The “Brain Milking” Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Animal breeding, maintenance, and experimental procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986, authorized by the Home Office, and with the Presidential Decree 56/2013 of the Hellenic Republic, scrutinized by the Animal Welfare and Ethical Review Bodies of the Universities of Cambridge and Patras, as well as approved and scrutinized by the local Prefectural Animal Care and Use Committee (Protocol number: 5675/39/18-01-2021). Male and female Sprague-Dawley, Wistar, and Long-Evans rats, with ages varying between 2 and 4 months and with body weights between 150 g and 250 g, were used. The protocol is graphically summarized in Figure 1.

1. Release cocktail preparation

NOTE: Prepare fresh on the day of the procedure and keep on ice. The quantities are given per 2 µL to be i.c.v. injected in each lateral ventricle. Prepare an additional 1 µL per intended injection.

1. Prepare 0.5 µL of 500 mU neuraminidase from Clostridium perfringens (Clostridium welchii).
   NOTE: Store this as a 1 U/µL stock diluted in sterile water at -20 °C. Other types of neuraminidases have not been tested.
2. Prepare 1 µL containing 1 µg of integrin-β1-blocking antibody.
   NOTE: Store it at 4 °C.
3. Prepare 0.5 µL containing 0.5 µg of basic fibroblast growth factor (recombinant human FGF-basic).
   ​NOTE: Store it as a 1 µg/µL stock diluted in sterile water at -20 °C.

2. Injection of release cocktail

NOTE: The whole process can be performed within 20 min. Take care to perform the surgery in aseptic conditions. Clean all surfaces with antiseptic (e.g., 3% or 6% hydrogen peroxide). Use autoclaved or readily sterile tools, gloves, gowns, and drapes.

1. Anesthetize the experimental animal by isoflurane inhalation (2.5% for induction and 2% for maintenance). Confirm the depth of anesthesia by checking the corneal reflex, the reaction to stimuli (hind limb and tail pinch test), and the mode of breathing.
   NOTE: Surgical procedures can be performed under general anesthesia induced by intraperitoneally injectable anesthesia (e.g., ketamine [40 mg/kg] and xylazine [10 mg/kg]). Deep anesthesia was confirmed by checking the corneal reflex, the reaction to stimuli (hind limb and tail pinch test), and the mode of breathing.
2. Administer analgesia subcutaneously (e.g., 0.3 mg/mL buprenorphine) upon the induction of anesthesia.
3. Mount the rat on the stereotaxic frame.
4. Shave the fur of the head with a razor and clean the skin with antiseptic, such as 10% povidone-iodine solution and then alcohol. Apply the antiseptic three times using sterile cotton swabs. Rub in a circular motion for 3-5 s each time. Apply eye ointment to prevent dryness.
5. Make a 2 cm incision in the head's skin along the middle line using a sterile scalpel.
6. Meticulously clear the skull using sterile swabs and sterile saline.
7. Identify the bregma using the edge of the needle of a 10 µL Hamilton syringe mounted on the stereotaxic device. Set the bregma as "point 0,0".
   NOTE: The bregma is identified as the point of intersection between the sagittal and the coronal sutures. More details can be found in the rat brain atlas of Paxinos and Watson⁵.
8. Move the device to the coordinates anterioposterior (AP) = 0.3 mm, lateral (L) = +1.2 mm (to target the SEZ), or AP = 1.5 mm, L = +2.0 mm (to target the CC).
9. Drill a 1 mm burr hole using a dental drill.
   NOTE: When drilling by hand, take care not to injure the cortical surface. The hemorrhage is not expected to be considerable. Clear any blood with saline and apply pressure to stop more persistent bleeding.
10. Load 4 µL of the release cocktail in the 10 µL Hamilton syringe.
11. Lower the needle (preferably blunt or conical edge) of the Hamilton syringe in order to get in contact with the dura.
12. Insert the needle at the desired depth (D = 3.5 mm).
    NOTE: Pour saline on the surface of the dura to keep the tissue hydrated.
13. Infuse 2 µL of the release cocktail at a rate of 1 µL/min.
14. Leave the needle in place for another 2 min before slow retrieval to avoid surfacing of the release cocktail.
15. Repeat steps 2.8-2.14 for the other hemisphere at the coordinates AP = 0.3 mm, L = -1.2 mm (to target the SEZ), or AP = 1.5 mm, L = -2.0 mm (to target the CC).
16. Suture the incision with 5-0 nylon (diameter 0.1 mm) and clean the sutured area with antiseptic.
17. Remove the mask supplying the anesthetic and transfer the animal to the post operation monitoring area.
    NOTE: Recovery from anesthesia and maintenance of recumbency should occur within 5-10 min, and full recovery (normal behavior) within 25 min.
    1. Closely monitor the recovery (vivid and uninterrupted movement, frequent access to water) in a well heated (24-25 °C), quiet space without small-particle bedding, which can block the airways. Return the animal to the company of its cage mates (not to new animals) only after confirming full recovery.
    2. Monitor the animal at the maintenance facility for at least 48 h after surgery. Address signs of pain (hiding of head, abnormal head or body posture, hypersensitivity, and hyperexcitability to handling) by administering analgesia (e.g., 0.3 mg/mL buprenorphine, subcutaneously). Refer animals with discolored or erected fur to the responsible vet.

3. Cerebrospinal fluid (CSF) liquid biopsy

NOTE: The whole process can be performed within 10 min. The liquid biopsy described here is performed 3 days post injection of the release cocktail but can be performed in exactly the same way whenever required. Take care to perform the surgery in aseptic conditions. Clean all the surfaces with antiseptic (e.g., 3% or 6% hydrogen peroxide). Use autoclaved or readily sterile tools, gloves, gowns, and drapes.

1. Anesthetize the animal by isoflurane inhalation (2.5% for induction and 2% for maintenance). Confirm the depth of anesthesia by checking the corneal
   reflex, the reaction to stimuli (hind limb and tail pinch test), and the mode of breathing.
   NOTE: The surgical procedures can be performed under general anesthesia induced by intraperitoneally injectable anesthesia (e.g., ketamine [40 mg/kg] and xylazine [10 mg/kg]). However, this is not advisable as the procedure is short, especially if biopsies will be performed multiple times on consecutive days.
2. Administer analgesia subcutaneously (e.g., 0.3 mg/mL buprenorphine) upon the induction of anesthesia.
3. Mount the experimental animal on the stereotaxic frame. Use ear bars to fix the head without obstructing rotational movement toward the front and the back.
4. Stabilize the head at a downward 40° angle so that a good extension of the back of the neck can be achieved.
   NOTE: One way to do this is by using the mouth bar of the device⁶.
5. Shave the fur in the neck area using a razor and clean the skin with antiseptic, such as 10% povidone-iodine solution and then alcohol. Apply the antiseptic three times using sterile cotton swabs. Rub in a circular motion for 3-5 s each time.
6. With the fingertip, find the rhomb-shaped depressible area positioned in between the occipital protuberance and the spine of the atlas⁶.
7. Draw a point-mark (e.g., using a marker).
8. Fix a 1 mL or 0.5 mL syringe on the stereotaxic frame.
   NOTE: It is advisable to use syringes with non-detachable needles as they produce better suction and have less dead volume.
9. Bring the needle at the point of contact with the skin at the point-mark.
10. With a pair of forceps, lift the skin while lowering the syringe through the skin layers.
11. Retrieve the plunger slightly to generate negative pressure in the syringe.
12. Restart to dip the needle very slowly until liquid (CSF) appears in the syringe.
13. Settle the needle at this position and allow the slow flow of CSF.
    NOTE: The needle can be lowered or elevated slightly if CSF flow is very slow.
14. Start removing the CSF slowly (in steps of 40 µL/min) up to 120 µL.
15. Slowly retrieve the syringe.
16. Intraperitoneally administer 1 mL of normal serum to support the experimental animal's replenishment of fluids.
17. Mix the liquid biopsy (CSF) with 400 µL of NSC medium and keep the microcentrifuge tube at 4 °C until further use.
    NOTE: Liquid biopsies should be processed within 3 h if not frozen.
18. Remove the mask that supplies the anesthetic and transfer the animal to the post operation monitoring area.
    NOTE: Recovery from anesthesia and maintenance of recumbency should occur within 5-10 min, and full recovery (normal behavior) within 25 min.
    1. Closely monitor the recovery (vivid and uninterrupted movement, frequent access to water) in a well heated (24-25 °C), quiet space without small-particle bedding, which can block the airways. Return the animal to the company of its cage mates (not to new animals) only after full recovery has been confirmed.
    2. Monitor the animal at the maintenance facility for at least 48 h after surgery. If signs of pain (hiding of head, abnormal head or body posture, hypersensitivity, and hyperexcitability to handling) are observed, administer analgesia (e.g., 0.3 mg/mL buprenorphine subcutaneously). Refer animals with discolored or erected fur to the responsible vet.

4. Processing of tissue for immunofluorescence

1. Euthanize the animal by intracardial infusion of 20 mL of ice-cold saline, followed by 50 mL of ice-cold 4% paraformaldehyde (PFA; in phosphate-buffered saline [PBS] of pH = 7.4).
2. Dissect the brain tissue.
   1. Separate the head from the body using scissors to make a cut in the cervical area. Use the scissors to remove the skin and then cut the skull's bone along the sagittal midline, with the tips of the scissors pointing upward in order not to damage the brain tissue. Aim to continue cutting as much as possible, passing the coronal midline.
   2. Use forceps to remove the bones, starting from the supraoccipital and the parietal bones and finally the frontal bones.
   3. Once the brain tissue is revealed, use the forceps or a spatula to lift it out of the skull. To facilitate this, cut the nerves at the base of the brain and the olfactory bulbs, if not wanted.
3. Post-fix the tissue overnight in 2% PFA at 4 °C.
4. Cryo-protect the tissue in 30% sucrose (in PBS) for at least 48 h at 4 °C until the tissue sinks to the bottom.
5. Freeze the tissue at -80 °C.
6. Cut the brain tissue into 14 μm thick coronal sections with a cryostat.
7. Perform immunofluorescence staining using standard protocols ^3,7
   1. Incubate the sections with blocking buffer (3% bovine serum albumin [BSA], 0.1% Triton X-100, in PBS) for 2 h at room temperature.
   2. Perform antigen retrieval (boiling for 15 min in 10 mM citrate buffer, pH = 6.0, using glass jars).
      ​NOTE: This step is optional, but necessary for nuclear antigens.
   3. Bring to room temperature and incubate with primary antibodies against glial fibrillary acidic protein (GFAP), doublecortin (Dcx), S100β, and β-catenin (see the Table of Materials) in blocking buffer overnight at 4 °C.
   4. Incubate for 2 h with secondary antibodies in PBS with 4',6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining at room temperature (see the Table of Materials).
   5. Mount the coverslips.

5. Processing isolated cells for immunofluorescence

1. Plate the cells into 96-well plates compatible for microscopy, coated with poly-D-lysine (100 µg/mL).
2. Fix the cells in ice-cold 2% PFA for 10 min and wash 3x with PBS.
3. Perform immunofluorescence using standard procedures^3,7for PDGFRα, GFAP, Dcx, SOX2, and ID3 (see the Table of Materials).
4. Keep the cells in PBS + NaN₃ (0.1%) at 4 °C in the dark.

6. Microscopy and image analysis

1. Take images using epifluorescence or confocal microscopy and perform cell counts using standard image analysis software tools, such as cell counters.