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 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

scholarly journals Microglia limit lesion expansion and promote functional recovery after spinal cord injury in mice

2018 ◽  
Author(s):  
Faith H. Brennan ◽  
Jodie C.E. Hall ◽  
Zhen Guan ◽  
Phillip G. Popovich
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Functional Recovery ◽  
Spontaneous Recovery ◽  
Recovery Of Function ◽  
Experimental Models ◽  
Post Injury ◽  
Thoracic Spinal ◽  
Cord Injury

AbstractTraumatic spinal cord injury (SCI) elicits a robust intraspinal inflammatory reaction that is dominated by at least two major subpopulations of macrophages, i.e., those derived from resident microglia and another from monocytes that infiltrate the injury site from the circulation. Previously, we implicated monocyte-derived macrophages (MDMs) as effectors of acute post-injury pathology after SCI; however, it is still unclear whether microglia also contribute to lesion pathology. Assigning distinct functional roles to microglia and MDMs in vivo has been difficult because these CNS macrophage subsets are morphologically and phenotypically similar. Here, to characterize the role that microglia play in experimental models of thoracic spinal contusion or lumbar crush injury, mice were fed vehicle chow or chow laced with a CSF1R receptor antagonist, PLX5622. Feeding PLX5622 depletes microglia. In both groups, spontaneous recovery of hindlimb motor function was evaluated for up to 8 weeks post-SCI using open-field and horizontal ladder tests. Histopathological assessment of intraspinal pathology was assessed in 8 week post-injury tissues. In both SCI models, microglia depletion exacerbated lesion pathology and impaired spontaneous recovery of hind limb function. Notably, the loss of microglia prevented astroglial encapsulation of the lesion core, which was associated with larger lesions, enhanced demyelination and neuron loss and a larger inflammatory response that was dominated by monocyte-derived macrophages. The neuroprotective and healing properties of microglia become obvious in the subacute phases of recovery; microglia depletion up to 7 days post-injury (dpi) had no apparent effect on recovery while delayed depletion from 8-28dpi exacerbated lesion pathology and significantly impaired functional recovery. These data suggest that microglia have essential tissue repair functions after SCI. Selective enhancement of microglial activities may be a novel strategy to preserve tissue and promote recovery of function after neurotrauma.

scholarly journals Combining Cell Therapy with Autologous Schwann Cell and Bone Marrow-derived Mesenchymal Stem Cell in Patients with Subacute Complete Spinal Cord Injury: Safety Considerations and Possible Outcomes

2020 ◽  
Author(s):  
Saeed Oraee Yazdani ◽  
Mohammadhosein Akhlaghpasand ◽  
Maryam Golmohammadi ◽  
Maryam Hafizi ◽  
Mina Soufi Zomorrod ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Bone Marrow ◽  
Spinal Cord Injury ◽  
Spinal Injury ◽  
Safety Evaluation ◽  
Autologous Bone Marrow ◽  
Spinal Cord Regeneration ◽  
Systemic Complications ◽  
Cord Injury ◽  
Safety Considerations

Abstract Background Cellular transplantations have promising effects on treating spinal cord injury (SCI) patients. Safety alongside the complementary characteristics of Mesenchymal stem cells (MSC) and Schwann cells (SC) are suggested to be the two of the best candidates in SCI treatment. In this study, we assessed the safety and efficacy of intrathecal co-transplantation of autologous bone marrow MSC and SC in the patients with subacute traumatic complete SCI.Methods Eleven patients with complete SCI (American Spinal Injury Association Impairment Scale (AIS); grade A) were enrolled in this study during the subacute period of injury. The patients received intrathecal autologous combination of MSC and SC and were followed-up for 12 months. We assessed neurological changes by American Spinal Injury Association’s (ASIA) sensory-motor scale, functional recovery by spinal cord independence measure (SCIM-III), and subjective changes along with adverse events (AE) with our checklist. Furthermore, electromyography (EMG), nerve conduction velocity (NCV), magnetic resonance imaging (MRI), and urodynamic study (UDS) were conducted for all the patients at the baseline, 6 months, and one year after the intervention.Results Light touch ASIA score alterations were approximately the same as the pinprick changes (11.6 ± 13.1 and 12 ± 13, respectively) in 50% of the cervical and 63% of the lumbar-thoracic patients and both were more than the motor score alterations (9.5 ± 3.3 in 75% of the cervical and 14% of the lumbar-thoracic patients). SCIM III total scores (21.2 ± 13.3) and all its sub-scores (“respiration and sphincter management” (15 ± 9.9), “mobility” (9.5 ± 13.3), and “self-care” (6 ± 1.4)) had significant changes after cell injection. Our findings support that the most remarkable positive, subjective improvements were in trunk movement, equilibrium in standing/sitting position, sensation of the bladder and rectal filling, and ability of voluntary voiding. Our safety evaluation revealed no systemic complications and radiological images showed no neoplastic overgrowth, syringomyelia, or pseudo-meningocele.Conclusion The present study showed that autologous SC and bone marrow-derived MSC transplantation at the subacute stage of SCI could reveal significant improvement in sensory and neurological functions among the patients. It appears that using this combination of cells is safe and effective for clinical application to the spinal cord regeneration during the subacute period.

scholarly journals Bazedoxifene, a Selective Estrogen Receptor Modulator, Promotes Functional Recovery in a Spinal Cord Injury Rat Model

2021 ◽  
Vol 22 (20) ◽  
pp. 11012
Author(s):  
Yiyoung Kim ◽  
Eun Ji Roh ◽  
Hari Prasad Joshi ◽  
Hae Eun Shin ◽  
Hyemin Choi ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Therapeutic Potential ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Sprague Dawley ◽  
Post Injury ◽  
Static Compression ◽  
Cord Injury

In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.

scholarly journals Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Gabriela Edwards-Faret ◽  
Karina González-Pinto ◽  
Arantxa Cebrián-Silla ◽  
Johany Peñailillo ◽  
José Manuel García-Verdugo ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Extracellular Matrix ◽  
Xenopus Laevis ◽  
Cellular Response ◽  
Central Canal ◽  
Spinal Cord Regeneration ◽  
Post Injury ◽  
Cord Injury ◽  
Regenerative Stage

Abstract Background The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. Methods We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP+ cells for RNAseq analysis. Results In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. Conclusions The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.

scholarly journals Relationship of American Spinal Injury Association Impairment Scale Grade to Post-injury Hospitalization and Costs in Thoracic Spinal Cord Injury

Neurosurgery ◽  
2017 ◽  
Vol 83 (3) ◽  
pp. 445-451 ◽  
Author(s):  
Ellen M Dukes ◽  
Steven Kirshblum ◽  
Alex A Aimetti ◽  
Sarah S Qin ◽  
Rebecca K Bornheimer ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Spinal Injury ◽  
American Spinal Injury Association ◽  
Economic Costs ◽  
Present Value ◽  
Post Injury ◽  
Thoracic Spinal Cord ◽  
Thoracic Spinal ◽  
Cord Injury

Abstract BACKGROUND The lifetime economic burden of thoracic spinal cord injury (SCI) is known to be high, but evidence of variability of costs in relation to the American Spinal Injury Association Impairment Scale (AIS) grade is limited. OBJECTIVE To estimate lifetime economic costs of hospitalization by AIS grade in thoracic SCI. METHODS Using SCI Model Systems data from January 2000 to March 2016 from the National Spinal Cord Injury Statistical Center, we estimated mean total annual days of all-cause hospitalization by AIS grade among persons with thoracic SCI, based on assessments 1, 5, and 10 yr post-injury. We combined this information with secondary cost data and projections of life expectancy to estimate lifetime economic costs of hospitalization by AIS grade in persons aged 35 yr at time of thoracic SCI. Future costs were discounted to present value at 3% annually. RESULTS One year post-injury, mean total annual days of hospitalization ranged from 2.1 for persons with AIS-D injuries to 5.9 for those who were AIS-A. Similar differences were noted 5 and 10 yr post-SCI. The estimated net present value of expected lifetime costs of hospitalization following thoracic SCI at age 35 yr was $321 534, $249 514, $188 989, and $68 120 (2015 US$) for AIS-A, AIS-B, AIS-C, and AIS-D injuries, respectively. CONCLUSION Persons with less severe thoracic SCI, as reflected in AIS grade, spend fewer days in hospital over their lifetimes, leading to lower costs of inpatient care. Therapies improving AIS grade following thoracic SCI may provide cost savings in addition to addressing substantial unmet need.

scholarly journals Cellular Response to Spinal Cord Injury in Regenerative and Non-Regenerative Stages in Xenopus Laevis

2020 ◽  
Author(s):  
Gabriela Edwards-Faret ◽  
Karina González-Pinto ◽  
Arantxa Cebrián-Silla ◽  
Johany Peñailillo ◽  
José Manuel García-Verdugo ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Extracellular Matrix ◽  
Xenopus Laevis ◽  
Cellular Response ◽  
Central Canal ◽  
Spinal Cord Regeneration ◽  
Post Injury ◽  
Cord Injury ◽  
Regenerative Stage

Abstract Background The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration.Methods We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP+ cells for RNAseq analysis.Results In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPC). RNASeq of GFAP-EGFP cells allowed a clear demonstration that indeed these cells are NSPC. On the contrary, the GFAP-EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPC, and we found that are mainly fated to form neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPC cells are necessary for functional regeneration of the spinal cord. Conclusions The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPC, which massively proliferate to reconstitute the spinal cord, and are differentiated into neurons. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.

scholarly journals Combining cell therapy with human autologous Schwann cell and bone marrow-derived mesenchymal stem cell in patients with subacute complete spinal cord injury: safety considerations and possible outcomes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saeed Oraee-Yazdani ◽  
Mohammadhosein Akhlaghpasand ◽  
Maryam Golmohammadi ◽  
Maryam Hafizi ◽  
Mina Soufi Zomorrod ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Bone Marrow ◽  
Spinal Cord Injury ◽  
Spinal Injury ◽  
Safety Evaluation ◽  
Autologous Bone Marrow ◽  
Spinal Cord Regeneration ◽  
Systemic Complications ◽  
Cord Injury ◽  
Safety Considerations

Abstract Background Cellular transplantations have promising effects on treating spinal cord injury (SCI) patients. Mesenchymal stem cells (MSCs) and Schwann cells (SCs), which have safety alongside their complementary characteristics, are suggested to be the two of the best candidates in SCI treatment. In this study, we assessed the safety and possible outcomes of intrathecal co-transplantation of autologous bone marrow MSC and SC in patients with subacute traumatic complete SCI. Methods Eleven patients with complete SCI (American Spinal Injury Association Impairment Scale (AIS); grade A) were enrolled in this study during the subacute period of injury. The patients received an intrathecal autologous combination of MSC and SC and were followed up for 12 months. We assessed the neurological changes by the American Spinal Injury Association’s (ASIA) sensory-motor scale, functional recovery by spinal cord independence measure (SCIM-III), and subjective changes along with adverse events (AE) with our checklist. Furthermore, electromyography (EMG), nerve conduction velocity (NCV), magnetic resonance imaging (MRI), and urodynamic study (UDS) were conducted for all the patients at the baseline, 6 months, and 1 year after the intervention. Results Light touch AIS score alterations were approximately the same as the pinprick changes (11.6 ± 13.1 and 12 ± 13, respectively) in 50% of the cervical and 63% of the lumbar-thoracic patients, and both were more than the motor score alterations (9.5 ± 3.3 in 75% of the cervical and 14% of the lumbar-thoracic patients). SCIM III total scores (21.2 ± 13.3) and all its sub-scores (“respiration and sphincter management” (15 ± 9.9), “mobility” (9.5 ± 13.3), and “self-care” (6 ± 1.4)) had statistically significant changes after cell injection. Our findings support that the most remarkable positive, subjective improvements were in trunk movement, equilibrium in standing/sitting position, the sensation of the bladder and rectal filling, and the ability of voluntary voiding. Our safety evaluation revealed no systemic complications, and radiological images showed no neoplastic overgrowth, syringomyelia, or pseudo-meningocele. Conclusion The present study showed that autologous SC and bone marrow-derived MSC transplantation at the subacute stage of SCI could reveal statistically significant improvement in sensory and neurological functions among the patients. It appears that using this combination of cells is safe and effective for clinical application to spinal cord regeneration during the subacute period.

scholarly journals PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure

2018 ◽  
Author(s):  
Arnau Hervera ◽  
Luming Zhou ◽  
Ilaria Palmisano ◽  
Eilidh McLachlan ◽  
Guiping Kong ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Axonal Regeneration ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Spinal Injury ◽  
Gene Expression Response ◽  
Cord Injury ◽  
Expression Response

The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small molecule inhibitors targeting key epigenetic enzymes in DRG neurons we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3 thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of ex vivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

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