What are some therapeutic strategies to promote neurogenesis? I found a 2022 publishment by Chaplygina et al. that looks at cell therapy as a way to correct impaired neurogenesis in the adult brain, and they found that the paracrine signaling effect of human mesenchymal stromal cells (isolated from umbilical cord) can improve memory and increase neuronal and glial density in the temporal cortex and hippocampal regions of the brain (mice model). https://doi.org/10.1134/S0022093022010112

Prism is great for generating graphs and doing statistical analysis, it also helps you with determining the parameters so you perform the correct test.

This, coupled with other methods they employed (e.g. testing different fixation times for the best result) is really impressive to me. The researchers were very meticulous and tried to eliminate all confounding variables.

Oil reduces light refraction, which allows for a better resolution!

Interesting! Not only are DCX+ cells decreasing as a result of AD, looking at Figure 4, we see that these cells are also not maturing properly: there is a lack of expression of important proteins for maturation.

We can see in (b) and (c) that the harmful proteins are building up as AD progresses through the stafes, and in d-l that DCX and DCX+ cells decrease as AD progresses compared to control subjects.

The build up of beta-amyloid forms plaques that hinder cell communication and triggers immune responses that kill brain cells.

Important mark of AD, tau is abnormally phosphorylated, which causes neuronal and synaptic loss in the brain.

This justifies why the authors chose to measure DCX expression as a marker of adult hippocampal neurogenesis. If we refer to Figure 2 (c), I believe the fourth image down shows the double labeled cells, which corroborates with (i) which shows that 91% of DCX+ cells are Prox1+. Prox1 specifies the granule cell identity.

Unfortunately, to keep the multiple methodological factors and constraints controlled, I think studying a smaller population is the best choice for a relatively new study like this. Being able to work closely with the local brain bank is especially beneficial, both for keeping in compliance with policies, and for gathering good samples. As of now, I think quality is more important over quantity. I did, however, find an interesting old meta-analysis paper that looks at geographical variation in dementia (Russ et al., 2012). They did find that there are variations in dimentia across countries and between urban and rural areas! However, there are many limitations and confounding factors that make it hard to make any conflusions. If interested, here is thee DOI: 10.1093/ije/dys103

I was curious about why DAPI was used as a counterstain because we do not use that when staining Drosophila melanogaster brains. I found an article that does a good job of briefing why we need DAPI by Loesel et al. They state that the fluorescent chromophore DAPI binds to double-stranded DNA in the nuclei of cells, which tells you the total number of cells. Then, since you double stained the cells with specific antibodies, the double labeled cells are neuronal cells, while the DAPI only cells are not (Loesel et al., 2006). 10.1016/j.jneumeth.2006.01.006

I admire the rigorousness of the immunostaining protocol. If we refer to Supplmental Figure 4., we will see that the reason they used four different antibodies is because they were raised against different domains of the protein. With this, the researchers can see which antibody works the best, and they can make sure no DCX protein expression is missed!