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 post-occlusive reactive hyperemia and iontophoresis, which are useful but allow for 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. 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 two to three 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.

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 Ringer's 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 thermoneutral 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's solution syringes with the syringe containing the lowest concentration of acetylcholine.

After securing the new syringes and confirming the fluid perfusion through the probe, turn the microinfusion pumps on again. Enter Start 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 Ringer's 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 perfusion with sodium nitroprusside after the RBC flux plateaus at 43 degrees Celsius, allowing the flux to reach its maximal plateau. Once the perfusion for each dose began, a continual increase in the RBC flux to a peak was observed, followed by a heat and SNP-induced plateau. Perfusion 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.

The 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. 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.