Name: intradermal microdialysis: an approach to investigating novel mechanisms of microvascular dysfunction in humans
Uploaded: 2023-07-21T15:00:00.000+00:00
Duration: 8 min 21 s
Description: Watch this Scientific Journal Video about A Pharmacodissection Approach to Uncover Mechanisms in Cardiovascular Disease Risk Populations at JoVE.com

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><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&#039;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>N&lt;sup&gt;G&lt;/sup&gt;-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 &quot;B&quot; (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.

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

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.

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 ...

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Medicine

1.2K 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.

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Biology

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an ...

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

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Chapters in this video

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Non-invasive Assessment of Microvascular and Endothelial Function

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

Custom-made Microdialysis Probe Design

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes