This study investigates the role of oligodendrocytes in neuronal death through the mechanism of demyelination. Using transgenic mouse models, the interaction between activated CD8+ T cells and oligodendrocytes is explored to understand the apoptotic pathways involved.
Oligodendrocytes, which are specialized cells of the central nervous system, form myelin sheaths around neurons. Myelin insulates the axon and facilitates the rapid conduction of electrical impulses. Demyelination — the destruction of myelin — leads to neuronal death.
To study neuronal death, take a transgenic mouse brain fixed on a vibratome plate. Immerse the brain in an ice-cold buffer, preserving the structural integrity while sectioning.
Using a vibratome, obtain thin coronal sections. Transfer the sections to a culture well containing an appropriate medium.
The oligodendrocytes in the transgenic mouse brain express a foreign antigen, which is displayed on the cell surface via the major histocompatibility complex.
Add a solution of activated CD8+ T cells — specific for the foreign antigen — onto the sections and incubate.
The T cell receptor binds to its antigen displayed on the oligodendrocyte. The co-stimulatory molecule binds to its ligand. The binding initiates a signaling cascade inside the T cell, causing the release of perforin and granzymes — cytotoxic mediators.
Perforin creates pores in the oligodendrocyte membrane, through which granzymes enter the cytoplasm. Further, Fas ligands on the T cells bind to their receptors on the oligodendrocyte surface. Granzymes, along with the Fas ligand binding, induce the apoptotic pathway in oligodendrocytes, leading to demyelination and neuronal death.
Post-incubation, the treated brain sections are ready for the assessment of cell death.
For this experiment, have 8- to 10-week-old transgenic ODC-OVA mice and age- and sex-matched C57Bl/6 controls. As usual, house the mice in a pathogen-free environment with free access to food and water. After anesthetizing a mouse, use a back paw pinch to assess the level of anesthesia, and then immediately euthanize it.
Now position a mouse ventrally, and disinfect the scalp, and decapitate it. Next, quickly, cut the scalp, open the cranium, and scoop out the brain. Fix the brain in place using glue, and transfer it to a vibratome plate, then, fill the plate with ice-cold placedine physiological saline. Once prepared, cut 300-micron slices. Transfer the slices to individual wells of a 12- or 24-well plate filled with aCSF. Then carefully add 1/2 a million OT-I T cells to each slice.
Half a million activated cells per slice provides good cell-cell interaction. Once the cells start migrating into the slice, at the same time, the amount of cells is not too high to induce an overreaction. So single-cell counting is feasible.
After loading all the wells, incubate the co-culture for up to eight hours. After the incubation, harvest the slices, and embed them in OCT compound, then, freeze the embedded slices in liquid nitrogen and store them at negative 20 Celsius for further histological studies.
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