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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of the materials
- Magnetic-activated cell sorting (MACS) buffer: Prepare 2 mM ethylene diamine tetraacetic acid (EDTA) and 0.5% bovine serum albumin (BSA) (Table of Materials) in PBS-/-.
- Collagenase IV stock solution (20 U/μL): Resuspend the powder in PBS+/+ (Table of Materials), aliquot, and store at -20 °C.
NOTE: Collagenase IV requires MgCl2 to be fully active; do not freeze-thaw Collagenase IV solution.
- Anesthetic-analgesic solution: Mix 150 μL of ketamine (150 mg/kg), 25 μl of xylazine (5 mg/kg), and 330 μl of buprecare (0.1 mg/kg) in 1 mL of PBS-/-.
NOTE: Prepare anesthetic-analgesic solution extemporaneously and do not keep it longer than a day.
- Heparin solution (100 U/mL): Resuspend the powder in PBS-/-.
- Prepare the infusion inset system connecting a tube to a decanter Erlenmeyer filled with PBS-/-. Connect a 23 G needle to the extremity of the infusion tube. Open the infusion inset and let the PBS run until there are no bubbles in the tube.
- Prepare the binocular loupe equipped with a light.
2. Housing of C57BL/6 mice
- For the analysis of CP (choroid plexuses) myeloid or T cells, use four mice for each and pool the four CP (two from each lateral ventricle, the third ventricle, and the fourth ventricle CP; for eight mice in total). Not pooling CP carries the risk of failing to detect the rare immune populations in CP due to their low abundance.
- Let the mice acclimatize for at least 7 days prior to any experimentation. Keep the mice under pathogen-free conditions at constant temperature and humidity, in a 12/12 h or 14/10 h light/dark cycle, with water and standard pellet food ad libitum.
3. PBS perfusion and brain dissection
- Weight each mouse and inject 10 μL/g of the anesthetic-analgesic mix solution (step 1.3) intraperitoneally.
- Wait around 30 min for efficient analgesia (check for the depth of anesthesia by pinching the mouse's digits).
- Position the anesthetized mouse flat on its back on the dissection support and tape its palms to the dissection support.
- Pinching the skin of the animal with forceps and using scissors, open the abdomen, the diaphragm, and the thorax to expose the heart.
NOTE: When the thorax is opened, the brain becomes anoxic. One must proceed precisely and swiftly through the next steps of the perfusion.
- Inject 20 μL of 100 U/mL heparin solution directly into the left ventricle.
- With fine scissors, make an incision of at least 3 mm in the right atrium to allow the blood to flow out of the body.
- Immediately after, insert the 23 G needle placed at the extremity of the infusion tube through the tip of the left ventricle.
- Open the infusion system at the maximum and wait for complete perfusion: Using a 23 G needle at a flow rate of about 6 mL/min, the perfusion is complete in 3 min.
NOTE: The even discoloration of organs such as the liver attests to perfusion efficacy.
- Close the infusion system and remove the needle from the heart ventricle.
- Remove the tape from the mouse's palms. Place the mouse in a ventral position.
- Pinching the animal's skin with forceps and using scissors, remove the skin from the top of the head, from the eyes to the ears.
- With the help of scissors, cut the skull first between the eyes and then laterally from each eye to the spinal cord just above the masseter muscles. For this, use the extremity of scissors and proceed gently to avoid damaging the brain with the scissors.
- Open the skull with forceps by pinching it from the extremity between the eyes.
- Use scissors to cut the spinal cord and extract the brain with forceps, placing their two points on the lateral side of the brain to tilt it and place it in a Petri dish filled with ice-cold PBS+/+. The CPs are then immediately collected.
NOTE: Check for discoloration of the brain to verify perfusion efficacy.
4. Dissection of the Choroid Plexus from the brain
- Position the brain dorsal side up in the Petri dish and below the objectives of the binocular loupes.
- With the help of forceps to maintain the brain in place, insert the two ends of another forceps down through the midline between the hemispheres.
- Use the forceps to pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle.
- Identify the lateral CP as a long veil lining the lateral ventricle that is flaring at both ends. Use the two ends of a thin forceps to catch the lateral CP. Be careful to collect the triangular posterior part that may be hidden by the posterior fold of the hippocampus.
- Pull the cortex, corpus callosum, and hippocampus of the contralateral hemisphere away from the septum to expose the entire third ventricle and the opposite lateral ventricle.
- Collect the third CP with fine forceps, which can be identified as a short structure with a granular surface aspect.
- Collect the other lateral CP.
- Insert the two ends of a forceps down between the cerebellum and midbrain. Detach the cerebellum from the pons and medulla to expose the fourth ventricle.
- Identify the fourth CP as a long globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and the medulla. Collect the fourth CP with fine forceps.
NOTE: Silane-base coating of forceps may prevent stickiness of CP on forceps.
- Repeat steps 3.1-4.9 for each of the other seven mice. In the meantime, the collected CP can be kept temporally in a tube placed on ice.
5. Preparation of samples for flow cytometry analysis
- Fill the tube containing all dissected CP to 750 μL of PBS+/+.
NOTE: The deposition of dissected CP with thin forceps in the tube produces a small volume of PBS+/+. To avoid a possible loss of the CP tissue, it is better to complete the existing volume to 750 μL than remove and replace it with fresh 750 μL of PBS+/+.
- Add 15 μL of 20 U/μL Collagenase IV stock solution (see step 1.2) for a final concentration of 400 U/mL.
NOTE: DNAse I (150 μg/mL) may prevent excessive cell clumping.
- Incubate CP with Collagenase IV at 37 °C under mild agitation (300 RPM) for 45 min.
- Gently pipette up and down around 10 times to finalize the CP dissociation.
- Fill the CP tube to 1.5 mL with MACS buffer (see recipe step 1.1) to stop collagenase IV activity.
NOTE: Collagenase IV activity is stopped at low temperatures and inhibited by serum albumin.
- Centrifuge the cells at 500 x g, for 5 min, at 4 °C. Discard the supernatant and wash the cells with 1.5 mL of MACS buffer.
- Centrifuge the cells at 500 x g for 5 min, at 4 °C. Discard the supernatant and resuspend cells in 220 μL of MACS buffer.