Name: assessment of vascular tone responsiveness using isolated mesenteric arteries with a focus on modulation by perivascular adipose tissues
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Description: Watch this Scientific Journal Video about Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues at JoVE.com

This work was financially support by the grants from Research Grant Council of Hong Kong [17124718 and 17121714], Hong Kong Health and Medical Research Fund [13142651 and 13142641], Collaborative Research Fund of Hong Kong [C7055-14G], and the National Basic Research Program of China [973 Program 2015CB553603].

All animals used for the following study were provided by the Laboratory Animal Unit of the Faculty of Medicine, The University of Hong Kong. Ethical approval was obtained from the departmental Committee on Use of Laboratory Animals for Teaching and Research (CULATR, no.: 4085-16).
1. Preparations
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	<li>Preparation of drugs
	<ol>
		<li>Store drugs appropriately as stated in the Material Safety Data Sheet (MSDS) immediately after receiving them. Dissolve the drugs in powd...

<ol>
	<li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetylcholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.</a> Nature. 288 (5789), 373-376 (1980).</li><li>Furchgott, R. F., Vanhoutte, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+relaxing+and+contracting+factors.">Endothelium-derived relaxing and contracting factors.</a> The FASEB Journal. 3 (9), 2007-2018 (1989).</li><li>Feletou, M., Kohler, R., Vanhoutte, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+vasoactive+factors+and+hypertension:+possible+roles+in+pathogenesis+and+as+treatment+targets.">Endothelium-derived vasoactive factors and hypertension: possible roles in pathogenesis and as treatment targets.</a> Current Hypertension Reports. 12 (4), 267-275 (2010).</li><li>Vanhoutte, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction:+the+first+step+toward+coronary+arteriosclerosis.">Endothelial dysfunction: the first step toward coronary arteriosclerosis.</a> Circulation Journal. 73 (4), 595-601 (2009).</li><li>Feletou, M., Huang, Y., Vanhoutte, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-mediated+control+of+vascular+tone:+COX-1+and+COX-2+products.">Endothelium-mediated control of vascular tone: COX-1 and COX-2 products.</a> British Journal of Pharmacology. 164 (3), 894-912 (2011).</li><li>Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+endothelial+cell+dysfunction.">Cellular and molecular mechanisms of endothelial cell dysfunction.</a> Journal of Clinical Investigation. 100 (9), 2153 (1997).</li><li>Vanhoutte, P. M., Shimokawa, H., Tang, E. H., Feletou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction+and+vascular+disease.">Endothelial dysfunction and vascular disease.</a> Acta physiologica. 196 (2), 193-222 (2009).</li><li>Kl&#246;&#223;, S., Bouloumi&#233;, A., M&#252;lsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+chronic+hypertension+decrease+expression+of+rat+aortic+soluble+guanylyl+cyclase.">Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase.</a> Hypertension. 35 (1), 43-47 (2000).</li><li>Csiszar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging-induced+phenotypic+changes+and+oxidative+stress+impair+coronary+arteriolar+function.">Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function.</a> Circulation Research. 90 (11), 1159-1166 (2002).</li><li>Guo, 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=Endothelial+SIRT1+prevents+age-induced+impairment+of+vasodilator+responses+by+enhancing+the+expression+and+activity+of+soluble+guanylyl+cyclase+in+smooth+muscle+cells.">Endothelial SIRT1 prevents age-induced impairment of vasodilator responses by enhancing the expression and activity of soluble guanylyl cyclase in smooth muscle cells.</a> Cardiovascular Research. , (2018).</li><li>Auch-Schwelk, W., Katusic, Z. S., Vanhoutte, P. M. <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+inactivates+endothelium-derived+contracting+factor+in+the+rat+aorta.">Nitric oxide inactivates endothelium-derived contracting factor in the rat aorta.</a> Hypertension. 19 (5), 442-445 (1992).</li><li>Tang, E. H., Feletou, M., Huang, Y., Man, R. Y., Vanhoutte, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+and+sodium+nitroprusside+cause+long-term+inhibition+of+EDCF-mediated+contractions.">Acetylcholine and sodium nitroprusside cause long-term inhibition of EDCF-mediated contractions.</a> American Journal of Physiology - Heart and Circulation Physiology. 289 (6), H2434-H2440 (2005).</li><li>Ghiadoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+common+carotid+artery+wall+thickening+in+patients+with+essential+hypertension.">Endothelial function and common carotid artery wall thickening in patients with essential hypertension.</a> Hypertension. 32 (1), 25-32 (1998).</li><li>Xu, X., 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=Age-related+Impairment+of+Vascular+Structure+and+Functions.">Age-related Impairment of Vascular Structure and Functions.</a> Aging and Disease. 8 (5), 590-610 (2017).</li><li>Tabit, C. E., Chung, W. B., Hamburg, N. M., Vita, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction+in+diabetes+mellitus:+Molecular+mechanisms+and+clinical+implications.">Endothelial dysfunction in diabetes mellitus: Molecular mechanisms and clinical implications.</a> Reviews in Endocrine &amp; Metabolic Disorders. 11 (1), 61-74 (2010).</li><li>Tanaka, K., Sata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+perivascular+adipose+tissue+in+the+pathogenesis+of+atherosclerosis.">Roles of perivascular adipose tissue in the pathogenesis of atherosclerosis.</a> Frontiers in Physiology. 9, 3 (2018).</li><li>Brown, N. 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=Perivascular+adipose+tissue+in+vascular+function+and+disease:+a+review+of+current+research+and+animal+models.">Perivascular adipose tissue in vascular function and disease: a review of current research and animal models.</a> Arteriosclerosis Thrombosis and Vascular Biology. 34 (8), 1621-1630 (2014).</li><li>Lohn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periadventitial+fat+releases+a+vascular+relaxing+factor.">Periadventitial fat releases a vascular relaxing factor.</a> The FASEB Journal. 16 (9), 1057-1063 (2002).</li><li>G&#225;lvez-Prieto, B., 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=A+reduction+in+the+amount+and+anti-contractile+effect+of+periadventitial+mesenteric+adipose+tissue+precedes+hypertension+development+in+spontaneously+hypertensive+rats.">A reduction in the amount and anti-contractile effect of periadventitial mesenteric adipose tissue precedes hypertension development in spontaneously hypertensive rats.</a> Hypertension research. 31 (7), 1415 (2008).</li><li>Gao, Y. J., Lu, C., Su, L. Y., Sharma, A., Lee, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+vascular+function+by+perivascular+adipose+tissue:+the+role+of+endothelium+and+hydrogen+peroxide.">Modulation of vascular function by perivascular adipose tissue: the role of endothelium and hydrogen peroxide.</a> British Journal of Pharmacology. 151 (3), 323-331 (2007).</li><li>Gao, Y. -. 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=Perivascular+adipose+tissue+promotes+vasoconstriction:+the+role+of+superoxide+anion.">Perivascular adipose tissue promotes vasoconstriction: the role of superoxide anion.</a> Cardiovascular Research. 71 (2), 363-373 (2006).</li><li>Szasz, T., Webb, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+adipose+tissue:+more+than+just+structural+support.">Perivascular adipose tissue: more than just structural support.</a> Clinical Science (London). 122 (1), 1-12 (2012).</li><li>Ramirez, J. G., O'Malley, E. J., Ho, W. S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-contractile+effects+of+perivascular+fat+in+health+and+disease.">Pro-contractile effects of perivascular fat in health and disease.</a> Brish Journal of Pharmacology. 174 (20), 3482-3495 (2017).</li><li>Hajer, G. R., van Haeften, T. W., Visseren, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+dysfunction+in+obesity,+diabetes,+and+vascular+diseases.">Adipose tissue dysfunction in obesity, diabetes, and vascular diseases.</a> European Heart Journal. 29 (24), 2959-2971 (2008).</li><li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+small+arterial+resistance+vessels+in+spontaneously+hypertensive+and+normotensive+rats.">Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats.</a> Circulation Research. 41 (1), 19-26 (1977).</li><li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+vascular+smooth+muscle+cells+in+situ.">Mechanical properties of vascular smooth muscle cells in situ.</a> Nature. 260 (5552), 617-619 (1976).</li><li>del Campo, L., Ferrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wire+myography+to+study+vascular+tone+and+vascular+structure+of+isolated+mouse+arteries.">Wire myography to study vascular tone and vascular structure of isolated mouse arteries.</a> Methods in Molecular Biology. 1339, 255-276 (2015).</li><li>Dobrin, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+initial+length+on+length-tension+relationship+of+vascular+smooth+muscle.">Influence of initial length on length-tension relationship of vascular smooth muscle.</a> American Journal of Physiology. 225 (3), 664-670 (1973).</li><li>Xu, C., 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=Calorie+restriction+prevents+metabolic+aging+caused+by+abnormal+SIRT1+function+in+adipose+tissues.">Calorie restriction prevents metabolic aging caused by abnormal SIRT1 function in adipose tissues.</a> Diabetes. 64 (5), 1576-1590 (2015).</li><li>Sheykhzade, M., Nyborg, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caliber+dependent+calcitonin+gene-related+peptide-induced+relaxation+in+rat+coronary+arteries:+effect+of+K++on+the+tachyphylaxis.">Caliber dependent calcitonin gene-related peptide-induced relaxation in rat coronary arteries: effect of K+ on the tachyphylaxis.</a> European Journal of Pharmacology. 351 (1), 53-59 (1998).</li><li>Soltis, E. E., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+perivascular+adipose+tissue+on+rat+aortic+smooth+muscle+responsiveness.">Influence of perivascular adipose tissue on rat aortic smooth muscle responsiveness.</a> Clinical and Experimental Hypertension A. 13 (2), 277-296 (1991).</li><li>Lohn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periadventitial+fat+releases+a+vascular+relaxing+factor.">Periadventitial fat releases a vascular relaxing factor.</a> FASEB Journal. 16 (9), 1057-1063 (2002).</li><li>Fesus, 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=Adiponectin+is+a+novel+humoral+vasodilator.">Adiponectin is a novel humoral vasodilator.</a> Cardiovascular Research. 75 (4), 719-727 (2007).</li><li>Greenstein, A. 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=Local+inflammation+and+hypoxia+abolish+the+protective+anticontractile+properties+of+perivascular+fat+in+obese+patients.">Local inflammation and hypoxia abolish the protective anticontractile properties of perivascular fat in obese patients.</a> Circulation. 119 (12), 1661-1670 (2009).</li><li>Yudkin, J. S., Eringa, E., Stehouwer, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Vasocrine"+signalling+from+perivascular+fat:+a+mechanism+linking+insulin+resistance+to+vascular+disease.">"Vasocrine" signalling from perivascular fat: a mechanism linking insulin resistance to vascular disease.</a> Lancet. 365 (9473), 1817-1820 (2005).</li><li>Xia, N., 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=Uncoupling+of+endothelial+nitric+oxide+synthase+in+perivascular+adipose+tissue+of+diet-induced+obese+mice.">Uncoupling of endothelial nitric oxide synthase in perivascular adipose tissue of diet-induced obese mice.</a> Arteriosclerosis Thrombosis and Vascular Biology. 36 (1), 78-85 (2016).</li><li>Xia, N., Forstermann, U., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+resveratrol+on+eNOS+in+the+endothelium+and+the+perivascular+adipose+tissue.">Effects of resveratrol on eNOS in the endothelium and the perivascular adipose tissue.</a> Annals of the New York Academy of Sciences. 1403 (1), 132-141 (2017).</li><li>Schinzari, F., Tesauro, M., Cardillo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+and+perivascular+adipose+tissue+abnormalities+in+obesity-related+vascular+dysfunction:+novel+targets+for+treatment.">Endothelial and perivascular adipose tissue abnormalities in obesity-related vascular dysfunction: novel targets for treatment.</a> Journal of Cardiovascular Pharmacology. 69 (6), 360-368 (2017).</li><li>Liu, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipocalin-2+deficiency+prevents+endothelial+dysfunction+associated+with+dietary+obesity:+role+of+cytochrome+P450+2C+inhibition.">Lipocalin-2 deficiency prevents endothelial dysfunction associated with dietary obesity: role of cytochrome P450 2C inhibition.</a> British Journal of Pharmacology. 165 (2), 520-531 (2012).</li><li>Martinez-Quinones, P., 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=Hypertension+induced+morphological+and+physiological+changes+in+cells+of+the+arterial+wall.">Hypertension induced morphological and physiological changes in cells of the arterial wall.</a> American Journal of Hypertension. 31 (10), 1067-1078 (2018).</li><li>Outzen, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+value+of+mechanical+and+vasomotor+properties+of+mouse+isolated+mesenteric+resistance-sized+arteries.">Translational value of mechanical and vasomotor properties of mouse isolated mesenteric resistance-sized arteries.</a> Pharmacology Research and Perspectives. 3 (6), e00200 (2015).</li><li>Sheykhzade, M., Simonsen, A. H., Boonen, H. C., Outzen, E. M., Nyborg, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ageing+on+the+passive+and+active+tension+and+pharmacodynamic+characteristics+of+rat+coronary+arteries:+age-dependent+increase+in+sensitivity+to+5-HT+and+K+.">Effect of ageing on the passive and active tension and pharmacodynamic characteristics of rat coronary arteries: age-dependent increase in sensitivity to 5-HT and K+.</a> Pharmacology. 90 (3-4), 160-168 (2012).</li></ol>

Apart from the endothelial cells, signals derived from PVAT play an important role in the regulation of smooth muscle tone reactivity30. Healthy PVAT releases NO and anti-inflammatory adiponectin to exert an anti-contractile effect on arteries, which is lost under pathological conditions such as obesity and metabolic syndrome31,32. In disease states, PVAT contributes to the development of endothelial dysfunction and other cardiovascular ab...

Dilatations and constrictions of arteries are achieved by relaxations and contractions, respectively, of their vascular smooth muscle cells. Changes in vascular responsiveness of small arteries contribute to the homeostatic regulation of arterial blood pressure by autonomic nerves and hormones present in the blood (e.g., catecholamines, angiotensin II, serotonin, vasopressin). At the local level, the vascular responses of smooth muscle cells are modulated by signals from both the endothelial cells of the intima and the adipose tissue surrounding the arteries (Figure 1).
The endothelium is not only a passive barrier, but also serves as a surface to exchange signals between the blood and the underlying vascular smooth muscle cells. By releasing various vasoactive substances, the endothelium plays a critical role in the local control of vascular tone responses1. For example, in response to acetylcholine, endothelial nitric oxide synthase (eNOS) is activated in the endothelium to produce nitric oxide (NO), which induces relaxation of the underlying vascular smooth muscle by activating soluble guanylyl cyclase (sGC)2. Other vasoactive substances include the products of cyclooxygenases (e.g., prostacyclin and thromboxane A2), lipoxygenase (e.g., 12-hydroxyeicosatetraenoic acids, 12-HETE), and cytochrome P450 monooxygenases (HETEs and epoxyeicosatrienoic acids, EETs), reactive oxygen species (ROS), and vasoactive peptides (e.g., endothelin-1 and angiotensin II), and endothelium-derived hyperpolarizing factors (EDHF)3. A delicate balance between endothelium-derived vasodilators and vasoconstrictors maintain the local vasomotor tone4,5.
Endothelial dysfunction is characterized by the impairment in endothelium-dependent vasodilatation6, a hallmark of vascular aging7. With age, the ability of endothelium to promote vasodilatation is progressively reduced, due largely to a decreased NO bioavailability, as well as the abnormal expression and function of eNOS in the endothelium and sGC in the vascular smooth muscle cells8,9,10. Reduced NO bioavailability potentiates the production of endothelium-dependent vasoconstrictors11,12. In aged arteries, endothelial dysfunction causes hyperplasia in the media, as reflected by the marked increases in wall thickness, number of medial nuclei, which are reminiscent of the arterial thickening in hypertension and atherosclerosis observed in human patients13,14. In addition, pathophysiological conditions such as obesity, diabetes or hypertension accelerate the development of endothelial dysfunction15,16.
Perivascular adipose tissue (PVAT) releases numerous adipokines to regulate vascular structure and function17. The anti-contractile effect of PVAT is mediated by relaxing factors, such as adiponectin, NO, hydrogen peroxide and hydrogen sulphide18,19,20. However, depending on the location and pathophysiological condition, PVAT also can enhance contractile responses&#160;in various arteries21. The pro-contractile substances produced by PVAT include angiotensin-II, leptin, resistin, and ROS22,23.&#160; In most of the studies on isolated blood vessels, PVAT has been considered as a simple structural support for the vasculature and thus removed during the preparation of blood vessel ring segments. Since adipose dysfunction represents an independent risk factor for hypertension and associated cardiovascular complications24, the PVAT surrounding the blood vessels should be considered when investigating the vascular responsiveness of different arteries.
The multi wire myograph systems have been widely used to investigate the vasomotor functions of a variety of blood vessels, including the aorta, mesenteric, renal, femoral, cerebral and coronary arteries25,26. The protocols described herein will use wire myography to evaluate vascular responsiveness in mesenteric arteries isolated from genetically modified mouse models, with a special focus on the modulation by PVAT.

<table>
	<tbody>
		<tr>
			<td>Acetylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>A6625</td>
			<td>Stock concentration: 10-1 M 
			Working concentration: 10-10 to 10-5 M</td>
		</tr>
		<tr>
			<td>L-NAME (N&#969;-nitro-L-arginine methyl ester)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751</td>
			<td>Stock concentration: 3 x 10-2 M 
			Working concentration: 10-4 M</td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich</td>
			<td>P6126</td>
			<td>Stock concentration: 10-2 M 
			Working concentration: 10-10 to 10-5 M</td>
		</tr>
		<tr>
			<td>U46619 (9,11-dideoxy-9&#945;,11&#945;methanoepoxy prostaglandin F2&#945;)</td>
			<td>Enzo</td>
			<td>BML-PG023-0001</td>
			<td>Stock concentration: 10-5 M 
			Working concentration: 1-3 x 10-8 M</td>
		</tr>
		<tr>
			<td>Multiwire myograph</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td>620M</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab 4/26</td>
			<td>ADInstruments</td>
			<td>ML848</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart7</td>
			<td>ADInstruments</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipo-SIRT1 wild type mice</td>
			<td>Laboratory Animal Unit, The University of Hong Kong</td>
			<td>CULATR NO.: 4085-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon-coated Petri dishes</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wires</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td>300331</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Examination of the length/tension relationships to obtain the normalization factor k 
The amount of stretch applied to a vessel segment influences the extent of the actin-myosin interaction and hence the maximal active force developed. Thus, for every type of blood vessel, determining the amount of stretch needed for maximal active force is required for proper myography studies. Here, normalization of t...

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

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 ...

Very strict protocol is required so that we can validly compare preparations from the same animals or from different animals or arteries in the presence or absence of the fat that surrounds them or in the presence or absence of the endothelium. The main advantage of this experimental technique is that we can compare rings of the same artery under isometric condition and thus validly make conclusions concerning the impact of the experimental factors that we investigate. This protocol provides insights into modulation of vascular responsiveness by perivascular adipose tissues of different genetically modified animals.Adipose tissue dysfunctions represent an independent risk factor for hypertension and its associated cardiovascular complications. Our myograph involves multiple complicated steps that require extreme caution at each point to avoid damaging the vessel, especially during mounting, and to ensure that the vessel responds appropriately to various stimuli. Begin this procedure with preparations and dissection of the mesenteric arterial rings as described in the text protocol.Switch on the computer and open the data recording software. Save the experiment as a lab chart data file with a new name to avoid overwriting the original setting file. Open the normalization settings window and set the K factor to one.Accept the default values for IPs calibration, target pressure, online averaging time, and delay time. Click okay to save the settings. Select channels of interest and input the wire diameter, tissue endpoints, and initial micrometer reading in the normalization window.To start the normalization procedure, apply the first passive stretch to the blood vessel by turning the micrometer screw counterclockwise. After waiting three minutes for the vessel to stabilize, input the new micrometer reading in the normalization window. The wall tension is automatically calculated and shown as a point on the graph.After each passive stretch, replace the control krebs buffer with an iso-osmotic high potassium krebs containing 150 millimolar potassium chloride. When the contraction reaches a plateau at about three minutes, record the active force by subtracting the passive force at each stretch from the potassium activated force. Calculate the wall tension, as well as the internal circumference values.Remove the high potassium condition by replacing the high potassium buffer with fresh krebs buffer. Repeat washing for three times over five minutes. Repeat the normalization steps by inducing passive stretches followed by active contraction in alternate turns until the active tension starts to decrease.Thoroughly wash out the high potassium krebs buffer and equilibrate the preparations for another 30 to 45 minutes. Reset the basal tensions to zero so that only active contractile responses will be recorded during the subsequent experiment. Prepare and mount paired arterial rings as described in the text protocol from the adjacent sections of each artery.Prepare one arterial ring with PVAT intact and the other with PVAT removed. After normalization, precontract the arterial segments with high potassium krebs buffer by adding 115 millimolar potassium chloride solution to the chamber containing krebs. Following contraction to the plateau period, wash out the high potassium and replace the buffer with fresh aerated krebs buffer.Repeat washing three times over five minutes. Repeat the potassium chloride stimulation and washing three times and record the maximal contractile response to potassium chloride by subtracting the baseline tension from the tension due to potassium chloride stimulation. After the last contraction and washing, refill the chamber with warm, aerated krebs buffer and allow the artery to recover for about 30 minutes before performing the next task.To each chamber, add cumulative amounts of phenylephrine to induce the concentration-dependent increases and isometric tension of the quiescent preparations. After adding the last dose of agonist, wash out the drug thoroughly and refill the chamber with fresh krebs buffer. Plot the concentration dependent responses as increasing percentages of the potassium chloride induced maximal contractions.To assess the contribution of nitric oxide, incubate the preparations with the nitric oxide synthase inhibitor L-NAME for 30 minutes prior to the addition of phenylephrine. For performing a second concentration response curve sequentially, wash the chamber completely and repeatedly to remove all of the previous agonists until no further changes in tone are observed. Normalization of the length-tension relationship was performed for mesenteric arteries isolated from mouse models fed with standard chow or a high-fat diet.A passive length-tension relationship was established by incremental stretching of the artery segments until an internal circumference corresponding to 100 millimeters of mercury transmural pressure, or IC100, was obtained. After each stretch, 115 millimolar potassium chloride was applied to stimulate contractions. The active length tension curves were plotted from the active force data on the Y-axis and the calculated IC values from the micrometer data on the X-axis.An IC value lying within the peak plateau is IC1. The normalization factor, K, was calculated as the ratio of IC1 and IC100, which could then be applied to samples of the same vessel type in the subsequent experiment. Shown here are output recordings of the vasoconstrictor responses to phenylephrine in mesenteric arteries with or without surrounding PVAT.Cumulative concentrations of phenylephrine were applied to stimulate the contractions of mesenteric arteries collected from 16-week-old adiposet one mice. The contractile responses were recorded and calculated as a percentage of 150 millimolar potassium chloride induced maximal contraction. The area under the contraction curves were plotted for comparison.Note that PVAT from adiposet one mice elicited an anti-contractile effect on the response to phenylephrine. When obtaining KCL-induced contractions, the responses have to be stable and steady. If the response is still increasing after three incremental administrations of KCL, KCL stimulation may be needed.To achieve optimal conditions of the tensions, the normalization step is very important, as this determines the force to open the blood vessels. If it's not done properly, it will affect the whole experiment. This protocol can be applied to other settings in addition to phenylephrine-induced contraction or relaxation.For example, in the presence or absence of perivascular adipose tissue, autonomic nerves, or endothelium. With this protocol, researchers can examine small blood vessels from diseased animal models under different pathophysiological conditions, such as obesity, diabetes, and cardiovascular diseases.

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Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6 However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.

This video describes a right hemihepatectomy with intrahepatic transection of the right hemipedicle leaving the hepatoduodenal ligament completely untouched.

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.