Neuroinflammation - Science topic

Hi Paula

Hi Mickael Pereira, I usually use CD64'CD68'CD32 and CD11b to in both cases and agree with Louis-Chales Beland

Please see my new Preprint from Preprints.org regarding:

Brain endothelial cells play a central role in the development of enlarged perivascular spaces in the metabolic syndrome. Preprints.org 2023 May 19

The paper also appears in my research section as a PDF preprint.

Hayden, M.R. Brain Endothelial Cells Play a Central Role in the Development of Enlarged Perivascular Spaces in the Metabolic Syndrome. Preprints.org 2023, 2023051406. https://doi.org/10.20944/preprints202305.1406.v1.

Melvin R Hayden

University of Missouri School of Medicine

Columbia, Missouri

There is also IMA 2.1, see:

And I just found out that they are now distributed:

Why not start with serial observations on how the different forms of neuroinflammation develop, Sneha Majumder?

In possibly considering the famous pieces of evidence presented at www.ms-info.net on the best-studied kind of neuroinflammation?

Thank you for the answer. It was helpful. Actually, the paper talked about the bad side of lipocalin-2

Try this one.

It's easy to apply, the only thing you need is FIJI.

Based on: https://pubmed.ncbi.nlm.nih.gov/34375551/

Regards

Good question

This will be very exciting and make content more visible and available to a wider audience.

Dear Gokhan Gokhaner

I suggest the following articles:

1. https://www.cancer.iu.edu/research-trials/facilities/mac/documents/bioplexarticle-rat-kit-lysis-conditions.pdf

2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801052/

3.

4. https://www.nature.com/articles/s41598-019-50708-w?proof=t

5. https://www.frontiersin.org/articles/10.3389/fphar.2020.562084/full

Best Regards

So these words aren't replaceable for one another, yes in case of neuroinflammation their is immunoreactivity but immunoreactivity exclusively not the way of neuroinflammation.

Thank you very much

Interesting detail, Gert Fricker - but how to recognize these biomarkers' pathogenic/clinical relevance?

Fakhruddin Masse Originally thought to be immunologically privileged, now it is accepted that immune cells are present in the meninges and provide immune surveillance of the CNS. However, the unique properties of these lymphatic systems, including their location and size, may explain why immune responses to CNS antigens are often slower than in peripheral tissues.

Reference: doi: 10.1016/j.it.2015.08.006

Dear Caleb Bailey

This depends on some variables, e.g., the kit you are using (Invitrogen, Merck, anyway), besides the cell culture you are going to analyze, and the solvent used in the treatment. If the kit manual does not indicate, the ideal is initially to perform a dose-response curve (6h, 12, 24 h ...). I treated overnight with EtOH extract in human neuroblastoma cells (SH-SY5Y) and there was a response. I hope you helped even after a certain time of you question.

Regards,

Three D - Modelling of SARS-COV-2 - https://www.kolabtree.com/find-an-expert/subject/3d-printing . But whether this kind of 3-D Molecular mapping of SARS-COV-2 will lead to some advantages for Vaccine developement ?

Dear Marko,

I have to admit that I'm not experienced with cochlea tissue. What exactly is the problem that prevents you from isolating single lymphocytes or myeloid cells? Are these cell types difficult to distinguish under the microscope? Can you do IHC or immunofluorescence staining to visualize and detect the cells of interest? Or is your microdissection system not precise enough to cut single cells? Which one are you using? Is the laser calibrated well?

Kind regards,

Heidi

try vector trueview, autofluorescence quenching kit with DAPI (Cat# SP-8500)

Yes. Please take a look at the following RG links.

Expression of TLR4 in neurons is mostly in non-detectable range (see ). So if you are treating pure neuronal cultures with LPS, you would not see a significant alteration in cyto/chemokine profile. However, in many cases, primary neuronal cultures are contaminated with astrocytes, which does respond to LPS stimulation. A better model would be to co-culture neurons with mixed glia.

dear...maybe the article can help you

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825946/

Hope this Helps!

Are you sure you want to block inflammation in the brain by an anti-inflammatory compound? The new paradigm in inflammation research is inflammation resolution which is an active process to resolve inflammation (instead of just blocking it). Inflammation resolution mediators are resolvins, maresins, protectins etc.

Hi Inaki,

LFB may be to colorful to visualize the fiber or demyelination. Better to use silver staining if there is more demylination as it may be less sensitive if there is suttle demylination.

Following article might help you

thanks

Dear Silvia,

Actually, not all children and adolescents with childhood adversity/trauma have an increased risk of PTSD. The magic word is "individual-environment-genetic-interaction". The following papers might give you further hints:

Belsky, J., Pasco Fearon, R.M., Bell, B., 2007. Parenting, attention and externalizing problems: testing mediation longitudinally, repeatedly and reciprocally. Journal of child psychology and psychiatry, and allied disciplines 48(12), 1233-1242.

Belsky, J., Steinberg, L., Draper, P., 1991. Childhood experience, interpersonal development, and reproductive strategy: and evolutionary theory of socialization. Child Dev 62(4), 647-670.

Quach, J.L., Nguyen, C.D., Williams, K.E., Sciberras, E., 2018. Bidirectional Associations Between Child Sleep Problems and Internalizing and Externalizing Difficulties From Preschool to Early Adolescence. JAMA Pediatr 172(2), e174363.

Check this out:

Int Immunopharmacol. 2002 Mar;2(4):487-97.

Silberman DM, Wald M, Genaro AM.

"Stress produces changes in various immune processes. Some of these changes may be due to neurochemical and hormonal alterations including thyroid hormones levels. This work was carried out to study the impact of chronic mild stress (CMS) exposure on proliferative responses and its correlation with serum thyroid hormone levels. In addition, the influence of serum corticosterone levels on these responses was also studied. For this purpose, mice were submitted from1 to 6 weeks to a CMS model. After undergoing the stress schedule for 4 weeks, an alteration in the proliferative response was observed. Lymphocytes from exposed animals showed a decrease in T-cell response to concanavalin-A (Con A) and phytohemagglutinin (PHA) and an increase in B-cell proliferation to lipopolysaccharides (LPS). In parallel, a reduction in T3 and T4 serum levels was observed. On the contrary, serum corticosterone levels increased in animals exposed to CMS for 1 or 2 weeks and then return to normal values. Lowering serum thyroid hormone levels by propylthiouracil (PTU) treatment negatively modulates T-cell response without affecting B-cell response. On the other hand, the substitutive T4 treatment in stressed animals improved significantly the proliferative T-cell response. Non-significative changes in CD4/CD8 ratio were observed neither in stressed, PTU- or T4-treated animals. Taken together, our results suggest an impact of chronic stress on thyroid function that in turn alters T-cell response. "

Please consider Glial fibrillary acidic protein (GFAP). It is not definitely neuroinflammation specific but is related to glial cells. We previously found good data in stroke patients.

Amino-acid deprivation

I agree with Clarissa's suggestion. M-CSF (10ng/ml) is very important for microglia proliferation. We did not find the many difference between the 1st and other harvest by using the marker labeling and patch clamp.

Hi Noor,

Although it is not totally necessary, I would recommend it or I would try both. Non-mature neuronal cells can behave very different and present diverse antigens. However, some differentiation protocols can be a little... "tricky". You can obtain some aberratic cells that can interfere with your results.

Overall, I would do some testing using both to characterize your in vitro model.

Regards,

Jordi.

Even if stereotactical Injections are the same, there may be anatomical differences between animals that will affect distribution. Imaging during injections may help.

i agree with Nemanja Zdravkovic

hi

see this

https://www.hindawi.com/journals/mi/2014/861231

Studying the inflammatory phenomena provoked by progressive and recurrent traumatizations of brain tissues can no doubt further our understanding of unexplained neurodegenerative diseases/cryptic neuroinflammatory conditions. Which might hardly be the case the other way round!

hi Eva,

I followed 6 LPS injections (1 mg/Kg/day), sample collection 24h after the last administration. I didnt check the cytokine markers yet.. but i checked the expression of nfkb, ikk, amyloid beta which was expressed high and also the hippocampal LTP was significant reduced. based on these, it is pretty much sure that TNF alpha and IL-6 will be higher in the brain.

could you tell me which LPS you are using?

I am using LPS from sigma

If you are still struggling with the marker expression, then i recommend you to initially check the blood serum cytokine levels.

have a nice day

I think it is very real the hypothesis that EBV is implicated in the neuroinflammation process, not by chance EBV is inserted into lymphocytes B that are likely to be hijacked to produce abnormal antibodies or proteins. Good work and good thoughts.

 Nlrp3−/− mice exhibit  defect in development of Th1 response(Gris et al. 2010) and NLRP3 seem to trigger a Th2 bias (Gurung et al. 2015; Daley et al. 2017). But my guess is this behaviour is tissue/cell dependent and so the jury is still out.

1.       Daley, D., et al. (2017). "NLRP3 signaling drives macrophage-induced adaptive immune suppression in pancreatic carcinoma." J Exp Med 214(6): 1711-1724.

2.       Gris, D., et al. (2010). "NLRP3 Plays a Critical Role in the Development of Experimental Autoimmune Encephalomyelitis by Mediating Th1 and Th17 Responses." The Journal of Immunology 185(2): 974-981.

3.       Gurung, P., et al. (2015). "An NLRP3 inflammasome-triggered Th2-biased adaptive immune response promotes leishmaniasis." Journal of Clinical Investigation 125(3): 1329-1338.

Dear Mir Alam,

Oxidative stress can be measured using any C. elegans strain. Our group uses DCF-DA as a fluorescent probe to measure ROS accumulation, not specifically H2O2. I just tried the previous method for N2, CF1038 and GR1307, but I guess it would also work for Alzheimer’s-related-strains, such as CL2006, CL4176, etc.

Stress to endoplasmic reticulum (ER) can cause UPR and aggresome formation. Aggressome accumulation has been shown in AD. The ER stress is also related to autophagy. So theoretically autophagy can be a target for AD treatment. The thing is when you start to study a new signaling pathway in a disease there is always some association. The issue becomes how to decipher your findings.

Other processes are related to increased levels of glutamate in extracellular space (ECS).

Under physiological conditions, glutamate is taken up by astrocytes (AS), which convert it into glutamine and deliver it back to neurons as an alternative energy source. However, under pathologic conditions, is excessively produced and AS cannot remove glutamate from the ECS. That produces excitotoxicity. Consequently, free glutamate binds to neuronal receptors, e.g.NMDA, inducing the influx of Ca²+ and Na+ and the efflux of K+ (ionic disturbance). This ion imbalance causes depolarization of the cell membrane and overload of intracellular Ca²+, which leads to mitochondrial dysfunction, decreased ATP synthesis, energy failure, and cell death. Mitochondrial dysfunction is followed by ROS production; wich causes oxidative stress. 

With all due respect, please use primary cells. BV-2 are, at best, poor replacements for the real thing. Can you gather lots of data quickly with cell lines? Sure. Is any of that data relevant? Not much. Just because a cell produces cytokines with TLR stimulation doesn't make it an actual factual immune cell.

Thank you very much, I will try and to see if it works for my cells, in addition, I plan to use add the TGF-β and IFN-γ to induce the inflammation response, I cultured the HUMAN vascular smooth muscles, so what kind of TGF-β and IFN-γ I need to choose,  I mean HUMAN recombinant or MOUSE recombinant one? Thank you!

There is a hypothetical target, a subgroup of

B lymphocytes (David Hafler, Kevin O Connor

et al.) which ripen in the lymph nodes of

the cervix. They can be fought outside

the central nervous system.

Regards,

Joachim

I used Lymphoprep for PBMC isolation, and the PBMC layer was easily collectable after centrifugation.

Hi Sagar,

Here is the protocol I use for BV2 or primary microglia cultures:

Prepare Griess reagent:

2.3ml Phosphoric acid (85%)

1g Sulfanilamide

0.1g Naphtylethylenediamine

97.7ml water

Vortex. Prepare Griess reagent at least half a day before use. Protect from light.

Keep media from cell cultures on ice.

Prepare standards from sodium nitrite (NaNO2): from 0 to 1000 µM. Use the culture media for dilutions.

Pipette media and standards 50µL/well on a 96w-plate.

Add 50µL of Griess reagent on top of each sample. Shake for 10min and protect from light.

Measure absorbance at 540nm in the reader machine.

Good luck,

Sighild

what do you mean by "extract" and "challenged by lps" ? I don't understand what you are asking.

Hi Cooper,

Glad the papers have been somewhat helpful in researching glial changes under inflammatory conditions.

It's one of the references within the papers you've already linked, but have you looked at Hickman's 2013 paper (http://www.ncbi.nlm.nih.gov/pubmed/24162652)? Could serve as another resource regarding how microglia specifically change responses at different ages (with the underlying implications of different inflammatory responses).

Good luck in your search! 

There is also an excellent new paper out from Marie Eve Tremblay showing a new type of microglia (called "dark" microglia based on their EM appearance) that becomes abundant during pathological states such as chronic stress, aging, Alzheimer's, etc. They display signs of oxidative stress, but also have highly ramified, thin processes, so it sounds like they may be relevant for your question. See paper below:

Glia. 2016 May;64(5):826-39. doi: 10.1002/glia.22966. Epub 2016 Feb 5.

Dark microglia: A new phenotype predominantly associated with pathological states.

Bisht K1, Sharma KP1, Lecours C1, Gabriela Sánchez M1, El Hajj H1, Milior G2, Olmos-Alonso A3, Gómez-Nicola D3, Luheshi G4, Vallières L1, Branchi I5, Maggi L2, Limatola C2, Butovsky O6, Tremblay MÈ1.

Hi Marc. That is interesting! I have never noticed the microglia being yellow. I wonder if it is due to their high expression of the NADPH oxidase, which contains FAD that should be yellow. I think this contributes to the yellowness of neutrophils. It might be worth taking a spectrum, and comparing it to FAD and FMN.  These are also fluorescent, so might be worth taking a fluorescensce spectrum.  If it does not fit a flavin spectrum, you could compare it to lipofuscin?

Dear Gaurav,

I'm actually performing RT-PCR, and I think that if you got primers, mixes and homogenate tissues, you are ready to work; In a couple of weeks you can already have some results. Maybe amygdala takes longer, because it is a very little area, with not too much RNA; so it request lyophilization before working.

Anyway I could help you.

Wishes

Maria Rosanna

There is a rather quick & yet informative review, downloadable as a lecture in the following link. I hope you find it useful.

Dear Fulvio.

Sorry, I asked you, is it other program in EU or the first, you got grant ?After that you are looking for placement?

Thanks

Hi Lalita,

Since more than ten years, our lab's members work on glial cells (microglia and astrocytes) in neuroinflammatory conditions. On what samples are you working: primary cultures ? in vivo experiments ? brain slices ?...

Also consider the type of immune reaction you are after (e.g. involvement of specific cells and acute inflammation versus chronic/prolonged). I have found that microglia react very quickly and easily to single cytokines or other toxins, while astrocytes often require a cytokine cocktail (e.g. TNF-alpha + IL-1beta). If you are after a chronic inflammation, then initial microglial activation can also lead to astroctye activation over time. 

Dear Diana,

As mentioned above i recommend the dark agouti rats. They are an invred strain that are great for reliable EAE research as immunisation with IFA and recombinant MOG is sufficient, you dont require further addition of pertussis or other complete adjuvants. 

আমার পক্ষে এক্কেবারেই অসম্ভব ব্যাপার...দুঃখিত ভাই...!!

Foamy macrophages (gitter cells) do migrate out of the MS plaque into the perivascular space and then into the blood stream.

Microglia cells are specialized macrophages that exist only in the brain. Raw 264.7, in contrast, are general mouse macrophages. There are numerous differences between the different types of macrophages. I think if your disease doesn't cause infection of the brain, but rather other tissue macrophages, then the RAW cells would be the better option

If you also have IBA-1 stained control sample then you can compare IBA-1 staining intensity between them. Activated microglia show increased IBA-1 staining. You can also quantify staining intensity in ImageJ. This is one of the simplest ways.

Yes. I agree with Dr. Lewis Alan Opler. 

I think the general answer is yes, everything a phagocyte will recognize and internalize will affect the cellular activity and in this case presumably its polarity. How it will affect the cells depend on the size of the particles, how many particles are taken up, particle surface composition, and of course external stimulations e.g. from other cells. This being said, many are using these SPIOs with no or little effect on cells, or at least on the measures they have set. Viability and/or apoptosis assays should be used.

What will happen in your case is difficult to predict, and why a control must always be included,with your specific purpose in mind.

Dear Katrina,

Hopefully some references cited in the article of Najjar et al. might be helpful for you.

Najjar S et al. Neurovascular unit dysfunction with blood-brain barrier hyperpermeability contributes to major depressive disorder: a review of clinical and experimental evidence. Journal of Neuroinflammation 2013;10:142.

Kind regards, Matheus Arts

Dear Gaurav Please find protccol and composition of gel preparation. Protocol is attached.

Good luck

Hello there

I personally studied this topic. By injecting LPS 2 mg / kg ip to C57BL6 micee, you obtain a clear  neuroinflammation 24-28hrs after injection. Microglia are activated, proinflammatory cytokines increased, in different brain regions.

In my experiments I have used CD11b to show immunologically active microglia where as  IBA-1 is constitutively produced in both ramified and amoeboid microglia that may or may not be immunologically active. If you are looking to quantify numbers of microglia I would choose IBA1. However, if you are looking to quantify numbers of activated microglia I would choose CD11b. Just a caution-- I have had issues getting a good IHC with CD11b from Abcam.

I was interested to read that not only is α-synuclein found in colon of PwP but so are Lewy Bodies (http://www.ncbi.nlm.nih.gov/pubmed/23017648).   It also seems that diarrhoea is often associated with PD (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012728#s1).  In an RG thread on Serotonin and Dopamine William Wilson introduced me to the role of enzyme co-factors, which links serotonin synthesis to dopamine (see file).  (William warned me that one of the authors had been struck off for self medication and that urinary levels of neurotransmitters are not a reliable indicator of CSF levels but this does not invalidate the interconnectedness of the cofactors.)  It is this that offers potentially a way to understand why PD has so many manifestations.  It still leaves us (me, anyway) unsure as to whether misfolded α-synuclein is a cause or a symptom of PD and, if it is a cause, then its role in the process.

Hi Pete,

I am pretty sure that NGF, CNTF, MAP2, VCAM, TNFalpha, TGFbeta, are not specific to astrocytes. If you want Astrocyte specific, stick with GFAP, S100B (which is constiutively expressed in the healthy adult brain), and AldhL1, Glutamine Synthetase, EAAT1 and or EAAT2.

Hello all

I count all type of cells using stereological techniques. Based on stereology rules, it is best to start with tissues that are 40um. why? becuase tissue get dihydrated 50%, so you end up with 20um thickness of your tissue once it have been stained IHC. then, I use either optical fractionator probe in my software or the cavalieri probe. If you dont have access to a software like that, you can do count of 1 in every 6 sections, for a total of 8 to 10 section per animal. Do a total count in a 5 ROI of 50um by 50um or 100 um by 100 um depends on how big is the hole area you want to count then average the count. the bigger the area the bigger the ROI.

If interested in more details, you are welcome to look at my publications.

i hope this helps.

thanks all

Sandra

I think that this will basically depend on the pattern of expression of TLR4 in each animal and the cascade initiated after it. So in theory, it can be very different.

There are multiple ways to assess neuroinflammation. You can perform immunohistochemistry by using specific markers for microglial activation (Iba1) , astrocytic response (GFAP), and neuronal loss (NeuN or Fluorojade for dying neurons). You can also look at the levels of various cytokines that are upregulated during inflammation (TNF-alpha, Interleukins, TGF-beta, and so on).

As you know LPS mediated inflammation is closely associated with nitric oxide production and up regulation of iNOS production at most sites all of which has considerable publications documenting LPS effect on inflammation. Other mediators also play a role. Systemic administration of LPS also produces change in the Nucleus Tractus Solitarii. There is a lot of literature supporting communication pathways between the immune system and brain. I have attached one for your consideration. You are treading on unknown territory as best I know and very interesting work, I look forward to hearing more.

Thanks for your kind answer.

According to (Santello 2011 and 2012), very low concentration of TNF-alpha (about 100 to 600 pM) controls glutamergic gliotransmission in the Dentate Gyrus and control synaptic plasticity which is underling for learning and memory. Furthermore lack of TNF-alpha lead to suppress the astrocyte effect and impair LTP.

thank for your kind answer. the problem is that. increasing AMPA receptors lead to more excitation of post synaptic neuron and result of that is exit more K+ from neuron which have effects on astrocyte. I think that may saturate astrocyte.

Is a radioligand important? If not, you could use Brillian Blue G (IV) as a selective P2X7 blocker and then do immunohisto for analysis. BBG is very similar to blue food coloring so it is rather safe. Sorry I don't know more about your specific compound. All the best in your research.

Jiang L-H, Mackenzie AB, North RA, Surprenant A. Brilliant Blue G Selectively Blocks ATP-Gated Rat P2X7 Receptors. Mol Pharmacol 2000; 58: 82–8.

Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, et al. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. PNAS 2009; 106: 12489–93.

Look into publications of Jens Zimmer

Thanks!

Multiple genetic models of Alzheimer have been described using gene knockouts, missense alleles and expressed transgenes:

Many of these are commercially available (including from non-USA repositories) or were developed by academic researchers who may be willing to send you some animals privately or as part of a collaboration. I can help you find a good one if you are interested.

well, neuroinflammation comes in may flavours and it depends on what you really want to study. systemic administration of lipopolysaccaride from bacterial sources causes a strong inflammatory response through the body, including blood-brain barrier leakage and among others, triggers microglia activation and infiltration of immune cells into the brain (see for example: http://www.ncbi.nlm.nih.gov/pubmed/23468966). otherwise, you have to go for specific disease models (EAE for myelin, coriomeningitic virus for encephalitis, anyone for neurodegeneration....). good luck...