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Evaluation Of Axonal Regeneration Across A Long Cross-face Nerve Graft In Rats
Lauren J. Kelsey, BS1, Lindsay E. Janes, MD2, Daniel Sasson, BA3, Joanna K. Ledwon, PhD1,2, Arun K. Gosain, MD1,2.
1Department of Surgery Plastic Division Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA, 2Northwestern University Feinberg School of Medicine Department of Surgery Plastic Division, Chicago, IL, USA, 3Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

PURPOSE:Cross-face nerve grafting is a preferred technique in facial reanimation due to its capability in providing spontaneous action and symmetry to a patient following regeneration. The length of graft used is thought to be involved in differing functional outcomes. The present study evaluates changes in axonal regeneration at different lengths along a cross-face nerve graft.
METHODS:The sciatic nerve of sixteen adult Sprague Dawley rats was harvested as a 2-cm graft and coapted end-to-end to the proximal main trunk of the facial nerve. Thirteen weeks later, the graft site was re-exposed for sectioning. 5mm segments were taken from the native facial nerve trunk (Group 1), in the nerve graft, just distal to coaptation (Group 2), midway (Group 3), and just proximal to the distal end of the graft (Group 4). The nerve segments were preserved and embedded in paraffin, cut in 1um cross-sections, and stained with 1% toluidine blue for axon counting. After segmentation, the nerve stump was immersed in 4% Fluoro-Gold (FG) for one hour, returned to the head for retrograde labeling over one week. The brains were preserved in 4% PFA/30% sucrose, frozen in OCT, and sectioned in 40um cross-sections. Every third section was taken to avoid double-counting cells, and counter-stained with Fluoroshield PI (PI). Images were taken with a fluorescent microscope using a combined filter, and cells with double-positive stain were counted as regenerated motoneurons. Statistical analysis was performed using unpaired t-tests in GraphPad Prism7.
RESULTS: Analysis of the retrograde-labeled regenerated motoneuron counts showed no significant difference between the proximal portion of the graft (3,538 1,969) and the facial trunk (4,128 731) (p=0.67) (Figure 1). However, the motoneuron count at the midway point of the graft (1,517 335) and at the distal end (269 293) were significantly lower than in the facial trunk (p=0.01 and 0.006, respectively). Analysis of the nerve biopsies along the length of the graft demonstrated no significant differences in myelinated axon count across the graft with a mean count of 6,207 310 at the nerve trunk, and 6,606 2399 (p=0.76), 6,405 2340 (p=0.88), and 7,179 744 (p=0.07) moving farther down the graft segment.

CONCLUSION:These findings show that although myelinated axon counts in the graft tissue remain consistent along the entire length, the number of axons making connections back to the facial nerve nucleus demonstrates significant drop-off the more distal one samples along the nerve graft. Although axon sprouting may compensate for motoneuron drop-off in longer grafts, this reinforces that determination of functional axonal regeneration along a nerve graft by counting axons in peripheral nerve tissue is questionable, and instead requires staining of motor nuclei in the brain. Future investigation is needed to determine the optimal length for a cross-face nerve graft, including evaluating the impact of axon sprouting on functional outcomes and muscle strength after reinnervation through a long nerve graft.


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