Hello Lien...

I am also working on neurodegeneration (related to seizures) in rats using IHC (NeuN, BrdU and apoptotic markers) and behavioral studies.

You can analyse different stages of neurons like mature and immature neurons and neurodegeneration of newly formed neurons between a time period using BrdU staining. You can distinguish apoptosis and necrosis, quantify their ratio in neurodegeneration.

If you are working on animal model/s, you can do behavioral studies like cognitive impairment tests to determine/correlate neurodegeneration.

You can also tag different types neurons undergoing neurodegeneration related to your objectives (e.g. I am targeting GABA and gluatamate neurons related to seizures)

If you can brief yours study, one can suggest you better......

Hope this will help you....

Thanks 

Thank you Hemant for your reply and suggestions! We applied TUNEL and Caspase 3 (IHC) and analyzed behavioral studies. However, we would like to proof that specific neurons degenerate and were wondering about different methods than TUNEL staining by IHC or quantification of neuronal cell bodies. Are there other methods (preferably IHC) to show that neurons are degenerating? 

Hello,

Yes there are other methods like.....

1) Magnetic resonance imaging (MRI)  or positron emission tomography (PET) at different time points can be used to determine ongoing neurodegeneration in various regions of brain....

Reference:

Glia. 2013 Jan;61(1):10-23. 

Molecular imaging of microglia/macrophages in the brain. DOI: 10.1002/glia.22357

2) Dendritic markers can also be used as a preceding neurodegeneration marker.

Dendritic damage or loss will indicate a preceding neurodegerative event in certain situations.

References:

The cellular basis of dendrite pathology in neurodegenerative diseases. doi: 10.5483/BMBRep.2017.50.1.131

Loss of Dendritic Complexity Precedes Neurodegeneration in a Mouse Model with Disrupted Mitochondrial Distribution in Mature Dendrites. doi.org/10.1016/j.celrep.2016.09.004

May be this information will help you.....

regards

Hemant

Hi Lien! :) 

Have you though about using something like FluoroJade which is often used to detect degenerating neurons?  http://www.sciencedirect.com/science/article/pii/S0006899300025130

maybe this in combination with different neuronal markers to detect sub populations of neurons?

Good luck! 

Claire 

That is a great idea, Claire!

Thank you so much for your suggestion!

Lien

Hi Lien

In addition to dendritic morphology and FluoroJade stainings, you can also study the microcircuitry of your neuronal subpopulation, focusing on synapse numbers and/or location. Deficits in microcircuitry could be preceding more severe neurodegeneration like morphology changes or cell death.

Good luck!

Cleo

Hi Lien!

Neurodegeneration is a multistage process, which depends on various factors. First of all, you have to decide what a model you want to use and what kind of cellular changes you want to see. Neurodegeneration may start from peripheral parts of neuron or may be initiated in the body. One more important question is how long it takes to develop neurodegenerative changes. When you will be able to answer these questions for yourself you can start to choose appropriate IHC or IF methods of detection.  

Hi Lien,

In some cases staining for dystrophic neurons might help SMI32 for instance is commonly used in MS for instance. This paper also used cleaved actin fragment in addition to cleaved caspase. doi: 10.1016/j.stem.2010.08.014. Do update what you found to be useful in your model.

Thank you all for your input! I really appreciate all your comments.

An update: I ordered Fluoro-Jade C and this works very well. I didn't need to optimize the procedure (only shortened the incubation time of KMnO4 solution). The cleaved caspase 3 and TUNEL staining were also successful. In my previous research project, I also used SMI32 (IHC).

 Hey Lien,

I am also working on Pig brain tissue and I  also want to do the TUNEL for the brain tissue. Can you give information like which kit you have used for TUNEL and how you have done that?

Thanks

Monika

Hi Lien

I have not read the previous answers but a suitable way to quantify neurodegeneration is by cell counting. You can stain the tissues with a cell nuclei marker such as DAPI or even POPO or BOBO (the last two are very bright and nice under a confocal microscope). You can obtain images of entire sections with a a widefield or a confocal instrument (10x objective using the tiling function available in most softwares), and then quantify cell nuclei using the cell nuclei counter function in FIJI or ImageJ. You can even define regions of interest (e.g. hippocampus or cortex) and count cell nuclei in these regions, and compare with your controls thereafter.

I hope this helps.