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 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.
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.
All animal studies were performed according to the guidelines of UCLA'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
2. Preparation of Animals
3. Parabiosis
4. Postoperative Recovery
5. Confirmation and Reverse Procedure
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).
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.
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.
Figure 3. Blood chimerism of parabiotic partners. Flow cytometric analysis of blood sample collected from the WT parabiotic partner two weeks following surgical joining.
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.
The authors have nothing to disclose.
The authors would like to thank Adriane Mosley and Libuse Jerabek (Stanford) for assistance with the surgical technique.
Name of equipment | Company | Catalog number |
Isoflurane-Phoenix | Clipper | NDC 57319-559-06 |
Posi-Seal Induction Chamber | Molecular Imaging Products | AS-01-0530-SM |
Portable Anesthesia System | Molecular Imaging Products | AS-01-0007 |
Gaymer T Pump | Gaymar Industries, Inc | TP650 |
Warming Blanket (Heating pad) | Kent Scientific Corp | TP-22G |
Curved forceps | Roboz | RS-5101 |
Scissors | Fine Science Tools (FST) | FST 14063-09 |
Needle holder | FST | FST 12501-13 |
Electrical shaver | Oster | Golden A5 |