Abstract
The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. Reports from salamander models of appendage regeneration (Singer, 1952; Singer, 1947; Kumar et al., 2007), and of human clinical skin and nail problems associated with spinal cord injury patients (Stover et al., 1994) suggest that appendage regeneration may have an important nerve component. To explore this question, we have generated hind limb tissues devoid of nerve supply. This protocol, combined with multi-color ‘Rainbow’ reporter mouse lines permits single cell clonal analysis and genetic lineage tracing studies in the absence of nerve supply (Rinkevich et al., 2014), exposing nerve requirements on cellular replacement and differentiation during tissue growth, maintenance, and regeneration.
Keywords: Denervation, Peripheral Nervous System, Tissue Maintenance, Limb Regeneration
Materials and Reagents
Equipment
Procedure
Notes
This protocol completely removes any sciatic nerve input to the hind limb. However, femoral nerve branches innervate cutaneous tissues of the leg and foot, as well as ligaments, joints and blood vessels and may overlap with territories supplied by the sciatic nerve. We therefore wished to ensure that complete denervation on hind limb tissues took place, and that femoral nerve supply does not substitute sciatic nerve requirements (electrically, chemically, or functionally) after sciatic denervation. To accomplish this, surgical denervation of both sciatic and femoral nerves was performed, and two weeks prior to digit tip amputations to allow full nerve degeneration to take place. Complete nerve degeneration can be verified by monitoring beta 3 tubulin immunoreactivity on histological sections of denervated hind limbs. While ablation of sciatic innervation alone denervates the distal limb, femoral ablation was performed to ensure that no nerve regeneration from the proximal limb would occur during the regeneration time interval. Additionally, an incision was made in the ventral skin of the thigh and the femoral fascial sheath. Femoral denervation was achieved by cutting a 5 mm section of the femoral nerve, leaving the femoral artery intact. Sham surgeries were performed by exposing the nerves without cutting them.
Acknowledgments
Y.R. was supported by the Machiah Foundation Fellowship, the Siebel Stem Cell Institute and the Thomas and Stacey Siebel Foundation (1119368-104-GHBJI), the Human Frontier Science Program (HFSP) Long Term Fellowship, and partly by the HFSP Career Development Award (CDA00017/2016) and from the German Research Foundation (DFG, RI 2787/1-1 AOBJ: 628819). C.D.M. is supported by the American College of Surgeons Resident Research Scholarship.
References
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