Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair
Introduction
The peripheral nervous system (PNS) is generally considered to have significant regenerative potential, allowing recovery after injury with varying degrees of success. PNS regeneration can be improved by nerve conduits, cell therapy, application of neurotrophic factors and other techniques (de Boer et al., 2011, Jubran and Widenfalk, 2003, Kemp et al., 2009, Midha et al., 2003, Tannemaat et al., 2008, Walsh et al., 2009). Cell-based therapy is a potent tool to guide and support nerve regeneration. Schwann cells (SCs) are valid candidates for cell therapy of PNS injuries, being the glial as well as myelinating cells in the PNS and playing a crucial role both in supporting peripheral axon regeneration and re-myelination after injury (Krick et al., 2011, Lehmann and Hoke, 2010). They contribute to axonal regeneration by secreting molecules of the extracellular matrix like laminin and collagen, cell adhesion molecules, and trophic factors including neurotrophins (Brushart, 2011, Snyder and Senut, 1997). Schwann cells also guide axons and provide physical support to regenerating axons by their alignment in the so-called bands of Bungner that define pathways for axonal regrowth (Ide, 1996).
Therapeutic potential of SCs can be further enhanced by over-expression of genes encoding proteins beneficial for cell function and/or involved in creating a local regenerative microenvironment. For improved nerve recovery, neurotrophin-encoding genes appear to be promising candidates, since exogenous systemic application of neurotrophins is hampered by rapid protein degradation and unwanted side effects. Neurotrophins are essential for survival, differentiation and maintenance of neurons (Brushart, 2011, Ide, 1996, Nakamura et al., 2011, Spencer et al., 2008). They play an important role in myelin formation in the peripheral nerve, prevent motoneuron atrophy, enhance re-myelination, and improve behavioral and electrophysiological recovery following nerve injuries (Boyd and Gordon, 2003, Fu and Gordon, 1997, Gordon, 2009, Gordon, 2010, Jubran and Widenfalk, 2003, Kemp et al., 2011, Midha et al., 2003, Simpson et al., 2003). Neurotrophin-transduced cells have been previously shown to stimulate regeneration in the central nervous system (Blesch et al., 2004, Grill et al., 1997, Hu et al., 2005, Nakahara et al., 1996, Tuszynski et al., 1997, Weidner et al., 1999). In a recent study, we demonstrated that application of the prototypical neurotrophin, nerve growth factor (NGF), to a nerve repair site in vivo induced improved early axonal regeneration in a dose-dependent manner, and 3 weeks of NGF therapy at the optimal dose had a dramatic effect on improving locomotor recovery (Kemp et al., 2011).
In the present study, we supplemented Schwann cell therapy of peripheral nerve injury with over-expression of NGF in SCs. Such a combined approach may exert dual effect, including both guidance and myelination of regenerating axons by SCs and trophic support by over-produced NGF. NGF is known to guide axons (Yu et al., 2010), promote axonal sprouting (Brushart, 2011), stimulate myelination by SCs (Chan et al., 2004) and eventually improve functional recovery after injury (Kemp et al., 2011). Endogenous NGF in transected peripheral nerve increases within the first few weeks, implying that initial basal level of NGF could be a limiting factor and engineered NGF over-expression could have a beneficial effect on regeneration of injured peripheral axons. We hypothesize that supplementation of NGF immediately post-injury by physiologically relevant Schwann cell therapy may elicit a heightened general and/or specific regenerative improvement. We have looked at early effects of the transplantation of isogenic NGF-transduced SCs, which are designed to compensate the relatively low neurotrophin levels in the early stages of nerve regeneration. Our results demonstrate that NGF-transduced SCs are able to maintain high NGF level in vivo, significantly improve axonal regrowth including a dramatic effect on the regeneration of a sub-class of sensory neurons population and reduce denervated muscle atrophy.
Section snippets
Lentiviral transduction of Schwann cells in vitro
Lentiviral vectors used in this study were earlier characterized as highly efficient in various models of neural regeneration (Hu et al., 2005, Tannemaat et al., 2007, Tannemaat et al., 2008). Lentiviruses encoding NGF or GFP reporter were used to transduce SCs isolated from postnatal inbred Lewis rats. Following LV-GFP transduction, CMV promoter-driven expression of reporter GFP gene was observed rapidly in over 99% transduced SCs in vitro. Immunostaining with an anti-NGF antibody revealed
Discussion
The effects of NGF on peripheral nerve regeneration has been studied in several experimental paradigms, including direct NGF application (Kemp et al., 2011), NGF-containing fibrin sealants (Jubran and Widenfalk, 2003), injection of NGF-encoding lentiviral (Tannemaat et al., 2007, Tannemaat et al., 2008) or adenoviral (Hu et al., 2010) particles, microspheres (de Boer et al., 2011), modified scaffolds (Chung et al., 2011) and other methods (Ahmed et al., 1999, Derby et al., 1993, Lindsay, 1988,
Conclusion
Our approach supports the use of NGF in combination with cell therapy — a supplementation of injured peripheral nerve with isogeneic Schwann cells. The latter convey support in multiple aspects such as cleansing the axonal debris, release of trophic factors and ultimately the re-myelination of regenerating axons. Although isogeneic or autologous Schwann cells are routinely obtained from the nerves, they can also be derived from adult stem cells, including mesenchymal bone marrow (Shea et al.,
Schwann cell culture
SCs were isolated from sciatic nerves of P2 Lewis rats according to modifications of established protocols (Komiyama et al., 2003, Walsh et al., 2009). Briefly, sciatic nerves were excised, stripped of the epineurium, and cut into 1 mm2 pieces. Nerve segments were placed on poly-d-lysine coated 35 mm culture dishes in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin/streptomycin and 0.25 μg/ml Fungizon for 3 days, allowing fibroblasts migration out from the nerve. Media was then changed to
Acknowledgments
This research was supported by grants to Rajiv Midha from the Canadian Institute for Health Research (Regenerative medicine and nanomedicine team grant #163322) and the Center for excellence in nerve regeneration (partnership between the Hotchkiss Brain Institute, University of Calgary and Integra LifeSciences). Post-doctoral fellowship support to A.S. was provided by Alberta Innovates-Health Solutions (AI-HS). The authors would like to thank Bhagat Singh for his kind assistance with
References (85)
- et al.
Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF
Neuron
(1991) - et al.
Neuroprotection of spinal motoneurons following targeted transduction with an adenoviral vector carrying the gene for glial cell line-derived neurotrophic factor
Exp. Neurol.
(1998) - et al.
Axonal responses to cellularly delivered NT-4/5 after spinal cord injury
Mol. Cell. Neurosci.
(2004) - et al.
NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes
Neuron
(2004) - et al.
Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation
Brain Res.
(1997) - et al.
Promoting regeneration of peripheral nerves in-vivo using new PCL-NGF/Tirofiban nerve conduits
Biomaterials
(2011) - et al.
Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms
Mol. Cell. Neurosci.
(1998) - et al.
Nerve growth factor facilitates regeneration across nerve gaps: morphological and behavioral studies in rat sciatic nerve
Exp. Neurol.
(1993) - et al.
Adipose-derived stem cells enhance peripheral nerve regeneration
J. Plast. Reconstr. Aesthet. Surg.
(2010) - et al.
Neuroregenerative effects of lentiviral vector-mediated GDNF expression in reimplanted ventral roots
Mol. Cell. Neurosci.
(2008)
Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons
Neuron
The physiology of neural injury and regeneration: the role of neurotrophic factors
J. Commun. Disord.
Lentiviral-mediated transfer of CNTF to Schwann cells within reconstructed peripheral nerve grafts enhances adult retinal ganglion cell survival and axonal regeneration
Mol. Ther.
Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve
Exp. Neurol.
Peripheral nerve regeneration
Neurosci. Res.
Repair of peripheral nerve transections with fibrin sealant containing neurotrophic factors
Exp. Neurol.
Dose and duration of nerve growth factor (NGF) administration determine the extent of behavioral recovery following peripheral nerve injury in the rat
Exp. Neurol.
A novel technique to isolate adult Schwann cells for an artificial nerve conduit
J. Neurosci. Methods
Signaling cue presentation and cell delivery to promote nerve regeneration
Curr. Opin. Biotechnol.
Grafts of fibroblasts genetically modified to secrete NGF, BDNF, NT-3, or basic FGF elicit differential responses in the adult spinal cord
Cell Transplant.
NGF activation of TrkA induces vascular endothelial growth factor expression via induction of hypoxia-inducible factor-1alpha
Mol. Cell. Neurosci.
Nerve growth factor enhances regeneration through silicone chambers
Exp. Neurol.
The effect of endoneurial nerve growth factor on calcitonin gene-related peptide expression in primary sensory neurons
Brain Res.
Effects of nerve crush and transection on mRNA levels for nerve growth factor receptor in the rat facial motoneurons
Brain Res. Mol. Brain Res.
Bone marrow-derived Schwann cells achieve fate commitment — a prerequisite for remyelination therapy
Exp. Neurol.
Neurotrophin-3 signaling maintains maturational homeostasis between neuronal populations in the olfactory epithelium
Mol. Cell. Neurosci.
The use of nonneuronal cells for gene delivery
Neurobiol. Dis.
BDNF activates CaMKIV and PKA in parallel to block MAG-mediated inhibition of neurite outgrowth
Mol. Cell. Neurosci.
From microsurgery to nanosurgery: how viral vectors may help repair the peripheral nerve
Prog. Brain Res.
Functional characterization of NGF-secreting cell grafts to the acutely injured spinal cord
Cell Transplant.
Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture
Mol. Cell. Neurosci.
Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration
Neuroscience
The nerve regenerative microenvironment: early behavior and partnership of axons and Schwann cells
Exp. Neurol.
Nerve growth factor enhances nerve regeneration through fibronectin grafts
J. Hand Surg. Br.
The use of immobilized neurotrophins to support neuron survival and guide nerve fiber growth in compartmentalized chambers
Biomaterials
Chapter 20: gene therapy perspectives for nerve repair
Int. Rev. Neurobiol.
Nerve growth factor enhances peripheral nerve regeneration in non-human primates
Scand. J. Plast. Reconstr. Surg. Hand Surg.
Implantation of Schwann cells in rat tendon autografts as a model for peripheral nerve repair: long term effects on functional recovery
Scand. J. Plast. Reconstr. Surg. Hand Surg.
Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons
Eur. J. Neurosci.
Cellular localization of nerve growth factor synthesis by in situ hybridization
EMBO J.
Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny
Nat. Protoc.
Skin-derived precursors generate myelinating Schwann cells that promote remyelination and functional recovery after contusion spinal cord injury
J. Neurosci.
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