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Schwann Cell-Derived Desert Hedgehog has a Neuroprotective Effect Against Mechanical Stimuli
James Jung; Derek Frump; Tahseen Mozaffar; Ranjan Gupta
University of California, Irvine
Hypothesis: Defining the molecular mechanisms responsible for the changes induced by chronic nerve compression (CNC) injuries is crucial to develop novel adjunct treatment. Desert hedgehog (dhh) is a Schwann cell produced protein critical for the formation of nerve perineurium. As Schwann cells have been implicated as vital in the pathogenesis of CNC injuries, we hypothesized that desert hedgehog is crucial in the neuroprotective effect offered by Schwann cells and may possibly serve as a potential therapeutic target. Methods: An in-vivo murine model of CNC injury was created in dhh-/- mice (Jackson Labs) by atraumatically placing a 3mm inert tube around the sciatic nerve. CNC injury was confirmed by electrophysiology and protein analysis was performed to detail changes in the basal lamina and fibro-proliferative response. Toluidine blue staining was performed for axon histology and g-ratio measurements and analyzed stereologically using VisioPharm. Moreover, nerves were further analyzed via electron microscopy (EM) for myelin debris. A Mann-Whitney test was performed with p-value<0.05 constituting significance. Results: Nerve conduction velocities (NCV) showed a marked rapid decline in dhh-/- relative to wildtype mice. NCV declined from 52.15±0.5 m/s at baseline to 15.06±0.578 m/s at 2-weeks (p<0.0001). There was a slight improvement at 4 and 6 weeks to 25.63±1.514 m/s and 26.13±1.21 m/s, respectively (p<0.0001). Wildtype animals show the slowest NCV at 6-weeks but never reached the profound slowing seen in dhh-/- animals. Immunohistochemical analysis for collagen IV, laminin-α-2, and fibronectin showed abnormally profuse scarring in dhh-/- at 2-weeks. Interestingly, this response was less profound in the dhh-/- mice at six weeks. G-ratios were measured after 2-weeks CNC injury in normal and compressed nerves (0.64±0.004 vs. 0.67±0.005; p<0.0001). The percentage of large caliber axons was significantly reduced after CNC in dhh-/- (0.404±0.009 vs. 0.153±0.035; p<0.001) but there was a substantial increase in the percentage smaller caliber fibers (0.543±0.007 vs 0.727±0.011; p<0.05). Summary: CNC injuries can be characterized by a progressive decline in NCV. In dhh-/- mice, NCV shows a more rapid and severe decline relative to wildtype after CNC injury as well as a significant loss of large caliber myelinated axons. These data point to a vital role for the dhh protein in the physiological function of myelinated axons. Our studies suggest a possible neuroprotective role of desert hedgehog in peripheral nerve as the lack of this protein accelerates the peripheral nerve demyelination and dysfunction secondary to the sustained mechanical stimuli of CNC injury. 
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