Transplanted neural stem cells promote axonal regeneration through chronically denervated peripheral nerves
Introduction
Compared to the central nervous system (CNS), regeneration of injured nerves in the peripheral nervous system (PNS) is robust. The capacity to extend axonal sprouts persists for many months in axotomized neurons even if the axon does not innervate a target organ (Fu and Gordon, 1995a). In contrast, the capacity of a chronically denervated nerve segment to support axonal regeneration decreases dramatically as the duration of denervation increases Fu and Gordon, 1995b, Li et al., 1997, Sulaiman and Gordon, 2000, Sunderland, 1952, Woodhall and Beebe, 1956. Many local factors probably influence this decreased regenerative capacity. These include a reduction in the expression of regeneration-associated Schwann cell molecules such as p75 (You et al., 1997), neuregulin receptors c-erb2 and c-erb4 (Li et al., 1997), and growth factors such as glial cell-line derived neurotrophic factor (GDNF) (Hoke et al., 2002). Loss of growth support is compounded by the eventual atrophy of Schwann cells and breakdown of the bands of Büngner, with their Schwann cell basal lamina scaffoldings that isolate axons from the endoneurial extracellular matrix (ECM) Griffin and Hoffman, 1993, Griffin et al., 1996.
Stem cells are being investigated as replacement therapy for a variety of disorders including central nervous system degenerative diseases Bjorklund and Lindvall, 2000, Lovell-Badge, 2001, Martinez-Serrano et al., 2001, Parenteau and Hardin-Young, 2002, Watt and Hogan, 2000. Stem cells can be engineered to deliver appropriate support to the intrinsic cells in a diseased organ system Akerud et al., 2001, Akerud et al., 2002, Castellanos et al., 2002, Himes et al., 2001, Park et al., 2002. In this paper, we show that transplantation of C17.2 NSCs into chronically denervated peripheral nerves resulted in both morphological and electrophysiological improvement after prolonged periods of denervation. Furthermore, we show that this improved regeneration in the NSC-transplanted animals is associated with a decrease in the expression of one of the inhibitory ECM components, chondroitin sulphate proteoglycans (CSPGs). The mechanism of this decrease in CSPGs is unknown, but secretion of CSPG degrading matrix metalloprotease-2 (MMP-2) by NSCs is likely to play a role. In addition to a decline in the expression of one of the inhibitory ECM components (CSPGs), secretion of a variety of neurotrophic factors is likely to have played an important role in the improved regeneration observed with the transplanted NSCs.
Section snippets
Materials and methods
All animal surgeries were conducted under the protocols approved by the Johns Hopkins University Animal Care and Use Committee according to guidelines established by National Institutes of Health and American Association for the Accreditation of Laboratory Animal Care. Tissue culture supplies were obtained from Invitrogen unless noted otherwise. C17.2 cells stably overexpressing GDNF or a control gene (bleomycin resistance) were kindly provided by Dr. E. Arenas of the Karolinska Institute,
Results
Out of the eight animals in each group, seven animals in the vehicle group and five animals each in the stem cell-transplanted groups (C17.2-GDNF and C17.2-Bleo) were available for complete analysis. One animal in the vehicle group and two animals each in the stem cell-transplanted groups died from complications of anesthesia during repeated motor nerve conduction studies. Furthermore, one animal each in both stem cell transplanted groups developed axillary tumors, and they were sacrificed to
Discussion
We think of nerve regeneration in the PNS as being excellent, but that is because of many studies done in young rat PNS Goldberg and Frank, 1981, Navarro et al., 1988, Tanaka and Webster, 1991, Verdu et al., 1995. In man, functional recovery after a peripheral nerve injury is often suboptimal (Woodhall and Beebe, 1956). Even in the rat, the outcome is poor following prolonged axotomy and denervation Fu and Gordon, 1995a, Fu and Gordon, 1995b, Li et al., 1997. Using both electrophysiological and
Acknowledgements
This work was supported by The Robert Packard Center for ALS Research at Johns Hopkins (AH).
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