Name: protocol for three-dimensional confocal morphometric analysis of astrocytes
Uploaded: 2015-12-11T14:00:00
Description: Watch this Scientific Journal Video about Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes at JoVE.com

The authors have nothing to disclose.

This study was carried out in accordance with the policies set forth in the Guide for the Care and Use of Laboratory Animals (NIH) approved by Ethic Committee of Iran University of Medical Sciences (Tehran, Iran).
1. Animals, Surgery and Specimen Preparations
NOTE: Prepare brain tissue for 3D confocal microscopic analysis.
<ol>
	<li>Divide animals randomly into two groups: A&#946;1-40&#8210;injection (n = 8), and A&#946;1-40&#8210;injection w...

<ol>
	<li>Barres, 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=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Pellerin, L., 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=Activity-dependent+regulation+of+energy+metabolism+by+astrocytes:+an+update.">Activity-dependent regulation of energy metabolism by astrocytes: an update.</a> Glia. 55 (12), 1251-1262 (2007).</li><li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119 (1), 7-35 (2010).</li><li>Pekny, M., Nilsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+activation+and+reactive+gliosis.">Astrocyte activation and reactive gliosis.</a> Glia. 50 (4), 427-434 (2005).</li><li>Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dissection+of+reactive+astrogliosis+and+glial+scar+formation.">Molecular dissection of reactive astrogliosis and glial scar formation.</a> Trends Neurosci. 32 (12), 638-647 (2009).</li><li>Anderova, 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=Cell+death/proliferation+and+alterations+in+glial+morphology+contribute+to+changes+in+diffusivity+in+the+rat+hippocampus+after+hypoxia-ischemia.">Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia-ischemia.</a> J Cereb Blood Flow Metab. 31 (3), 894-907 (2011).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+astroglial+morphology+and+proliferation+in+vitro.">Neuronal regulation of astroglial morphology and proliferation in vitro.</a> J Cell Biol. 100 (2), 384-396 (1985).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A stereotaxic atlas of the rat brain. , (1998).</li><li>Kirby, E. D., Jensen, K., Goosens, K. A., Kaufer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+surgery+for+excitotoxic+lesion+of+specific+brain+areas+in+the+adult+rat.">Stereotaxic surgery for excitotoxic lesion of specific brain areas in the adult rat.</a> J Vis Exp. (65), e4079 (2012).</li><li>Bagheri, 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=Amyloid+beta(1-40)-induced+astrogliosis+and+the+effect+of+genistein+treatment+in+rat:+a+three-dimensional+confocal+morphometric+and+proteomic+study.">Amyloid beta(1-40)-induced astrogliosis and the effect of genistein treatment in rat: a three-dimensional confocal morphometric and proteomic study.</a> PloS One. 8 (10), e76526 (2013).</li><li>Chvatal, A., Anderova, M., Kirchhoff, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+confocal+morphometry+-+a+new+approach+for+studying+dynamic+changes+in+cell+morphology+in+brain+slices.">Three-dimensional confocal morphometry - a new approach for studying dynamic changes in cell morphology in brain slices.</a> J Anat. 210 (6), 671-683 (2007).</li><li>Kulkarni, P. 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=Quantitative+3-D+analysis+of+GFAP+labeled+astrocytes+from+fluorescence+confocal+images.">Quantitative 3-D analysis of GFAP labeled astrocytes from fluorescence confocal images.</a> J Neuroscie Methods. 15 (246), 38-51 (2015).</li><li>Wagner, D. C., 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=Object-based+analysis+of+astroglial+reaction+and+astrocyte+subtype+morphology+after+ischemic+brain+injury.">Object-based analysis of astroglial reaction and astrocyte subtype morphology after ischemic brain injury.</a> Acta Neurobiol Exp. 73 (1), 79-87 (2013).</li></ol>

In the current protocol, we employed 3D confocal morphometry to evaluate 12 different parameters that were associated with astrocyte morphology. For this purpose, hippocampal tissue of rats with A&#946;1-40&#8210;induced astrogliosis, with or without genistein pretreatment as an anti-inflammatory agent were used. By using 3D images and morphometric software, we were able to show the effect of genistein on astrogliosis i.e. the morphology of astrocytes.
Changes in...

In healthy central nervous system (CNS), astrocytes play an important role in the regulation of blood flow, energy metabolism, synaptic function and plasticity, and extracellular ion and neurotransmitter homeostasis1-3. In addition, astrocytes respond to different harmful stimuli and abnormal conditions such as trauma, infection, ischemia or neurodegeneration via reactive astrogliosis which is characterized by hypertrophy, proliferation and functional remodeling of astrocytes4,5.
Reactive astrogliosis can engineer the inflammatory response and repair process in the tissue and, therefore, can affect the clinical outcome of therapeutic interventions. Accordingly, astrocytes have received attention from neuroscientist during the last decades as potential targets for therapeutic interventions for a variety of diseases affecting the CNS.
Astrocytes normally have a stellate shape with well-defined branches that spread around the soma6. In a diseased condition in the brain, astrocyte branches become convoluted and show swollen ends7, for example in the presence of amyloid beta (A&#946;).
This article presents a protocol for analyzing 3D images of astrocytes acquired by confocal microscopy. Twelve different quantitative parameters for each astrocyte were measured: the surface areas and volumes of the astrocyte territory (the tissue covered by an astrocyte), entire cell (including branches), cell body, and nucleus; the total length and number of branches; the fluorescence intensity of antibodies used for astrocyte detection; and the density of astrocytes (number/1,000 &#181;m2). For this purpose, we used brain sections from rats exposed to intrahippocampal injection of A&#946;1-40 with or without genistein treatment as an anti-inflammatory substance. The described protocol can be used for morphometric analysis of different cell types in vitro or in vivo in different conditions.

<table border="0">
	<tbody>
		<tr>
			<td>Amyloid beta 1-40</td>
			<td>Sigma Aldrich</td>
			<td>79793</td>
			<td>Keep in -70 &#176;C</td>
		</tr>
		<tr>
			<td>Genistein</td>
			<td>Sigma Aldrich</td>
			<td>446-72-0</td>
			<td>keep in -20 &#176;C</td>
		</tr>
		<tr>
			<td>polycolonal rabbit antibodies against glial fibrillary acidic protein</td>
			<td>DAKO</td>
			<td>Z0334</td>
			<td></td>
		</tr>
		<tr>
			<td>alkaline phosphate-conjugated swine anti-rabbit IgG antibodies</td>
			<td>DAKO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Permanent Chromogen</td>
			<td>DAKO</td>
			<td>K0640</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid permanent Red Substrate Buffer</td>
			<td>DAKO</td>
			<td>K0640</td>
			<td></td>
		</tr>
		<tr>
			<td>Cremophor EL</td>
			<td>Sigma Aldrich</td>
			<td>27963</td>
			<td>Polyethoxylated castor oil - Step 1.1</td>
		</tr>
		<tr>
			<td>LSM 700 Confocal Laser Scanning Microscopy</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity 6.3</td>
			<td>Perkin Elmer Inc.,</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis 2000</td>
			<td>Tekno Optic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streotaxic apparatus</td>
			<td>Stoelting</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graph pad Prism 5</td>
			<td>Graph pad software Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for three-dimensional confocal morphometric analysis of astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

This section presents some examples of the qualitative and quantitative observations produced by 3D morphometric analysis. For complete results of all 12 parameters mentioned earlier, please see our previous publication10.
Qualitative Observations
Astrocytes exhibited thin or thick branches that were usually long in A&#946;1-40 injected rats (Figure 1). A few small stellate-shaped astrocytes with short branches were observed in...

Watch this Scientific Journal Video about Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes at JoVE.com

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

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