Work was supported by grants from the Smith Family Foundation (B.S.), Dana Foundation (B.S.), John Merck Scholars Program (B.S.), NINDS (RO1-NS-07100801; B.S.), NRSA (F32-NS-066698; D.P.S.), Nancy Lurie Marks Foundation (D.P.S.), NIH (P30-HD-18655; MRDDRC Imaging Core).

1. Anterograde Labeling of RGC Presynaptic Inputs
Note: All experiments involving the use of animals were reviewed and overseen by the institutional animal care and use committee (IACUC) in accordance with all NIH guidelines.
<ol>
	<li>Sterilize field and instruments.</li>
	<li>Anesthetize mouse with 4 vol % isoflurane in a Plexiglas induction chamber (this vol % isoflurane works for neonatal-adult mice). Observe mice closely to avoid over-anesthetizing. CAUTION: Avoid inhalation with a vacuum waste gas evacuation.</li>
	<li>After 1-3 min, (older mice will require less time) ensure appropriate level of anesthesia is achieved by pinching the tail (no reaction should be observed) and observing breathing rate (rate should be slow and steady).</li>
	<li>Place mouse under the stereo microscope on its side and place nose cone delivering 3-4 vol % isoflurane over the snout (neonates &#60; postnatal day 10 (P10) ~4 vol %; &#62;P10 ~3 vol %).</li>
	<li>Expose the sclera. For injection of neonates prior to eye opening, use a pair of small spring scissors to open the eyelid and pull back the skin to expose the sclera. For older mice, use fingers to pull back the skin surrounding the eye to expose the sclera. CAUTION: In neonates, sometimes another perpendicular cut at the corner of the eye is necessary. Take care when cutting the corner of the eyelid as there is a blood vessel that, if cut, will bleed excessively.</li>
	<li>Use a sterile 30.5 G&#160;needle to puncture a small hole in the side of the eye at the line where the sclera begins. Take care to avoid damaging the lens by inserting the needle just far enough that the bevel goes into the eye.</li>
	<li>Allow the vitreous to flow out of the hole and use a sterile cotton-tipped applicator to absorb the liquid.</li>
	<li>Once the vitreous has stopped flowing out of the hole, insert a blunt ended needle attached to a Hamilton syringe preloaded with anterograde tracing dye into the hole. CAUTION: Insert needle slowly to avoid puncturing the other side of the eye or damaging the lens.</li>
	<li>Slowly inject dye into the eye. Typically, cholera toxin &#946; subunit conjugated to Alexa 594, 647, or 488 (CTB-594, CTB-647, or CTB-488, 5-6 &#181;g/&#181;l) is used to anterogradely trace RGC inputs. For neonates (&#8804;P10) 1-2 &#181;l is sufficient and for mice &#62;P10 ~3 &#181;l is sufficient. Note: Alexa dyes are particularly resistant to lysosomal degradation</li>
	<li>Leave the needle in the hole for a few seconds and then slowly remove.</li>
	<li>Use a cotton tipped applicator to absorb excess fluid and prevent dye from leaking out.</li>
	<li>Apply a small amount of antibiotic ointment to the eye. If eye was surgically opened, gently reposition the eyelids together.</li>
	<li>If injecting both eyes, repeat procedure on other eye.</li>
	<li>Following injection(s), leave mouse under a heat lamp or on a heat pad until it begins to recover from the anesthesia.</li>
	<li>Return mouse to a clean home cage and monitor to make sure it is fully awake before returning to the colony.</li>
</ol>
2. Prepare Tissue for Imaging
This tissue preparation protocol is used for reporter mice in which the phagocytes are labeled with fluorescent markers (e.g., CX3CR1-EGFP for microglia). If a reporter line is not available, the investigator can immunostain tissue sections (see Discussion).
<ol>
	<li>~24 hr after the injection, sacrifice the mouse and dissect the brain.</li>
	<li>Drop fix the brain in a Falcon tube filled with 4% paraformaldehyde (PFA) overnight at 4 &#176;C. CAUTION: PFA is toxic. Avoid inhalation and skin exposure by using in a fume hood or on a downdraft table and wearing personal protective equipment. Notes: Fixation can be shorter (&#8805;4 hr). In addition, instead of drop fixing, 4% PFA perfusion can be used followed by a 2-24 hr drop fix. However, comparison of perfused to drop fixed neonatal and adult brains revealed no difference in image quality or quantification.</li>
	<li>Rinse the brain 3x in phosphate buffered saline (PBS).
	<ol>
		<li>Pour the brain and PFA into an empty weigh boat.</li>
		<li>Use a spatula to transfer the brain to another weigh boat filled with PBS.</li>
		<li>Wash brain twice&#160;more&#160;in PBS (transfer brain to two more weigh boats filled with PBS).</li>
	</ol>
	</li>
	<li>Transfer the brain to a Falcon tube filled with a 30% sucrose solution. Leave the brain in sucrose at 4 &#176;C until the brain sinks to the bottom of the tube (24-48 hr).</li>
	<li>Once the brain sinks, prepare the brain for sectioning. The following steps are preparation of 40 &#181;m floating sections using a sliding microtome with a freezing stage (a cryostat can also be used).
	<ol>
		<li>Use a razor blade to remove parts of the brain that are not needed (for the dLGN, remove the rostral and caudal parts of the brain).</li>
		<li>Freeze the brain on aluminum foil over dry ice. During this time, freeze the microtome stage and fill each well of a 24-well plate with 0.5 ml of 0.1 M phosphate buffer (PB).</li>
	</ol>
	</li>
	<li>Mount the frozen brain (it should appear opaque) on the freezing stage.
	<ol>
		<li>Apply a small amount of optimal cutting temperature compound (OCT) to the stage.</li>
		<li>Once the OCT begins to freeze, lay the brain in the OCT. The end of the brain that will be cut should be facing up (e.g., for the dLGN, face the caudal side up).</li>
		<li>Cover the brain and OCT with very finely crushed dry ice.</li>
		<li>Leave dry ice on the brain/OCT for ~30 sec.</li>
		<li>Use a large paint brush to remove the dry ice. The brain and OCT should be frozen to the stage.</li>
	</ol>
	</li>
	<li>Begin sectioning through the tissue until the region of interest (ROI) is reached.</li>
	<li>Once the ROI is reached, use a small paint brush to remove sections from the blade and transfer them to the 24-well plate containing 0.1 M PB (step 2.5.2). Keep the paint brush moist by wetting it in the 0.1 M PB prior to removing sections from the blade. Note: Sections can be left in 0.1 M PB overnight at 4 &#176;C.</li>
	<li>Once sections have been collected, visualize the anterograde labeling under a fluorescent dissecting microscope and choose sections that contain the ROI.</li>
	<li>Mount sections on a slide with a small paint brush.
	<ol>
		<li>Apply a small pool of 0.1 M PB to a charged microscope slide.</li>
		<li>Transfer the tissue section to the pool of PB.</li>
		<li>Use the PB and paint brush to orient and spread the tissue.</li>
		<li>Use a Kimwipe to wick off excess PB. Take care to avoid wicking off the section.</li>
		<li>Allow to completely air dry.</li>
		<li>Apply a small drop of mounting medium to each section and mount a coverslip on top (22 x 50 mm, No. 1.5).</li>
		<li>Seal edges of slide with nail polish and store at -20 &#176;C until imaging session. Note: Although imaging within a week of preparation is advised, slides can be maintained at -20 &#176;C for several weeks prior to imaging.</li>
	</ol>
	</li>
</ol>
3. Imaging Tissue
All images are acquired on a spinning disk confocal microscope (UltraView Vox spinning disk confocal microscope equipped with diode lasers (405 nm, 445 nm, 488 nm, 514 nm, 561 nm, and 640 nm)). Images can also be acquired on any microscope with the ability to acquire high resolution z-stacks (e.g., laser scanning confocal microscope, epifluorescent microscope followed by deconvolution, etc.). Frame size is typically 1,000 x 1,000 pixels.
<ol>
	<li>Find ROI at 10X magnification and acquire an image. For imaging microglia in the retinogeniculate system, the ROI is the most medial dLGN sections.</li>
	<li>Shift to higher magnification (a 60X Plan Apo objective, NA = 1.4, is typically used).</li>
	<li>Acquire first field of view containing phagocytes using 0.2 &#181;m z-steps.</li>
	<li>Acquire 15-19 more fields containing phagocytes per animal.</li>
</ol>
4. Prepare Images for Quantification (ImageJ)
<ol>
	<li>Open z-stack in ImageJ.</li>
	<li>Go to the Image menu, select Color and Split Channels.</li>
	<li>Subtract background from the channel(s) containing the engulfed material.
	<ol>
		<li>Select the channel window containing the engulfed material (e.g., presynaptic inputs).</li>
		<li>Go to the Process menu and select Subtract Background. Use a rolling ball radius of 10 pixels for anterograde tracing of presynaptic inputs in the dLGN. Note: The rolling ball radius is determined empirically. However, it&#160;should generally be as large as the radius of the largest object in the image that is not part of the background.</li>
	</ol>
	</li>
	<li>Smooth the channel containing the phagocyte.
	<ol>
		<li>Select the channel window containing the phagocyte (e.g., microglia).</li>
		<li>Go to the Process menu and select Filters, Mean. Use a mean filter of 1.5 (this smoothes the image for surface rendering in later steps).</li>
	</ol>
	</li>
	<li>Subtract background from the channel containing the phagocyte.
	<ol>
		<li>Select the channel window containing the phagocyte (e.g., microglia).</li>
		<li>Go to the Process menu, select subtract background (settings will need to be empirically determined). Use a rolling ball radius of 50 pixels for EGFP-positive microglia.</li>
	</ol>
	</li>
	<li>Merge channels by going to the Image menu and selecting Color, Merge Channels.</li>
	<li>Crop ROI containing phagocyte from merged file (this makes surface rendering faster, see step 5).
	<ol>
		<li>Draw a box around the ROI using the rectangular selections tool in the tool bar.</li>
		<li>Go to the Image menu and select Crop.</li>
	</ol>
	</li>
	<li>Split channels again (see step 4.2).</li>
	<li>Save each channel as its own TIFF file.</li>
</ol>
5. Prepare Image for Quantification in Imaris
<ol>
	<li>Open Imaris and make sure that the Volume icon is selected in the top left menu (Figure 2).</li>
	<li>Open first channel of cropped image (any of the channels can be opened first, just be consistent).</li>
	<li>Add the other channel(s). Go to the Edit menu, select Add Channels and select the next channel in the file. (Repeat this step for any remaining channels). Note: To change the color, go to the Display Adjustment window and click on the channel name (Figure 3A). This will bring up a dialogue box to adjust the color. If the Display Adjustment window is not visible, go to the Edit menu and select Display Adjustment.</li>
	<li>Adjust the pixel values. Go to the Edit menu and select Image Properties. In this window, adjust x, y, and z pixel values (These values depend on the microscope, objective, camera, and z-step acquisition and can be found in the software used to acquire the image).</li>
	<li>Step 5.5 will result in a very small image. To resize, click on the Fit icon in the bottom right corner (Figure 2).</li>
	<li>In the Display Adjustment window, adjust the brightness and contrast for each channel using the left and middle triangles.</li>
</ol>
6. 3 Dimensional (3D) Surface Rendering of the Phagocyte
<ol>
	<li>In the top tool bar, be sure the Surpass mode is selected (Figure 3C).</li>
	<li>In the Display Adjustment window, uncheck all channels except the phagocyte channel. This will remove them from the field of view.</li>
	<li>Click the Surface Rendering icon (Figure 2).</li>
	<li>A create window will be displayed in the bottom left (Figure 4A), make sure Segment ROI is unchecked in this first window. Click the blue forward button at the bottom.</li>
	<li>Adjust the smoothing.
	<ol>
		<li>In the next window, choose the phagocyte channel from the pull down menu (Figure 4B). This adjusts the amount of detail that will be surfaced and should be determined empirically (0.1 &#181;m smoothing is typically used for microglia).</li>
		<li>Select Absolute thresholding.</li>
		<li>Click on the blue forward button at the bottom.</li>
	</ol>
	</li>
	<li>Threshold the image.
	<ol>
		<li>Click on histogram and drag until image is surfaced appropriately (Figure 4C). This is determined empirically by rotating the image to ensure the grey surfaces overlaid on the fluorescent image are an accurate surface representation. Notes: To rotate, select Navigate in the Pointer menu on the upper right side of the screen (Figure 3B), click and hold image to rotate. To return image to original orientation, select Origin Bottom Left from the View menu.</li>
		<li>Click on the blue forward button at the bottom.</li>
	</ol>
	</li>
	<li>In the next window, there is an option to filter out any surfaces by size, etc. Unless image is very noisy, do not filter and delete any filters that automatically appear. Click the green arrow at the bottom to finish surface rendering the phagocyte. A new set of tabs will appear (Figure 5)</li>
	<li>If necessary, delete any surfaces that are not part of the phagocyte of interest.
	<ol>
		<li>Make sure that the Select option is selected in the Pointer menu (Figure 3B).</li>
		<li>Click on any surface to be deleted so that it turns yellow. To delete multiple surfaces, click on the surfaces while holding down the control button.</li>
		<li>Click Delete under the Pencil tab.</li>
	</ol>
	</li>
	<li>Select and merge all the surfaces of the phagocyte into one surface.
	<ol>
		<li>Click on the Funnel (i.e. Filter tab; Figure 5C).</li>
		<li>Click Add.</li>
		<li>Choose any filter, drag the histogram to the far left (all surfaces should be yellow).</li>
		<li>Go back to the Pencil tab and click Unify.</li>
	</ol>
	</li>
	<li>Go to the Graph tab (Figure 5A) and click on Details and select any parameters to display. Select the volume (Figure 5D). For displaying multiple parameters, go to the Wrench icon at the bottom left (Figure 2) and select parameters to record.</li>
	<li>Record the volume of the phagocyte and save the file before continuing.</li>
</ol>
7. 3D Surface Rendering of Engulfed Material
<ol>
	<li>Create a new channel for material that has been engulfed by the phagocyte.
	<ol>
		<li>While the phagocyte surface is selected, click on the pencil tab.</li>
		<li>Click Mask All.</li>
		<li>In the dialogue that appears, select the channel corresponding to engulfed material (e.g., anterograde tracing of presynaptic inputs; Figure 5B).</li>
		<li>Check duplicate channel before applying mask and hit OK.</li>
		<li>A new channel will appear in the Display Adjustment window that contains only the material that has been engulfed and is inside the phagocyte.</li>
	</ol>
	</li>
	<li>Surface render the channel containing total engulfed and nonengulfed material.
	<ol>
		<li>Uncheck all channels in the Display Adjustment window except for the channel to be surfaced and uncheck the surface created for the phagocyte.</li>
		<li>Click on the Surface rendering icon again (see step 6.3) and create a surface for the channel using the same steps as described for the phagocyte (steps 6.4-6.11). Notes: Be sure to select the correct channel prior to smoothing and thresholding (step 6.5; Figure 4B). Make note of the threshold value (see step 6.6; Figure 4C). This value can also be obtained after the surface rendering is complete (Wand tab; Figure 5A).</li>
	</ol>
	</li>
	<li>Record the volume of the total engulfed and non-engulfed material and save the file before continuing.</li>
	<li>Surface render engulfed material (material within the phagocyte).
	<ol>
		<li>Visualize fluorescence of only the new channel of engulfed material (see step 7.2.1).</li>
		<li>Follow the same steps as above (steps 7.2-7.3). Notes: Be sure to select the correct channel from the pull down menu prior to smoothing (choose the new channel created in step 7.1). In the threshold window, manually enter the threshold value that was set for rendering the total engulfed and non-engulfed material (see step 7.2.2).</li>
	</ol>
	</li>
	<li>Record the volume of the engulfed material and save file before continuing.</li>
</ol>
8. Calculate the Total Volume of the Field of View
<ol>
	<li>Create a new surface for any channel.</li>
	<li>In the thresholding window (step 6.6), slide the histogram all the way to the left so that the entire field is surfaced.</li>
	<li>Finish surfacing the entire field and record the volume (steps 6.7-6.11).</li>
	<li>Save the file.</li>
</ol>
9. Calculate the Amount of Phagocytosed Material
<ol>
	<li>Calculate the density of the total engulfed and non-engulfed material channel using the following algorithm: 
	Volume of Total Engulfed and Non-Engulfed Material (step 7.3) / Volume of Field (step 8). </li>
	<li>Calculate % engulfment per cell using the following algorithm: 
	(Volume of Engulfed Material (step 7.5) / Total Volume of the Phagocyte (step 6.11)) x 100 
	Notes: To account for variations in cell size, data are normalized to the total volume of the phagocyte. If numbers from step 9.1 are highly variable across fields, it may be necessary to normalize the data from step 9.2 to the calculation in 9.1.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+synaptic+pathophysiology+in+neurodegeneration:+insights+from+Huntington's+disease.">Early synaptic pathophysiology in neurodegeneration: insights from Huntington's disease.</a> Trends Neurosci. 33 (11), 513-523 (2010).</li><li>Noda, M., Suzumura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweepers+in+the+CNS:+Microglial+Migration+and+Phagocytosis+in+the+Alzheimer+Disease+Pathogenesis.">Sweepers in the CNS: Microglial Migration and Phagocytosis in the Alzheimer Disease Pathogenesis.</a> Int J Alzheimers Dis. , (2012).</li><li>Rotshenker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+macrophage+activation+and+the+regulation+of+complement-receptor-3+(CR3/MAC-1)-mediated+myelin+phagocytosis+in+injury+and+disease.">Microglia and macrophage activation and the regulation of complement-receptor-3 (CR3/MAC-1)-mediated myelin phagocytosis in injury and disease.</a> J Mol Neurosci. 21 (1), 65-72 (2003).</li><li>Smith, 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=Phagocytosis+of+myelin+in+demyelinative+disease:+a+review.">Phagocytosis of myelin in demyelinative disease: a review.</a> Neurochem Res. 24 (2), 261-268 (1999).</li></ol>

No conflicts of interest declared.

In order to accurately measure phagocytosis, engulfed material must be labeled in such a way that the researcher can visualize it once lysosomal degradation has occurred. In addition, high resolution imaging is required, followed by the use of software that will enable the researcher to visualize the volume of the entire cell and quantify its contents. In this protocol, we describe a highly reliable and quantitative method for measuring phagocyte-mediated engulfment using CTB conjugated to Alexa dyes to label engulfed ma...

Synaptic circuits remodel throughout the life of an animal. In the developing brain, synapses form in excess and must undergo synaptic pruning which involves the selective removal of a subset of synapses and the maintenance and strengthening of those synapses that remain8-10. This process is necessary to achieve the precise connectivity characteristic of the adult nervous system. In the adult, synapses can also be plastic, particularly in the context of learning and memory. The structural correlates of this plasticity are thought to include the addition and/or elimination of dendritic spines and presynaptic boutons11-13. In addition to these roles in the healthy nervous system, synaptic remodeling is also involved in nervous system disease/injury12,14,15. For example, following spinal cord injury, severed axons must subsequently remodel and form new synapses to achieve functional recovery16-19.
Emerging as an important aspect of synaptic plasticity is the process of phagocytosis or engulfment of synapses destined for removal3,5,20. We recently showed this phenomenon in the context of synaptic pruning in the healthy, postnatal mouse brain7. Specifically, microglia, the resident CNS immune cells and phagocytes, were shown to engulf presynaptic inputs during a peak period and in a region of developmental synaptic pruning, the postnatal dorsal lateral geniculate nucleus (dLGN) of the thalamus. Genetic or pharmacological blockade of this engulfment resulted in sustained deficits in synaptic connectivity.
In this protocol, we describe a reliable and highly quantitative assay to measure phagocyte-mediated engulfment of presynaptic inputs. For the purposes of this article, this assay will be presented in the context of the developing retinogeniculate system, which includes retinal ganglion cells (RGCs) residing in the retina that project presynaptic inputs to the dLGN (Figure 1A). To begin, a lysosomal degradation-resistant anterograde labeling strategy will be described, which is used to visualize RGC-specific presynaptic inputs in the dLGN (Figure 1)7,21. Following this description, a detailed methodology for imaging and quantitatively measuring engulfment using confocal microscopy combined with 3 dimensional (3D) surface volume rendering will be given. This methodology is based on fixed tissue preparation but may also be adapted for use in live imaging studies. Importantly, while the assay has been validated in the context of the healthy, postnatal retinogeniculate system, one could apply the same techniques to assess other phagocyte-neuron interactions throughout the brain and during disease, as well as phagocyte function in other organ systems.

<table border="0" cellpadding="0" cellspacing="0" width="533">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:144px;">Heat pad</td>
			<td style="width:159px;">Vet Equip, Inc.</td>
			<td style="width:168px;">965500&#160;</td>
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			<td height="38" style="height:38px;width:144px;">Warm water source for heat pad</td>
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			<td height="39" style="height:39px;width:144px;">Stereo microscope</td>
			<td style="width:159px;">DSC Optical</td>
			<td style="width:168px;">Zeiss Opmi -6 Surgical Microscope</td>
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			<td height="40" rowspan="2" style="height:40px;width:144px;">Sliding microtome with freezing stage</td>
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			<td style="width:159px;">Nikon</td>
			<td style="width:168px;">SMZ800 with Epi-fluorescence attachment</td>
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			<td height="57" style="height:57px;width:144px;">Spinning disk confocal microscope</td>
			<td style="width:159px;">Perkin Elmer</td>
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			<td height="51" style="height:51px;width:131px;">10 &#181;l Hamilton gas tight syringes</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">80030</td>
			<td style="width:144px;">Use a different syringe for each color dye/tracer</td>
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		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hamilton needles</td>
			<td style="width:131px;">Hamilton</td>
			<td style="width:116px;">7803-05, specifications: blunt, 1.5&#34;</td>
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			<td height="54" rowspan="3" style="height:54px;width:131px;">Alexa-conjugated cholera toxin &#946; subunit (CTB)</td>
			<td rowspan="3" style="width:131px;">Invitrogen</td>
			<td style="width:116px;">488: C22841</td>
			<td rowspan="3" style="width:144px;">Reconstitute in sterile saline, 80 &#181;l (488), 100 &#181;l (594), 20 &#181;l (647)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:116px;">594: C22842</td>
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			<td height="17" style="height:17px;width:116px;">647: C34778</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Phosphate Buffered Saline&#160;(PBS)</td>
			<td style="width:131px;">Sigma</td>
			<td>P4417-50TAB</td>
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			<td height="73" style="height:73px;width:131px;">Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment USP (antibiotic ointment)</td>
			<td style="width:131px;">Bausch &#38; Lomb</td>
			<td style="width:116px;">24208-780-55</td>
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			<td height="17" style="height:17px;width:131px;">30.5 gauge needle</td>
			<td style="width:131px;">Becton Dickinson</td>
			<td style="width:116px;">305106</td>
			<td rowspan="3" style="width:144px;"></td>
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			<td height="17" style="height:17px;width:131px;">Spring scissors</td>
			<td style="width:131px;">Roboz</td>
			<td style="width:116px;">RS-5630</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Cotton-tipped applicator</td>
			<td style="width:131px;">Fisher</td>
			<td style="width:116px;">23-400-125</td>
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			<td height="104" rowspan="2" style="height:104px;width:131px;">Paraformaldeyde (PFA)</td>
			<td rowspan="2" style="width:131px;">Electron Microscopy Sciences</td>
			<td rowspan="2" style="width:116px;">15710</td>
			<td rowspan="2" style="width:144px;">Dilute 16%to 4% in PBS. Paraformaldehye is toxic, use&#160; in a fume hood and wear personal protective equipment.</td>
		</tr>
		<tr height="83">
		</tr>
		<tr height="36">
			<td height="138" rowspan="4" style="height:138px;width:131px;">Dissection tools &#8211; scissors, forceps, spatula</td>
			<td style="width:131px;">Small scissors: Fine Science Tools</td>
			<td style="width:116px;">Small scissors:14370-22</td>
			<td rowspan="4" style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Large scissors: Roboz</td>
			<td style="width:116px;">Large scissors: RS-6820</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">#55 forceps: Fine Science Tools</td>
			<td style="width:116px;">#55 forceps: 11255-20</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Spatula: Ted Pella, Inc.</td>
			<td style="width:116px;">Spatula: 13504</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:131px;">Sucrose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S8501-5KG</td>
			<td style="width:144px;">Make 30% sucrose in PBS (weight/vol)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">OCT Compound</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">25608-930</td>
			<td style="width:144px;"></td>
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			<td height="17" style="height:17px;width:131px;">Weigh boat</td>
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			<td style="width:116px;">2347-1426</td>
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			<td height="17" style="height:17px;width:131px;">24-well plates</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:116px;">353047</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Sodium phosphate monobasic</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:116px;">S6566-500G</td>
			<td rowspan="2" style="width:144px;">Make 0.2 M sodium phosphate monobasic (PB-A) in ddH20 and 0.2 M sodium phosphate dibasic (PB-B) in ddH20.&#160; To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH20&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:131px;">Sodium phosphate dibasic&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td>S5136-500G</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Coverslips, 22 X 50 mm, No. 1.5</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48393 194</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:131px;">Charged microscope slide</td>
			<td style="width:131px;">VWR</td>
			<td style="width:116px;">48311-703</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:131px;">Vectasheild</td>
			<td style="width:131px;">Vector Laboratories</td>
			<td style="width:116px;">H-1200</td>
			<td style="width:144px;"></td>
		</tr>
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</table>

an engulfment assay: a protocol to assess interactions between cns phagocytes and neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Recently, we used this engulfment assay to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing retinogeniculate system (Figure 1)7. RGCs from CX3CR1-EGFP heterozygous mice were anterogradely traced with CTB-594 and CTB-647 into the left and right eyes, respectively. Following this tracing, EGFP-positive microglia within the dLGN were imaged. These images were subsequently surface-rendered for volume measurements.
Using this t...

Watch this Scientific Journal Video about An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons at JoVE.com

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

an-engulfment-assay-protocol-to-assess-interactions-between-cns

Research

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Neuroscience

18.2K Views.  Boston Children's Hospital, Harvard Medical School.  מיקרוגליה הם התאים חיסוניים תושב של מערכת העצבים המרכזית (CNS) עם קיבולת גבוהה לphagocytose או לבלוע את חומר בסביבה תאית שלהם. הנה, assay החלים רחב, אמין, וכמותי מאוד להדמיה ומדידת היבלעות בתיווך מיקרוגליה של רכיבים סינפטיים מתואר. 

Video: An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

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

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

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

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

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

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...