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
<ol>
	<li>Place the mouse in a dropbox containing a supratheraputic dose of isoflurane and allow to inhale to effect. Once the mouse is euthanized, remove from the box.</li>
	<li>Cervically dislocate as a second means of euthanasia.</li>
	<li>Sterilize the external surface of the mouse by cleansing the animal with 70% ethanol.</li>
</ol>
2. Identification and Isolation of Three Different Adipose Depots
<ol>
	<li>Brown Adipose Tissue (BAT) Isolation:
	<ol>
		<li>Make sure the fur is wet from the alcohol cleansing to aid in cutting through the epidermal and dermal layers.</li>
		<li>Position the mouse in a supine position with its back against the table.</li>
		<li>Grab the skin just below the diaphragm with forceps, lift and incise with scissors.</li>
		<li>Cut transversely around the circumference of the mouse to expose the peritoneum.</li>
		<li>Reveal the butterfly-shaped BAT depot by degloving the top half of the mouse. Hold the lower appendages and abdomen in one hand and pulling the skin up towards the head.</li>
		<li>Orient the mouse, so that it is positioned prone on the table. Be careful not to contaminate the exposed depot with hair.</li>
		<li>Clean surgical instruments and change to a fresh pair of gloves.</li>
		<li>Locate the scapulae and corresponding depot. Carefully remove any superficial white adipose atop the butterfly and then dissect the butterfly of interscapular brown fat. Be careful to avoid the muscle closely associated with the brown fat. 
		NOTE: The use of a dissection microscope is recommended for removal of the white adipose, as well as in the separation of the brown adipose from the scapulae.</li>
		<li>Remove fat depot and transfer to a 2 ml microcentrifuge tube.</li>
		<li>If RNA or protein is to be extracted, freeze the tissue by immersion in liquid nitrogen and store at -80 &#176;C. Freeze the sample at once to prevent degradation. If culturing, cover the tissue in DMEF-12 and set on ice till all samples are collected for culture (supplemental info).</li>
	</ol>
	</li>
	<li>Isolation of Subcutaneous Adipose Tissue (SQ), a White Adipose Tissue Depot (WAT):
	<ol>
		<li>Put on a fresh pair of gloves as to not cross-contaminate between fat depots.</li>
		<li>Reveal the inguinal, triangular SQ depots by de-gloving the bottom half of the mouse. Hold the upper appendages and thorax in one hand and pulling the skin down towards the feet with the other hand.</li>
		<li>Orient the mouse in a supine position while being careful not to contaminate the exposed depot with hair.</li>
		<li>Clean surgical instruments and change to a fresh pair of gloves.</li>
		<li>Carefully dissect the triangles of SQ fat. Be careful not to contaminate the sample with muscle, neighboring fat, mammary glands or by cutting any vessels and contaminate the sample with blood. 
		NOTE: The use of a dissection microscope is recommended if borders are not clearly defined.</li>
		<li>Remove fat depots and transfer to 2 ml microcentrifuge tubes.</li>
		<li>If RNA or protein is to be extracted, freeze the tissue by immersion in liquid nitrogen and store at -80 &#176;C. Freeze the sample at once to prevent degradation.</li>
		<li>For staining, fix or embed in OCT. If culturing, cover the tissue in DMEF-12 and set on ice till all samples are collected for culture (supplemental info).</li>
	</ol>
	</li>
	<li>Isolation of Gonadal Fat a Visceral Adipose Tissue (VAT) and White Adipose Tissue (WAT) Depot:
	<ol>
		<li>Put on a fresh pair of gloves as to not cross-contaminate between fat depots.</li>
		<li>With scissors, cut the peritoneum transversely, directly below the diaphragm. Cut the peritoneum from the diaphragm to the rectum mid coronally to expose abdominal organs.</li>
		<li>Locate the testes or ovaries and identify the attached white adipose tissue, known as the epididymal adipose in males or gonadal adipose in females.</li>
		<li>Clean surgical instruments and change to a fresh pair of gloves</li>
		<li>Carefully dissect both epididymal fat depots from the testes, epididymides, and vasa deferentia. Or if female, carefully dissect both gonadal fat pads from the ovaries.</li>
		<li>Remove fat depots and transfer them to 2 ml microcentrifuge tubes.</li>
		<li>If RNA or protein is to be extracted, freeze the tissue by immersion in liquid nitrogen and store at -80 &#176;C. Freeze the sample at once to prevent degradation.</li>
		<li>For staining, fix or embed in OCT. If culturing, cover the tissue in DMEF-12 and set on ice till all samples are collected for culture (supplemental info).</li>
	</ol>
	</li>
</ol>
3. Isolation of Perivascular Adipose Tissue (PVAT)
<ol>
	<li>Isolation of the Heart:
	<ol>
		<li>Lay the mouse out in a supine position with upper and lower appendages extended outward.</li>
		<li>Secure appendages using surgical tape.</li>
		<li>After positioning the mouse as listed above, create tension by lifting up on the xiphoid process with forceps. Cut horizontally through the diaphragm, exposing the lower portion of the thoracic cavity.</li>
		<li>While maintaining tension, by lifting up on the xiphoid process, cut through the ribcage superiorly towards the head, just to the side of the sternum.</li>
		<li>Pick up the rib cage just inferior to the clavicle, and cut along the inferior length of the clavicle out toward the axilla in both directions. The thoracic cavity and its contents (heart, lungs, etc.) should now be clearly visible.</li>
		<li>Clean the thoracic cavity of extraneous blood and fluid by using sterile gauze to absorb the fluid. If collecting organs or vessels be sure to profuse (supplemental info).</li>
		<li>Once the area is cleared of fluid, cut the bronchi and attaching vessels to remove the lungs, allowing better exposure of the heart.</li>
	</ol>
	</li>
	<li>Localization and Excision of Aortic Perivascular Adipose Tissue (PVAT):
	<ol>
		<li>Remove the following organs as to better identify the inferior portions of the aorta: liver, stomach, spleen, pancreas, intestines, and colon.</li>
		<li>First start with identification of the stomach and the esophagus. Cut the esophagus at the gastro-esophageal junction to free the stomach from the body.</li>
		<li>Next, identify the intestines and the surrounding mesentery. Cut superficially through mesentery, as it lies very closely to the renal portion of the aorta, then &#8220;run the bowel.&#8221;</li>
		<li>Cut the colon free as close to the rectum as possible. Thus, freeing the stomach, intestine and colon from the mouse.</li>
		<li>Remove the stomach, intestine, colon, pancreas and spleen by cutting through the attaching mesentery and vessels. The pancreas and spleen should come free with the stomach, intestine and colon.</li>
		<li>Remove the liver by cutting through the hepatic veins and attaching mesentery, remove all lobes.</li>
		<li>Cut away the visceral layer and fat surround the kidneys. Leave the kidneys attached to the aorta in vivo to serve as geographic markers for different segments of the aorta.</li>
		<li>Rinse the area with sterile 1x PBS and remove all fluid by absorption with a sterile gauze.</li>
		<li>Using micro-scissors and micro-forceps, separate the aorta from its dorsal attachment to the spine and its ventral attachment to the esophagus.</li>
		<li>Isolate the aorta by following and detaching the aorta the length of the descending aorta from the origin in the heart to the bifurcation in the iliac region.</li>
		<li>Identify and isolate the subclavian vessels. Isolate these vessels from the neck to the aortic root to better expose the aortic junction and root in the heart.</li>
		<li>Remove the thymus then cut the brachiocephalic artery, the left common carotid artery, and the left subclavian artery, allowing movement of the heart.</li>
		<li>With the assistance of a dissection microscope, view the perivascular adipose tissue (PVAT) layer surrounding the aorta.</li>
		<li>Taking great care to not pinch or squeeze the PVAT, gently pull the PVAT way from the aorta with micro-forceps. Gently cut the attachment of the PVAT to the aorta with micro-scissors starting at the thoracic region just superior to where the diaphragm is located. 
		NOTE: A dissection microscope is recommended.</li>
		<li>Repeat this process the entire length of the aorta, finishing at the infrarenal aortic region, which is located just superior to the iliac bifurcation of the aortic vessel.</li>
		<li>If aortic arch PVAT is desired, used the same method to remove the PVAT from the lesser curvature of the arch.</li>
		<li>Place PVAT samples into 2 ml microcentrifuge tube(s).</li>
		<li>If RNA or protein is to be extracted, freeze the tissue by immersion in liquid nitrogen and store at -80 &#176;C. Freeze the sample at once to prevent degradation. If culturing, cover the tissue in DMEF-12 and set on ice till all samples are collected for culture (supplemental info). 
		NOTE: Additional adipose depots to consider if interested in a comprehensive adipose depot analysis include: retroperitoneal, mesenteric, omental, pericardial and popliteal.</li>
	</ol>
	</li>
</ol>

<ol>
	<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. 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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. 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The authors declare that they have no competing financial interests or other conflicts of interest.

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.

<table border="0" cellpadding="0" cellspacing="0" width="730">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane</td>
			<td>Med-Vet International&#160;</td>
			<td>#RXISO-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
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</table>

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 ...

localization-identification-and-excision-of-murine-adipose-depots

Research

JoVE Journal

Medicine

40.9K Views.  University of Cincinnati College of Medicine. 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.

Video: Localization, Identification, and Excision of Murine Adipose Depots

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

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

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

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

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

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

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

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

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.

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. ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...