<meta charset="utf8"></head><body>Elucidando o comportamento da microglia no desenvolvimento do tecto óptico do peixe-zebra

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat Neurosci. 10 (12), 1538-1543 (2007).</li><li>Mildner, A., 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=Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2++monocytes+only+under+defined+host+conditions.">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat Neurosci. 10 (12), 1544-1553 (2007).</li><li>Cook, Z. T., Brockway, N. L., Weissman, T. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L Breeding tanks</td>
			<td>Tecniplast</td>
			<td>ZB10BTE</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>Diluted to 0.2 mM in E3 to prevent embryo pigmentation</td>
		</tr>
		<tr>
			<td>Bottom glass imaging dish</td>
			<td>FluoroDish</td>
			<td>FD3510-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Graduated transfer pipette</td>
			<td>avantor</td>
			<td>16001-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry block heater</td>
			<td>Novolab</td>
			<td>Grant QBD4</td>
			<td>To keep low melting agarose at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 10.0</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>analysis software</td>
		</tr>
		<tr>
			<td>Imaris File Converter</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>https://imaris.oxinst.com/big-data</td>
		</tr>
		<tr>
			<td>Laser-scanning confocal microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti2-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>NuSieve GTG Agarose</td>
			<td>Lonza</td>
			<td>&#160;50081</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (90 mm)</td>
			<td>avantor</td>
			<td>391-0559</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma-Aldrich</td>
			<td>11459643001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Stainless Steel Forceps Dumont No. 5</td>
			<td>FineScienceTools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>Leica M80</td>
			<td>To mount the embryos</td>
		</tr>
		<tr>
			<td>teasing needle</td>
			<td>avantor</td>
			<td>76549-024</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging and characterization of microglia dynamics in the zebrafish embryo

As resident macrophages in the central nervous system (CNS), microglia represent a distinct non-neuronal population that accounts for up to 15% of all glial cells in the adult brain. Studying microglia biology has gained increasing attention in recent years due to their established importance in development, physiology, and disease1. Under physiological conditions, microglial cells are highly dynamic, continuously surveying the brain parenchyma2,3. This behavior allows microglia to colonize the brain and play pivotal roles in its development such as shaping neuronal circuitry4, synaptic pruning5, and vasculogenesis6. Furthermore, this inherent dynamic nature allows microglia to constantly monitor the CNS for signs of infection, injury, or any deviations from homeostasis7. To dissect these intricate cell dynamics, live imaging of microglia across space and time is indispensable. Fortunately, the optical transparency of externally developing zebrafish embryos, coupled with the availability of well-characterized transgenic reporter lines that fluorescently label microglia, positions zebrafish as an ideal vertebrate model for such investigations. Live imaging in zebrafish embryos offers a non-invasive approach that does not require surgery or extensive tissue manipulation, minimizing potential perturbations to the CNS status. This is a critical consideration when studying microglial cells, as they are highly sensitive to even subtle changes in the extracellular environment8.
Here, we provide a guideline to successfully track 3D microglial cell movements in the zebrafish embryo, allowing for an unprecedented view of microglia behavior within the intact architecture of the developing brain parenchyma (see Figure 1&#160;for a graphical overview of the protocol). This step-by-step protocol details how to set up and image zebrafish microglia at different developmental stages and how to extract high-resolution data on microglia cell motility to provide valuable insights into their migratory patterns and responses to environmental cues. We also demonstrate this protocol can be adapted to perform live multi-color imaging, thus extending its applicability to studying microglia in combination with transgenic lines that mark neighboring cells, including neurons3, oligodendrocytes9, and endothelial cells10&#160;(as shown in Figure 2). By adding to the toolbox that allows one to directly observe and characterize the dynamics of microglia behavior&#160;in real time and in their natural environment, this protocol will likely contribute to better elucidating microglia functionality during early development, both in physiology and disease.

<meta charset="utf8"></head><body>Autor em destaque: Usando o peixe-zebra para explorar a migração da microglia durante o desenvolvimento do cérebro

Imagem ao vivo e caracterização da dinâmica da microglia no embrião de peixe-zebra

My research project focuses on characterizing microglia dynamics, morphology, and function during the first week of zebra fish embryonic development. Microglia are highly dynamic cells, and we believe that a deeper characterization of their behavioral patterns and morphological changes could give us an interesting insight into their maturation process and activity in the developing brain. The major challenge in this type of imaging lies in capturing multiple channels with eye quality and an excellent temporary resolution.However, advancement in confocal technology are now enabling us to meet this challenge effectively. We believe our protocol to be a potent tool that could facilitate the characterization of microglia dynamics in diverse settings. This includes not only different stages of development, but also very smooth background where microglia homeostasis is compromised.

Read this Scientific Article Protocol about Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo at JoVE.com

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and ...

live-imaging-characterization-microglia-dynamics-zebrafish

Research

JoVE Journal

Immunology and Infection

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

653 visualizações.  Université Libre de Bruxelles (ULB). Meu projeto de pesquisa se concentra na caracterização da dinâmica, morfologia e função da microglia durante a primeira semana de desenvolvimento embrionário do peixe-zebra. As microglias são células altamente dinâmicas, e acreditamos que uma caracterização mais profunda de seus padrões comportamentais e mudanças morfológicas pode nos dar uma visão interessante de seu processo de maturação e atividade no cérebro em desenvolvimento. O maior d...

Vídeo: Autor em destaque: Usando o peixe-zebra para explorar a migração da microglia durante o desenvolvimento do cérebro

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

Imunologia e Infecção

Confocal Imaging and Tracking Analysis of Microglial Dynamics in Zebrafish Embryos

To begin, obtain anesthetized zebrafish embryos in E3 buffer. After selecting the embryos with the desired fluorescence, visualize their heartbeat under a bright field stereo microscope. Using a wide tip transfer pipette, draw up to three embryos, and transfer them to a bottom glass imaging plate.Under a stereo microscope, using a pipette, replace the medium around the embryos with a 1%low melting agarose solution. Using a plastic thin tapered tip attached to a teasing needle, gently push each embryo to the bottom of the imaging plate, and orient the dorsal side to face the glass. Mount the embryo in an inverted confocal microscope.After setting the time-lapse acquisition parameters, acquire the images. Open the Imaris file converter software, and drag and drop the files into the input area. In the Output menu, designate the location for the converted files.Click on the set voxel size to verify, and press the Start All button to start the file conversion to IMS format. Once launched, let the analysis software start in the arena. Click on Observe Folder to open the previously converted file.Once the file is loaded into the software, click the Slice view to scroll through the z-stack. Using the slider in the Slice toolbar, measure cell diameters after drawing the diameter of the selected cells with the pointer. Click and drag the mouse to draw segments spanning the cell core, and observe the length of the segment drawn in the measure toolbar situated on the right of the view area.To automatically detect the cells, go back to the 3D view, and click on the Spot icon in the object toolbar to add a new Spots object in the Object list, and open the Automatic Spots Creation Wizard. In the first step of the Wizard, take the option to segment only a region of interest. Then enable the Track Spots Over Time option to compute the tracking data, as well as the Object-Object Statistics to allow comparison between spots and expand the range of data.Click the next button in the lower right of the Wizard window. Specify a region of interest, or ROI area, that encloses the optic tectum of the embryo. Click and drag the small white arrows present on each face of it to modify the size of the ROI.Then extend it to all the recorded frames with time interval adjustments. Then select a source channel, and set the cell diameter measurement obtained previously as the estimated XY diameter. Enable the Background Subtraction option, and click on Next.Enter the intensity threshold values directly into the lower and upper threshold boxes to ensure the detection of all cells. Click on View Area to observe all these changes in real time. Once satisfied, click on Next to validate the selected quality thresholding, and click on View Area to visualize spots satisfying these two parameters superimposed on the source channel.Next, select the Autoaggressive Motion as the tracking algorithm to track cells that have a more or less continuous motion. Observe the motion of the spots in between two frames to judge the longest distance a cell moves between two time points, and enter an estimated number in the max distance field. To set the maximum gap size, if the interval between frames is below 60 seconds, use a size of three, and for longer time intervals, increase the same.Enable the fill gaps with all detected objects to drop the detection threshold and connect tracks. Then click on Next, and let the software automatically generate tracks for all the previously generated spots. If the obtained tracks are numerous or fragmented, return to the Creation Wizard with the Previous button, and adjust the maximum distance previously defined.To exclude the tracks shorter than three to eight minutes, enter the value directly in the data field for the lower limit of the track duration filter. Click on Next to validate the filter, and move on to the Creation Wizard. Manually remove the tracks generated from skin macrophages from the analysis to focus on brain parenchymal microglia.Using the Track Editor, manually correct other potential mistakes in the tracks. On the right of the view area, activate the circle Select Mode button, and highlight tracks that require modifications or adjustments. Once the problematic track is selected, using the Connect and Disconnect buttons, edit it to correctly represent the cell movements.With the Track Editor, select Circle Select Mode to delete duplicated single spots. If using microglia reporter with other fluorescently labeled parenchymal cells, adjust the estimated XY diameter and max distance to the new cell population. Modify the source channel input to the image.Finally, using the Statistic tab, extract the desired tracking statistics. Click on the Settings button, and select the statistics for the computation of each spot object or surface object previously created. The brain of the embryonic zebrafish was imaged in its entirety, and the location of neurons with respect to microglia was visualized.The spatial data obtained using this protocol depicts the average speed of microglial cells, the average distance they cover in one hour, their means square displacement, and their distribution in space at different times.