Parabiosis modeling: protocol, application and perspectives
摘要: 并联共生（parabiosis）是通过手术方法将两只实验动物进行外科连接的一种历史悠久的动物模型。在过去的二十年里，该模型被广泛应用于生物医学研究，特别是在衰老、干细胞、神经科学和免疫等领域。虽然并联共生模型已经得到了不断改进和完善，但从手术操作到围术期管理，仍有诸多细节值得研究者们关注，以降低死亡率和提高并联共生的稳定性。尽管存在着自身的局限性，但作为一种不可替代的研究手段，并联共生动物模型在生物医学研究领域仍有广阔的应用前景。Abstract: Parabiosis is a surgical method of animal modeling with a long history. It has been widely used in medical research, particularly in the fields of aging, stem cells, neuroscience, and immunity in the past two decades. The protocols for parabiosis have been improved many times and are now widely accepted. However, researchers need to consider many details, from surgical operation to perioperative management, to reduce mortality and maintain the parabiosis union. Although parabiosis has certain inevitable limitations, it still has broad application prospects as an irreplaceable animal model in the medical research field.
Figure 1. Surgical preparation of parabiosis mouse model
A: Frequently used surgical instruments. B: Anesthesia of paired mice (showing connection between animal anesthesia machine and paired mice). C: Dorsal positions of paired mice are adjusted after anesthesia, which can be used as a reference for dorsal skin incisions. D: Ventral positions of paired mice are adjusted after anesthesia, which can be used as a reference for ventral skin incisions. E: Dorsal positions of paired mice are adjusted again after skin preparation. F: Ventral positions of paired mice are adjusted again after skin preparation. G: Surgical region is disinfected.
Figure 2. Formation of surgical incisions and management of scapulae and femurs (left mouse taken as example)
A: Left mouse is in lateral decubitus position. Upper and lower limbs of left side and tail are fixed by adhesive tape, respectively. B: Skin incision is made from the back of the ear to back of the thigh. A skin section on the dorsal side of the incision is then removed. C: A skin section on the ventral side of the incision is then removed. D: A skin incision approximately 0.8–1.0 cm wide is formed. E: Right scapula is carefully separated and raised above shoulder muscles. F–H: Muscles attached to bone surface of right scapula are removed. I: Right thigh muscles are separated bluntly to expose right femur. Muscles covering femurs are partly removed. J: Right mouse undergoes the same surgical procedures at corresponding sites.
Figure 3. Suturing scapulae and securing femurs together
A: Relative positions of paired mice are adjusted again to align the scapulae and femurs on both sides. Two ends of the incisions are threaded without sutures, which is helpful to assess relative positions of incisions throughout the operation. B: Suture needle passes through the left scapula of right mouse. C: Suture needle passes through the right scapula of left mouse. D: Relative positions of both scapulae are confirmed. E: Surfaces of both scapulae are overlapped, and first suture of the scapula connection is completed. F–H: Second suture of the scapula connection is completed. I: Suture needle passes around left femur of right mouse. J: Suture needle passes around right femur of left mouse. K: Relative positions of both femurs and thighs are confirmed, respectively. L: Both femurs are overlapped and tied together. M: Corresponding femoral muscle tips of paired mice are sutured together. N: Alignment of dorsal skin incisions, scapula sutures, and femoral sutures are re-checked.
Figure 4. Suturing dorsal skin incisions
A: Relative positions of mice are adjusted, and skin alignment is determined before skin incision suturing. B: First suture of dorsal skin incisions. C: Dorsal skin incisions are sutured from tail-end to head-end. D: Remaining head-end incisions are aligned and sutured with the assistance of the preset head-end suture thread. E: Remaining tail-end incisions are aligned and sutured with the assistance of the preset tail-end suture thread.
Figure 6. Suturing ventral skin incisions
A: Ventral skin incisions are sutured from head-end to tail-end. B: Remaining head-end incisions are aligned and sutured with the assistance of the preset head-end suture thread. C, D: Remaining tail-end incisions are aligned and sutured with the assistance of the preset tail-end suture thread. E, F: Two ends of incisions are finally sutured.
Figure 7. Re-checking all sutured incisions
A: Dorsal view of parabiotic mice after surgical operation. All dorsal skin sutures are checked. B: Ventral view of parabiotic mice after surgical operation. All ventral skin sutures are checked. C: Sutures close to upper limbs are checked carefully. D: Sutures close to lower limbs are checked carefully.
 Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FMV. 2007. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nature Neuroscience, 10(12): 1538−1543. doi: 10.1038/nn2014  Ajami B, Bennett JL, Krieger C, McNagny KM, Rossi FMV. 2011. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nature Neuroscience, 14(9): 1142−1149. doi: 10.1038/nn.2887  Bert P. 1864. Experiences et considerations sur la greffe animale. Journal de l'Anatomie et de la Physiologie, 1: 69−87.  Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, Keller C, et al. 2007. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science, 317(5839): 807−810. doi: 10.1126/science.1144090  Bu XL, Xiang Y, Jin WS, Wang J, Shen LL, Huang ZL, et al. 2018. Blood-derived amyloid-β protein induces Alzheimer's disease pathologies. Molecular Psychiatry, 23(9): 1948−1956. doi: 10.1038/mp.2017.204  Castellano JM, Palner M, Li SB, Freeman GM Jr, Nguyen A, Shen B, et al. 2016. In vivo assessment of behavioral recovery and circulatory exchange in the peritoneal parabiosis model. Scientific Reports, 6: 29015. doi: 10.1038/srep29015  Chimoskey JE, Spielman WS, Brandt MA, Heidemann SR. 1984. Cardiac atria of BIO 14.6 hamsters are deficient in natriuretic factor. Science, 223(4638): 820−822. doi: 10.1126/science.6538050  Coleman DL. 2010. A historical perspective on leptin. Nature Medicine, 16(10): 1097−1099. doi: 10.1038/nm1010-1097  Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. 2005. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature, 433(7027): 760−764. doi: 10.1038/nature03260  Conboy MJ, Conboy IM, Rando TA. 2013. Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity. Aging Cell, 12(3): 525−530. doi: 10.1111/acel.12065  Davison J. 1966. Chimeric and ex-parabiotic frogs (Rana pipiens): specificity of tolerance. Science, 152(3726): 1250−1253. doi: 10.1126/science.152.3726.1250  Dick SA, Macklin JA, Nejat S, Momen A, Clemente-Casares X, Althagafi MG, et al. 2019. Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction. Nature Immunology, 20(1): 29−39. doi: 10.1038/s41590-018-0272-2  Eggel A, Wyss-Coray T. 2014. A revival of parabiosis in biomedical research. Swiss Medical Weekly, 144: w13914.  Finerty JC, Panos TC. 1951. Parabiosis intoxication. Experimental Biology and Medicine, 76(4): 833−835. doi: 10.3181/00379727-76-18647  Garzia L, Kijima N, Morrissy AS, De Antonellis P, Guerreiro-Stucklin A, Holgado BL, et al. 2018. A hematogenous route for medulloblastoma leptomeningeal metastases. Cell, 172(5): 1050−1062.e14. doi: 10.1016/j.cell.2018.01.038  Goldmann T, Wieghofer P, Jordão MJC, Prutek F, Hagemeyer N, Frenzel K, et al. 2016. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nature Immunology, 17(7): 797−805. doi: 10.1038/ni.3423  Harris RB, Martin RJ. 1984. Specific depletion of body fat in parabiotic partners of tube-fed obese rats. American Journal of Physiology, 247(2): R380−R386.  Hervey GR. 1959. The effects of lesions in the hypothalamus in parabiotic rats. The Journal of Physiology, 145(2): 336−352. doi: 10.1113/jphysiol.1959.sp006145  Im SJ, Konieczny BT, Hudson WH, Masopust D, Ahmed R. 2020. PD-1+ stemlike CD8 T cells are resident in lymphoid tissues during persistent LCMV infection. Proceedings of the National Academy of Sciences of the United States of America, 117(8): 4292−4299. doi: 10.1073/pnas.1917298117  Kadoki M, Patil A, Thaiss CC, Brooks DJ, Pandey S, Deep D, et al. 2017. Organism-level analysis of vaccination reveals networks of protection across tissues. Cell, 171(2): 398−413.e21. doi: 10.1016/j.cell.2017.08.024  Kamran P, Sereti KI, Zhao P, Ali SR, Weissman IL, Ardehali R. 2013. Parabiosis in mice: a detailed protocol. Journal of Visualized Experiments, (80): 50556.  Karin O, Alon U. 2020. Senescent cell accumulation mechanisms inferred from parabiosis. GeroScience, doi: 10.1007/s11357-020-00286-x.  Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, et al. 2014. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science, 344(6184): 630−634. doi: 10.1126/science.1251141  Kiss T, Tarantini S, Csipo T, Balasubramanian P, Nyúl-Tóth Á, Yabluchanskiy A, et al. 2020. Circulating anti-geronic factors from heterochonic parabionts promote vascular rejuvenation in aged mice: transcriptional footprint of mitochondrial protection, attenuation of oxidative stress, and rescue of endothelial function by young blood. GeroScience, 42(2): 727−748. doi: 10.1007/s11357-020-00180-6  Liu K, Waskow C, Liu XT, Yao KH, Hoh J, Nussenzweig M. 2007. Origin of dendritic cells in peripheral lymphoid organs of mice. Nature Immunology, 8(6): 578−583. doi: 10.1038/ni1462  Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, et al. 2013. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell, 153(4): 828−839. doi: 10.1016/j.cell.2013.04.015  Mehdipour M, Skinner C, Wong N, Lieb M, Liu C, Etienne J, et al. 2020. Rejuvenation of three germ layers tissues by exchanging old blood plasma with saline-albumin. Aging (Albany NY), 12(10): 8790−8819.  Middeldorp J, Lehallier B, Villeda SA, Miedema SSM, Evans E, Czirr E, et al. 2016. Preclinical assessment of young blood plasma for alzheimer disease. JAMA Neurology, 73(11): 1325−1333. doi: 10.1001/jamaneurol.2016.3185  Mordes JP, Rossini AA. 1981. Tumor-induced anorexia in the Wistar rat. Science, 213(4507): 565−567. doi: 10.1126/science.6941477  Nisbet NW. 1968. Mechanisms in early and late phases of tolerance induced by parabiosis in adult mice. Nature, 217(5125): 265−267. doi: 10.1038/217265a0  Perera J, Meng LP, Meng FY, Huang HC. 2013. Autoreactive thymic B cells are efficient antigen-presenting cells of cognate self-antigens for T cell negative selection. Proceedings of the National Academy of Sciences of the United States of America, 110(42): 17011−17016. doi: 10.1073/pnas.1313001110  Rieux M, Alpaugh M, Sciacca G, Saint-Pierre M, Masnata M, Denis HL, et al. 2020. Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology. Molecular Psychiatry, doi: 10.1038/s41380-020-0787-4.  Rinkevich Y, Lindau P, Ueno H, Longaker MT, Weissman IL. 2011. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. Nature, 476(7361): 409−413. doi: 10.1038/nature10346  Rossi FMV, Corbel SY, Merzaban JS, Carlow DA, Gossens K, Duenas J, et al. 2005. Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1. Nature Immunology, 6(6): 626−634. doi: 10.1038/ni1203  Sinha M, Jang YC, Oh J, Khong D, Wu EY, Manohar R, et al. 2014. Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science, 344(6184): 649−652. doi: 10.1126/science.1251152  Villeda SA, Luo J, Mosher KI, Zou BD, Britschgi M, Bieri G, et al. 2011. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature, 477(7362): 90−94. doi: 10.1038/nature10357  Villeda SA, Plambeck KE, Middeldorp J, Castellano JM, Mosher KI, Luo J, et al. 2014. Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nature Medicine, 20(6): 659−663. doi: 10.1038/nm.3569  Walker DG. 1973. Osteopetrosis cured by temporary parabiosis. Science, 180(4088): 875. doi: 10.1126/science.180.4088.875  Wang J, Jin WS, Bu XL, Zeng F, Huang ZL, Li WW, et al. 2018. Physiological clearance of tau in the periphery and its therapeutic potential for tauopathies. Acta Neuropathologica, 136(4): 525−536. doi: 10.1007/s00401-018-1891-2  Wang XF, Sun R, Hao XL, Lian ZX, Wei HM, Tian ZG. 2019. IL-17 constrains natural killer cell activity by restraining IL-15-driven cell maturation via SOCS3. Proceedings of the National Academy of Sciences of the United States of America, 116(35): 17409−17418. doi: 10.1073/pnas.1904125116  Xiang Y, Bu XL, Liu YH, Zhu C, Shen LL, Jiao SS, et al. 2015. Physiological amyloid-beta clearance in the periphery and its therapeutic potential for Alzheimer's disease. Acta Neuropathologica, 130(4): 487−499. doi: 10.1007/s00401-015-1477-1  Yousefzadeh MJ, Wilkinson JE, Hughes B, Gadela N, Ladiges WC, Vo N, et al. 2020. Heterochronic parabiosis regulates the extent of cellular senescence in multiple tissues. GeroScience, 42(3): 951−961. doi: 10.1007/s11357-020-00185-1  Zhang YY, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. 1994. Positional cloning of the mouse obese gene and its human homologue. Nature, 372(6505): 425−432. doi: 10.1038/372425a0