Casey A Spencer1 and Thomas Leung1,2,3 Research Techniques Made Simple: Parabiosis to Elucidate Humoral Factors in Skin Biology Casey A Spencer1 and Thomas Leung1,2,3 1Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, US. 2Corporal Michael Crescenz Veterans Affairs Medical Center, Philadelphia, PA, 19104, US. 3Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, 19104, US.

Parabiosis Two living animals are surgically conjoined and share a single circulatory system Surgical union of skin causes formation of vascular anastomoses 150+ years of use Led to advances in endocrinology, neurology, musculoskeletal biology, and dermatology

Selected History of Parabiosis Sauerbruche and Heyde coin the term “parabiosis” and modify the technique by extending the length of the incision and adding an intestinal anastomosis. Hervey uses parabiosis to demonstrate that the circulating factor leptin is involved in energy balance. Villeda and Wyss-Coray uses parabiosis to study impact of aging on neurons and brain function. 1908 1959 2014 1864 1933 2018 French biologist Bert tests the viability of skin allografts by joining two rats together and attaching flaps of skin from one animal to another animal. Bunster and Meyer improve on the technique by joining the skin, muscle layers, and abdominal wall together. Nishiguchi uses parabiosis to show that the circulating factor, SDF1, is responsible for scar formation in skin. 2005 Conboy and Rando shows that the aged environment impairs muscular regenerative potential of older individuals.

Example of parabiosis experiment to test age-defined phenotypes Pairings Intervention Observation Conclusion Both parabionts maintain phenotype A Parabiont B adopts phenotype A Systemic circulating factor Parabiont A adopts phenotype B Wound Both parabionts maintain individual phenotypes Local cell signaling Both parabionts maintain phenotype B

Basic Principles of Parabiosis Standard aseptic technique used Elbows and knee joints are sutured together Left and right flanks of parabiont skin are sutured with single, interrupted stitches Surgery length: 30-60 minutes Post-procedure care is critical Treat with antibiotics, subcutaneous normal saline, meloxicam and buprenorphine hydrochloride Monitor for recovery characteristics Incisions are healed 2 weeks post-surgery Additional skin wounding performed 4 weeks post-surgery

Technical Challenges Background matched animals have best chance of survival Perioperative mortality 90% + pairs recover from procedure Parabiotic disease 1-2 weeks post-surgery Likely represents underlying graft-versus-host disease, where the rejected “organ” is the anastomoses Affects 20-30% of pairs in inbred mouse strains Affects 60-70% of pairs in outbred mouse strains

Kinetic Considerations Circulatory system anastomosis requires 2 weeks to mature before steady-state is reached Use a reporter mouse (e.g. mTmG mouse) to determine level of blood exchange Consider rate of clearance of factors from circulation Rate of blood exchange in parabiosis is slow Potential for a false negative result if circulating factor cannot reach the other parabiont

Applications Test for a circulating factor involved in skin physiological processes (i.e. differentiation, neoplastic formation, wound healing) Mice can vary in physiological condition: Parabiont A Parabiont B Potential physiological conditions Aged Mutant Wounded Irradiated Infected Obese Young Wildtype Unwounded Non-irradiated Non-infected Lean

Applications Answer questions about systemic regulation of cell and tissue aging in multiple organs Use genetically altered mouse strains for direct testing of signaling pathways/networks involved in regulating an identified process