analyzing purkinje cell survival in organotypic cerebellar slice cultures

Analyzing Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

To harvest the cerebellum, use straight dressing forceps to grasp the pup head by the nose, and use straight eye scissors to cut open the scalp from the posterior end laterally to the midline. Cut open the skull in the same manner, pointing the scissor tips outward to avoid damaging the cerebellum, and use a spatula to transfer the brain to a 60-millimeter dish containing cold HBSS and 5 milligrams per milliliter of glucose.
Use the straight dressing forceps to carefully dissect out the cerebellum, and use sterile curved fine forceps to place the tissue onto a plastic disk on the cutting table of a tissue chopper. Rotate the cutting table to orient the tissue to allow the acquisition of parasagittal sections, and pull the table release knob to the right to move the cutting table until the blade is positioned at the edge of the tissue. Then, adjust the slice thickness to 350 micrometers and the blade speed to medium and start the chopper. When the whole cerebellum has been sliced, turn off the chopper.
Using sterile forceps, hold the disk over a new 60-millimeter dish. Use a transfer pipette to flush HBSS plus glucose over the disk so that the slices fall into the dish. Then, touching the samples as minimally as possible, use a microprobe to separate the slices.
Use the transfer pipette to select the sections of the cerebellum close to the vermis onto individual cell culture inserts in the six-well plate, and use the microprobe to position the slices into the center of each insert. When all of the slices have been placed, carefully aspirate any excess HBSS plus glucose and return the plate to the cell culture incubator.
For immunofluorescence staining, remove the supernatant from each well and wash the inserts with PBS. Fix the slices with 1 milliliter of cold 4% paraformaldehyde in the well of each insert, and 500 microliters on top of each insert for 1 hour.
At the end of the fixation, wash the inserts four times for 10 minutes with 1 milliliter of fresh PBS under each insert and 500 microliters of fresh PBS on top of each insert per wash on an orbital shaker. Prefill each well of a 24-well plate with 500 microliters of PBS-TB, and use a paintbrush to transfer the cerebellar slices from each cell culture insert into individual wells of a 24-well plate.
Permeabilize and block the slices at room temperature for one hour. At the end of the incubation, label the cells with 200 microliters of the primary antibody of interest diluted in PBS-TB per well overnight at 4 degrees Celsius on the orbital shaker.
The next morning, wash the slices four times for 10 minutes and 500 microliters of fresh PBS per wash on the shaker before labeling the samples with the appropriate fluorophore-conjugated secondary antibodies for two hours at room temperature on the shaker protected from light.
At the end of the incubation, counter-stain the sections with 500 microliters of an appropriate nuclear stain per well for 10 minutes at room temperature protected from light, and use a transfer pipette to mount the slices onto glass slides.
Then, let the sections air-dry completely before rehydrating with PBS and mounting the tissues with coverslips coated with approximately 80 microliters of mounting medium. Once the mounting medium has cured, the cerebellar sections are ready to be imaged.

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1. Preparation prior to organotypic cerebellar slice cultures
<ol>
	<li>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&#39; 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 &#176;C for up to a month.</li>
	<li>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 &#956;L of sterile 3 M KCl solution to the treated well (30 mM final) and 1 &#956;L of sterile water to the control wells.</li>
	<li>Bring inside the cell culture hood the needed number of individually wrapped cell culture inserts with hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size = 0.4 &#956;m). 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 &#176;C, 5% CO2 incubator for at least 2 h before culture. 
	NOTE: The tissue chopper permanently resides in the cell culture hood.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Dissection and Cerebellar Organotypic Slice Cultures
<ol>
	<li>Bring the mouse pup inside the cell culture hood to perform the dissection.</li>
	<li>Decapitate the pup quickly using straight operating scissors (Figure 1A).</li>
	<li>While holding the pup&#39;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.</li>
	<li>Proceed identically for the skull. 
	NOTE: Make sure to point scissor tips outwards to avoid damaging the cerebellum during dissection.</li>
	<li>Take out the brain and transfer it to a 60-mm dish filled with cold HBSS + 5 mg/mL glucose.</li>
	<li>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.</li>
	<li>Orient the tissue by rotating the cutting table to perform parasagittal sections.</li>
	<li>Move the cutting table by pulling the table release knob to the right, so the blade is positioned at the edge of the tissue.</li>
	<li>Adjust the slice thickness to 350 &#181;m and the blade speed to medium.</li>
	<li>Start the chopper. Once the whole cerebellum has been sliced (Figure 1C), turn off the chopper.</li>
	<li>Remove the plastic disc with sterile forceps.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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&#8211;8 slices can be cultured for a P0&#8211;P4-old animal, and 4&#8211;6 slices for animals older than P5.</li>
	<li>Place the culture dish in the incubator, changing the medium completely every 2&#8211;3 days.</li>
	<li>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).</li>
</ol>
3. Immunofluorescence
<ol>
	<li>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 &#181;L on top of the cell culture insert.</li>
	<li>Wash the inserts 4x 10 min with 1x PBS, with 1 mL under and 500 &#181;L on top of the insert, on an orbital shaker.</li>
	<li>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 &#181;L/well.</li>
	<li>Permeabilize and block the slices by incubation in PBS-TB for 1 h at room temperature.</li>
	<li>Incubate with primary antibodies diluted in the PBS-TB solution, overnight at + 4 &#176;C, on an orbital shaker, 200 &#181;L/well.</li>
	<li>On the following day, perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 &#181;L/well.</li>
	<li>Incubate with secondary antibodies diluted in the PBS-TB solution, two hours at room temperature, on an orbital shaker, and protected from light, 200 &#181;L/well.</li>
	<li>Perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 &#181;L/well.</li>
	<li>Counterstain the slices using Hoechst 33342, diluted in 1x PBS (2 &#181;g/mL), for 10 min at room temperature, protected from light, 500 &#181;L/well.</li>
	<li>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 &#181;L). Avoid making any bubbles.</li>
	<li>Once the mounting medium has cured, cerebellar sections are ready to be imaged.</li>
</ol>
4. Imaging and Quantification of Cell Survival
<ol>
	<li>Turn on the microscope and lasers according to the manufacturer.</li>
	<li>Start the acquisition software.</li>
	<li>Using a 10X objective, locate non-altered cerebellar slices that present 9&#8211;10 lobules (usually cerebellar sections close to the vermis).</li>
	<li>Activate z-stack acquisition. Define the range of the z-stack to include all Purkinje cells present in the cerebellar slice. Set a 2 &#181;m step size and start the acquisition. Save the acquired picture in the format of the acquisition software manufacturer to preserve the associated metadata.</li>
	<li>Open the acquired file in ImageJ using the Bio-formats plugin.</li>
	<li>Go to Image &#62; Stacks &#62; Z-project&#8230; (Figure 2A).</li>
	<li>Choose Max Intensity in the Projection type dropdown menu and click OK (Figure 2B).</li>
	<li>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&#8211;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.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Donkey anti- Mouse IgG secondary antibody</td>
			<td>ThermoFisher scientific</td>
			<td>A21203</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal Medium Eagle (BME)</td>
			<td>ThermoFisher scientific</td>
			<td>21010046</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet Class II, Type A2</td>
			<td>NuAire</td>
			<td>NU-540-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin D-28K antibody (CB-955)</td>
			<td>Abcam</td>
			<td>ab82812</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NuAire</td>
			<td>NU-5700</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blades</td>
			<td>Fisher Scientific</td>
			<td>50949411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-127</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>ThermoFisher scientific</td>
			<td>25030149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose solution</td>
			<td>ThermoFisher scientific</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution</td>
			<td>ThermoFisher scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated, New Zealand origin</td>
			<td>ThermoFisher scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Fisher Scientific</td>
			<td>NC9914528</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>Fisher Scientific</td>
			<td>08-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore Sigma</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td>
			<td>ThermoFisher scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser microscope system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>41678-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8403</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker Belly Dancer</td>
			<td>IBI Scientific</td>
			<td>BDRLS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish, 60 mm w/ grip ring</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Falcon/Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes, sterile</td>
			<td>ThermoFisher scientific</td>
			<td>13-711-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Rakotomamonjy, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60353">Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures.</a> J. Vis. Exp. (2019)
This video illustrates a technique for obtaining cerebellar organotypic slices from a mouse pup&#39;s brain and culturing the slices in media supplemented with nutrients and growth factors to mimic cerebellar development in vitro. Additionally, specific cerebellar slices are treated with a test compound, and the compound&#39;s neuroprotective ability is analyzed using an immunofluorescence staining technique.

<img alt="Figure 1" src="/files/ftp_upload/22420/22420_Figure1.jpg" /> 
Figure 1: Illustration of the protocol from dissection to Purkinje cell survival quantification. The mouse pup is quickly decapitated (A). Following brain dissection, the cerebellum is isolated (B), and then chopped into 350 &#181;m-width slices (C). The cerebellar sections are placed onto a control or treated cell culture insert (D) and maintained in culture for at least 5 days in vitro. The tissue is then fixed in 4% paraformaldehyde, immunostaining is performed, and pictures are acquired with a confocal microscope. Lastly, Purkinje cell number is quantified in each slice using the Multipoint Tool in ImageJ (E). (F) Schematic of the time course of KCl treatment during culture.
<img alt="Figure 2" src="/files/ftp_upload/22420/22420_Figure2.jpg" /> 
Figure 2: ImageJ settings used to flatten the 3D-stack prior to quantification.
<img alt="Figure 3" src="/files/ftp_upload/22420/22420_Figure3.jpg" /> 
Figure 3: Settings used to count Purkinje cell number using the Multipoint tool in ImageJ with an example of a cerebellar slice being quantified.

1. Preparation prior to organotypic cerebellar slice cultures

	In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture ...

Begin with a mouse pup brain.&#160;
Isolate the cerebellum and obtain tissue slices of appropriate thickness.
Transfer the slices onto membrane inserts placed within culture plate wells containing media.
In treated wells, the media contains a neuroprotective test compound. Control wells lack the compound. Incubate.
Under control conditions, the cerebellar Purkinje cells undergo apoptosis, mimicking early postnatal development in vivo.
In treated wells, depending on its potential, the compound may protect the cells from apoptosis.
Wash the slices and fix them to preserve cellular integrity.
Transfer the slices into a permeabilization-blocking solution to permeabilize cell membranes and block non-specific binding sites.
Overlay with primary antibodies targeting a calcium-binding protein specifically expressed in Purkinje cells.
Wash and introduce fluorophore-conjugated secondary antibodies targeting the primary antibodies.
Counterstain the nuclei with a DNA-binding dye and mount the slices.
Using a confocal microscope, quantify the viable Purkinje cells to identify the test compound&#39;s neuroprotective potential

34 Views. Analisi della sopravvivenza delle cellule di Purkinje in colture organotipiche di fette cerebellari

Video: Analisi della sopravvivenza delle cellule di Purkinje in colture organotipiche di fette cerebellari - Experiment

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Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

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In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

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A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Analyzing Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

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Analyzing Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures