Amy M Moore1, Gregory H Borschel2, Katherine B Santosa1, Eric R Flagg1, Alice Y Tong1, Rahul Kasukurthi1, Piyaraj Newton1, Ying Yan1, Daniel A Hunter1, Philip J Johnson1, Susan E Mackinnon3. 1. Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA. 2. The Hospital for Sick Children Division of Plastic and Reconstructive Surgery, Toronto, Canada M5G1X8; University of Toronto Department of Surgery, Toronto, Canada M5G1X8; University of Toronto Institute of Biomaterials and Biomedical Engineering, Toronto, Canada M5G1X8. 3. Division of Plastic and Reconstructive Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: mackinnon@wudosis.wustl.edu.
Abstract
BACKGROUND: In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. METHODS: Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. RESULTS: In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. CONCLUSION: The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances.
BACKGROUND: In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. METHODS: Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. RESULTS: In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. CONCLUSION: The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances.
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