All procedures are approved by the Institutional Review Board of the Pennsylvania State University prior to participant recruitment.
1. Equipment setup
<ol>
	<li>Turn on the local heating unit and the laser Doppler flowmeter. 
	NOTE: Both should be calibrated prior to data collection according to the manufacturer&#39;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.</li>
	<li>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.</li>
</ol>
2. Microdialysis fiber placement
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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 &#8805;2.5 cm apart and positioned prior to microdialysis probe insertion. Probes should not be placed along the same major vessel.</li>
	<li>Leaving the needle in place, connect the probe (via the Luer lock) to a syringe containing lactated Ringer&#39;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&#39;s solution through the fiber until the solution is visibly perfused through the pores of the membrane to confirm the integrity of the membrane.</li>
	<li>If using a Harvard Bioscience microdialysis probe and introducer needle, follow steps 2.5.1-2.5.2.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>If using a Bioanalytical Systems microdialysis probe and introducer needle, follow steps 2.6.1-2.6.2.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Hyperemia
<ol>
	<li>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&#39;s solution, saline, or vehicle solution (the solution in which the experimental pharmacological agent is dissolved; 2 &#181;L/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&#39;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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Place an automatic blood pressure cuff on the arm of a subject which has not been instrumented.</li>
	<li>Set the local heaters to 33 &#176;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.</li>
</ol>
4. Acetylcholine dose-response protocol
<ol>
	<li>Once the RBC flux has stabilized in response to the 33 &#176;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.</li>
	<li>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.</li>
	<li>Turn off the microinfusion pumps, and exchange the syringes full of lactated Ringer&#39;s solution for the syringe filled with the lowest concentration of ACh (10&#8722;10 M).</li>
	<li>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 &#8722;10 into the data acquisition software recording.</li>
	<li>Each concentration of ACh will be perfused for 5-10 min at 2 &#181;L/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&#8722;10 M ACh solution is exchanged for 10&#8722;9 M ACh solution), as described in steps 4.2-4.4.</li>
	<li>Immediately after perfusing the final concentration of ACh (10&#8722;1 M), replace the ACh syringe with one containing Ringer&#39;s solution, and increase the local heater temperature to 43 &#176;C. Once the RBC flux has stabilized, replace the Ringer&#39;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.</li>
	<li>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.</li>
</ol>
5. Local heating protocol
<ol>
	<li>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.</li>
	<li>Increase the local heaters to either 39 &#176;C or 42 &#176;C, depending on the protocol&#39;s needs (explained in the discussion section).</li>
	<li>Once the RBC flux has plateaued in response to local heat application (~40-60 min heating), perfuse NG-nitro-l-arginine methyl ester (L-NAME; 15 mM dissolved in Ringer&#39;s solution; 2 &#181;L/min; a NO&#160;synthase inhibitor) through the microdialysis probe(s).</li>
	<li>Once the RBC flux has plateaued in response to L-NAME (~15-25 min of perfusion), increase the local heaters to 43 &#176;C.</li>
	<li>Once the RBC flux has plateaued in response to 43 &#176;C (a ~2-5 min plateau occurs after ~20-45 min of heating), perfuse sodium nitroprusside (28 mM dissolved in Ringer&#39;s solution) through the microdialysis probe(s).</li>
	<li>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.</li>
</ol>
6. Removing the microdialysis probes
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place sterile gauze over any bleeding that results from the probe removal, and apply pressure.</li>
</ol>

Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Greater+daily+psychosocial+stress+exposure+is+associated+with+increased+norepinephrine-induced+vasoconstriction+in+young+adults.">Greater daily psychosocial stress exposure is associated with increased norepinephrine-induced vasoconstriction in young adults.</a> Journal of the American Heart Association. 9 (9), e015697 (2020).</li><li>Nakata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+catecholamine+neurotransmitters+released+from+cutaneous+vasoconstrictor+nerve+endings+in+men+with+cervical+spinal+cord+injury.">Quantification of catecholamine neurotransmitters released from cutaneous vasoconstrictor nerve endings in men with cervical spinal cord injury.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 324 (3), R345-R352 (2023).</li><li>Tucker, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+cutaneous+vasodilation+and+sweating:+Influence+of+adiposity+and+hydration+status.">Postsynaptic cutaneous vasodilation and sweating: Influence of adiposity and hydration status.</a> European Journal of Applied Physiology. 118 (8), 1703-1713 (2018).</li><li>Craighead, D. H., Alexander, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menthol-induced+cutaneous+vasodilation+is+preserved+in+essential+hypertensive+men+and+women.">Menthol-induced cutaneous vasodilation is preserved in essential hypertensive men and women.</a> American Journal of Hypertension. 30 (12), 1156-1162 (2017).</li><li>Brunt, V. E., Minson, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCa+channels+and+epoxyeicosatrienoic+acids:+Major+contributors+to+thermal+hyperaemia+in+human+skin.">KCa channels and epoxyeicosatrienoic acids: Major contributors to thermal hyperaemia in human skin.</a> Journal of Physiology. 590 (15), 3523-3534 (2012).</li><li>Choi, P. J., Brunt, V. E., Fujii, N., Minson, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approach+to+measure+cutaneous+microvascular+function:+An+improved+test+of+NO-mediated+vasodilation+by+thermal+hyperemia.">New approach to measure cutaneous microvascular function: An improved test of NO-mediated vasodilation by thermal hyperemia.</a> Journal of Applied Physiology. 117 (3), 277-283 (2014).</li><li>Johnson, J. M., Kellogg, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+thermal+control+of+the+human+cutaneous+circulation.">Local thermal control of the human cutaneous circulation.</a> Journal of Applied Physiology. 109 (4), 1229-1238 (2010).</li><li>Jung, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Doppler+flux+measurement+for+the+assessment+of+cutaneous+microcirculation-Critical+remarks.">Laser Doppler flux measurement for the assessment of cutaneous microcirculation-Critical remarks.</a> Clinical Hemorheology and Microcirculation. 55 (4), 411-416 (2013).</li></ol>

The authors have no conflicts of interest and nothing to disclose.

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 approaches26,27,28,</sup...

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 vasodilation3,4,5. 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 arteries6,7,8. 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 survival9,10,11. 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 disorder14,15,16,17,18,19, and hypertension20,21,22,23,24. 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 &#176;C or 42 &#176;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringes</td>
			<td>BD Syringes</td>
			<td>302100</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetlycholine</td>
			<td>United States Pharmacopeia</td>
			<td>1424511</td>
			<td>Pilot data collected in our lab indicate drying acetylcholine increases variability of CVC response; do not dry, store in desiccator</td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Mckesson</td>
			<td>191089</td>
			<td></td>
		</tr>
		<tr>
			<td>Baby Bee Syringe Drive</td>
			<td>Bioanalytical Systems, Incorporated</td>
			<td>MD-1001</td>
			<td>In this study the optional 3-syringe bracket (catalg number MD-1002) was utilized</td>
		</tr>
		<tr>
			<td>CMA 30 Linear Microdialysis Probes</td>
			<td>Harvard Apparatus</td>
			<td>CMA8010460</td>
			<td></td>
		</tr>
		<tr>
			<td>Connex Spot Monitor</td>
			<td>WelchAllyn</td>
			<td>74CT-B</td>
			<td>automated blood pressure monitor</td>
		</tr>
		<tr>
			<td>Hive Syringe Pump Controller</td>
			<td>Bioanalytical Systems, Incorporated</td>
			<td>MD-1020</td>
			<td>Controls up to 4 Baby Bee Syringe Drives</td>
		</tr>
		<tr>
			<td>LabChart 8</td>
			<td>AD Instruments</td>
			<td></td>
			<td>**PowerLab hardware and LabChart software must be compatible versions</td>
		</tr>
		<tr>
			<td>Lactated Ringer&#39;s Solution</td>
			<td>Avantor (VWR)</td>
			<td>76313-478</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler Blood FlowMeter</td>
			<td>Moor Instruments</td>
			<td>MoorVMS-LDF</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler probe calibration kit</td>
			<td>Moor Instruments</td>
			<td>CAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler VP12 probe</td>
			<td>Moor Instruments</td>
			<td>VP12</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Microdialysis Probes</td>
			<td>Bioanalytical Systems, Inc.</td>
			<td>MD-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>NG-nitro-l-arginine methyl ester</td>
			<td>Sigma Aldrich</td>
			<td>483125-M</td>
			<td>L-NAME</td>
		</tr>
		<tr>
			<td>Povidone-iodine / betadine</td>
			<td>Dynarex</td>
			<td>1202</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab C Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PLC01</td>
			<td>**</td>
		</tr>
		<tr>
			<td>PowerLab C Instrument Interface</td>
			<td>AD Instruments</td>
			<td>PLCI1</td>
			<td>**</td>
		</tr>
		<tr>
			<td>Probe adhesive discs</td>
			<td>Moor Instruments</td>
			<td></td>
			<td>attach local heating unit to skin</td>
		</tr>
		<tr>
			<td>Skin Heater Controller</td>
			<td>Moor Instruments</td>
			<td>moorVMS-HEAT 1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Small heating probe</td>
			<td>Moor Instruments</td>
			<td>VHP2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile drapes</td>
			<td>Halyard</td>
			<td>89731</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Dukal Corporation</td>
			<td>2085</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>Esteem Cardinal Health</td>
			<td>8856N</td>
			<td>catalogue number followed by the initials of the glove size, then the letter &#34;B&#34; (e.g., 8856NMB for medium)</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Cole-Parmer</td>
			<td>UX-06287-26</td>
			<td></td>
		</tr>
	</tbody>
</table>

intradermal microdialysis: an approach to investigating novel mechanisms of microvascular dysfunction in humans

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.

Author Spotlight: A Pharmacodissection Approach to Uncover Mechanisms in Cardiovascular Disease Risk Populations 

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

My research examines mechanisms of vascular dysfunction in humans with traditional and non-traditional cardiovascular disease risk. I have developed a model to examine microvascular dysfunction mechanisms in humans, which allows pharmaco dissection that was previously impossible. We have explored several molecular targets, mediating vascular dysfunction, translating preclinical research to humans using this novel bioassay.One of the current challenges in experiments is that vasodilator and vasoconstrictor responses are caused by multiple mechanisms. To isolate the mechanisms of interest, pharmacological agents must be used. While this approach is limited, it does allow for a more comprehensive understanding of cutaneous vascular responses.In clinical cohorts such as hypertension and major depressive disorder, or MDD, we have discovered microvascular dysfunction. The possible reasons for this dysfunction in these groups are increased arginase and reactive oxygen species activity, respectively. Short-term salicylate treatment has been found to be effective in improving microvascular function and MDD.This technique is minimally invasive and allows for well controlled pharmacological dissection of the mechanisms of function of a local vascular bed. This is opposed to techniques like postocclusive reactive hyperemia and iontophoresis, which are useful, but allow for a little control of a pharmacologic tool. Our laboratory is currently investigating the contribution of inflammation and mediating microvascular dysfunction in women with endometriosis.In addition, we plan to investigate similar mechanisms in non-Hispanic black women.

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...

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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.

The cutaneous vasculature is an accessible tissue that can be used to assess microvascular function in humans. Intradermal microdialysis is a minimally ...

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Equipment Setup and Microdialysis Fiber Placement for Intradermal Microdialysis

To begin, turn on the local heating unit and the laser Doppler flowmeter. Open a LabChart software file. Next, identify the large visible blood vessels on the forearm's ventral aspect, and mark them with a permanent marker.Clean the marked area with Betadine followed by alcohol. Place a sterile drape over the sterilized skin and apply ice for approximately five minutes. Then, remove the ice and insert an introducer needle with the bevel facing upwards into the skin's dermal layer at 2-3 millimeters depth.Carefully advance the needle within the dermal layer and exit the skin around 20 millimeters from the insertion point. Connect the probe to a syringe containing lactated Ringer's solution, leaving the needle in place. Carefully insert the opposite end of the probe into the introducer needle until the semipermeable membrane is close, but not inside of the opening of the needle.Then, slowly perfuse a small amount of Ringer's solution through the fiber until the solution visibly flows through the membrane's pores. Insert the Harvard Bioscience microdialysis probe into the introducer needle until the membrane is fully inside the dermal layer of the skin within the needle. Next, secure the probe using a finger proximal to the needle.Carefully withdraw the needle in the opposite insertion direction, and tape the external portion of the fiber in place on the skin. Adjust the probe and tape the external fiber on the skin to prevent displacement of the semipermeable membrane during the experiment.

Exploring Mechanisms of Vasodilation and Vasoconstriction in the Cutaneous Circulation

After placing the microdialysis fiber in the forearm, place the single-use syringe into the syringe holder tray on the microinfusion pumps. During the hyperemia phase, perfuse lactated ringers, saline, or vehicle solution. Attach the local heating unit to the skin, ensuring it covers the semipermeable membrane, using the probe adhesive disc.Insert the laser doppler flowmetry probe into the opening at the center of the local heater, and begin recording red blood cell or RBC flux values by clicking Start on the data acquisition software. Place an automatic blood pressure cuff on the arm of an uninstrumented subject. Set the local heaters to 33 degrees Celsius to maintain a thermo neutral skin temperature.To add a comment to the continuous recording in the data acquisition software, click on the text box in the top right corner of the screen. Type a comment, select which channel should receive the comment, and click on Add. Start baseline data collection once the RBC flux stabilizes at 33 degrees Celsius local heat by entering the comment, start baseline.Measure and record the baseline blood pressure after five to 10 minutes of baseline data collection. Enter the comment, end baseline in the data acquisition software. Turn off the microinfusion pumps and replace the lactated ringer solution syringes with the syringe containing the lowest concentration of acetylcholine.After securing the new syringes and confirming fluid perfusion through the probe, turn the microinfusion pumps on again. Enter start minus 10 as a comment in the data acquisition software recording. During the last minute of perfusion, measure and record the blood pressure.Once the perfusion for a given concentration ends, replace the syringe with the next highest concentration. After the final perfusion, raise the local heater temperature to 43 degrees Celsius. Allow the RBC flux to stabilize, and replace the ringers solution with sodium nitroprusside.Measure and record the blood pressure approximately every three minutes during this maximal vasodilation phase. Once a maximal RBC flux plateau has occurred, select Stop in the bottom right corner of the data acquisition software. Record the baseline data collection as demonstrated earlier.Based on the protocol's requirements, increase the local heaters to 39 or 42 degrees Celsius. After the RBC flux plateaus in response to local heat application, perfuse L-Name through the microdialysis probe. Next, set local heaters to 43 degrees Celsius.Then start microdialysis probe profusion with sodium nitroprusside after the RBC flux plateaus at 43 degrees Celsius, allowing the flux to reach its maximal plateau. Once the profusion for each dose began, a continual increase in the RBC flux to a peak was observed, followed by a heat and SNP-induced plateau. Profusion of sodium nitroprusside produced maximal local vasodilation, depicted as a rise in the RBC flux.A relatively lower RBC flux was observed in the presence of a nitric oxide synthase inhibitor. During maximal local vasodilation, there will be an exponential rise in the RBC flux, due to the previous nitric oxide synthase inhibition. In the local heating protocol, L-Name was perfused after achieving a stable plateau in the RBC flux.A rapid decline was observed in the RBC flux until it reached a new plateau in response to L-Name. The heating produced an additional peak and nadir response in the RBC flux.

Removing the Microdialysis Probes After Performing the Dose-Response Experiments

After performing the dose-response experiment, use surgical scissors to cut the microdialysis probes. Carefully remove the laser doppler flowmetry probes from the heaters and detach the heaters from the skin. Gently remove the tape securing the probes to the skin.Inspect the puncture sites on either side of the probe to identify the smallest blood clot. Cut the probe near the site with the smaller clot, leaving approximately one inch of the probe outside the skin uncut. Then clean the skin area around the probe's entry and exit sites using an alcohol swab, including the one-inch length on the less-clotted side.Grasp the probe extending from the puncture site with the larger clot and gently pull it towards the clot. Lastly, cover any bleeding from the probe's removal with sterile gauze and apply pressure.

761 Views.  The Pennsylvania State University.  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.

Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

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