Name: Parabiosis in Mice: A Detailed Protocol (Video) | JoVE Journal
Uploaded: 2013-10-06T21:30:00.000+00:00
Duration: 7 min 56 s
Description: Learn parabiosis surgery in mice to create a shared circulatory system for neuroscience research. This protocol demonstrates surgical joining techniques and blood chimerism analysis using flow cytometry.

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5 minutes

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/pubmed?term=((Exp%C3%A9riences+et+consid%C3%A9rations+sur+la+greffe+animale%5BTitle%5D)+AND+%22Journal+de+l%27Anatomie+et+de+la+Physiologie%22%5BJournal%5D)">Expériences et considérations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 Forthcoming.</li>
<li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+methods+of+parabiosis%5BTitle%5D)+AND+%22Anat.+Rec%22%5BJournal%5D)">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/pubmed?term=((Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">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/pubmed?term=((Parabiosis+in+physiological+studies%5BTitle%5D)+AND+%22Physiol.+Rev%22%5BJournal%5D)">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/pubmed?term=((Nonbone+marrow-derived+endothelial+progenitor+cells%3A+what+is+their+exact+location%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">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/pubmed?term=((Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury%5BTitle%5D)+AND+%22Am.+J.+Respir.+Crit.+Care+Med%22%5BJournal%5D)">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/pubmed?term=((Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((A+transient+parabiosis+skin+transplantation+model+in+mice%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">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/pubmed?term=((Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Tolerance+and+the+H-Y+antigen%3A+Requirement+for+male+T+cells%2C+but+not+B+cells%2C+to+induce+tolerance+in+neonatal+female+mice%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">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 prev...

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

parabiosis in mice: a detailed protocol

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 ...

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 ...

The overall goal of this procedure is to perform para biotic joining of two mice to study a shared circulatory system. This is accomplished by first performing an incision to the adjacent sides of the animals. After the fascia are separated, the two mice are firmly connected at the elbow and knee joints.The final step is to connect the skin of the two animals by a continuous suture. Ultimately, flow cytometric analysis can show blood chimerism within 10 to 14 days after the procedure. The main advantage of this technique over existing method, like bone marrow transplantation or cell injection, is that it does not require immunosuppression and also allows the maintenance of CX circulation for a long period of time.Demonstrating the procedure will be punctu jaw. The animal surgeon from my laboratory At least two weeks in advance, set up several cages of mouse pairs of the same sex, preferably female, same genetics, and similar size and weight. Once a harmonious pair has been selected, perform the parabiosis in a clean animal surgery room.Be sure all contact surfaces tools and gloves are sterile. Anesthetize the mice in a PAI seal induction chamber connected to an iso fluorine vaporizer. Once anesthesia is induced, maintain it throughout the procedure.Using a nose cone, delivering a lower concentration of isof fluorine, now shave their abdominal fur on opposite sides and apply ophthalmic ointment. Then aseptically. Prepare the shaved areas by thoroughly wiping them with betadine soaked wipes followed by alcohol wipes.Next, transfer the mice to a heated pad covered by a sterile pad. For analgesia, use carprofen and buprenorphine. Position the mice on their sides back to back with the shaved areas upwards.Then cover the mice with a sterile drape, exposing only the operation area. Using a sharp scissor, perform longitudinal skin incisions to the sides of each animal, starting half a centimeter below the knee joint, and finishing half of a centimeter above the elbow. Following the incision, gently detached the skin from the subcutaneous fascia by holding the skin up with a pair of curved forceps and separating the fascia with a second pair.To create half a centimeter of free skin, the LECs and knee joints are clearly distinguishable. Following the skin incision, begin joining the left and right LECs. First bend the elbow of the first mouse and pass the needle of the 3.0 suture under Theon.Second, bend the elbow of the second mouse and pass the same suture under it. Attach the joints tightly by a double surgical knot. Next, connect the knee joints following the same procedure.Following the attachment of the joints. Connect the skin of the two animals with a continuous absorbable five oh Vicryl suture. Starting ventrally from the elbow to prevent skin rupture and separation.Perform tight suture closures of the skin in the area around the elbows and knees. Connecting the skin mice is a critical step of this procedure. It is important to ensure that the mice are tightly joined to avoid rupture and separation after the surgery.Therefore, every four to five months, we confirm that the skin is tightly joined. Once the ventral skin attachment has been completed, 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 that it lacks openings.Finish the surgery by administering half a milliliter of 0.9%Sodium chloride subcutaneously to each animal to prevent dehydration. Keep the animals on the heated pad until they recover. House each para biotic pair in a clean cage with moistened food pellets to minimize the strain of reaching for food while adjusting to para biotic existence.Place the food pellets on the cage floor. Also, be sure to provide nesting material over the first 48 postoperative hours. Administer carprofen every 24 hours and administer buprenorphine every 12 hours daily for two weeks.Monitor the animals for signs of pain and distress, such as shaking lethargy, tail chewing, arched back, lack of grooming, and so forth. To prevent bacterial infections over the first 10 days post-op, treat the mice with an oral suspension of sulfamethoxazole and trimethoprim in their water bottle. Blood cism will occur 10 to 14 days following the surgery.So after two weeks, draw blood from their tail veins to confirm that this has occurred. Use flow cytometry to analyze the blood. Dilute two to three drops in 500 microliters of 10 millimolar EDTA.Then thoroughly mix the dilution with 500 microliters of 2%Dextrin in PBS. Incubate the blood mixture at 37 degrees Celsius for 30 minutes, and then spin it down to collect the supernatant. For in practice.Probiotic pairs can be kept together for nine months and separated without any complications. Perform the reverse procedure on a sterile surgical surface. Use the same anesthesia protocol as previously described, and remove the hair from the area surrounding the initial suture.After aseptically, preparing the shaved skin, administer the analgesics and cover the surgical area with a drape using sharp scissors. Separate the mice through a longitudinal incision along the lateral suture to separate the joints, cut the knots of the suture connecting them on each mouse. Trim off the skin along the incision to smoothen the edges.Then reattach the skin with an absorbable continuous five oh coated Vicryl suture to prevent dehydration, administer half a milliliter of 0.9%sodium chlorides subcutaneously, and then keep the animals on a heated pet until they recover from the anesthesia. Use the same analgesic and antibacterial postoperative treatments as previously described, the anticipated outcome of a parabiosis of two organisms is the equal contribution of each animal circulatory system to a common blood circulation. Equilibration of the blood from Parabios Wild type and GFP positive mice was investigated by flow cytometric analysis consistent with previous studies.Blood from the wild type mouse revealed presence of chimerism as indicated by approximately half GFP positive and half wild type blood cells Once mastered. This technique can be done in 45 to 60 minutes if it performed properly.

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Learn parabiosis surgery in mice to create a shared circulatory system for neuroscience research. This protocol demonstrates surgical joining techniques and blood chimerism analysis using flow cytometry.

Parabiosis in Mice: A Detailed Protocol

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