It is widely accepted that the adult mammalian central nervous system (CNS) is unable to regenerate axons. In addition to physical or molecular barriers presented by glial scarring at the lesion site, it has been suggested that the normal myelinated CNS environment contains potent growth inhibitors or lacks growth-promoting molecules. Here we investigate whether adult CNS white matter can support long-distance regeneration of adult axons in the absence of glial scarring, by using a microtransplantation technique that minimizes scarring to inject minute volumes of dissociated adult rat dorsal root ganglia directly into adult rat CNS pathways. This atraumatic injection procedure allowed considerable numbers of regenerating adult axons immediate access to the host glial terrain, where we found that they rapidly extended for long distances in white matter, eventually invading grey matter. Abortive regeneration correlated precisely with increased levels of proteoglycans within the extracellular matrix at the transplant interface, whereas successfully regenerating transplants were associated with minimal upregulation of these molecules. Our results demonstrate, to our knowledge for the first time, that reactive glial extracellular matrix at the lesion site is directly associated with failure of axon regrowth in vivo, and that adult myelinated white matter tracts beyond the glial scar can be highly permissive for regeneration.

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.

Previous studies have correlated the failure of axon regeneration after spinal cord injury with axons contacting scar tissue rich in chondroitin sulfate proteoglycans (CSPGs; Davies et al., 1999). In the present study, we have conducted immunohistochemical and quantitative Western blot analysis of five axon-growth-inhibitory CSPGs and tenascin-C within stab injuries of adult rat spinal cord at time points ranging from 24 hr to 6 months post injury. Quantitative Western blot analysis showed robust increases in neurocan, tenascin-C, and NG2 levels by 24 hr, suggesting that these molecules play a role in preventing axon regeneration across acutely forming scar tissue. Peak levels of 245/130 kD neurocan, NG2, and 250/200 kD tenascin-C were reached at 8 days, with maximum levels of phosphacan and 140/80 kD brevican attained later, at 1 month post injury. Versican V2 protein levels, however, displayed an opposite trend, dropping below unlesioned spinal cord values at all time points studied. Confocal microscopy at 8 days post injury revealed heightened immunoreactivity for phosphacan, NG2, and tenascin-C, particularly within fibronectin(+) scar tissue at lesion centers. In contrast, neurocan was displayed within lesion margins on the processes of stellate NG2(+) cells and, to a much lesser extent, by astrocytes. At 6 months post injury, 130 kD neurocan, brevican, and NG2 levels within chronic scar tissue remained significantly above control. Our results show novel expression patterns and cell associations of inhibitory CSPGs and tenascin-C that have important implications for axon regeneration across acute and chronic spinal cord scar tissue.

Fibrosis is a characteristic feature in the pathogenesis of a wide spectrum of diseases. Recently, it was suggested that IL-13-dependent fibrosis develops through a TGF-β1 and matrix metalloproteinase-9-dependent (MMP-9) mechanism. However, the significance of this pathway in a natural disorder of fibrosis was not investigated. In this study, we examined the role of TGF-β in IL-13-dependent liver fibrosis caused by Schistosoma mansoni infection. Infected IL-13−/− mice showed an almost complete abrogation of fibrosis despite continued and undiminished production of TGF-β1. Although MMP-9 activity was implicated in the IL-13 pathway, MMP-9−/− mice displayed no reduction in fibrosis, even when chronically infected. To directly test the requirement for TGF-β, studies were also performed with neutralizing anti-TGF-β Abs, soluble antagonists (soluble TGF-βR-Fc), and Tg mice (Smad3−/− and TGF-βRII-Fc Tg) that have disruptions in all or part of the TGF-β signaling cascade. In all cases, fibrosis developed normally and with kinetics similar to wild-type mice. Production of IL-13 was also unaffected. Finally, several genes, including interstitial collagens, several MMPs, and tissue inhibitors of metalloprotease-1 were up-regulated in TGF-β1−/− mice by IL-13, demonstrating that IL-13 activates the fibrogenic machinery directly. Together, these studies provide unequivocal evidence of a pathway of fibrogenesis that is IL-13 dependent but TGF-β1 independent, illustrating the importance of targeting IL-13 directly in the treatment of infection-induced fibrosis.

BackgroundTwo critical challenges in developing cell-transplantation therapies for injured or diseased tissues are to identify optimal cells and harmful side effects. This is of particular concern in the case of spinal cord injury, where recent studies have shown that transplanted neuroepithelial stem cells can generate pain syndromes.ResultsWe have previously shown that astrocytes derived from glial-restricted precursor cells (GRPs) treated with bone morphogenetic protein-4 (BMP-4) can promote robust axon regeneration and functional recovery when transplanted into rat spinal cord injuries. In contrast, we now show that transplantation of GRP-derived astrocytes (GDAs) generated by exposure to the gp130 agonist ciliary neurotrophic factor (GDAsCNTF), the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDACNTF cells promoted the onset of mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted through 5 weeks post-injury. Delayed onset of similar neuropathic pain was also caused by transplantation of undifferentiated GRPs. In contrast, rats transplanted with GDAsBMP did not exhibit pain syndromes.ConclusionOur results show that not all astrocytes derived from embryonic precursors are equally beneficial for spinal cord repair and they provide the first identification of a differentiated neural cell type that can cause pain syndromes on transplantation into the damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells to cause allodynia before initiating clinical trials. They also confirm the particular promise of GDAs treated with bone morphogenetic protein for spinal cord injury repair.

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.

The formation of misaligned scar tissue by a variety of cell types expressing multiple axon growth inhibitory proteoglycans presents a physical and molecular barrier to axon regeneration after adult spinal cord injuries. Decorin is a small, leucine-rich proteoglycan that has previously been shown to reduce astrogliosis and basal lamina formation in acute cerebral cortex stab injuries. We have therefore tested whether mini pump infusion of hr-decorin into acute stab injuries of the adult rat spinal cord can not only inhibit formation of an astroglial limitans but also deposition of the axon growth inhibitory proteoglycans neurocan, NG2, phosphacan and brevican. Combined immunohistochemical and quantitative Western blot analysis revealed major reductions in levels of core protein expression (>80% for 130-kDa neurocan, 145/80-kDa brevican, 300-kDa phosphacan) and immunoreactivity for all four chondroitin sulfate proteoglycans (CSPGs) within decorin-treated injuries compared with untreated controls. Astrogliosis within lesion margins and the accumulation of OX42+ macrophages/microglia within lesion centres were also significantly reduced. These decorin-induced changes in scar formation combined to promote the striking ability of axons from microtransplanted adult sensory neurons to enter, grow within and exit decorin-infused spinal cord injuries, in sharp contrast to the complete failure of axons to cross untreated, CSPG-rich lesions. Decorin pretreatment of meningial fibroblasts in vitro also resulted in a three-fold increase in neurite outgrowth from co-cultured adult sensory neurons and suppression of NG2 immunoreactivity. The ability of decorin to promote axon growth across acute spinal cord injuries via a coordinated suppression of inflammation, CSPG expression and astroglial scar formation make decorin treatment a promising component of future spinal cord regeneration strategies.

We have identified an alternate developmental pathway in the life cycle of the trematode pathogen Schistosoma mansoni. This pathway is used in immunodeficient hosts in which the parasite fails to receive appropriate signals from the host immune system. Helminth development is altered at an early stage during infection, resulting in the appearance of attenuated forms that prolong survival of host and parasite. Hepatic CD4+ T lymphocyte populations are an integral component of the immune signal recognized by the parasite.