Characterization Of The Macrophage Response To Peripheral Nerve Injury
Recovery after peripheral nerve injury (PNI) is slow and often incomplete. Nerve graft offers a promising therapy for improving functional recovery after repair. Increasing Schwann cell migration and modulating macrophage phenotype can improve nerve regeneration by using endogenous tissue repair mechanisms to create a more robust growth-permissive environment. Here we investigate the modulation of macrophage phenotype using a peripheral nerve-specific extracellular matrix (NSECM) at the site of peripheral nerve injury. The effects of NSECM on macrophage gene expression were evaluated in vitro. Results demonstrate that NSECM promotes a distinct phenotype compared to known phenotypes of classically (M1) and alternatively (M2) activated macrophages. The effects of NSECM on macrophage accumulation in the injured nerve, motor neuron regeneration, and in vivo macrophage gene expression were also evaluated. Murine sciatic nerves were transected and repaired using nerve conduits alone or conduits filled with NSECM or 0.7% agarose. Immunohistochemical evaluation of macrophage accumulation in the injured nerve at 5 days after repair showed that M1 macrophage populations decreased with NSECM treatment, but M2 macrophage populations did not change, compared to control (empty). FACS was used to isolate macrophages from the injured nerve at different time points after repair and gene expression analysis was performed to characterize changes in macrophage phenotype associated with time after injury and experimental group. Our results showed that macrophages express M2 genes early after injury, followed by development of M1-like genes later. These changes were more profound in macrophages treated with agarose. Macrophages treated with NSECM showed less variation over time. We also evaluated functional recovery by quantifying motor neuron regeneration at different time points after injury, and although we saw progression of recovery from 2-8 weeks after injury, experimental groups did not affect functional regeneration in the mouse. Taken together, our results show improvement over previously used immunohistochemical methods for evaluating macrophage populations by use of FACS and high throughput gene expression analyses. In addition, we found that, in contrast to previous findings, NSECM does not improve functional regeneration of the murine peripheral nerve.
Van de Walle,Gerlinde
M.S., Veterinary Medicine
Master of Science
dissertation or thesis