removing the microdialysis probes after performing the dose-response experiments

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 Removing the Microdialysis Probes After Performing the Dose-Response Experiments at JoVE.com

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.

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79 Views.  The Pennsylvania State University. Dopo aver eseguito l'esperimento dose-risposta, utilizzare le forbici chirurgiche per tagliare le sonde di microdialisi. Rimuovere con cautela le sonde di flusso laser doppler dai riscaldatori e staccare i riscaldatori dalla pelle. Rimuovere delicatamente il nastro che fissa le sonde alla pelle.Ispezionare i siti di puntura su entrambi i lati della sonda per identificare il coagulo di sangue più piccolo. Tagliare la sonda vicino al sito con il coagulo più piccolo, lasciando circa un ...