Research PaperMyelination of axons emerging from neural progenitor grafts after spinal cord injury
Introduction
Recent studies have suggested a potential therapeutic role of neural stem cell therapies for spinal cord injury (SCI) (Yamane et al., 2010, Yan et al., 2007, Salazar et al., 2010, Ogawa et al., 2002, Haas and Fischer, 2013, Lu et al., 2014, Lu et al., 2012). While many neural stem cell approaches focus on remyelination of spared axons (Kobayashi et al., 2012; Keirstead et al., 2005, our recent findings indicate that neural progenitor cells grafted into sites of SCI can establish novel synaptic relays across lesion sites that support functional improvement (Lu et al., 2014, Lu et al., 2012, Kadoya et al., 2016). Because 40–60% of all axons in the spinal cord are estimated to be myelinated (Chung and Coggeshall (1983b), (Chung and Coggeshall, 1983a), axons emerging from neural progenitor cell grafts will likely require some degree of myelination to optimize functional recovery. We directly investigated this question in the present study.
Neural progenitor cell grafts to the injured spinal cord encounter a markedly different environment from the developing CNS, suggesting that intact, mature patterns of myelination may not be re-established by axons emerging from neural progenitor cell grafts and extending through the adult, lesioned spinal cord. Indeed, even mild perturbations of glia-neuronal signaling can lead to dysfunction and neurodegeneration (Alizadeh et al., 2015). For instance, developmentally altered communication between oligodendrocytes and axons results in a lack of myelination, axonal swelling and neurodegeneration (Edgar et al., 2009, Griffiths et al., 1998).
Central myelination during development is the result of a complex multiphasic crosstalk between oligodendrocytes and axons (Taveggia et al., 2008, Bercury et al., 2014), and myelination in the adult CNS following injury has classically been characterized by thinner axonal remyelination compared to the intact state (Blakemore and Murray, 1981), (Gledhill and McDonald, 1977). Thus, it is important to understand whether axons emerging from neural progenitor cells grafted to sites of SCI undergo myelination and the extent to which this occurs.
To address these questions, we grafted E14-derived multipotent neural progenitor cells (NPCs) ubiquitously expressing GFP into rats with cervical level 5 (C5) right hemisection lesions (Lu et al., 2012, Kadoya et al., 2016) and examined graft-derived axon myelination in the host spinal cord at C8-T2, 3–5 segments below the lesion site. 24% of graft-derived axons became myelinated; surprisingly, these axons were of normal axon caliber and recapitulated normal G-ratios of myelinated axons in the intact spinal cord.
Section snippets
Materials and methods
This study was done in strict accordance with laboratory animal care and safety guidelines of the National Institutes of Health. Animals were provided food and water ad libitum throughout the duration of this study. Animal surgery, embryonic spinal cord dissociation and grafting were performed as summarized below (described previously by Lu et al., 2012). For all surgeries and perfusions, animals were deeply anesthetized using a combination of (2 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 g/ml),
Axon diameter
GFP-expressing grafts survived well and filled the lesion cavity when assessed three months post-transplantation (Fig. 1a). Large numbers of GFP-labeled axons emerged from the lesion site and into the host spinal cord, consistent with previous observations (Fig. 1b) (Lu et al., 2014, Lu et al., 2012, Kadoya et al., 2016). At the ultrastructural level, GFP-labeled axons appeared electron-dense (Fig. 1c) and were readily distinguished from shrunken, electron-dense degenerating host axons (Fig. 1
Discussion
The present study reveals that approximately one-quarter of all axons emerging from grafts of neural progenitor cells placed into sites of spinal cord injury are myelinated by host oligodendrocytes. Moreover, the thickness of the myelin sheaths is normal, reflected by a G-ratio that is equal to that of axons in the intact spinal cord.
Following injury to the adult CNS, several reports have indicated that host oligodendrocytes remyelinate host axons, but that host axons are typically
Acknowledgments
This work was supported by the Veterans Administration (Gordon Mansfield Spinal Cord Injury Consortium 1I50RX001706-01), the NIH (NS042291), The Craig H. Neilsen Foundation, and the Bernard and Anne Spitzer Charitable Trust to M.H.T.
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