Promoting survival, migration, and integration of transplanted Schwann cells by over-expressing polysialic acid

The poor survival and migration of transplanted Schwann cells (SCs) are major drawbacks for their clinical application in cell therapy for neurotrauma. To overcome such drawbacks we genetically modified SCs to over-express polysialic acid (PSA) by lentiviral delivery of polysialyltransferase (PST) to study whether over-expression of PSA could enhance their survival, migration, and integration when transplanted into the spinal cord. It was found that more PSA-expressing SCs (PST/SCs) survived than GFP-expressing SCs (GFP/SCs) after transplantation, although cell loss was still quite significant. PSA expression did not enhance the motility of transplanted SCs in uninjured spinal cord. However, in a spinal cord crush injury model PST/SCs transplanted caudal to the lesion showed that increased number of PST/SCs migrated to the injury site compared with that of GFP/SCs. Induced expression of PSA in spinal cord can further facilitate the infiltration of PST/SCs into the lesion site. PST/SCs were also shown to intermingle well with host spinal cells while GFP/SCs formed boundaries with host tissue. This was confirmed by an in vitro confrontation assay showing that more PST/SCs crossed over to astrocyte territory than GFP/SCs. Furthermore, PST/SCs induced much less expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycan in the surrounding tissues than GFP/SCs, indicating that expression of PSA on SCs do not cause significant stress response of astrocytes. These results demonstrate that expression of PSA on SCs significantly changes their biological properties and makes them more feasible for neural repair after neurotrauma.     

Transplantation of Schwann cells and/or olfactory ensheathing glia into the contused spinal cord: Survival, migration, axon association, and functional recovery

Schwann cells (SCs) and olfactory ensheathing glia (OEG) have shown promise for spinal cord injury repair. We sought their in vivo identification following transplantation into the contused adult rat spinal cord at 1 week post-injury by: (i) DNA in situ hybridization (ISH) with a Y-chromosome specific probe to identify male transplants in female rats and (ii) lentiviral vector-mediated expression of EGFP. Survival, migration, and axon-glia association were quantified from 3 days to 9 weeks post-transplantation. At 3 weeks after transplantation into the lesion, a 60-90% loss of grafted cells was observed. OEG-only grafts survived very poorly within the lesion (<5%); injection outside the lesion led to a 60% survival rate, implying that the injury milieu was hostile to transplanted cells and or prevented their proliferation. At later times post-grafting, p75(+)/EGFP(-) cells in the lesion outnumbered EGFP(+) cells in all paradigms, evidence of significant host SC infiltration. SCs and OEG injected into the injury failed to migrate from the lesion. Injection of OEG outside of the injury resulted in their migration into the SC-injected injury site, not via normal-appearing host tissue but along the pia or via the central canal. In all paradigms, host axons were seen in association with or ensheathed by transplanted glia. Numerous myelinated axons were found within regions of grafted SCs but not OEG. The current study details the temporal survival, migration, axon association of SCs and OEG, and functional recovery after grafting into the contused spinal cord, research previously complicated due to a lack of quality, long-term markers for cell tracking in vivo.     

Transduced Schwann cells promote axon growth and myelination after spinal cord injury

We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.     

Embryonic and adult stem cells promote raphespinal axon outgrowth and improve functional outcome following spinal hemisection in mice

Spinal cord injury (SCI) often results in permanent neurological deficits below the injury site. Serotonergic raphespinal projections promote functional recovery after SCI, but spontaneous regeneration of most severed axons is limited by the glial cyst and scar that form at the lesion site. Stem cell (SC) transplantation offers a promising approach for inducing regeneration through the damaged area. Here we compare the effects of transplantation of embryonic neural precursors (NPs) or adult mesenchymal SCs, both of which are potential candidates for SC therapy. The spinal cord was hemisected at the L2 neuromer in adult mice. Two weeks post-injury, we transplanted neural precursors or mesenchymal SCs into the cord, caudal to the hemisection. Injured mice without a graft served as controls. Mice were tested for functional recovery on a battery of motor tasks, then killed and analysed for survival of grafted cells, for effects of engraftment on the local cellular environment and for the sprouting of serotonergic axons. Both types of SCs survived and were integrated into the host tissue, but only the NPs expressed neuronal markers. All transplanted animals displayed an increased number of serotonin-positive fibres caudal to the hemisection, compared with untreated mice. And both cell types led to improved motor performance. These results point to a therapeutic potential for such cell grafting.     

Human Schwann cells exhibit long‐term cell survival,are not tumorigenic and promote repair when transplanted into the contused spinal cord

The transplantation of rodent Schwann cells (SCs) provides anatomical and functional restitution in a variety of spinal cord injury (SCI) models, supporting the recent translation of SCs to phase 1 clinical trials for human SCI. Whereas human (Hu)SCs have been examined experimentally in a complete SCI transection paradigm, to date the reported behavior of SCs when transplanted after a clinically relevant contusive SCI has been restricted to the use of rodent SCs. Here, in a xenotransplant, contusive SCI paradigm, the survival, biodistribution, proliferation and tumorgenicity as well as host responses to HuSCs, cultured according to a protocol analogous to that developed for clinical application, were investigated. HuSCs persisted within the contused nude rat spinal cord through 6 months after transplantation (longest time examined), exhibited low cell proliferation, displayed no evidence of tumorigenicity and showed a restricted biodistribution to the lesion. Neuropathological examination of the CNS revealed no adverse effects of HuSCs. Animals exhibiting higher numbers of surviving HuSCs within the lesion showed greater volumes of preserved white matter and host rat SC and astrocyte ingress as well as axon ingrowth and myelination. These results demonstrate the safety of HuSCs when employed in a clinically relevant experimental SCI paradigm. Further, signs of a potentially positive influence of HuSC transplants on host tissue pathology were observed. These findings show that HuSCs exhibit a favorable toxicity profile for up to 6 months after transplantation into the contused rat spinal cord, an important outcome for FDA consideration of their use in human clinical trials.     

Macrophage depletion and Schwann cell transplantation reduce cyst size after rat contusive spinal cord injury

Schwann cell transplantation is a promising therapy for the treatment of spinal cord injury(SCI) and is currently in clinical trials.In our continuing efforts to improve Schwann cell transplantation strategies,we sought to determine the combined effects of Schwann cell transplantation with macrophage depletion.Since macrophages are major inflammatory contributors to the acute spinal cord injury,and are the major phagocytic cells,we hypothesized that transplanting Schwann cells after macrophage depletion will improve cell survival and integration with host tissue after SCI.To test this hypothesis,rat models of contusive SCI at thoracic level 8 were randomly subjected to macrophage depletion or not.In rat subjected to macrophage depletion,liposomes filled with clodronate were intraperitoneally injected at 1,3,6,11,and 18 days post injury.Rats not subjected to macrophage depletion were intraperitoneally injected with liposomes filled with phosphate buffered saline.Schwann cells were transplanted 1 week post injury in all rats.Biotinylated dextran amine(BDA) was injected at thoracic level 5 to evalute axon regeneration.The Basso,Beattie,and Bresnahan locomotor test,Gridwalk test,and sensory test using von Frey filaments were performed to assess functional recovery.Immunohistochemistry was used to detect glial fibrillary acidic protein,neurofilament,and green fluorescent protein(GFP),and also to visulize BDA-labelled axons.The GFP labeled Schwann cell and cyst and lesion volumes were quantified using stained slides.The numbers of BDA-positive axons were also quantified.At 8 weeks after Schwann cell transplantation,there was a significant reduction in cyst and lesion volumes in the combined treatment group compared to Schwann cell transplantation alone.These changes were not associated,however,with improved Schwann cell survival,axon growth,or locomotor recovery.Although combining Schwann cell transplantation with macrophage depletion does improve histopathology of the injury site,the effect on axon growth and behavioral recovery appears no better than what can be achieved with Schwann cell transplants alone.     

GDNF modifies reactive astrogliosis allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury

Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft–host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10) level. Within the graft, migrated host astrocytes were in close association with regenerated axons. Astrocyte processes extended parallel to the axons, implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons. In vitro, GDNF induced migration of SCs and astrocytes toward each other in an astrocyte–SC confrontation assay. GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay, suggesting that this effect is mediated by direct cell–cell contact between the two cell types. Morphologically, GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells, similar to what was observed in vivo. Notably, GDNF treatment significantly reduced production of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs), two hallmarks of astrogliosis, in both the in vivo and in vitro models. Thus, our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats.     

Labeled Schwann cell transplantation: cell loss, host Schwann cell replacement, and strategies to enhance survival

Although transplanted Schwann cells (SCs) can promote axon regeneration and remyelination and improve recovery in models of spinal cord injury, little is known about their survival and how they interact with host tissue. Using labeled SCs from transgenic rats expressing human placental alkaline phosphatase (PLAP), SC survival in a spinal cord contusion lesion was assessed. Few PLAP SCs survived at 2 weeks after acute transplantation. They died early due to necrosis and apoptosis. Delaying transplantation until 7 days after injury improved survival. A second wave of cell death occurred after surviving cells had integrated into the spinal cord. Survival of PLAP SCs was enhanced by immunosuppression with cyclosporin; delayed transplantation in conjunction with immunosuppression resulted in the best survival. In all cases, transplantation of SCs resulted in extensive infiltration of endogenous p75+ cells into the injury site, suggesting that endogenous SCs may play an important role in the repair observed after SC transplantation.     

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.     

Schwann cell dependence of regenerating rat sensory neurons is inversely related to the quality of axon growth substratum

It is still controversial to what extent elongation of regenerating sensory axons depends on proliferating Schwann cells (SCs) in an injured peripheral nerve. We hypothesized that such regeneration was independent of SC support early after nerve injury, but later became SC-dependent. The sural nerve in rats was crushed, and freezing destroyed cells but not their basal laminae (BL) in the distal nerve segment. Sensory axon elongation was assessed by the nerve pinch test and their abundance was examined immunohistochemically. Sensory axons regenerated fairly rapidly during the first week even if SC migration was prevented. Thereafter, they ceased to elongate and withdrew until their terminals contacted the SCs migrating from the proximal nerve segment. Intrinsic neuronal capacity for growth without cell support, however, had not been lost. Rather, progressive degradation of the former SC BL and loss of laminin in the acellular segment arrested axon growth. Further elongation occurred only when SC migration was possible, corroborating our hypothesis. Sensory neurons continued to elongate and maintain their axons in spite of deteriorating growth substratum if, prior to injury the axons had been allowed to sprout into the denervated skin. Previous sprouting exposed the sensory neurons to high levels of NGF.     

Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts

Background

Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process.

Results

We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response.

Conclusions

These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for meningeal and glial cells in facilitating axon regeneration. Given that these cell types interact to form inhibitory barriers in mammals, identifying the mechanisms underlying their permissive behaviors in the newt will provide new insights for improving spinal cord regeneration in mammals.     

Transplanting neural progenitors into a complete transection model of spinal cord injury

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron‐ and glia‐restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft‐derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar‐staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft‐derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin‐positive fibers and biotinylated dextran amine‐traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI. © 2014 Wiley Periodicals, Inc.     

BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury

Bone marrow stromal cells (MSCs) constitute a heterogeneous cell layer in the bone marrow, supporting the growth and differentiation of hematopoietic stem cells. Recently, it has been reported that MSCs harbor pluripotent stem cells capable of neural differentiation and that simple treatment of MSCs with chemical inducing agents leads to their rapid transdifferentiation into neural cells. We examined whether native or neurally induced MSCs would reconstitute an axonal growth-promoting milieu after cervical spinal cord injury (SCI), and whether such cells could act as vehicles of growth factor gene delivery to further augment axonal growth. One month after grafting to cystic sites of SCI, native MSCs supported modest growth of host sensory and motor axons. Cells "neurally" induced in vitro did not sustain a neural phenotype in vivo and supported host axonal growth to a degree equal to native MSCs. Transduction of MSCs to overexpress brain-derived neurotrophic factor (BDNF) resulted in a significant increase in the extent and diversity of host axonal growth, enhancing the growth of host serotonergic, coerulospinal, and dorsal column sensory axons. Measurement of neurotrophin production from implanted cells in the lesion site revealed that the grafts naturally contain nerve growth factor (NGF) and neurotrophin-3 (NT-3), and that transduction with BDNF markedly raises levels of BDNF production. Despite the extensive nature of host axonal penetration into the lesion site, functional recovery was not observed on a tape removal or rope-walking task. Thus, MSCs can support host axonal growth after spinal cord injury and are suitable cell types for ex vivo gene delivery. Combination therapy with other experimental approaches will likely be required to achieve axonal growth beyond the lesion site and functional recovery.     

Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion SCI

Accumulation of intracellular sodium through voltage-gated sodium channels (VGSCs) is an important event in the cascade leading to anatomic degeneration of spinal cord axons and poor functional outcome following traumatic spinal cord injury (SCI). In this study, we hypothesized that phenytoin, a sodium channel blocker, would result in protection of axons with concomitant improvement of functional recovery after SCI. Adult male Sprague-Dawley rats underwent T9 contusion SCI after being fed normal chow or chow containing phenytoin; serum levels of phenytoin were within therapeutic range at the time of injury. At various timepoints after injury, quantitative assessment of lesion volumes, axonal degeneration, axonal conduction, and functional locomotor recovery were performed. When compared to controls, phenytoin-treated animals demonstrated reductions in the degree of destruction of gray and white matter surrounding the lesion epicenter, sparing of axons within the dorsal corticospinal tract (dCST) and dorsal column (DC) system rostral to the lesion site, and within the dorsolateral funiculus (DLF) caudal to the lesion site, and enhanced axonal conduction across the lesion site. Improved performance in measures of skilled locomotor function was observed in phenytoin-treated animals. Based on these results, we conclude that phenytoin provides neuroprotection and improves functional outcome after experimental SCI, and that it merits further examination as a potential treatment strategy in human SCI.     

Degradation of chondroitin sulfate proteoglycans potentiates transplant-mediated axonal remodeling and functional recovery after spinal cord injury in adult rats

Transplantation of growth-permissive cells or tissues was used to bridge a lesion cavity and induce axonal growth in experimental spinal cord injury (SCI). Axonal interactions between host and transplant may be affected by upregulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) following various transplantation strategies. The extent of axonal growth and functional recovery after transplantation of embryonic spinal cord tissue decreases in adult compared to neonatal host. We hypothesized that CSPGs contribute to the decrease in the extent to which transplant supports axonal remodeling and functional recovery. Expression of CSPGs increased after overhemisection SCI in adult rats but not in neonates. Embryonic spinal cord transplant was surrounded by CSPGs deposited in host cord, and the interface between host and transplant seemed to contain a large amount of CSPGs. Intrathecally delivered chondroitinase ABC (C'ase) improved recovery of distal forelimb usage and skilled motor behavior after C4 overhemisection injury and transplantation in adults. This behavioral recovery was accompanied by an increased amount of raphespinal axons growing into the transplant, and raphespinal innervation to the cervical motor region was promoted by C'ase plus transplant. Moreover, C'ase increased the number of transplanted neurons that grew axons to the host cervical enlargement, suggesting that degradation of CSPGs supports remodeling not only of host axons but also axons from transplanted neurons. Our results suggest that CSPGs constitute an inhibitory barrier to prevent axonal interactions between host and transplant in adults, and degradation of the inhibitory barrier can potentiate transplant-mediated axonal remodeling and functional recovery after SCI.     

GDNF‐enhanced axonal regeneration and myelination following spinal cord injury is mediated by primary effects on neurons

We previously demonstrated that coadministration of glial cell line‐derived neurotrophic factor (GDNF) with grafts of Schwann cells (SCs) enhanced axonal regeneration and remyelination following spinal cord injury (SCI). However, the cellular target through which GDNF mediates such actions was unclear. Here, we report that GDNF enhanced both the number and caliber of regenerated axons in vivo and increased neurite outgrowth of dorsal root ganglion neurons (DRGN) in vitro, suggesting that GDNF has a direct effect on neurons. In SC‐DRGN coculture, GDNF significantly increased the number of myelin sheaths produced by SCs. GDNF treatment had no effect on the proliferation of isolated SCs but enhanced the proliferation of SCs already in contact with axons. GDNF increased the expression of the 140 kDa neural cell adhesion molecule (NCAM) in isolated SCs but not their expression of the adhesion molecule L1 or the secretion of the neurotrophins NGF, NT3, or BDNF. Overall, these results support the hypothesis that GDNF‐enhanced axonal regeneration and SC myelination is mediated mainly through a direct effect of GDNF on neurons. They also suggest that the combination of GDNF administration and SC transplantation may represent an effective strategy to promote axonal regeneration and myelin formation after injury in the spinal cord. © 2009 Wiley‐Liss, Inc.     

Axon regeneration through scars and into sites of chronic spinal cord injury

Cellular and extracellular inhibitors are thought to restrict axon growth after chronic spinal cord injury (SCI), confronting the axon with a combination of chronic astrocytosis and extracellular matrix-associated inhibitors that collectively constitute the chronic "scar." To examine whether the chronically injured environment is strongly inhibitory to axonal regeneration, we grafted permissive autologous bone marrow stromal cells (MSCs) into mid-cervical SCI sites of adult rats, 6 weeks post-injury without resection of the "chronic scar." Additional subjects received MSCs genetically modified to express neurotrophin-3 (NT-3), providing a further local stimulus to axon growth. Anatomical analysis 3 months post-injury revealed extensive astrocytosis surrounding the lesion site, together with dense deposition of the inhibitory extracellular matrix molecule NG2. Despite this inhibitory environment, axons penetrated the lesion site through the chronic scar. Robust axonal regeneration occurred into chronic lesion cavities expressing NT-3. Notably, chronically regenerating axons preferentially associated with Schwann cell surfaces expressing both inhibitory NG2 substrates and the permissive substrates L1 and NCAM in the lesion site. Collectively, these findings indicate that inhibitory factors deposited at sites of chronic SCI do not create impenetrable boundaries and that inhibition can be balanced by local and diffusible signals to generate robust axonal growth even without resecting chronic scar tissue.     

Astrocyte progenitor transplantation promotes regeneration of bulbospinal respiratory axons,recovery of diaphragm function,and a reduced macrophage response following cervical spinal cord injury

Stem/progenitor cell transplantation delivery of astrocytes is a potentially powerful strategy for spinal cord injury (SCI). Axon extension into SCI lesions that occur spontaneously or in response to experimental manipulations is often observed along endogenous astrocyte “bridges,” suggesting that augmenting this response via astrocyte lineage transplantation can enhance axon regrowth. Given the importance of respiratory dysfunction post-SCI, we transplanted glial-restricted precursors (GRPs)—a class of lineage-restricted astrocyte progenitors—into the C2 hemisection model and evaluated effects on diaphragm function and the growth response of descending rostral ventral respiratory group (rVRG) axons that innervate phrenic motor neurons (PhMNs). GRPs survived long term and efficiently differentiated into astrocytes in injured spinal cord. GRPs promoted significant recovery of diaphragm electromyography amplitudes and stimulated robust regeneration of injured rVRG axons. Although rVRG fibers extended across the lesion, no regrowing axons re-entered caudal spinal cord to reinnervate PhMNs, suggesting that this regeneration response—although impressive—was not responsible for recovery. Within ipsilateral C3-5 ventral horn (PhMN location), GRPs induced substantial sprouting of spared fibers originating in contralateral rVRG and 5-HT axons that are important for regulating PhMN excitability; this sprouting was likely involved in functional effects of GRPs. Finally, GRPs reduced the macrophage response (which plays a key role in inducing axon retraction and limiting regrowth) both within the hemisection and at intact caudal spinal cord surrounding PhMNs. These findings demonstrate that astrocyte progenitor transplantation promotes significant plasticity of rVRG-PhMN circuitry and restoration of diaphragm function and suggest that these effects may be in part through immunomodulation.     

Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2

In the peripheral nervous system, Schwann cells (SCs) promote nerve regeneration by the secretion of trophic support molecules and the establishment of a supportive growth matrix. Elucidating factors that promote SC outgrowth following nerve injury is an important strategy for improving nerve regeneration. We identified the Netrin-1 receptors, Deleted in Colorectal Cancer (DCC) and Uncoordinated (Unc)5H2 as SC receptors that influence nerve regeneration by respectively promoting or inhibiting SC outgrowth. Significantly, we show both DCC and Unc5H2 receptors are distributed within SCs. In adult nerves, DCC is localized to the paranodes and Schmidt-Lantermann incisures of myelinating SCs, as well as along unmyelinated axons. After axotomy, DCC is prominently expressed in activated SCs at the regenerating nerve front. In contrast, Unc5H2 receptor is robustly distributed in myelinating SCs of the intact nerve and it is found at low levels in the SCs of the injury site. Local in vivo DCC siRNA mRNA knockdown at the growing tip of an injured nerve impaired SC activation and, in turn, significantly decreased axon regeneration. This forced DCC inhibition was associated with a dramatic reciprocal upregulation of Unc5H2 in the remaining SCs. Local Unc5H2 knockdown at the injury site, however, facilitated axon regrowth, indicating it has a role as an intrinsic brake to peripheral nerve regeneration. Our findings demonstrate that in adult peripheral nerves, SCs respond to DCC and Unc5H2 signaling, thereby promoting or hindering axon outgrowth and providing a novel mechanism for SC regulation during nerve regeneration.     

Increased expression of the putative axon growth-repulsive extracellular matrix molecule,keratan sulphate proteoglycan,following traumatic injury of the adult rat spinal cord

Keratan sulphate proteoglycan (KSPG) is a developmentally regulated barrier molecule, directing axonal growth during central nervous system (CNS) formation. The possible re-expression and functional significance of KSPG in preventing axon regeneration following spinal cord injury (SCI) is poorly understood. In the present investigation, the spatio-temporal expression of KSPG was studied following experimental SCI. There was no indication of sparing of axons at the lesion epicentre following severe compression injury. By 7 days post operation (p.o.) a diffuse increase of KSPG immunoreactivity (KSPG-IR) was observed in the parenchyma surrounding the lesion. This was followed by a delayed (21-28 days p.o.) and largely heterogeneous increase of KSPG-IR in the lesion epicentre, which revealed both cellular and extracellular matrix-like distribution patterns. Although no re-growth of anterogradely labelled corticospinal axons was observed, many 200-kDa neurofilament (NF)-positive axons could be detected growing into the connective tissue scar. This phase of spontaneous axonal re-growth was closely associated with a framework of glial cells (including Schwann cells from damaged local spinal nerve roots) that had migrated into the lesion site. The spontaneous nerve fibre re-growth could be detected in both KSPG-rich and KSPG-poor territories. The present data suggest that the lesion-induced up-regulation of KSPG-IR may have contributed to the lack of corticospinal axon re-growth. However, the lack of any direct spatio-temporal correlation between the distribution of raised KSPG-IR and spontaneous NF-positive axonal regeneration suggests that at least some populations of axons can resist the putative inhibitory effects of this extracellular matrix molecule.