fluorescence-based monitoring of mitochondrial stress in astrocytes

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

Assess the astrocytic mitochondrial system, at least three to five days after the lentiviral infections with LV-G1-MitoTimer. Select five astrocytes per well with a mitochondrial network expressing sufficient levels of LV-G1-MitoTimer using a magnification of 40 times, taking care to select astrocytes as flat and large as possible and not located in clusters of cells. Then, capture fluorescence images using sequential excitation at 490 nanometers for the green channel and 550 nanometers for the red channel with green and red fluorescent signals, and using a magnification of 150 times for each coordinate.
Select the first frame for red and green channels for each image sequence by clicking on ND Processing and Select Frame. Then, merge the red and the green channels by selecting conversions and Merge Channel. To correct image shading, select Pre-Processing and then Auto-Shading Correction. Apply the rolling ball algorithm by selecting Pre-Processing and Rolling Ball.
To generate binary masks for each mitochondrion, select Segmentation and Threshold. Remove any objects truncated by the border by selecting Binary Processing and Touching Border. Select Measurement, and then Object Area, EQ Diameter, Length, Width, Roughness, Circularity, or Elongation to measure surface area, diameter, length, width, roughness, circularity, or elongation respectively.
Compose a group with these measurements and rename it as morpho data. Then select Measurement and then select Mean Intensity to measure the mean green and red intensities, and Ratio to measure the red by green ratio.
Now, compose a group with these measurements and rename it as ratio data. Finally, export the table to a CSV file by selecting Reference and Table to CSV, and then save the GA 3 script of analysis by selecting Save As.

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1. Rat hippocampal astrocyte primary culture
<ol>
	<li>Sacrifice five rat pups (Wistar IGS Rat) by decapitation at postnatal days 1-2.</li>
	<li>Remove the brain and keep it in a Petri dish containing 5 mL of fresh astrocyte medium (Dulbecco&#39;s Modified Eagle Medium or DMEM with GlutaMAX supplemented with 1% Penicillin/Streptomycin and 10% fresh Horse Serum).</li>
	<li>Isolate the hippocampus. Dissociate in 5 mL of the astrocytic medium by three passages through a 21 G needle and three passages through a 25 G needle.</li>
	<li>Transfer the dissociated cells to a 15 mL centrifuge tube and count in a hemocytometer.</li>
	<li>Plate 20,000-25,000 cells/cm2 in multiwell dishes (9.4 mm x 10.7 mm x 9.3 mm) and store them at 37 &#176;C under an atmosphere containing 5% CO2 for the rest of the experiment.</li>
	<li>Replace the medium entirely at 3 days in vitro (Division or DIV 3).</li>
	<li>At DIV 8, add 0.6 pg of p24 antigen per cell of lentiviral vector coding for mitochondrial biosensor MitoTimer (LV-G1-MitoTimer) diluted in phosphate-buffered saline (Phosphate-buffered saline or PBS + 0.01% Bovine serum albumin or BSA).</li>
	<li>At DIV 9, wash with pre-warmed 1x sterile PBS (37 &#176;C) and add fresh astrocyte medium.</li>
	<li>At DIV 11, 2 washes were performed with pre-warmed 1x sterile PBS (37 &#176;C), and fresh astrocyte medium was added without phenol red. 
	&#160;NOTE: The choice of the coating depends on the intended assay. As a standard coating for astrocyte primary culture, it is recommended to use 0.2 mg/mL poly-D-lysine or 8.7 &#181;g/cm2 of basement membrane matrix (e.g., Matrigel). To visualize the mitochondrial system of astrocytes, it is essential to work on flattened and stretched cells. Combining the basement membrane matrix on IBIDI u-slides is the most suitable in this context. It is also crucial not to work with phenol red, which is toxic for the cell under repeated exposure to light.</li>
</ol>
2. Long-term monitoring of the mitochondrial system.
<ol>
	<li>Assess the astrocytic mitochondrial system at least 3-5 days after the lentiviral infections with a lentiviral vector (LV-G1-MitoTimer) to allow a sufficient level of fluorescence in the mitochondria.</li>
	<li>Image the cells with an inverted microscope controlled by the acquisition and analysis software and a module for complete automation of acquisition. 
	NOTE: Refer to the Table of Materials for the details of the software and module used in this study.</li>
	<li>Ensure the microscope is equipped with a cage incubator (see Table of Materials) to maintain astrocyte culture at 5% CO2 and 37 &#176;C throughout the experiment.</li>
	<li>Capture fluorescence images using sequential excitation at 490 nm (for the green channel) and 550 nm (for the red channel), detecting green (500-540 nm for the green channel) and red (550-600 nm for the red channel) fluorescence signals.</li>
	<li>Select 5 astrocytes per well with a mitochondrial network expressing sufficient levels of LV-G1-MitoTimer using a 40x magnification. Take care to select astrocytes that are as flat and large as possible and not located in clusters of cells (to work on individual cells).</li>
	<li>Save the coordinates of the 5 selected cells in an xlm file (map.xlm). This map.xlm allows the user to return to the same cells over time.</li>
	<li>Acquire image sequences (1 image/s for 60 s) using a 150x magnification (100x oil immersion objective, 1.5x intermediate magnification) for each coordinate.</li>
	<li>Repeat image acquisition (1 image/s for 60 s) for the same astrocytes 6 h, 12 h, and 24 h after treatments. 
	NOTE: Use a microscope equipped with a fast and efficient autofocus system. In this context, the hardware solution Perfect Focus System (PFS) was used. PFS uses near-infrared 870-nm LED and CCD line sensors to combat axial focus fluctuations in real-time during long-term imaging investigations.</li>
</ol>
3. Analysis of individual mitochondrial morphology and LV-G1-MitoTimer ratio
NOTE: NIS General Analysis 3 (GA3) from Nikon was used to automatize morphometric analysis in this study.
<ol>
	<li>For each image sequence (baseline, 6 h, 12 h, 24 h), select the first frame for red and green channels by clicking on ND Processing &#62; Select Frame.</li>
	<li>Merge the red and the green channels by selecting Conversions &#62; Merge Channel.</li>
	<li>To correct image shading, select Preprocessing &#62; Auto Shading Correction.</li>
	<li>Apply the Rolling Ball algorithm by selecting Preprocessing &#62; Rolling Ball.</li>
	<li>To generate binary masks for each mitochondrion, select Segmentation &#62; Threshold.</li>
	<li>Remove any objects truncated by the border by selecting Binary processing &#62; Touching Border.</li>
	<li>To measure the surface area, select Measurement &#62; Object Area.</li>
	<li>To measure the diameter, select Measurement &#62; Eq Diameter.</li>
	<li>To measure the length, select Measurement &#62; Length.</li>
	<li>To measure the width, select Measurement &#62; Width.</li>
	<li>To measure roughness, select Measurement &#62; Roughness.</li>
	<li>To measure circularity, select Measurement &#62; Circularity.</li>
	<li>To measure elongation, select Measurement &#62; Elongation.</li>
	<li>Compose a group (right-click) with the above measurement and rename it MorphoData.</li>
	<li>Measure mean green intensity by selecting Measurement &#62; Mean Intensity.</li>
	<li>Measure mean red intensity by selecting Measurement &#62; Mean Intensity.</li>
	<li>To measure the Red/Green ratio, select Measurement &#62; Ratio.</li>
	<li>Compose a group (right-click) with the above measurement and rename it as RatioData.</li>
	<li>Export the table to a CSV file by selecting Reference &#62; Table to CSV.</li>
	<li>Save the GA3 script of analysis by selecting Save As 
	NOTE: A non-exhaustive list of the criteria used in this procedure is summarized in Figure 1, Table 1, and Table 2. The thresholds used were adapted to individualize as many mitochondria as possible (excluding large mitochondrial networks where possible). Wherever possible, mitochondrial networks are either individualized manually or excluded from the analyses. Due to intercellular variability, these analyses are performed on at least 20-25 cells per condition (with a minimum of 50 mitochondria by cell).</li>
</ol>
Table 1: Summary of selected parameters for mitochondrial morphology.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Morphology criteria</td>
			<td>Range</td>
			<td>Remarks</td>
		</tr>
		<tr>
			<td>Surface Area (SA)</td>
			<td>0.5&#8211;4 &#181;m2</td>
			<td rowspan="7">These criteria inform on the fragmented-elongated features of mitochondria. They generally evolve in the same direction. Fragmented mitochondria will have decreased surface area, diameter, length, and elongation factor, while roundness, sphericity, and width may be unchanged or increased.</td>
		</tr>
		<tr>
			<td>Diameter (D)</td>
			<td>0.5&#8211;1.5 &#181;m</td>
		</tr>
		<tr>
			<td>Length (L)</td>
			<td>0.5&#8211;5 &#181;m</td>
		</tr>
		<tr>
			<td>Width (W)</td>
			<td>0.5&#8211;2 &#181;m</td>
		</tr>
		<tr>
			<td>Roundness (R)</td>
			<td>0&#8211;1</td>
		</tr>
		<tr>
			<td>Sphericity (S)</td>
			<td>0&#8211;1</td>
		</tr>
		<tr>
			<td>Elongation factor (EF = L/W)</td>
			<td>1&#8211;10</td>
		</tr>
	</tbody>
</table>
Table 2: Summary of selected parameters for mitochondrial redox state.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Redox State criteria</td>
			<td>Range</td>
			<td>Remarks</td>
		</tr>
		<tr>
			<td>Individual ratio (G/R)</td>
			<td>0&#8211;10</td>
			<td rowspan="3">The ratio indicates the result of the redox state. It informs about the general state and age of the mitochondria in the cell. It is essential to consider that this ratio is the balance of biogenesis and degradation of mitochondria and the fission/fusion of oxidized mitochondria with reduced mitochondria. Therefore, evaluating the number of green and red puncta can powerfully help interpret the results.&#160; Green puncta mitochondria are determined when the intensity of green is 10 times that of red. Red puncta mitochondria are determined when the intensity of red is 10 times greater than green&#39;s. The redox state of an astrocyte is the average of all the mitochondria ratios of that cell.</td>
		</tr>
		<tr>
			<td>Percentage of green puncta mitochondria (Gpuncta)</td>
			<td>0%&#8211;100%</td>
		</tr>
		<tr>
			<td>Percentage of red puncta mitochondria (Rpuncta)</td>
			<td>0%&#8211;100%</td>
		</tr>
	</tbody>
</table>

Source: Espourteille, J., et al. <a href="https://app.jove.com/v/62957">Live-imaging of Mitochondrial System in Cultured Astrocytes.</a> J. Vis. Exp. (2021)
This video demonstrates the evaluation of mitochondrial health in genetically modified astrocytes, which express a fluorescent protein within their mitochondria. The protein helps to track changes in mitochondrial health as its fluorescence changes from green to red, indicating stress or damage. Images are captured and analyzed using various parameters.

<img alt="Figure 1" src="/files/ftp_upload/22981/22981_Figure1.jpg" /> 
Figure 1: Astrocyte culture expressing LV-G1-MitoTimer biosensor. (A) Confocal photographs of astrocytes expressing LV-G1-MitoTimer. (B) Selection of confocal photographs of reduced (green), balanced (orange), and oxidized (red) mitochondria with different levels of fragmentation. (C) Summary diagram of the different criteria available for analysis in an astrocyte expressing LV-G1-MitoTimer. Scal...

1. Rat hippocampal astrocyte primary culture

	Sacrifice five rat pups (Wistar IGS Rat) by decapitation at postnatal days 1-2.
	Remove the brain and keep ...

Begin with an astrocyte culture transduced to express a Mito-timer protein localized to their mitochondria.
Mito-timer acts as a mitochondrial biosensor, emitting green fluorescence in healthy mitochondria and transitioning to red fluorescence in stressed or damaged ones.
Microscopically select large and isolated astrocytes for imaging.
Using different excitation wavelengths, capture images for green and red fluorescence.
To analyze the images, select frames for both red and green channels, then choose merge channels.
Apply auto shading correction and run the image enhancement algorithm to enhance image clarity.
Select the desired morphological parameters of mitochondria for analysis.
Using the mean intensity tab, measure the mean intensities of red and green fluorescence.
Calculate the ratio between red and green intensities, which indicates the overall mitochondrial health of the astrocyte.

11 Views. Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

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