3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

The overall goal of this procedure is to fully visualize neurons infected with Toxoplasma Gandhi cysts. This is accomplished by first intraperitoneal infecting Cree reporter mice with parasites that express M cherry and are engineered to inject CRE recombinase into host cells. Once CNS infection is established, the second step is to perform trans cardial perfusion with heparin saline, followed by 4%paraldehyde.

Next, harvest the brain, remove and discard the olfactory bulbs and cerebellum cut the remaining brain into two halves, then section the halves into 160 micron thick sections. The final step is to clear the sections with graded glycerol PBS tween solutions and mount them onto slides for imaging with confocal micro microscopy. Ultimately, the images can be analyzed with software that allows 3D rendering and analysis, and this method can be used to show the cellular relationship between the cyst and the infected neurons.

The main advantage of this technique over existing methods such as serially, sectioning, staining, and reconstructing thick sections is that it eliminates the requirements for staining and reconstruction, both of which can be technically difficult and time consuming. Demonstrating this procedure will be Carla Cabral, a research specialist from my laboratory. Begin this procedure by placing the mouse brain infected with Toxoplasma Gandhi in a brain matrix for precise and even cuts trim off the cerebellum and the olfactory bulbs.

Then cut the brain into two coronal sections. A fix each brain section onto a specimen mounting block with Sano acrl. Subsequently affix a small block of 5%Aros gel behind the brain section for support.

Cut the tissue into 160 micron fix sections with a viome set at a speed of four and amplitude of nine. Then transfer the tissue sections in a scintillation vial filled with fresh chilled one XPBS if they're going to be cleared within one week. If the sections are not going to be cleared within one week, place them in 1.5 milliliter micro centrifuge tubes filled with cryo preservative solution and store at minus 20 degrees Celsius.

Now prepare 25%50%75%and 90%volume per volume glycerol in one XPBS plus 2%between 20. Next, remove the sections from one XPBS, or if they are in cryo preservative, rinse them in one XPBS and transfer them to a scintillation vial containing 10 milliliters of 25%Glycerol PBST. Place the covered vial on a shaker.

Cover the vial to avoid light exposure and incubate at four degrees Celsius for 12 hours. After 12 hours, confirm all the sections have sunk to the bottom of the vial. Aspirate the 25%glycerol PBST, leaving just enough to cover the sections.

Then build a vial with 10 milliliters of 50%glycerol PBST. Place the vial on a shaker and protect from light incubate at four degrees Celsius for another 12 hours. Repeat this process for 75%glycerol PBST and 90%glycerol PBST, in which the sections will remain floating in the middle of the vial.

Leave the sections in the 90%glycerol PBST for 24 hours to reach maximal clearing. Now create a spacer by cutting a number 1.5 cover slip into five pieces with a ruler and a diamond tipped pencil. Discard the centerpiece and arrange the four outer pieces on a plain glass slide to create a window.

Then brush clear nail polish onto the seams to adhere the pieces of the spacer to the slide in the correct configuration. Next, transfer the sections to a slide using a plastic pipette with the tip cut off. Make sure to fill in the surrounding area with 90%glycerol PBST.

Then carefully place a cover slip over the sections and be careful not to introduce any bubbles. Excess glycerol PBST may be wicked away with a lint-free wipe.Afterward. Image GFP positive neurons containing RFP positive Toxoplasma Gandhi cysts with a confocal microscope when finished, store the slides flat and covered from light at four degrees Celsius.

Shown here are the maximum projection images of GFP positive neurons containing RFP positive cysts. This is a 3D rotational movie of the cell. This image shows the iris generated direct line measurement of the distance between the edge of the cyst and the edge of the cell body.

In this example, the distance is measured as 149 microns, and here is an IMS generated 3D movie showing the middle to middle and edge to edge measurement between the cyst and the cell body Once mastered, this technique can be done in four to five days if performed properly following this procedure. Additional analysis such as counting the number of batons between infected dendrites and uninfected dendrites can be done to answer such questions as whether or not cyst disrupt normal neuronal function.