Hello!
Does anybody know what would be a good in vitro model (in the context of primary DRG culture calcium imaging experiments) of CFA-induced inflammatory pain behavioral model?
I have seen some literature doing the following: 1) inject CFA in vivo and culture only on L4, L5 DRG, 2) flood with inflammatory cytokine soup, 3) take the media of co-culture (neuron and glia) after introducing inflammatory cytokines and transfer the media into the new primary DRG culture and 4) just flooding the culture with CFA at lower dose.
After many years, I have finally seen some behavioral difference n WT vs KO (and cKO) mice in CFA-induced inflammatory model. And the change in expression of the target receptor was in tandem with the resolution of CFA-induced hypersensitivity. I was hoping to investigate the functionality changes of the target receptor as the CFA-induced hypersensitivity is resolved using calcium imaging. However, not sure which in vtiro model (or inflammatory inducing agent) would represent the in vivo CFA inflammatory model. I do wanted this to be high-throughput experiment. That being said, I was worried that it would take forever to do experiments if I am only collecting L4, L5 of DRG to do primary culture after injecting CFA in vivo.
Any advice would be really helpful!!! Thank you in advance!
Thanks for the question. I think this is really tough. The issue with in vitro is that the culturing process itself is a massive stimulus to DRG neurons. You’re axotomizing them and causing injury-induced changes. So the dissociation process itself will likely overwhelm what effect you might have seen from the CFA application in vivo. Perhaps some in vivo effects will be retained in the DRG even after dissociation, and if you have a control, you could see it.
It’s also unclear what inflammatory milieu is produced by CFA in the paw such that you could try to artificially recreate it in vitro using mediators.
I’d say the highest fidelty readout would be to try to do in vivo calcium imaging in mice after CFA. That’s technically challenging but some labs do it.
@dmolliver @tberta @thicunha
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Thank you for your kind reply!
I feel so unfortunate that we’ve just changed our scope to inverted (from upright, although now I get better images). But I do see that in vivo calcium imaging would be a better representation of what’s going on with cells in response to the stimulus in CFA-induced pain states. The quick search came up with a protocol for in vivo DRG calcium imaging (In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia) although not sure how I would be proceeding this with inverted scope.
I will first try with in vitro calcium imaging on DRG that may retain CFA effect to compare with the control (This still would be a caveate for the study, though). Secondly, I would contact with the core to see if they have photon available.
Again, thank you for your kindness and thoughts!!!
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I also found this articel by Dr. M. Gold about doing invitro Ca2+ on dissociated rat DRG collected after CFA-induced inflammation (Contribution of endoplasmic reticulum Ca2+ regulatory mechanisms to the inflammation-induced increase in the evoked Ca2+ transient in rat cutaneous dorsal root ganglion neurons - PMC. and original article that sees increase in evoked calcium transients in iB4+ cells: https://www.sciencedirect.com/science/article/pii/S030645220800211X ). But still not sure if this would be same for mice model.