Hi Pete,

I believe the real question is whether or not the M1/M2 cells accurately recapitulate the microglial biology (in the brain) that you are investigating. In general, as you are aware, cell lines have some artifacts that do not accurately reflect all bioligcal processes, and it is at the descression of the investigotrs to determine if the system is too articifial for their experiment.

I believe Ricahrd Ransohoff's arguement is that there is an enourmous differences in gene expression profiles between M1/M2 cells and in vivo brain microglia. In this case, then M1/M2 cells would almost always be too articifial to be useful in modeling brain microglia. Some people would disagree with these statements.

The systems that do more accurately reflect brain mircoglia are more technically challenging. As a result there is a resistance to utilizing these systems over M1/M2 cells. If someone disagress with the Ransohoff arguement completely, then there is no logical arguement to moving away from M1/M2 cells. In my opinion, results generated in M1/M2 cells are being more highly scrutinized.

Best,

Justin

Justin,

Thank you for your response to my posed question.

Microglia are indeed a unique cell in the provision of the brains innate immune cell of the brain- which are derived - originate from erythromyeloid progenitors in the yolk sac and not from bone marrow monocytes like resident innate immune macrophaes i.e. microglia are an ontogenically distinct population [*Ginhoux et al., 2010/ **Kierdorf et al., 2013] . So, right up front we know that the brain microglia are very unique cells and may diversify to the beat of a different drummer from birth on as compared to

their companion - peripheral innate immune macrophages.

*Science. 2010 Nov 5;330(6005):841-5. doi: 10.1126/science.1194637. Epub 2010 Oct 21.

Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Ginhoux F1, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M.

**Nat Neurosci. 2013 Mar;16(3):273-80. doi: 10.1038/nn.3318. Epub 2013 Jan 20.

Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.

Kierdorf K1, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, Hölscher C, Müller DN, Luckow B, Brocker T, Debowski K, Fritz G, Opdenakker G, Diefenbach A, Biber K, Heikenwalder M, Geissmann F, Rosenbauer F, Prinz M.

I do believe in Ransohoffs discussion, and that there are spectrums of remodeling and functional changes regarding the gene expression profiles in microglia .

If you place the M1-like Microglia on one extreme very polarized position and the M2-like microglia at the other end of a very polarized spectrum of microglia that this concept is still one of helping in

the visualization of the spectrum of changes in between polarization that may occur with great diversity amongst different regions and different conditions of type of injury that the older system still has an important point at this current time, at least for teaching purposes. While it is simple, it still provides the possibility of the two opposing extremes as a reactive-activated spectrum that the microglia undergoes in each region and when exposed to different mechanisms of injury or "brain wounding" via different toxicities or disease states.

Hopefully, others will chime in on this question of diversity regarding the

microglia and I think your comments will be very helpful to others as it was for me.

Thank YOU so much for sharing your thoughts and ideas !

M.R. (Pete) Hayden, MD

University of Missouri

Columbia, MIssouri

addendum:

Could the increase chromatin condensation remodeling that seems to be a

reoccuring abnormality in the db/db microglia be related to the TLRs constant activation and translocation of NFkappaB to the nucleus be a result of or association with increased injury signals from DAMPS or RAGE via the diabetic toxic hyperglycemic state in the db/db models?

Open to discussion...

Please feel free to interact regarding this possibility as presented in the original question placed. MRH.

This topic is very hotly debated. I think the title of M1/M2 is indeed outdated as it was largely based on in vitro work with macrophages where stimuli are isolated and highly controlled. That's no so in an in vivo environment. I really do not prefer to discuss microglia in papers as M1/M2 because it only emphasizes and prolongs that we still use these terms.

I think it's important to remember that microglia are cells responding to signals in their microenvironment, which I believe to be related to their concentrations. The old terms were primarily M0 (resting), M1, and M2. I think it's fine to discuss microglia as surveillant, and then either predominantly pro-inflammatory or anti-inflammatory in general terms. I think these terms open up one to think of a predominant phenotype but understand that there is a spectrum of phenotypes. More specifically, I think the stimulus needs to be specifically outlined and defined because microglia phenotypes can change based on stimulus, model, duration, and all kinds of various factors. I think doing this is important because if one lab does a 60 minute model of ischemic stroke and another lab does 30 minutes, there may be differences in the data they acquire based on differences in their models.

These are just my general thoughts. But overall I think M1 and M2 are important to avoid because it's limiting in how we think about these cells.

Edward and all co-authors I just finished reading your MS and I must share with you and readers that this is must read paper- it is a beautifully crafted paper and so well written.

I mush highly recommend this paper to all readers and to share and reference. I am sure over time this will be highly cited by so many!

Edward C. Koellhoffer 1 , Louise D. McCullough 1,* and Rodney M. Ritzel: Old Maids: Aging and Its Impact on Microglia Function.

Int. J. Mol. Sci. 2017, 18, 769. doi: 10.3390/ijms18040769.

Thanks for sharing it on Research Gate

pete hayden

Is there any research that ties Parkinson pathology to type2 Diabetes and dopaminergic neurons' mitochondria?

Dear Daniel,

Please read my paper

Ultrastructural Remodeling of the Neurovascular

Unit in the Female Diabetic db/db Model—Part

I: Astrocyte which is a open access online Journal devoted to the Neuroglia.

Article Ultrastructural Remodeling of the Neurovascular Unit in the ...

you will be free to download or read in PDF as well as in HTML.

https://www.mdpi.com/journal/neuroglia

Here there are ultrastructural TEM images that demonstrate the different cells plus

there will be Part II Microglia and Mitochondria probably available tomorrow or MOnday of next week. and soon thereafter Part III Oligodendrocyte and Myelin

paper to wrap up this Triology of ultrastructural changes of diabetes type II and gluco toxic effects on the brain.

One will be able to note that there are many differences between the Neurons and

glial cells in the brain each with their own morphologies in addition to their different roles in their secretome and their receptors when some commonalities just as there are differences between the horse and the cow... we defined them as humans define all things they encounter as they always have and will continue to do. It is we physicians that have defined what glucose levels should be before we diagnose diabetes yet our own cells know that the diabetes is present long before what glucose levels are at a clinical predefined level of blood glucose as an example .. i.e. the cells know they are being exposed to toxic injurious changes long before we diagnose in their cellular response to injury.

Each cell in the brain has a very different morphological appearance and each responds to an injury in completely different set of remodeling changes...

So morphologically each of the cells are different quite like horses and cows.

So we humans have determined what each cell should look like. I like to draw a comparison to a blind man describing and elephant when he can only examine a certain part of an elephant like the legs or the trunk... since he is only able to describe what he can feel and reach. Whereas the sighted man can describe the part to the whole animal that can now be seen in totality...

So even though we may be like the blinded man in some aspect ... but we are capable of relating the morphology to the physiological functions now so we are making headway between structure and function... When ask by others and directed toward me people often ask which come first or which one is more important the structure or the function... One would think that since I study structure more than function that I might say structure ... but that is not the case as I think these two are inseparable as in some cells the function comes first then the structural change and in other instances the structure changes and then the function changes. For me with a more structural background, I still find these two modalities inseparable...

Personally, I think the brain was created such that we can respond to the sensory input and then react to that sensory input due to our Neurons actions and their action potential to respond via its connections and subsequently to call into actions the other aspects of our body to react to these sensory inputs i.e. sights ,spounds, pain and taste and thus memory was created in order for us to remember these sensory inputs and learn from them.

However, I am known to try or attempt to oversimplify any biological situation...!!

Each of these cells respond in quite different a different mechanism when the brain is injured via blunt injury, a sharp injury and its location is important too... and chemical injury such as glucotoxicity or lipotoxicity. Each response calls into play individualistic cellular responses and probably our genetic backgrounds are equally important as to how our cells respond to injury...

Type 2 diabetes is now know to increase the risk of age-related neurodegnerative diseases such as Spontaneous AD and PD ... I think this is due to a chronic wounding of the brain tissues with chronic remodeling such that these brains are highly more susceptible or vulnerable as the model ages and highly sensitive to the age-related associated neurodegenerative diseases like sporadic AD or PD.

there are references to this in the fist part of this triology regarding the neuroglia.

I apologize for my running on or rambling on this subject but there is a definite link for increased risk for type 2 diabetes patients to develop either sporadic AD or PD. and I personally think that it is the mitochondria and reactive oxygen species - reactive nitrogen species are at the very roots or core of these age-related neurodegenertive diseases.

with gratitude for your very important questions.

I remain,

M.R. (pete) Hayden, MD