Dual-functional hydrogel system for spinal cord regeneration with sustained release of arylsulfatase B alleviates fibrotic microenvironment and promotes axonal regeneration.

Traumatic damage to the spinal cord does not spontaneously heal, often leading to permanent tissue defects. We have shown that injection of imidazole-poly(organophosphazene) hydrogel (I-5) bridges cystic cavities with the newly assembled fibronectin-rich extracellular matrix (ECM). The hydrogel-created ECM contains chondroitin sulfate proteoglycans (CSPGs), collagenous fibrils together with perivascular fibroblasts, and various fibrotic proteins, all of which could hinder axonal growth in the matrix. In an in vitro fibrotic scar model, fibroblasts exhibited enhanced sensitivity to TGF-ß1 when grown on CSPGs. To alleviate the fibrotic microenvironment, the I-5 hydrogel was equipped with an additional function by making a complex with ARSB, a human enzyme degrading CSPGs, via hydrophobic interaction. Delivery of the I-5/ARSB complex significantly diminished the fibrotic ECM components. The complex promoted serotonergic axonal growth into the hydrogel-induced matrix and enhanced serotonergic innervation of the lumbar motor neurons. Regeneration of the propriospinal axons deep into the matrix and to the lumbar spinal cord was robustly increased accompanied by improved locomotor recovery. Therefore, our dual-functional system upgraded the functionality of the hydrogel for spinal cord regeneration by creating ECM to bridge tissue defects and concurrently facilitating axonal connections through the newly assembled ECM.

Role of Myc Proto-Oncogene as a Transcriptional Hub to Regulate the Expression of Regeneration-Associated Genes following Preconditioning Peripheral Nerve Injury.

Preconditioning peripheral nerve injury enhances the intrinsic growth capacity of DRGs sensory axons by inducing transcriptional upregulation of the regeneration-associated genes (RAGs). However, it is still unclear how preconditioning injury leads to the orchestrated induction of many RAGs. The present study identified Myc proto-oncogene as a transcriptional hub gene to regulate the expression of a distinct subset of RAGs in DRGs following the preconditioning injury. We demonstrated that c-MYC bound to the promoters of certain RAGs, such as Jun, Atf3, and Sprr1a, and that Myc upregulation following SNI preceded that of the RAGs bound by c-MYC. Marked DNA methylation of the Myc exon 3 sequences was implicated in the early transcriptional activation and accompanied by open histone marks. Myc deletion led to a decrease in the injury-induced expression of a distinct subset of RAGs, which were highly overlapped with the list of RAGs that were upregulated by Myc overexpression. Following dorsal hemisection spinal cord injury in female rats, Myc overexpression in DRGs significantly prevented the retraction of the sensory axons in a manner dependent on its downstream RAG, June Our results suggest that Myc plays a critical role in axon regeneration via its transcriptional activity to regulate the expression of a spectrum of downstream RAGs and subsequent effector molecules. Identification of more upstream hub transcription factors and the epigenetic mechanisms specific for individual hub transcription factors would advance our understanding of how the preconditioning injury induces orchestrated upregulation of RAGs.

Insulin-like Growth Factor-1 Receptor Dictates Beneficial Effects of Treadmill Training by Regulating Survival and Migration of Neural Stem Cell Grafts in the Injured Spinal Cord.

Survival and migration of transplanted neural stem cells (NSCs) are prerequisites for therapeutic benefits in spinal cord injury. We have shown that survival of NSC grafts declines after transplantation into the injured spinal cord, and that combining treadmill training (TMT) enhances NSC survival via insulin-like growth factor-1 (IGF-1). Here, we aimed to obtain genetic evidence that IGF-1 signaling in the transplanted NSCs determines the beneficial effects of TMT. We transplanted NSCs heterozygous (+/-) for Igf1r, the gene encoding IGF-1 receptor, into the mouse spinal cord after injury, with or without combining TMT. We analyzed the influence of genotype and TMT on locomotor recovery and survival and migration of NSC grafts. In vitro experiments were performed to examine the potential roles of IGF-1 signaling in the migratory ability of NSCs. Mice receiving +/- NSC grafts showed impaired locomotor recovery compared with those receiving wild-type (+/+) NSCs. Locomotor improvement by TMT was more pronounced with +/+ grafts. Deficiency of one allele of Igf1r significantly reduced survival and migration of the transplanted NSCs. Although TMT did not significantly influence NSC survival, it substantially enhanced the extent of migration for only +/+ NSCs. Cultured neurospheres exhibited dynamic motility with cytoplasmic protrusions, which was regulated by IGF-1 signaling. IGF-1 signaling in transplanted NSCs may be essential in regulating their survival and migration. Furthermore, TMT may promote NSC graft-mediated locomotor recovery via activation of IGF-1 signaling in transplanted NSCs. Dynamic NSC motility via IGF-1 signaling may be the cellular basis for the TMT-induced enhancement of migration.

An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling.

The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle for tissue repair in central nervous system (CNS). Here we report that injection of imidazole-poly(organophosphazenes) (I-5), a hydrogel with thermosensitive sol-gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model. Cystic cavities are bridged by fibronectin-rich extracellular matrix. The fibrotic extracellular matrix remodeling is mediated by matrix metalloproteinase-9 expressed in macrophages within the fibrotic extracellular matrix. A poly(organophosphazenes) hydrogel lacking the imidazole moiety, which physically interacts with macrophages via histamine receptors, exhibits substantially diminished bridging effects. I-5 injection improves coordinated locomotion, and this functional recovery is accompanied by preservation of myelinated white matter and motor neurons and an increase in axonal reinnervation of the lumbar motor neurons. Our study demonstrates that dynamic interactions between inflammatory cells and injectable biomaterials can induce beneficial extracellular matrix remodeling to stimulate tissue repair following CNS injuries.The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle. Here the authors show an injection of imidazole poly(organophosphazenes), a hydrogel with thermosensitive sol-gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model.

CCL2 Mediates Neuron-Macrophage Interactions to Drive Proregenerative Macrophage Activation Following Preconditioning Injury.

CNS neurons in adult mammals do not spontaneously regenerate axons after spinal cord injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate beyond the injury site by promoting expression of regeneration-associated genes. We have previously shown that peripheral nerve injury increases the number of macrophages in the DRGs and that the activated macrophages are critical to the enhancement of intrinsic regeneration capacity. The present study identifies a novel chemokine signal mediated by CCL2 that links regenerating neurons with proregenerative macrophage activation. Neutralization of CCL2 abolished the neurite outgrowth activity of conditioned medium obtained from neuron-macrophage cocultures treated with cAMP. The neuron-macrophage interactions that produced outgrowth-promoting conditioned medium required CCL2 in neurons and CCR2/CCR4 in macrophages. The conditioning effects were abolished in CCL2-deficient mice at 3 and 7 d after sciatic nerve injury, but CCL2 was dispensable for the initial growth response and upregulation of GAP-43 at the 1 d time point. Intraganglionic injection of CCL2 mimicked conditioning injury by mobilizing M2-like macrophages. Finally, overexpression of CCL2 in DRGs promoted sensory axon regeneration in a rat spinal cord injury model without harmful side effects. Our data suggest that CCL2-mediated neuron-macrophage interaction plays a critical role for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury. Manipulation of chemokine signaling mediating neuron-macrophage interactions may represent a novel therapeutic approach to promote axon regeneration after CNS injury.

Survival of neural stem cell grafts in the lesioned spinal cord is enhanced by a combination of treadmill locomotor training via insulin-like growth factor-1 signaling.

Combining cell transplantation with activity-based rehabilitation is a promising therapeutic approach for spinal cord repair. The present study was designed to investigate potential interactions between the transplantation (TP) of neural stem cells (NSCs) obtained at embryonic day 14 and treadmill training (TMT) in promoting locomotor recovery and structural repair in rat contusive injury model. Combination of TMT with NSC TP at 1 week after injury synergistically improved locomotor function. We report here that combining TMT increased the survival of grafted NSCs by >3-fold and >5-fold at 3 and 9 weeks after injury, respectively. The number of surviving NSCs was significantly correlated with the extent of locomotor recovery. NSCs grafted into the injured spinal cord were under cellular stresses induced by reactive nitrogen or oxygen species, which were markedly attenuated by TMT. TMT increased the concentration of insulin-like growth factor-1 (IGF-1) in the CSF. Intrathecal infusion of neutralizing IGF-1 antibodies, but not antibodies against either BDNF or Neurotrophin-3 (NT-3), abolished the enhanced survival of NSC grafts by TMT. The combination of TP and TMT also resulted in tissue sparing, increased myelination, and restoration of serotonergic fiber innervation to the lumbar spinal cord to a larger extent than that induced by either TP or TMT alone. Therefore, we have discovered unanticipated beneficial effects of TMT in modulating the survival of grafted NSCs via IGF-1. Our study identifies a novel neurobiological basis for complementing NSC-based spinal cord repair with activity-based neurorehabilitative approaches.

Contribution of macrophages to enhanced regenerative capacity of dorsal root ganglia sensory neurons by conditioning injury.

Although the central branches of the dorsal root ganglion (DRG) sensory neurons do not spontaneously regenerate, a conditioning peripheral injury can promote their regeneration. A potential role of macrophages in axonal regeneration was proposed, but it has not been critically addressed whether macrophages play an essential role in the conditioning injury model. After sciatic nerve injury (SNI) in rats, the number of macrophages in DRGs gradually increased by day 7. The increase persisted up to 28 d and was accompanied by upregulation of inflammatory mediators, including oncomodulin. A macrophage deactivator, minocycline, reduced the macrophage number and expressions of the inflammatory mediators. Molecular signatures of conditioning effects were abrogated by minocycline, and enhanced regenerative capacity was substantially attenuated both in vitro and in vivo. Delayed minocycline infusion abrogated the SNI-induced long-lasting heightened neurite outgrowth potential, indicating a role for macrophages in the maintenance of regenerative capacity. Intraganglionic cAMP injection also resulted in an increase in macrophages, and minocycline abolished the cAMP effect on neurite outgrowth. However, conditioned media (CM) from macrophages treated with cAMP did not exhibit neurite growth-promoting activity. In contrast, CM from neuron-macrophage cocultures treated with cAMP promoted neurite outgrowth greatly, highlighting a requirement for neuron-macrophage interactions for the induction of a proregenerative macrophage phenotype. The growth-promoting activity in the CM was profoundly attenuated by an oncomodulin neutralizing antibody. These results suggest that the neuron-macrophage interactions involved in eliciting a proregenerative phenotype in macrophages may be a novel target to induce long-lasting regenerative processes after axonal injuries in the CNS.

Implantation of polymer scaffolds seeded with neural stem cells in a canine spinal cord injury model.

BACKGROUND AIMS: Combinatorial approaches employing diverse therapeutic modalities are required for clinically relevant repair of injured spinal cord in human patients. Before translation into the clinic, the feasibility and therapeutic potential of such combinatorial strategies in larger animal species need to be examined. METHODS: The present study tested the feasibility of implanting polymer scaffolds via neural stem cell (NSC) delivery in a canine spinal cord injury (SCI) model. The poly(lactic-co-glycolic acid) (PLGA) scaffolds seeded with human NSC were implanted into hemisected canine spinal cord. RESULTS: The PLGA scaffolds bridged tissue defects and were nicely integrated with residual canine spinal cord tissue. Grafted NSC survived the implantation procedure and showed migratory behavior to residual spinal cord tissue. Ectopic expression of a therapeutic neurotrophin-3 gene was also possible in NSC seeded within the PLGA scaffolds. CONCLUSIONS: Our description of a canine SCI model would be a valuable resource for pre-clinical trials of combinatorial strategies in larger animal models.

Ex vivo VEGF delivery by neural stem cells enhances proliferation of glial progenitors, angiogenesis, and tissue sparing after spinal cord injury.

The present study was undertaken to examine multifaceted therapeutic effects of vascular endothelial growth factor (VEGF) in a rat spinal cord injury (SCI) model, focusing on its capability to stimulate proliferation of endogenous glial progenitor cells. Neural stem cells (NSCs) can be genetically modified to efficiently transfer therapeutic genes to diseased CNS. We adopted an ex vivo approach using immortalized human NSC line (F3 cells) to achieve stable and robust expression of VEGF in the injured spinal cord. Transplantation of NSCs retrovirally transduced to overexpress VEGF (F3.VEGF cells) at 7 days after contusive SCI markedly elevated the amount of VEGF in the injured spinal cord tissue compared to injection of PBS or F3 cells without VEGF. Concomitantly, phosphorylation of VEGF receptor flk-1 increased in F3.VEGF group. Stereological counting of BrdU+ cells revealed that transplantation of F3.VEGF significantly enhanced cellular proliferation at 2 weeks after SCI. The number of proliferating NG2+ glial progenitor cells (NG2+/BrdU+) was also increased by F3.VEGF. Furthermore, transplantation of F3.VEGF increased the number of early proliferating cells that differentiated into mature oligodendrocytes, but not astrocytes, at 6 weeks after SCI. F3.VEGF treatment also increased the density of blood vessels in the injured spinal cord and enhanced tissue sparing. These anatomical results were accompanied by improved BBB locomotor scores. The multifaceted effects of VEGF on endogenous gliogenesis, angiogenesis, and tissue sparing could be utilized to improve functional outcomes following SCI.

Stem cell-based cell therapy for spinal cord injury.

Traumatic injuries to the spinal cord lead to severe and permanent neurological deficits. Although no effective therapeutic option is currently available, recent animal studies have shown that cellular transplantation strategies hold promise to enhance functional recovery after spinal cord injury (SCI). This review is to analyze the experiments where transplantation of stem/progenitor cells produced successful functional outcome in animal models of SCI. There is no consensus yet on what kind of stem/progenitor cells is an ideal source for cellular grafts. Three kinds of stem/progenitor cells have been utilized in cell therapy in animal models of SCI: embryonic stem cells, bone marrow mesenchymal stem cells, and neural stem cells. Neural stem cells or fate-restricted neuronal or glial progenitor cells were preferably used because they have clear capacity to become neurons or glial cells after transplantation into the injured spinal cord. At least a part of functional deficits after SCI is attributable to chronic progressive demyelination. Therefore, several studies transplanted glial-restricted progenitors or oligodendrocyte precursors to target the demyelination process. Directed differentiation of stem/progenitor cells to oligodendrocyte lineage prior to transplantation or modulation of microenvironment in the injured spinal cord to promote oligodendroglial differentiation seems to be an effective strategy to increase the extent of remyelination. Transplanted stem/progenitor cells can also contribute to promoting axonal regeneration by functioning as cellular scaffolds for growing axons. Combinatorial approaches using polymer scaffolds to fill the lesion cavity or introducing regeneration-promoting genes will greatly increase the efficacy of cellular transplantation strategies for SCI.