RetroDISCO: Clearing technique to improve quantification of retrograde labeled motor neurons of intact mouse spinal cords.
Quantification of the number of axons reinnervating a target organ is often used to assess regeneration after peripheral nerve repair, but because of axonal branching, this method can overestimate the number of motor neurons regenerating across an injury. Current methods to count the number of regenerated motor neurons include retrograde labeling followed by cryosectioning and counting labeled motor neuron cell bodies, however, the process of sectioning introduces error from potential double counting of cells in adjacent sections.



We describe a method, retroDISCO, that optically clears whole mouse spinal cord without loss of fluorescent signal to allow imaging of retrograde labeled motor neurons using confocal microscopy.



Complete optical clearing of spinal cords takes four hours and confocal microscopy can obtain z-stacks of labeled motor neuron pools within 3-5min. The technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair compared to intact mice and is highly repeatable.



RetroDISCO is inexpensive, simple, robust and uses commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site, avoiding the need for labor-intensive cryosectioning and potential double counting of motor neuron cell bodies in adjacent sections.



RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Emilija
E
Žygelytė
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Megan E
ME
Bernard
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Joy E
JE
Tomlinson
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Matthew J
MJ
Martin
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Allegra
A
Terhorst
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States; The Royal Veterinary College, University of London, North Mymms, Hertfordshire, UK.
Harriet E
HE
Bradford
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
Sarah A
SA
Lundquist
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States; The Royal Veterinary College, University of London, North Mymms, Hertfordshire, UK.
Michael
M
Sledziona
Department of Clinical Sciences, Cornell College of Veterinary Medicine, Cornell University, Ithaca, NY, United States. Electronic address: jc485@cornell.edu.
Jonathan
J
Cheetham
D000818:Animals; D005240:Feasibility Studies; D005260:Female; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods*; D009046:Motor Neurons / Q000166:cytology*; D056347:Neuroanatomical Tract-Tracing Techniques / Q000379:methods*; D056325:Neuronal Tract-Tracers; D061848:Optical Imaging / Q000379:methods; D010543:Peroneal Nerve / Q000166:cytology / Q000254:growth & development / Q000293:injuries / Q000473:pathology; D012584:Sciatic Nerve / Q000166:cytology / Q000254:growth & development / Q000293:injuries / Q000473:pathology; D013116:Spinal Cord / Q000166:cytology* / Q000254:growth & development / Q000473:pathology; D013997:Time Factors; D046509:Tissue Culture Techniques
D005456:Fluorescent Dyes; D056325:Neuronal Tract-Tracers
Confocal microscopy; Mouse; Optical clearing; Regeneration; Retrograde tracer