Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Podsumowanie

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Streszczenie

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Wprowadzenie

In vitro modeling is an essential tool in biomedical research. It allows investigators to study and tightly control specific mechanisms in restricted cell types, or in isolated systems/organs. Organotypic slice culture is a widely used in vitro technique, especially in the field of neuroscience¹. The method was first established by Gähwiler, who cultured brain slices using the roller tube technique², and later modified by Yamamoto et al., who introduced the use of a microporous membrane to perform cortical slice cultures³. Compared to primary cell cultures, organotypic slice cultures present the advantage of preserving the cytoarchitecture of the tissue, as well as native cell-cell connections in the plane of the tissue section.

Organotypic slices have been cultured from many parts of the central nervous system, such as the hippocampus⁴, cortex⁵, striatum⁶, cerebellum^4,7, and spinal cord^8,9, among others. They have been proven to be a powerful tool in drug discovery studies¹⁰. The effects of neuroactive molecules can be assessed in many ways: survival and neurodegeneration using immunostaining and biochemistry assays, neuronal circuit formation, or disruption using electrophysiology and live-imaging.

The goal of this work is to describe a simple method to perform organotypic cerebellar slice culture, which is known to be a relevant model to mimic cerebellar development in vitro. Particularly, we focused on the study of Purkinje cell developmental death. In vivo, Purkinje cells undergo apoptosis during the first postnatal week, peaking at postnatal day 3 (P3)¹¹. The same pattern is observed in cerebellar slice culture, with Purkinje neurons dying by apoptosis when cerebella are taken from animals between P1 and P8, with a peak at P3^4,12. The use of the organotypic cerebellar slice cultures has allowed to identify several neuroprotective molecules^7,13, as well as understanding part of the mechanisms involved in this programmed cell death^14,15,16. Here, we describe a protocol based upon the study of Stoppini et al.¹⁷ in hippocampus, and adapted to cerebellum by Dusart et al.⁴ It includes rapid dissection and chopping of postnatal cerebella; slicing culture onto a cell culture insert containing a microporous membrane, with or without neuroprotective treatment; and immunofluorescence staining to assess neuronal survival.

Protokół

All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.

1. Preparation prior to organotypic cerebellar slice cultures

1. In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture medium in a 250 mL bottle-top vacuum filter attached to a sterile bottle receiver. Add 100 mL of Basal Medium Eagle (BME), 50 mL of Hanks' Balanced Salt Solution (HBSS), 50 mL of heat-inactivated horse serum, 1 mL of 200 mM L-Glutamine (final concentration 1 mM), and 5 mL of 200 g/L glucose (final concentration 5 mg/mL). Vacuum-filter the culture medium and keep it at +4 °C for up to a month.
2. In the sterilized biosafety cabinet, take out a 6-well culture plate from its package. Fill each well with 1 mL of culture medium. If a treatment is tested, add the pharmacological agent to the treated well, and the same volume of vehicle solution for the control well. In the present study, we added 1 µL of sterile 3 M KCl solution to the treated well (30 mM final) and 1 µL of sterile water to the control wells.
3. Bring inside the cell culture hood the needed number of individually wrapped cell culture inserts with hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size = 0.4 µm). Carefully unwrap them and take out the cell culture inserts with sterile forceps. Drop them one by one in a well of a 6-well plate, avoiding bubbles at the interface between the insert membrane and the cell culture medium. Let the inserts equilibrate in the medium in a 37 °C, 5% CO₂ incubator for at least 2 h before culture.
   NOTE: The tissue chopper permanently resides in the cell culture hood.
4. Prepare two 200 mL beakers, one filled with 150 mL sterile water, and the other filled with 150 mL 70% ethanol. Dip the surgical tools in the 70% ethanol bath and then in water prior to using them.
   NOTE: Do not use surgical tools immediately following contact with ethanol.
5. Disinfect the double-edged blade in the 70% ethanol beaker. Place it on the blade holder using sterile forceps and hold it into place using the blade clamp wrench. Let it dry until use.
6. Disinfect the plastic disc provided with the tissue chopper in the 70% ethanol beaker. Spray the cutting table with 70% ethanol prior to placing the disc on it. Let dry until use.

2. Dissection and Cerebellar Organotypic Slice Cultures

1. Bring the mouse pup inside the cell culture hood to perform the dissection.
2. Decapitate the pup quickly using straight operating scissors (Figure 1A).
3. While holding the pup's head by the nose with straight dressing forceps, cut open the scalp using straight eye scissors, starting from the posterior end, and going lateral to midline.
4. Proceed identically for the skull.
   NOTE: Make sure to point scissor tips outwards to avoid damaging the cerebellum during dissection.
5. Take out the brain and transfer it to a 60-mm dish filled with cold HBSS + 5 mg/mL glucose.
6. Carefully dissect out the cerebellum using sterile straight fine forceps (Figure 1B). Transfer it onto the plastic disc placed on the cutting table of the tissue chopper using sterile curved fine forceps.
7. Orient the tissue by rotating the cutting table to perform parasagittal sections.
8. Move the cutting table by pulling the table release knob to the right, so the blade is positioned at the edge of the tissue.
9. Adjust the slice thickness to 350 µm and the blade speed to medium.
10. Start the chopper. Once the whole cerebellum has been sliced (Figure 1C), turn off the chopper.
11. Remove the plastic disc with sterile forceps.
12. Carefully bring the plastic disc close to a 60-mm dish containing HBSS + 5 mg/mL glucose. Pipette cold HBSS + 5 mg/mL glucose onto the tissue using a sterile transfer pipette to allow harvesting of cerebellar slices in the buffer-filled dish.
13. Separate the cerebellar slices from each other using a microprobe. Take out good quality sections with the transfer pipette (it is recommended to use tissue sections that are close to the vermis) and drop them off in a pre-equilibrated cell culture insert (Figure 1D).
14. Position the cerebellar slices on the cell culture insert using the microprobe, then carefully remove the excess HBSS + 5 mg/mL glucose solution with the transfer pipette.
    NOTE: At this stage the tissue is extremely tender and prone to physical damage. The maximum number of cerebellar slices per cell culture insert depends on the age of the donor animal. 6–8 slices can be cultured for a P0–P4-old animal, and 4–6 slices for animals older than P5.
15. Place the culture dish in the incubator, changing the medium completely every 2–3 days.
16. To assess Purkinje cell survival, slices can be collected as early as 5 days in vitro. For other purposes, they can be maintained in culture for several weeks (Figure 1F).
    NOTE: In the case of Purkinje cell survival experiments, there is no need to renew the pharmacological treatment when changing cell culture medium (Figure 1F).

3. Immunofluorescence

1. Take out the 6-well plate from the incubator. Remove cell culture medium, wash the cell culture insert with 1x PBS, and fix the slices with cold 4% paraformaldehyde solution for 1 h, with 1 mL under the cell culture insert and 500 µL on top of the cell culture insert.
2. Wash the inserts 4x 10 min with 1x PBS, with 1 mL under and 500 µL on top of the insert, on an orbital shaker.
3. With a brush, transfer the cerebellar slices from 1 cell culture insert to 1 well of a 24-well pre-filled with 1x PBS, 0.25% Triton X-100, and 3% BSA (PBS-TB), 500 µL/well.
4. Permeabilize and block the slices by incubation in PBS-TB for 1 h at room temperature.
5. Incubate with primary antibodies diluted in the PBS-TB solution, overnight at + 4 °C, on an orbital shaker, 200 µL/well.
6. On the following day, perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 µL/well.
7. Incubate with secondary antibodies diluted in the PBS-TB solution, two hours at room temperature, on an orbital shaker, and protected from light, 200 µL/well.
8. Perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 µL/well.
9. Counterstain the slices using Hoechst 33342, diluted in 1x PBS (2 µg/mL), for 10 min at room temperature, protected from light, 500 µL/well.
10. Mount the cerebellar slices on a microscopy slide with a transfer pipette. Let them air-dry completely, protected from light. Re-humidify them with 1x PBS and apply a coverslip covered with few drops of mounting medium (~80 µL). Avoid making any bubbles.
11. Once the mounting medium has cured, cerebellar sections are ready to be imaged.

4. Imaging and Quantification of Cell Survival

1. Turn on the microscope and lasers according to the manufacturer and/or the imaging core facility's instructions.
2. Start the acquisition software.
3. Using a 10X objective, locate non-altered cerebellar slices that present 9–10 lobules (usually cerebellar sections close to the vermis).
4. Activate z-stack acquisition. Define the range of the z-stack to include all Purkinje cells present in the cerebellar slice. Set a 2 µm step size and start the acquisition. Save the acquired picture in the format of the acquisition software manufacturer to preserve the associated metadata.
5. Open the acquired file in ImageJ using the Bio-formats plugin¹⁸.
6. Go to Image > Stacks > Z-project… (Figure 2A).
7. Choose Max Intensity in the Projection type dropdown menu and click OK (Figure 2B).
8. A flattened 2-D image will be generated. Double click on the Multi-point tool to let the Point Tool window appear. Uncheck the Label points option to ease counted cells visualization. Then, click directly on a soma of a Purkinje cell to begin counting (Figure 1E). The total number of counted cells will be indicated at the bottom of the Point Tool window (Figure 3).
   NOTE: Usually, at least 3 non-damaged sections containing 9–10 lobules per cell culture insert can be quantified. To assess the neuroprotective effect of a pharmacological agent, at least 3 independent experiments should be performed.

Wyniki

As shown in Figure 4, this protocol produces organotypic cerebellar slice cultures in which Purkinje cell survival can be assessed following the immunofluorescence and image acquisition steps. Purkinje cells were labeled with a combination of anti-Calbindin D-28K (dilution 1/200) and Alexa594 anti-mouse (dilution 1/300) antibodies. Image stitching was done automatically by the microscope acquisition software (NIS-Elements) to obtain a picture of the whole cerebellar slice. Purkinje cell numb...

Dyskusje

Cerebellar slice culture is a powerful tool to study programmed Purkinje cell death during postnatal development. This technique can be used to rapidly screen candidate molecules for their neuroprotective potential. The main advantage is that the setup is simple and very cost effective, and only requires a modest investment in equipment (a vibratome can cost up to 3 times more than a tissue chopper). Moreover, 10 to 15 healthy slices can be generated from one mouse pup, allowing for different assays to be performed in pa...

Materiały

Name	Company	Catalog Number	Comments
Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody	ThermoFisher scientific	A21203
Basal Medium Eagle (BME)	ThermoFisher scientific	21010046
Biosafety cabinet Class II, Type A2	NuAire	NU-540-400
Bovine serum albumin	Millipore Sigma	A2153
Brush
anti-Calbindin D-28K antibody (CB-955)	Abcam	ab82812
CO2 Incubator	NuAire	NU-5700
Corning Costar Flat Bottom 6-well Cell Culture Plates	Fisher Scientific	07-200-83
Coverslips, 22 x 50 mm	Fisher Scientific	12-545-E
Dressing forceps, straight	Harvard Apparatus	72-8949
Double edge blades	Fisher Scientific	50949411
Ethanol 200 proof	Decon Labs, Inc	2701
Eye Scissors, straight	Harvard Apparatus	72-8428
Fine forceps	Fisher Scientific	16-100-127
L-Glutamine 100X	ThermoFisher scientific	25030149
Glucose solution	ThermoFisher scientific	A2494001
Hanks’ Balanced Salt Solution	ThermoFisher scientific	14025092
Hoechst 33342, Trihydrochloride, Trihydrate	Fisher Scientific	H21492
Horse Serum, heat inactivated, New Zealand origin	ThermoFisher scientific	26050088
ImageJ
McIlwain Tissue Chopper	Fisher Scientific	NC9914528
Microprobes	Fisher Scientific	08-850
Millicell Cell Culture Inserts	Millipore Sigma	PICM0RG50
Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL	ThermoFisher scientific	168-0045
Nikon A1R confocal laser microscope system	Nikon
NIS-Elements Imaging Software	Nikon
Paraformaldehyde	Acros Organics	41678-0010
Pasteur pipets	Fisher Scientific	13-678-20D
Potassium Chloride	Fisher Scientific	BP366-500
ProLong Gold Antifade Mountant	ThermoFisher scientific	P10144
Operating Scissors, straight	Harvard Apparatus	72-8403
Orbital shaker Belly Dancer	IBI Scientific	BDRLS0003
Prism 8	GraphPad
Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Tissue Culture Dish, 60 mm w/ grip ring	Fisher Scientific	FB012921
Tissue culture plate, 24 well	Falcon/Corning	353047
Transfer pipettes, sterile	ThermoFisher scientific	13-711-21
Triton X-100	ThermoFisher scientific	BP151-500