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

Overview

This video demonstrates an ex vivo technique to induce brain cell death mediated through cytotoxic CD8⁺ T cells. Upon obtaining brain slices, activated oligodendrocyte-directed CD8⁺ T cells are transferred onto the slices to target the myelin sheath and oligodendrocytes. Post-incubation, the damage to myelin and neuronal death can be assessed histologically in the regions of interest.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. General Comments for Mouse Experiments

1. Keep the mice under pathogen-free conditions and enable them access to food and water ad libitum.
   NOTE: It is important to use age- and sex-matched mice in experimental groups because immunological patterns can vary with age and gender.

2. Preparation and Activation of OVA-specific CD8⁺ T-cells (OT-I)

1. Perform stimulation of OT-I T cells as described below 5 days before preparing brain slices.
   NOTE: 5 x 10⁵ activated effector CD8⁺ OT-I T-cells (CD8⁺ T-cells of transgenic mice recognizing only the OVA_257-264 peptide in the context of MHC-I molecules) are needed per slice The concentration 5 x 10⁵ was chosen experimentally as optimal one to favor a good cell-cell interaction once that the cells start migrating into the slice and, at the same time, the amount of cells is not too high to induce an overreaction allowing single cell counting.
2. Prepare the medium for culturing OT-I T-cells. Supplement 500 ml DMEM with 5% fetal calf serum (FCS), 10 mM HEPES, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 1% nonessential amino acids, and 25 µg/ml gentamicin. Store the medium at 4 °C until use.
3. Remove the spleen of OT-I transgenic mice as per reference. Transfer them into the prepared medium.
4. Generate a single-cell suspension by mashing spleens through a 70 µm strainer and centrifuge suspension at 300 x g for 5 min, 4 °C.
5. Incubate cell suspension with 5 ml ammonium-chloride-potassium (ACK) buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.3) for 5 min to lyse red blood cells.
6. Stop incubation with a 10 ml medium and centrifuge it again as described above.
7. Dilute cell suspension in a medium and calculate numbers. Plate cells at a density of 3 x 10⁷ cells/well in a 12-well plate and prime them by incubation with OVA_257–264 (SIINFEKL; 1 nM) and interleukin 2 (IL-2) (500 IU/ml) for 5 days.
8. After 4 days, add IL-2 at a concentration of 500 IU/ml again.
9. Subsequent to stimulation, purify OT-I T-cells from cell suspension by using a negative selection-based mouse CD8⁺ T-cell isolation kit following the manufacturer’s instructions. Purification is based on the depletion of all non-CD8⁺ cells by using a cocktail of antibodies against CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC Class II, Ter-119, and TCRγ/δ which are then discarded by using magnetic columns for the elution of the purified CD8⁺ T-cells.
   NOTE: Critical steps in this procedure are indicated by the manufacturers; it is important that the reaction involves only single cells because the presence of clumps could block the column during the elution step; counting the cell before adding the Byotin cocktail is important for the degree of purity of the sample and the procedure should be performed on ice to minimize unspecific antibody bindings.

3. Preparation of Acute Brain Slices and Co-culture with OT-I T-cells

1. Prior to the preparation of acute brain slices, prepare placedine ice-cold physiological saline solution using 200 mM sucrose, 20 mM PIPES, 2.5 mM KCL, 1.25 mM NaH₂PO₄, 10 mM MgSO₄, 0.5 CaCl_2, and 10 mM dextrose and artificial cerebrospinal fluid (ACSF) by using 125 mM NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 24 mM NaHCO₃, 2 mM MgSO₄, 2 mM CaCl₂, 10 mM dextrose. Adjust the pH of each solution to 7.35 by using NaOH. Before adjusting the pH of ACSF, the solution must be bubbled with a mixture of 95% O₂ and 5% CO₂.
2. Pre-cool the solutions.
3. Anesthetize 8 - 10 week-old mice (e.g., C57Bl/6 as control or transgenic ODC-OVA mice with inhalation using 5.0% isoflurane, 5.0% halothane or inject 100 mg/kg ketamine and 10 mg/kg body weight xylazine via i.p. Wait for anesthesia and use a toe pinch to assess the level of anesthesia and decapitate them at once. Euthanize the mice as per institutional animal care and use committee criteria for euthanasia.
4. Put the mouse in the ventral position. Disinfect and cut scalp sagittally. Open the cranial bone sagittally, remove the brain quickly with the help of a scoop, and fix it on the plate of a vibratome by using glue.
5. Fill the plate with the prepared placedine ice-cold physiological saline solution.
6. Cut 300 µm coronal slices with the vibratome.
   NOTE: This procedure is known to yield viable intact tissue specimens appropriate for functional cellular studies within a time interval of at least 8 hr. Indeed, after this time period, already starting after 6 hr, the preparation shows reduced quality, namely changes in basic and functional properties like action potential generation and propagation.
7. After sectioning, transfer one slice immediately into each well of a 12-well plate filled with ACSF.
8. Add carefully 5 x 10⁵ OT-I T cells per slice.
9. Incubate slices for up to 8 hr in an incubator (37 °C, 5% CO₂).
10. After the incubation period, harvest slices and embed them by using OCT compound tissue-tek and freeze them in liquid nitrogen. Store them at -20 °C for further histological studies to e.g., evaluate neuronal structure (e.g., using anti-MAPII or anti-synaptophysin antibodies) or apoptosis (e.g., using anti-Caspase-3 antibodies and anti-NeuN antibodies) according to standard procedures.

Materials

Name	Company	Catalog Number	Comments
12-Well-plate	Corning	3513
2-Mercaptoethanol	Gibco	31350-010
2-Methylbutan	Roth	3927.1
70 µm strainer	Falcon	352350
CaCl2	Merck	1.02382.0500	Calcium chloride
CD8+-isolation kit	Miltenyi Biotech	130-090-859
D(+)-glucose	Merck	1.08337.1000
DMEM	Gibco	31966-021	Warm in 37 °C water bath before use
EDTA	Sigma	E5134
FCS	PAA Laboratories	A15-151	Fetal calve serum
Gentamicin	Gibco	15750-060
HEPES 1M	Gibco	15630-050
IL-2	Peprotech	212-12
Isofluran	Abbott	05260-05
KCl	Merck	1.04933.0500	Potassium chloride
KHCO3	Sigma	P9144	Potassium hydrogen carbonate
L-Glutamine	Gibco	35050-038
MgSO4	Merck	1.05886.0500	Magnesium sulfate
NaCl	Sigma	31434	Sodium chloride
NaH2PO4 * H2O	Merck	1.06346.0500	Sodium hydrogen phosphate
NaHCO3	Merck	1.06329.0500	Sodium hydrogen carbonate
NaOH	Merck	1.09137.1000	Sodium hydroxide
NH4Cl	Sigma	213330	Ammonium chloride
Non-Essential Amino Acid	Gibco	11140-050
OVA (257-264)	Genscript	RP10611	Ovalbumin
PIPES	Sigma	P6757
Sukrose	Merck	1.07687.1000
Tissue-Tek OCT	Sakura	4583