This work was supported in part by grants from the National Multiple Sclerosis Society (RG 5001-A-3 to S.J.C.), the National Institutes of Health (NS065808 to E.R.B.; NS078392 to S.J.C.), start-up funds from the UConn Health Center (to SJC) and the Kim Family Fund (UCHC in support of K.I.C.).

All procedures involving animals were performed in accordance with the Policy on Humane Care and Use of Laboratory Animals set forth by the Office of Laboratory Animal Welfare (NIH) and only with approval from the Institutional Animal Care and Use Committee (IACUC) of the University of Connecticut Health Center.
1. Preparation of Mixed Glial Cultures
<ol>
	<li>Sterilize all instruments prior to use. Add 3 ml of ice-cold sterile Hank&#8217;s balanced salt solution (HBSS) containing no cations (Mg2+ or Ca2+) to three sterile 60 mm Petri dishes maintained on ice to keep cold.</li>
	<li>Isolate the cortices from postnatal P0-P2 mouse pups, as previously described17,18. 
	NOTE: Subcortical structures, such as hippocampi, are not included in these cultures.</li>
	<li>Carefully remove the meninges and then transfer the isolated cortices to fresh HBSS and dissociate the tissues enzymatically, using a neural tissue dissection kit according to the manufacturer&#39;s protocol.</li>
	<li>Plate the cells into sterile T-175 flasks and incubate overnight in media [Dulbecco&#39;s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) containing 1x penicillin/streptomycin].</li>
	<li>Replace all media the following day with fresh media. Continue to incubate cultures, with regular media changes, for approximately 3 weeks or until a cellular monolayer is established. 
	NOTE: This monolayer will be comprised of astrocytes and microglia with an approximately 10:1 ratio19.</li>
</ol>
2. Globoid Cell Induction in Mixed Glial Cultures
<ol>
	<li>Prepare the ECM-coated coverslips.
	<ol>
		<li>Soak circular coverslips with sterile 1 N hydrochloric acid (HCl) in a sterile Petri dish for 30 min. Wash coverslips with sterile water 3x and then soak in 95% EtOH for at least 20 min. and then let air-dry.</li>
		<li>Place droplets of diluted extracellular matrix (ECM) protein coating materials onto a sheet of Parafilm (e.g. 150-250 &#181;l/droplet of laminin 10 &#956;g/ml dissolved in sterile phosphate buffer solution [PBS]). Distribute the droplets so that coverslips will not touch when placed onto these droplets. Gently place each dried coverslip onto a droplet using forceps.</li>
		<li>Leave the coverslips on the droplets for approx. 4-6 hr inside the culture hood with the sash closed.</li>
		<li>Place the ECM-coated coverslips (ECM-coated side up) into each of the wells of a 24-well plate. Wash each well with sterile PBS 3x and keep at 4 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Preparing Glial Cells for Plating onto Coverslips.
	<ol>
		<li>Aspirate the media from the glial cultures and gently wash the glial monolayer 3x with 10 ml of sterile PBS. Next, add 8-10 ml trypsin-EDTA solution to the T-175, and incubate for 5-10 min at 37 &#176;C.</li>
		<li>When most of the monolayer has been detached, add 20 ml of complete media to the flask to stop trypsinization. Collect the media containing the detached cells into a 50 ml conical tube and spin for 10 min at 300 x g at room temperature.</li>
		<li>Aspirate the supernatant without disturbing the pellet, and then re-suspend the cells in 2 ml of fresh complete media by gently pipetting the solution using a P1000 pipet.</li>
		<li>Determine the number of cells using a hemocytometer and then re-suspend cells in complete medium at the desired density (e.g. 105 cells/ml). Plate the cells onto the ECM-coated coverslips (e.g. 500 &#956;l into each well of the 24-well plate). Add additional fresh complete media to each well for 3-5 days of incubation at 37 &#176;C, or until cells grow to the desired visual confluence.</li>
	</ol>
	</li>
	<li>Treatment with Psychosine.
	<ol>
		<li>Reconstitute the psychosine to a stock concentration (108.3 mM) in dimethyl sulfoxide (DMSO). Dilute this stock in 100% EtOH to make a working stock that can then be added into each well of 24-well plate to achieve a final concentration of 10 &#956;M.</li>
		<li>At the time of adding the psychosine, gently swirl the culture plate to mix the psychosine into culture media. Supplement the psychosine by adding the equivalent of 10 &#181;M every other day for one week.</li>
	</ol>
	</li>
	<li>After 7 days of psychosine treatment, collect the media and pipet 500&#160;&#181;l of cold 4% paraformaldehyde (PFA) into each well. Incubate at room temperature for 20 min to fix the cells. Then, wash each well 3x with PBS and then keep plates at 4 &#176;C until processed for immunocytochemistry (see below). 
	NOTE: cell culture media can be collected for enzymatic or ELISA assay at this point if desired.</li>
</ol>
3. Immunocytochemistry (ICC) to Visualize Globoid Cells
<ol>
	<li>Replace PBS with blocking buffer [PBS + 0.3% Tween-20 + 5% normal goat serum (NGS)], and incubate the cells for 1 hr at room temperature.</li>
	<li>Incubate the fixed cells in primary antisera against Iba-1 [(1:1,000) diluted in PBS + 0.3% Tween-20 + 2% NGS] for at least 1 hr at room temperature, then wash each well with PBS 3x 5 min/wash.</li>
	<li>Add the appropriate secondary fluorescence-conjugated antibody to identify the primary antibody labeling and counter-stain with 4&#39;,6-diamidino-2-phenylindole (DAPI, 1:1,000) to identify nuclei. Incubate in secondary antibody solution for 1 hr at room temperature and wash with PBS 3x 5 min.</li>
	<li>Mount each coverglass to microscope slides using a mounting medium. View slides on a fluorescent microscope for visual and quantitative analyses.</li>
</ol>
4. Analysis and Characterization of Globoid Cells in Primary Culture
<ol>
	<li>Use the following three morphological criteria to identify a globoid cell in culture:
	<ol>
		<li>Identify globoid cells using fluorescent microscopy by immunopositive labeling for the microglial marker, Iba-1+.</li>
		<li>Identify globoid cells as polynucleated (i.e. containing two or more DAPI positive nuclei).</li>
		<li>Identify globoid cells by a rounded morphology distinct from the highly branched appearance of resting microglial cells. 
		NOTE: Cells may also be collected for cell surface marker analysis and quantification by flow cytometry from these cultures.</li>
	</ol>
	</li>
	<li>Measuring the phagocytic activation of microglia and globoid cells
	<ol>
		<li>To assess the phagocytic activity of psychosine-treated microglia, add FITC-labeled latex beads to the wells in accordance with the manufacturer&#8217;s instructions. Add fluorescent-conjugated latex beads 48 hr prior to fixation with PFA. Fix cells treated with beads (as mentioned in section 2.4), and process for immunocytochemistry (as described in section 3).</li>
		<li>Select fluorophore-conjugated secondary antibodies so that they do not overlap the fluorescent emission spectra of the fluorophore labeled beads.</li>
		<li>Analyze the phagocytotic profiles of Iba-1+ microglia by identifying those cells that co-localize with the fluorescently-labeled beads. 
		NOTE: Psychosine will activate microglia. Phagocytosed beads will be identified within mononucleated and multinucleated Iba-1+ cells in this culture setting.</li>
	</ol>
	</li>
</ol>
5. Induction of Globoid Cells from Purified Microglial Cultures
NOTE: An alternate approach to decipher intercellular signaling between the astrocyte and microglia is another advantage of this in vitro globoid cell model. Purification of primary microglial cells for replating or co-culturing can be achieved by their lower adherence property in culture, as described in the following steps (see Section 5.2).
<ol>
	<li>Collect the media from the confluent mixed glial cultures into 50 ml conical tube and maintain it at 37 &#176;C in the incubator.</li>
	<li>Microglial Isolation from Primary Mixed Glial Cultures.
	<ol>
		<li>After removing the media from a confluent mixed glial culture T175 flask, add warm shaking media [consisting of complete media + 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) + 25 mM sodium bicarbonate (Sigma)]. Shake the flasks for 3-4 hr in an orbital shaker at 100 rpm (37 &#176;C). Collect the media from these shaken flasks into 50 ml conical tubes. 
		NOTE: This media will include the less adherent microglia from the flasks.</li>
		<li>Spin the media at 300 x g for 10 min at room temperature. Aspirate the supernatant and then re-suspend the cells in 2 ml of the astrocyte-conditioned media (collected in step 5.1).</li>
		<li>Establish the number of cells acquired using a hemocytometer and then plate the microglial cells onto substrate-coated coverslips (see section 2.1) for immunocytochemistry, or plate onto UltraLow adherence well plates for future collection of globoid cells for co-culture with other cell types, such as oligodendrocytes (as described in the following section). 
		NOTE: Once plated the microglia can be treated with psychosine (10 &#181;M) as described above (see section 2.3).</li>
	</ol>
	</li>
	<li>Co-culture with Primary Oligodendrocytes for Analysis of Cytotoxicity. 
	NOTE: Collection of psychosine-treated microglia from the low adherence plates for subsequent co-culture onto primary cultures of oligodendrocytes, or oligodendrocyte progenitor cells, can be used to examine the potential cytotoxic function of these globoid-like cells in vitro.
	<ol>
		<li>Develop cultures of primary oligodendrocytes using established methods18,20. To collect the psychosine-treated microglia, gently pipet to detach the loosely adherent cells from the bottom of the wells. Collect the cells, centrifuge at 300 x g for 10 min, and then carefully resuspend in oligodendrocyte differentiation media [composed of Neurobasal media, L-glutamine (1x), B-27 supplement (1x), and Triiodothyronine (T3, 10 ng/ml)].</li>
		<li>Count the number of cells using a hemocytometer and then seed the microglia onto the oligodendrocyte culture at 1 x 105 cells/ml, resulting in an approximate 1:2 microglia/oligodendrocyte ratio. Incubate as a co-culture for up to 3 days.</li>
		<li>Fix the cells for immunocytochemistry (see section 2.4). Collect cell culture media for ELISA or chemical analyses as needed.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Rafi, M. A., Luzi, P., Chen, Y. Q., Wenger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+deletion+together+with+a+point+mutation+in+the+GALC+gene+is+a+common+mutant+allele+in+patients+with+infantile+Krabbe+disease.">A large deletion together with a point mutation in the GALC gene is a common mutant allele in patients with infantile Krabbe disease.</a> Hum Mol Genet. 4, 1285-1289 (1995).</li><li>Duffner, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+outcomes+of+presymptomatic+infants+transplanted+for+Krabbe+disease:+report+of+the+workshop+held+on+July+11+and+12.">The long-term outcomes of presymptomatic infants transplanted for Krabbe disease: report of the workshop held on July 11 and 12.</a> Genet Med. 11, 450-454 (2008).</li><li>Duffner, P. K., Jalal, K., Carter, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hunter's+Hope+Krabbe+family+database.">The Hunter's Hope Krabbe family database.</a> Pediatr Neurol. 40, 13-18 (2009).</li><li>Duffner, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newborn+screening+for+Krabbe+disease:+the+New+York+State+model.">Newborn screening for Krabbe disease: the New York State model.</a> Pediatr Neurol. 40, 245-252 (2009).</li><li>Krabbe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+familial,+infantile+form+of+brain+sclerosis.">A new familial, infantile form of brain sclerosis.</a> Brain. 39, 74-114 (1916).</li><li>Percy, A. K., Odrezin, G. T., Knowles, P. D., Rouah, E., Armstrong, D. 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A morphologic study of two cases.</a> Acta Neuropathol. 53, 87-91 (1981).</li><li>Ijichi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-3+mediates+psychosine-induced+globoid+cell+formation:+Implications+for+leukodystrophy+pathology.">MMP-3 mediates psychosine-induced globoid cell formation: Implications for leukodystrophy pathology.</a> Glia. , (2013).</li><li>Pellegatta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+potential+of+neural+stem/progenitor+cells+in+murine+globoid+cell+leukodystrophy+is+conditioned+by+macrophage/microglia+activation.">The therapeutic potential of neural stem/progenitor cells in murine globoid cell leukodystrophy is conditioned by macrophage/microglia activation.</a> Neurobiol Dis. 21, 314-323 (2006).</li><li>Crocker, S. 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The authors declare no conflicts of interest.

The protocol described herein provides a new model system in which to study the development and functional characterization of activated microglia and globoid cells. Prior work by Im et al. using a HEK293 cell line provided a template for the development of the present protocol for the study of globoid cell formation21. It is also important to point out that the globoid cells derived in the model differ from the native globoid cells identifiable in GLD. For instance, we have routinely observed quadran...

Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a fatal demyelinating disease resulting from loss of function mutations in the galatocerebrosidase (galc) gene1. The most prevalent form of GLD is the infantile variant which is typified by onset in early childhood and characterized by an aggressive clinical course of motor and cognitive decline leading to premature death often before five years of age2,3. Genetic testing is used to verify a diagnosis of GLD4. Neuropathology of GLD reveals widespread demyelination, neuronal atrophy, astrogliosis and presence of engorged multi-nucleated microglia called globoid cells5-7. The identification of globoid cells, often containing tubulous inclusions in their cytoplasm, has been a defining feature of GLD for the past 97 years, although the specific function of these conspicuous cells has remained elusive.
The involvement of non-myelinating glia (microglia and astrocytes) in the pathogenesis of GLD has long been considered a secondary response to the profound demyelination in this disease8. Interestingly, the first description of this disease, made by Knud Krabbe in 19165, reported formation of multinucleated phagocytes containing lipid debris that have been named &#39;globoid cells&#39; and are a defining characteristic of this disease.
Globoid cells are the hallmark feature of GLD pathology, although their role in GLD has long been ignored. Interestingly, these cells are among the earliest characteristic changes in CNS tissue of GLD. This lack of knowledge may have been due to the assumption that the formation of multinucleated phagocytes, called giant cells in other diseases, are typically considered as a consequence of pathology rather than an initial pathogenic driving force9. Therefore, there have been few studies investigating the mechanism by which globoid cells are formed from phagocytes, particularly in the CNS of GLD. The procedure described in this report focuses on the importance of globoid cell formation in the CNS and our previous demonstration that psychosine-induced multinucleation of microglia in vitro and these cells exhibited higher levels of phagocytic activity. Consistent with these observations, globoid cells in twitcher brains frequently contain PAS-positive debris, suggesting high levels of phagocytic activity. Globoid cells are also found to be immunopositive for ferritin (a microglia marker)10, KP-1/CD68 (a monocyte marker), and some are also positive for vimentin (an intermediate filament protein and marker of astrocytes and activated microglia)11, HLA-DRa (an MHCII surface receptor), and TNF-&#945;7, and Iba-1 (a calcium binding protein used to identify microglia)12. Based on this collection of markers, globoid cells originate from microglia that develop a unique phenotype.
Despite their uniqueness, the specific function and contribution of GCs to GLD pathogenesis has been largely overlooked. Globoid cells have been thought to be a secondary consequence of chronic demyelination. However, past studies examining the temporal association of globoid cells to the white matter pathology of GLD have identified the presence of globoid cells in the late embryonic to early postnatal periods; times preceding oligodendrocyte apoptosis and overt demyelination13. Thus, the temporal sequence of development of the neuropathology in GLD suggests that globoid cells are formed in advance of demyelination in this disease14. This led to our hypothesis that the early formation of globoid cells in GLD may represent a defining pathogenic event rather than a secondary, reactive response to oligodendrocyte damage15. Additionally, dysregulation of microglial activity in GLD has been considered a factor limiting the long-term efficacy of hematopeotic stem cell therapies for treating this disease16. Thus, investigating the cellular functions and regulation of microglia, and globoid cells, in response to psychosine is expected to provide new insights in the pathogenesis of GLD.
Until recently, the lack of an appropriate model in which to study globoid cell formation had limited the understanding of the precise function and contribution of these cells to the pathology of GLD. In recent studies, it was determined that globoid-like cells can be formed in direct response to psychosine, a pathogenic lipid toxin that accumulates in GLD. We found that microglia, but not macrophages, are activated and transformed into globoid cells in primary glial cultures in response to psychosine15. This transformation into globoid cells was found to be mediated by the extracellular protease, matrix metalloproteinase (MMP)-315. More recently, we have extended these findings and determined that psychosine-activated microglia and globoid cells developed in this in vitro model system are potently toxic to oligodendrocytes and oligodendrocyte progenitor cells. Hence, when considered in the context of GLD, the early accumulation of psychosine and formation of globoid cells prior to demyelination would support an emerging primary and possibly pathogenic role for microglia in this disease.
We propose that study of globoid cell formation will reveal new information about the pathogenesis of GLD that will contribute to our understanding of this disease. Moreover, this new cellular model of GLD may provide a new format from which novel therapeutic approaches to address pathological changes in this disease could be tested. Hence, in this report we provide a detailed protocol for the in vitro development of psychosine-induced globoid cells from primary cultures of non-myelinating glia.

<table><tbody><tr><td>Hank&amp;rsquo;s balanced salt solution (HBSS) containing no cations (Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;).</td><td>Life technologies</td><td>14175-095</td><td></td></tr><tr><td>Neural Tissue Dissociation Kit</td><td>Miltenyi</td><td>130-092-628</td><td></td></tr><tr><td>40 &amp;#956;m cell-strainer</td><td>Fisherbrand</td><td>22363547</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution (HBSS) containing cations (Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;).</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Life technologies</td><td>15070-063</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Life technologies</td><td>25299-056</td><td></td></tr><tr><td>Psychosine</td><td>Sigma</td><td>P9256</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Science</td><td>19208</td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td>Invitrogen</td><td>PCN5000</td><td></td></tr><tr><td>Iba-1</td><td>WAKO</td><td>019-19741</td><td></td></tr><tr><td>Alexa Fluor conjugated antisera</td><td>Life Technologies</td><td>Various</td><td></td></tr><tr><td>Mounting Media</td><td>Southern Biotech</td><td>OB100-01</td><td></td></tr><tr><td>Phagocytic Assay Kit</td><td>Cayman Chemicals</td><td>500290</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>BP310-500</td><td></td></tr></tbody></table>

an in vitro model for the study of cellular pathophysiology in globoid cell leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

This protocol, as written, is expected to take approximately 36 days to complete from start to finish (See Figure 1: Experimental Workflow Scheme). It has been our experience that the development of &#39;globoid-like&#39; cells in this primary culture system is both reliable and reproducible: the formation of multinucleated cells in response to psychosine is consistently observed with 7 days of treatment.
Immunocytochemical staining of microglia using Iba-1 in conjunction with...

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An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

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Research

JoVE Journal

Neuroscience

7.8K Views.  University of Connecticut Health Center. Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells. 

Video: An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Developmental Biology

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunology and Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Medicine

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Biology

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Summary

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Chapters in this video

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Derivation of Glial Restricted Precursors from E13 mice

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions