Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons Zubair Ahmed, * Russell G. Dent, Wendy E. Leadbeater, Conrad Smith, Martin Berry, and Ann Logan Molecular Neuroscience Group, Department of Medicine, University of Birmingham, Birmingham B15 2TT, UK Received 28 June 2004; accepted 23 August 2004 Available online 28 September 2004 After injury to the central nervous system, a glial/collagen scar forms at the lesion site, which is thought to act as a physicochem- ical barrier to regenerating axons. We have shown that scar formation in the transected optic nerve (ON) is attenuated when robust growth of axons is stimulated. Matrix metalloproteases (MMP), modulated by tissue inhibitors of MMP (TIMP), degrade a wide variety of extracellular matrix components (ECM) and may be activated by growing axons to remodel the ECM to allow regeneration through the inhibitory environment of the glial or collagen scar. Here, we investigate whether MMP levels are modulated in a nonregenerating (scarring) versus a regenerating (nonscarring) model of ON injury in vivo. Western blotting and immunohistochemistry revealed that MMP-1, -2, and -9 levels were higher and TIMP-1 and TIMP-2 levels were lower in regenerating compared to nonregenerating ON and retinae. In situ zymography demonstrated significantly greater MMP-related gelatinase activity in the regenerating model, mainly colocalized to astrocytes in the proximal ON stump and around the lesion site. These results suggest that activation of MMP and coincident down-regulation of TIMP may act to attenuate the inhibitory scarring in the regenerating ON, thus transforming the ON into a noninhibitory pathway for axon regrowth. D 2004 Elsevier Inc. All rights reserved. Introduction Injury to the central nervous system (CNS) induces inflam- mation and scar deposition at the lesion site. The sequelae of neuron death and abortive regeneration of severed axons causes permanent loss of function (Berry et al., 1999, 2001; Logan and Berry, 2002; Logan et al., 1994b, 1999). If appropriately stimulated, CNS neurons can, however, extend axons for long distances through growth permissive peripheral nerve grafts, demonstrating underlying growth potential (David and Aguayo, 1981, 1985). The failure of regeneration of axons in the injured adult mammalian CNS is probably multifactorial; including the unavailability of essential neurotrophic factors and/or their receptors and the arrest of axon growth by the interaction of axon growth inhibitory ligands in the CNS environment with their cognate receptors on axonal growth cones (Bandtlow, 2003; Berry and Logan, 2000; Berry et al., 1998; Bulsara et al., 2002; Sandvig et al., 2004; Schwab, 2002; Strittmatter, 2002; Tang, 2003; Woolf, 2003; Woolf and Bloechlinger, 2002). The formation of a glial/collagen scar within CNS lesions is probably, on balance, detrimental to successful regeneration (Berry et al., 1983; Maxwell et al., 1990; Windle et al., 1953) since it contains a dense array of extracellular matrix (ECM) proteins, including collagen, laminin, fibronectin, and chondroitin sulphate proteoglycans (CSPG), which are deposited by reactive astrocytes in the scar and by invading meningeal fibroblasts (Berry et al., 1999; Logan and Berry, 2002). It was once thought that the glial scar exerted a mechanical barrier to axon regrowth (Clemente, 1964; Windle et al., 1953). However, the astroglial or fibrotic scar more likely provides a deleterious molecular environment, comprising either an incompatible growth substrate and/or axon growth inhibitory ligands, e.g. Semaphorins and ephrins (Fawcett and Asher, 1999; Pasterkamp et al., 1998, 1999, 2001; Sandvig et al., 2004). A large number of regulatory cytokines and growth factors, including transforming growth factor-h (TGF-h), are released and/or activated in CNS wounds by blood cells and platelets and endogenous glia (Logan and Berry, 1993; McPherron and Lee, 1996). TGF-h1 and -h2 induce CNS scar formation (Lagord et al., 2002; Logan et al., 1992, 1994a, 1999), while neutralization of TGF-h1 significantly reduced the deposition of a scar ECM in a CNS lesion without concomitant enhanced axon regeneration (Logan et al., 1994a, 1999; Davies et al., 2004). 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2004.08.013 * Corresponding author. Molecular Neuroscience Group, Department of Medicine, 3rd Floor Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, UK. Fax: +44 121 4720499. E-mail address: z.ahmed.1@bham.ac.uk (Z. Ahmed). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 28 (2005) 64 – 78