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>

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	<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. 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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><tbody><tr><td>&lt;strong&gt;Chemical Name&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetylcholine</td><td>Sigma Aldrich</td><td>A6625</td><td></td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>223506</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich</td><td>G5767</td><td></td></tr><tr><td>Endothelin-1</td><td>Sigma Aldrich</td><td>E7764</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetra acetic acid (EGTA)</td><td>Sigma Aldrich</td><td>E3889</td><td></td></tr><tr><td>Ethylene diamine tetra acetic acid (EDTA)</td><td>Sigma Aldrich</td><td>E9884</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3784</td><td></td></tr><tr><td>N&lt;sub&gt;&lt;em&gt;w&lt;/em&gt;&lt;/sub&gt;-nito-L-arginine methyl ester hydrochloride</td><td>Sigma Aldrich</td><td>N5751</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>Potassium chloride (KCL)</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Papaverine</td><td>Sigma Aldrich</td><td>P3510</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt; )</td><td>Sigma Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate (NaH2Po4)</td><td>Sigma Aldrich</td><td>S9638</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>Finescience tools</td><td>15000-08</td><td></td></tr><tr><td>Inverted microscope</td><td>Zeiss Achromat</td><td></td><td></td></tr><tr><td>Laboratory tubing</td><td>Euro-Pharm</td><td>250100306F999</td><td></td></tr><tr><td>Needle/pippette puller</td><td>David kopf instruments</td><td>720</td><td></td></tr><tr><td>Ophthalmic monofilament nylon suture</td><td>Surgical specialties</td><td>A7756N</td><td></td></tr><tr><td>Scissors</td><td>Finescience tools</td><td>150000-08</td><td></td></tr><tr><td>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

Universidad de Cartagena UDEC

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 ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Immunology and Infection

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Bioengineering

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...