Name: a technique for subcutaneous abdominal adipose tissue biopsy via a non-diathermy method
Uploaded: 2017-09-30T13:00:00
Description: Watch this Scientific Journal Video about A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method at JoVE.com

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...

<ol>
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E., Malik, M., Ahmed, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-blind+controlled+study+of+electrocautery+and+ultrasonic+scalpel+smoke+plumes+in+laparoscopic+surgery.">A single-blind controlled study of electrocautery and ultrasonic scalpel smoke plumes in laparoscopic surgery.</a> Surg Endosc. 26, 337-342 (2012).</li><li>Memon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Diathermy.">Surgical Diathermy.</a> Br J Hosp Med. 52, (1994).</li><li>Tomita, 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=Mutagenecity+of+smoke+condensates+induced+by+CO-laser+irradiation+and+electrocauterization.">Mutagenecity of smoke condensates induced by CO-laser irradiation and electrocauterization.</a> Mutat Res. 89, 145-149 (1989).</li><li>Brandon, H., Young, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+and+removal+of+electrosurgical+smoke.">Characterisation and removal of electrosurgical smoke.</a> Surg Serv Manag. 3, 14-16 (1997).</li><li>Attner, P., Hemlin, C., Soderman, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligasure+versus+diathermy+scissors+tonsillectomy:+A+controlled+randomized+study.">Ligasure versus diathermy scissors tonsillectomy: A controlled randomized study.</a> Acta Otolaryngol. 130, 1180-1184 (2010).</li><li>Ecker, T., Carvalho, A. L., Choe, J. H., Walosek, G., Preuss, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemostasis+in+thyroid+surgery:+harmonic+scalpel+versus+other+techniques--a+meta-analysis.">Hemostasis in thyroid surgery: harmonic scalpel versus other techniques--a meta-analysis.</a> Otolaryngol Head Neck Surg. 143, 17-25 (2010).</li><li>Campbell, K. 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+pilot+study+of+sampling+subcutaneous+adipose+tissue+to+examine+biomarkers+of+cancer+risk.">A pilot study of sampling subcutaneous adipose tissue to examine biomarkers of cancer risk.</a> Cancer Prev Res. 2, 37-42 (2009).</li><li>Jamurtas, A. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+a+single+bout+of+exercise+on+resting+energy+expenditure+and+respiratory+exchange+ratio.">The effects of a single bout of exercise on resting energy expenditure and respiratory exchange ratio.</a> Eur J Appl Physiol. 92, 393-398 (2004).</li><li>Adriaens, P. E. M., Schoffelen, F. M. P., Westerterp, K. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltrative+anesthesia+in+office+practice.">Infiltrative anesthesia in office practice.</a> Am Fam Phys. 89, 956-962 (2014).</li><li>Jurevic, R., 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=Plasma+levels+of+2%+lidocaine+with+1:100,000+epinephrine+with+young+children+undergoing+dental+procedures.">Plasma levels of 2% lidocaine with 1:100,000 epinephrine with young children undergoing dental procedures.</a> Anesth Prog. 45, 87-90 (1998).</li><li>Kale, S. 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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/particip...

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 d...

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 ...

Subcutaneous adipose tissue biopsies are very important for research and health because they can reveal mechanisms of adipose tissue related to the disease. In this paper we describe a technique for obtaining subcutaneous adipose tissue biopsies in the abdominal area. To obtain such samples, researchers and physicians use either a needle-based technique or a surgical-based technique.However, it is important to note that the surgical technique provides a more complete overview of the biological indices in white adipose tissue compared to the needle-based technique, which may lead to misdiagnosis. This is true, for example, for histological analysis, which is vital for both research and health purposes. Overall, it is important to note that the surgical technique has been performed in both men and women and can be safely performed in the bedside.The participant undertaking a surgical fat biopsy must undergo an eight hour fast and refrain from exercise, excessive stressors, alcohol, as well as active and passive smoking in the 72 hours prior to the biopsy procedure. These precautions are meant to minimize the risk for misleading results when the white adipose tissue sample is analyzed. We first prepare the materials that are needed for the adipose tissue biopsy procedure.On a wheeled surgical Mayo we position a sterile surgical sheet. The materials are placed on Mayo and are the following. One pair of operating scissors, straight, 15 centimeters.One scalpel, number 11. One scalpel handle. One pair of scissors, curved, 14 centimeters.One Mosquito forceps. One pair of tweezers Kocher. One surgical tweezers.Povidone iodine on a sterile gauze. One suture, 4.0. One pair of scissors, straight, 11 centimeters.One needle holder, 15 centimeters. Five sterile gauzes. One adhesive sterile gauze.One 10 milliliter syringe with a disinfected needle. 10 mL Xylocaine, 2%no adrenaline. And one pair of disinfected surgical gloves.On a separate bench we prepare the following materials that are needed for the tissue collection. Two Eppendorf tubes. Using a sterile needle, we open a small hole on the cap of each Eppendorf.This is because we want to avoid the cap failure when we immerge the Eppendorf in liquid nitrogen in minus 190 degrees Celsius. One tube in which we put five mL of formalin, 10%One small container with liquid nitrogen, in minus 190 degrees Celsius. We need this to immediately immerse the Eppendorf tubes containing the tissue to ensure tissue's integrity until final deposition.Following preparation of all materials and consumables, we position the patient on a surgical bed in a supine position. The surgeon's position is on the one side of the surgical bed. The surgeon first performs disinfection of the abdominal region using povidone iodine, 15 centimeters around the point of the incision.10 mL of Xylocaine, 2%no adrenaline, is then injected in the point of the incision for local anesthesia. A sterile surgical field is then positioned in the area. Once this is completed, we confirm that local anesthesia has been achieved.This requires approximately three to five minutes. Thereafter, the surgeon connects the scalpel number 11 with the scalpel handle and performs an incision of the skin and the subcutaneous tissue until the underlying adipose tissue is revealed. The incision is approximately two to two and a half centimeters, and it is performed three to five centimeters away from the navel.Thereafter, with a pair of operating scissors, straight, 15 centimeters, the surgeon removes the subcutaneous tissue. Once the adipose tissue is revealed, it is captured with a pair of tweezers and a pair of curved scissors, 14 centimeters. Then the adipose tissue is prepared and cut.The total amount of adipose tissue removed depends on the analysis that will be performed. Based on our experience, approximately one gram of adipose tissue will be enough for the purposes of most histological and gene expression analyses. Once the adipose tissue is removed, the surgeon places sterile gauzes on the incision area for approximately 30 seconds to ensure hemostasis.Once hemostasis is confirmed, the surgeon captures a 4-0 suture with a 14 centimeters needle and the skin with the surgical tweezers until subcutaneous tissue is visible. The suture needle enters the skin towards the subcutaneous tissue while the skin and subcutaneous are then brought together. The suture needle then follows and continues zig-zag technique into the subcutaneous tissue until being driven externally off the skin.This continues until the skin incision is closed. Finally, the suture tying is done externally. Usually the stitches fall off within eight to 12 days, and this whole technique reduces the possibility of a post-operation scar on the skin.The trauma is then cleaned with saline, and it is covered with adhesive sterile gauze. You may have noticed that we did not use the tweezers Mosquito. This instrument would be used in the case that the vessel is bleeding.If so, the vessel is captured with a tweezers Mosquito to ligate and close it until achieving hemostasis. Remember that for our purposes the sample that we collected weighed approximately one gram. The collected tissue is cut into three pieces and weighed 350 milligrams, 150 milligrams, and 500 milligrams.The 350 milligram sample is placed into an Eppendorf tube and will be used for protein levels analysis. The 150 milligram sample is placed in another Eppendorf tube and will be used for mRNA expression analysis. These two Eppendorf tubes are immediately immersed in a small container with liquid nitrogen at minus 190 degrees Celsius to ensure the integrity of the samples until final deposition in a minus 80 degrees freezer.Finally, the 500 milligram sample is immersed in a tube containing 5 mL of formalin, 10%and will be used for histological analysis. Our technique is based on a non-diathermy method. We perform hemostasis by using sterile gauzes in order to eliminate the risk for a skin lesion, such as a scar after the surgery.Overall, our non-diathermy method has insignificant side effects. Indeed, we performed in this study a total of 115 biopsies and we occurred only three excessive bleedings. These occurred in the second day following the biopsy procedure.We treated these individuals by reopening the trauma, draining, applying hemostasis, and placing surgical stapling. No further complications were observed and normal healing occurred in the following days. Finally, it's important to note that three months after each biopsy procedure for all subjects no side effects were observed, including skin scars.Our non-diathermy surgical biopsy technique showed minimal side effects, and it's proposed to be used when a complete overview of the biological indices of white adipose tissue is required.

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Medicine

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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.

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

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 ...

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 ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...