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. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globoid+cell+leukodystrophy:+comparison+of+neuropathology+with+magnetic+resonance+imaging.">Globoid cell leukodystrophy: comparison of neuropathology with magnetic resonance imaging.</a> Acta Neuropathol. 88, 26-32 (1994).</li><li>Itoh, M., 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=Immunohistological+study+of+globoid+cell+leukodystrophy.">Immunohistological study of globoid cell leukodystrophy.</a> Brain Dev. 24, 284-290 (2002).</li><li>Reddy, A. S., Patel, J. R., Vogler, C., Klein, R. S., Sands, M. 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 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...

Watch this Scientific Journal Video about An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy at JoVE.com

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 ビュー.  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. 

ビデオ: 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.

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An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

development of an insert co-culture system of two cellular types in the absence of cell-cell contact

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell...

an in vitro model for measuring immune responses to malaria in the context of hiv co-infection

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Transfer the plates to a laminar flow hood, and use a P1000 pipette to vigorously aspirate and dispense the coated-beads solutions to detach the cells from the plate bottoms, and transfer the supernatants into individual...

an in vitro hemodynamic loop model to investigate the hemocytocompatibility and host cell activation of vascular medical devices

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...