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.
