An Ex Vivo Technique to Induce Neuronal Death via Oligodendrocyte-Specific CD8+ T Cells

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.