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Skin Derived Precursor Cells (Skps) Improve Myelination Within a Model of Focal Adriamycin-Induced Tibial Nerve Demyelination
Joey Grochmal, MD; Sundeep Dhaliwal; Rajiv Midha, MD
University of Calgary, Calgary, AB, Canada
Introduction: Skin derived precursor cells (SKPs) can mimic the phenotypic appearance of Schwann cells when predifferentiated in vitro (SKP-SCs), and are a reliable autologous source of Schwann cells that survive in considerable numbers when micro-injected into nerve grafts and denervated nerve. Our hypothesis is that SKP-SCs can produce morphologically and electrophysiologically functional myelin as they ensheath axons. We are testing this hypothesis in a model of focal demyelination of the rat tibial nerve.
Methods: We unilaterally injected 500,000 GFP labeled SKP-SCs into the tibial nerves of 10 adult Lewis rats, while the contralateral tibial nerve received media injection. This was done one week after a demyelinating bilateral tibial nerve lesion was created using a 30ul injection of 12.5ug/ml Adriamycin (1). This model provides a reproducible timeline of demyelination that begins after nine days, followed by remyelination over the course of several weeks. All animals are followed for compound motor action potentials (CMAPs) every three days until 50 days post-initial injection. Animals from identical cohorts of each group are being periodically sacrificed for teased fibre, morphological, and immunohistological analysis using anti-voltage gated sodium channel, anti-voltage gated potassium channel, as well as anti-CASPAR antibodies. Also, these animals are being periodically sacrificed for immunohistological analysis of axial tibial nerve sections looking for myelination of tibial nerve axons by labeled SKP-SCs, using a combination of frozen sections, semi-thin Epon sections, as well as ultrathin EM cryosections.
Results and Conclusions: In our preliminary work, we have demonstrated that SKP-SCs promoted a significantly lower G-ratio when analyzed against either media or Schwann cell injection in this model (Day 33 Epon morphometry, N=5/group). We have also demonstrated preliminary evidence of myelination by DiI labeled SKP-SCs (Day 33 teased nerve fibre). Results concerning the electrophysiological data are pending. We expect to see a more rapid return to baseline function in the SKP-SC injected group. We also predict that SKP-SCs will participate in functional nodes of Ranvier formation, as demonstrated by confocal microscopy. Finally, we predict that prelabeled SKP-SCs will be seen to conclusively myelinate tibial nerve axons on immunohistological analysis.
1. England, JD., Rhee, EK., Said,G., Sumner, AJ: Schwann cell degeneration induced by doxorubicin (adriamycin). Brain 111: 901-913, 1988
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