma, has subsided, attach the local heating unit to the skin covering the semipermeable membrane via the probe adhesive disc, ensuring that the center of the heater aligns with the path of the microdialysis probe.
3. Place the LDF probe into the opening in the center of the local heater so that the laser is directly perpendicular to the surface of the skin. Once the LDF probes are placed and secured, click on start on the data acquisition software to continuously record and display the red blood cell flux values (RBC flux; perfusion units, PUs). If hyperemia has completely subsided, the RBC flux will be stable at ~5-20 PU (the pulsatility of the vessels below the LDF probe may be reflected by slight elevations in the PU that coincide with the heartbeats).
4. Place an automatic blood pressure cuff on the arm of a subject which has not been instrumented.
5. Set the local heaters to 33 °C to clamp the skin temperature within a thermoneutral range²⁵, thus removing any variations in the influence of thermal stimuli. To add a comment to the continuous recording in the data acquisition software to denote events in the experiment, click on the text box in the top-right corner of the screen, type a comment, select which channels should receive the comment, and click on add.

4. Acetylcholine dose-response protocol

1. Once the RBC flux has stabilized in response to the 33 °C local heat, begin baseline data collection, distinguished in the data acquisition software file by a comment begin baseline. At least a minimum of 5-10 min of stable baseline is required for data analysis; restart the baseline at any time during this point in data collection if needed, and mark this in the LabChart file. In the final minute of baseline, collect a blood pressure measurement, and enter the values in a comment in the LabChart file.
2. At the end of the 5-10 min of baseline data collection, measure and record the baseline blood pressure, and enter the comment end baseline into the data acquisition software.
3. Turn off the microinfusion pumps, and exchange the syringes full of lactated Ringer's solution for the syringe filled with the lowest concentration of ACh (10⁻¹⁰ M).
4. Secure the new syringes in place, and confirm the perfusion of the fluid through the end of the probe before turning the microinfusion pumps on again. Enter the comment begin −10 into the data acquisition software recording.
5. Each concentration of ACh will be perfused for 5-10 min at 2 µL/min. In the final minute of perfusion, for every concentration, measure and record the blood pressure. Once the perfusion time for a given concentration has ended, replace the syringe with the next highest concentration (e.g., 10⁻¹⁰ M ACh solution is exchanged for 10⁻⁹ M ACh solution), as described in steps 4.2-4.4.
6. Immediately after perfusing the final concentration of ACh (10⁻¹ M), replace the ACh syringe with one containing Ringer's solution, and increase the local heater temperature to 43 °C. Once the RBC flux has stabilized, replace the Ringer's solution with sodium nitroprusside (28 mM) to produce both a heat-induced and pharmacologically induced maximal local vasodilation. Measure and record the blood pressure every ~3 min during this maximal vasodilation phase.
7. Once a maximal RBC flux plateau has occurred (~5 min stable PU), end the experiment. Select stop in the bottom-right corner of the data acquisition software to end the continuous data collection.

5. Local heating protocol

1. Once the RBC flux has stabilized following hyperemia, begin the baseline data collection, and indicate this in the data acquisition software file with a comment. In the final minute of baseline, collect a blood pressure measurement, and enter the values in a comment into the data acquisition software file.
2. Increase the local heaters to either 39 °C or 42 °C, depending on the protocol's needs (explained in the discussion section).
3. Once the RBC flux has plateaued in response to local heat application (~40-60 min heating), perfuse N^G-nitro-l-arginine methyl ester (L-NAME; 15 mM dissolved in Ringer's solution; 2 µL/min; a NO synthase inhibitor) through the microdialysis probe(s).
4. Once the RBC flux has plateaued in response to L-NAME (~15-25 min of perfusion), increase the local heaters to 43 °C.
5. Once the RBC flux has plateaued in response to 43 °C (a ~2-5 min plateau occurs after ~20-45 min of heating), perfuse sodium nitroprusside (28 mM dissolved in Ringer's solution) through the microdialysis probe(s).
6. Once a maximal RBC flux plateau has occurred (~5 min stable PU), end the experiment. Select stop in the bottom-right corner of the data acquisition software to end the data collection.

6. Removing the microdialysis probes

1. Following the termination of the experiment, use a pair of surgical scissors to cut the microdialysis probes. Carefully remove the LDF probes from the heaters, and remove the heaters from the skin. Gently remove the tape holding the probes in place on the skin.
2. Visually identify which puncture site on either side of the probe has formed the smallest blood clot. Cut the portion of the probe near the site with the smaller clot, leaving ~1 in of the probe outside of the skin uncut.
3. Clean the portion of the skin surrounding the entry and exit sites of the probe with an alcohol swab, as well as the ~1 in length of probe left on the less-clotted site.
4. Allow the alcohol to dry on the skin. Then, grasp the portion of the probe extending from the puncture site with the greater clot, opposite the ~1 in portion on the less-clotted end. Slowly pull the probe toward the larger blood clot.
5. Place sterile gauze over any bleeding that results from the probe removal, and apply pressure.

Results

Acetylcholine dose-response protocol

Figure 1A depicts a schematic detailing the ACh dose-response protocol. Figure 1B illustrates representative tracings of the RBC flux values (perfusion units, PU; 30 s averages) from the standardized ACh dose-response protocol for one subject over time. Figure 1C illustrates a raw data file of an ACh dose-response protocol. Additional baseline measurem...

Discussion

The intradermal microdialysis technique is a versatile tool in human vascular research. Investigators may alter the protocol to further diversify its applications. For example, we describe an ACh dose-response protocol, but other investigations into the mechanisms of vasoconstriction or vasomotor tone, rather than vasodilation alone, have utilized norepinephrine or sodium nitroprusside dose-response approaches^26,27,28,

Materials

Name	Company	Catalog Number	Comments
1 mL syringes	BD Syringes	302100
Acetlycholine	United States Pharmacopeia	1424511	Pilot data collected in our lab indicate drying acetylcholine increases variability of CVC response; do not dry, store in desiccator
Alcohol swabs	Mckesson	191089
Baby Bee Syringe Drive	Bioanalytical Systems, Incorporated	MD-1001	In this study the optional 3-syringe bracket (catalg number MD-1002) was utilized
CMA 30 Linear Microdialysis Probes	Harvard Apparatus	CMA8010460
Connex Spot Monitor	WelchAllyn	74CT-B	automated blood pressure monitor
Hive Syringe Pump Controller	Bioanalytical Systems, Incorporated	MD-1020	Controls up to 4 Baby Bee Syringe Drives
LabChart 8	AD Instruments		**PowerLab hardware and LabChart software must be compatible versions
Lactated Ringer's Solution	Avantor (VWR)	76313-478
Laser Doppler Blood FlowMeter	Moor Instruments	MoorVMS-LDF
Laser Doppler probe calibration kit	Moor Instruments	CAL
Laser Doppler VP12 probe	Moor Instruments	VP12
Linear Microdialysis Probes	Bioanalytical Systems, Inc.	MD-2000
NG-nitro-l-arginine methyl ester	Sigma Aldrich	483125-M	L-NAME
Povidone-iodine / betadine	Dynarex	1202
PowerLab C Data Acquisition Device	AD Instruments	PLC01	**
PowerLab C Instrument Interface	AD Instruments	PLCI1	**
Probe adhesive discs	Moor Instruments		attach local heating unit to skin
Skin Heater Controller	Moor Instruments	moorVMS-HEAT 1.3
Small heating probe	Moor Instruments	VHP2
Sterile drapes	Halyard	89731
Sterile gauze	Dukal Corporation	2085
Sterile surgical gloves	Esteem Cardinal Health	8856N	catalogue number followed by the initials of the glove size, then the letter "B" (e.g., 8856NMB for medium)
Surgical scissors	Cole-Parmer	UX-06287-26