Name: localization, identification, and excision of murine adipose depots
Uploaded: 2014-12-04T14:00:00
Description: Watch this Scientific Journal Video about Localization, Identification, and Excision of Murine Adipose Depots at JoVE.com

The authors have no acknowledgements.

NOTE: All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati and in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).
1. Euthanize and Sterilize the Mouse
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	<li>Place the mouse in a dropbox containing a supratheraputic dose of isoflurane and allow to inhale to effect. Once the mouse is euthanized, r...

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	<li>Ogden, C. L., Carroll, M. D., Kit, B. K., Flegal, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+in+the+United+States,+2009-2010.">Prevalence of obesity in the United States, 2009-2010.</a> NCHS Data Brief. 82, 1-8 (2012).</li><li>Flegal, K. M., Carroll, M. D., Kit, B. K., Ogden, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+and+trends+in+the+distribution+of+body+mass+index+among+US+adults,+1999-2010.">Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999-2010.</a> 307 (5), 491-497 (2012).</li><li>Cawley, J., Meyerhoefer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medical+care+costs+of+obesity:+an+instrumental+variables+approach.">The medical care costs of obesity: an instrumental variables approach.</a> Journal of Health Economics. 31 (1), 219-230 (2012).</li><li>Marder, W., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+Obesity:+Costs,+Treatment+Patterns,+Disparities+in+Care,+and+Prevalent+Medical+Conditions.">Childhood Obesity: Costs, Treatment Patterns, Disparities in Care, and Prevalent Medical Conditions.</a> Thomson Medstat Research Brief. , (2006).</li><li>Cortez, M., Carmo, L. S., Rogero, M. M., Borelli, P., Fock, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-fat+diet+increases+IL-1,+IL-6,+and+TNF-&#945;+production+by+increasing+NF-&#954;B+and+attenuating+PPAR-&#947;+expression+in+bone+marrow+mesenchymal+stem+cells.">A high-fat diet increases IL-1, IL-6, and TNF-&#945; production by increasing NF-&#954;B and attenuating PPAR-&#947; expression in bone marrow mesenchymal stem cells.</a> Inflammation. 36 (2), 379-386 (2013).</li><li>Sanchez-Gurmaches, J., Hung, C. M., Sparks, C. A., Tang, Y., Li, H., Guertin, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+loss+in+the+Myf5+lineage+redistributes+body+fat+and+reveals+subsets+of+white+adipocytes+that+arise+from+Myf5+precursors.">PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors.</a> Cell Metab. 16 (3), 348-362 (2012).</li><li>Sethi, J. K., Vidal-Puig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+Adipose+tissue+function+and+plasticity+orchestrate+nutritional+adaptation.">Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation.</a> J Lipid Res. 48 (6), 1253-1262 (2007).</li><li>Aarsland, A., Chinkes, D., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+and+whole-body+fat+synthesis+in+humans+during+carbohydrate+overfeeding.">Hepatic and whole-body fat synthesis in humans during carbohydrate overfeeding.</a> Am J Clin Nutr. 65 (6), 1774-1782 (1997).</li><li>Park, A., Kim, W. K., Bae, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+of+white,+beige+and+brown+adipocytes+derived+from+mesenchymal+stem+cells.">Distinction of white, beige and brown adipocytes derived from mesenchymal stem cells.</a> World J. Stem Cells. 6 (1), 33-42 (2014).</li><li>Wu, 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=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and.</a> 150 (2), 366-376 (2012).</li><li>Giralt, M., Villarroya, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White,+brown,+beige/brite:+different+adipose+cells+for+different+functions.">White, brown, beige/brite: different adipose cells for different functions.</a> Endocrinology. 154 (9), 2992-3000 (2013).</li><li>Casteilla, L., P&#233;nicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity.">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity.</a> Methods Mol. Biol. 456, 23-38 (2008).</li><li>Grant, R., Youm, Y. H., Ravussin, A., Dixit, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+adipose+tissue+leukocytosis+in+obesity.">Quantification of adipose tissue leukocytosis in obesity.</a> Methods Mol. Biol. 1040, 195-209 (2013).</li></ol>

Obesity can lead to a large host of morbidities and the full understanding of the role that adipose plays is not fully understood; therefore continued research in the field of adipose is necessary. Animal models, specifically murine models are ideal for initial research in the progression of diseases and testing of potential pharmaceutical treatments. In using these models, precise isolation and excision of adipose depots is an extremely important and necessary tool in the study of pathology of adipose affected diseases....

Adipose made a notable appearance in the media spotlight, due to obesity&#8217;s dramatic increase during the last few decades of the 20th century. Obesity currently affects more than one-third of adults and 17% of children and adolescents in the United States (US)1. Spanning all ethnic groups, statistical research surrounding the obesity epidemic has shown that non-Hispanic blacks have the highest age-adjusted rate of obesity (49.5%) compared with Mexican Americans (40.4%), all Hispanics (39.1%), and non-Hispanic whites (34.3%)2. The economic effect of obesity is also a growing concern for the healthcare system. In 2012, it was estimated that the annual medical cost of care for obesity in the US in 2005 was $190.2 billion, nearly 21% of the overall medical spending budget. Sadly, childhood obesity was estimated to be responsible for $14 billion in direct medical costs alone. Statistically, it was determined that the average medical cost of individuals with obesity was $2,741 higher a year than those without this morbidity3-5.
Obesity is a major risk factor for a variety of conditions such as: type 2 diabetes, dyslipidemia, cardiovascular disease, cancer, muscular skeletal disorders and chronic inflammation. Obesity is deeply tied to the pathogenesis of metabolic syndrome and other chronic diseases6-8. With such drastic and negative connections between obesity and other comorbidities, scientific research has focused attention to better understand the current epidemic and the diverse and pivotal roles played by adipose.
Historically, adipose tissue was considered inconsequential and was viewed merely as a simple filling tissue. Currently, adipose has been shown to play many essential roles in the body&#8217;s function in: metabolism, hormone regulation, inflammation, protection and insulation9. Adipose tissue is composed primarily of adipocytes but also contains pericytes, endothelial cells, monocytes, macrophages and pluripotent stem cells8. Adipose tissue is distributed throughout the body in distinct depots. The principal depots can be found subdermally, subcutaneously, intramuscularly, and viscerally10. Adipose depots have been shown to have depot specific metabolic profiles, which have shown a depot specific susceptibility to obesity and related disorders8.
Traditionally, adipose tissue has been classified into two major types: white adipose tissue (WAT) and brown adipose tissue (BAT); although recent literature indicates the presences of a third group christened brite or beige adipose11. Adipose tissue has been shown to have different colors, morphologies, metabolic functions, biochemical features and genetic patterns of expression10. Adipocytes in WAT have a single, large lipid droplet and variable amounts of mitochondria. WAT is dominantly found in subcutaneous and visceral localities of the body. WAT functions primarily as a site of energy storage and organ protection. Adipocytes in BAT have a multilocular morphology and abundant mitochondria. BAT is located primarily in the neck and large blood vessels of the thorax, as well as the scapulae12. BAT primarily functions in energy-expending behaviors that regulate thermogenesis7. Brite or beige adipose has been shown to share an analogous morphology and expression to BAT but has been found to be originated from white adipocytes11.
The described surgical method in this manuscript provides researchers with the capacity to analyze different effects that factors such as: environment, pharmaceuticals, and genetics, have on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Also, providing a way to identify and isolate different types of adipose tissue to allow for better understand of the biochemical relationships and differences between depots. This can aid in determining the relationship between location, function, and types of fat within the body. This described method accomplishes this by providing the means for gross visualization, gene expression analysis, protein expression analysis, histological examination, and isolation of primary cell lines for in vitro studies. Currently there are many articles that provide insight into the metabolic behavior of different adipose depots, as well as their anatomic locations; but do not provide an in-depth method on how to specifically localize, identify, and isolate these depots. This surgical method provides a precise technique that allows for isolation of multiple depots with a minimal quantity of dissection and contamination compared to others methods designed for the isolate of one or two depots13-14.
The goal of this protocol is to provide a precise method for the identification and isolation of different types of fat depots from multiple anatomic locations.

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			<td style="width:175px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
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			<td height="21" style="height:21px;">isoflurane</td>
			<td>Med-Vet International&#160;</td>
			<td>#RXISO-250</td>
			<td></td>
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			<td height="21" style="height:21px;width:243px;">70% Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:125px;">07-678-001</td>
			<td></td>
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			<td height="21" style="height:21px;width:243px;">DMEF-12</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">D-6421</td>
			<td>Warm in waterbath before putting on tissue</td>
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			<td height="21" style="height:21px;width:243px;">2 mL microcentrifuge tubes</td>
			<td style="width:175px;">Midsci</td>
			<td style="width:125px;">MCT-200-C-S</td>
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			<td height="21" style="height:21px;width:243px;">Phosphate buffered saline</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:125px;">P5368-10PAK</td>
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localization, identification, and excision of murine adipose depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Identification, and localization of inguinal subcutaneous adipose, interscapular brown adipose, visceral epididymal adipose (Figure 1), as well as the aortic arch perivascular adipose, thoracic aortic adipose, suprarenal aortic adipose and the infrarenal aortic adipose (Figure 2) was achieved successfully using the described surgical method. Histological examination and differentiation between BAT and WAT samples were positively evaluated using Hematoxylin and Eosin (H&#38;E) staining (<...

Watch this Scientific Journal Video about Localization, Identification, and Excision of Murine Adipose Depots at JoVE.com

Localization, Identification, and Excision of Murine Adipose Depots

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

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