Depolarization and electrical stimulation enhance in vitro and in vivo sensory axon growth after spinal cord injury

Activity dependent plasticity is a key mechanism for the central nervous system (CNS) to adapt to its environment. Whether neuronal activity also influences axonal regeneration in the injured CNS, and whether electrical stimulation (ES) can activate regenerative programs in the injured CNS remains incompletely understood. Using KCl-induced depolarization, in vivo ES followed by ex-vivo neurite growth assays and ES after spinal cord lesions and cell grafting, we aimed to identify parameters important for ES-enhanced neurite growth and axonal regeneration. Using cultures of sensory neurons, neurite growth was analyzed after KCl-induced depolarization for 1-72h. Increased neurite growth was detected after short-term stimulation and after longer stimulation if a sufficient delay between stimulation and growth measurements was provided. After in vivo ES (20Hz, 2× motor threshold, 0.2ms, 1h) of the intact sciatic nerve in adult Fischer344 rats, sensory neurons showed a 2-fold increase in in vitro neurite length one week later compared to sham animals, an effect not observed one day after ES. Longer ES (7h) and repeated ES (7days, 1h each) also increased growth by 56-67% one week later, but provided no additional benefit. In vivo growth of dorsal column sensory axons into a graft of bone marrow stromal cells 4weeks after a cervical spinal cord lesion was also enhanced with a single post-injury 1h ES of the intact sciatic nerve and was also observed after repeated ES without inducing pain-like behavior. While ES did not result in sensory functional recovery, our data indicate that ES has time-dependent influences on the regenerative capacity of sensory neurons and might further enhance axonal regeneration in combinatorial approaches after SCI

Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats

Following a spinal cord injury (SCI) a growth aversive environment forms, consisting of a fibroglial scar and inhibitory factors, further restricting the already low intrinsic growth potential of injured adult central nervous system (CNS) neurons. Previous studies have shown that local administration of the microtubule-stabilizing drug paclitaxel or epothilone B (Epo B) reduce fibrotic scar formation and axonal dieback as well as induce axonal growth/sprouting after SCI. Likewise, systemic administration of Epo B promoted functional recovery. In this study, we investigated the effects of epothilone D (Epo D), an analog of Epo B with a possible greater therapeutic index, on fibrotic scarring, axonal sprouting and functional recovery after SCI. Delayed systemic administration of Epo D after a moderate contusion injury (150 kDyn) in female Fischer 344 rats resulted in a reduced number of footfalls when crossing a horizontal ladder at 4 and 8 weeks post-injury. Hindlimb motor function assessed with the BBB open field locomotor rating scale and Catwalk gait analysis were not significantly altered. Moreover, formation of laminin positive fibrotic scar tissue and 5-HT positive serotonergic fiber length caudal to the lesion site were not altered after treatment with Epo D. These findings recapitulate a functional benefit after systemic administration of a microtubule-stabilizing drug in rat contusion SCI

Mesenchymal Stem Cells Promote Oligodendroglial Differentiation in Hippocampal Slice Cultures

We have previously shown that soluble factors derived from mesenchymal stem cells (MSCs) induce oligodendrogenic fate and differentiation in adult rat neural progenitors (NPCs) in vitro. Here, we investigated if this pro-oligodendrogenic effect is maintained after cells have been transplanted onto rat hippocampal slice cultures, a CNS-organotypic environment. We first tested whether NPCs, that were pre-differentiated in vitro by MSC-derived conditioned medium, would generate oligodendrocytes after transplantation. This approach resulted in the loss of grafted NPCs, suggesting that oligodendroglial pre-differentiated cells could not integrate in the tissue and therefore did not survive grafting. However, when NPCs together with MSCs were transplanted in situ into hippocampal slice cultures, the grafted NPCs survived and the majority of them differentiated into oligodendrocytes. In contrast to the prevalent oligodendroglial differentiation in case of the NPC/MSC co-transplantation, naive NPCs transplanted in the absence of MSCs differentiated predominantly into astrocytes. In summary, the pro-oligodendrogenic activity of MSCs was maintained only after co-transplantation into hippocampal slice cultures. Therefore, in the otherwise astrogenic milieu, MSCs established an oligodendrogenic niche for transplanted NPCs, and thus, co-transplantation of MSCs with NPCs might provide an attractive approach to re-myelinate the various regions of the diseased CNS. Copyright (C) 2009 S. Karger AG, Base

Co-transplantation of adult neural stem, progenitor cells together with cells of mesenchymal origin into the injured spinal cord

The irreversible loss of spinal cord parenchyma including astroglia, oligodendroglia and neurons is one of the key factors responsible for the severe functional impairment in individuals suffering from spinal cord injury Therefore, adequate cell replacement strategies might be one means to promote structural and functional recovery. Neural stem/ progenitor cells (NPC), which have been identified in the adult mammalian nervous system including the spinal cord, represent one promising source to replace astrocytes, oligodendrocytes and neurons within the injured spinal cord. Intrinsic NPC at and around the lesion site can be stimulated by the application of appropriate molecules to replace lost spinal cord tissue intrinsically. Alternatively, neural stem cells can be isolated from small brain/spinal cord biopsies, propagated in vitro and ultimately transplanted into the injured spinal cord. Recently it has been published that mesenchymal stem cells (MSC) secrete a yet unidentified factor, which strongly promotes oligodendroglial differentiation of hippocampus derived adult neural progenitor cells in vitro under co-culture conditions, whereas the astrogenic commitment of NPC is inhibited. Based on these findings, I investigated whether the region of isolation (origin) of NPC will influence the expression pattern of specific differentiation markers after incubation with MSC-conditioned media (MSC-CM). I could show that MSC-derived soluble factors induce the expression of oligodendrocyte markers in NPC in vitro regardless of the origin of the NPC. Furthermore, incubation of NPC with conditioned media derived from fibroblasts resulted in an even higher number of cells expressing the oligodendroglial marker MBP at the expense of cells expressing the astroglial marker GFAP. In the next step, NPC or NPC pre-differentiated towards an oligodendroglial lineage were co-seeded with MSC onto hippocampal slice cultures. Under CNS-organotypic conditions MSC still promoted an oligodendroglial fate of seeded NPC. While the survival of the seeded NPC was good, the survival of oligodendroglial pre-differentiated NPC was very limited after seeding onto hippocampal slices. To see if the pro-oligodendrogenic activity of MSC is maintained in vivo, NPC or pre-differentiated NPC were co-transplanted with MSC into the intact spinal cord of adult rats. Although the survival of pre-differentiated NPC was very low, a significantly increased oligodendroglial differentiation was observed when compared to NPC co-grafted with MSC. In subsequent experiments, NPC were co-grafted with MSC or fibroblast into the injured spinal cord. Histological analysis demonstrated that fibroblast as well as MSC containing grafts filled the cystic lesion after SCI and provided a supporting scaffold to sustain adult NPC within the lesion cavity. Interestingly, fibroblasts but not MSC increased the oligodendroglial differentiation of co-grafted NPC in the injured spinal cord. In vitro data demonstrated that BMP2 and BMP4 (bone morphogenic protein 2 and 4), which are strongly up-regulated after spinal cord injury completely counteracted effects of MSC, on oligodendroglial differentiation of NPC. Moreover, my studies revealed that the transplantation of MSC into the injured spinal cord does not alter the proliferation or survival of endogenous NPC. Rather MSC influence the differentiation of endogenous oligodendroglial progeny as early as three days after SCI and shift the differentiation pattern of NPC towards an oligodendroglial phenotype four weeks after SCI at the expense of astroglial differentiation. In summary, these studies demonstrate that MSC provide a pro-oligodendrogenic microenvironment for NPC seeded onto hippocampal slices or transplanted into the intact spinal cord. In contrast, MSC do not influence the differentiation of co-transplanted NPC in the acutely injured spinal cord, but profoundly affect the differentiation of endogenous NPC. For any cell-based therapy to be translated into the clinic appropriate monitoring tools need to be established to visualize morphological changes caused by cell transplantation into the injured spinal cord. Magnetic resonance imaging (MRI) represents the gold-standard to non-invasively visualize the spinal cord parenchyma. As a first step to validate cell-therapy induced morphological changes, I performed analysis using a routine clinical 3T MRI-scanner. The referring study demonstrated that a routine clinical 3T MRI-scanner can be used for small animal imaging to noninvasively visualize pathological changes occurring after rat spinal cord injury. Changes in 3T MRI signals correlate with histological, structural and behavioral outcomes after SCI

Thoracic rat spinal cord contusion injury induces remote spinal gliogenesis but not neurogenesis or gliogenesis in the brain.

After spinal cord injury, transected axons fail to regenerate, yet significant, spontaneous functional improvement can be observed over time. Distinct central nervous system regions retain the capacity to generate new neurons and glia from an endogenous pool of progenitor cells and to compensate neural cell loss following certain lesions. The aim of the present study was to investigate whether endogenous cell replacement (neurogenesis or gliogenesis) in the brain (subventricular zone, SVZ; corpus callosum, CC; hippocampus, HC; and motor cortex, MC) or cervical spinal cord might represent a structural correlate for spontaneous locomotor recovery after a thoracic spinal cord injury. Adult Fischer 344 rats received severe contusion injuries (200 kDyn) of the mid-thoracic spinal cord using an Infinite Horizon Impactor. Uninjured rats served as controls. From 4 to 14 days post-injury, both groups received injections of bromodeoxyuridine (BrdU) to label dividing cells. Over the course of six weeks post-injury, spontaneous recovery of locomotor function occurred. Survival of newly generated cells was unaltered in the SVZ, HC, CC, and the MC. Neurogenesis, as determined by identification and quantification of doublecortin immunoreactive neuroblasts or BrdU/neuronal nuclear antigen double positive newly generated neurons, was not present in non-neurogenic regions (MC, CC, and cervical spinal cord) and unaltered in neurogenic regions (dentate gyrus and SVZ) of the brain. The lack of neuronal replacement in the brain and spinal cord after spinal cord injury precludes any relevance for spontaneous recovery of locomotor function. Gliogenesis was increased in the cervical spinal cord remote from the injury site, however, is unlikely to contribute to functional improvement

Thoracic Rat Spinal Cord Contusion Injury Induces Remote Spinal Gliogenesis but Not Neurogenesis or Gliogenesis in the Brain

After spinal cord injury, transected axons fail to regenerate, yet significant, spontaneous functional improvement can be observed over time. Distinct central nervous system regions retain the capacity to generate new neurons and glia from an endogenous pool of progenitor cells and to compensate neural cell loss following certain lesions. The aim of the present study was to investigate whether endogenous cell replacement (neurogenesis or gliogenesis) in the brain (subventricular zone, SVZ; corpus callosum, CC; hippocampus, HC; and motor cortex, MC) or cervical spinal cord might represent a structural correlate for spontaneous locomotor recovery after a thoracic spinal cord injury. Adult Fischer 344 rats received severe contusion injuries (200 kDyn) of the mid-thoracic spinal cord using an Infinite Horizon Impactor. Uninjured rats served as controls. From 4 to 14 days post-injury, both groups received injections of bromodeoxyuridine (BrdU) to label dividing cells. Over the course of six weeks post-injury, spontaneous recovery of locomotor function occurred. Survival of newly generated cells was unaltered in the SVZ, HC, CC, and the MC. Neurogenesis, as determined by identification and quantification of doublecortin immunoreactive neuroblasts or BrdU/neuronal nuclear antigen double positive newly generated neurons, was not present in non-neurogenic regions (MC, CC, and cervical spinal cord) and unaltered in neurogenic regions (dentate gyrus and SVZ) of the brain. The lack of neuronal replacement in the brain and spinal cord after spinal cord injury precludes any relevance for spontaneous recovery of locomotor function. Gliogenesis was increased in the cervical spinal cord remote from the injury site, however, is unlikely to contribute to functional improvement

Peptides and Astroglia Improve the Regenerative Capacity of Alginate Gels in the Injured Spinal Cord

Anisotropic alginate hydrogels with microchannels can provide a substrate for cotransplanted cells and for axons in the injured spinal cord by physically guiding regenerating axons across a lesion. However, alginate gels alone only result in modest axonal growth responses. To determine whether modification of negatively charged alginate hydrogels with positively charged polyamino acids (poly-l-ornithine [PLO]) and laminin enhance axonal growth, cell adhesion, and neurite growth were examined in vitro and in vivo. Up to 400 mu g peptide/mg alginate dry weight could be electrostatically bound for at least 2 weeks in vitro significantly increasing cell adhesion and neurite outgrowth from dorsal root ganglion neurons. In vivo, PLO/laminin-coated hydrogels grafted into a cervical lateral hemisection in adult female Fischer 344 rats resulted in increased host cell migration into alginate channels and a slight increase in neurite growth. To further enhance integration of scaffolds, syngeneic postnatal astrocytes isolated from GFP-transgenic rats were seeded into coated alginate channels before grafting. Astrocytes survived, filled the majority of alginate channels, and served as a cellular bridge for axons. Regenerating axons, including descending serotonergic fibers, preferentially extended into astrocyte-containing channels, which contained a higher number of axons over the entire length of the hydrogel. Thus, alginate hydrogel scaffolds can be stably modified with bioactive peptides and cografts of postnatal astrocytes further promote scaffold integration and neurite extension. Impact Statement Axonal bridging across a lesion in the injured spinal cord requires a growth substrate and guidance cues. Using alginate hydrogels with capillary channels we show that poly-l-ornithine and laminin can be stably bound and improve cell adhesion and neurite growth in vitro, and axon growth in vivo by enhancing host cell infiltration in the injured spinal cord. Filling of coated hydrogels with postnatal astrocytes further increases short-distance axon growth and results in a continuous astroglial substrate across the host/graft interface. Thus, positively charged bioactive molecules can be stably bound to anisotropic capillary alginate hydrogels and early astrocytes further promote tissue integration

Post SCI cell renewal in motor cortex, subventricular zone, corpus callosum and hippocampus.

<p>(A, D, G, J) Quantification of BrdU positive cells in control (Intact) versus spinal cord injured (SCI) animals in the motor cortex (MC; A), subventricular zone (SVZ; D), corpus callosum (CC; G) and hippocampus (HC; H). Brightfield micrographs display cell renewal represented by BrdU immunoreactive nuclei in the MC (B, C), SVZ (E, F), CC (H, I) and HC (K, L) of control (Intact; B, E, H, K) and injured animals (C, F, I, L). Scale bar: 50 µm in (L).</p