TGF-α increases astrocyte invasion and promotes axonal growth into the lesion following spinal cord injury in mice
Introduction
Astrocytes play multifaceted roles in the developing and mature central nervous system, and contribute to repair after central nervous system (CNS) injury. During development, radial glial cells, precursors to astrocytes, are produced alongside neurons and are essential for the support of neuronal migration and axon guidance (Rakic, 1978, Vaccarino et al., 2007). In the mature intact CNS, astrocytes regulate synaptic activity (Norenberg, 1979, Anderson and Swanson, 2000, Schousboe et al., 2004, Tanaka, 2007), modulate the extracellular ionic environment (Walz et al., 1984, Walz, 2000), control volume homeostasis (Sykova et al., 1992), and maintain the characteristics of the blood–brain barrier (Vise et al., 1975, Haseloff et al., 2005, Abbott et al., 2006). After injury, mature astrocytes respond rapidly to local environmental cues by undergoing hypertrophy, proliferating, and migrating to the edge of the lesion site (Buffo et al., 2008). One consequence of these responses is the production of the glial scar, a physical and chemical barrier to regeneration (Luizzi and Lasek, 1987, Rudge and Silver, 1990, Fitch and Silver, 1997).
Within the local environment of a CNS injury, the pro-inflammatory and growth inhibitory effects of reactive astrocytes are offset by the essential role of the astrocyte response in neuroprotection and the potential for astrocytes to support axon growth. Astrocytes contribute to restoring the extracellular ionic environment (Sykova et al., 1992), sequestering extracellular glutamate (Rothstein et al., 1996), and producing neurotrophic factors (Lee et al., 1998, Krenz and Weaver, 2000, Ikeda et al., 2001, do Carmo Cunha et al., 2007) after injury. Although reactive astrocytes produce chondroitin sulfate proteoglycans (CSPGs) (Zuo et al., 1998, Morgenstern et al., 2002, Fitch and Silver, 2008), molecules that are inhibitory to axon growth, they also produce adhesive extracellular matrix (ECM) molecules, such as laminin, that provide a substrate supportive to growth (Frisen et al., 1995, Costa et al., 2002). Thus, the astrocyte response to injury is necessary for successful homeostasis and tissue repair. Indeed, if astrocyte proliferation (Faulkner et al., 2004) or migration (Okada et al., 2006) is disrupted after spinal cord injury, wound healing and recovery is diminished. Understanding the balance between the protective and inhibitory functions of astrocytes is important in order to devise strategies that will prompt beneficial astrocytic repair following injury.
TGF-α is an endogenous, mitogenic ligand that can promote changes in astrocytes and other cells via activation of the epidermal growth factor receptor (EGFR) (Lee et al., 1995, Junier, 2000). TGF-α administration increases glial proliferation and survival both in vitro (Sharif et al., 2006b) and in vivo (Fallon et al., 2000). In vitro, TGF-α alters astrocyte phenotype by increasing expression of the radial glial markers BLBP and RC2 and inducing an immature, bipolar astrocytic morphology (Zhou et al., 2001, Sharif et al., 2006a). In vivo, TGF-α overexpression induces astrocyte hypertrophy (Rabchevsky et al., 1998) and neuroprotection (Boillee et al., 2001), and administration of exogenous TGF-α induces the migration of neural and glial progenitors from the subventricular zone (Fallon et al., 2000). Induction of the ErbB2 EGFR subunit, which upregulates TGF-α and EGFR (Xie et al., 1999), also promotes a neural supportive radial glial phenotype in the adult cerebral cortex (Ghashghaei et al., 2007). These bipolar and radial glial phenotypes are supportive to neuronal migration in the developing brain (Vaccarino et al., 2007) and axonal growth in the developing spinal cord (Joosten and Gribnau, 1989), suggesting that this pattern of protein expression and morphology is a growth-supportive phenotype.
Based on these findings, we hypothesized that administration of TGF-α to the injured spinal cord would alter the glial response to injury and create a glial environment that would support axonal growth. Following a two-week infusion of human recombinant TGF-α, we detected a striking astrocyte-rich matrix that extended into the lesion site of the TGF-α-treated mice. The treated injury site included an enhanced axonal plexus that extended throughout and beyond the edges of the glial border into the center of the lesion. The increased axonal growth in the lesion core prevailed despite production of the inhibitory CSPG neurocan by surrounding astrocytes. The axon-rich matrix was associated with increased laminin immunoreactivity throughout the lesion site. Thus, administration of exogenous TGF-α alters the evolution of the local environment after spinal cord injury, resulting in the production of an extracellular matrix that is permissive to both astrocyte invasion and axonal growth.
Section snippets
Subjects
Adult female C57BL/6 mice 10 weeks of age weighing 17 to 20 g (Jackson Laboratories, Bar Harbor, ME) were housed in barrier cages in a temperature and humidity controlled room with ad libidum access to food and water. After 1 week of acclimation, all mice were evaluated for normal locomotor function using the Basso Mouse Scale (BMS, Basso et al., 2006, see below). All animal experimentation procedures were performed according to approved protocols and in accordance with the NIH Guide to the
TGF-α infusion increases the number of newborn cells in the lesion center and dorsal horn after contusion injury
TGF-α can stimulate proliferation of a variety of cell types. To determine the effects of TGF-α infusion on cell proliferation after SCI, BrdU was administered daily for the first week after laminectomy or contusion injury. At 3 weeks after laminectomy, there were very few BrdU+ nuclei in TGF-α- or vehicle-infused animals and there was no effect of TGF-α on the total number of BrdU+ nuclei or BrdU+ nuclei in gray or white matter regions (not shown). However, after SCI, BrdU+ nuclei were
Discussion
The astrocytes of the glial scar comprise a major impediment to recovery after SCI by forming a physical and chemical barrier that inhibits axon growth into and beyond the lesion edge. A common strategy to improve axonal growth and regeneration is to try to reduce the glial and astrocytic response to injury while maintaining the supportive functions of these cells. However, through an alternative approach, by stimulating astrocytes and other cells to divide, migrate into the lesion and modify
Conclusions
The development of a glial scar at the edge of an injury to the CNS is a significant and well-established barrier to axonal growth and an impediment to maximizing functional recovery. However, it is clear that the principle cells that contribute to the scar are heterogeneous in structure and function, highly influenced by local cues, and exhibit plasticity in their phenotype and protein synthesis capacity. This study demonstrates the first step in the investigation of a unique and paradoxical
Acknowledgments
The authors gratefully acknowledge the assistance of Patricia Walters with surgeries and animal care. We thank Megan Detloff and Dr. Dana McTigue for critical review of the manuscript. This work was funded by R01 NS043246 and P30-NS045748.
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