The overall goal of this quantitative 3-D in silico modeling, or q3DISM technique, is to quantify the uptake of amyloid-beta by mononuclear phagocytes in the brains of rodent models of Alzheimer's disease. This method addresses a pressing need in the field of Alzheimer's research for quantifying the clearance of amyloid-beta that is deposited in the brains of Alzheimer's patients forming senile plaques. For the first time, this technique enables true 3-D reconstruction and quantitation of amyloid-beta by macrophages in vivo We began to use cued 3-Dism to study amyloid-beta deposition because we needed a robust tool to visualize and quantify microphage phagocytosis in vivo.
This technique may prove useful in testing experimental Alzheimer's disease therapeutics to assess which drugs stimulate macrophages for the efficient clearance of amyloid-beta. When the tissue samples are ready for immunostaining fill the hydrophobic barrier pen-encircled tissue region with blocking buffer for a one hour incubation at room temperature in a humidified chamber. At the end of incubation, replace the blocking buffer with the first primary antibody of interest overnight at 4 degrees Celsius in the humidified chamber.
The next morning, wash the samples in three five-minute PBS baths at room temperature with light agitation. After the third wash, incubate the slides with the appropriate fluorescent secondary antibody at room temperature protected from light. After one hour, wash the sections three more times in PBS protected from light.
Then label the cells with the other tissue-appropriate primary and secondary antibodies of interest over the next two to four days as just demonstrated. To efficiently stain cellular and supercellular compartments and amyloid-beta deposits, it is crucial to label with only one primary antibody at a time followed by the corresponding secondary antibody. A time-consuming but result-optimizing process.
Following the last wash of the secondary antibody staining, allow the sections to dry completely overnight at room temperature in the dark. Then, cover the dried specimens with a cover slip sealed by fluorescent mounting medium containing DAPI. To image the samples by confocal microscopy, select the 60x microscope objective and add immersion oil to the lens.
Place the sample onto the microscope stage slide holder and raise the objective until the oil makes contact with the slide. Using epifluorescent illumination, adjust the focal plane to locate the amyloid plaques in the hippocampus or cerebral cortex. Then, acquire confocal images of the activated mononuclear phagocytes surrounding the amyloid deposits in the hippocampus or the cortex.
For qualitative 3-D in silico modeling, first analyze the confocal datasets with scientific 3-D image processing in an analysis software colocalization module for spatial proximity of the activated macrophage marker staining in all Z planes simultaneously. Create colocalization channels that correspond to the phagolysosome staining within the activated mononuclear phagocytes. Select TRITC for Channel A, and FITC for Channel B.In the Mode Check window, select Threshold.
For the colocalization intensities, select Source Channels. Click Edit to select the colocalization color. Then adjust the thresholds of each channel independently to include the specific staining and to exclude the background and nonspecific signals.
The colocalized voxels will appear in the selected colocalization color in all z-stacks simultaneously. Next, click Build Colocalization Channel. The created colocalizaton channel will appear in the Display Adjustment window.
Click on the colocalization channel to open the channel statistics. The percentage of volume material A above threshold colocalized represents the monocyte volume occupied by the phagolysosomes. Using the Colocalization Module, analyze the colocalization channel for spatial proximity with the amyloid-beta signals to quantify the amyloid-beta encapsulated within the phagolysosomes.
Select the Channel A colocalization data set and Cy5 for Channel B and build a colocalization channel as just demonstrated. Click on the colocalization channel to open the channel statistics again. The percentage of volume material A above threshold colocalized represents the phagolysosomal volume occupied by the amyloid-beta.
Using the Surpass Module, reconstruct the confocal image stacks and generate 3-D models of the encapsulated amyloid-beta within the monocyte phagolysosomes. In the Display Adjustment window, select TRITC to show only the macrophage staining, and click add new surface"in the volume properties. Under Settings, in step one of five, algorithm, select Surface.
In color, select the color type of interest and adjust the transparency as appropriate. Then check the select region of interest box and click next. In step two of five, region of interest, adjust the X, Y, and Z coordinates to draw a window around the cell of interest and click next.
In step three of five, source channel, select the TRITC source channel and check the Smooth box. Set the surface area detail level to 0.4 microns and click next. In step four of five, threshold, adjust the threshold so that the created volume overlaps perfectly with the TRITC channel signal and click next.
Finally, in step five of five, classify surfaces, in the Filter Types section, select the objects depending on their size to be included or excluded from the volumes to be created. Repeat the same steps to create 3-D surfaces for phagolysosomes with the FITC channel and amyloid-beta with the Cy5 channel. After qualitative 3-D in silico modeling, as just demonstrated, amyloid-beta uptake into the monocyte phagolysosomes in the brains of transgenic mice and rats can be analyzed.
Interestingly, the volume occupied by CD68 positive phagolysosomes is significantly increased in Iba1 positive mononuclear phagocytes, associated with, compared to distant from plaques, in both mice and rats, demonstrating that the mononuclear phagocytes near plaques are poised for phagocytosis, compared to the cells located away from the plaques. It is also noteworthy that plaque-associated mononuclear phagocytes exhibit an increased amyloid-beta uptake in transgenic mice compared to cells distant from plaques whereas in transgenic rats a very modest uptake of amyloid-beta fibrils is observed overall, with no apparent change in amyloid-beta fibril phagocytosis as a function of the distance from the plaques. While attempting this procedure, it's important to remember that the quality of the confocal images is crucial and that similar thresholds should be applied for all channels during the colocalization analysis.
Since its development, this technique has paved the way for researchers in the field of neuroinflammation to explore phagocytic events in the brains of rodent models of Alzheimer's disease. After watching this video, you should have a good understanding of how to label rodent brain sections for mononuclear phagocytes, phagolysosome, and amyloid-beta. To quantify the phagocytosis of cerebral amyloid-beta in 3-D in vivo.
