Induction of regional collateral sprouting following muscle denervation.

OBJECTIVES/HYPOTHESIS: Anecdotal clinical findings suggest that denervated muscle may regain modest functional recovery via spontaneous collateral sprouts from intact adjacent nerve fibers. The current study evaluates the conditions needed for the denervated masseter muscle to induce axonal sprouting from the facial nerve. We hypothesize that epineurial injury is required to induce collateral sprouting toward a neighboring denervated muscle. STUDY DESIGN: Twelve thy1-yellow fluorescent protein-16 (thy1-YFP-16) transgenic mice whose axons express yellow fluorescent protein were allocated into six groups, with four degrees of facial nerve injury (intact, crush, transection, removed segment) with or without masseter denervation. METHODS: Animals underwent serial in vivo imaging analyses under the fluorescent microscope weekly for 5 or 7 weeks and were subsequently perfused for analysis. Masseter muscle acetylcholine receptors (AChRs) were stained with Alexa Fluor 594 conjugated alpha-bungarotoxin, and whole mounts were imaged with confocal microscopy. RESULTS: In groups with intact or crushed facial nerves, no evidence of collateral sprouting was demonstrated. Mice with transected facial nerve branches or removed segments demonstrated sprouting from the proximal stump into the denervated masseter. Staining of the AChRs confirmed that new neuromuscular junctions were established between the facial nerve and the denervated masseter. CONCLUSIONS: This study suggests that epineurial injury is required to stimulate axonal sprouting into adjacent denervated muscle. Nerves with compromised epineurium may be useful in promoting neo-neurotization after muscle denervation.

A double-transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves.

We propose that double-transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1-cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163+/-15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388+/-107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431+/-31) and unmyelinated (64+/-9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts.