We have recently reported that minimally disturbed adult CNS white matter can support regeneration of adult axons by using a novel microtransplantation technique to inject minute volumes of dissociated adult rat dorsal root ganglion neurons directly into adult rat CNS pathways (Davies et al., 1997). This atraumatic injection procedure minimized scarring and allowed considerable numbers of regenerating adult axons immediate access to the adult CNS glial terrain where they rapidly extended for long distances. A critical question remained as to whether degenerating white matter at acute and chronic stages (up to 3 months) after injury could still support regeneration. To investigate this, we have microtransplanted adult sensory neurons into degenerating white matter of the adult rat spinal cord several millimeters rostral to a severe lesion of the dorsal columns. Regeneration of donor sensory axons in both directions away from the site of transplantation was robust even within white matter undergoing fulminant Wallerian degeneration despite intimate contact with myelin. Along their route, the regrowing axons extended large numbers of collaterals into the adjacent dorsal horn. However, after entering the lesion, the rapidly extending growth cones stopped and became dystrophic within high concentrations of reactive glial matrix. Our results offer compelling evidence that the major environmental impediment to regeneration in the adult CNS is the molecular barrier that forms directly at the lesion site, and that degenerating white matter beyond the glial scar has a far greater intrinsic ability to support axon regeneration than previously thought possible.

Post-traumatic cystic cavitation, in which the size and severity of a CNS injury progress from a small area of direct trauma to a greatly enlarged secondary injury surrounded by glial scar tissue, is a poorly understood complication of damage to the brain and spinal cord. Using minimally invasive techniques to avoid primary physical injury, this study demonstrates in vivo that inflammatory processes alone initiate a cascade of secondary tissue damage, progressive cavitation, and glial scarring in the CNS. An in vitro model allowed us to test the hypothesis that specific molecules that stimulate macrophage inflammatory activation are an important step in initiating secondary neuropathology. Time-lapse video analyses of inflammationinduced cavitation in our in vitro model revealed that this process occurs primarily via a previously undescribed cellular mechanism involving dramatic astrocyte morphological changes and rapid migration. The physical process of cavitation leads to astrocyte abandonment of neuronal processes, neurite stretching, and secondary injury. The macrophage mannose receptor and the complement receptor type 3 ␤2-integrin are implicated in the cascade that induces cavity and scar formation. We also demonstrate that anti-inflammatory agents modulating transcription via the nuclear hormone receptor peroxisome proliferator-activated receptor-␥ may be therapeutic in preventing progressive cavitation by limiting inflammation and subsequent secondary damage after CNS injury. Injury to the adult mammalian C NS leads to a complex series of cellular and molecular events, as cells respond to trauma and attempt to repair damaged regions of the brain or spinal cord (for review, see Fitch and Silver, 1999a). Unlike the successf ul healing responses in the peripheral nervous system, adult C NS injury leads to permanent disability, because most severed axons fail to regenerate (Ramon y C ajal, 1928;Guth, 1975;Reier et al., 1983). A phenomenon that adds to the complexity of regenerative failure is the process of progressive cavitation in which, after days to weeks, a C NS injury can expand in size leading to a scarencapsulated cavity many times the size of the initial wound (Balentine, 1978). Although various hypotheses suggest that this secondary process of cavitation is related to ischemia (Balentine, 1978), hemorrhage (Ducker et al., 1971;Wallace et al., 1987), lysozyme activity (Kao et al., 1977), pulsatile hydrodynamics (Williams et al., 1981), or macrophage infiltration and inflammation (Blight, 1991a(Blight, , 1994Szczepanik et al., 1996;Fitch and Silver, 1997a;Z hang et al., 1997), the underlying causes of progressive axon damage and the cellular mechanisms that lead to cyst formation are poorly understood. Insights into this process will provide direction for therapeutic intervention designed to minimize secondary damage and lead to enhanced function after a debilitating injury.In this study we have used both in vivo and in vitro models to test our hypothesis that post-traumatic inflammation can lead to ...

We have developed a novel in vitro model of the glial scar that mimics the gradient of proteoglycan found in vivo after spinal cord injury. In this model, regenerated axons from adult sensory neurons that extended deeply into the gradient developed bulbous, vacuolated endings that looked remarkably similar to dystrophic endings formed in vivo. We demonstrate that despite their highly abnormal appearance and stalled forward progress, dystrophic endings are extremely dynamic both in vitro and in vivo after spinal cord injury. Time-lapse movies demonstrated that dystrophic endings continually send out membrane veils and endocytose large membrane vesicles at the leading edge, which were then retrogradely transported to the rear of the "growth cone." This direction of movement is contrary to membrane dynamics that occur during normal neurite outgrowth. As further evidence of this motility, dystrophic endings endocytosed large amounts of dextran both in vitro and in vivo. We now have an in vitro model of the glial scar that may serve as a potent tool for developing and screening potential treatments to help promote regeneration past the lesion in vivo.

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