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...

<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. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+pathology+progresses+independently+of+KC+and+CXCR2+in+globoid-cell+leukodystrophy.">Central nervous system pathology progresses independently of KC and CXCR2 in globoid-cell leukodystrophy.</a> PLoS One. 8, (2013).</li><li>McNally, A. K., Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+fusion+and+multinucleated+giant+cells+of+inflammation.">Macrophage fusion and multinucleated giant cells of inflammation.</a> Adv Exp Med Biol. 713, 97-111 (2011).</li><li>Kaneko, Y., Kitamoto, T., Tateishi, J., Yamaguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferritin+immunohistochemistry+as+a+marker+for+microglia.">Ferritin immunohistochemistry as a marker for microglia.</a> Acta Neuropathol. 79, 129-136 (1989).</li><li>Graeber, M. B., Streit, W. J., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microglial+cytoskeleton:+vimentin+is+localized+within+activated+cells+in+situ.">The microglial cytoskeleton: vimentin is localized within activated cells in situ.</a> J Neurocytol. 17, 573-580 (1988).</li><li>Kondo, Y., Adams, J. M., Vanier, M. T., Duncan, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+counteract+demyelination+in+a+mouse+model+of+globoid+cell+leukodystrophy.">Macrophages counteract demyelination in a mouse model of globoid cell leukodystrophy.</a> J Neurosci. 31, 3610-3624 (2011).</li><li>Pollanen, M. S., Brody, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+globoid+cell+leukodystrophy.">Fetal globoid cell leukodystrophy.</a> Arch Pathol Lab Med. 114, 213-216 (1990).</li><li>Martin, J. J., 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=Fetal+Krabbe+leukodystrophy.+A+morphologic+study+of+two+cases.">Fetal Krabbe leukodystrophy. 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. J., Milner, R., Pham-Mitchell, N., Campbell, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+agonist-specific+regulation+of+genes+for+matrix+metalloproteinases+and+their+tissue+inhibitors+by+primary+glial+cells.">Cell and agonist-specific regulation of genes for matrix metalloproteinases and their tissue inhibitors by primary glial cells.</a> Journal of Neurochemistry. 98, 812-823 (2006).</li><li>Schildge, S., Bohrer, C., Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+cortical+astrocytes.">Isolation and culture of mouse cortical astrocytes.</a> J Vis Exp. , (2013).</li><li>Crocker, S. J., Frausto, R. F., Whitton, J. L., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+establish+microglia-free+astrocyte+cultures:+comparison+of+matrix+metalloproteinase+expression+profiles+in+pure+cultures+of+astrocytes+and+microglia.">A novel method to establish microglia-free astrocyte cultures: comparison of matrix metalloproteinase expression profiles in pure cultures of astrocytes and microglia.</a> Glia. 56, 1187-1198 (2008).</li><li>Moore, C. 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=Astrocytic+Tissue+Inhibitor+of+Metalloproteinase-1+(TIMP-1)+Promotes+Oligodendrocyte+Differentiation+and+Enhances+CNS+Myelination.">Astrocytic Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes Oligodendrocyte Differentiation and Enhances CNS Myelination.</a> J Neurosci. 31, 6247-6254 (2011).</li><li>Im, D. S., Heise, C. E., Nguyen, T., O'Dowd, B. F., Lynch, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+molecular+target+of+psychosine+and+its+role+in+globoid+cell+formation.">Identification of a molecular target of psychosine and its role in globoid cell formation.</a> J Cell Biol. 153, 429-434 (2001).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="884">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:500px;">Hank&#8217;s balanced salt solution (HBSS) containing no cations (Mg2+ and Ca2+).&#160;</td>
			<td style="width:297px;">Life technologies</td>
			<td style="width:87px;">14175-095</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neural Tissue Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>130-092-628</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 uM cell-strainer</td>
			<td>Fisherbrand</td>
			<td>22363547</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s balanced salt solution (HBSS) containing cations (Mg2+ and Ca2+).&#160;</td>
			<td>Gibco</td>
			<td>14025-092</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>11995-065</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilin/Streptomycin</td>
			<td>Life technologies</td>
			<td>15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA solution</td>
			<td>Life technologies</td>
			<td>25299-056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Psychosine</td>
			<td>Sigma</td>
			<td>P9256</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Science</td>
			<td>19208</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum (NGS)</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iba-1</td>
			<td>WAKO</td>
			<td>019-19741</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor conjugated antisera</td>
			<td>Life Technologies</td>
			<td>Various</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting Media</td>
			<td>Southern Biotech</td>
			<td>OB100-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phagocytic Assay Kit</td>
			<td>Cayman Chemicals</td>
			<td>500290</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma</td>
			<td>BP310-500</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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7.8K Görüntüleme.  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. 