scholarly journals Isolation of Mature Spinal Motor Neurons and Single-cell Analysis Using the Comet Assay of Early Low-level DNA Damage Induced In Vitro and In Vivo

2001 ◽  
Vol 49 (8) ◽  
pp. 957-972 ◽  
Author(s):  
Zhiping Liu ◽  
Lee J. Martin
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Motor Neuron ◽  
Single Cell ◽  
Motor Neurons ◽  
Ventral Horn ◽  
Motor Neuron Degeneration ◽  
Spinal Motor Neurons

We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in α-motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from naïve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration. (J Histochem Cytochem 49:957–972, 2001)

scholarly journals Modeling motor neuron resilience in ALS using stem cells

2018 ◽  
Author(s):  
Ilary Allodi ◽  
Jik Nijssen ◽  
Julio Aguila Benitez ◽  
Christoph Schweingruber ◽  
Andrea Fuchs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
Spinal Motor Neurons ◽  
Neuronal Progenitors ◽  
Oculomotor Neurons ◽  
Bona Fide

SUMMARYOculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

scholarly journals Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing

2000 ◽  
Vol 35 (3) ◽  
pp. 206-221 ◽  
Author(s):  
R. R. Tice ◽  
E. Agurell ◽  
D. Anderson ◽  
B. Burlinson ◽  
A. Hartmann ◽  
...  
Keyword(s):  
Comet Assay ◽  
Single Cell ◽  
Genetic Toxicology

DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single-cell gel test (comet assay)

1995 ◽  
Vol 25 (3) ◽  
pp. 180-187 ◽  
Author(s):  
Andreas Hartmann ◽  
Kathleen Herkommer ◽  
Michael Glück ◽  
Günter Speit
Keyword(s):  
Comet Assay ◽  
Single Cell ◽  
Human Blood ◽  
Blood Cells ◽  
Human Blood Cells

scholarly journals Comet assay measures of DNA damage are predictive of bladder cancer cell treatment sensitivity in vitro and outcome in vivo

2013 ◽  
Vol 134 (5) ◽  
pp. 1102-1111 ◽  
Author(s):  
Karen J. Bowman ◽  
Manar M. Al‐Moneef ◽  
Benedict T. Sherwood ◽  
Alexandra J. Colquhoun ◽  
Jonathan C. Goddard ◽  
...  
Keyword(s):  
Dna Damage ◽  
Bladder Cancer ◽  
Comet Assay ◽  
Cancer Cell ◽  
Bladder Cancer Cell ◽  
Treatment Sensitivity

scholarly journals “Axonal Length Determines distinct homeostatic phenotypes in human iPSC derived motor neurons on a bioengineered platform”

2021 ◽  
Author(s):  
Cathleen Hagemann ◽  
Carmen Moreno Gonzalez ◽  
Ludovica Guetta ◽  
Giulia Tyzack ◽  
Ciro Chiappini ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Systematic Investigation ◽  
Threshold Size ◽  
Spinal Motor Neurons ◽  
Mitochondrial Homeostasis ◽  
Conventional Culture ◽  
Human Ipsc ◽  
First Time

AbstractStem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). The axons of these neurons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. It follows that shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here we directly address this issue by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, permitting systematic investigation of the effects of length on human axonal biology for the first time. With this approach, we reveal a link between length and metabolism in human MNs in vitro, where axons above a “threshold” size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation and mitochondrial homeostasis. Our findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs in order to sustain long axons. Our findings have critical implications for in vitro modelling of several neurodegenerative disorders and reinforce the importance of modelling cell shape and biophysical constraints with fidelity and precision in vitro.

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 Study of DNA damage induced by dental bleaching agents in vitro

2006 ◽  
Vol 20 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Daniel Araki Ribeiro ◽  
Mariângela Esther Alencar Marques ◽  
Daisy Maria Fávero Salvadori
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Comet Assay ◽  
Single Cell ◽  
Chinese Hamster ◽  
Dental Bleaching ◽  
Dna Breakage ◽  
The Mean ◽  
Positive Controls

Dental bleaching is a simple and conservative procedure for aesthetic restoration of vital and non-vital discolored teeth. Nevertheless, a number of studies have demonstrated the risk of tissue damage from the contact of these agents with the oral mucosa. In the current study, the genotoxic potential associated with exposure to dental bleaching agents was assessed by the single cell gel (comet) assay in vitro. Chinese hamster ovary (CHO) cells in vitro were exposed to six commercial dental bleaching agents (Clarigel Gold - Dentsply; Whitespeed - Discus Dental; Nite White - Discus Dental; Magic Bleaching - Vigodent; Whiteness HP - FGM and Lase Peroxide - DMC). The results pointed out that all dental bleaching agents tested contributed to DNA damage as depicted by the mean tail moment, being the strongest effect observed with the highest dose of hydrogen peroxide (Whiteness HP and Lase Peroxide, at a 35% concentration). On the other hand, Magic Bleaching (Vigodent) induced the lowest level of DNA breakage. Negative and positive controls displayed absence and presence of DNA-damaging, respectively. Taken together, these results suggest that dental bleaching agents may be a factor that increases the level of DNA damage. A higher concentration of hydrogen peroxide produced higher noxious activities in the genome as detected by single cell gel (comet) assay.

scholarly journals Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (comet) assay

2006 ◽  
Vol 17 (2) ◽  
pp. 91-94 ◽  
Author(s):  
Daniel Araki Ribeiro ◽  
Patrícia Lepage Alves de Lima ◽  
Mariângela Esther Alencar Marques ◽  
Daisy Maria Fávero Salvadori
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Single Cell ◽  
Human Fibroblast ◽  
Fibroblast Cells ◽  
Potential Health ◽  
Human Fibroblast Cells ◽  
Genotoxicity Tests ◽  
Mouse Lymphoma

Fluoride has widely been used in Dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on genetic apparatus. Genotoxicity tests constitute an important part of cancer research for risk assessment of potential carcinogens. In this study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Mouse lymphoma and human fibroblast cells were exposed to sodium fluoride (NaF) at final concentration ranging from 7 to 100 µg/mL for 3 h at 37ºC. The results pointed out that NaF in all tested concentrations did not contribute to DNA damage as depicted by the mean tail moment and tail intensity for both cellular types assessed. These findings are clinically important because they represent a valuable contribution for evaluation of the potential health risk associated with exposure to agents usually used in dental practice.

Genotoxicity of Endosseous Implants Using Two Cellular Lineages In Vitro

2014 ◽  
Vol 40 (1) ◽  
pp. 25-29 ◽  
Author(s):  
Mariza Matsumoto ◽  
Hugo Nary Filho ◽  
Raquel Ferrari ◽  
Kristianne Fernandes ◽  
Ana Claudia Renno ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acetic Acid ◽  
Comet Assay ◽  
Single Cell ◽  
Dental Implant ◽  
Genetic Damage ◽  
Endosseous Implants ◽  
Murine Fibroblasts ◽  
Murine Fibroblast

The genotoxic potential of corrosion eluates obtained from a single dental implant using murine fibroblasts or osteoblasts cells in vitro by the single-cell gel (comet) assay was examined. A single commercially available dental implant (Biotechnology) was eluted in a solution consisting of equal amounts of acetic acid and sodium chloride (0.1 M) for 1, 3, 7, 14, and 21 days. Murine fibroblast or osteoblast cultures were then exposed to all corrosion eluates obtained from endosseous dental implants for 30 minutes at 37°C. The results suggest that none of the eluates produced genotoxic changes in murine fibroblasts regardless of the length of exposure to the eluate. Similarly, no genotoxicity was found in osteoblasts. The results suggest that the dental implant eluates tested in this study did not induce genetic damage as depicted by the single-cell gel (comet) assay. Because DNA damage is an important event during oncogenesis, this study represents a relevant contribution to estimate the real risks to the cellular system induced by the corrosion products of a dental implant.

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