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

<ol>
	<li>Xu, J. Q., Murphy, S. L., Kochanek, K. D., Arias, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+in+the+United+States,+2021.">Mortality in the United States, 2021.</a> NCHS Data Brief. 456, (2022).</li><li>Tsao, C. W., 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=Heart+disease+and+stroke+statistics-2023+update:+A+report+from+the+American+heart+association.">Heart disease and stroke statistics-2023 update: A report from the American heart association.</a> Circulation. 147 (8), e93 (2023).</li><li>Cohuet, G., Struijker-Boudier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+target+organ+damage+caused+by+hypertension:+Therapeutic+potential.">Mechanisms of target organ damage caused by hypertension: Therapeutic potential.</a> Pharmacology &amp; Therapeutics. 111 (1), 81-98 (2006).</li><li>Park, K. H., Park, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction:+Clinical+implications+in+cardiovascular+disease+and+therapeutic+approaches.">Endothelial dysfunction: Clinical implications in cardiovascular disease and therapeutic approaches.</a> Journal of Korean Medical Science. 30 (9), 1213-1225 (2015).</li><li>Levy, B. I., Ambrosio, G., Pries, A. R., Struijker-Boudier, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculation+in+hypertension:+a+new+target+for+treatment.">Microcirculation in hypertension: a new target for treatment.</a> Circulation. 104 (6), 735-740 (2001).</li><li>Sara, J. D., 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=Prevalence+of+coronary+microvascular+dysfunction+among+patients+with+chest+pain+and+nonobstructive+coronary+artery+disease.">Prevalence of coronary microvascular dysfunction among patients with chest pain and nonobstructive coronary artery disease.</a> Journal of the American College of Cardiology: Cardiovascular Interventions. 8 (11), 1445-1453 (2015).</li><li>Weis, M., Hartmann, A., Olbrich, H. G., H&#246;r, G., Zeiher, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+coronary+flow+reserve+on+left+ventricular+ejection+fraction+in+cardiac+transplant+recipients.">Prognostic significance of coronary flow reserve on left ventricular ejection fraction in cardiac transplant recipients.</a> Transplantation. 65 (1), 103-108 (1998).</li><li>Rossi, M., 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=Investigation+of+skin+vasoreactivity+and+blood+flow+oscillations+in+hypertensive+patients:+Effect+of+short-term+antihypertensive+treatment.">Investigation of skin vasoreactivity and blood flow oscillations in hypertensive patients: Effect of short-term antihypertensive treatment.</a> Journal of Hypertension. 29 (8), 1569-1576 (2011).</li><li>Pepine, C. J., 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=Coronary+microvascular+reactivity+to+adenosine+predicts+adverse+outcome+in+women+evaluated+for+suspected+ischemia+results+from+the+National+Heart,+Lung+and+Blood+Institute+WISE+(Women's+Ischemia+Syndrome+Evaluation)+study.">Coronary microvascular reactivity to adenosine predicts adverse outcome in women evaluated for suspected ischemia results from the National Heart, Lung and Blood Institute WISE (Women's Ischemia Syndrome Evaluation) study.</a> Journal of the American College of Cardiology. 55 (25), 2825-2832 (2010).</li><li>Matsuda, J., 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=Prevalence+and+clinical+significance+of+discordant+changes+in+fractional+and+coronary+flow+reserve+after+elective+percutaneous+coronary+intervention.">Prevalence and clinical significance of discordant changes in fractional and coronary flow reserve after elective percutaneous coronary intervention.</a> Journal of the American Heart Association. 5 (12), e004400 (2016).</li><li>Gupta, 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=Integrated+noninvasive+physiological+assessment+of+coronary+circulatory+function+and+impact+on+cardiovascular+mortality+in+patients+with+stable+coronary+artery+disease.">Integrated noninvasive physiological assessment of coronary circulatory function and impact on cardiovascular mortality in patients with stable coronary artery disease.</a> Circulation. 136 (24), 2325-2336 (2017).</li><li>Roustit, M., Cracowski, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+endothelial+and+neurovascular+function+in+human+skin+microcirculation.">Assessment of endothelial and neurovascular function in human skin microcirculation.</a> Trends in Pharmacological Sciences. 34 (7), 373-384 (2013).</li><li>Low, D. A., Jones, H., Cable, N. T., Alexander, L. M., Kenney, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+reviews+of+the+assessment+of+human+cardiovascular+function:+interrogation+and+understanding+of+the+control+of+skin+blood+flow.">Historical reviews of the assessment of human cardiovascular function: interrogation and understanding of the control of skin blood flow.</a> European Journal of Applied Physiology. 120 (1), 1-16 (2020).</li><li>Kenney, W. L., Cannon, J. G., 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=Cutaneous+microvascular+dysfunction+correlates+with+serum+LDL+and+sLOX-1+receptor+concentrations.">Cutaneous microvascular dysfunction correlates with serum LDL and sLOX-1 receptor concentrations.</a> Microvascular Research. 85, 112-117 (2013).</li><li>Holowatz, L. A., Thompson, C. S., Minson, C. T., Kenney, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+acetylcholine-mediated+vasodilatation+in+young+and+aged+human+skin.">Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skin.</a> Journal of Physiology. 563, 965-973 (2005).</li><li>Sokolnicki, L. A., Roberts, S. K., Wilkins, B. W., Basu, A., Charkoudian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+nitric+oxide+to+cutaneous+microvascular+dilation+in+individuals+with+type+2+diabetes+mellitus.">Contribution of nitric oxide to cutaneous microvascular dilation in individuals with type 2 diabetes mellitus.</a> American Journal of Physiology - Endocrinology and Metabolism. 292 (1), E314-E318 (2007).</li><li>DuPont, J. J., Ramick, M. G., Farquhar, W. B., Townsend, R. R., Edwards, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase-derived+reactive+oxygen+species+contribute+to+impaired+cutaneous+microvascular+function+in+chronic+kidney+disease.">NADPH oxidase-derived reactive oxygen species contribute to impaired cutaneous microvascular function in chronic kidney disease.</a> American Journal of Physiology - Renal Physiology. 306 (12), F1499-F1506 (2014).</li><li>Sprung, V. S., 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=Nitric+oxide-mediated+cutaneous+microvascular+function+is+impaired+in+polycystic+ovary+syndrome+but+can+be+improved+by+exercise+training.">Nitric oxide-mediated cutaneous microvascular function is impaired in polycystic ovary syndrome but can be improved by exercise training.</a> Journal of Physiology. 591 (6), 1475-1487 (2013).</li><li>Greaney, J. L., Saunders, E. F. H., Santhanam, L., 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=Oxidative+stress+contributes+to+microvascular+endothelial+dysfunction+in+men+and+women+with+major+depressive+disorder.">Oxidative stress contributes to microvascular endothelial dysfunction in men and women with major depressive disorder.</a> Circulatory Research. 124 (4), 564-574 (2019).</li><li>Stanhewicz, A. E., Jandu, S., Santhanam, L., 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=Increased+angiotensin+II+sensitivity+contributes+to+microvascular+dysfunction+in+women+who+have+had+preeclampsia.">Increased angiotensin II sensitivity contributes to microvascular dysfunction in women who have had preeclampsia.</a> Hypertension. 70 (2), 382-389 (2017).</li><li>Greaney, J. L., 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=Impaired+hydrogen+sulfide-mediated+vasodilation+contributes+to+microvascular+endothelial+dysfunction+in+hypertensive+adults.">Impaired hydrogen sulfide-mediated vasodilation contributes to microvascular endothelial dysfunction in hypertensive adults.</a> Hypertension. 69 (5), 902-909 (2017).</li><li>Dillon, G. A., Stanhewicz, A. E., Serviente, C., Greaney, J. L., 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=Hydrogen+sulfide-dependent+microvascular+vasodilation+is+improved+following+chronic+sulfhydryl-donating+antihypertensive+pharmacotherapy+in+adults+with+hypertension.">Hydrogen sulfide-dependent microvascular vasodilation is improved following chronic sulfhydryl-donating antihypertensive pharmacotherapy in adults with hypertension.</a> Journal of Physiology. 321 (4), H728-H734 (2021).</li><li>Wong, B. J., 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=Sensory+nerve-mediated+and+nitric+oxide-dependent+cutaneous+vasodilation+in+normotensive+and+prehypertensive+non-Hispanic+blacks+and+whites.">Sensory nerve-mediated and nitric oxide-dependent cutaneous vasodilation in normotensive and prehypertensive non-Hispanic blacks and whites.</a> American Journal of Physiology - Heart and Circulatory Physiology. 319 (2), H271-H281 (2020).</li><li>Dillon, G. A., Greaney, J. L., Shank, S., Leuenberger, U. A., 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=AHA/ACC-defined+stage+1+hypertensive+adults+do+not+display+cutaneous+microvascular+endothelial+dysfunction.">AHA/ACC-defined stage 1 hypertensive adults do not display cutaneous microvascular endothelial dysfunction.</a> American Journal of Physiology - Heart and Circulatory Physiology. 319 (3), H539-H546 (2020).</li><li>Gagge, A. P., Stolwijk, J. A., Hardy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comfort+and+thermal+sensations+and+associated+physiological+responses+at+various+ambient+temperatures.">Comfort and thermal sensations and associated physiological responses at various ambient temperatures.</a> Environmental Research. 1 (1), 1-20 (1967).</li><li>Greaney, J. L., Stanhewicz, A. E., Kenney, W. L., 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=Lack+of+limb+or+sex+differences+in+the+cutaneous+vascular+responses+to+exogenous+norepinephrine.">Lack of limb or sex differences in the cutaneous vascular responses to exogenous norepinephrine.</a> Journal of Applied Physiology. 117 (12), 1417-1423 (2014).</li><li>Greaney, J. L., Stanhewicz, A. E., Kenney, W. L., 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=Impaired+increases+in+skin+sympathetic+nerve+activity+contribute+to+age-related+decrements+in+reflex+cutaneous+vasoconstriction.">Impaired increases in skin sympathetic nerve activity contribute to age-related decrements in reflex cutaneous vasoconstriction.</a> Journal of Physiology. 593 (9), 2199-2211 (2015).</li><li>Alba, B. K., Greaney, J. L., Ferguson, S. B., 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=Endothelial+function+is+impaired+in+the+cutaneous+microcirculation+of+adults+with+psoriasis+through+reductions+in+nitric+oxide-dependent+vasodilation.">Endothelial function is impaired in the cutaneous microcirculation of adults with psoriasis through reductions in nitric oxide-dependent vasodilation.</a> American Journal of Physiology - Heart and Circulatory Physiology. 314 (2), H343-H349 (2018).</li><li>Greaney, J. L., Surachman, A., Saunders, E. F. H., Alexander, L. M., Almeida, D. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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>NG-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 &#34;B&#34; (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...

Watch this Scientific Journal Video about A Pharmacodissection Approach to Uncover Mechanisms in Cardiovascular Disease Risk Populations at JoVE.com

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 ...

intradermal-microdialysis-an-approach-to-investigating-novel

Research

JoVE Journal

Medicine

1.1K 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 

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances.
Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a &#39;sepsis-like&#39; state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

Intravenous administration of endotoxin reliably elicits dose-dependent physiological and immunological alterations consistent with several pathological states. This reductionist approach permits the investigation, modeling and experimental modification of systemic inflammation and its downstream effects in man.

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or ...

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or intraosseous routes, but in many patients, particularly in the emergency or critical care settings, these routes are often unavailable or time-consuming to access. A novel device is now available that offers an easy route for administration of medications or fluids via rectal mucosal absorption (also referred to as proctoclysis in the case of fluid administration and subsequent absorption). Although originally intended for the palliative care market, the utility of this device in the emergency setting has recently been described. Specifically, reports of patients being treated for dehydration, alcohol withdrawal, vomiting, fever, myocardial infarction, hyperthyroidism, and cardiac arrest have shown success with administration of a wide variety of medications or fluids (including water, aspirin, lorazepam, ondansetron, acetaminophen, methimazole, and buspirone). Device placement is straightforward, and based on the observation of expected effects from the medication administrations, absorption is rapid. The rapidity of absorption kinetics are further demonstrated in a recent report of the measurement of phenobarbital pharmacokinetics. We describe here the placement and use of this device, and demonstrate methods of pharmacokinetic measurements of medications administered by this method.

This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration.

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Cardiopulmonary Bypass in a Mouse Model: A Novel Approach

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization and for minimizing organ damage resulting from prolonged extracorporal circulation. The goal of this paper was to demonstrate a fully functional and clinically relevant model of cardiopulmonary bypass in a mouse. We report on the device design, perfusion circuit optimization, and microsurgical techniques. This model is an acute model, which is not compatible with survival due to the need for multiple blood drawings. Because of the range of tools available for mice (e.g., markers, knockouts, etc.), this model will facilitate investigation into the molecular mechanisms of organ damage and the effect of cardiopulmonary bypass in relation to other comorbidities.

This paper describes how to perform cardiopulmonary bypass in mice. This novel model will facilitate the investigation of the molecular mechanisms involved in organ damage.

As prolonged cardiopulmonary bypass becomes more essential during cardiac interventions, an increasing clinical demand arises for procedure optimization ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...