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G10 Article Summary, Tri-culture response to excitotoxicity, Discussion,…
G10 Article Summary
Background
Neurons, astrocytes and microglia are key players in regulating inflammatory responses in the central nervous system, but little is known about their interaction with each other.
Neurons, nastrocytes, and microglia are all involved in neuroinflammation. We demonstrate that multiple "tri-culture" can be maintained for at least 14 days (DIV) without any deleterious effect on the overall health of the neurons.
New multicellular culture models are required to better understand the role they play in neuroinflammation.
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Discussion
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1) IL-34, TGF-β, and cholesterol have been identified as key factors in supporting serum-free cultures of isolated microglia
activation of the colony-stimulating factor 1
receptor (CSF1R) via r IL-34 is required for microglia viability both in vitro and in vivo
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excess cholesterol in the culture media may also be beneficial for maintaining a functional microglia gene expression profile
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5) In response to lipopolysaccharide (LPS), the tri-culture displays many classic hallmarks of neuroinflammation
increase in caspase 3/7 activity,
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7) The tri-culture model is especially useful in modeling the glial response to glutamate-induced excitotoxic
events.
tri-culture recapitulates the complex interactions between the microglia and neurons during excitotoxicity
morphologically resting/ramified microglia can serve a neuroprotective role during excitotoxic events
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8) Tri-culture model has multiple advantages over other methods used to study neuroinflammation in vitro
The presence of neurons, astrocytes, and microglia in the same cell culture, allow researchers to study the complex interplay between these cells that leads to different responses to inflammatory stimuli.
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General Cell Culture
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4) Neocortices from all pups in the litter were pooled, dissociated and plated
- density - 650 cells/mm2
- precoated with 0.5 mg/mL poly-L-lysine
- incubate 4h at 37 celcius and 5% CO2
7) Half-media changes performed at DIV 3, 7 and 10.
6) Primary cortical cells plated in plating medium, allowed to adhere for 4h before medium changed to co- or tri- culture medium.
1) All procedures involving animals were conducted in accordance with the National Institutes of Health Guide
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Cytokine Profile
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1) Incubate DIV 7 media change, co- or tri-culture with 5 μg/mL LPS or vehicle control for 48 hours
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Morphological Analysis
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2) Zeiss Observer D1 inverted microscope 100x or 200x magnification -> obtain sample images -> analyzed by ImageJ
3) Manual counting the number of nuclei that were co-localized with b-III tubulin (neurons) /GFAP(astrocytes) / Iba1 (microglia) -> using 100x magnification images -> determine cell number/mm² of different cell types
4) Manual tracing the outline of astrocytes/microglia -> using 200x magnification images -> determine the average areas & area inside the trace.
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Statistical Methods
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1) >3 biological replicates was used, >3 technical replicates / biological replicate (all experiments)
2) >3 predetermined fields were analyzed / technical replicate. (experiments require image analysis)
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4) Analysis of main effects used to compare two treatments (interaction not determined (p<0.05)); simple main effects conducted via a post hoc Tukey test. (significant interaction was found)
5) One-way ANOVA test: comparing multiple groups against a single treatment.
Two tailed Student's t-test: when only two groups were analyzed.
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Calcium Imaging
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3) For each well, a 200x magnification fluorescence image was taken, after the light source was closed, glutamate solution was added.
4) Following a 2 min incubation (glutamate solution), second fluorescence image was taken (same field of view and exposure time) and compared the change in fluorescence intensity.
Immunostaining
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2)For NG2 staining, cultures were incubated for 1 h with mouse anti-NG2 (1:200 dilution, Abcam) alongwith the other primary antibodies. The slides were restained with phalloidin conjugated toAlexaFluor-555 and washed 3 times with DPBS+.
3)Biopsy slides were stained with Alexa Fluor-555 (1:25 dilution, ThermoFisher) for 1 h following an overnight wash in PBS+ at 37 °C to remove antifade mountant and 5 minpermeabilization with Triton X-100 solution.
1) Cell cultures were washed with 0.1% v/v Triton X-100 (ThermoFisher) solution in DPBS+. They were incubated for 1 h in primary antibody, mouse anti-β-III tubulin, rabbit anti-GFAP and chicken anti-Iba1.
The tri-culture supports neurons, astrocytes, and
microglia in vitro
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a significant population of microglia at both DIV 7 and 14 in
the cultures maintained in the tri-culture media
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all three factors (IL-34, cholesterol, and
TGF-β) are required to support a healthy tri-culture of neurons, astrocytes, and microglia
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