This study investigates the neurotoxic effects of silver nanoparticles on hypothalamic neurons through the activation of microglial cells. The research demonstrates how microglial activation leads to the release of pro-inflammatory cytokines, which subsequently induce neuronal death.
Take microglial cultures in a multi-well plate containing media.
Replace half of the media in the test well with media containing silver nanoparticles, and in the control well with vehicle media. Incubate.
The microglia internalize the nanoparticles, become activated, and release pro-inflammatory cytokines.
Transfer the vehicle media and the conditioned media containing these cytokines to filters fitted onto collection tubes.
Centrifuge to remove the nanoparticles, retaining the cytokines.
Invert the filters into new collection tubes, centrifuge, and collect the media. Add fresh media.
Next, take hypothalamic neuronal cultures.
Replace half of the control well media with vehicle media and the test well media with conditioned media. Incubate.
The cytokines bind to receptors on hypothalamic neurons, activating signaling pathways that induce neuronal death.
Add resazurin and incubate.
In live cells, dehydrogenase enzymes reduce the weakly fluorescent resazurin to fluorescent resorufin.
Measure the fluorescence.
Reduced fluorescence indicates increased hypothalamic neuronal death, suggesting nanoparticle-induced neurotoxicity.
To begin, collect 200 microliters of supernatant medium from each plate well, and load it onto a filter that will remove the nanoparticles, placed over a microcentrifuge tube.
It's important to remember that when filtering the nanoparticles, their size, shape, and concentration will affect the filtration efficiency, so it is advisable to test the filtration beforehand.
Now, spin the tubes down at 14,000 g at room temperature for 15 minutes. Then, discard the flowthrough, which contains the nanoparticles, and place the filters upside down in new collection tubes. Spin the filters upside down at 1,000 g for two minutes to collect concentrated, conditioned medium containing cytokines.
Next, increase the volume of the collection to 400 microliters with freshly made supplemented DMEM and store the tubes on ice. For this assay, establish hypothalamic cells from frozen stocks using the same methods described for establishing microglial cultures. Ultimately, use them to seed black-walled, clear-bottom plates with 5,000 cells per well in 200 microliters of supplemented DMEM.
After 24 hours of culturing, remove 100 microliters of medium and replace it with 100 microliters of filtered and concentrated medium collected from the activated microglial cell cultures. Then, incubate the plate for 24 hours under standard conditions. The next day, add 22 microliters of resazurin reagent to each well, and continue the incubation for 20 minutes. Then, with a multi-mode spectrophotometer, take a fluorescence measure to estimate the cell's viability in terms of relative fluorescence units.
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