This work received funding from the European Union&#39;s Horizon 2020 (grant agreement no. 645710) and 7th Framework (grant agreements no. 612547 and 319010) Programmes. The&#160;authors wish to thank Dr. Fotini Sourli-Chasioti for the assistance with the biopsy procedure. The authors also thank Mr. Yiorgos Paterakis for his valuable help in the preparation of the video.

The study conformed to the standards set by the Declaration of Helsinki and was approved by the University of Thessaly, Department of Exercise Science, &#914;i&#959;ethics Committee.
1. Preparation of the Surgery Equipment and Consumables
<ol>
	<li>Position a disinfected surgical field on a 4-wheel surgical Mayo stand.</li>
	<li>Position the following consumables and surgical equipment on a 4-wheel surgical Mayo stand: 
	One pair of straight operating scissors (15 cm) 
	One scalpel (No. 11) 
	One scalpel handle 
	One pair of curved scissors (14 cm) 
	One mosquito forceps 
	One pair of Kocher tweezers 
	One surgical tweezer 
	Povidone-iodine (on a sterile gauze) 
	One suture (4.0) 
	One pair of straight scissors (11 cm) 
	One needle holder (15 cm) 
	Five sterile gauzes 
	One adhesive sterile gauze 
	One 10-mL syringe with a disinfected needle 
	10mL of 2% xylocaine (no adrenaline) 
	One pair of disinfected surgical gloves</li>
	<li>Position the following materials on a separate bench: 
	Two microcentrifuge tubes 
	One tube containing 5 mL of 10% formalin 
	One small container containing 50-100 mL of liquid nitrogen at -190 &#176;C.</li>
</ol>
2. Preparation of the Participants
<ol>
	<li>Have participants follow an 8 h fast15 prior to the biopsy procedure.</li>
	<li>Have participants refrain from physical exercise, excessive stressors, alcohol, and active and passive smoking 72 h prior the biopsy16,17.</li>
</ol>
3. Performing the Subcutaneous Adipose Tissue Biopsy
<ol>
	<li>Position the participant on the surgical bed in a supine position.</li>
	<li>Disinfect the region of the incision using povidone-iodine.</li>
	<li>Inject 10 mL of 2% xylocaine (no adrenaline) slowly and steadily18 into the selected region to serve as local anesthesia.</li>
	<li>Position a sterile surgical field over the area to cover the body of the participant, excluding the region of the incision.</li>
	<li>At least 3-5 min afterwards, apply pressure with a pair of tweezers to the area of local anesthesia and ask the participant if he feels any pain19 to ensure that local anesthesia has been achieved.</li>
	<li>Attach the No. 11 scalpel to the scalpel handle.</li>
	<li>Create an incision of 2-2.5 cm in length, 3-5 cm to the left or right of the navel.</li>
	<li>Remove the subcutaneous tissue with a pair of straight 15 cm operating scissors.</li>
	<li>Once the adipose tissue is revealed, capture it with a pair of tweezers and a pair of curved 14 cm scissors.</li>
	<li>Cut and remove approximately 1 g of adipose tissue.</li>
	<li>Following tissue removal, position sterile gauzes over the incision to achieve hemostasis.</li>
	<li>When hemostasis is confirmed, capture the 4.0 suture with a 14 cm needle and the skin with a pair of surgical tweezers until the subcutaneous tissue is visible.</li>
	<li>Insert the suture-needle into the skin, moving towards the subcutaneous tissue while the skin and subcutaneous are closely aligned.</li>
	<li>With the suture-needle, follow a continuous zig-zag technique into the subcutaneous tissue until driven externally off the skin. Continue until the skin incision is closed.</li>
	<li>Externally tie the suture.</li>
	<li>Clean the area of incision with saline and cover it with an adhesive sterile gauze.</li>
</ol>
4. Deposition of the Tissue
<ol>
	<li>Separate the collected adipose tissue into three pieces: 150, 350, and 500 mg.</li>
	<li>Position the 150 and 350 mg pieces of sample tissue in microcentrifuge tubes.</li>
	<li>Immerse the two microcentrifuge tubes in the small container with the liquid nitrogen at -190 &#176;C until final deposition in a freezer at -80 &#176;C. 
	NOTE: In advance, use a sterile needle to puncture a small hole in the microcentrifuge tubes to avoid cup failure during the immersion of the microcentrifuge tubes in the liquid nitrogen at -190 &#176;C.</li>
	<li>Immerse the 500 mg piece of sample tissue-to be used for histological analysis-in a tube containing 5 mL of 10% formalin. 
	NOTE: Use the mosquito forceps only in cases of excessive bleeding during the surgery. In such cases, capture the vessel with the mosquito forceps to ligate and close until hemostasis is achieved.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sampling+and+long-term+storage+of+subcutaneous+adipose-tissue+biopsies+for+determination+of+fatty+acid+composition.">Rapid sampling and long-term storage of subcutaneous adipose-tissue biopsies for determination of fatty acid composition.</a> Am J Clin Nutr. 42, 317-322 (1985).</li><li>Wang, 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=Muscle+and+adipose+tissue+biopsy+in+older+adults+with+type+2+diabetes+.">Muscle and adipose tissue biopsy in older adults with type 2 diabetes .</a> J Diabetes Mellitus. 1, 27-35 (2011).</li><li>Mutch, D. 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=Needle+and+surgical+biopsy+techniques+differentially+affect+adipose+tissue+gene+expression+profiles.">Needle and surgical biopsy techniques differentially affect adipose tissue gene expression profiles.</a> Am J Clin Nutr. 89, 51-57 (2009).</li><li>Ikeda, S., Sekijima, Y., Tojo, K., Koyama, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value+of+abdominal+wall+fat+pad+biopsy+in+senile+systemic+amyloidosis.">Diagnostic value of abdominal wall fat pad biopsy in senile systemic amyloidosis.</a> Amyloid. 18, 211-215 (2011).</li><li>Alderete, T. 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=A+novel+biopsy+method+to+increase+yield+of+subcutaneous+abdominal+adipose+tissue.">A novel biopsy method to increase yield of subcutaneous abdominal adipose tissue.</a> Int J Obes. 39, 183-186 (2015).</li><li>Padoin, A. V., 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+comparison+of+wedge+and+needle+hepatic+biopsy+in+open+bariatric+surgery.">A comparison of wedge and needle hepatic biopsy in open bariatric surgery.</a> Obes Surg. 16, 178-182 (2006).</li><li>Kral, J. 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The authors declare that they have no competing financial interests.

The aim of this study was to standardize a surgical technique for subcutaneous abdominal adipose tissue biopsy, performed under local anesthesia with a non-diathermy method in healthy men. Diathermy is commonly used in the operating room; however, possible side effects (i.e., flash fires and environmental toxicity)9 and delays in healing10 may occur. These unwanted effects of diathermy can generate health risks for both the operating staff and the patients/participants. Therefore, a surgical non-diathermy method for subcutaneous adipose tissue biopsy is warranted.
We observed excessive bleeding in three of our participants two days after the surgery. These three individuals reported pain in the area of the incision. To treat these cases, the area of the skin incision was reopened, any excess blood was removed, and the area was cleaned with saline and closed using the same technique described above. No complications were reported after that, and normal healing was observed in the subsequent days. Our results have shown minimal side effects after the non-diathermy method: three cases of excessive post-operation bleeding out of 115 operations (2.61%). Previous evidence from needle-based adipose tissue biopsies showed minor bleeding in one out of 10 participants 12 h after the surgery15, while no significant bleeding occurred in healthy obese individuals after a side-cutting adipose tissue biopsy needle technique6. Based on our results, we anticipate that a non-diathermy surgical adipose tissue biopsy method may eliminate the risk for skin lesion, flash fires, and toxicity released in the operating environment, which are caused by diathermy during surgical adipose tissue biopsy.
Previous reports have indicated that aging is positively associated with abnormalities in hemostasis20. Interestingly, the three individuals who experienced excessive bleeding in the current study were significantly older (by eight years, on average) than those who did not experience any side effects. Therefore, it may be noted that older individuals have a higher risk for side effects when the non-diathermy surgical adipose tissue biopsy method is applied. However, we believe that the prevalence of such side effects is too low (i.e., 2.61%) to cause any significant concerns. It should be noted that our participants filled in advance a medical history questionnaire to determine their health status. No previous chronic disease conditions were reported by the participants, including the three participants who showed excessive post-operation bleeding. Similarly, no previous chronic medication treatment was reported by both the participants who showed and those who did not show any excessive bleeding. Therefore, the post-operation excessive bleeding we observed in the current study cannot be attributed to previous chronic disease conditions and/or previous chronic medication treatment. In this light, the prevalence of this side effect may be different if the technique is applied to clinical populations.
A critical step during the proposed surgical adipose tissue biopsy is to ensure local anesthesia before the incision, which prevents any pain and discomfort in the participant. Furthermore, a pre-screening of allergic history to the anesthetic must be done to prevent allergic complications during the anesthetic injection. Also, it is crucial to ensure hemostasis before the incision is closed to prevent post-operation bleeding, while it is important to keep in contact with the participants for several days after the procedure is performed until complete healing is achieved to discover any potential abnormalities and to prevent infection and/or cases of post-operation bleeding.
Limitations of the proposed biopsy technique include the discomfort that area of the incision may cause to the participants, particularly relating to their ability to comfortably sit down and take a shower/bath, until complete healing is achieved. The participants should be instructed to avoid washing the area of the incision to prevent infection, as well as to replace the adhesive sterile gauze over the incision area every day until complete healing is achieved. This daily procedure may cause discomfort. Finally, it is important to note that our purpose was to introduce and standardize a new technique. In this light, we did not use a control group undertaking needle-based subcutaneous fat biopsies in order to quantify the differences between this technique and the newly-introduced non-diathermy method.
As previously suggested4,7,8, the proposed surgical biopsy technique may offer tissue samples with higher integrity than the needle-based technique. Indeed, using this technique, we were able to perform mRNA and protein analyses on the obtained tissue and, in addition, to form paraffin cubes for use in histological analyses. Also, we suggest avoiding diathermy in surgical subcutaneous adipose tissue biopsies, given that hemostasis and normal healing can be achieved with our technique.
In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely performed in healthy men at the bedside, with minimal side effects.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide an insightful overview of mechanisms relating to metabolism and disease. For instance, they are used for breast cancer tumor identification1, fatty acid composition examination2, and research on important metabolic and hormonal disease pathways3. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of complex biological mechanisms [e.g., messenger ribonucleic acid (mRNA) expression, histological analysis, etc.]4. They have previously been used in healthy adults4 and can be safely performed at the bedside5.
The needle-based biopsy technique requires intense liposuction that may destroy the integrity of the tissue, and it usually retrieves a small amount of tissue (100-500 mg)6. These tissue samples may not be appropriate for complex research studies. Furthermore, compared to the needle-based technique, surgical subcutaneous fat biopsies can offer tissue samples that may draw a more complete picture of several inflammatory indices (e.g., B- and T-cell signaling); cytoskeleton regulation; cellular pathways; metabolic pathways of lipids, carbohydrates and amino acids; and oxidative phosphorylation pathways4. Also, the needle-based technique includes a higher risk for blood cell contamination than the surgical one4, while providing little information about the fibrotic regions of the tissue4,7,8.
Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding9. A diathermy is also used to burn and destroy tissues affected by neoplasms and warts or tissues that are infected by disease9. This technique is particularly applicable in neurosurgery and eye surgery9.
The excessive use of diathermy may lead to the formation of necrotic tissue, causing infections and delaying the healing process10. Indeed, side effects such as flash fires and skin lesions in the tissue have been reported following diathermy9. Additionally, concerns have been raised about the toxicity of surgical smoke released by diathermy into the surgery environment, which may be inhaled by the participant/patient and the operating staff9. Indeed, the use of diathermy may cause air toxicity in the operating room equal to that produced by smoking six cigarettes11 and, although operating rooms typically have good ventilation systems, the contamination can last for at least 20 min12. However, in patients undertaking a tonsillectomy surgery, the diathermy method has shown less, but non-significant, post-operative pain13 and bleeding14 than the classic ligation hemostasis method. Overall, evidence regarding differences in the post-operative pain and bleeding rates after adipose tissue biopsies in healthy individuals barely exists. Given the lack of evidence and the side effects of diathermy, standardization of a non-diathermy method for adipose tissue biopsy is warranted. Accordingly, the aim of this study was to standardize a surgical technique for subcutaneous abdominal adipose tissue biopsy, performed under local anesthesia using a non-diathermy method in healthy men. Moreover, we outline the procedures for tissue collection to determine mRNA expression and protein concentration and to perform histological analyses.

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">&#160;Disinfected surgical field&#160;</td>
			<td style="width:183px;">Hartmann</td>
			<td style="width:175px;">23516-00</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Instrument Stands (surgical Mayo)</td>
			<td>Medical Device Depot Inc.</td>
			<td>32000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Operating Scissors straight 15 cm</td>
			<td>Karl Storz</td>
			<td>791903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel No 11&#160;</td>
			<td>Swann-Morton</td>
			<td>203</td>
			<td>Bipolar, catalog number 429011</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scarpel handle No 3</td>
			<td>Karl Storz</td>
			<td>488090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors curved 14 cm</td>
			<td>Karl Storz</td>
			<td>511514, 752918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mosquito forceps</td>
			<td>Karl Storz</td>
			<td>535012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezers Kocher&#160;</td>
			<td>Karl Storz</td>
			<td>530416</td>
			<td>Bipolar, catalog number 830316</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical tweezers</td>
			<td>Karl Storz</td>
			<td>793216</td>
			<td>Bipolar, catalog number 831016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-iodine&#160;</td>
			<td>Various</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture 4.0 Vicryl</td>
			<td>Johnsons</td>
			<td>V4970H&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors straight 11 cm</td>
			<td>Karl Storz</td>
			<td>512511 DS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holder 15 cm</td>
			<td>Karl Storz</td>
			<td>515515</td>
			<td>Bipolar, catalog number 213015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Sterile gauzes</td>
			<td>Hartmann</td>
			<td>3021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#913;dhesive sterile gauze&#160;</td>
			<td>Digas medical equipment</td>
			<td>2889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL syringe with disinfected needle</td>
			<td>Alpha medical solutions</td>
			<td>10310-55-0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2%-xylocaine (no adrenaline)</td>
			<td>Various</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical gloves&#160;</td>
			<td>Sempermed</td>
			<td>10001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf tubes</td>
			<td>Kartell</td>
			<td>16283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% formalin&#160;</td>
			<td>Alpha medical solutions</td>
			<td>11016-30-2909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubes 120 mL</td>
			<td>Digas medical equipment</td>
			<td>9025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid nitrogen at -190&#176;C</td>
			<td>Revival</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique for subcutaneous abdominal adipose tissue biopsy via a non-diathermy method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

The characteristics of the participants are provided in Table 1. The stitches in the area of the incision fell off within the next 8-12 days. We completed a total of 115 subcutaneous adipose tissue biopsies using the non-diathermy abdominal surgical biopsy method in healthy adult men (age: 35.2 &#177; 6.9 years; body mass index: 27.3 &#177; 4.4; and body fat percentage: 27.0 &#177; 9.0%). Post-biopsy excessive bleeding was observed in three cases (prevalence: 2.61%) two days after the surgery. The three individuals who experienced excessive bleeding were older than their counterparts who experienced no side effects [t(113)= -2.115, p = 0.037], and no other differences (i.e., body mass index and body fat percentage) were observed (p &#62;0.05) between the two groups. Finally, three months after each operation, none of our participants reported any side effects (including skin wounds).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55593/55593tbl1.jpg" /> 
Table 1: Characteristics of the participants.

Watch this Scientific Journal Video about A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method at JoVE.com

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

14.9K Views.  University of Thessaly. We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Video: A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

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 ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

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 ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta.
The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum. After laparotomy with a ventral midline abdominal incision, exfoliation of the aorta is performed, which blocks blood flow from the perivascular tissue. Aortotomy involving a small incision adjacent to the renal artery branches is performed, and a polyurethane catheter is inserted using an 18-gauge indwelling needle. After repairing the incision, tight ligation of the aorta over the catheter blocks VV blood flow from the proximal direction through the aortic wall without disturbing the aortic blood flow. This technique can induce an AAA with progressive aortic dilatation.
The greatest benefit of this model is that VV hypoperfusion causes tissue hypoxia and the development of an infrarenal AAA, which has morphological and pathological characteristics similar to those of a human AAA.

Polyurethane catheter insertion into the aortic lumen and suture ligation of the aorta induce chronic hypoxia due to hypoperfusion of the adventitial vasa vasorum. This article describes a novel animal model of abdominal aortic aneurysm (AAA) with characteristics similar to those of AAA in humans.

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic ...

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 ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...