Studies on the Development and Behavior of the Dystrophic Growth Cone, the Hallmark of Regeneration Failure, in anIn VitroModel of the Glial Scar and after Spinal Cord Injury

Abstract

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.

Inhibiting Glycosaminoglycan Chain Polymerization Decreases the Inhibitory Activity of Astrocyte-Derived Chondroitin Sulfate Proteoglycans

“…This indicates that siRNA transfection decreased the inhibitory activity of the CSPGs present in the CM. Although one study has shown that the concentration of CSPG at a spot border is consistently higher if the spot is allowed to dry (Tom et al, 2004), we found that the concentration of CSPG on the spot is uniform if the spot is incubated in a humidified chamber (Fig. 5C).…”

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering

“…In addition, our confocal results indicate preservation of CSPGs after decellularization. CSPGs are known to be potent neurite inhibitors [11,12,60,64]. By maintaining CSPG presence in the matrix, this has the potential to prevent nerve growth into the nucleus pulposus.…”

CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure

“…These sprouting tips of severed axons then remain stalled in place without further functional growth. Interestingly, these dystrophic growth cones are actually dynamic structures with continuing turnover of membrane components and are extremely dynamic in vitro and in vivo (Kerschensteiner et al, 2005;Tom et al, 2004b). Time-lapse video of these dystrophic endings illustrates repeated episodes of endocytosis and efforts to send out membrane veils (Tom, et al, 2004b).…”

“…Interestingly, these dystrophic growth cones are actually dynamic structures with continuing turnover of membrane components and are extremely dynamic in vitro and in vivo (Kerschensteiner et al, 2005;Tom et al, 2004b). Time-lapse video of these dystrophic endings illustrates repeated episodes of endocytosis and efforts to send out membrane veils (Tom, et al, 2004b). The failure of these dystrophic growth cones to continue growing to produce long-distance regeneration appears to be related to the inhibitory and nonpermissive nature of the injured brain and spinal cord.…”

“…The rapid and long lasting upregulation of proteoglycans within the vicinity of the glial scar has implicated them in the creation of nonpermissive growth environments in the CNS, similar to their role in boundary formation within the CNS during development (Fitch and Silver, 1997b;Grimpe and Silver, 2004;Silver, 1994;Snow et al, 1990). It is now well established that proteoglycans associated with reactive astrocytes clearly inhibit neurite outgrowth in vitro (Bovolenta et al, 1993;Canning et al, 1993;Dou and Levine, 1994;McKeon, et al, 1991;Snow et al, 1990;Tom, et al, 2004b), and these molecules also appear to play a key role in creating an environment that is not appropriate for successful long-distance regeneration of adult neurons after injury in vivo, since their modification allows successful regeneration to occur (Bradbury et al, 2002;Houle et al, 2006;Steinmetz et al, 2005).…”