Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair

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Abstract

Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a lentiviral vector encoding NGF (NGF-SCs). Transplantation of NGF-SCs in a rat sciatic nerve transection/repair model led to significant increase of NGF levels 2 weeks after injury and correspondingly to substantial improvement in axonal regeneration. Numbers of NF200, ChAT and CGRP-positive axon profiles, as well as the gastrocnemius muscle weights, were significantly higher in the NGF-Schwann cell group compared to the animals that received control SCs transduced with a lentiviral vector encoding GFP (GFP-SCs). Comparison with other models of NGF application signifies the important role of this neurotrophin during the early stages of regeneration, and supports the importance of developing combined gene and cell therapy for 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

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