Multiplex array proteomics detects increased MMP-8 in CSF after spinal cord injury.

INTRODUCTION: A variety of methods have been used to study inflammatory changes in the acutely injured spinal cord. Recently novel multiplex assays have been used in an attempt to overcome limitations in numbers of available targets studied in a single experiment. Other technical challenges in developing pre-clinical rodent models to investigate biomarkers in cerebrospinal fluid (CSF) include relatively small volumes of sample and low concentrations of target proteins. The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic spinal cord injury (SCI). Our other aim was to test a microarray proteomics platform specifically for this application. METHODS: A 34 cytokine sandwich ELISA microarray was used to study inflammatory changes in CSF samples taken 12 days post-cervical SCI in adult rats. The difference between the median foreground signal and the median background signal was measured. Bonferroni and Benjamini-Hochburg multiple testing corrections were applied to limit the False Discovery Rate (FDR), and a linear mixed model was used to account for repeated measures in the array. RESULTS: We report a novel subacute SCI biomarker, elevated levels of matrix metalloproteinase-8 protein in CSF, and discuss application of statistical models designed for multiplex testing. CONCLUSIONS: Major advantages of this assay over conventional methods include high-throughput format, good sensitivity, and reduced sample consumption. This method can be useful for creating comprehensive inflammatory profiles, and biomarkers can be used in the clinic to assess injury severity and to objectively grade response to therapy.

Decorin, erythroblastic leukaemia viral oncogene homologue B4 and signal transducer and activator of transcription 3 regulation of semaphorin 3A in central nervous system scar tissue.

Scar tissue at sites of traumatic injury in the adult central nervous system presents a combined physical and molecular impediment to axon regeneration. Of multiple known central nervous system scar associated axon growth inhibitors, semaphorin 3A has been shown to be strongly expressed by invading leptomeningeal fibroblasts. We have previously demonstrated that infusion of the small leucine-rich proteoglycan decorin results in major suppression of several growth inhibitory chondroitin sulphate proteoglycans and growth of adult sensory axons across acute spinal cord injuries. Furthermore, decorin treatment of leptomeningeal fibroblasts significantly increases their ability to support neurite growth of co-cultured adult dorsal root ganglion neurons. In the present study we show that decorin has the ability to suppress semaphorin 3A expression within adult rat cerebral cortex scar tissue and in primary leptomeningeal fibroblasts in vitro. Infusion of decorin core protein for eight days resulted in a significant reduction of semaphorin 3A messenger RNA expression within injury sites compared with saline-treated control animals. Both in situ hybridization and immunostaining confirmed that semaphorin 3A messenger RNA expression and protein levels are significantly reduced in decorin-treated animals. Similarly, decorin treatment decreased the expression of semaphorin 3A messenger RNA in cultured rat leptomeningeal fibroblasts compared with untreated cells. Mechanistic studies revealed that decorin-mediated suppression of semaphorin 3A critically depends on erythroblastic leukaemia viral oncogene homologue B4 and signal transducer and activator of transcription 3 function. Collectively, our studies show that in addition to suppressing the levels of inhibitory chondroitin sulphate proteoglycans, decorin has the ability to suppress semaphorin 3A in the injured central nervous system. Our findings provide further evidence for the use of decorin as a potential therapy for promoting axonal growth and repair in the injured adult mammalian brain and spinal cord.

Cell therapies for the central nervous system: how do we identify the best candidates?

PURPOSE OF REVIEW: Central to the obstacles to be overcome in moving promising cell-based therapies from the laboratory to the clinic is that of determining which of the many cell types being examined are optimal for repairing particular lesions. RECENT FINDINGS: Our studies on astrocyte replacement therapies demonstrate clearly that some cells are far better than others at promoting recovery in spinal cord injury and that, at least in some cases, transplanting undifferentiated precursor cells is far less useful than transplanting specific astrocytes derived from those precursor cells. But further comparison between different approaches is hindered by the difficulties in replicating results between laboratories, even for well defined pharmacological agents and bioactive proteins. These difficulties in replication appear most likely to be due to unrecognized nuances in lesion characteristics and in the details of delivery of therapies. SUMMARY: We propose that the challenge of reproducibility provides a critical opportunity for refining cell-based therapies. If the utility of a particular approach is so restricted that even small changes in lesions or treatment protocols eliminate benefit, then the variability inherent in clinical injuries will frustrate translation. In contrast, rising to this challenge may enable discovery of refinements needed to confer the robustness needed for successful clinical trials.

Decorin promotes plasminogen/plasmin expression within acute spinal cord injuries and by adult microglia in vitro.

Spinal cord scar tissue presents a combined physical and molecular barrier to axon regeneration. Theoretically, spinal cord injuries (SCIs) can be rendered more permissive to axon growth by either suppressing synthesis of misaligned, fibrotic scar tissue and associated axon growth inhibitors, or enzymatically degrading them. We have previously shown that acute infusion of human recombinant decorin core protein into discreet stab injuries of the rat dorsal column pathways effected a major suppression of inflammation, astrogliosis, and multiple axon growth inhibitory chondroitin sulfate proteoglycans, which combined to promote rapid axon growth across the injury site. The high efficiency of chondroitin sulfate proteoglycan (CSPG) core protein suppression (approximately 90%) suggested that decorin may promote CSPG degradation in addition to suppressing CSPG synthesis. As the serine protease plasmin can degrade axon growth inhibitory CSPGs (neurocan and phosphacan) and its zymogen, plasmininogen is synthesized by microglia, we have investigated whether decorin treatment of acute SCIs and cultured adult spinal cord microglia can increase plasminogen/ plasmin synthesis. Infusion of hr-decorin over the first 8 days post-SCI induced 10- and 17-fold increases in plasminogen and plasmin protein levels, respectively, within sites of injury and a threefold increase in microglial plasminogen mRNA in vitro. In addition to potentially degrading multiple axon growth inhibitory components of the glial scar, plasmin is known to play major roles in activating neurotrophins and promoting central nervous system (CNS) plasticity. The wider implications of decorin induction of plasmin in the injured spinal cord for axon regeneration, and recovery of function at acute and chronic time points post-SCI are reviewed.

Transplantation of specific human astrocytes promotes functional recovery after spinal cord injury.

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.

Precursor cell biology and the development of astrocyte transplantation therapies: lessons from spinal cord injury.

This review summarizes current progress on development of astrocyte transplantation therapies for repair of the damaged central nervous system. Replacement of neurons in the injured or diseased central nervous system is currently one of the most popular therapeutic goals, but if neuronal replacement is attempted in the absence of appropriate supporting cells (astrocytes and oligodendrocytes), then the chances of restoring neurological functional are greatly reduced. Although the past 20 years have offered great progress on oligodendrocyte replacement therapies, astrocyte transplantation therapies have been both less explored and comparatively less successful. We have now developed successful astrocyte transplantation therapies by pre-differentiating glial restricted precursor (GRP) cells into a specific population of GRP cell-derived astrocytes (GDAs) by exposing the GRP cells to bone morphogenetic protein-4 (BMP) prior to transplantation. When transplanted into transected rat spinal cord, rat and human GDAs(BMP) promote extensive axonal regeneration, rescue neuronal cell survival, realign tissue structure, and restore behavior to pre-injury levels on a grid-walk analysis of volitional foot placement. Such benefits are not provided by GRP cells themselves, demonstrating that the lesion environment does not direct differentiation in a manner optimally beneficial for the restoration of function. Such benefits also are not provided by transplantation of a different population of astrocytes generated from GRP cells exposed to ciliary neurotrophic factor (GDAs(CNTF)), thus providing the first transplantation-based evidence of functional heterogeneity in astrocyte populations. Moreover, lessons learned from the study of rat cells are strongly predictive of outcomes using human cells. Thus, these studies provide successful strategies for the use of astrocyte transplantation therapies for restoration of function following spinal cord injury.

Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury.

BACKGROUND: Two 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. RESULTS: We 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 (GDAs(CNTF)), the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDA(CNTF) 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 GDAs(BMP) did not exhibit pain syndromes. CONCLUSION: Our 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.

Astrocytes derived from glial-restricted precursors promote spinal cord repair.

BACKGROUND: Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair. RESULTS: Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery. CONCLUSION: Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.

Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue.

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.

Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries.

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.