Name: primary cultures of rat astrocytes and microglia and their use in the study of amyotrophic lateral sclerosis
Uploaded: 2022-06-23T12:30:00
Description: Watch this Scientific Journal Video about Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis at JoVE.com

This work was supported by the Ministry of Education Science and Technological Development Republic of Serbia Contract No. 451-03-9/2021-14/ 200178, the FENS - NENS Education and Training Cluster project &#34;Trilateral Course on Glia in Neuroinflammation&#34;, and the EC H2020 MSCA RISE grant #778405. We thank Marija Ad&#382;i&#263; and Mina Peri&#263; for supplying the immunohistochemistry images and Danijela Batavelji&#263; for help with paper writing.

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&#39;s consent in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The protocol ...

This paper presents the method of primary cell culturing as a fast and &#34;on the budget&#34; tool for studying different aspects of cell (patho)physiology such as ALS in the rat hSOD1G93A 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 ...

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 disease4,5,6, 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 hSOD1G93A ALS rat model (containing multiple copies of the human SOD1 mutation10) showed a number of markers of oxidative stress in cultured neuroglial cells11, tissues12,13,14, 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 ALS16,17. 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 cord18,19,20,21. This is also true for microglial primary cultures, although to a lesser extent than astrocytes and mostly from brain regions at the embryonic stage22,23,24.
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 animals25,26,27,28,29, which then showed intracellular Ca2+ elevation and IgG depositions. A line of studies explored the effect of ALS IgGs on the motor synapse in vitro30,31,32. 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 hSOD1G93A 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.

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			<td>Eppendorf, Germany</td>
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			<td>Biowest, France</td>
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			<td>Incubator</td>
			<td>Memmert GmbH + Co. KG, Germany</td>
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			<td>Math Works, USA</td>
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			<td>Merck Millipore, USA</td>
			<td>MAB360</td>
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			<td>Mowiol 40-88</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>324590</td>
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			<td>Sigma-Aldrich, Germany</td>
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			<td>Sigma-Aldrich, Germany</td>
			<td>158127</td>
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			<td>Sigma-Aldrich, Germany</td>
			<td>P5280</td>
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			<td>Thapsigargine</td>
			<td>Tocris Bioscience, UK</td>
			<td>1138</td>
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			<td>Triton X - 100</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>T8787</td>
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			<td>Sigma-Aldrich, Germany</td>
			<td>T4799</td>
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			<td>Vapro Vapor Pressure Osmometer 5520</td>
			<td>Wescor, ELITechGroup Inc., USA</td>
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			<td>ViiFluor Imaging System</td>
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			<td>VisiChrome Polychromator System</td>
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			<td>VisiView high performance setup</td>
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			<td></td>
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		<tr>
			<td>Xenon Short Arc lamp</td>
			<td>Ushio, Japan</td>
			<td></td>
			<td></td>
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primary cultures of rat astrocytes and microglia and their use in the study of amyotrophic lateral sclerosis

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 hSOD1G93A 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 hSOD1G93A 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 hSOD1G93A 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.

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...

Watch this Scientific Journal Video about Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis at JoVE.com

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

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 hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

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