Delay modulates the immune response to nerve repair.

Effective regeneration after peripheral nerve injury requires macrophage recruitment. We investigated the activation of remodeling pathways within the macrophage population when repair is delayed and identified alteration of key upstream regulators of the inflammatory response. We then targeted one of these regulators, using exogenous IL10 to manipulate the response to injury at the repair site. We demonstrate that this approach alters macrophage polarization, promotes macrophage recruitment, axon extension, neuromuscular junction formation, and increases the number of regenerating motor units reaching their target. We also demonstrate that this approach can rescue the effects of delayed nerve graft.

Evaluation of two methods to isolate Schwann cells from murine sciatic nerve.

BACKGROUND: Schwann cells (SC) and macrophages play key roles in the response to peripheral nerve injury (PNI). Accurate isolation of such cells is essential for further analyses that can lead to better understanding of the repair process after PNI. Separation of live SC from the injury site without culture enrichment is necessary for targeted gene expression analysis. NEW METHODS: Two flow cytometric techniques are presented for rapid enrichment of live SC and macrophages from injured murine peripheral nerve without the need for culture. RESULTS: SC were isolated by fluorescent activated cell sorting (FACS) using transgenic expression of eGFP in SC, or by exclusion of other cell types collected from the injury site. COMPARISON WITH EXISTING METHOD(S): Gene expression analyses of peripheral nerve repair have commonly used whole nerve lysates. Isolating SC allows more accurate understanding of their specific role in repair. SC are commonly enriched from nerve by culture, however this changes gene expression patterns and limits the utility for transcriptomic analysis. The surface marker p75-NTR has variable expression in different SC phenotypes and during the course of injury and repair. Using p75-NTR for SC isolation might enrich only a subset of SC. More stably expressed lineage markers for SC are intracellular and not suitable for sorting for gene expression. The methods used here avoid the requirement for surface marker labeling of SC. CONCLUSION: Gene expression analysis of sorted cells from both methods showed successful enrichment of SC. Lineage markers such as Map1b, p75-NTR and S100b were enriched in the sorted SC population. SC sorting by eGFP expression showed improved enrichment, particularly of mature myelinating genes, although this could represent sampling of a subset of SC.

High-Efficiency Gene Transfection of Cells through Carbon Nanotube Arrays.

Introducing nucleic acids into mammalian cells is a crucial step to elucidate biochemical pathways, and to modify gene expression and cellular development in immortalized cells, primary cells, and stem cells. Current transfection technologies are time consuming and limited by the size of genetic cargo, the inefficient introduction of test molecules into large populations of target cells, and the cytotoxicity of the techniques. A novel method of introducing genes and biomolecules into tens of thousands of mammalian cells has been developed through an array of aligned hollow carbon nanotubes, manufactured by template-based nanofabrication processes, to achieve rapid high-efficiency transfer with low cytotoxicity. The utilization of carbon nanotube arrays for gene transfection overcomes molecular weight limits of current technologies and can be adapted to deliver drugs or proteins in addition to nucleic acids.