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Correlation Between Regenerated Motor Neurons And Graft Length In Rat Cross-face Nerve Grafts­
Lauren J. Kelsey, BS1, Joanna K. Ledwon, PhD2, Lindsay E. Janes, MD3, Daniel C. Sasson, BA4, Arun K. Gosain, MD2.
1Department of Surgery Plastic Division, Stanley Manne Children's Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA, 2Northwestern University Feinberg School of Medicine, Department of Surgery Plastic Division, Stanley Manne Children's Research Institute, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA, 3Northwestern University Feinberg School of Medicine, Department of Surgery Plastic Division, Chicago, IL, USA, 4Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Purpose
Changes in axonal regeneration across the length of a cross-face nerve graft are not well understood. The present study evaluates the survival of regenerated motoneurons from different locations along a cross-face nerve graft after a 12-week period of regeneration.
Methods
The left sciatic nerve of mature Sprague Dawley rats was harvested as a 30-mm nerve graft, and coapted end-to-end to the main trunk of the facial nerve. 12 weeks later, the nerve graft was sectioned within the proximal 5 mm (Proximal group), middle 5 mm (Middle group), or the distal 5 mm (Distal group). The facial nerve trunk of grafted rats was also sectioned (Control). The proximal end of the cut nerve was immersed in 4% Fluoro-Gold (FG) in sterile saline for 1 hour, and then returned to the head. Allowing 1 week for the FG retrograde labeler to trace back to the facial nucleus, the brain was then harvested and preserved in 4% PFA/30% sucrose and frozen in OCT. The whole brain was sectioned at 40 μm to capture the entire facial nucleus, and counter-stained with Fluoroshield PI (PI) to detect nuclei. Images were taken using a Zeiss fluorescent microscope with filters for FG and PI, and merged in Photoshop. The percentage of survived motoneurons was calculated as a ratio of double-positive cells to all motoneurons detected with FG. Statistical analysis was performed using unpaired t-tests in GraphPad Prism 7. P-values of < 0.05 were considered significant.
Results
In the Control group, 80% of regenerated motoneurons showed positive staining for nuclei (Fig.1), which was not significantly different from the Proximal group of nerve grafts (p=0.433). However, traveling from the proximal to the distal end of the graft, a decreased percentage of surviving regenerated motoneurons was observed. The Middle group showed 2.2 times less percent survival (p<0.0001) when compared to Control. The Distal group showed the most drastic decline in motoneuron survival, reaching 8.9 times less percent survival (p<0.0001) compared to Control.

Conclusions
These findings indicate that, despite ample time for complete regeneration, the survival of regenerated motoneurons diminishes along the length of a cross-face nerve graft. Although a longer cross-face nerve graft may be of greater clinical utility in providing more options for end-target reinnervation, the middle and distal areas of a long nerve graft may not provide adequate axonal regeneration for this benefit to be realized. Analyzing the tissue of the nerve itself will allow us to explore how the biological environment that encapsulates the nerve graft impacts its survival.


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