Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

Podsumowanie

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca²⁺ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1^G93A rat model.

Streszczenie

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1^G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca²⁺ changes in video imaging experiments on live cells.

The examples of video recordings consist of: (1) cases of Ca²⁺ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1^G93A astrocytes compared to non-transgenic controls; (2) Ca²⁺ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca²⁺ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca²⁺ store operation in hSOD1^G93A and in non-transgenic astrocytes; (3) Ca²⁺ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca²⁺ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca²⁺ dye and dye-loading techniques.

Wprowadzenie

Cell culture techniques have given rise to numerous advancements in diverse fields of cellular neurophysiology in health and disease. Particularly, primary cell cultures, freshly isolated from the neuronal tissue of a lab animal, allow the experimenter to closely study the behavior of diverse cells in different biochemical media and physiological setups. Using different fluorescent physiological indicators such as the Ca²⁺-sensitive dyes in combination with time-lapse video microscopy provides better insight into the cellular biophysical and biochemical processes in real time.

ALS is a devastating neurodegenerative disease that affects upper and lower motor neurons¹. The disease has a complex pathogenesis of the familial type but mostly of the sporadic form (90% of cases)². It is well known that non-cell autonomous mechanisms contribute to ALS pathophysiology, primarily due to the essential role of glial cells³. ALS is also well characterized as a neuroinflammatory disease with involvement of humoral and cellular factors of inflammation.

Immunoglobulin G is widely used as a molecular marker in ALS and other neurodegenerative diseases. Studying the serum level of this marker can indicate the presence and stage of neuroinflammation in the disease^4,5,6, while its presence in the cerebrospinal fluid can indicate a breach of the blood brain barrier⁷. IgGs were also identified as deposits in the spinal cord motor neurons of ALS patients⁷. Nevertheless, this approach has shown some inconsistencies in the correlation of the level of IgGs with the stage and characteristics of the disease⁶.

IgG isolated from the sera of ALS patients (ALS IgG) can induce a calcium response in naive astrocytes⁸ and glutamate release in neurons, pointing to an excitotoxic effect-a hallmark of ALS pathology⁹. However, studies on the hSOD1^G93A ALS rat model (containing multiple copies of the human SOD1 mutation¹⁰) showed a number of markers of oxidative stress in cultured neuroglial cells¹¹, tissues^12,13,14, or live animals¹³. It is noteworthy that the astrocytes cultured from the ALS rat model were more prone to oxidative stress induced by peroxide than the astroglia from non-transgenic littermates¹¹.

Microglial cells in culture are affected by ALS IgG in a less apparent way. Namely, a BV-2 microglial cell line displayed a rise in the signal from fluorescent markers of oxidative stress in response to the application of only 4/11 ALS IgG patient samples¹⁵. It is well known that microglia participate in many neuroinflammatory pathologies, adding to oxidative stress and late progression phase in the non-cell autonomous mechanism of ALS^16,17. Nevertheless, the data with ALS IgGs indicated that these cells may not be as reactive as astrocytes to these humoral factors of ALS inflammation. Several studies have been conducted with primary astrocytes from ALS murine models, not only in pups but also in symptomatic animals, either on the brain or on the spinal cord^18,19,20,21. This is also true for microglial primary cultures, although to a lesser extent than astrocytes and mostly from brain regions at the embryonic stage^22,23,24.

We use time-lapse video imaging of Ca²⁺ on cells in culture primarily as a means to follow intracellular transients of this ion as a physiological marker of excitotoxicity. Thus, by biophysical characterization of these transients (amplitude, area under transient, rise-time, frequency) the researcher can obtain experimental diagnostic parameters from diverse cellular models of neurodegeneration. This technique thus offers an advantage of a quantitative physiological assessment of IgGs as disease biomarkers. There is a large body of literature on the role of IgGs and Ca²⁺ in the induction of ALS. Most of these studies were performed by inducing ALS by injecting patient IgGs into experimental animals^{25,26,27,28,29}, which then showed intracellular Ca²⁺ elevation and IgG depositions. A line of studies explored the effect of ALS IgGs on the motor synapse in vitro^30,31,32. In the above context, the technique presented here puts the focus on the glial cells as important players in the non-cell autonomous mechanism of ALS and quantifies their potential excitotoxic response to IgGs as humoral factors of neuroinflammation. This approach may have a wider application in testing other humoral factors such as whole sera, CSF, or cytokines in different cell culture systems and in cellular models of general inflammation.

This paper describes how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to further use these cells to study ALS pathophysiology with patient sera-derived IgG. Protocols are detailed for the dye-loading of cultured cells (Figure 1) and the recordings of Ca²⁺ changes in time-lapse video imaging experiments. Examples of video recordings will show how glial cells react to ALS IgG as compared to ATP, the latter activating purinergic membrane receptors. Shown for the first time is an example on how astrocytes isolated from the hSOD1^G93A ALS rat brain react with a more pronounced Ca²⁺ response to ALS IgG compared to non-transgenic controls and how to relate this process to the differences in Ca²⁺ store operation. Also shown is an example of calcium imaging in microglial cells acutely challenged with ALS IgG, with only a modest response of intracellular calcium.

Protokół

All experiments were performed in accordance with the EU directives on the protection of animals for scientific purposes and with permission from the Ethical Commission of the Faculty of Biology, University of Belgrade (approval number EK-BF-2016/08). Regarding patient material (sera for IgGs), it was collected for routine clinical examination with informed patient's consent in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The protocol was approved by the Ethics committee of the Clinical Center of Serbia (No. 850/6).

1. Primary cell culture preparation

1. Brain tissue isolation
   NOTE: Isolation should be performed on ice, using ice cold solutions.
   1. Use newborn pups 1-3 days old for primary neonatal cell culture³³.
      NOTE: For the study presented here, Sprague Dawley hSOD1^G93A and non-transgenic rats were used. For genotyping, rat tails were used for later DNA extraction and PCR.
   2. Sprinkle the pup's head with 70% ethanol and immediately decapitate it fast using scissors.
   3. Cut open the skin using small-angled scissors to expose the skull. Open the skull by making a cut from the foramen magnum toward the orbits. Next, make a perpendicular midline cut. Remove the brain from the skull and place it in a Petri dish containing phosphate-buffered saline (PBS).
      NOTE: Clean the tools between pups to prevent cross-contamination between transgenic and non-transgenic pups. Isolation of the cortices from the rest of the brain should be done under a stereomicroscope.
   4. Using the tip of the forceps, tear the connections between both hemispheres. Then, separate the hemispheres with curved forceps by gently pushing a hemisphere from the center to the side.
   5. Remove the meninges by carefully tearing them with straight and curved forceps.
   6. Remove the hippocampus by pinching it with a curved forceps. Discard the hippocampus or use it for another cell culture preparation.
      NOTE: Further steps should be performed under the laminar flow hood to ensure sterile conditions.
2. Tissue homogenization and dissociation
   1. Transfer one cortex into a 15 mL tube filled with 3 mL of cold PBS. Pipet the suspension up and down with a 1 mL tip, completing 10-15 strokes until the suspension becomes homogeneous.
      NOTE: Be very cautious not to produce bubbles in this process.
   2. Centrifuge at 500 × g for 5 min. Remove the supernatant and resuspend the pellet in 3 mL of cold PBS by pipetting it up and down with a 1 mL tip. Repeat the centrifugation step.
      NOTE: All solutions used from this point should be prewarmed to 37 °C.
   3. Discard the supernatant and resuspend the pellet in 2 mL of complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Transfer the homogenate to a 2 mL tube.
   4. Pass the homogenate through 21 G and 23 G needles (three times each) to make a suspension of single cells.
      NOTE: Be very cautious not to produce bubbles in this process.
3. Pour the cell suspension prepared from one cortex into a 60 mm diameter Petri dish or a T25 flask (with the surface pretreated with a polypeptide coating for the growth of adherent cells) containing 3 mL of complete DMEM. Lightly shake the Petri dish so that the suspension is uniformly distributed.
4. Grow the cells in the incubator in a humidified atmosphere of 5% CO₂/95% air at 37 °C.
5. Change the medium 48 h after isolation and replace the medium every 3 days.
6. To promote astrocyte growth, when cells reach 70%-80% confluency, wash them with prewarmed PBS to remove the loosely attached glial cells and traces of FBS in the medium.
   1. Trypsinize the underlying layer of astrocytes by adding 1 mL of prewarmed trypsin solution (0.25% trypsin, 0.02% EDTA in PBS, sterile-filtered).
   2. Place the dish in the incubator at 37 °C for 2-5 min.
   3. Check the cells under the microscope. When they start to detach, add 4 mL of complete DMEM.
   4. Collect the cell suspension and transfer it to a 15 mL tube. Centrifuge at 500 × g for 5 min.
   5. Discard the supernatant and resuspend the pellet in 1 mL of complete medium. Count the cells using a hemocytometer.
   6. Replate the cells at a density of 10⁴ cells/cm² in 5 mL of fresh complete DMEM.
7. Change the medium every 3 days. To minimize the presence of other glial cell types, after astrocytes reach 50% confluence and prior to each media replacement, wash the cells with complete DMEM.
   1. Aspirate the supernatant medium with a 1 mL pipette and gently dispense it onto the layer of cells several times. Ensure that the whole surface of the cell layer is covered during this washing step.
8. Once the cells reach 80% confluence (usually after 14 days), repeat the trypsinization and the collection of astrocytes as described in step 1.6.
9. Seed 5 × 10³ of astrocytes on a 7 mm circular glass coverslip coated with poly-L-lysine (50 µg/mL). Use them in experiments after 48 h.
10. To promote microglia, after step 1.7, allow the glial cells to reach a confluent layer³⁴. When microglial cells appear on top of the astrocyte layer (after 10-15 days, recognized by their smaller, more oval bodies and shorter processes), shake the Petri dish on an orbital shaker for 2 h at 220 rpm.
11. Dispersing and seeding microglial cells
    1. Lightly wash the detached and loosely attached cells by aspirating the supernatant medium with a 1 mL pipette tip and gently dispense it onto the layer of cells several times. Be sure to cover the whole surface of the cell layer during this washing step, collect the medium with the detached cells, and transfer it to a 15 mL tube.
    2. Centrifuge at 500 × g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of medium.
    3. Pass the cell suspension through a 21 G needle to obtain a single-cell suspension.
       NOTE: Most published methods use trypsin or papain to dissociate the tissue; however, 21 G needles have the advantage of preventing the overdigestion of cells, allowing for a gentler dissociation.
    4. Count the cells using a hemocytometer. Seed 5 × 10³ microglial cells on a 7 mm circular glass coverslip coated with poly-L-lysine (50 µg/mL). Use them in experiments after 48 h.
       ​NOTE: It is difficult to obtain a pure microglial culture by shaking, since the oligodendrocyte precursor cells and astrocytes may still be present in a variable number depending on the experimenter's experience. The immunocytochemical protocol used to estimate the presence of different cell populations in glial cultures has been described elsewhere³⁵.

2. Immunocytochemistry

1. Rinse the cells plated on coverslips in PBS, 2 x 1 min.
2. Fix the cells in 4% paraformaldehyde for 20 min at room temperature (RT).
3. Wash in PBS 3 x 5 min.
4. Incubate in a blocking solution containing 10% normal donkey serum (NDS)/1% bovine serum albumin (BSA)/0.1% Triton X-100 for 45 min at RT.
   1. Add 50 µL of a blocking solution per 10 mm diameter coverslip.
5. Prepare primary antibodies in 1% NDS/1% BSA/0.1% TritonX-100 in the following dilutions: mouse anti-GFAP 1:300, goat anti-Iba1 1:500. Incubate the cells with primary antibodies overnight at +4 °C.
6. Rinse in PBS, 3 x 10 min.
7. Incubate with the secondary antibodies conjugated with a fluorophore: donkey anti-mouse (excitation 488 nm [1:200)) or donkey anti-goat (excited at 647 nm [1:200)). Incubate the cells for 2 h at RT in the dark.
8. Wash in PBS, 7 x 5 min.
9. Incubate the cells with 4',6-diamidino-2-phenylindole (DAPI, 1:4,000) for 10 min at RT.
10. Wash in PBS 5 x 5 min.
11. Mount the coverslips on microscope slides using a mounting solution; use one drop of it per coverslip.
    NOTE: Antibody dilutions may vary depending on the producer. Users must determine optimal dilutions for their experiments.

3. Time-lapse video imaging

NOTE: Solutions containing the fluorescent dye should be protected from direct light. Before starting the imaging experiment, make sure that the glass coverslip does not move when turning on the perfusion.

1. Preparation of solutions and cells
   1. Prepare extracellular solution (ECS) for imaging: 140 mM NaCl, 5 mM KCl, 2 mM CaCal₂, 2 mM MgCl₂, 10 mM D-glucose, and 10 mM HEPES. Adjust the pH to 7.4. Ensure that the osmolarity is 280-300 mOsm/kg and prepare ECS without Ca²⁺: 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 10 mM D-glucose, 10 mM HEPES, and 0.1 mM EGTA.
   2. Prepare the test solutions: 100 µM ATP dissolved in ECS, 1 µM Thg in ECS without Ca²⁺, and 0.1 mg/mL ALS IgG in ECS⁸.
   3. Transfer one coverslip to a dish with ECS to wash out the complete DMEM. Prepare the dye loading solution by diluting 1 mM stock solution of Fluo-4 AM with ECS to the final concentration of 5 µM Fluo-4 AM.
      1. Place the coverslip with cells in the dye loading solution for 30 min at RT in the dark. Wash the cells in ECS for 20 min.
   4. If cells are not adequately loaded (i.e., if the basal level of fluorescence is too low-<5% of the dynamic range of the camera with exposure time greater than 400 ms), increase the loading time to up to 45 min to 1 h at 37 °C. Alternatively, mix the Fluo-4 AM stock solution with an equal volume of 20% (w/v) detergent (Pluronic) in DMSO before diluting in ECS, making the final Pluronic concentration ~0.02%.
      NOTE: The experimental examples of this study were performed with human IgG isolated from ALS patients' blood sera as described previously^4,5,11. Each experiment was performed with IgG samples of a single patient.
2. Video imaging
   NOTE: In this protocol, the Video Imaging System was combined with a xenon short arc lamp, a polychromator system, and an inverted epifluorescence microscope equipped with water, glycerin, and oil immersion objective. Timelapse imaging was performed using a Digital Camera System.
   1. Switch on the components of the imaging setup 15 min before the experiment to allow the system to reach the working temperature.
   2. Place the coverslip in the recording chamber with 1 mL of working solution (ECS or ECS without Ca²⁺).
   3. To choose the correct imaging protocol, open the imaging software and in the Acquisition panel, choose the filter pairs for Fluo4-AM with an excitation wavelength at 480 nm, a dichroic mirror at 505 nm, and an emission wavelength at 535 nm.
   4. Choose the field of view taking care to have a consistent number of cells throughout the experiment. Adjust the exposure time and detector gain appropriately, in such a manner that the signal is not saturated. Choose the pseudocolor mode for acquisition. For more detailed instructions, refer to the manual provided by the manufacturer.
   5. Adjust the sampling rate to 1 Hz (1 frame per second).
   6. Start the imaging by acquiring the basal level of fluorescence for 3-5 min for the baseline determination (F₀). Ensure a constant flow of the working solution.
   7. Stop the flow of the working solution and switch to the test solution for the desired length of time (also see time bars in examples of Figure 2 and Figure 3). Between each test solution, wash the cells with a constant flow of the working solution for 3-5 min.
   8. Keep the solution volume in the recording chamber at ~1 mL by arranging a constant suction from the top of the solution (see Figure 2E).
      ​NOTE: To apply the treatment directly to the imaged cells, a customized delivery system made of a glass pipette (0.8 mm inner diameter) was positioned ~350 µm away and ~1 mm above the cells, at an angle of 45° combined with the solution exchange system containing pinch valves and an electronic valve controller (see Figure 2E).

4. Data analysis

1. Define a region of interest (ROI) that corresponds to the individual cell.
   1. Choose the frame with the highest signal intensity (i.e., from the application of ATP) and encircle one cell by using the application Polygonal Tool. Repeat for all the cells in the acquired field. Choose five ROIs in the background using the Circle Tool.
   2. To measure the mean signal intensity of a single cell and the background for each time frame, select all ROIs and use the Multi Measure command in ROI Manager in ImageJ or any equivalent command in commercial software (see the Table of Materials).
2. Export the mean signal intensity values of the ROIs as a spreadsheet.
3. Average five background ROIs and subtract the averaged background of each frame from the mean ROI intensity of the frame acquired at the same time.
4. To normalize the obtained data to the baseline signal, use equation (1):
   ΔF/F₀ = (F - F₀)/F₀   (1)
   where F is the signal intensity of each time frame after background subtraction, and F₀ is the baseline Fluo-4 fluorescence.
   NOTE: A custom-made code was used in this study.
5. To analyze calcium activity, determine several parameters⁴: the amplitude of the calcium peak (ΔF/F₀), the integrated change (time integral, surface under the response recording) of the calcium transient (ΔF/F₀ × s), time-to-peak-elapsed time from the onset of stimulation to the maximum of calcium transient (s), rise time-time elapsed from the onset of the stimulation to the value of ΔF/F₀ that reaches 50%-80% of the maximal amplitude (s), half-width-full width of a transient at half-maximal amplitude (s), frequency of responses if they are repetitive (Hz).

Wyniki

Characterization of different glial cell types in culture
It usually takes 15-21 days to produce astrocytes for experiments, while microglial cells take 10-15 days to grow. Immunostaining was performed to assess the cell purity of the culture. Figure 1 shows the expression of double labeling of the astrocytic marker GFAP and the microglial marker Iba1 in respective cultures.

Calcium imaging is known to reveal the differences in cell physiolo...

Dyskusje

This paper presents the method of primary cell culturing as a fast and "on the budget" tool for studying different aspects of cell (patho)physiology such as ALS in the rat hSOD1^G93A model. The technique is thus suitable for studies at the single-cell level that can be extrapolated and further investigated at a higher level of organization (i.e., in tissue slices or in a live animal). Cell culturing as a technique, however, has a few caveats. It is most critical to do the brain tissue isolation and the ...

Materiały

Name	Company	Catalog Number	Comments
15 mL tube	Sarstedt, Germany	62 554 502
2 mL tube	Sarstedt, Germany	72.691
21 G needle	Nipro, Japan	HN-2138-ET
23 G needle	Nipro, Japan	HN-2338-ET
5 mL syringe	Nipro, Japan	SY3-5SC-EC
6 mm circular glass coverslip	Menzel Glasser, Germany	630-2113
60 mm Petri dish	ThermoFisher Sientific, USA	130181
ATP	Sigma-Aldrich, Germany	A9062
AxioObserver A1	Carl Zeiss, Germany
Bovine serum albumine	Sigma-Aldrich, Germany	B6917
Calcium chloride	Sigma-Aldrich, Germany	2110
Centrifuge	Eppendorf, Germany
DAPI	Sigma-Aldrich, Germany	10236276001
D-glucose	Sigma-Aldrich, Germany	158968
DMEM	Sigma-Aldrich, Germany	D5648
Donkey-anti goat AlexaFluor 647 IgG antibody	Invitrogen, USA	A-21447
Donkey-anti mouse AlexaFluor 488 IgG antibody	Invitrogen, USA	A-21202
EDTA	Sigma-Aldrich, Germany	EDS-100G
EGTA	Sigma-Aldrich, Germany	E4378
”evolve”-EM 512 Digital Camera System	Photometrics, USA
Fetal bovine serum (FBS)	Gibco, ThermoFisher Scientific, USA	10500064
Fiji ImageJ Software	Open source under the GNU General Public Licence
FITC filter set	Chroma Technology Inc., USA
Fluo-4 AM	Molecular Probes, USA	F14201
Goat anti-Iba1	Fujifilm Wako Chemicals, USA	011-27991
HEPES	Biowest, France	P5455
HighSpeed Solution Exchange System	ALA Scientific Instruments, USA
Incubator	Memmert GmbH + Co. KG, Germany
Magnesium chloride	Sigma-Aldrich, Germany	M2393
Matlab software	Math Works, USA
Mouse anti-GFAP	Merck Millipore, USA	MAB360
Mowiol 40-88	Sigma-Aldrich, Germany	324590
Normal donkey serum	Sigma-Aldrich, Germany	D9663
Paraformaldehyde	Sigma-Aldrich, Germany	158127
Penicilin and Streptomycin	ThermoFisher Sientific, USA	15140122
Poly-L-lysine	Sigma-Aldrich, Germany	P5899
Potassium chloride	Sigma-Aldrich, Germany	P5405
Potassium dihydrogen phosphate	Carlo Erba Reagents, Spain	471686
Shaker DELFIA PlateShake	PerkinElmer Life Sciencies, USA
Sodium bicarbonate	Sigma-Aldrich, Germany	S3817
Sodium chloride	Sigma-Aldrich, Germany	S5886
Sodium phosphate dibasic heptahydrate	Carl ROTH GmbH	X987.2
Sodium pyruvate	Sigma-Aldrich, Germany	P5280
Thapsigargine	Tocris Bioscience, UK	1138
Triton X - 100	Sigma-Aldrich, Germany	T8787
Trypsin	Sigma-Aldrich, Germany	T4799
Vapro Vapor Pressure Osmometer 5520	Wescor, ELITechGroup Inc., USA
ViiFluor Imaging System	Visitron System Gmbh, Germany
VisiChrome Polychromator System	Visitron System Gmbh, Germany
VisiView high performance setup	Visitron System Gmbh, Germany
Xenon Short Arc lamp	Ushio, Japan