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

Summary

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

Abstract

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.

Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail "brain milking", a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are "released" from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a "release cocktail". The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-β1-blocking antibody, and fibroblast growth factor-2. At a second "collection" step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.

Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well  by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Introduction

Tissue-specific stem cells are partially committed cells that can give rise to all cell populations that constitute the respective tissues. Apart from being multipotent, they are self-renewing cells and crucial for maintaining the homeostasis and the regenerative capacity of tissues¹. Some tissue-specific stem cells remain in an active, strongly proliferative state, such as intestinal or hematopoietic stem cells. Others, such as brain stem cells, remain largely quiescent or dormant². In the adult brain, neural stem cells (NSCs) can be found in specialized areas, often called niches. Two such well described areas exist in the subependymal zone (SEZ) of the lateral ventricles and in the dentate gyrus of the hippocampus. The SEZ niche generates the highest numbers of cells, primarily neuroblasts that migrate toward the olfactory bulbs and contribute to the local interneuron population; in contrast, generated oligodendroblasts migrate to the adjacent corpus callosum (CC)³. Oligodendrocyte progenitor cells (OPCs) are mitotically active cells, widely distributed throughout the central nervous system, that: i) are committed to the oligodendroglial lineage, ii) can migrate to sites of demyelination, and iii) can differentiate into myelinating oligodendrocytes. OPCs also exhibit self-renewal potential and quiescence⁴.

Until now, the isolation and study of NSCs and OPCs required postmortem dissociation of the dissected brain and spinal cord tissue. To circumvent this experimental limitation, we established a method that allows, for the first time, the isolation of brain NSCs and OPCs from live animals. We call this method "milking", because it enables multiple collections of cells as their pools are not depleted. The protocol was developed in rats, due to their large brain size, targeting mainly the SEZ, or the CC, and includes two major steps. First, NSCs or OPCs are "removed" from the tissue via i.c.v. injection of a "release cocktail" containing neuraminidase, a toxin that induces ventricular wall denudation, an integrin-β1-blocking antibody, and fibroblast growth factor 2 (FGF2). The cocktail is stereotaxically injected bilaterally within the lateral ventricles. If the intended use is the isolation of NSCs, rostral areas of the lateral ventricles are targeted. If the aim is to isolate OPCs more purely, the cocktail is injected caudally in the area of the hippocampal fimbria. At a second "collection" step, liquid biopsies of cerebrospinal fluid (CSF) are performed from the cisterna magna of anesthetized rats, without the need of an incision. The liquid biopsy is mixed with NSC culture medium and can be kept at 4 °C until plating.

Protocol

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.

Results

Release and collection of NSCs
NSCs of the SEZ are separated from the CSF only by the monolayer of ependymal cells, albeit they remain in direct contact with the ventricular content via intercalating mono-ciliated processes^8,9. Neuraminidase acts specifically on ependymal cells via cleavage of sialic acid residues and can induce denudation of the ventricular wall. This leads to neuroblast clustering on the surface of the ven...

Discussion

Stem and progenitor cells are relatively sparse in mammalian brain tissue. In addition, NSCs are located in areas inaccessible for easy and safe biopsies (ventricular walls, hippocampus). Therefore, the only way to work experimentally with such cells, so far, has been their postmortem isolation. A method allowing the single or repeated collection of NSCs and OPCs from live rats, named milking, is described here step by step. The method is based on two key features: i) NSCs or OPCs are separated by the ependymal cell mono...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Release cocktail
β1-integrin-blocking antibody	BD Biosciences	#555002	purified NA/LE Hamster Anti-Rat CD29 Clone Ha2/5, 1 mg/mL. Any abntibody with blocking activity should be appropriate.
Neuraminidase from Clostridium perfringens (Clostridium welchii)	Sigma-Aldrich	#N2876	Neuraminidases fromother sources (e.g., from Vibrio cholerae) have not been tested.
Recombinant Human FGF-basic (154 a.a.)	Peprotech	#100-18B	kept as a 1 μg/μL stock, diluted in sterile water at -20 °C
Surgical procedures
10 µL Syringe	Hamilton	#80330	Model 701 RN, Small Removable Needle, 26s gauge, 2 in., point style 2
BD Micro-fine 1 mL insulin syringes	BD biosciences	04085-00	29 G x 12.7 mm
BETADINE CUT.SOL 10% FLx30ML	LAVIPHARM-CASTALIA	SKU: 5201048131168
Bupaq	RICHTERPHARMA	1021854AF	10 mL (buprenophine 0.3 mg/mL)
Digital New Standard Stereotaxic, Rat and Mouse	Stoelting	51500D
Homeothermic Monitoring System	Harvard Apparatus	55-7020
ISOFLURIN 1,000 mg/g inhalation vapour, liquid	Vetpharma Animal Health	32509/4031
Ketamidor	RICHTER PHARMA	SKU: 9004114002531	Ketamine 100 mg/mL
Nylon suture, Ethilon	Ethicon	D9635	Clear , size 5-0
Rechargeable Cordless Surgical Trimmers	Stoelting	Item:51472
Scalpel blades, sterile	Swann Morton	AW050
Scopettes Jr.  8-inch Swabs	Birchwood Laboratories	34-7021-12P
Stereotaxic High Speed Drill	Foredom	1474w/o1464
Stoelting’s Stereotaxic Instrument Kit	Stoelting	Item: 52189
Xylan 2%	Chanelle Pharmaceuticals	13764/03/19-5-2004	Xylazine, 25 mL
Tissue and cells handling and immunostainings
96-well plates appropriate for microscopy	Greiner	#655866	Screen star microplate
B27 supplement	ThermoFisher Scientific	A1486701
Bovine Serum Albumin (BSA)	Merck	P06-1391100	Fraction V, heat shock
Citrate	Merck	71497	Sodium citrate monobasic
Cryostat	Leica	CM1510S
DAPI	Merck, Calbiochem	28718-90-3	Nuclear staining, Dilution: 1/1,000
DMEM	ThermoFisher Scientific	11995065	High glucose, pyruvate
donkey anti-goat	Biotium	20016 or 20106 or 20048	Dilution: 1/1,000
donkey anti-mouse	Biotium	20014 or 20105 or 20046	Dilution: 1/1,000
donkey anti-rabbit	Biotium	20015 or 20098 or 20047	Dilution: 1/1,000
EGF	Peprotech	315-09
FGF-2 (or bFGF)	Peprotech	100-18B
goat anti-GFAP	Abcam	ab53554	Dilution: 1/500
goat anti-SOX2	Santa Cruz Biotecnology	sc-17320	Dilution: 1/200
mouse anti-ID3	Santa Cruz Biotecnology	sc-56712	Dilution: 1/200
mouse anti-S100β	Sigma	S2532	Dilution: 1/200
Mowiol	Merck, Calbiochem	475904	Mounting medium
N2 supplement	ThermoFisher Scientific	17502048
Parafolmadehyde	Merck	158127
Poly-D-Lysine	Merck, Millipore	A-003-E	Solution, 1.0 mg/mL
rabbit anti-Doublecortin (DCX)	Abcam	ab18723	Dilution: 1/500
rabbit anti-PDGFRα	Abcam	ab51875	Dilution: 1/200
rabbit anti-β- catenin	Abcam	ab16051	Dilution: 1/500
Triton X-100	Merck	X100
Microscopy and image analysis
Confocal microscope	Leica	SP6 and SP8
Image analysis	NIH, USA	ImageJ
Image analysis	Leica	LasX