promotion-survival-differentiation-neural-stem-cells-with-fibrin

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Previous studies indicate that peripheral nerve conditioning lesions significantly enhance central axonal regeneration via modulation of cAMP-mediated mechanisms. To gain insight into the nature and temporal dependence of neural mechanisms underlying conditioning lesion effects on central axonal regeneration, we compared the efficacy of peripheral sciatic nerve crush lesions to cAMP elevations (in lumbar dorsal root ganglia) on central sensory axonal regeneration when administered either before or after cervical spinal cord lesions. We found significantly greater effects of conditioning lesions compared to cAMP elevations on central axonal regeneration when combined with cellular grafts at the lesion site and viral neurotrophin delivery; further, these effects persisted whether conditioning lesions were applied prior to or shortly after spinal cord injury. Indeed, conditioning lesions recruited extensively greater sets of genetic mechanisms of possible relevance to axonal regeneration compared to cAMP administration, and sustained these changes for significantly greater time periods through the post-lesion period. We conclude that cAMP-mediated mechanisms account for only a portion of the potency of conditioning lesions on central axonal regeneration, and that recruitment of broader genetic mechanisms can extend the effect and duration of cellular events that support axonal growth.

Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects.

BACKGROUND: Reliable outcome measures are essential for preclinical modeling of spinal cord injury (SCI) in primates. MEASURES: need to be sensitive to both increases and decreases in function in order to demonstrate potential positive or negative effects of therapeutics. OBJECTIVES: To develop behavioral tests and analyses to assess recovery of function after SCI in the nonhuman primate. METHODS: In all, 24 male rhesus macaques were subjected to complete C7 lateral hemisection. The authors scored recovery of function in an open field and during hand tasks in a restraining chair. In addition, EMG analyses were performed in the open field, during hand tasks, and while animals walked on a treadmill. Both control and treated monkeys that received candidate therapeutics were included in this report to determine whether the behavioral assays were capable of detecting changes in function over a wide range of outcomes. RESULTS: The behavioral assays are shown to be sensitive to detecting a wide range of motor functional outcomes after cervical hemisection in the nonhuman primate. Population curves on recovery of function were similar across the different tasks; in general, the population recovers to about 50% of baseline performance on measures of forelimb function. CONCLUSIONS: The behavioral outcome measures that the authors developed in this preclinical nonhuman primate model of SCI can detect a broad range of motor recovery. A set of behavioral assays is an essential component of a model that will be used to test efficacies of translational candidate therapies for SCI.

Methods for Functional Assessment After C7 Spinal Cord Hemisection in the Rhesus Monkey.

Hintergrund: Zuverl&auml;ssiges Ergebnis Ma&szlig;nahmen sind unabdingbar f&uuml;r die pr&auml;klinische Modellierung einer R&uuml;ckenmarksverletzung (SCI) bei Primaten. Ma&szlig;nahmen: m&uuml;ssen Sie sensibel auf Erh&ouml;hungen und Verringerungen der Funktion sein, um m&ouml;gliche positive oder negative Auswirkungen der Therapeutika zu demonstrieren. Ziele: Verhaltens Versuche und Analysen zur Wiederherstellung der Funktion nach SCI in die nichtmenschlichen Primaten Bewertung zu entwickeln. Methoden: Insgesamt wurden 24 m&auml;nnlich Rhesus-Makaken komplette C7 seitliche Hemisektion unterworfen. Die Autoren erzielte Wiederherstellung der Funktion in ein offenes Feld und w&auml;hrend der Hand Aufgaben in einem d&auml;mpfenden Stuhl. Dar&uuml;ber hinaus wurden EMG Analysen durchgef&uuml;hrt in das Feld &Ouml;ffnen ein, w&auml;hrend Hand Aufgaben und w&auml;hrend Tiere auf einem Laufband ging. Kontrolle und behandelten Affen, die Kandidaten-Therapeutika empfangen wurden in diesem Bericht um festzustellen, ob die verhaltensbasierte Assays feststellen k&ouml;nnen &Auml;nderungen in der Funktion &uuml;ber einen weiten Bereich der Ergebnisse wurden aufgenommen. Ergebnisse: Die verhaltensbasierte Assays sind empfindlich auf die Aufdeckung von vielf&auml;ltige motorische funktionelle Ergebnisse nach zervikaler Hemisektion in die nichtmenschlichen Primaten dargestellt. Bev&ouml;lkerung Kurven auf Wiederherstellung der Funktion &auml;hnelten sich &uuml;ber die verschiedenen Aufgaben; im allgemeinen gewinnt die Bev&ouml;lkerung auf etwa 50 % der Basisleistung &uuml;ber Ma&szlig;nahmen der Vorderbein-Funktion. Schlussfolgerungen: Die verhaltensbasierte ergebnisorientierten Ma&szlig;nahmen, die die Autoren in diesem pr&auml;klinische nichtmenschlichen Primaten-Modell der SCI entwickelt k&ouml;nnen eine breite Palette von motorischen Wiederherstellung erkennen. Eine Reihe von Verhaltens-Assays ist ein wesentlicher Bestandteil eines Modells, die Leistungsnachteile translational Kandidat Therapien f&uuml;r Sci testen verwendet werden

Methoden zur funktionellen Beurteilung nach C7 R&uuml;ckenmarks Hemisektion in der Rhesus-Affen.

Primates are an important and unique animal resource. We have developed a nonhuman primate model of spinal cord injury (SCI) to expand our knowledge of normal primate motor function, to assess the impact of disease and injury on sensory and motor function, and to test candidate therapies before they are applied to human patients. The lesion model consists of a lateral spinal cord hemisection at the C7 spinal level with subsequent examination of behavioral, electrophysiological, and anatomical outcomes. Results to date have revealed significant neuroanatomical and functional differences between rodents and primates that impact the development of candidate therapies. Moreover, these findings suggest the importance of testing some therapeutic approaches in nonhuman primates prior to the use of invasive approaches in human clinical trials. Our primate model is intended to: 1) lend greater positive predictive value to human translatable therapies, 2) develop appropriate methods for human translation, 3) lead to basic discoveries that might not be identified in rodent models and are relevant to human translation, and 4) identify new avenues of basic research to "reverse-translate" important questions back to rodent models.

Animal models of neurologic disorders: a nonhuman primate model of spinal cord injury.

Primaten sind eine wichtige und einzigartige Tier-Ressource. Wir entwickelten ein nichtmenschlichen Primaten-Modell einer R&uuml;ckenmarksverletzung (SCI) erweitern unsere Kenntnisse der normalen Primas Motorik, zur Bewertung der Auswirkungen von Erkrankungen und Verletzungen auf die sensorischen und motorischen Funktion und Kandidat Therapien zu testen, bevor sie auf menschliche Patienten angewendet werden. Das L&auml;sion-Modell besteht aus einer seitlichen R&uuml;ckenmarks Hemisektion auf spinaler Ebene C7 mit anschlie&szlig;enden Untersuchung der Verhaltens- und elektrophysiologische anatomische Ergebnisse. Die bisherigen Ergebnisse haben gezeigt, dass erhebliche neuroanatomischen und funktionale Unterschiede zwischen Nagetiere und Primaten, die Auswirkungen auf die Entwicklung der Kandidat Therapien. Diese Erkenntnisse empfehlen dar&uuml;ber hinaus die Bedeutung von Tests einige therapeutische Ans&auml;tze in nicht-menschlichen Primaten vor der Verwendung der invasive Ans&auml;tze in den menschlichen klinischen Studien. Unser Primas-Modell soll: 1) verleihen mehr positiven Vorhersagewert zu menschlichen &uuml;bersetzbare Therapien, 2) entwickeln geeignete Methoden f&uuml;r menschliche &Uuml;bersetzung, 3) dazu f&uuml;hren, dass grundlegende Entdeckungen, die m&ouml;glicherweise nicht im Nagetier Modelle und menschliche &Uuml;bersetzung relevant sind, und 4) identifizieren neue Wege der Grundlagenforschung auf &quot;umgekehrt-&uuml;bersetzen&quot; wichtige Fragen zur&uuml;ck zu Nagetier-Modelle.

Tiermodellen f&uuml;r neurologische Erkrankungen: ein nicht-menschlichen Primaten-Modell einer R&uuml;ckenmarksverletzung.

Progress in the field of axonal regeneration research has been like the process of axonal growth itself: there is steady progress toward reaching the target, but there are episodes of mistargeting, misguidance along false routes, and connections that must later be withdrawn. This primer will address issues in the study of axonal growth after central nervous system injury in an attempt to provide guidance toward the goal of progress in the field. We address definitions of axonal growth, sprouting and regeneration after injury, and the research tools to assess growth.

Concepts and methods for the study of axonal regeneration in the CNS.

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.

Motor axonal regeneration after partial and complete spinal cord transection.

Although Aβ peptides are causative agents in Alzheimer's disease (AD), the underlying mechanisms are still elusive. We report that Aβ42 induces a translational block by activating AMPK, thereby inhibiting the mTOR pathway. This translational block leads to widespread ER stress, which activates JNK3. JNK3 in turn phosphorylates APP at T668, thereby facilitating its endocytosis and subsequent processing. In support, pharmacologically blocking translation results in a significant increase in Aβ42 in a JNK3-dependent manner. Thus, JNK3 activation, which is increased in human AD cases and a familial AD (FAD) mouse model, is integral to perpetuating Aβ42 production. Concomitantly, deletion of JNK3 from FAD mice results in a dramatic reduction in Aβ42 levels and overall plaque loads and increased neuronal number and improved cognition. This reveals AD as a metabolic disease that is under tight control by JNK3.

JNK3 perpetuates metabolic stress induced by Aβ peptides.

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.

Significant advances have been made in understanding the mechanisms that limit axon regeneration in the adult mammalian central nervous system and in addressing some of the obstacles for axon growth. Despite this progress numerous challenges remain to achieve regeneration of a large number of axons sufficient to mediate functional improvement. Given the complexity of injury-induced changes in axon, cell body, and parenchyma surrounding a spinal cord lesion, it seems likely that multiple factors both intrinsic and extrinsic to injured neurons have to be addressed to augment axon regeneration and useful reorganization of spared circuitry. Neurotrophic factors have been shown to be one potent means to increase the number and range of regenerating axons, to guide regenerating axons across a lesion site, and to augment regenerative cell body responses to injury. In this chapter we will review the potential and current limitations of neurotrophic factors and gene therapy, in combination with cellular transplants, for axon regeneration and sprouting in the injured spinal cord.

Gene therapy, neurotrophic factors and spinal cord regeneration.

Bioengineered scaffolds have the potential to support and guide injured axons after spinal cord injury, contributing to neural repair. In previous studies we have reported that templated agarose scaffolds can be fabricated into precise linear arrays and implanted into the partially injured spinal cord, organizing growth and enhancing the distance over which local spinal cord axons and ascending sensory axons extend into a lesion site. However, most human injuries are severe, sparing only thin rims of spinal cord tissue in the margins of a lesion site. Accordingly, in the present study we examined whether template agarose scaffolds seeded with bone marrow stromal cells secreting Brain-Derived Neurotrophic Factor (BDNF) would support regeneration into severe, complete spinal cord transection sites. Moreover, we tested responses of motor axon populations originating from the brainstem. We find that templated agarose scaffolds support motor axon regeneration into a severe spinal cord injury model and organize axons into fascicles of highly linear configuration. BDNF significantly enhances axonal growth. Collectively, these findings support the feasibility of scaffold implantation for enhancing central regeneration after even severe central nervous system injury.

Templated agarose scaffolds for the support of motor axon regeneration into sites of complete spinal cord transection.

Regeneration of injured CNS neurons was once thought to be an unachievable goal. Most patients with significant damage to the spinal cord suffer from permanently impaired neurological function. A century of research, however, has led to an understanding of multiple factors that limit CNS regeneration and from this knowledge experimental strategies have emerged for enhancing CNS repair. Some of these approaches have undergone human translation. Nevertheless, translating experimental findings to human trials has been more challenging than anticipated. In this chapter, we will review the current state of knowledge regarding central axonal growth failure after injury, and approaches taken to enhance recovery after SCI.

Frontiers of spinal cord and spine repair: experimental approaches for repair of spinal cord injury.

Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders.

Low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cell signaling promotes axonal regeneration.

Adult CNS neurons exhibit a reduced capacity for growth compared to developing neurons, due in part to downregulation of growth-associated genes as development is completed. We tested the hypothesis that SnoN, an embryonically regulated transcription factor that specifies growth of the axonal compartment, can enhance growth in injured adult neurons. In vitro, SnoN overexpression in dissociated adult DRG neuronal cultures significantly enhanced neurite outgrowth. Moreover, TGF-β1, a negative regulator of SnoN, inhibited neurite outgrowth, and SnoN over-expression overcame this inhibition. We then examined whether SnoN influenced axonal regeneration in vivo: indeed, expression of a mutant form of SnoN resistant to degradation significantly enhanced axonal regeneration following cervical spinal cord injury, despite peri-lesional upregulation of TGF-β1. Thus, a developmental mechanism that specifies extension of the axonal compartment also promotes axonal regeneration after adult CNS injury.

SnoN facilitates axonal regeneration after spinal cord injury.

Brain-derived neurotrophic factor (BDNF) improves molecular, cellular, and behavioral measures of neural dysfunction in genetic models of Alzheimer's disease (Blurton-Jones et al., 2009; Nagahara et al., 2009). However, BDNF treatment after disease onset has not been reported to improve neuronal survival in these models. We now report prevention of neuronal loss with early life BDNF treatment in mutant mice expressing two amyloid precursor protein (APP) mutations associated with early-onset familial Alzheimer's disease. APP transgenic mice underwent lentiviral BDNF gene delivery into the entorhinal cortices at age 2 months and were examined 5 months later. BDNF-treated mice exhibited significant improvements in hippocampal-dependent contextual fear conditioning compared with control-treated APP mice (p < 0.05). Stereological analysis of entorhinal cortical cell number demonstrated ∼20% reductions in neuronal number in layers II-VI of the entorhinal cortex in untreated APP mutant mice compared with wild-type mice (p < 0.0001), and significant amelioration of cell loss by BDNF (p < 0.001). Moreover, BDNF gene delivery improved synaptophysin immunoreactivity in the entorhinal cortex and, through anterograde BDNF transport, in the hippocampus (p < 0.01). Notably, BDNF did not affect amyloid plaque numbers, indicating that direct amyloid reduction is not necessary to achieve significant neuroprotective benefits in mutant amyloid models of Alzheimer's disease.

Early BDNF treatment ameliorates cell loss in the entorhinal cortex of APP transgenic mice.

Guest Editorial: Opportunities in rehabilitation research.

Neural stem cell dissemination after grafting to CNS injury sites.

Spinal cord injury (SCI) damages both gray matter and white matter, but white matter damage is responsible for the vast majority of the subsequent functional loss. Neural stem cells (NSCs) have been investigated as a means of improving outcomes after SCI, either through neuroprotective properties that limit secondary damage or by direct cell replacement. This review will focus on cell replacement strategies, and the ability of multipotent NSCs to form new functional synaptic relays across sites of even severe SCI. The ability of these early stage neurons to extend axons from the lesion site in large numbers and over long distances constitutes an important mechanism underlying their potential to promote neural repair.

Article	Year
Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics\| PubMed ID: 21904786	2011
Conditioning lesions before or after spinal cord injury recruit broad genetic mechanisms that sustain axonal regeneration: superiority to camp-mediated effects. Experimental neurology\| PubMed ID: 22227059	2012
Methods for Functional Assessment After C7 Spinal Cord Hemisection in the Rhesus Monkey. Neurorehabilitation and neural repair\| PubMed ID: 22331214	2012
Animal models of neurologic disorders: a nonhuman primate model of spinal cord injury. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics\| PubMed ID: 22427157	2012
Concepts and methods for the study of axonal regeneration in the CNS. Neuron\| PubMed ID: 22681683	2012
Motor axonal regeneration after partial and complete spinal cord transection. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 22699902	2012
JNK3 perpetuates metabolic stress induced by Aβ peptides. Neuron\| PubMed ID: 22958823	2012
Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell\| PubMed ID: 22980985	2012
Gene therapy, neurotrophic factors and spinal cord regeneration. Handbook of clinical neurology\| PubMed ID: 23098737	2012
Templated agarose scaffolds for the support of motor axon regeneration into sites of complete spinal cord transection. Biomaterials\| PubMed ID: 23182350	2013
Frontiers of spinal cord and spine repair: experimental approaches for repair of spinal cord injury. Advances in experimental medicine and biology\| PubMed ID: 23281510	2012
Low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cell signaling promotes axonal regeneration. The Journal of biological chemistry\| PubMed ID: 23867460	2013
SnoN facilitates axonal regeneration after spinal cord injury. PloS one\| PubMed ID: 23936531	2013
Early BDNF treatment ameliorates cell loss in the entorhinal cortex of APP transgenic mice. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24068826	2013
Guest Editorial: Opportunities in rehabilitation research. Journal of rehabilitation research and development\| PubMed ID: 24203548	2013
Neural stem cell dissemination after grafting to CNS injury sites. Cell\| PubMed ID: 24485445	2014
Axonal growth and connectivity from neural stem cell grafts in models of spinal cord injury. Current opinion in neurobiology\| PubMed ID: 24709371	2014

Mark Tuszynski

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