Effects of Gynostemma pentaphyllum on spinal cord motor neurons and microglial cells in vitro

2021 ◽  
Vol 123 (6) ◽  
pp. 151759
Author(s):  
Gerburg Keilhoff ◽  
Christina Ludwig ◽  
Josephine Pinkernelle ◽  
Benjamin Lucas
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Microglial Cells ◽  
Gynostemma Pentaphyllum

scholarly journals Knockdown of long non-coding RNA LEF1-AS1 attenuates apoptosis and inflammatory injury of microglia cells following spinal cord injury

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Sheng-Yu Cui ◽  
Wei Zhang ◽  
Zhi-Ming Cui ◽  
Hong Yi ◽  
Da-Wei Xu ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Cell Viability ◽  
Microglial Cells ◽  
Protective Effects ◽  
Loss Of Function ◽  
Sprague Dawley ◽  
Cord Injury ◽  
Apoptosis And Inflammation

Abstract Background Spinal cord injury (SCI) is associated with health burden both at personal and societal levels. Recent assessments on the role of lncRNAs in SCI regulation have matured. Therefore, to comprehensively explore the function of lncRNA LEF1-AS1 in SCI, there is an urgent need to understand its occurrence and development. Methods Using in vitro experiments, we used lipopolysaccharide (LPS) to treat and establish the SCI model primarily on microglial cells. Gain- and loss of function assays of LEF1-AS1 and miR-222-5p were conducted. Cell viability and apoptosis of microglial cells were assessed via CCK8 assay and flow cytometry, respectively. Adult Sprague-Dawley (SD) rats were randomly divided into four groups: Control, SCI, sh-NC, and sh-LEF-AS1 groups. ELISA test was used to determine the expression of TNF-α and IL-6, whereas the protein level of apoptotic-related markers (Bcl-2, Bax, and cleaved caspase-3) was assessed using Western blot technique. Results We revealed that LncRNA LEF1-AS1 was distinctly upregulated, whereas miR-222-5p was significantly downregulated in LPS-treated SCI and microglial cells. However, LEF1-AS1 knockdown enhanced cell viability, inhibited apoptosis, as well as inflammation of LPS-mediated microglial cells. On the contrary, miR-222-5p upregulation decreased cell viability, promoted apoptosis, and inflammation of microglial cells. Mechanistically, LEF1-AS1 served as a competitive endogenous RNA (ceRNA) by sponging miR-222-5p, targeting RAMP3. RAMP3 overexpression attenuated LEF1-AS1-mediated protective effects on LPS-mediated microglial cells from apoptosis and inflammation. Conclusion In summary, these findings ascertain that knockdown of LEF1-AS1 impedes SCI progression via the miR-222-5p/RAMP3 axis.

scholarly journals Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives

Development ◽  
2021 ◽  
Vol 148 (6) ◽  
Author(s):  
Matthew Wind ◽  
Antigoni Gogolou ◽  
Ichcha Manipur ◽  
Ilaria Granata ◽  
Larissa Butler ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Lumbosacral Spinal Cord ◽  
Bona Fide ◽  
Promising Solution ◽  
Bmp Signalling ◽  
Crucial Parameter ◽  
Ventral Spinal Cord

ABSTRACT The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus, it is a crucial parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine/disease modelling applications. However, the in vitro generation of posterior MNs corresponding to the thoracic/lumbosacral spinal cord has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well defined. Here, we determine the signals guiding the transition of human NMP-like cells toward thoracic ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal pre-/early neural state, whereas suppression of TGFβ-BMP signalling pathways promotes a ventral identity and neural commitment. Based on these results, we define an optimised protocol for the generation of thoracic MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the comparison of hPSC-derived spinal cord cells of distinct axial identities.

scholarly journals Fully Characterized Mature Human iPS- and NMP-Derived Motor Neurons Thrive Without Neuroprotection in the Spinal Contusion Cavity

2022 ◽  
Vol 15 ◽  
Author(s):  
Zachary T. Olmsted ◽  
Cinzia Stigliano ◽  
Brandon Marzullo ◽  
Jose Cibelli ◽  
Philip J. Horner ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Progenitor Cells ◽  
Motor Neurons ◽  
Ex Vivo ◽  
Cervical Spinal Cord ◽  
Neural Cell ◽  
Oligodendrocyte Progenitor Cells ◽  
Limited Information ◽  
Cord Injury

Neural cell interventions in spinal cord injury (SCI) have focused predominantly on transplanted multipotent neural stem/progenitor cells (NSPCs) for animal research and clinical use due to limited information on survival of spinal neurons. However, transplanted NSPC fate is unpredictable and largely governed by injury-derived matrix and cytokine factors that are often gliogenic and inflammatory. Here, using a rat cervical hemicontusion model, we evaluate the survival and integration of hiPSC-derived spinal motor neurons (SMNs) and oligodendrocyte progenitor cells (OPCs). SMNs and OPCs were differentiated in vitro through a neuromesodermal progenitor stage to mimic the natural origin of the spinal cord. We demonstrate robust survival and engraftment without additional injury site modifiers or neuroprotective biomaterials. Ex vivo differentiated neurons achieve cervical spinal cord matched transcriptomic and proteomic profiles, meeting functional electrophysiology parameters prior to transplantation. These data establish an approach for ex vivo developmentally accurate neuronal fate specification and subsequent transplantation for a more streamlined and predictable outcome in neural cell-based therapies of SCI.

scholarly journals “Stretch-Growth” of Motor Axons in Custom Mechanobioreactors to Generate Long-Projecting Axonal and Axonal-Myocyte Constructs

2019 ◽  
Author(s):  
Kritika S. Katiyar ◽  
Laura A. Struzyna ◽  
Suradip Das ◽  
D. Kacy Cullen
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Motor Axon ◽  
Central Feature ◽  
Motor Axons ◽  
Ganglion Neurons

AbstractThe central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to an often-distant target muscle. However, to date in vitro models have not replicated this central feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of micro-tissue engineering and mechanically assisted growth techniques to create long-projecting centimeter-scale motor axon tracts. Here, primary motor neurons were isolated from the spinal cords of rats and induced to form engineered micro-spheres via forced aggregation in custom micro-wells. This three-dimensional micro-tissue yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom-built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were systematically optimized to generate parameters for robust and efficient “stretch-growth” of motor axons. We found that axons projecting from motor neuron aggregates were able to respond to axon displacement rates at least 10 times greater than that tolerated by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch-grown motor axons were compared to benchmark stretch-grown axons from sensory dorsal root ganglion neurons, revealing similar axon densities yet increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and then stretch-grown to create novel long-projecting axonal-myocyte constructs that better recreate characteristic dimensions of native nerve-muscle anatomy. This is the first demonstration of mechanical elongation of spinal cord motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue engineered “living scaffolds” for targeted axon tract reconstruction following nervous system injury or disease.Significance StatementWe have developed novel axon tracts of unprecedented lengths spanning either two discrete populations of neurons or a population of neurons and skeletal myocytes. This is the first demonstration of “stretch-grown” motor axons that recapitulate the structure of spinal motor neurons in vivo by projecting long axons from a pool of motor neurons to distant targets, and may have applications as anatomically inspired in vitro test beds to study mechanisms of axon growth, development, and neuromuscular function in anatomically accurate axo-myo constructs; as well as serve as “living scaffolds” in vivo for targeted axon tract reconstruction following nervous system trauma.

scholarly journals Chitosan polyplex mediated delivery of miRNA-124 reduces activation of microglial cells in vitro and in rat models of spinal cord injury

2016 ◽  
Vol 12 (3) ◽  
pp. 643-653 ◽  
Author(s):  
Andrew M. Louw ◽  
Mallappa K. Kolar ◽  
Liudmila N. Novikova ◽  
Paul J. Kingham ◽  
Mikael Wiberg ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Microglial Cells ◽  
Rat Models ◽  
Cord Injury

scholarly journals Expression of the ALS-Causing Variant hSOD1G93A Leads to an Impaired Integrity and Altered Regulation of Claudin-5 Expression in an in Vitro Blood–Spinal Cord Barrier Model

2015 ◽  
Vol 35 (7) ◽  
pp. 1112-1121 ◽  
Author(s):  
Sabrina Meister ◽  
Steffen E Storck ◽  
Erik Hameister ◽  
Christian Behl ◽  
Sascha Weggen ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Neurodegenerative Disorder ◽  
Cellular Systems ◽  
Apparent Permeability ◽  
Protein Levels ◽  
Forkhead Box ◽  
Altered Regulation ◽  
Blood Spinal Cord Barrier

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood–spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood–spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood–spinal cord barrier endothelial cells of presymptomatic hSOD1G93A mice and bEnd.3 cells stably expressing hSOD1G93A. In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the β-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1G93A cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood–spinal cord barrier.

scholarly journals CD34 Identifies a Subset of Proliferating Microglial Cells Associated with Degenerating Motor Neurons in ALS

2019 ◽  
Vol 20 (16) ◽  
pp. 3880 ◽  
Author(s):  
Mariángeles Kovacs ◽  
Emiliano Trias ◽  
Valentina Varela ◽  
Sofia Ibarburu ◽  
Joseph S. Beckman ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Human Subjects ◽  
Microglial Cells ◽  
Lumbar Spinal Cord ◽  
Reactive Microglia ◽  
Sporadic Als ◽  
Cd34 Cells ◽  
Misfolded Sod1 ◽  
Lumbar Spinal

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1G93A mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1G93A rats, but not in controls. Most CD34+ cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34+ cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34+ cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1G93A rat spinal cords yielded a large number of CD34+ cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34+ cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.

scholarly journals Experimental Porcine Polioencephalomyelitis in Germfree Pigs

1967 ◽  
Vol 4 (2) ◽  
pp. 186-198
Author(s):  
John F. Long ◽  
Adalbert Koestner ◽  
Leopold Liss
Keyword(s):  
Spinal Cord ◽  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Motor Neurons ◽  
Dorsal Root ◽  
Ganglion Cells ◽  
Silver Impregnation ◽  
Microglial Cells ◽  
Regressive Phase

The neuronal changes and glial response in the spinal cord were studied by silver impregnation techniques in 23 germfree pigs orally infected with a porcine polioencephalomyelitis viras. By the sixth day swelling occurred in motor neurons. During the next 24 to 96 hours this progressed to diffuse chromatolysis, vesiculation, necrosis, and neuronophagia in massive areas of the ventral horns. Massive degeneration of axons in tracts originating in the ventral horns and from dorsal root ganglia correlated well with the extensive destruction of both motor and sensory neurons. The initial responses to necrosis of ganglion cells were infiltration and proliferation of microglial cells. As active neuronal destruction ceased (about two weeks following infection) the microglial reaction began to decline and proliferative astrocytosis became the predominant feature. The paucity of surviving neurons in the ventral horns, and the density of the mesh of astrocytic processes marked the chronic regressive phase of the disease.

scholarly journals 1408. Enterovirus D68 RNA Visualized in the Anterior Horn of the Spinal Cord of a Pediatric Patient with Flaccid Paralysis

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S712-S712
Author(s):  
Matthew R Vogt ◽  
Peter Wright ◽  
William Hickey ◽  
James E Crowe ◽  
Kelli Boyd
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
The United States ◽  
Surface Proteins ◽  
Anterior Horn ◽  
Human Spinal Cord ◽  
Acute Flaccid Myelitis ◽  
Enterovirus D68 ◽  
Outstanding Question

Abstract Background Acute flaccid myelitis (AFM) is a polio-like paralyzing illness of children. AFM incidence is increasing during every other year outbreaks that occur in the United States simultaneously with outbreaks of enterovirus D68 (EV-D68) infection. Demonstrating that EV-D68 directly causes AFM has been challenging due to rare detection of the virus in the cerebrospinal fluid (CSF) of patients despite frequent detection at nonsterile sites. Murine studies have shown that EV-D68 can infect spinal cord anterior horn motor neurons and cause paralysis, similar to poliovirus. However, a key outstanding question is whether EV-D68 causes AFM in humans by direct viral pathogenesis or by indirect host immunopathogenesis. Methods We investigated the pathogenesis of AFM using tissues from a previously reported case of a 5-year-old boy who presented in fall 2008 with four days of progressive limb and voice weakness followed by incontinence, apnea, and death. He had a CSF pleocytosis of 2094/µL with EV-D68 identified in the CSF by sequencing of the VP1 gene. We designed probes for in situ hybridization (ISH) based on this sequence to stain formalin fixed paraffin embedded tissues from his autopsy. For immunohistochemistry (IHC) we used both commercial polyclonal anti-EV-D68 antibodies and our own human monoclonal antibodies that stain virus infected cells in vitro. Immunophenotyping was done by IHC. Results With ISH we identified EV-D68 RNA in the anterior horn of the patient’s spinal cord, corresponding to the location of motor neuron cell bodies. This area was highly inflamed, with an infiltrate of lymphocytes and macrophages. Viral RNA was in low abundance, and we could not detect viral surface proteins by IHC. Neither RNA nor viral antigen was detected in the lungs, which had extensive inflammatory infiltrate. Conclusion Deaths in AFM patients are rare and often distant from initial presentation, but this patient died four days after onset of weakness, allowing us to directly demonstrate that EV-D68 can infect the human spinal cord. Low abundance of virus suggests the virus either reached the spinal cord prior to weakness onset or was cleared rapidly by the immune response. Therefore, both direct viral pathology and immune factors likely contribute to AFM disease in EV-D68 infection. Disclosures James E. Crowe, Jr, MD, IDBiologics (Board Member, Consultant, Grant/Research Support)Vanderbilt University (Other Financial or Material Support, Inventor on patent related to this abstract)

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