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 with genistein treatment (n = 8); administer genistein (10 mg/kg diluted in a vehicle such as polyethoxylated castor oil) by gavage 1 hr before surgery.</li>
	<li>Anesthetize the animals with ketamine (100 mg/kg) and xylazine (10 mg/kg). Confirm proper anesthesia by lack of withdrawal reflex after pinching the toe. Use ointment on eyes during the surgery to prevent dryness.</li>
	<li>Place animals in stereotaxic apparatus and shave head. Apply iodine solution to clean the scalp before incision and keep the operation site sterile during the surgery to reduce the risk of infection. Inject A&#946;1-40 stereotaxically (4 &#181;l) in the hippocampus at &#8722;3.5 mm posterior to bregma, &#177; 2 mm lateral to midline, and &#8722;2.8 mm below dura, according to rat brain atlas8. For stereotaxic method please see previous publication9.</li>
	<li>Provide post-operative observation/care until the animals are fully conscious to ensure the comfort of the animals. Keep the animals warm during recovery, and do not return them to a cage with other animals until full recovery. Pay attention to any sign of infection in the animals after surgery.</li>
	<li>Anesthetize the animals deeply by ketamine (150 mg/kg) three weeks after surgery. Perform transcardial fixation by perfusing the animals with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS (pH = 7.4), then remove and fix brain as described in10.</li>
	<li>Post-fix the brains in 4% paraformaldehyde for 2-3 days at 4 &#730;C.</li>
	<li>Embed the tissue in paraffin blocks by use of tissue embedding equipment, and avoid under-filling or over-filling the cassette since it may interfere with correct alignment or sectioning. Pay attention to the orientation of the tissue in the paraffin block. 
	NOTE: The rat hippocampus, for example, is close to posterior part of the cerebral hemisphere. Thus, the tissue should be embedded in a way that sectioning starts at posterior part of the brain. Use the Paxinos atlas8 as a guide to reach the desired anatomical structure.</li>
	<li>Place the microtome away from air drafts or doorways since air movements make the handling of sections difficult.</li>
	<li>Use sections with a thickness &#8805;20 &#181;m as this depth with be necessary to produce Z-Stack images of complete astrocytes. Do not use the first couple sections, as they may have undesired thickness due to thermal expansion.</li>
	<li>Place the sections on the surface of warm water (5 &#177; 2 &#730;C, below the melting temperature for paraffin) just long enough to flatten the sections. 
	NOTE: Over expansion of the section can disturb the morphology of the structure. Use tiny paintbrush to carefully transfer sections. Collect two to three sections on each slide.</li>
	<li>Store slides in a rack in an upright position and let them dry at 37 &#730;C for several hr or O/N.</li>
</ol>
2. Immunohistochemistry
<ol>
	<li>Deparaffinize sections in xylene, 2 x 10 min, and rehydrate through alcohol gradient (99.8%, 95%, and 70%, 10 min each) and PBS.</li>
	<li>Incubate with antibodies against glial fibrillary acidic protein (GFAP) diluted 1:1,500 in PBS containing 0.25% bovine serum albumin (BSA), 0.25% Triton X-100 and 3.5% normal serum (O/N at 4 &#730;C).</li>
	<li>Wash sections in PBS for 3 x 5 min.</li>
	<li>Incubate with secondary alkaline phosphate-conjugated antibodies for 1h (1:100, 21 &#730;C, diluted in PBS).</li>
	<li>Wash the sections in PBS for 3 x 5 min. 
	NOTE: It is important to wash the sections properly after secondary antibodies to minimize background staining.</li>
	<li>Incubate sections in darkness with Liquid Permanent Red Chromogen (15-20 min). Note: Prepare the solution no more than 30 min before use.</li>
	<li>Wash sections in PBS for 3 x 5 min.</li>
	<li>Finally, stain the nuclei with 4-6-diamidino-2-phenylindole (DAPI; 1:500, diluted in PBS) prior to cover-slipping. Store the slides in the refrigerator 24-48 hr before microscopy.</li>
</ol>
3. Confocal Microscopy
NOTE: For quantitative evaluation (see below), select an astrocyte with a clearly visible DAPI-stained nucleus with a minimum of overlapping branches to reduce the risk of errors. Producing Z-Stack images is time consuming. Be patient and do not stop during processing. Upright confocal laser scanning microscope is used to create 3D images from astrocytes.
<ol>
	<li>Put the slide on the microscope-stage, and select the 63X objective.</li>
	<li>Open the imaging software and click on Start system. Click on the Locate tab. Go to Assign, and select GFP to adjust the proper light.</li>
	<li>Open the Shutter to reflect light. Find the Reflector Revolver on the right side of Shutter and choose the color that should be reflected by astrocytes (green, red or violet); red color (FSet20 wf) was used here. 
	NOTE: Do not forget to focus the picture directly in the ocular of the microscope.</li>
	<li>To find the best focus, click on All closed, put the microscope pin in screen mode, and click on the Acquisition tab.</li>
	<li>Click on Smart Setup under the Acquisition tab; a window opens. Select the proper fluorophore (i.e. DAPI and Alexa Fluor 555, in the current project) from the list. The color is automatically selected.</li>
	<li>Click on Best signal, and Apply, then click on Set Exposure; the computer will then automatically set the exposure parameters.</li>
	<li>For optimizing the image, go to the Channels window. Unmark Track2, highlight Track1 and click on Live. Focus the image if needed.</li>
	<li>In the Channels window adjust the following keys; click on&#160;1AU to automatically optimize the pinhole. It is very important to do this step otherwise the confocal images will be non-optimal. Adjust Gain to have the best intensity (keep this setting below 800 to reduce extra noise). Finally, set the Digital Gain between 2 and 3.</li>
	<li>Unmark Track1, click on Track2 and repeat Step 3.8 for Track2.&#160;Then stop Live.</li>
	<li>Go to the Acquisition Mode window. Improve the image by optimizing Frame Size and Averaging. In order to change frame size go to X*Y and select 1,024 x 1,024. Choose a slow Speed to create a better image.</li>
	<li>Go to Averaging and select a Number &#8805;4. This averaging value indicates the number of scans that will be averaged to produce the acquired image. Now click on the Snap button, and save the captured 2D image.</li>
	<li>For 3D imaging, mark the Z-Stack item under Smart Setup causing the Z-Stack window to open.</li>
	<li>Set the first and last positions for the Z-Stack, i.e. the depth of the cell. First click on Live. Then use the focus drive of the microscope to focus on the uppermost position of astrocyte in the tissue, where the Z-Stack is to start. Click on Set First. Then focus down to the lowest position of the astrocyte in the tissue, where Z-Stack scanning should be stopped. Click on Set Last. Then stop Live.</li>
	<li>Choose the Interval; 1.01 &#181;m is used in the current study; choosing interval can be done by clicking on Optimal to set the number of slices.</li>
	<li>Click on Start Experiment. Save the images once the scan is complete. This image will be used for the measurement of the following parameters: surface area and volume of astrocyte territory, the entire astrocyte including branches, cell body, and nucleus.</li>
	<li>Acquire a 2D image using a 10X or 20X objective as described in Steps 3.6-3.11. This image can be used for the measurement the intensity of fluorescence immunoreactivity of antibodies, and astrocyte density (number/1,000 &#181;m2).</li>
</ol>
4. Astrocyte Density and GFAP+ Fluorescence Intensity
<ol>
	<li>Open the 20X 2D image. Click on Histo (Hisogram) on the left side of image window.</li>
	<li>To calculate astrocyte density, select three consecutive visual fields under 20X objective. Count the number of astrocytes that exhibit a clear soma with minimum, overlapping branches in the fields.</li>
	<li>To quantify fluorescence intensity, select a proper tool (rectangle or circle) on the footer of image window, and choose an area for the measurement. Note the mean value and standard deviation appear automatically under the image. In the current study, a 0.7 mm2 rectangular area between striatum and medial blade of dentate gyrus was used.</li>
</ol>
5. Astrocyte Branches
<ol>
	<li>To quantify the length and total number of branches, use 3D images (acquired with 20X) in the image analyzing software. Draw a line manually along the length of each branch of the selected astrocyte. Then select the measurement-tool that is specialized for measuring line-length. In order to quantify the number of branches, select the count-tool to count the number of marked items.</li>
</ol>
6. Volume and Surface Area
NOTE: Marking a structure manually in 3D image analysis software requires focus and training. Practice several times before starting an experiment.
<ol>
	<li>To quantify the volume and surface area of the astrocyte nucleus, soma, cell body and territory, transfer the 3D images (acquired in Steps 3.1-3.16) to a 3D image analysis software such as Volocity 6.1.</li>
	<li>Open the software and create a new library. Drag all images to the left gray column in the new library. Then, select one 3D image. On the right, there are different channels with different colors. Turn on one channel of interest; for example the blue channel for DAPI when measuring the nucleus. Change the brightness manually to get an optimal contrast.</li>
	<li>Select the Tools menu, go to Make Volume and produce a single 3D image out of Z-stack images. Click on Properties in the Edit menu and be sure that X, Y and Z properties are presented; otherwise it is impossible to carry out 3D analysis.</li>
	<li>Click on Freehand RIO tool icon in the toolbar. To measure the volume and surface area of astrocyte nucleus draw a line around the DAPI&#8210;labeled nucleus.</li>
	<li>To measure the volume and surface area of astrocyte cell body draw a line around the soma excluding branches.</li>
	<li>To measure the volume and surface area of entire astrocyte (cell body including branches) draw a line around the soma following the surface of all branches.</li>
	<li>To assess the volume and surface area of astrocyte territory draw a line between tips of the branches. 
	NOTE: Press Delete on the keyboard to erase unwanted drawings.</li>
	<li>Click on the Measurements menu at the top of the image window to create a measurement protocol; a window containing different tools opens.</li>
	<li>Drag Find objects to top of the window (pane). Give a descriptive name to the Population&#160;1, select the associated color, and click on Measure. Another window opens.</li>
	<li>Choose a parameter i.e., volume or surface area. Click on OK. The data will appear in a table.</li>
	<li>To save the data click on the Measurement menu and select Make Measurement Item.</li>
	<li>Select A new measurement item called from the opened window. Then give a name to the data and click on OK.</li>
	<li>Finally, export the data to statistics software such as Graph pad or excel program. Use t-test to analyze the data, then plot 2D graphs.</li>
</ol>

<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>

This work was supported by grants from the County Council of &#214;sterg&#246;tland, (Sweden) and the Cellular and Molecular Research Center at Iran University of Medical Sciences (Tehran, Iran).

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 ...

protocol-for-three-dimensional-confocal-morphometric-analysis

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8.7K Views.  Linköping University. 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.

Video: Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.