Axon regeneration through scars and into sites of chronic spinal cord injury

Abstract

Cellular and extracellular inhibitors are thought to restrict axon growth after chronic spinal cord injury (SCI), confronting the axon with a combination of chronic astrocytosis and extracellular matrix-associated inhibitors that collectively constitute the chronic “scar.” To examine whether the chronically injured environment is strongly inhibitory to axonal regeneration, we grafted permissive autologous bone marrow stromal cells (MSCs) into mid-cervical SCI sites of adult rats, 6 weeks post-injury without resection of the “chronic scar.” Additional subjects received MSCs genetically modified to express neurotrophin-3 (NT-3), providing a further local stimulus to axon growth. Anatomical analysis 3 months post-injury revealed extensive astrocytosis surrounding the lesion site, together with dense deposition of the inhibitory extracellular matrix molecule NG2. Despite this inhibitory environment, axons penetrated the lesion site through the chronic scar. Robust axonal regeneration occurred into chronic lesion cavities expressing NT-3. Notably, chronically regenerating axons preferentially associated with Schwann cell surfaces expressing both inhibitory NG2 substrates and the permissive substrates L1 and NCAM in the lesion site. Collectively, these findings indicate that inhibitory factors deposited at sites of chronic SCI do not create impenetrable boundaries and that inhibition can be balanced by local and diffusible signals to generate robust axonal growth even without resecting chronic scar tissue.

Introduction

Damage to the central nervous system (CNS) induces a multicellular response to injury and the formation of a “glial scar.” Shortly after injury, astrocytes, oligodendrocyte precursor cells, microglia, macrophages, fibroblasts, leptomeningeal cells, and Schwann cells establish a dense cellular response surrounding the lesion site (Bunge et al., 1997, Fawcett and Asher, 1999, Fitch and Silver, 1999, Dawson et al., 2000). The cells express several inhibitory molecules which fill the extracellular matrix (ECM) surrounding the lesion site, including chondroitin sulfate proteoglycans (CSPGs) (Lemons et al., 1999, McTigue et al., 2001, Plant et al., 2001, Jones et al., 2002, Jones et al., 2003a, Jones et al., 2003b, Qi et al., 2003, Tang et al., 2003), keratan sulfate proteoglycans (KSPGs) (Jones and Tuszynski, 2002), tenascins (Deckner et al., 2000), ephrins (Miranda et al., 1999, Rodger et al., 2001), and semaphorins (Pasterkamp et al., 1999, De Winter et al., 2002, Moreau-Fauvarque et al., 2003). This extracellular and glial scar is thought to be a major factor limiting axon regeneration following CNS injury (Fawcett and Asher, 1999, Grimpe and Silver, 2002). Indeed, acute degradation of the scar improves axonal growth after SCI (Moon et al., 2001, Bradbury et al., 2002). Yet the scar is a relative rather than absolute impediment to axon growth: we recently reported that axon regeneration can readily occur through this inhibitory environment following acute SCI when a sufficient number of growth-promoting factors are also present in the injury site, including cell adhesion molecules and growth factors (Jones et al., 2003a). Thus, regeneration can proceed when local permissive signals balance and exceed inhibitory signals.

The glial and extracellular scar forms within days of SCI but becomes well established only after several weeks in rodents (Keirstead et al., 1998, Fawcett and Asher, 1999, Fitch and Silver, 1999, Jones et al., 2002). For this reason, the “chronic scar” is believed to constitute a greater impediment to axonal regeneration at subacute and chronic stages of injury than immediately post-injury. Based on the belief that the chronic scar must be removed to foster a more permissive environment for axon regeneration, inhibitory scar tissue is often surgically resected as a component of experimental investigations of chronic SCI (Houle, 1991, Grill et al., 1997b, Ye and Houle, 1997, Coumans et al., 2001, Woerly et al., 2001, Jin et al., 2002, Kwon et al., 2002, Lu et al., 2002, Dolbeare and Houle, 2003, Shumsky et al., 2003, Storer et al., 2003, Storer and Houle, 2003, Tobias et al., 2003, Tuszynski et al., 2003). A major caveat to this approach, however, is that resection of chronic scar tissue inevitably will introduce further tissue damage and elicit a second, unfavorable cellular response (Tuszynski et al., 2003). Thus, addressing the problem of the chronic scar represents a major challenge to the safety and efficacy of potential axon growth-promoting treatments for chronic SCI.

The present study further investigated the nature of the chronic scar and its ability to block axon growth by testing the hypothesis that chronically injured spinal cord axons can regenerate through chronic scar when local growth-stimulating factors are also present. Rats underwent cervical spinal cord injuries and 6 weeks later received cell suspension injections of syngenic bone marrow stromal cells into the cystic lesion site to provide a permissive environment for axon growth. The chronic scar was not removed. Separate subjects also received a positive stimulus for axon growth by injecting bone marrow stromal cells genetically modified to secrete neurotrophin-3 (NT-3) into the lesion site. A total of 3 months after the original lesion, we find that modest numbers of axons penetrate through chronic scar consisting of a mixture of inhibitory and growth-stimulating molecules and enter the lesion site; furthermore, robust axon growth can be induced by the local provision of neurotrophic factors, without resecting chronic scar.