Adult neural progenitor cells provide a permissive guiding substrate for corticospinal axon growth following spinal cord injury

Adult neural progenitor cells (NPC) are an attractive source for cell transplantation and neural tissue replacement after central nervous system (CNS) injury. Following transplantation of NPC cell suspensions into the acutely injured rat spinal cord, NPC survive; however, they migrate away from the lesion site and are unable to replace the injury-induced lesion cavity. In the present study we examined (i) whether NPC can be retained within the lesion site after co-transplantation with primary fibroblasts, and (ii) whether NPC promote axonal regeneration following spinal cord injury. Co-cultivation of NPC with fibroblasts demonstrated that NPC adhere to fibroblasts and the extracellular matrix produced by fibroblasts. In the presence of fibroblasts, the differentiation pattern of co-cultivated NPC was shifted towards glial differentiation. Three weeks after transplantation of adult spinal-cord-derived NPC with primary fibroblasts as mixed cell suspensions into the acutely injured cervical spinal cord in adult rats, the lesion cavity was completely replaced. NPC survived throughout the graft and differentiated exclusively into glial cells. Quantification of neurofilament-labeled axons and anterogradely labeled corticospinal axons indicated that NPC co-grafted with fibroblasts significantly enhanced axonal regeneration. Both neurofilament-labeled axons and corticospinal axons aligned longitudinally along GFAP-expressing NPC-derived cells, which displayed a bipolar morphology reminiscent of immature astroglia. Thus, grafted astroglial differentiated NPC promote axon regrowth following spinal cord injury by means of cellular guidance.     

Stimulation of corticospinal tract regeneration in the chronically injured spinal cord

Acute spinal cord injury models have proved popular in studies aimed at identifying factors capable of influencing axonal regeneration within the central nervous system. In these models, the test factors (e.g. graft tissues or cells, antibodies, growth factors, etc.) are typically administered at the time of spinal cord injury. In this study, we use a rat chronic spinal cord injury model to identify possible factors which can stimulate regeneration of the chronically lesioned corticospinal tract axons. We demonstrate that surgical grafting of segments of autologous, preligated sural nerve, into the syrinx, stimulates sprouting and regeneration of the corticospinal tract as evidenced by the presence of anterograde labelled corticospinal tract processes within the cavity walls two or more weeks after treatment. Regrowing corticospinal processes were not observed within control animals. The anterogradely labelled corticospinal tract axons were found exclusively within the central grey tissue comprising the cavity walls with no regrowing corticospinal process observed within the white matter. A similar pattern of regeneration was observed following injection into the cavity of a suspension of minced autologous preligated sural nerve. Evidence of corticospinal tract regeneration was seen when either wheat germ agglutinin--horseradish peroxidase or biotinylated--dextran was used as an anterograde tracer. These data demonstrate that the chronically injured cortical motor neurons retain the capacity to regenerate for extended periods and that regeneration can be stimulated using grafts of minced, preligated autologous peripheral nerve tissue.     

Enhanced viability and neuronal differentiation of neural progenitors by chromaffin cell co-culture

The transplantation of neural stem cells and progenitors has potential in restoring lost cellular populations following central nervous system (CNS) injury or disease, but survival and neuronal differentiation in the adult CNS may be insufficient in the absence of exogenous trophic support. Adrenal medullary chromaffin cells produce a trophic cocktail including basic fibroblast growth factor (FGF-2) and neurotrophins. The aim of this study was to evaluate whether chromaffin cells can provide a supportive microenvironment for neural progenitor cells. In order to assess this, the growth and differentiation of neural progenitor cell cultures from embryonic rat cortex were compared in standard FGF-2-supplemented neural progenitor growth media, in standard media but lacking FGF-2, or in media lacking FGF-2 but co-cultured with bovine chromaffin cells. Using bromodeoxyuridine (BrdU)-prelabeling, findings indicated poor survival of progenitor cultures in the absence of FGF-2. In contrast, the addition of chromaffin cells in co-culture appeared to 'rescue' the progenitor cultures and resulted in robust neurospheres containing numerous BrdU-labeled cells interspersed with and closely apposed to chromaffin cells. As indicated by H3 labeling, cells in co-cultures continued to proliferate, but at a substantially reduced rate compared with standard FGF-2 supplemented growth media. The co-cultures contained more beta-tubulin III-positive processes than parallel cultures maintained in FGF-2-supplemented media and these cells displayed a more mature phenotype with numerous varicosities and complex processes. These findings indicate that chromaffin cells can provide a supportive environment for the survival and neuronal differentiation of neural progenitor cells and suggest that their addition may be useful as a sustained source of trophic support to improve outcomes of neural stem cell transplantation.     

Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways

The main rationale for cell-based therapies following spinal cord injury are: (i) replacement of degenerated spinal cord parenchyma by an axon growth supporting scaffold; (ii) remyelination of regenerating axons; and (iii), local delivery of growth promoting molecules. A potential source to meet these requirements is adult neural progenitor cells, which were examined in the present study. Fibroblast growth factor 2-responsive adult spinal cord-derived syngenic neural progenitor cells were either genetically modified in vitro to express green fluorescent protein (GFP) using retroviral vectors or prelabelled with bromodeoxyuridine (BrdU). Neural progenitor cells revealed antigenic properties of neurons and glial cells in vitro confirming their multipotency. This differentiation pattern was unaffected by retroviral transduction. GFP-expressing or BrdU-prelabelled neural progenitor cells were grafted as neurospheres directly into the acutely injured rat cervical spinal cord. Animals with lesions only served as controls. Three weeks postoperatively, grafted neural progenitor cells integrated along axonal profiles surrounding the lesion site. In contrast to observations in culture, grafted neural progenitor cells differentiated only into astro- and oligodendroglial lineages, supporting the notion that the adult spinal cord provides molecular cues for glial, but not for neuronal, differentiation. This study demonstrates that adult neural progenitor cells will survive after transplantation into the acutely injured spinal cord. The observed oligodendroglial and astroglial differentiation and integration along axonal pathways represent important prerequisites for potential remyelination and support of axonal regrowth.     

IGF-I gene delivery promotes corticospinal neuronal survival but not regeneration after adult CNS injury

An unmet challenge of spinal cord injury research is the identification of mechanisms that promote regeneration of corticospinal motor axons. Recently it was reported that IGF-I promotes corticospinal axon growth during nervous system development. We therefore investigated whether IGF-I also promotes regeneration or survival of adult lesioned corticospinal neurons. Adult Fischer 344 rats underwent C3 dorsal column transections followed by grafts of IGF-I-secreting marrow stromal cell grafts into the lesion cavity. IGF-I secreting cell grafts promoted growth of raphespinal and cerulospinal axons, but not corticospinal axons, into the lesion/graft site. We then examined whether IGF-I-secreting cell grafts promote corticospinal motor neuron survival or axon growth in a subcortical axotomy model. IGF-I expression coupled with infusion of the IGF binding protein inhibitor NBI-31772 significantly prevented corticospinal motor neuron death (93% cell survival compared to 49% in controls, P < 0.05), but did not promote corticospinal axon regeneration. Coincident with observed effects of IGF-I on corticospinal survival but not growth, expression of IGF-I receptors was restricted to the somal compartment and not the axon of adult corticospinal motor neurons. Thus, whereas IGF-I influences corticospinal axonal growth during development, its application to sites of adult spinal cord injury or subcortical axotomy fails to promote corticospinal axonal regeneration under conditions that are sufficient to prevent corticospinal cell death and promote the growth of other supraspinal axons. We conclude that developmental patterns of growth factor responsiveness are not simply recapitulated after adult injury, potentially due to post-natal shifts in patterns of IGF-I receptor expression.     

Corticospinal regeneration into lumbar grey matter correlates with locomotor recovery after complete spinal cord transection and repair with peripheral nerve grafts, fibroblast growth factor 1, fibrin glue, and spinal fusion

Knowledge of which tracts are essential for the recovery of locomotor function in rats after repair is unknown. To assess the mechanism of recovery, we examined the correlation between functional recovery and axonal regeneration. All rats underwent complete cord transection and repair with peripheral nerves, fibroblast growth factor 1, fibrin glue, and spinal fixation. Repaired rats recovered both motor-evoked potentials recorded at the lumbar level and locomotor function. Cord retransection rostral to the repair abolished the recovery, indicating improvement was due to long tract regeneration. To determine which long tracts correlated with recovery, a novel technique of simultaneous bidirectional axonal tracing and immunohistochemical examination of axonal type was used to quantitate the regeneration of corticospinal, rubrospinal, reticulospinal, vestibulospinal, raphespinal, propriospinal, serotonergic, and calcitonin gene-related peptide containing axons. Multiple linear regression analysis revealed recovery of function correlated only with regeneration of corticospinal axons into the gray matter of the lumbar spinal cord (R = 0.977, p < 0.02). For the first time, we show that regeneration of the corticospinal tract into the lumbar gray matter is a mechanism of functional locomotor recovery after complete cord transection and repair.     

Elimination of basal lamina and the collagen "scar" after spinal cord injury fails to augment corticospinal tract regeneration

The production of specific extracellular matrix molecules is upregulated following injury to the adult CNS, and some of these molecules have been postulated to inhibit axonal regeneration. In particular, the deposition of collagen in conjunction with basal lamina formation has been correlated with the failure of CNS axons to extend beyond sites of injury. In the present experiment, the spatial and temporal distribution of fibrillar collagen type III and the main constituents of basal lamina (collagen type IV and laminin) were characterized after defined lesions of the adult spinal cord at cervical and thoracic levels. The deposition of collagen was then blocked in animals undergoing defined mid-thoracic spinal cord lesions by administration of the iron chelator 2,2'-bipyridine, and subsequent effects on corticospinal axonal growth were examined. At time points from 1 to 6 weeks postinjury, collagen and laminin were deposited at spinal cord lesion sites as a dense matrix at the host-lesion interface that extended for short distances into the surrounding spinal cord parenchyma. The failure of corticospinal axons to grow beyond the lesioned region correlated spatially and temporally with collagen III formation and basal lamina production. However, successful blockade of collagen and basal lamina formation with 2,2'-bipyridine injections failed to enhance corticospinal axon regeneration or sprouting. These results suggest either that collagen and basal lamina formation after CNS injury do not contribute to corticospinal axonal growth failure or, more likely, that molecules in addition to collagen and basal lamina contribute to axonal growth failure and must be collectively blocked to promote corticospinal regeneration.     

Schwann cell‐derived exosomes enhance axonal regeneration in the peripheral nervous system

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor‐like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano‐vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC‐derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro‐regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. GLIA 2013;61:1795–1806     

CD133 cells from human umbilical cord blood reduce cortical damage and promote axonal growth in neonatal rat organ co-cultures exposed to hypoxia

To evaluate the effect of CD133⁺ cells (endothelial progenitor cells) on the hypoxia-induced suppression of axonal growth of cortical neurons and the destruction of blood vessels (endothelial cells), we used anterograde axonal tracing and immunofluorescence in organ co-cultures of the cortex and the spinal cord from 3-day-old neonatal rats. CD133⁺ cells prepared from human umbilical cord blood were added to the organ co-cultures after hypoxic insult, and axonal growth, vascular damage and apoptosis were evaluated. Anterograde axonal tracing with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate was used to analyze axonal projections from the cortex to the spinal cord. Immunolabeling co-cultured tissues of the cortex and the spinal cord were used to investigate the effect of CD133⁺ cells on the survival of blood vessels and apoptosis in the brain cortex. Hypoxia remarkably suppressed axonal growth in organ co-cultures of the cortex and the spinal cord, and this suppression was significantly restored by the addition of CD133⁺ cells. CD133⁺ cells also reduced the hypoxia-induced destruction of the cortical blood vessels and apoptosis. CD133⁺ cells had protective effects on hypoxia-induced injury of neurons and blood vessels of the brain cortex in vitro. These results suggest that CD133⁺ cell transplantation may be a possible therapeutic intervention for perinatal hypoxia-induced brain injury.     

Lavendustin A enhances axon elongation in VHL gene-transfected neural stem cells

Axonal elongation is necessary for neuronal regeneration of the spinal cord after spinal injury. Recently neural stem cells have been proposed as hopeful graft donors for regeneration of the central nervous system. However, most grafted stem cells are not able to differentiate into neurons, and grafted stem cells cannot usually grow axons. Here, we show the effect of the protein tyrosine kinase inhibitor lavendustin A on axonal growth of neurons differentiated from neural stem cells obtained from adult rat hippocampal cells transfected with the von Hippel-Lindau (VHL) gene. Significantly greater axonal outgrowth was observed for the transfected cells treated with the inhibitor than for those not so treated. Thus, protein-tyrosine kinase inhibition is effective for axonal outgrowth of neurons differentiated from neural stem cells and may prove to be useful for neuronal regeneration via transplanted stem cells, particularly in the case of spinal cord injuries.     

Roles of neural stem cells in the repair of peripheral nerve injury

Currently, researchers are using neural stem cell transplantation to promote regeneration after peripheral nerve injury, as neural stem cells play an important role in peripheral nerve injury repair. This article reviews recent research progress of the role of neural stem cells in the repair of peripheral nerve injury. Neural stem cells can not only differentiate into neurons, astrocytes and oligodendrocytes, but can also differentiate into Schwann-like cells, which promote neurite outgrowth around the injury. Transplanted neural stem cells can differentiate into motor neurons that innervate muscles and promote the recovery of neurological function. To promote the repair of peripheral nerve injury, neural stem cells secrete various neurotrophic factors, including brain-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor and hepatocyte growth factor. In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways.     

Serum response factor is required for cortical axon growth but is dispensable for neurogenesis and neocortical lamination

Previous studies have shown that neuron-specific deletion of serum response factor (SRF) results in deficits in tangential cell migration, guidance-dependent circuit assembly, activity-dependent gene expression, and synaptic plasticity in the hippocampus. Furthermore, SRF deletion in mouse embryonic stem cells causes cell death in vitro. However, the requirement of SRF for early neuronal development including neural stem cell homeostasis, neurogenesis, and axonal innervations remains unknown. Here, we report that SRF is critical for development of major axonal tracts in the forebrain. Conditional mutant mice lacking SRF in neural progenitor cells (Srf-Nestin-cKO) exhibit striking deficits in cortical axonal projections including corticostriatal, corticospinal, and corticothalamic tracts, and they show a variable loss of the corpus callosum. Neurogenesis and interneuron specification occur normally in the absence of SRF and the deficits in axonal projections were not due to a decrease or loss in cell numbers. Radial migration of neurons and neocortical lamination were also not affected. No aberrant cell death was observed during development, whereas there was an increase in the number of proliferative cells in the ventricular zone from embryonic day 14 to day 18. Similar axonal tract deficits were also observed in mutant mice lacking SRF in the developing excitatory neurons of neocortex and hippocampus (Srf-NEX-cKO). Together, these findings suggest distinct roles for SRF during neuronal development; SRF is specifically required in a cell-autonomous manner for axonal tract development but is dispensable for cell survival, neurogenesis, neocortical lamination, and neuronal differentiation.     

Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM‐dependent mechanism

Olfactory ensheathing cells (OECs) support the ability of the olfactory neuraxis to continually retarget within the mature central nervous system. This has led many groups to transplant OECS into the lesioned rodent spinal cord (SCd) in vivo, with variable degrees of anatomical, physiological, and behavioral success. Some of the most conflicting results in OEC transplantation have come from the corticospinal tract (CST) which has shown a relatively poor regeneration response. Although spinal neurite sprouting occurs in response to OECs in vivo and in vitro, we do not know if OECs possess the molecular machinery to stimulate outgrowth of functionally important motor tracts like the CST. Here, we assay cultured postnatal day 8 mouse CST neurons expressing yellow fluorescent protein (YFP) for their ability to extend axons and dendrites in response to different glia, and show that CST axons elongate in response to proteins in OEC plasma membrane (PM). In contrast, CST dendritic branching preferentially occurs in response to factors secreted by both OECs and astrocytes. We identify the L1‐neural cell adhesion molecule (L1‐NCAM) as a major component of OEC‐induced corticospinal axon elongation, and have determined that OEC PM factors (including L1), can stimulate CST outgrowth even when inhibition is induced by myelin associated glycoprotein. Together, these results suggest that in the right context, OEC‐derived PM factors could enhance CST axonal regeneration, and potentially contribute to approaches to ameliorate recovery from SCd injury. GLIA 2013;61:1873–1889     

Neurotrophic factors,gene therapy,and neural stem cells for spinal cord repair

Several experimental strategies have been employed to minimize tissue damage and to enhance axonal growth and regeneration after spinal cord injury. The transplantation of suitable cell types to provide an axonal growth substrate and the application of growth factors have been shown to augment morphological and sometimes functional recovery. In this review we discuss the use of neural stem cell transplants and neurotrophic factor delivery by gene therapy to improve axonal regeneration in animal models of spinal cord injury.     

Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells

The neurogenic response to injury in the postnatal brain is limited and insufficient for restoration of function. Recent evidence suggests that transplantation of mesenchymal stem cells (MSCs) into the injured brain is associated with improved functional recovery, mediated in part through amplification in the endogenous neurogenic response to injury. In the current study we investigate the interactions between bone marrow-derived MSCs and embryonic neural stem cells (NSCs) plus their differentiated progeny using an in vitro co-culture system. Two populations of MSCs were used, MSCs induced to express neural antigens (nestin+, Tuj-1+, GFAP+) and neural antigen negative MSCs. Following co-culture of induced MSCs with differentiating NSC/progenitor cells a significant increase in Tuj-1+ neurons was detected compared to co-cultures of non-induced MSCs in which an increase in astrocyte (GFAP+) differentiation was observed. The effect was mediated by soluble interactions between the two cell populations and was independent of any effect on cell death and proliferation. Induced and non-induced MSCs also promoted the survival of Tuj-1+ cell progeny in long-term cultures and both promoted axonal growth, an effect also seen in differentiating neuroblastoma cells. Therefore, MSCs provide instructive signals that are able to direct the differentiation of NSCs and promote axonal development in neuronal progeny. The data indicates that the nature of MSC derived signals is dependent not only on their microenvironment but on the developmental status of the MSCs. Pre-manipulation of MSCs prior to transplantation in vivo may be an effective means of enhancing the endogenous neurogenic response to injury.     

Deletion of Krüppel-like factor-4 promotes axonal regeneration in mammals

Axonal regeneration plays an important role in functional recovery after nervous system damage. However, after axonal injury in mammals, regeneration is often poor. The deletion of Krüppel-like factor-4 (Klf4) has been shown to promote axonal regeneration in retinal ganglion cells. However, the effects of Klf4 deletion on the corticospinal tract and peripheral nervous system are unknown. In this study, using a mouse model of sciatic nerve injury, we show that the expression of Klf4 in dorsal root ganglion sensory neurons was significantly reduced after peripheral axotomy, suggesting that the regeneration of the sciatic nerve is associated with Klf4. In vitro, dorsal root ganglion sensory neurons with Klf4 knockout exhibited significantly enhanced axonal regeneration. Furthermore, the regeneration of the sciatic nerve was enhanced in vivo following Klf4 knockout. Finally, AAV-Cre virus was used to knockout the Klf4 gene in the cortex. The deletion of Klf4 enhanced regeneration of the corticospinal tract in mice with spinal cord injury. Together, our findings suggest that regulating KLF4 activity in neurons is a potential strategy for promoting axonal regeneration and functional recovery after nervous system injury. This study was approved by the Animal Ethics Committee at Soochow University, China (approval No. SUDA20200316A01).

Chinese Library Classification No.R456; R741; Q344+.14     

Axonal remodeling in the corticospinal tract after stroke: how does rehabilitative training modulate it?

Stroke causes long-term disability, and rehabilitative training is commonly used to improve the consecutive functional recovery. Following brain damage, surviving neurons undergo morphological alterations to reconstruct the remaining neural network. In the motor system, such neural network remodeling is observed as a motor map reorganization. Because of its significant correlation with functional recovery, motor map reorganization has been regarded as a key phenomenon for functional recovery after stroke. Although the mechanism underlying motor map reorganization remains unclear, increasing evidence has shown a critical role for axonal remodeling in the corticospinal tract. In this study, we review previous studies investigating axonal remodeling in the corticospinal tract after stroke and discuss which mechanisms may underlie the stimulatory effect of rehabilitative training. Axonal remodeling in the corticospinal tract can be classified into three types based on the location and the original targets of corticospinal neurons, and it seems that all the surviving corticospinal neurons in both ipsilesional and contralesional hemisphere can participate in axonal remodeling and motor map reorganization. Through axonal remodeling, corticospinal neurons alter their output selectivity from a single to multiple areas to compensate for the lost function. The remodeling of the corticospinal axon is influenced by the extent of tissue destruction and promoted by various therapeutic interventions, including rehabilitative training. Although the precise molecular mechanism underlying rehabilitation-promoted axonal remodeling remains elusive, previous data suggest that rehabilitative training promotes axonal remodeling by upregulating growth-promoting and downregulating growth-inhibiting signals.     

Factors intrinsic to the neuron can induce and maintain its ability to promote axonal outgrowth: a role for BCL2?

The adult CNS provides a poor environment for axonal growth and regeneration. The question of to what extent the loss of axonal growth occurring as the brain matures is dependent on factors intrinsic or extrinsic to the growing neuron is still unanswered. Examination of axonal growth from neural transplants provides insight into the roles of growth factors, inhibitory molecules, growth-promoting substrates and the differences between CNS and PNS environments in the regulation of neurite extension. The data that imply a role for BCL2 and related molecules in such processes are reviewed in this article, which analyzes the factors intrinsic to the neuron that control its capacity for axonal growth.