# Adult DRG explants or dissociated cultures for axonal degeneration studies? **URL:** \ **Category:** Uncategorized\ **Created:** [September 8, 2020, 12:09am UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751 "2020-09-08T00:09:38Z")\ **Posts on this page:** 9\ **Page:** 1
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 8, 2020, 12:09am UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/1 "2020-09-08T00:09:38Z")
Does anyone have any experience using adult DRGs, either as explants or dissociated cultures, for axonal degeneration studies? My preference is to use adult because the process/disease I want to study occurs in adults, and also I’d like the full mature repertoire of non-neuronal cells like SGCs, macrophages, etc. to be present. I don’t know if the neuropathic process I want to study is a ‘neuron intrinsic’ one or if it requires other cells, so I don’t want to just have neurons all alone in a dish. @adgaudet @liz @tberta @thicunha Most all papers use embryonic DRGs. I’m assuming because they grow more readily and so you have more axon to quantify. But apart from that, any other reason that adult DRG shouldn’t work? I did find a few recent papers using adult DRG: > **[Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve...](https://pubmed.ncbi.nlm.nih.gov/30712871/)** > > Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D,... ![image](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/34dc8343fb27e0380e244fc84258ae2907af7c94.jpeg) - They use adult DRG in Xona microfluid chambers and look at axon fragmentation > **[Dorsal root ganglion explants derived from chemotherapy-treated mice have...](https://pubmed.ncbi.nlm.nih.gov/30439399/)** > > Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and debilitating adverse effect of cancer therapy that results from treatment with neurotoxic agents. Although chemotherapy treatment has been shown to inhibit neurite outgrowth from... ![livni-2019-explant-drg](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/74eb6f663fdda8cd9393a0820597da757b326299.jpeg) But, in contrast, this paper from Neto et al. suggests adult explants don’t grow much axon > **[Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase...](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524368/)** > > Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root... ![image](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/835f09564031ead7fdf398bc7dc16574bdffe82b.jpeg) Latremoliere et al. adapted the “spot culture” method that is normally used with embryonic DRG and got it going with adult DRG > **[Neuronal-Specific TUBB3 Is Not Required for Normal Neuronal Function but Is...](https://pubmed.ncbi.nlm.nih.gov/30110642/)** > > We generated a knockout mouse for the neuronal-specific β-tubulin isoform Tubb3 to investigate its role in nervous system formation and maintenance. Tubb3-/- mice have no detectable neurobehavioral or neuropathological deficits, and upregulation of... ![image](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/84327a9c9a09aefdd8daf9153e268fd00594f319.jpeg) ---
**Author:** ![tberta](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/tberta/32/145_2.png) [@tberta](https://forum.painresearcher.net/u/tberta)\ **Post date:** [September 8, 2020, 1:51pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/2 "2020-09-08T13:51:23Z")
We use 8-weeks old mice and the Malin protocol ([https://pubmed.ncbi.nlm.nih.gov/17401349/](https://pubmed.ncbi.nlm.nih.gov/17401349/)) to prepare our cultures of dissociated DRG neurons. We obtain a good density of neurites (see image below, tubb3 staining, 3-4 days) in these cultures that we have successfully used to test neurodegeneration and potential neuroprotective drugs. ![image](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/ddf69331ecf1b8ef4dad030eb98478bd2055aac8.png) ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [September 8, 2020, 1:53pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/3 "2020-09-08T13:53:21Z")
Wow @tberta that’s really nice! Thanks for sharing with us. Are you counting neurites or axon length? Also are you using anti-mitotic agents? Unlike most axon degen people, I want the non-neuronal cells to be present, in case they are playing a role. ---
**Author:** ![tberta](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/tberta/32/145_2.png) [@tberta](https://forum.painresearcher.net/u/tberta)\ **Post date:** [September 8, 2020, 2:22pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/4 "2020-09-08T14:22:08Z")
For the moment, we have just measured the neurite length. We did not use anti-mitotic agents, so I suspect that we have several non-neuronal cells in these cultures, too. ![image](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/b09f6493219dba612c1c588912a11b0c11339521.png) ---
**Author:** ![thicunha](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/thicunha/32/386_2.png) [@thicunha](https://forum.painresearcher.net/u/thicunha)\ **Post date:** [September 16, 2020, 1:53pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/5 "2020-09-16T13:53:34Z")
Dear @achamess. We have some ongoing projects and we have established the protocol for DRG explants similar to [https://pubmed.ncbi.nlm.nih.gov/26431741/](https://pubmed.ncbi.nlm.nih.gov/26431741/). Good luck. best ---
**Author:** ![dmolliver](https://avatars.discourse-cdn.com/v4/letter/d/5e9695/32.png) [@dmolliver](https://forum.painresearcher.net/u/dmolliver)\ **Post date:** [March 1, 2021, 3:24pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/6 "2021-03-01T15:24:59Z")
I am late to this thread, but in case anyone is still reading this, I have an update to our publication (the Malin protocol cited above): simply increasing the duration of the second enzyme incubation from 10 minutes to 20 minutes increases the yield of healthy isolated neurons, just do as little trituration as possible (we do 3-5 strokes each with a larger bore glass pipet followed by a smaller bore pipet, as in the protocol), too much trituration winds up killing a lot of your cells. For postnatal day 2 to adult dissociated neurons, you don’t need trophic factors for small neuron survival, but if you are trying to study large diameter neurons in vitro you will probably need to add NT3 and/or BDNF to keep them healthy. ---
**Author:** ![tberta](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/tberta/32/145_2.png) [@tberta](https://forum.painresearcher.net/u/tberta)\ **Post date:** [March 1, 2021, 4:04pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/7 "2021-03-01T16:04:38Z")
Thank you, @dmolliver. This is very helpful and still of current interest. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [March 2, 2021, 3:46pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/8 "2021-03-02T15:46:15Z")
Thank you @dmolliver for sharing the updates on your widely used protocol. Thanks for joining Pain Researcher as well. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [March 26, 2021, 6:02pm UTC](https://forum.painresearcher.net/t/adult-drg-explants-or-dissociated-cultures-for-axonal-degeneration-studies/751/9 "2021-03-26T18:02:14Z")
@dmolliver Do you have any thoughts on how best to count neurons/cells accurately for plating? We’re doing some imaging assays where it’ll probably be important to consistently have the same number of cells on glass. What’s the best way to count? Hemocytometer and some live cell dye (calcein)?