Figures were created using Adobe Illustrator (version 2023) and Servier Medical Art (https://smart.servier.com). Antonia Henes was supported by J&#252;rgen Manchot Stiftung.

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

All authors declare to have no conflicts of interest.

So far, methods to map CNS-resident cells ex vivo by combining mass spectrometry and RNA sequencing offer a very precise cellular profiling in health and disease but require ambitious technical knowledge and expertise in this field50,51. Further, they do not allow functional analyses and are very expensive. Besides that, microfluidic brain-on-a-chip systems provide a rapid and affordable screening for disease mechanisms and testing of new therapeutic approaches with the restriction of cell growth and migration52,53,54,55. CNS organoids could also represent an equivalent alternative in the future for the investigation of cellular modeling, inter-cellular connections, and interactions during disease courses56,57,58,59. However, fluorescence- and magnetic-activated cell sorting are currently the most effective methods to generate pure and viable single-cell suspensions ex vivo35,60,61. Even if other established manufactural protocols for the isolation of CNS-resident cell types are similar regarding the individual steps of the magnetic isolation and the prior cell dissociation, they are intended to be performed for each cell type separately. By contrast, the current protocol integrates different isolation methods for each CNS-resident cell type into a logical context so that they can be performed simultaneously at once and from one single CNS cell suspension (Table 1, Table 2). Thus, it enables multi-omics analyses from one single CNS cell suspension and, eventually, the exploration of complex neuronal networks. Even if it is not a must to pool several tissues from multiple animals to perform this protocol, this pooling ensures an adequate number of isolated cells for further downstream analysis. The usage of different mice for the isolation of the single cell types would exclude the possibility of analyzing potential cellular interactions. Besides that, combining individual isolation methods for the different CNS cell types, which all follow a prior CNS dissociation, saves material costs by using one dissociated CNS cell suspension for all following magnetic isolation steps. Additionally, a potential technical bias caused by the usage of different mice is minimized.
One limitation of the protocol could be the nearly exclusive usage of female C57BL/6J mice. The EAE immunization protocol has been designed and established for female mice, so this cell isolation protocol was implemented in female C57BL/6J mice as well. Nevertheless, na&#239;ve male mice were also used during the development of this protocol, without recognizing any effect on the resulting cell number or purities. Another restriction affects the magnetic cell isolation of neurons as there exist no specific micro-beads for the isolation of neurons in terms of a positive selection. It was assumed that a pure single-cell suspension could be obtained via biotin labeling and depletion of all non-neuronal cells (Table 2). This assumption was verified by the usage of NeuN as a specific nuclear marker for neurons, integrated in the mentioned flow cytometry purity panel. Another limitation concerns the isolation of microglia in EAE mice. Here, resulting cell yields are decreased in comparison to the other cell types because of the additional sorting step after the MACS protocol. Moreover, one could argue that sorting increases the mechanical stress of the microglia compared to the other cell populations. Individual sorting strategies may lead to different amounts of cell yields. If isolated cell number is less than expected or desired, adjusting the gating set up and/or improving the live/dead discrimination is recommended.
A critical step in the protocol represents the debris removal. The gradient must be layered very slowly and gently to create the three wanted separate phases (Figure 2A). Only if the myelin and other debris residues in the two top phases are removed totally (Figure 2E), pure single-cell suspensions can be generated, and further contamination can be reduced. If the resulting cell suspensions lack purity, this is probably the section of the protocol that should be improved first next to the assurance of the right usage of all micro-beads.
Receiving high levels in purity and viability can be challenging in this type of experiment. Some recommendations for troubleshooting are:
-Working under sterile conditions is obligatory to prevent contamination of the different micro-beads and enable repeated use, especially for subsequent cultivation. 
-Labeling of each tube to prevent mix-ups is highly recommended. 
-Avoid the usage of uncooled reagents/buffers. Store all cell suspensions on ice during the whole experiment to ensure high viability. 
-Keep the time between the different working steps as short as possible. There is no specific part in the protocol where pausing the experiment is recommended. 
-It is highly relevant to adhere to the specified incubation periods.
In conclusion, this current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and neuroinflammatory pathways ex vivo from one CNS cell suspension. Thus, CNS-resident cells can be investigated during different stages of disease courses, e.g., during neuroinflammation, neurodegeneration and/or remission in EAE. Furthermore, cell-cell interactions and biochemical pathways can be studied on an individual level and variability within experimental groups can be reduced. There is also the opportunity to cultivate fractions of the isolated CNS cells in monocultures for further functional assays and validation. All in one, this protocol offers significant advances potentially affecting preclinical and clinical research approaches.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage, gliosis, and neurodegeneration. Despite numerous research approaches in this field, the pathophysiology of MS is still not fully understood1,2,3,4. The most common animal model for investigating MS is the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) which shares many of its clinical and pathophysiological features5,6,7,8,9. It is based on the response of the immune system against CNS-specific antigens leading to inflammation, demyelination, and neuroaxonal degeneration. Experimental autoimmune encephalomyelitis (EAE) is a suitable model for the investigation of neuroinflammatory pathways and signaling cascades found in MS.
Current therapy options for MS are only partially effective and focus primarily on the initial inflammatory phase of the disease. However, the neurodegenerative component of MS seems to be the major challenge for long-term therapeutic approaches. Therefore, reproducible, and precise cell isolation protocols are required to investigate molecular and cellular mechanisms in autoimmune diseases in a comprehensive way. Even if some protocols for the isolation of one single cell type exist10,11,12,13,14,15, there is an unmet need for the simultaneous isolation of several CNS-resident cell populations at once. Previous protocols for the isolation of CNS-resident cells lack in preserving cellular functionality and purity, resulting in co-cultivation with neighboring cells16,17,18 or the unsuitability for complex analyses of intracellular networks ex vivo19,20,21,22.
The aim of this protocol was to establish a reproducible and comprehensive method for the simultaneous isolation of pure viable single-cell suspensions of all principal CNS-resident cell types applicable in adult healthy and EAE mice. The different cell types were isolated using magnetic-activated cell sorting (MACS)23. The cell separation can be accomplished either by positive selection, i.e., magnetic labeling of cell-type-specific surface markers, or by negative selection via biotinylation and depletion of all undesired cells. Flow cytometry was applied to ensure a purity of above 90% and a viability of at least 80% of the isolated single-cell suspensions.
In conclusion, the main goal was to establish a protocol for the simultaneous isolation of all main CNS-resident cell types as a versatile tool for the investigation of neuroinflammatory pathways offering a comprehensive and precise analysis of complex cellular networks and biochemical signaling cascades in healthy and EAE mice.

<table><tbody><tr><td>70 &amp;mu;m cell strainers</td><td>Corning, MA, USA</td><td>352350</td><td>CNS tissue dissociation</td></tr><tr><td>ACSA-2 Antibody, anti-mouse, PE-Vio 615 (clone REA-969)</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-116-244</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>Adult Brain Dissociation Kit, mouse, and rat</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-107-677</td><td>Tissue dissociation,contains debris and red blood cell removal solutions; prepare aliquots of enzyme A and P upon arrival and store them at -20 &amp;deg;C; store the remaining kit at 4 &amp;deg;C</td></tr><tr><td>Anti-ACSA-2 MicroBead Kit, mouse</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-097-678</td><td>MACS of astrocytes, store at 4 &amp;deg;C</td></tr><tr><td>Anti-mouse CD16/32 antibody&amp;nbsp;</td><td>BioLegend, London, UK</td><td>101301</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>Anti-O4 MicroBeads, human, mouse, rat</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-094-543</td><td>MACS of oligodendrocytes, store at 4 &amp;deg;C</td></tr><tr><td>AstroMACS Separation buffer</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-091-221</td><td>MACS of astrocytes, store at 4 &amp;deg;C</td></tr><tr><td>Biotin Antibody, PE (clone Bio3-18E7)</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-113-853</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>BRAND Neubauer counting chamber</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>10195580</td><td>Cell counting&amp;nbsp;</td></tr><tr><td>Brilliant Violet 510 anti-mouse CD45 Antibody (clone 30-F11)</td><td>BioLegend, London, UK</td><td>103137</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>CD11b MicroBeads, human, mouse</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-049-601</td><td>MACS of microglia, store at 4 &amp;deg;C</td></tr><tr><td>DNAse I, recombinant, Rnase-free</td><td>Merck KGaA, Darmstadt, Germany</td><td>4716728001</td><td>Flow cytometry, store at -20&amp;deg; C</td></tr><tr><td>D-PBS with Calcium, Magnesium, Glucose, Pyruvat</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>14287080</td><td>Buffer, store at 4 &amp;deg;C</td></tr><tr><td>D-PBS, without calcium, without magnesium</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>14190250</td><td>Buffer, store at 4 &amp;deg;C</td></tr><tr><td>eBioscience Fixable Viability Dye eFluor 780</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>65-0865-14</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>eBioscience Foxp3/Transcription factor staining buffer set</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>00-5523-00</td><td>Flow cytometry, store at 4&amp;deg;C</td></tr><tr><td>Falcon (15 mL)</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>11507411</td><td>Cell tube</td></tr><tr><td>Falcon (50 mL)</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>10788561</td><td>Cell tube&amp;nbsp;</td></tr><tr><td>Falcon&amp;nbsp;Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mL</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>08-771-23</td><td>Flow cytometry</td></tr><tr><td>FcR Blocking Reagent, mouse&amp;nbsp;</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-092-575</td><td>MACS of oligodendrocytes, store at 4 &amp;deg;C</td></tr><tr><td>Female C57BL/6J mice</td><td>Charles River Laboratories, Sulzfeld, Germany</td><td></td><td>Active EAE induction&amp;nbsp;</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Merck KGaA, Darmstadt, Germany</td><td>F2442-50ML</td><td>Flow cytometry, store at -5 to -20 &amp;deg;C</td></tr><tr><td>FITC Rat Anti-CD 11b&amp;nbsp; (clone M1/70)</td><td>BD Biosciences, San Jose, CA, USA</td><td>553310</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>Freund&amp;rsquo;s Complete adjuvant</td><td>Merck KGaA, Darmstadt, Germany</td><td>AR001</td><td>Active EAE induction, store at 4 &amp;deg;C</td></tr><tr><td>GentleMACS C Tubes</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-093-237</td><td>CNS tissue dissociation</td></tr><tr><td>GentleMACS Octo Dissociator with Heaters</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-096-427</td><td>CNS tissue dissociation</td></tr><tr><td>Graphpad Prism 8.4.3</td><td>Graphpad by Dotmatics</td><td></td><td>Graphical Analysis</td></tr><tr><td>Isoflurane</td><td>AbbVie, North Chicago, IL, USA</td><td></td><td>Active EAE induction, store at 4 &amp;deg;C</td></tr><tr><td>Kaluza Analysis Software V2.1.1</td><td>Beckman Coulter, Indianapolis, IN, USA</td><td></td><td>Flow cytometry analysis</td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-042-401</td><td>MACS</td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-091-376</td><td>PB-buffer</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-042-303</td><td>MACS&amp;nbsp;</td></tr><tr><td>MOG&lt;sub&gt;35&amp;ndash;55&lt;/sub&gt; peptide</td><td>Charit&amp;eacute;, Berlin, Germany; alternatives: Genosphere Biotechnologies (Paris, France) or sb-Peptide (Saint Egr&amp;egrave;ve, France)</td><td></td><td>Active EAE induction, store at -20 &amp;deg;C</td></tr><tr><td>Mycobacterium tuberculosis strain H37 Ra</td><td>Becton, Dickinson and Company (BD),Franklin Lakes, NJ, USA</td><td></td><td>Active EAE induction, store at 4 &amp;deg;C</td></tr><tr><td>Neuron Isolation Kit, mouse&amp;nbsp;</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-115-390</td><td>MACS of neurons, store at 4 &amp;deg;C</td></tr><tr><td>O4 Antibody, anti-human/mouse/rat, APC, (clone REA-576)</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-119-897</td><td>Flow cytometry, store at 4 &amp;deg;C</td></tr><tr><td>Pertussis toxin in glycerol</td><td>Hooke Laboratories Inc., Lawrence, MA, USA</td><td>BT-0105</td><td>Active EAE induction; store at -20 &amp;deg;C</td></tr><tr><td>pluriStrainer Mini 100 &amp;mu;m&amp;nbsp;</td><td>pluriSelect Life Science UG, Leipzig, Sachsen, Germany</td><td>43-10100-40</td><td>Flow cytometry</td></tr><tr><td>QuadroMACS Separator&amp;nbsp;</td><td>Miltenyi Biotec, Bergisch Gladbach, NRW, Germany</td><td>130-090-976</td><td>MACS</td></tr><tr><td>Recombinant Alexa Fluor 647 Anti-NeuN antibody (clone EPR12763)</td><td>Abcam, Cambridge, UK</td><td>EPR12763</td><td>Flow cytometry, store at -20 &amp;deg;C</td></tr><tr><td>Stainless Steel Brain Matrices, 1 mm</td><td>Ted Pella, Redding, CA, USA</td><td>15067</td><td>CNS tissue dissection</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>15250061</td><td>Cell counting&amp;nbsp;</td></tr><tr><td>UltraPure 0.5&amp;nbsp;M EDTA, pH 8.0</td><td>Thermo Fisher Scientific,Waltham, MA, USA</td><td>15575020</td><td>Flow cytometry, store at room temperature</td></tr></tbody></table>

simultaneous isolation of principal central nervous system-resident cell types from adult autoimmune encephalomyelitis mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

The current protocol offers the possibility to simultaneously isolate all principal CNS-resident cells, i.e., microglia, oligodendrocytes, astrocytes, and neurons from one single CNS replicate. This is important for the reduction of mice numbers needed for these kinds of experiments and to ensure the comparability of molecular and biochemical analyses on cellular level. If the individual cell types are isolated from different CNS replicates, cellular interactions cannot be mapped truthfully and potential technical deviations during the isolation processes could bias further downstream analyses. In addition, molecular and biochemical findings from each cell type would not be comparable to each other as they are not derived from the same EAE context. A pre-existing MACS protocol using a commercial system/kit was adapted to enable simultaneous isolation of the above-mentioned cell types.
The isolation of microglia was performed using anti-CD11b micro-beads, oligodendrocytes were isolated via anti-O4 micro-beads (Table 1), and anti-ACSA-2 micro-beads were used to isolate astrocytes (Table 2). In contrast, the isolation of neurons represents a negative selection and was accomplished by biotinylation and magnetic labeling of all non-neuronal cells (Table 2). All non-neuronal cells (e.g., oligodendrocytes, microglia, astrocytes, endothelial cells, and fibroblasts) except for blood cells can be magnetically labeled by using a biotin-conjugated antibody, specifically directed against a surface antigen expressed on these non-neuronal cells (Table 2). By depletion of these magnetically labeled non-neuronal cells, highly pure and viable neuronal cell populations can be generated30,42,43.
Two new flow cytometry panels for the purity analyses of the generated single-cell suspensions were designed. Here, cell-type-specific surface and nuclear markers combined with live/dead cell discrimination were used.
The resulting cell yields per mouse and cell-type (Figure 3A) were analyzed and led to an average of 8. 4 x 105 &#177; 3 x 104 microglia, 3.23 x 106 &#177; 1 x 105 oligodendrocytes, 8.6 x 105 &#177; 2 x 104 astrocytes, and 4.5 x 105 &#177; 1 x 104 neurons per na&#239;ve mouse.
In the context of the aim to investigate disease models of neuroinflammation, the protocol was also applied to a mouse model of EAE. The mice were euthanized on day 16 after EAE induction representing the disease maximum. In this EAE setting, approximately 2.9 x 106 &#177; 6.7 x 105 oligodendrocytes, 5.2 x 105 &#177; 9 x 104 astrocytes and 4.4 x 105 &#177; 4 x 104 neurons were isolated. The microglia cell yield was decreased to approximately 1.7 x 105 &#177; 4 x 104 microglia per EAE mouse because of the additional cell sorting after the MACS steps (Figure 3A).
After isolation, phenotypic characterizations of the different cell populations via flow cytometry proved that viable single-cell suspensions with a purity of approximately 90% for all main CNS-resident cell types (Figure 3B) could be achieved. Microglia were gated as being CD45intCD11bhigh as defined in the literature44,45,46,47.
In EAE, microglia had to be sorted from all CD11b+ cells to differentiate them from other CD11b+ immune cells like monocytes, neutrophils, natural killer cells, granulocytes, and macrophages that immigrate into the CNS during neuroinflammation27,28,48. Therefore, the microglia were sorted as CD45intCD11high cells from the CD11b+ cell suspension. The whole microglia sorting strategy is depicted in Figure 4. In na&#239;ve mice, the microglia population was 91.16% of all live single cells (96% of the total CD11b+ population) (Figure 4A). In EAE mice, the microglia population was 46.85% of all live single cells (55% of the total CD11b+ population) (Figure 4B). Although both MACS and FACS procedures apply mechanical stress to the single cells, 75.41% &#177; 8.63% of the sorted purified microglia were viable (Figure 3B).
Astrocytes and neurons that were directly isolated from the initial CNS cell suspension showed relevant contamination with oligodendrocytes which led to the assumption that the simultaneous isolation of neurons and astrocytes from the negative flow-through of oligodendrocytes could prevent this contamination. Flow cytometry analyses confirmed that astrocytes isolated from the negative flow-through of oligodendrocytes had a purity of 89.23% &#177; 2.78% and demonstrated a viability of 80.56% &#177; 1.41%. Similar to these results, the purity of the neurons isolated from the O4- cell fraction was 81.25% &#177; 3.31% and the viability was 83.90% &#177; 2.61% (Figure 3B). These findings also confirm that the simultaneous isolation of these two cell types only subsequently to the isolation of oligodendrocytes has no impact on the amount of viable functional cells.
The results regarding the viability and purity of the isolated single-cell suspensions were very similar in EAE mice compared to those received in na&#239;ve mice confirming that this protocol is suitable for healthy mice as well as in the context of EAE (Figure 3B).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65735/65735fig03.jpg" /> 
Figure 3: Cell yields and flow cytometry-based validation of isolated CNS-resident cells. (A) Cell yields per mouse and cell-type after isolation of CNS-resident cells in na&#239;ve and EAE mice. Bar graphs visualize the amount of cell yields per mouse and cell-type after the implementation of the presented protocol. Five biological replicates were processed for the results in na&#239;ve mice, and four biological replicates were analyzed in EAE mice. Respective means &#177; SEMs are depicted. (B) Corresponding purity and viability analyses of the purified cell fractions. Bar graphs indicate the viability and purity of the resulting single-cell suspensions based on their expression of cell-type-specific markers. NeuN was used as a cell-type specific nuclear marker for neurons. Five biological replicates were acquired and compared for each cell type for both healthy and EAE mice. Respective means &#177; SEMs are indicated.&#160;Abbreviations: Anti-ACSA-2 = astrocyte cell surface antigen-2; CD11b = cyclin-dependent kinase 11B; CD45 = receptor-type tyrosine-protein phosphatase C; CNS = central nervous system; EAE = experimental autoimmune encephalomyelitis; MACS = magnetic-activated cell sorting; NeuN =RNA binding protein fox-1 homolog 3; O4 = oligodendrocyte marker O4; SEM = standard error of the mean. This figure has been modified from49. <a href="https://www.jove.com/files/ftp_upload/65735/65735fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65735/65735fig04.jpg" /> 
Figure 4: Gating strategy for cell sorting of microglia after isolation of CD11b+ cells. (A) Gating strategy in na&#239;ve and (B) EAE mice. The upper row of each panel shows dot plots before sorting and the lower row after sorting. After selection of live (SSC-A / FSC-A) and single cells (FCS-H / FSC-W), the population of CD45intCD11b+ cells were sorted as microglia population. <a href="https://www.jove.com/files/ftp_upload/65735/65735fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types at JoVE.com

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

simultaneous-isolation-principal-central-nervous-system-resident-cell

Research

JoVE Journal

Neuroscience

1.6K Views.  Heinrich Heine University Duesseldorf. Om moleculaire en cellulaire mechanismen bij auto-immuunziekten zoals multiple sclerose te onderzoeken, is het genereren van reproduceerbare en geavanceerde celisolatieprotocollen een onvervulde behoefte. De meeste van de huidige technologieën om CZS-residente cellen te isoleren en te analyseren, vertonen ernstige tekortkomingen, zoals het focussen op slechts één celtype of alleen postnatale muizen. Het huidige protocol voor de gelijktijdige isolatie van alle belangrijke CZS-residente celt...

Video: Auteur in de schijnwerpers: het bestuderen van neuro-inflammatoire routes door gelijktijdige isolatie van alle belangrijke celtypen van het CZS 

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Biology

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Immunology and Infection

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

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