Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair
Introduction
Injury to the peripheral nerve is common and debilitating. Such injuries may occur in the context of trauma, or in specific disorders of peripheral nerves known as neuropathies. A severe peripheral nerve injury 1(PNI) affects 2.8% of trauma patients (Noble et al., 1998), and approximately 360,000 people in the United States suffer from upper extremity PNI yearly, resulting in 8,648,000 and 4,916,000 restricted activity days and bed/disability days, respectively (Kelsey et al., 1997). It is assumed that people recover from PNI, as compared to those of the central nervous system (CNS). While axons in peripheral nerves do regenerate better than neurons within the CNS, recovery is very frequently incomplete, misdirected and/or associated with debilitating neuropathic pain (Sunderland, 1978). Regenerating peripheral axons face challenges including the obstacle of traversing a large gap between distal and proximal ends of a nerve, and regenerating within a distal denervated nerve whose Schwann cells (SCs) become increasingly less able to support axon regeneration over time. Nerve injury gaps, or scarring within the injured nerve, are currently managed by resecting the scarred segment and placement of interposed nerve grafts (Millesi et al., 1972), which provide a pathway for regenerating axons from the proximal nerve stump to innervate the distal stump. Yet recovery following nerve or nerve graft repair is extremely limited; for example after median nerve suture repair, only 25% of the patients recover full motor function and only 3% full sensory function (Kelsey et al., 1997). Poor outcome from peripheral nerve injury is especially evident when repair is delayed (Jivan et al., 2009, Kline and Hudson, 1995, Samii et al., 1997, Samii et al., 2003, Seddon, 1972, Sunderland, 1978, Sunderland, 1991). Even patients who have immediate nerve repair are subject to distal nerve denervation for considerable periods of time as the rate of regeneration is relatively low (approximately 1 mm/day in humans (Sunderland, 1947)), and the distances that regenerating nerve fibers need to grow are typically long. Hence, every severe human nerve injury creates a situation in which the SCs of the distal nerve are denervated and less able to support axonal growth (Fu and Gordon, 1997). The capacity of the denervated distal nerve to support axonal regeneration is normally highly dependent on de-differentiated and proliferating SCs within the basal lamina tubes (Bungner, 1891) that guide elongating axons to their denervated target (Ramon y Cajal, 1928). Unfortunately, denervated SCs progressively lose their ability to express regeneration-assisting genes (Li et al., 1997, You et al., 1997) and in effect become “turned off” (Fu and Gordon, 1997). This loss of vitality and functionality in distal SCs directly translates to poor behavioral recovery (Fu and Gordon, 1995). Hence, SC replacement or rejuvenation approaches are of considerable interest (Lehmann and Hoke, 2010, Sulaiman and Gordon, 2002).
The skin dermis, which can be obtained from patients with minimal morbidity, contains a reservoir of self-renewing precursors that exhibit remarkable similarity to embryonic neural crest cells, which is the tissue of origin for SCs (Biernaskie and Miller, 2010). These neural crest-related precursor cells (termed skin-derived precursors, or SKPs) readily differentiate into neural crest cell types in vitro when supplied with the appropriate cues (Fernandes et al., 2004, McKenzie et al., 2006, Toma et al., 2001, Toma et al., 2005). Specifically, SKPs respond to neuregulins in vitro to generate SCs (hereafter referred to as SKP-SCs) (McKenzie et al., 2006), highlighting their potential to serve as transplantable cells for nerve injury (Carroll et al., 1997, Mahanthappa et al., 1996). SKP-SCs survive and associate with axons within both normal and degenerating mouse sciatic nerves, where they express a myelinating phenotype (McKenzie et al., 2006). Moreover, SKPs appear to generate bona fide functional SCs given that they myelinate sensory neurons in dorsal root ganglion co-cultures, dysmyelinated shiverer mouse nerve axons in vivo (McKenzie et al., 2006), and normal rat axons in vivo within nerve (Walsh et al., 2012) and spinal cord (Biernaskie et al., 2007). As proof of principal that SKP-SCs could recapitulate the function of endogenous SCs, SKP-SC seeded acellular nerve grafts demonstrated significant histomorphometric and electrophysiological improvement as compared to diluent and even nerve derived-SC controls, with equivalent outcomes relative to nerve autograft controls (S. Walsh et al., 2009). In a more challenging regeneration paradigm of chronic nerve denervation, SKP-SC treated animals exhibited superior axonal regeneration compared to denervated nerve receiving media alone, with significantly higher counts of regenerated motor neurons, histological recovery and muscle re-innervation (S.K. Walsh et al., 2010). While these studies demonstrated the promise and potential of SKP therapy, convincing behavioral outcome data have been lacking. We hypothesized that SKP-SC therapy would improve behavioral outcomes for nerve injury repair, and tested this hypothesis in all three scenarios encountered in clinical practice: acute nerve repair, chronic nerve repair and within a nerve gap injury repaired with a nerve graft. SKP-SC therapy was used as an adjunct (and compared to all appropriate controls) to standard microsurgical nerve repair and indeed dramatically improved behavioral outcome following acute nerve repair, nerve graft repair, and even following delayed nerve repair.
Section snippets
Rat primary SKP culture and differentiation into SCs
SKPs were islolated and propagated as previously described (Biernaskie et al., 2007). Briefly, back skin was dissected from neonatal P2 Lewis rats and collected into Hanks balanced salt solution (HBSS; Gibco). Fat tissue, blood vessels and nerve endings were removed, chopped into 1 cm2 pieces and floated over dispase (2 mg/ml, Gibco) for 40 min at 37 °C. Epidermis was removed and dermis was minced and then digested using Collagenase type XI (1 mg/ml, Gibco) for 45 min at 37 °C. Primary dermal cells
GRAFT repair experiment
Rat's body weights were recorded weekly, and all rats remained healthy with only moderate weight gain at completion of the study, which was similar across all groups.
The efficacy and mechanistic considerations of SKP-SC therapy
In prior studies, we showed that SKP-SCs supported robust axon outgrowth and muscle reinnervation in both acute and chronic models of rodent nerve injury (Walsh and Midha, 2010, S. Walsh et al., 2009, S.K. Walsh et al., 2009). In the present work, our data confirm and extend these findings by demonstrating that SKP-SCs significantly improved behavioral recovery over standard nerve and nerve graft repair alone. Surprisingly, the effects were much earlier than would be expected following acute
Acknowledgments
This research was partially supported by grants to R.M. from the Canadian Institute for Health Research (Regenerative medicine and nanomedicine team grant #163322) to R.M. and A.W. by the Robertson Fund at the University of Calgary, and by a grant to J.B. by the Canadian Institutes of Health Research. Post-doctoral fellowship support awards to H.K. and F.S. were provided by the Hotchkiss Brain Institute (HBI) and Integra LifeSciences Foundation, respectively. Salary support to R.K. and A.I.
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