equipment setup and microdialysis fiber placement for intradermal microdialysis

Investigating Novel Mechanisms of Microvascular Dysfunction in Humans

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.

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.

Watch this Scientific Journal Video about Equipment Setup and Microdialysis Fiber Placement for Intradermal Microdialysis at JoVE.com

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.

equipment-setup-microdialysis-fiber-placement-for-intradermal

Research

JoVE Journal

Medicine

143 Views.  The Pennsylvania State University. Per iniziare, accendi l'unità di riscaldamento locale e il misuratore di portata laser Doppler. Aprire un file del software LabChart. Successivamente, identifica i grandi vasi sanguigni visibili sull'aspetto ventrale dell'avambraccio e contrassegnali con un pennarello indelebile.Pulire l'area contrassegnata con Betadine seguito da alcool. Posizionare un telo sterile sulla pelle sterilizzata e applicare del ghiaccio per circa cinque minuti. Quindi, rimuovere il ghiaccio e inserire un a...