RESUMO
OBJECTIVES: Axon tracers provide crucial insight into the development, connectivity, and function of neural pathways. A tracer can be characterized as a substance that allows for the visualization of a neuronal pathway. Axon tracers have previously been used exclusively with in vivo studies; however, newer methods of axon tracing can be applied to ex vivo studies. Ex vivo studies involve the examination of cells or tissues retrieved from an organism. These post mortem methods of axon tracing offer several advantages, such as reaching inaccessible tissues and avoiding survival surgeries. METHODS: In order to evaluate the quality of the ex vivo tracing methods, we performed a systematic review of various experimental and comparison studies to discern the optimal method of axon tracing. RESULTS: The most prominent methods for ex vivo tracing involve enzymatic techniques or various dyes. It appears that there are a variety of techniques and conditions that tend to give better fluorescent character, clarity, and distance traveled in the neuronal pathway. We found direct comparison studies that looked at variables such as the type of tracer, time required, effect of temperature, and presence of calcium, however, there are other variables that have not been compared directly. DISCUSSION: We conclude there are a variety of promising tracing methods available depending on the experimental goals of the researcher, however, more direct comparison studies are needed to affirm the optimal method.
Assuntos
Axônios/metabolismo , Indicadores e Reagentes/metabolismo , Vias Neurais/metabolismo , Técnicas de Rastreamento Neuroanatômico , Neurônios/citologia , Animais , HumanosRESUMO
PURPOSE: To determine the most effective method of dissociating neural stem and progenitor cells into a single-cell suspension. MATERIALS/METHODS: Induced pluripotent stem cells were differentiated toward the neural fate for 4 weeks before clusters were subjected to enzymatic (Accutase, trypsin, TrypLE, dispase, or DNase I) or mechanical (trituration with pipettes of varying size) or combined dissociation. Images of cells were analyzed for cluster size using ImageJ. RESULTS: Cells treated with the enzymes Accutase, TrypLE, or trypsin/EDTA, these enzymes followed by trituration, or a combination one of these enzymes followed by incubation with another enzyme, including DNase I, were more likely to be dissociated into a single-cell suspension. CONCLUSIONS: Cells treated with enzymes or combinations of methods were more likely to be dissociated into a single-cell suspension.
Assuntos
Separação Celular/métodos , Enzimas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Estresse Mecânico , Contagem de Células , Sobrevivência Celular , Humanos , Tamanho da PartículaRESUMO
Neural cell grafting is a promising therapy for stroke, but the optimal differentiation status of the cells prior to grafting is unclear. We grafted cells at different maturity stages (days 28, 42, or 56 of in vitro neural differentiation) into the brains of eight-week-old rats one week after subcortical ischemic stroke, and assessed motor and sensory behavioral recovery over one month. We did not find a difference between the grafted or control groups on behavioral recovery, or on brain tissue outcomes including infarct size, microgliosis, or astrocytosis. Further research is needed into mechanisms of benefit of neural cell grafting for stroke.