The authors would like to thank the volunteers for their participation in these studies and the surgical staff at the Boston Medical Center for providing adipose tissue biopsies. Dr. Gokce is supported by National Institutes of Health (NIH) grants HL081587, HL114675, and HL126141. Dr. Farb is supported by NIH grant K23 HL135394.

The protocol and examples described here were approved by Boston University School of Medicine Institutional Review Board (IRB, protocol #H-25644) and were conducted in accordance with the Declaration of Helsinki. All subjects provided written informed consent before participation.
1. Preparation of Solutions and Micro-glass Cannulas
<ol>
	<li>Prepare the 4-2-hydrosyethyl-1-piperazineethanesulfonic acidic saline (HEPES) solution. For 1 L, dissolve 8.059 g NaCl, 0.298 g KCl, 0.296 g MgSO4&#8226;7H2O, 0.235 g CaCl2&#8226;2H2O, 0.16 g KH2PO4, 0.01 g EDTA, 1.081 g D-Glucose, and 2.383 g HEPES acid in 950 mL of deionized water. Make up the volume to 1 L with deionized water. Adjust pH to 7.4 using NaOH. HEPES buffer can be stored up to 7 days at 4 &#176;C.</li>
	<li>Prepare the 20x Salt Buffer Stock. For 1 L, add 143.8 g NaCl, 7.0 g KCl, 5.9 g MgSO4, and 7.4 g CaCl2&#8226;2H2O into 900 mL of distilled water. Make up volume to 1 L with deionized water. Store stock solution at 4 &#176;C.</li>
	<li>Prepare the 20x Buffer Stock. For 1 L, add 26.8 g NaHCO3 and 0.2 g EDTA&#903;2H2O into 900 mL of deionized water. Make up volume to 1 L with deionized water. Store stock solution at 4 &#176;C.</li>
	<li>Prepare the KREBS Solution to be used during the physiological experiments. For 1 L, add 900 mL of deionized water, 50 mL of 20x Buffer Stock, 50 mL of 20x Salt Buffer Solution, 0.99 g D-Glucose and 0.16 g KH2PO4. Adjust pH to 7.4 using HCl. To maintain pH of the buffer, stir constantly and cover with paraffin or bubble continuously.
	<ol>
		<li>Check the pH every 20 min. Make the KREBS solution fresh daily.</li>
	</ol>
	</li>
	<li>Make micro-glass cannulas with an internal diameter of 40 - 240 &#181;m using a commercially available needle/pipette puller. The size of glass cannulas is determined by the size of the internal diameter of isolated blood vessels (50-350 &#181;m). Alternatively, purchase micro-glass cannulas of the pre-determined size.</li>
</ol>
2. Preparation of Reagents
<ol>
	<li>Prepare the acetylcholine (Ach, 10-2 M, stock solution). Dissolve 18.29 g in 10 mL deionized water. Store 1 mL aliquots of stock solution (10-2 M) at -80 &#176;C. On the day of the experiment serially dilute to obtain the following working concentrations: 10-5, 10-6, 10-7,10-8, 10-9 M.</li>
	<li>Prepare the papaverine (2 x 10-2 M, stock solution). Dissolve 0.07517 g in 10 mL deionized water. Prepare 10 &#181;L aliquots of stock solution and store at -80 &#176;C. Serially dilute to obtain 10-4, 10-5, 10-6, 10-7, 10-8 M on the day of the experiment.</li>
	<li>Prepare the endothelin-1 (ET-1, 2 x 10-5 M, stock solution). Dissolve 50 &#181;g endothelin-1 in 1 mL of 1% BSA/PBS. Store 1 mL aliquots of stock solution (2 x 10-5 M) at -80 &#176;C. On the day of the experiment dilute to obtain a working concentration of 10-10M on the day of experiment.</li>
	<li>Store reagents in either -20 &#176;C or up to -80 &#176;C depending on type of freezer available, but not longer than 6 months.</li>
</ol>
3. Adipose Tissue Collection and Vessel Preparation
<ol>
	<li>Recruit obese men and women (body mass index (BMI) &#8805; 35 kg/m&#178;, age &#8805;18 years) enrolled in the bariatric surgery program at Boston Medical Center (BMC). A total of 40 subjects participated in these experiments.</li>
	<li>Collect subcutaneous and visceral adipose tissue samples during planned bariatric surgeries by the surgeon performing the operation.
	<ol>
		<li>Harvest the subcutaneous adipose tissues from the lower abdominal wall and visceral fat from the greater omentum, respectively. Typical fat tissue sample size varies from 3 - 10 mg of tissue.</li>
		<li>Place tissues immediately in cold HEPES buffer solution with a pH of 7.4 and store at 4 &#176;C for up to 24 h. 
		NOTE: Additionally, adipose arterioles can be obtained from lean individuals as well during other types of surgeries such as hernia repairs or plastic surgery, and can also be biopsied from the subcutaneous depot via trans-cutaneous abdominal fat pad biopsy6.</li>
	</ol>
	</li>
	<li>Using a tissue dissection microscope, micro-scissors and micro-forceps, carefully remove surrounding fat and connective tissue from the small adipose arterioles (50 - 350 &#181;m intraluminal diameter, 2 - 3 mm in length).</li>
	<li>Tie off all branches on the arterioles using tiny nylon or silk sutures before cannulating on the glass capillary pipettes. Carefully dissect the arterioles since even minor damage of the arteriole wall causes significant functional changes.
	<ol>
		<li>Minimize vessel exposure to light and heat as it may cause vasodilation. Since it can be sometimes difficult to distinguish arterioles from venules, identify the size and smooth muscle tone of vessels by nipping the tips of the vessels. Arterioles are usually smaller and demonstrate greater tone compared to venules.</li>
	</ol>
	</li>
	<li>Prepare the organ chamber. Slowly fill the glass capillary pipettes and rubber tubing with KREBS solution using a 10 mL syringe.</li>
	<li>Once rubber tubing is filled and glass capillary pipettes are submerged in KREBS solution within the organ chamber, cannulate the arterioles securely on the secured pipettes and tie both ends of the arteriole with nylon or silk suture knots carefully.</li>
	<li>Fill up the organ chamber with KREBS solution up to 2 mL.</li>
	<li>Secure the organ chamber onto a stage with the inverted microscope (magnification 10X/0.25 objective) and video camera.</li>
	<li>Turn on the edge detection software that is streamed in real-time at a sampling rate of 1 kHz (1 frame/s).</li>
	<li>Connect one side of the pressurized tubing to the KREBS solution filled pressure reservoirs free of microbubbles, as bubbles can introduce experimental error. Connect the other side of pressurized tubing to the organ chamber.</li>
	<li>Connect the KREBS solution filled pressure reservoirs to the pressure transducer with clean tubing.</li>
	<li>Control intra-luminal flow by regulating the height of these two reservoirs; thus, when reservoirs are placed at the same height, no intra-luminal flow will occur.</li>
	<li>Once all these processes are complete, turn on heating block and software program to maintain temperature at 37 &#176;C. Continuously perfuse the vessel with KREBS solution and aerate with a gas mixture of 5% CO2, 21% O2 and 74% N2 7,8 throughout the entire experimental procedure.</li>
	<li>To achieve desired pressure inside the lumen of the isolated vessels gradually increase the intraluminal pressure (5 mmHg, every 5 min) via the pressure control unit in the myo-interface panel, doing so at a slow rate to avoid endothelial layer damage. 
	NOTE: Once the pressure reaches 60 mmHg, a 20-30 min equilibration period is required to stabilize the vessel. All vascular function studies are performed at 60 mmHg and 37 &#176;C at pH 7.4.7</li>
</ol>
4. Assessment of Adipose Microvascular Function
NOTE: In general, adipose arteriole endothelial-dependent vasodilation can be elicited in response to physiological (flow-induced) and pharmacological stimuli (Ach-induced).
<ol>
	<li>Flow-mediated, endothelial-dependent vasodilation
	<ol>
		<li>Following pressurization, record the diameter of the adipose arteriole at rest (Di). This is referred to as the resting baseline diameter.</li>
		<li>Pre-constrict blood vessels to ~55% of the resting baseline diameter (Dp) by adding 1 &#181;L of endothelin-1 (ET-1, 10&#8722;10 M) directly to the bath and wait 5 min for effect. Repeat this process until reaching the desired ~55% pre-constricted state.</li>
		<li>Following pre-constriction, induce continuous flow into the intraluminal space of adipose tissue arterioles, in equal and opposite directions so that a pressure difference can be developed across the vessel without altering the mean intraluminal pressure of 60 mmHg. 
		NOTE: For example, if one reservoir moves up by 10 cm in height, the other one needs to be moved down by 10 cm to alter pressure gradients and thus change intraluminal flow rate. If more accurate flow rate measurements are desired, a quantitative flow-meter system can be applied to the experimental set-up.</li>
		<li>Measure flow-mediated dilation 3 - 5 min after the initiation of the flow induction. The levels of intraluminal flow (pressure gradients) can be in the range of 0 - 100 cmH2O. Increase each increment of pressure gradient by &#916;10 cmH2O every 5-6 min, up to a maximum of 100 cmH2O. 
		NOTE: In order to induce stable laminar flow in the lumen, both micro glass cannula tip sizes need to be very close to inner diameter of arterioles; otherwise, it will produce turbulent flow inside of lumen and induce unwanted measurement errors.</li>
		<li>Following the flow- mediated dilation assessment, return pressure reservoirs to the same height (60 mmHg).</li>
		<li>Then, wash arteriole and chamber by immediately removing solution from chamber and replace with fresh KREBS solution. This is done with caution as to not disrupt the suspended arteriole within the chamber.</li>
		<li>Repeat this process 3 - 4 times for 20 - 30 min, or until the isolated vessel returns to the resting baseline diameter.</li>
	</ol>
	</li>
	<li>Acetylcholine-mediated, endothelial-dependent, vasodilation
	<ol>
		<li>When the adipose arteriole has returned to the resting baseline diameter, pre-constrict vessel ~55% by administering endothelin-1 (ET-1, 10&#8722;10 M) directly to the bath, as described above in section 4.1.2.</li>
		<li>Following pre-constriction, sequentially administer increasing doses (2 &#181;L) of acetylcholine which is a receptor-mediated, nitric oxide agonist (Ach, 10-9 to 10-5 M) directly to the bath. Record the change in arteriolar diameter 5 min after administration of each dose.</li>
		<li>Once the vessel has reached a plateau following administration of the final Ach dose, wash the vessel 3 - 4 times with KREBS solution. Allow 20 - 30 min for vessel to recover and return to resting baseline diameter. To help maintain pH in the organ camber, change KREBS solution every 15 min.</li>
	</ol>
	</li>
	<li>Incubate the arteriole with Nw-nitro-l-arginine methyl ester (L-NAME, 10-4 M), an inhibitor of nitric oxide synthase for 30 min, and then repeat 4.2.1 and 4.2.2 to characterize the relative contribution of nitric oxide to Ach-mediated vasodilation.</li>
	<li>Assess endothelium-independent vasodilation (vascular smooth muscle function) and vessel viability by a sequential administration of increasing doses of papaverine (10-8 to 10-4 M) directly to the bath. If the vessel diameter does not return to, or exceed the resting baseline (Di) diameter in response to papaverine (i.e. appropriate vasodilation), consider the vessel non-viable and discard data points.</li>
</ol>
5. Data Analysis and Calculation
<ol>
	<li>Calculate the vascular reactivity. Use percent vasodilation for data expression to account for baseline differences in vessel diameter and calculate using the following equation: 
	% vasodilation = (DT-Dp/Di-Dp) &#215;100 
	where DT is the recorded diameter at a given time point (maximum diameter), Dp is the diameter recorded after the addition of the vasoconstriction agent (i.e. ET-1 induced constriction diameter), and Di is the diameter recorded immediately before the addition of the vasoconstriction agent (resting baseline diameter).9</li>
	<li>Define vasodilation and vasoconstriction sensitivity (dose-response) to an agonist as the concentration of Ach, papaverine, or ET-1 that elicits 50% of the maximal response (EC50) which can be defined by a sigmoidal parameter and plotted, as described previously.10 
	NOTE: The inter-observer reproducibility studies of vasodilation of adipose microvessels has a high correlation coefficient (CC) of 0.99 (n = 10 vessel experiments) in our laboratory.</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Express continuous measurements as mean &#177; SEM. These tend to be normally distributed. Analyze vascular reactivity in adipose arterioles by repeated-measures two-way ANOVA.</li>
	<li>Alternatively, compare the area under the curve (AUC) of the plot for cumulative vasodilation to dose-responses between treatment groups. In all cases, consider P&#160;&#60;0.05 statistically significant.</li>
</ol>

<ol>
	<li>Schubert, R., Mulvany, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myogenic+response:+established+facts+and+attractive+hypotheses.">The myogenic response: established facts and attractive hypotheses.</a> Clin Sci (Lond). 96 (4), 313-326 (1999).</li><li>Jadeja, R. N., Rachakonda, V., Bagi, Z., Khurana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Myogenic+Response+and+Vasoactivity+In+Resistance+Mesenteric+Arteries+Using+Pressure+Myography.">Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography.</a> J Vis Exp. (101), e50997 (2015).</li><li>Gutterman, D. 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=The+Human+Microcirculation:+Regulation+of+Flow+and+Beyond.">The Human Microcirculation: Regulation of Flow and Beyond.</a> Circ Res. 118 (1), 157-172 (2016).</li><li>Fuster, J. J., Ouchi, N., Gokce, N., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity-Induced+Changes+in+Adipose+Tissue+Microenvironment+and+Their+Impact+on+Cardiovascular+Disease.">Obesity-Induced Changes in Adipose Tissue Microenvironment and Their Impact on Cardiovascular Disease.</a> Circ Res. 118 (11), 1786-1807 (2016).</li><li>Farb, M. G., 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=Reduced+adipose+tissue+inflammation+represents+an+intermediate+cardiometabolic+phenotype+in+obesity.">Reduced adipose tissue inflammation represents an intermediate cardiometabolic phenotype in obesity.</a> J Am Coll Cardiol. 58 (3), 232-237 (2011).</li><li>Farb, M. G., 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=WNT5A-JNK+regulation+of+vascular+insulin+resistance+in+human+obesity.">WNT5A-JNK regulation of vascular insulin resistance in human obesity.</a> Vasc Med. 21 (6), 489-496 (2016).</li><li>Durand, M. J., Phillips, S. A., Widlansky, M. E., Otterson, M. F., Gutterman, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+renin-angiotensin+system+contributes+to+blunted+vasodilation+induced+by+transient+high+pressure+in+human+adipose+microvessels.">The vascular renin-angiotensin system contributes to blunted vasodilation induced by transient high pressure in human adipose microvessels.</a> Am J Physiol Heart Circ Physiol. 307 (1), H25-H32 (2014).</li><li>Farb, M. G., 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=Cyclooxygenase+inhibition+improves+endothelial+vasomotor+dysfunction+of+visceral+adipose+arterioles+in+human+obesity.">Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity.</a> Obesity (Silver Spring). 22 (2), 349-355 (2014).</li><li>Park, S. Y., 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=Impact+of+age+on+the+vasodilatory+function+of+human+skeletal+muscle+feed+arteries.">Impact of age on the vasodilatory function of human skeletal muscle feed arteries.</a> Am J Physiol Heart Circ Physiol. 310 (2), H217-H225 (2016).</li><li>Ives, S. 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=Human+skeletal+muscle+feed+arteries+studied+in+vitro:+the+effect+of+temperature+on+alpha(1)-adrenergic+responsiveness.">Human skeletal muscle feed arteries studied in vitro: the effect of temperature on alpha(1)-adrenergic responsiveness.</a> Exp Physiol. 96 (9), 907-918 (2011).</li><li>Grizelj, I., 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=Reduced+flow-and+acetylcholine-induced+dilations+in+visceral+compared+to+subcutaneous+adipose+arterioles+in+human+morbid+obesity.">Reduced flow-and acetylcholine-induced dilations in visceral compared to subcutaneous adipose arterioles in human morbid obesity.</a> Microcirculation. 22 (1), 44-53 (2015).</li><li>Farb, M. G., 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=Arteriolar+function+in+visceral+adipose+tissue+is+impaired+in+human+obesity.">Arteriolar function in visceral adipose tissue is impaired in human obesity.</a> Arterioscler Thromb Vasc Biol. 32 (2), 467-473 (2012).</li><li>Tanner, M. 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=Dynamin-Related+Protein+1+Mediates+Low+Glucose-Induced+Endothelial+Dysfunction+in+Human+Arterioles.">Dynamin-Related Protein 1 Mediates Low Glucose-Induced Endothelial Dysfunction in Human Arterioles.</a> Am J Physiol Heart Circ Physiol. , (2016).</li><li>Ives, S. 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=alpha1-Adrenergic+responsiveness+in+human+skeletal+muscle+feed+arteries:+the+impact+of+reducing+extracellular+pH.">alpha1-Adrenergic responsiveness in human skeletal muscle feed arteries: the impact of reducing extracellular pH.</a> Exp Physiol. 98 (1), 256-267 (2013).</li><li>Dharmashankar, K., 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+synthase-dependent+vasodilation+of+human+subcutaneous+arterioles+correlates+with+noninvasive+measurements+of+endothelial+function.">Nitric oxide synthase-dependent vasodilation of human subcutaneous arterioles correlates with noninvasive measurements of endothelial function.</a> Am J Hypertens. 25 (5), 528-534 (2012).</li><li>Truran, 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=Adipose+and+leptomeningeal+arteriole+endothelial+dysfunction+induced+by+beta-amyloid+peptide:+a+practical+human+model+to+study+Alzheimer's+disease+vasculopathy.">Adipose and leptomeningeal arteriole endothelial dysfunction induced by beta-amyloid peptide: a practical human model to study Alzheimer's disease vasculopathy.</a> J Neurosci Methods. 235, 123-129 (2014).</li><li>Karki, 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=Forkhead+box+O-1+modulation+improves+endothelial+insulin+resistance+in+human+obesity.">Forkhead box O-1 modulation improves endothelial insulin resistance in human obesity.</a> Arterioscler Thromb Vasc Biol. 35 (6), 1498-1506 (2015).</li><li>Shahid, M., Buys, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+murine+resistance+artery+function+using+pressure+myography.">Assessing murine resistance artery function using pressure myography.</a> J Vis Exp. (76), (2013).</li></ol>

Authors have no disclosures or conflicts of interest to report.

The dissection and isolation of adipose arterioles from surrounding tissues can be a time-consuming and labor-intensive process with careful attention to detail and technical protocol. The microdissection procedure requires meticulous skills and specialized dissection utensils to prevent potential damage to the smooth muscle or endothelial cell layers of the microvasculature. Even tiny accidental punctures in the arteriolar wall can prevent intraluminal pressurization and results in an unsuccessful experiment. Additional...

Videomicroscopy is a useful technique utilized to examine the vasomotor function of small arterioles removed from living human subjects ex vivo. Our laboratory has focused on dissecting out tiny microvessels from different adipose tissue compartments to characterize the effects of various adipose microenvironments on the microvasculature. A major advantage of this technique is that blood vessels removed from the human body remain functional and can be examined readily within minutes to hours following biopsy. Physiological conditions are mimicked and steady transmural pressure maintained in the intraluminal space via micro-glass cannulas which recapitulate many in vivo characteristics.1,2 Additionally, a reliable videomicroscopy set-up with automated edge detection software allows for both qualitative and quantitative assessment of endothelium-dependent and -independent vasodilator and vasoconstrictor capacity of isolated vessels in real-time, permitting rapid physiological assessment in response to physical and pharmacological stimuli.3 Other microvascular techniques are also available such as wire myography which tends to be less time consuming and measure tension responses to various agonists by a force transducer.
Our laboratory has applied videomicroscopy to examine the relationship between obesity and vascular dysfunction, focusing on the effects of different adipose tissue domains on the vasculature. Central adiposity with accumulation of intra-abdominal visceral fat has been most closely linked to adipocytokine production, metabolic dysfunction, and cardiometabolic risk. It has been postulated that adipose tissue dysfunction with over-production of pro-atherogenic cytokines and adipokine dysregulation are strongly implicated in these processes, but specific regulatory molecules and treatment targets remain largely undiscovered.4 Moreover, at the local adipose tissue level, capillary rarefaction and impaired perfusion have been linked to adipose tissue pseudohypoxia and metabolic dysregulation. The hypothesis that cardiovascular disease may be the consequence of adipose tissue dysfunction is evolving. Pro-atherogenic mediators released from fat, particularly in association with central/visceral adiposity, likely promote endothelial dysfunction and pathogenic vascular changes that can be manifest locally in the adipose vasculature and detected using the herein described methodology.5
The functional assessment of isolated adipose tissue arterioles is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction in human obesity. To investigate mechanisms that contribute to fat depot-specific dysfunction, we have developed methods to examine endothelium-dependent and -independent vasodilatory responses of the microvasculature, and evaluate the expression of various regulatory candidates in paired visceral and subcutaneous (SC) fat tissue specimens obtained from obese subjects at the time of bariatric surgery.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Chemical Name</td>
			<td style="width:159px;"></td>
			<td style="width:132px;"></td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Acetylcholine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">A6625</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride (CaCl2)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td>223506</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">D-(+)-Glucose</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">G5767</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Endothelin-1</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E7764</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetra acetic acid (EGTA)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E3889</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Ethylene diamine tetra acetic acid (EDTA)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">E9884</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">HEPES</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">H3784</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:352px;">Nw-nito-L-arginine methyl ester hydrochloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">N5751</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">M7506</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride (KCL)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P3911</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Potassium phosphate (KH2PO4)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P5655</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Papaverine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">P3510</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Sodium bicarbonate (NaHCO3 )</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">S6014</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Sodium chloride (NaCl)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:132px;">S7653</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Sodium phosphate monobasic monohydrate (NaH2Po4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:127px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Finescience tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td>Zeiss Achromat</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laboratory tubing</td>
			<td>Euro-Pharm</td>
			<td>250100306F999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle/pippette puller</td>
			<td>David kopf instruments</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmic monofilament nylon suture</td>
			<td>Surgical specialties</td>
			<td>A7756N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Finescience tools</td>
			<td>150000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vessel Chamber</td>
			<td>DMT</td>
			<td>VAS v.2.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of human adipose tissue microvascular function using videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Our laboratory has used videomicroscopy to examine endothelium-dependent and -independent vasodilation, as well as vasocontractile function of adipose tissue arterioles isolated from subcutaneous and visceral fat of obese humans. The characteristic experimental set-up is displayed in Figure 1A. Adipose tissue arterioles are suspended between two glass capillary pipettes and secured in place with sutures within the organ chamber as shown in <strong class="xfig...

Watch this Scientific Journal Video about Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy at JoVE.com

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

assessment-human-adipose-tissue-microvascular-function-using

Research

JoVE Journal

Medicine

7.4K Views.  Boston University School of Medicine. Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

Video: Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a computerized functional skills training program that was paired in half of the participants with a commercially available cognitive training (CCT) program.
Non-impaired older individuals (NC) aged 60+ (n=45) and similarly aged individuals with mild cognitive impairment (MCI; n=50) were randomized to receive 12 weeks of twice-weekly computerized functional skills training (CFST) or 12 weeks of twice-weekly sessions split between CCT and CFST. Skills trained were use of an ATM; internet banking; ticket kiosk; telephone and internet prescription refill; medication management; and internet shopping. As with previous functional capacity assessments, we focus on completion time for each simulation.
51 participants completed the training program, either by mastering all 6 tasks (34) or completing 12 weeks of training. 44 more participants completed 4 or more training sessions so they were also analyzed for improvement up to their last training session. Completion time for all 6 tests significantly improved from the baseline assessment to the final training session in both groups of participants (all p&#60;0.001 with an average improvement in task completion time of 45%). Further, there was no differential improvement in MCI and NC in the 6 tests from baseline to end of training (all t&#60;1.66, all p&#62;0.12). Finally, combined CCT plus CFST did not differ from CSFT alone on any of the percent-change score measures (all t&#60;1.64, all p&#62;0.11).
Both NC and MCI groups evidenced substantial improvements in performance. CCT supplementation led to similar functional gains with half as many training sessions. The NC participants proceeded through the training fairly rapidly even without CCT supplementation; MCI participants required more training but learned equivalently. These findings suggest that even in cases with memory impairments, functional skills can be efficiently learned with training.

This training protocol uses computerized training to teach technology-related everyday functional skills. These skills include financial skills, travel and transit, as well as medication management.

Today, many functional skills are technology-based, so development of a technology-based training program has broad importance. Here we present a ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...