Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

Summary

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

Abstract

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Introduction

The defining characteristic of vertebrates is the vertebral column or spine, in which the notochord has been replaced by a sequence of segmented bones called vertebrae, divided by intervertebral discs. This succession of osseous material shapes the spinal canal, a cavity that encloses and protects the spinal cord¹. In the genus Rodentia, the spine is usually formed by seven cervical vertebrae, thirteen thoracic vertebrae, six lumbar vertebrae, and a variable number of caudal vertebrae^2,3. The length of the spinal cord is similar to that of the spine, and the terminal filum is a non-nervous structure that anchors the spinal cord to the sacrum. Additionally, nerve fibers exit through the intervertebral foramen¹.

The development and proper function of the central nervous system in mammals critically depend on the activity of the nervous system's resident macrophages, called microglia⁴. Although microglia were initially described as brain resident phagocytes, recent research has attributed many dynamic functions to these cells^5,6. Microglia's size ranges from 7 to 10 µm in homeostasis; they are considered among the most versatile cells in the body and can adapt morphologically and functionally to their constantly changing environment⁷. These cells exhibit high heterogeneity during both the embryonic and adult stages^8,9, while in the adult stage, they also display complex functional heterogeneity based on their spatiotemporal context¹⁰. The heterogeneity and multiple functions of microglia allow for differential gene expression and behavior in the spinal cord and brain. It has been shown that CD11b, CD45, CD86, and CCR9 expression is higher in the spinal cord compared to the brain^8,9.

Multiple protocols exist for cerebral microglia isolation^11,12; however, only a few exist for spinal cord microglia^13,14. Optimizing a method for purifying microglia from the spinal cord facilitates the development of multiple studies focused on discovering microglia physiology. This protocol aims to describe a simple and highly reproducible extraction of the mouse spinal cord and the purification of microglia (Figure 1).

Protocol

The study was conducted in accordance with the official Mexican standard NOM-062-ZOO-1999 and the guide for the care and use of laboratory animals. Approval for the study was obtained from the Research, Ethics, and Biosafety Committees of the Mexico Children's Hospital (HIM/2023/006) and the Research and Bioethics Committee of the General Hospital of Mexico Eduardo Liceaga (DI/21/501/04/62). Three C57BL/6 mice aged 6 to 8 weeks were obtained from the Mexico Children's Hospital, where they were raised under isolated conditions in ventilated racks, in compliance with institutional animal care and use guidelines.

1. Preparation of materials and reagents

1. Pre-warm Ca²⁺ and Mg²⁺ free PBS as well as Hank's balanced salt solution (HBSS) to 37 °C.
2. Supplement the HBSS solution with 100 µg/mL of Liberase and 4 µg/mL of DNase (HBSS-LD). Ensure that the solution maintains a physiological pH of 7.4.
   NOTE: 1 mL of working solution per spinal cord will be necessary.
3. Prepare an isotonic 90% solution for density gradient centrifugation (commercially available, see Table of Materials) and prewarm it to 37 °C.
4. Prepare the staining buffer by combining 5% heat-inactivated fetal bovine serum (FBS) with PBS, and store the mixture on ice.
5. Pre-warm Dulbecco's Modified Eagle's Medium-high glucose (1g/L) (DMEM) to 37 °C.
6. Supplement the DMEM medium with 10% FBS, 2 mM L-glutamine, 200 U/mL penicillin, 200 µg/mL of streptomycin, and 500 pg/mL of amphotericin B (see Table of Materials) to prepare DMEM-S.

2. Dissection and preparation of the spinal cord

1. Euthanize the mouse with an intraperitoneal overdose of pentobarbital at a dose of 150 mg/kg. Sterilize the mouse's thorax and back using 70% ethanol.
   NOTE: Confirm euthanasia was successful before proceeding with the mouse dissection.
2. Shave the thoracolumbar region and secure the extremities to the dissection board using adhesive tape.
3. Using a scalpel, make a careful incision along the dorsal midline from the occipital to the sacral region (Figure 2).
4. With the assistance of a stereoscopic surgical microscope, dissect in layers until reaching the lumbar spine and identifying the last costal arch.
   NOTE: The 13 costal arches are articulated in the thoracic vertebrae; when dissected, they become visible superior to the L1 vertebra².
5. Determine the last cervical vertebra and the sacrum, then make a cut to separate the spine (Figure 2).
6. Utilizing dissecting forceps, perform a dorsal laminectomy of the vertebrae to extract the spinal cord (Figure 3A-D).
   NOTE: Except for the first two cervical vertebrae (atlas and axis), all movable vertebrae share a common morphological design in various regions (cervical, thoracic, or lumbar). Each typical movable vertebra features a cylindrical vertebral body at its anterior end. Attached to the back of the body is a bony arch known as the vertebral arch (neural arch), consisting of laminae and spinous processes. Between these structures lies the vertebral foramen¹⁵.
7. Remove the nerves emerging through the foramina (dorsal and ventral roots) that constitute the peripheral nervous system and may contain infiltrated macrophages (Figure 3D).
   NOTE: It is unnecessary to remove the pia mater or meninges as the density gradient medium effectively eliminates contaminating astrocytes. Additionally, retaining the meninges reduces purification time and enhances viability.

3. Tissue digestion and isolation of microglia

1. Place the spinal cord on the dissecting board, and using Vannas scissors, cut the spinal cord into 2 mm pieces, yielding an average of 10 pieces.
2. Transfer the sections of the spinal cord to a 2 mL flat-bottom microtube (Figure 3E,F).
3. Add 1 mL of the HBSS-LD working solution (step 1.2). Incubate at 37 °C for 25 min, vigorously vortexing every 5 min.
4. Pass the contents of each digestion through a 40 µm strainer, and then add 7 mL of HBSS with 2% FBS to neutralize the digestion.
5. Crush the remaining tissue using a 1 mL syringe plunger. Centrifuge the suspension in a 50 mL conical tube for 5 min at 220 x g, 4 °C.
6. Discard the supernatant using a 1 mL micropipette, and resuspend the pellet in 2 mL of HBSS with 2% FBS in a 15 mL conical tube. Combine with 2 mL of 90% isotonic density gradient medium. Centrifuge for 25 min at 160 x g, 18 °C.
   NOTE: Utilize slow acceleration and avoid using the brake.
7. Use a transfer pipette to retrieve the interface and transfer it to a 15 mL conical tube. Wash the cells with 10 mL of PBS and centrifuge for 5 min at 220 x g, 4 °C.
8. Resuspend the cells in DMEM-S (step 1.6) and keep them stored on ice.

4. Determining purity/viability

1. For quantifying viable cells using a hemocytometer chamber, employ a 0.4% Trypan blue stain (see Table of Materials).
   NOTE: On average, a single mouse provides 4-6 million cells.
2. To quantify viable cells via a flow cytometer, employ commercially available amine-reactive fluorescent dye staining (see Table of Materials).
   1. Transfer 1 million cells into a 5 mL polystyrene round-bottom (FACS) tube. Dilute the fluorescent dye with PBS at a ratio of 1:1000 (creating the viability working solution).
   2. Incubate the samples with the viability solution in the dark at 4 °C for 30 min. Wash the cells with ice-cold PBS twice. Resuspend the cells in 400 µL of staining buffer (step 1.4).
   3. Block the cells with 2.4G2 anti-FcRIII/I (see Table of Materials) in the ice-cold staining buffer for 15 min at 4 °C.
   4. Formulate the antibody staining cocktail by adding the fluorescent-labeled monoclonal antibodies CD45-PerCP and CD11b-eFluor 450 to the staining buffer (at a dilution of 1:300) (see Table of Materials).
      NOTE: While both techniques are available, choose the one that best aligns with the subsequent protocol.
   5. Incubate the samples with the antibody staining cocktail in the dark at 4 °C for 30 min. Wash the cells with ice-cold staining buffer twice. Resuspend the cells in 400 µL of staining buffer.
   6. Acquire 250,000 events on a flow cytometer, as outlined by the gating strategy in step 6 (Figure 4).

5. Immunofluorescent staining

1. Place a pretreated coverslip into a well of a 6-well plate.
   NOTE: To treat coverslips, place them in a Petri dish with 500 µL of L-polylysine (0.01 mg/mL, see Table of Materials) solution and incubate for 1 h. Afterward, wash them three times with distilled water and allow them to dry.
2. Seed 200,000 cells onto the treated coverslip using 500 µL of DMEM-S solution. Incubate for 24 h.
3. Remove the coverslips and fix the cells using 3.7% paraformaldehyde (PFA) for 20 min at room temperature. Wash the coverslips three times with ice-cold PBS.
4. Permeabilize the cells with 0.2% Triton X-100 in PBS for 15 min at room temperature. Subsequently, wash the coverslips three times with cold PBS. Block the cells with 0.2% BSA in PBS for 1 h at room temperature.
5. Dilute the fluorescence-labeled monoclonal antibodies (at a 1:300 dilution, see Table of Materials) in the blocking solution and incubate for 1 h at room temperature. Wash the coverslips three times with ice-cold PBS.
6. Apply mounting medium containing DAPI onto the slide, and then seal it with a coverslip.
   NOTE: The slides can be immediately analyzed using a fluorescence or confocal microscope, or they can be stored for up to 3 months at -20°C.

6. Gating strategy for spinal cord microglia

1. Generate a dot plot and establish a gate to isolate viable cells based on viability staining and an empty fluorescent channel¹⁶. Exclude non-viable cells.
2. Produce another plot utilizing forward and side scatter parameters to eliminate debris¹⁶.
3. Develop an additional dot plot and analyze the expression of CD11b and CD45 to differentiate microglia.

Representative Results

Utilizing mouse spinal cord tissue, enzymatic digestion was performed using a mixture highly enriched with collagenase and thermolysin. The resulting digested tissue underwent passage through a 40 µm filter to eliminate undigested material. The collected cells were enriched through a Percoll density gradient, with 90% in the lower portion and 45% in the upper portion. The microglia-enriched cells within the interface were then stained with CD45 and CD11b antibodies and subjected to flow cytometric analysis (Figure 1).

The dissection of the vertebral column extending from the occipital region to the sacrum, involves the exposure and dissection of the paravertebral musculature. To release the vertebral column, the costal arches were exposed, and their measurement was performed from vertebra T13 to T1. The vertebral column, articulating with the skull and sacrum, became visible, and a cut was executed on each side to liberate it completely (Figure 2).

The extraction of the spinal cord was achieved through a lateral incision into the vertebral bodies, forming a canal. During the removal of the spinal cord, the peripheral nerves were severed to prevent contamination from peripheral macrophages. Subsequently, the obtained spinal cord was spread across the dissection table and sectioned into 2 mm pieces, which were added to the digestion solution (Figure 3). The use of liberase for spinal cord digestion is reported for the first time, yielding 4 to 6 million highly viable microglial cells per adult mouse. The optimal viability achieved through this process ranges from 80% to 99%.

To confirm the purification process, FACS staining of microglia was performed using CD45 and CD11b antibodies. The purification process demonstrated efficiency, yielding up to 99% microglial cells (Figure 4). Furthermore, immunofluorescence staining was conducted on the obtained microglia using the same antibodies, observing double-positive cells with an average size of 10 µm - consistent with the reported size of non-activated microglia. This suggests that these cells are suitable for activation studies (Figure 4E). It is plausible that other immune cells may be present during inflammatory processes or that microglia might undergo morphological changes under different stimuli. In such cases, the incorporation of specific markers for the identification of each cell population is recommended.

Figure 1: Schematic overview of the spinal cord microglia purification protocol. Please click here to view a larger version of this figure.

Figure 2: Spinal cord dissection procedure. Following euthanasia, the mouse's spinal column was isolated and dissected. (A) Stepwise skin dissection extending from the occipital to caudal regions, revealing the para-vertebral musculature. (B) Sequential dissection through the layers of paravertebral muscles. (C) Removal of the spine following rib cutting. (D) Visualization of an intact spinal cord within the vertebral canal. Please click here to view a larger version of this figure.

Figure 3: Spinal cord extraction and digestion process. (A) Sequential spinal laminectomy involving two approximately 2 mm cuts. (B) Exposed spinal cord. (C) Removal of the spinal cord. (D) Peripheral nerve sectioning. (E) Spinal cord was divided into 2 mm sections and placed in the digestion medium. (F) Standard 37 °C incubation for tissue digestion. Please click here to view a larger version of this figure.

Figure 4: Assessment of cell purity and viability. Immunostaining of purified cells using microglia-specific markers. (A) Identification of live cells (fluorescent dye-negative cells). (B) Cell size-based selection. (C) CD45low and CD11b+ cells, demonstrating purity exceeding 99%. (D) Average viability (87.46 ± SD 1.468) and average purity (88.51 ± SD 3.948; n = 5). (E) Immunofluorescence depicting CD45+ and CD11b+ staining in purified cells. Images were captured using a confocal microscope with a white light laser at 60x magnification (scale bar = 5 µm). Please click here to view a larger version of this figure.

Discussion

Numerous protocols have been developed for the study of microglia due to their significance in brain homeostasis. In these methods, microglia are typically sourced from the cerebral hemispheres of embryonic or neonatal rats and mice¹⁷. A limited number of studies have addressed the purification of microglia from the spinal cords of adult mice^13,14. These techniques involve enzymatic digestion using collagenase and/or papain along with DNAse, often combined with gradient separation using Percoll or OptiPrep. However, employing papain-based spinal cord digestion without gradient separation can lead to astrocyte contamination, even after the removal of the pia mater^14,17.

This study presents a protocol for purifying microglia from the adult mouse spinal cord. The protocol utilizes a precise mixture of collagenase I and II along with thermolysin, a combination known for efficiently digesting diverse tissues with controlled degradation¹⁸, thus reducing apoptosis due to its low content of neutral proteases. Meninges or the pia mater, often sources of astrocyte contamination, can be eliminated through the use of a Percoll gradient in this procedure, negating the necessity to remove these layers (Figure 1).

Microglial heterogeneity arises from distinct morphologies in the spinal cord and brain⁸. However, this protocol was also applied to cerebral hemispheres with similar outcomes (data not presented), highlighting its robustness for analyzing both spinal cord and cerebral microglia.

High levels of purity (ranging from 80% to 99%) and viability (85% to 98%) can be consistently achieved with this protocol. Processing cells from the nervous system is intricate, primarily because cell viability is substantially affected by processing duration; hypoxia exceeding 20 min can be lethal. For optimal viability, it is recommended to limit processing time from spinal cord extraction to digestion to no more than 10 min. This description is tailored to healthy mice aged 8 to 10 weeks, necessitating further standardization for younger or older mice. Suboptimal viability implies over-digestion of tissue, mandating repetition. Alternatively, FACS sorting can salvage cells from over-digested samples.

The application of liberase for digesting and purifying spinal cord microglia is novel. Its limited usage in nervous system tissues has historically been due to viability challenges^18,19. This study, however, demonstrates heightened viability when using this meticulously purified collagenase. Alternatively, other collagenases with lower potency could be explored.

Several crucial considerations must be acknowledged, such as the presence of neuroinflammation, blood-brain barrier compromise, and mouse age. These factors alter both microglial morphology and frequency. In instances of neuroinflammation or blood-brain barrier disruption, immune cells expressing CD45 and CD11b can infiltrate, diminishing microglial purity. As microglia are extensively explored in various neuroinflammatory conditions, this protocol holds potential for pathological and neurophysiological investigations.

Materials

Name	Company	Catalog Number	Comments
15 mL collection tubes	Corning, USA	430790
2 mL microtubes	Axygen, USA	MCT-200-G
2.4G2 anti-FcR	BioLegend, USA	101302
50 mL collection tubes	Corning, USA	430829
70% ethanol
Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin b)	Gibco, USA	15240062
Antibody CD11b eFluor 450 anti-mouse	eBioscience, USA	48-0112
Antibody CD45 PerCP anti-mouse	BioLegend, USA	103130
Balanced salt solution (PBS) calcium- magnesium-free	Corning, USA	46-013-CM
Blue Cell Strainer 40 μm	Corning, USA	352340
Costar 6-well Clear Not Treated	Corning, USA	CLS3736
Coverslips
Digital Heating Shaking Drybath	Thermo Scientific Digital HS Drybath, USA	88870001
Dissecting forceps for microsurgery	FT by DUMONT
DNase	Roche, USA	4536282001
Dulbecco´s Modified Eagle´s Medium-high glucose (DMEM)	Merck, USA	D6429
Electric shaver
FACS tube	Thermo, USA	352058
Fetal bovine serum (FBS)	PAN Biotech, Alemania	P30-3306
Flow cytometer Cytoflex	Beckman Coulter
Hank’s balanced salt solution	Merck, USA	H2387
L-glutamine	Corning, USA	15393631
Liberase TM	Roche, USA	5401119001
Neubauer chamber Counting Chambers	China	1103
Pentobarbital
Percoll	Merck, USA	17089101	density gradient centrifugation
Poly-L-lysine solution	Merck, USA	P8920
Scalpel No. 25	HERGOM, Mexico	H23
Snaplock Microcentrifuge Tubes 2 mL	Axygen, USA	10011-680
Stereoscopic microscope	Velab, Mexico	HG927831
Straight surgical scissors (10 cm)	HERGOM, Mexico
Straight Vannas scissors	HERGOM, Mexico
Triton X100	Merck, USA	X100
Trypan blue Stain 0.4%	Merck, USA	15250-061
Vortex mixer	DLAB, China	8031102000
Zombie Aqua Fixable Viability Kit	BioLegend, USA	423102	amine-reactive fluorescent dye staining