Cellular Dynamics during Spinal Cord Regeneration in Larval Zebrafish

2019 ◽  
Vol 41 (1-2) ◽  
pp. 112-122 ◽  
Author(s):  
Consuelo Anguita-Salinas ◽  
Mario Sánchez ◽  
Rodrigo A. Morales ◽  
María Laura Ceci ◽  
Diego Rojas-Benítez ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Immune Cells ◽  
Cell Types ◽  
Regenerative Process ◽  
Spinal Cord Regeneration ◽  
Cord Injury ◽  
Complete Reversal ◽  
Transgenic Lines ◽  
Functional Regeneration

The study of spinal cord regeneration using diverse animal models, which range from null to robust regenerative capabilities, is imperative for understanding how regeneration evolved and, eventually, to treat spinal cord injury and paralysis in humans. In this study, we used electroablation to fully transect the spinal cord of zebrafish larvae (3 days postfertilization) and examined regeneration of the tissue over time. We used transgenic lines to follow immune cells, oligodendrocytes, and neurons in vivo during the entire regenerative process. We observed that immune cells are recruited to the injury site, oligodendrocytes progenitor cells (olig2-expressing cells) invade, and axons cross the gap generated upon damage from anterior to reinnervate caudal structures. Together with the recovery of cell types and structures, a complete reversal of paralysis was observed in the lesioned larvae indicating functional regeneration. Finally, using transplantation to obtain mosaic larvae with single-labeled neurons, we show that severed spinal axons exhibited varying regenerative capabilities and plasticity depending on their original dorsoventral position in the spinal cord.

scholarly journals Dynamic control of proinflammatory cytokines Il-1β and Tnf-α by macrophages is necessary for functional spinal cord regeneration in zebrafish

2018 ◽  
Author(s):  
Themistoklis M. Tsarouchas ◽  
Daniel Wehner ◽  
Leonardo Cavone ◽  
Tahimina Munir ◽  
Marcus Keatinge ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Immune Cells ◽  
Dynamic Control ◽  
Spinal Cord Regeneration ◽  
Tnf Α ◽  
Cord Injury ◽  
Functional Regeneration ◽  
Necessary And Sufficient ◽  
Early Regeneration

ABSTRACTSpinal cord injury leads to a massive response of innate immune cells (microglia, macrophages, neutrophils) both, in non-regenerating mammals and in successfully regenerating zebrafish, but the role of these immune cells in functional spinal cord regeneration in zebrafish has not been addressed. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in larval zebrafish. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary and sufficient for full regeneration. Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Il-1β and Tnf-α. Decreasing Il-1β levels or number of Il-1β+ neutrophils rescues functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1β function impairs regeneration in irf8 and wildtype animals. Inhibiting Tnf-α does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. Hence, inflammation is tightly and dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

scholarly journals Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury

2020 ◽  
Author(s):  
Marcus Keatinge ◽  
Themistoklis M. Tsarouchas ◽  
Tahimina Munir ◽  
Juan Larraz ◽  
Davide Gianni ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Rapid Assessment ◽  
Limiting Factor ◽  
Polymorphism Analysis ◽  
List Type ◽  
Spinal Cord Regeneration ◽  
Highly Active ◽  
Cord Injury ◽  
Crispr Screen

ABSTRACTAcute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by prescreening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest.HIGHLIGHTS- Synthetic CRISPR gRNAs are highly active- in vivo pre-screening allows rapid assessment of CRISPR gRNA activity- Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration- tgfb1a promotes spinal regeneration by controlling inflammation

Potential of combination of bone marrow nucleated and mesenchymal stem cells in complete spinal cord injury

2020 ◽  
Vol 15 ◽  
Author(s):  
Shojiro Katoh ◽  
Vidyasagar Devaprasad Dedeepiya ◽  
Satoshi Kuroda ◽  
Masaru Iwasaki ◽  
Rajappa Senthilkumar ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Animal Studies ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Definitive Treatment ◽  
Spinal Cord Regeneration ◽  
Cord Injury

Background: Cell-based therapies represent one of the definitive treatment approaches to SCI which to become a routine clinical application is marred by several known unknowns. The bone marrow mononuclear cells (BMMNCs) and mesenchymal stem cells (MSCs) represent the most clinically applied cell types for SCI in humans, with safety established and to an extent, efficacy reported. Methods: In this review we have analysed the clinical studies done using BMMNC and MSC for complete SCI separately and the potential for applying those cells in combination. We have also analysed those factors whose outcome in animal studies of SCI could be evaluated in depth but the clinical outcome cannot be evaluated intrinsically owing to practical difficulties. Conclusion: A combination of these two cell types, BMMNC and MSC has been proven to be advantageous than applying them separately. Therefore, a thorough evaluation including rationale and potential implications of applying these two therapies has been presented here and we hypothesize that such a combination is likely to improvise the outcome of a wholesome approach to spinal cord regeneration after SCI.

scholarly journals Survival and Integration of Transplanted Olfactory Ensheathing Cells are Crucial for Spinal Cord Injury Repair: Insights from the Last 10 Years of Animal Model Studies

2019 ◽  
Vol 28 (1_suppl) ◽  
pp. 132S-159S ◽  
Author(s):  
Ronak Reshamwala ◽  
Megha Shah ◽  
James St John ◽  
Jenny Ekberg
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Three Dimensional ◽  
Cell Types ◽  
Olfactory Nerve ◽  
Olfactory Ensheathing Cells ◽  
Spinal Cord Regeneration ◽  
Key Factors ◽  
Cord Injury ◽  
Ensheathing Cells

Olfactory ensheathing cells (OECs), the glial cells of the primary olfactory nervous system, support the natural regeneration of the olfactory nerve that occurs throughout life. OECs thus exhibit unique properties supporting neuronal survival and growth. Transplantation of OECs is emerging as a promising treatment for spinal cord injury; however, outcomes in both animals and humans are variable and the method needs improvement and standardization. A major reason for the discrepancy in functional outcomes is the variability in survival and integration of the transplanted cells, key factors for successful spinal cord regeneration. Here, we review the outcomes of OEC transplantation in rodent models over the last 10 years, with a focus on survival and integration of the transplanted cells. We identify the key factors influencing OEC survival: injury type, source of transplanted cells, co-transplantation with other cell types, number and concentration of cells, method of delivery, and time of transplantation after the injury. We found that two key issues are hampering optimization and standardization of OEC transplantation: lack of (1) reliable methods for identifying transplanted cells, and (2) three-dimensional systems for OEC delivery. To develop OEC transplantation as a successful and standardized therapy for spinal cord injury, we must address these issues and increase our understanding of the complex parameters influencing OEC survival.

scholarly journals CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009515
Author(s):  
Marcus Keatinge ◽  
Themistoklis M. Tsarouchas ◽  
Tahimina Munir ◽  
Nicola J. Porter ◽  
Juan Larraz ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Spinal Injury ◽  
Spinal Cord Regeneration ◽  
Post Injury ◽  
Highly Active ◽  
Cord Injury ◽  
Mechanistic Basis ◽  
Acute Injection

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

scholarly journals Transneuronal delivery of designer-cytokine enables functional recovery after complete spinal cord injury

2019 ◽  
Author(s):  
Marco Leibinger ◽  
Charlotte Zeitler ◽  
Philipp Gobrecht ◽  
Anastasia Andreadaki ◽  
Dietmar Fischer
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Brain Stem ◽  
Functional Recovery ◽  
Spinal Cord Regeneration ◽  
Serotonergic Neurons ◽  
Cord Injury ◽  
Functional Regeneration ◽  
Complete Spinal Cord Injury ◽  
The Brain

AbstractSpinal cord injury (SCI) often causes severe and permanent disabilities. The current study uses a transneuronal approach to stimulate spinal cord regeneration by AAV-hyper-IL-6 (hIL-6) application after injury. While preinjury PTEN knockout in cortical motoneurons fails to improve functional recovery after complete spinal cord crush, a single, postinjury injection of hIL-6 into the sensorimotor cortex markedly promotes axon regeneration in the corticospinal and, remarkably, raphespinal tracts enabling significant locomotion recovery of both hindlimbs. Moreover, transduced cortical motoneurons directly innervate serotonergic neurons in both sides of the raphe nuclei equally, enabling the synaptic release of hIL-6 and the transneuronal stimulation of raphe neurons in the brain stem. Functional recovery depends on the regeneration of serotonergic neurons as their degeneration induced by a toxin abolishes the hIL-6-mediated recovery. Thus, the transneuronal application of highly potent cytokines enables functional regeneration by stimulating neurons in the deep brain stem that are otherwise challenging to access, yet highly relevant for functional recovery after SCI.

Further studies on free-radical pathology in the major central nervous system disorders: effect of very high doses of methylprednisolone on the functional outcome, morphology, and chemistry of experimental spinal cord impact injury

1982 ◽  
Vol 60 (11) ◽  
pp. 1415-1424 ◽  
Author(s):  
H. B. Demopoulos ◽  
E. S. Flamm ◽  
M. L. Seligman ◽  
D. D. Pietronigro ◽  
J. Tomasula ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Central Nervous System ◽  
Nervous System ◽  
Free Radical ◽  
Tissue Injury ◽  
Functional Restoration ◽  
Radical Reactions ◽  
Free Radical Reactions ◽  
Cord Injury

The hypothesis that pathologic free-radical reactions are initiated and catalyzed in the major central nervous system (CNS) disorders has been further supported by the current acute spinal cord injury work that has demonstrated the appearance of specific, cholesterol free-radical oxidation products. The significance of these products is suggested by the fact that: (i) they increase with time after injury; (ii) their production is curtailed with a steroidal antioxidant; (iii) high antioxidant doses of the steroidal antioxidant which curtail the development of free-radical product prevent tissue degeneration and permit functional restoration. The role of pathologic free-radical reactions is also inferred from the loss of ascorbic acid, a principal CNS antioxidant, and of extractable cholesterol. These losses are also prevented by the steroidal antioxidant. This model system is among others in the CNS which offer distinctive opportunities to study, in vivo, the onset and progression of membrane damaging free-radical reactions within well-defined parameters of time, extent of tissue injury, correlation with changes in membrane enzymes, and correlation with readily measurable in vivo functions.

scholarly journals A Collagen-Based Scaffold for Promoting Neural Plasticity in a Rat Model of Spinal Cord Injury

Polymers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2245
Author(s):  
Jue-Zong Yeh ◽  
Ding-Han Wang ◽  
Juin-Hong Cherng ◽  
Yi-Wen Wang ◽  
Gang-Yi Fan ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Rat Model ◽  
Neural Plasticity ◽  
Collagen Scaffold ◽  
Glial Scar ◽  
Beneficial Effects ◽  
Cord Injury

In spinal cord injury (SCI) therapy, glial scarring formed by activated astrocytes is a primary problem that needs to be solved to enhance axonal regeneration. In this study, we developed and used a collagen scaffold for glial scar replacement to create an appropriate environment in an SCI rat model and determined whether neural plasticity can be manipulated using this approach. We used four experimental groups, as follows: SCI-collagen scaffold, SCI control, normal spinal cord-collagen scaffold, and normal control. The collagen scaffold showed excellent in vitro and in vivo biocompatibility. Immunofluorescence staining revealed increased expression of neurofilament and fibronectin and reduced expression of glial fibrillary acidic protein and anti-chondroitin sulfate in the collagen scaffold-treated SCI rats at 1 and 4 weeks post-implantation compared with that in untreated SCI control. This indicates that the collagen scaffold implantation promoted neuronal survival and axonal growth within the injured site and prevented glial scar formation by controlling astrocyte production for their normal functioning. Our study highlights the feasibility of using the collagen scaffold in SCI repair. The collagen scaffold was found to exert beneficial effects on neuronal activity and may help in manipulating synaptic plasticity, implying its great potential for clinical application in SCI.

scholarly journals Immunosuppressants Affect Human Neural Stem Cells In Vitro but Not in an In Vivo Model of Spinal Cord Injury

2013 ◽  
Vol 2 (10) ◽  
pp. 731-744 ◽  
Author(s):  
Christopher J. Sontag ◽  
Hal X. Nguyen ◽  
Noriko Kamei ◽  
Nobuko Uchida ◽  
Aileen J. Anderson ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Stem Cells ◽  
Spinal Cord Injury ◽  
Neural Stem Cells ◽  
In Vivo Model ◽  
Human Neural Stem Cells ◽  
Cord Injury
Sign in / Sign up

Export Citation Format

Share Document