Hi everyone,
Here is a call to immunology and/or neurobiology experts: I recently observed these cells (see video, showing at least 4 of them in the field of view) when performing an experiment I have performed almost daily for over a year, and was very surprised by the discovery. Does anyone have a clue what these cells are--if they are even cells? This is basically out of naive curiosity but I did find it very intriguing.
More context:
- Optical parameters are: 40x objective, infrared illumination. The reading speed of this time-lapse acquisition was increased about 2.5 times. The focus is changed a few times during the video (starting above and lowering the focus) to give an idea in depth. It is still possible to see the cells' processes move in real time, which is what attracted my eye in the first place.
- Tissue is a living, acute ex vivo horizontal slice of a newborn wild-type Swiss mouse brain (about 4 hours old at the time of tissue preparation). Under the microscope is a slice of one brain hemisphere. The camera is looking at the hippocampus, close to the lateral ventricle.
- The focus of the camera is set at the surface of the slice. Anything blurry is focused on the supporting medium maintaining the slice alive, i.e. artificial cerebral spinal fluid (ACSF). In other words, no tissue there, only liquid. Anything not blurry is the slice surface proper, admittedly harmed by the cutting process (dead and dying cells, debris, possibly some still alive and repairing neuronal processes).
- Since the whole brain was cut, the blood-brain barrier (BBB) obviously did not maintain its integrity. In fact I routinely observe red blood cells hanging around at the surface of the slice. This means it is possible that cells which were in the blood --or in the lymphatic fluid, for that matter-- were spread at the surface of the slice when the slice was cut or later when it was resting before being put under the microscope. If these cells have some sort of adherence mechanism, then they could stay at the slice surface.
- Finally a few leads: macrophages? Immune dendritic cells? Activated microglia? Anything neuronal? Anything exogenous (contaminated in the process of tissue preparation/storage)?
Many thanks for your input!
https://goo.gl/photos/L5kLekvgNGJN11DJ8
I see 4 ordinary polymorph nuclear cells (macrophages? basophiles?) over which is moving solution in which some artefacts are floating, probably smooth muscle cells or debris of connective or nerve tissue?
looks like microglia/ macrophages. These are the only cells that will send and communicate so fast
Hi,
I recorded the attached video of microglia in culture. They appear to be in their ameboid (activated) form and resemble the cell type in your video. I speculate that the 'mystery cell' is microglia.
Thanks,
Kelly
Many thanks to all three of you Dragan, Eliseo and Kelly for your helpful replies.
Based on Eliseo's and Kelly's replies it appears we might be looking at microglia/macrophages from either side of the BBB. Kelly, indeed the cultured microglia video looks a lot like the cells I encountered! For illustrating purpose, may I upload your video on the Google page I created for mine?
Before reading your replies though, and based on Dragan's, I looked for pictures of immune dendritic cells (DCs) especially in infrared light, and I thought they looked a lot like the cells on the video. Here are a few figures I found in articles published in the journals FASEB, Blood and JCB:
- http://www.fasebj.org/content/22/11/3908/F3.expansion.html
- http://www.bloodjournal.org/content/112/13/5037.figures-only
- http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172164/figure/fig3/
However these are still pictures. Having done just a quick search I don't know how fast the dendritic cells' veils can move. Eliseo's reply seems to imply they could not move as fast as the protrusions captured on my video.
So just to confirm: do you agree that these cells' protrusions move too fast to be dendritic cells' veils, leaving only the possibility of macrophages/microglial cells? Or could it be both?
Many thanks again!
http://www.fasebj.org/content/22/11/3908/F3.expansion.html
http://www.bloodjournal.org/content/112/13/5037.figures-only
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172164/figure/fig3/
Hi Damien,
Yes, you may use my microglia video however you see fit.
Good luck!
Kelly
Dear Damien,
I think your cells look different than activated microglia that I see in brain slices after fixation. The length and shape (not as smooth) of the processes, and the soma shape looks very different. Please have a look at activated microglia shape/size in Fig. 9C-D in my following paper and compare.
best wishes, Refik
Article Emerging Roles of Filopodia and Dendritic Spines in Motoneur...
Dear Refik,
Many thanks for your reply. Indeed, the two look very different. However the two environments are also very different, with the main difference I see being that one cell is more or less in a liquid environment (ACSF) with very little surrounding physical constraint, whereas the other (in your figure) must adapt to relatively packed surrounding tissue. If you have encountered microglia -- or other cell types -- in less constraining environment though, and if they resemble the mysterious cells I now see on every slice I put under the microscope... I am very interested!
Dear Damien,
Given the morphological differences, It may be that your cells are macrophages or monocytes originating from circulation, while the cells that I filled with dye in the brain slices are resident microglia, which are not as mobile as your cells.
best wishes, Refik
Cool movies, bro.
Look like monocytes-->macs. Macs don't circulate. Monocytes do. They appear to be phagocytic, extending membrane out. I'd venture they look a LOT like DC, but that would be a load of DC for a small area. If they popped out of choroid plexus, maybe, though. Don't look much like microglia I've seen in tissue, dishes, or suspended, but, to be fair, almost any cell rounds up in liquid, because....physics.
Wish I could be more help. Almost 100% sure they are myeloid, not lymphocytes, and 99% sure they're not microglia. Microglia wouldn't leave the tissue to run around on top. Which leaves DC, Granulocytes (I seriously doubt basophils, which are rare as hens teeth, or eos; neutrophils have a very distinctive nucleus with lobes--did you check that?) or monocytes. Monocytes are crazy mobile. Macs are differentiated (often from monocytes), and way less mobile.
OK, I'm going with monocyte. That's my final guess.....
Many thanks for a great reply, Noel. This clarifies things a lot for me who has close to no knowledge in immunology.
The thought process you expose appears very logical, so reading you I'm inclined to agree and go with monocytes. There are only a few elements that I'm not sure hold in the reasoning, but I don't know if they change the conclusion. Specifically, the fact that microglia "wouldn't leave the tissue to run around on top": you have to remember that the slice surface used to be in the middle of the tissue, before it became a slice. As for the lobe nucleus of neutrophils I don't see it, but then again I don't have much more info than what you see on the video.
I would have liked to be able to say with 100% certainty "this is cell X" but what I discover is that immune cells can look like each other, I guess, and without further tests it can be impossible to clearly tell one apart from another. And as you say in a liquid environment, it's tricky. So, in short, many thanks!
Sure--I don't know how helpful I was, but I tried to dump the contents of my thoughts in a stream for you. :) In general, tissue-resident macrophages would be loathe to leave their tissue for something relatively chemotactically neutral like ACSF (or PBS, or medium). They'll move towards purinergic signals, for example, or perhaps a powerful chemokine gradient, but in an injured tissue (a slice) one would hypothesize that injured self cells would provide a massive "stay here and fix it" signal via ATP, etc. That's why I'd guess against microglia. But, I am happy to be wrong--I'd be excited to hear what you conclude.
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