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 the intra- and extra cellular environment of the brain tissue can alter volume and/or size of cells/tissue. These alterations are considered pathological hallmarks in various brain injuries3,11. In order to quantify these alterations, a number of techniques are used. Most investigations are based on using 2D low resolution images captured by light- or electron microscopy (EM) that give information only about a small fraction of the cell. To overcome this limitation, 3D confocal morphometry on high resolution images was developed11. Another advantage of confocal microscopy is that the architecture of a cell can be studied since several consecutive 2D images are obtained while Z-Stack imaging is performed. In addition, 3D confocal microscopy raises the possibility to improve the quality of the images by filtering background noise11.
The method presented here is time-consuming; taking Z-Stack images with a confocal microscope takes several minutes for each single cell. In addition, manually marking a structure in software requires some practice, and the researcher may need to redo the drawing several times to be sure that the structure is correctly marked. It should be mentioned that the &#8216;Magic&#8217; tab in 3D image analysis software such as Volocity, can be used to automatically mark all DAPI&#8210;stained nuclei in a single image. This option can be used when astrocytes are cultured. The brain sections, however, contain not only astrocytes but also many other cells such as microglia and neurons. This means that many of the DAPI-stained nuclei do not belong to astrocytes. The nuclei associated with astrocytes should be manually marked using images having both DAPI-labeled nuclei and GFAP-immunoreactive astrocytes.
In this protocol, antibodies against GFAP were used to visualize astrocytes in paraffin sections. Astrocytes can also be visualized by using other antigens12,13, or transgenic mice. The investigators should be aware of limitations of the methods, and use the best possible experimental design based on their aims. The limitation of using antibodies against GFAP, for example, is that only 10-15% of the cytoskeleton can be detected. This limitation, however, can become an advantage when studying astrogliosis; the increase number of astrocytes and a dense network of astrocytes&#8217; branches in astrogliosis can make it difficult to identify a single cell if a larger area of the cell is detected. Although this protocol describe the quantification of 12 different parameters but the investigator can choose the ones that suit their aim. According to our experience, the measurements of all parameters can give valuable information about the parameters that are significantly affected in a certain condition or by a treatment. The quantification method performed on 3D pictures captured in confocal microscope can also be performed on frozen sections. The issue is to treat the samples (fixation, dehydration, embedding and sectioning) in a similar way since these steps can lead to shrinkage or swelling of the tissue.
In conclusion, 3D confocal imaging, in combination with morphometric software, makes it possible to quantify several parameters associated with the morphology of a cell. The protocol we presented here can be used for evaluation of morphological changes that appear in a cell, in different pathological conditions, or as the effect of an intervention strategy.

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><tbody><tr><td>Amyloid beta 1-40</td><td>Sigma Aldrich</td><td>79793</td><td>Keep in -70 &amp;deg;C</td></tr><tr><td>Genistein</td><td>Sigma Aldrich</td><td>446-72-0</td><td>keep in -20 &amp;deg;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 the cerebral cortex, and a compact network of astrocytes occurred in the corpus callosum.
Astrocytes in the brain tissues of animals with A&#946;1-40&#8210;injection followed by genistein treatment exhibited a stellate form with short and thin branches (Figure 2)10. A few astrocytes showed an atrophic appearance.
Astrocyte Density and GFAP+ Fluorescence Intensity
The mean number of astrocytes/1,000 &#181;m2 was significantly higher in rats with A&#946;1-40&#8210;injection in comparison with animals in A&#946;1-40&#8210;injection and genistein treatment group (P &#60;0.0001). In addition, the GFAP+ fluorescence intensity was significantly lower in the brain section with genistein treatment compared with non-treated A&#946;1-40 injected animals (Figure 3).
Morphometric Analysis of Astrocytes Volume
The volume of the astrocyte nucleus, cell body, entire astrocyte and astrocyte territory decreased significantly in the genistein treatment group compared with untreated animals. This result suggests that genistein ameliorated the astrogliosis caused by the presence of amyloid (Figure 4A-D).
<img alt="Figure 1" src="/files/ftp_upload/53113/53113fig1.jpg" /> 
Figure 1.&#160;Confocal image of an astrocyte. An individual astrocyte with thick and long branches (arrowhead) and DAPI-labeled nucleus (arrow) in a rat brain subjected to hippocampal injection of A&#946;1-40. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53113/53113fig2.jpg" /> 
Figure 2.&#160;Confocal image of an astrocyte. An astrocyte with short branches (arrowhead). In a rat brain subjected to hippocampal A&#946;1-40&#8210;injection, and genistein pre-treatment administered by gavage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53113/53113fig3.jpg" /> 
Figure 3.&#160;GFAP+ fluorescence intensity decreased significantly in animals with A&#946;1-40&#8210;injection that received genistein pre-treatment. Values are mean &#177; SEM. Fifty astrocytes per animal were evaluated. P &#60;0.05 was regarded as significant, and T-test was used to compare the data between groups with or without treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53113/53113fig4.jpg" /> 
Figure 4.&#160;The mean volume of nucleus (A), cell body (B), astrocyte (including branches; (C), and territory (D) in brain samples taken from rats subjected to A&#946;1-40&#8210;injection, with or without genistein pre-treatment. Genistein significantly ameliorated the enlargement of nucleus, cell body, the entire astrocyte, and astrocyte territory (see ref10). Values are mean &#177; SEM. Fifty astrocytes per animal were evaluated. P &#60;0.05 was regarded as significant, and T-test was used to compare the data between groups before and after treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53113/53113fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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8.8K 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

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 ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Biology

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network level. Astrocytes are strongly connected to each other through gap junctions and coupling through these junctions is dynamic and highly regulated. An emerging concept is that astrocytic functions are specialized and adapted to the functions of the neuronal circuit with which they are associated. Therefore, methods to measure various parameters of astrocytic networks are needed to better describe the rules governing their communication and coupling and to further understand their functions.
Here, using the image analysis software (e.g., ImageJFIJI), we describe a method to analyze confocal images of astrocytic networks revealed by dye-coupling. These methods allow for 1) an automated and unbiased detection of labeled cells, 2) calculation of the size of the network, 3) computation of the preferential orientation of dye spread within the network, and 4) repositioning of the network within the area of interest.
This analysis can be used to characterize astrocytic networks of a particular area, compare networks of different areas associated to different functions, or compare networks obtained under different conditions that have different effects on coupling. These observations may lead to important functional considerations. For instance, we analyze the astrocytic networks of a trigeminal nucleus, where we have previously shown that astrocytic coupling is essential for the ability of neurons to switch their firing patterns from tonic to rhythmic bursting1. By measuring the size, confinement, and preferential orientation of astrocytic networks in this nucleus, we can build hypotheses about functional domains that they circumscribe. Several studies suggest that several other brain areas, including the barrel cortex, lateral superior olive, olfactory glomeruli, and sensory nuclei in the thalamus and visual cortex, to name a few, may benefit from a similar analysis.

Here we present a protocol to assess the organization of astrocytic networks. The described method minimizes bias to provide descriptive measures of these networks such as cell count, size, area, and position within a nucleus. Anisotropy is assessed with a vectorial analysis.

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...

Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

Source: Eaker, A. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63804">Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for immunostaining a fixed mouse brain tissue section with fluorescently labeled astrocytes using primary and secondary antibodies and capture the images of the stained astrocytes through confocal microscopy.

Source: Eaker, A. R., et al. Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections. J. Vis. Exp. (2022)
This video ...