A. N. Hoffman2, R. Bamba1, M. Stephanides1, A. C. Pollins1, W. P. Thayer1 1Vanderbilt University Medical Center,Plastic Surgery,Nashville, TN, USA 2Vanderbilt University School Of Medicine,Nashville, TN, USA
Introduction:
The degree of functional recovery after a peripheral nerve injury is rarely satisfactory. Peripheral nerves have the capacity to regenerage, but unfortunately this regeneration is very slow, and there is usually significant loss of function. Current methods in nerve repair depend on the type of nerve injury and include direct repair, nerve conduits, nerve autografts and allografts. These techniques focus on optimizing the process of axonal regeneration. Polyethylene glycol (PEG) fusion is a new technique in peripheral nerve repair that aims to fuse damaged axons in order to provide some immediate restoration of axonal function. The PEG fusion technique involves reconnecting the cut ends of severed axons with microsutures and applying a sequence of solutions including PEG. It is thought to delay Wallerian degeneration, and animal studies have shown immediate restoration of compound action potentials and improved behavioral outcomes.
Methods:
Rat B35 neuroblastoma cells were grown using standard cell culture techniques. The cells were labeled separately with CellTracker Green CMFDA and CellTrace Violet fluorescent dyes. These dyes were chosen because they freely diffuse across cell membranes, and are not transferable between cells except to daughter cells. Equal concentrations and volumes of the green- and violet-labeled cells were then combined and PEG solutions were added to the cell pellet at concentrations of 50%, 75% or 100% PEG by weight with 5% DMSO. A control sample consisted of the two populations of fluorescently labeled cells combined without addition of PEG. Other controls included unstained and single stain groups. The samples were analyzed using the BD LSRFortessa flow cytometer. The amount of fusion was assessed by the percent of double positive cells.
Results:
Results show that there is increasing cell fusion with increasing concentrations of PEG. Viability tests were also performed with the viability factor PI, and they showed decreasing viability with increasing concentrations of PEG (Table 1).
Conclusion:
The results of this experiment provide evidence of PEG fusion at the cellular level. Using the double-positive population as an indicator of cell fusion is not a perfect measure, as cells are equally likely to fuse with other cells of the same color. Therefore, it is most likely an underestimate of the amount of fusion. Assessment of cell viability indicated that there is increasing cell death with increasing concentration of PEG. This trade-off between increased fusion and increased cell death represents a significant balance that must be assessed for applicability in patients with nerve injuries. Future directions of this experiment include studying the long term viability of fused cells.
