Background: 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.
New method: 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.
Results: 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.
Comparison with existing method: 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.
Conclusions: RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.
Keywords: Confocal microscopy; Mouse; Optical clearing; Regeneration; Retrograde tracer.
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