The authors would like to thank Adriane Mosley and Libuse Jerabek (Stanford) for assistance with the surgical technique.

All animal studies were performed according to the guidelines of UCLA&#39;s animal care and use committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The duration of the procedure described below is approximately 45-60 min from beginning to end.
1. Preparation of Surgical Field
<ol>
	<li>Perform procedure in a clean animal surgery room.</li>
	<li>Equipment: isoflurane Vaporizer, Gaymer T Pump with heating pad.</li>
	<li>Sterile tools: two curved forceps, fine scissors, needle holder.</li>
	<li>Sterile gloves must be used during the entire procedure.</li>
</ol>
2. Preparation of Animals
<ol>
	<li>Place two male or female mice, from same genetic background, of similar weight and size in the same cage and monitor for at least two weeks to ensure harmonious cohabitation. Female mice are preferred due to their less aggressive behavior.</li>
	<li>Anesthetize animals by using an isoflurane vaporizer. Place mice in a Posi-Seal Induction Chamber connected to the isoflurane vaporizer (4-5% v/v). Once anesthesia is induced, transfer the animal to the fur shaving area and maintain the anesthesia throughout the procedure through a nose cone connected to isoflurane (1.5-2% v/v). Apply ophthalmic ointment with a Q-tip to prevent dry eyes.</li>
	<li>Place the animal on the supine position. Thoroughly shave the left side of the mouse placed on the left and the right side of the mouse placed on the right starting at approximately 1 cm above the elbow to 1 cm below the knee.</li>
	<li>Aseptically prepare the shaved areas by thoroughly wiping (2-3x) with Betadine-soaked wipes followed by alcohol wipes. Place the mice on a heated pad covered by a sterile pad.</li>
	<li>For analgesia, administer Carprofen and Buprenorphine intraperitoneally or subcutaneously at a dose of 10 mg/kg and 0.1 mg/kg respectively.</li>
	<li>Place animals on their side, back to back, with adjacent shaved areas facing up. To avoid any contamination of the surgical area, cover the mice with a sterile drape exposing only the operation area. Create a small drape opening to stay sterile when performing the surgery. We made the drape window large to have a better viewing during videotaping.</li>
</ol>
3. Parabiosis
<ol>
	<li>Using a sharp scissor, perform longitudinal skin incisions to the shaved sides of each animal starting at 0.5 cm above the elbow all the way to 0.5 cm below the knee joint (Figure 1). Following the incision, gently detach the skin from the subcutaneous fascia by holding the skin up with a pair of curved forceps and separate the fascia with a second pair to create 0.5 cm of free skin. Perform this separation along the entire incision.</li>
	<li>Begin the joining by attaching the left olecranon of one animal to the right olecranon of the other. Both olecranons and knee joints are clearly distinguishable following the skin incision. To facilitate the joining, bend the elbow of the first mouse and pass the needle of the non-absorbable 3-0 suture under the olecranon. Similarly, bend the elbow of the second mouse and pass the same suture under it. Attach joints tightly by a double surgical knot.</li>
	<li>Connect the knee joints following the same procedure.</li>
	<li>Following the attachment of the joints, connect the skin of the two animals with a continuous absorbable 5-0 Vicryl suture starting ventrally from the elbow towards the knee. To prevent skin rupture and separation perform a tight suture closure of the skin in the area around the elbows and knees. Once the ventral skin attachment has been completed, perform a double surgical knot. Place the mice in the prone position and continue the suture dorsally ending with a double surgical knot. Verify the continuity of the suture and confirm the lack of openings.</li>
	<li>Administer 0.5 ml of 0.9% NaCl subcutaneously to each mouse to prevent dehydration.</li>
</ol>
4. Postoperative Recovery
<ol>
	<li>Keep animals on heated pad until recovery.</li>
	<li>Following recovery, provide analgesics carprofen and buprenorphine. Repeat intraperitoneally or subcutaneously every 24 and 12 hr, respectively, for 48 hr at the same doses described above (step 2.5). Monitor animals for signs of pain and distress such as shaking, lethargy, chewing of tail, arched back, lack of grooming, etc. daily for two weeks.</li>
	<li>Prophylactically, treat mice with Sulfamethoxazole /Trimethoprim oral suspension in their water bottle 2 mg sulfa/ml +0.4 mg trim/ml for 10 days to prevent bacterial infections.</li>
	<li>House each parabiotic pair in a clean cage with monolithic bedding material (e.g. paper towel or absorbent sterile pad) to prevent aspiration of bedding material. Return the animals to a bedding filled cage when they are able to maintain sternal recumbency with their head up. To minimize the strain of reaching for food while adjusting to parabiotic existence, place the moistened food pellets on the cage floor. Provide nesting material. In 1-2 weeks parabiotic mice have the ability to ambulate normally on surgically paired fore-and hind-limbs.</li>
	<li>Blood chimerism occurs 10-14 days following the surgery.</li>
</ol>
5. Confirmation and Reverse Procedure
<ol>
	<li>After two weeks draw blood from the tail veins of each mouse for flow-cytometric analysis to confirm blood chimerism (Figure 3).</li>
	<li>Blood processing for flow cytometric analysis: dilute 2-3 drops of blood in 500 &#956;l of 10 mM EDTA. Add 500 &#956;l of 2% dextran (diluted in PBS), mix thoroughly and incubate at 37 &#176;C for 30 min. Transfer the supernatant in a new tube, spin at 1,200 rpm for 5 min and resuspend the pelleted cells in PBS (or other buffer) for flow cytometric analysis.</li>
	<li>Depending on experimental design, the parabiotic pair can be separated at later time points. In our experience, we have maintained parabiosed pairs for up to 9 months without any complications.</li>
	<li>Perform reverse procedure on a sterile surgical surface. Anesthetize mice by placing them in an induction chamber connected to an isoflurane vaporizer (4-5%) and maintain anesthesia with 1.5-2% isoflurane, as described above (step 2.2).</li>
	<li>Remove hair from the area surrounding the initial suture and aseptically prepare the shaved areas as described above (steps 2.3, 2.4).</li>
	<li>For analgesia, administer Carprofen and Buprenorphine intraperitoneally or subcutaneously at a dose of 10 mg/kg and 0.1 mg/kg, respectively.</li>
	<li>Cover surgical area with a sterile drape as described above (step 2.6).</li>
	<li>Using sharp scissors, separate the mice through a longitudinal incision along the lateral suture and gently detach the newly formed fascia between the two mice with a pair of curved forceps. To separate the joints, cut the knots of the suture connecting them. Trim off the skin along the incision to achieve smooth edges and reattach skin with an absorbable continuous 5-0 coated Vicryl suture.</li>
	<li>To prevent dehydration, administer 0.5 ml of 0.9% NaCl subcutaneously to each mouse. Keep animals on heated pad until recovery.</li>
	<li>After recovery, repeat analgesic administration, carprofen (10 mg/kg) every 24 hr and buprenorphine (0.1 mg/kg) every 12 hr for 48 hr.</li>
	<li>To prevent bacterial infections treat mice with Sulfamethoxazole /Trimethoprim oral suspension (2 mg sulfa/ml +0.4 mg trim/ml) in their water bottle for 10 days.</li>
</ol>

<ol>
	<li>Bert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exp&#233;riences+et+consid&#233;rations+sur+la+greffe+animale.">Exp&#233;riences et consid&#233;rations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 .</li><li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+of+parabiosis.">Improved methods of parabiosis.</a> Anat. Rec. 57, 339-380 (1933).</li><li>Waskow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation.">Generation of parabiotic mice for the study of DC and DC precursor circulation.</a> Methods Mol. Biol. 595, 413-428 (2010).</li><li>Finerty, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+in+physiological+studies.">Parabiosis in physiological studies.</a> Physiol. Rev. 32, 277-302 (1952).</li><li>Aicher, A., Heeschen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow-derived+endothelial+progenitor+cells:+what+is+their+exact+location.">Nonbone marrow-derived endothelial progenitor cells: what is their exact location.</a> Circ. Res. 101, e102 (2007).</li><li>Abe, S., Boyer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury.">Cells derived from the circulation contribute to the repair of lung injury.</a> Am. J. Respir. Crit. Care Med. 170, 1158-1163 (2004).</li><li>Donskoy, E., Goldschneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice.">Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.</a> J. Immunol. 148, 1604-1612 (1992).</li><li>Powell, A. E., Anderson, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming.">Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.</a> Cancer Res. 71, 1497-1505 (2011).</li><li>Duyverman, A. M., Kohno, 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=A+transient+parabiosis+skin+transplantation+model+in+mice.">A transient parabiosis skin transplantation model in mice.</a> Nat. Protoc. 7, 763-770 (2012).</li><li>Ajami, B., Bennett, J. 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=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat. Neurosci. 10, 1538-1543 (2007).</li><li>Weissman, I. L., Jerabek, 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=Tolerance+and+the+H-Y+antigen:+Requirement+for+male+T+cells,+but+not+B+cells,+to+induce+tolerance+in+neonatal+female+mice.">Tolerance and the H-Y antigen: Requirement for male T cells, but not B cells, to induce tolerance in neonatal female mice.</a> Transplantation. 37, 3-6 (1984).</li></ol>

The authors have no competing financial interests.

The parabiosis method discussed here presents minimal technical difficulties and results in low mortality rates. The attachment of knee and elbow joints is a significant improvement of the Bunster and Meyer technique. However, the procedure remains invasive thus maintenance of sterile conditions throughout the surgery is imperative. To further prevent infection of the surgical site, it is important that the parabiosed animals receive a combination of antibiotics and be monitored regularly. To ensure firm support and prevent pain, the suture connecting the elbows and knees should surround the joints rather than passing through the tissue.
As an alternative to isoflurane inhalation, anesthesia may be induced with the use of intraperitoneal injection of ketamine/xylazine or other anesthetic drugs approved by institutional committees. The advantage of using isoflurane is that it can rapidly induce anesthesia and significantly reduce the recovery time. Furthermore, the level of anesthesia can be precisely controlled.
Parabiosis, which allows circulatory systems from two animals to commingle and equilibrate, is a powerful experimental procedure for physiological studies. It presents several advantages to other commonly used techniques such as bone marrow transplantation or cell injection. Cell delivery procedures, at times, provide a short window to examine the effect of transplanted cells. In addition, immunosuppression increases the risk of infection and subsequent morbidity and mortality. However, with parabiosis one can maintain a chimeric circulation for long periods of time and study circulating factors (cells, cytokines, etc.) independent of their origin. This system can be used to determine the role of circulating cells in wound healing, tumor formation, aging, regeneration, and inflammatory response, among many others.

Parabiosis, the surgical joining of two organisms, was first described in 1864 by Paul Bert as a way to develop a model to study shared circulatory systems and consisted of the joining of the skin and muscular walls of two rats1. Parabiosis promotes formation of microvasculature at the site of inflammation3 and has had several applications in physiological studies, such as the hormonal communication between the pituitary gland and gonads as well as the role of the kidney in hypertension4. It has been further employed to investigate the recruitment and integration of progenitor cells in neovascularization5, migration of hematopoietic stem cells6, and lymphocyte trafficking7, as well as the role and kinetics of circulating inflammatory or stem cells in tumor metastasis8,9, and neurodegenerative disease10.
One significant advantage of parabiosis lies in that the partnered animals share common circulating antigens, allowing cell migration and neovascularization without triggering an immunological reaction. Importantly, Weissman et al. have shown that parabiosis between male and female mice does not lead to formation of anti H-Y antibodies11.
In the original protocol described by Paul Bert the two animals were joined together through connection of the skin and muscle walls1. This method however, caused significant strain to the animals and resulted in high mortality due to infection of the wound. Since then the parabiosis technique has been revised by several groups with the most predominant being the protocol proposed by Bunster and Meyer in 19332. Their method included joining of the scapula joints, body cavities, and skin, permitting better support and less pain for the animals. At the same time, the new method resulted in minimal post-operative care and significantly decreased mortality rates. The protocol described herein is a modification of the Bunster and Meyer technique that is less invasive and allows firmer joining. Namely, mice are connected through the elbow and knee joints as well as the skin. This joining prevents extension of the skin and therefore causes less pain and complications. Here we describe the joining of a wild type (WT) adult mouse to a constitutive GFP expressing mouse. We show that two weeks following surgery we can achieve 50% of blood chimerism demonstrating the efficacy of this surgical procedure to create a shared circulatory system.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Isoflurane-Phoenix</td>
			<td>Clipper</td>
			<td>NDC 57319-559-06</td>
		</tr>
		<tr>
			<td>Posi-Seal Induction Chamber</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0530-SM</td>
		</tr>
		<tr>
			<td>Portable Anesthesia System</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0007</td>
		</tr>
		<tr>
			<td>Gaymer T Pump</td>
			<td>Gaymar Industries, Inc</td>
			<td>TP650</td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp</td>
			<td>TP-22G</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Roboz</td>
			<td>RS-5101</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>FST 14063-09</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>FST 12501-13</td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Oster</td>
			<td>Golden A5</td>
		</tr>
	</tbody>
</table>

parabiosis in mice: a detailed protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

The anticipated outcome of parabiosis of two organisms is the equal contribution of each animal's circulatory system to a common blood circulation (Figure 2). One can easily verify the successful equilibration of the blood of the parabiosed WT and GFP positive mice by flow cytometric analysis. Here, venous blood was obtained from the tails of both parabionts at 2 weeks after the surgery and was fractionated for peripheral blood cells (to exclude erythrocytes). The fractionated hematopoietic-derived cell fraction was subsequently analyzed by flow cytometry for the presence of GFP positive and WT cells. Consistent with previous studies, blood from the WT mouse revealed presence of chimerism, as indicated by approximately half GFP positive and half WT blood cells (53% WT cells and 47% GFP cells) (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/50556/50556fig1.jpg" /> 
Figure 1. Parabiosis surgery: Incision site. The lateral sides of anesthetized animals are shaved and longitudinal skin incisions are performed (doted area) from 0.5 cm above the elbow to 0.5 cm below the knee joint.
<img alt="Figure 2" src="/files/ftp_upload/50556/50556fig2.jpg" /> 
Figure 2. Schematic representation of the parabiosis method. A) Joining of a WT mouse (left) to a GFP positive mouse (right). B) Approximately two weeks following surgical attachment, new microvasculature is formed at the location where the two mice are connected allowing joining of the blood circulation.
<img alt="Figure 3" src="/files/ftp_upload/50556/50556fig3.jpg" /> 
Figure 3. Blood chimerism of parabiotic partners. Flow cytometric analysis of blood sample collected from the WT parabiotic partner two weeks following surgical joining.

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Parabiosis in Mice: A Detailed Protocol

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

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73.9K Views.  University of California, Los Angeles. Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Video: Parabiosis in Mice: A Detailed Protocol

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

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 ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

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 ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...