Name: Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice (Video) | JoVE Journal
Uploaded: 2017-03-01T10:10:00.000+00:00
Duration: 8 min 5 s
Description: Learn bioluminescence imaging to visualize and quantify optic neuritis and brain inflammation in the EAE model of multiple sclerosis in living mice. This method enables real-time monitoring of disease progression and therapeutic effects.

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5 minutes

This research was supported by the Deutsche Forschungsgemeinschaft (CRC1039 A3) and the research funding program "Landesoffensive zur Entwicklung wissenschaftlich-&#246;konomischer Exzellenz" (LOEWE) of the State of Hessen, Research Center for Translational Medicine and Pharmacology TMP and the Else Kr&#246;ner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation - Pharma (TRIP).

1. EAE Induction in SJL/J Mice
<ol>
	<li>Mice
	<ol>
		<li>Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = 10 mice per group.</li>
		<li>For the assessment of medication effects, administer the drug and placebo for the control group continuously via the drinking water or via food pellets starting 3 or 5 days after immunization (n = 10 per group). During the peak of the disease, administer medication or placebo with milk or 3% sugar water-soaked corn flakes.</li>
	</ol>
	</li>
	<li>Immunization material
	<ol>
		<li>Use an EAE induction kit consisting of antigen (peptide of proteolipid protein, PLP139-151, 1 mg/mL emulsion) in an emulsion with complete Freund&#39;s adjuvant (CFA, heat-killed Mycobacterium tuberculosis H37 Ra) and 2 vials (5 &#181;g each) of lyophilized pertussis toxin (PTX).</li>
		<li>Dissolve the PTX (2 &#181;g/mL) in 1x phosphate-buffered saline (PBS; i.e., add 1.5 mL of PBS to each PTX tube, mix well, remove with the same pipet tip, and add to 1 mL of PBS in a 50-mL tube); mix well.</li>
	</ol>
	</li>
	<li>Immunization
	<ol>
		<li>Inject PLP/CFA subcutaneously in 2 portions, each 100 &#181;L, both at the base of the tail. Do not inject into the back of the neck, because any immune reactions in the skin in the upper back or neck will disturb imaging of the head and spinal cord.</li>
		<li>Inject 100 &#181;L of PTX intraperitoneally (i.p.), twice per mouse, the first 1 - 2 h after immunization and the second at 24 h.</li>
		<li>For the control mice, inject CFA without PLP (2 portions of 100 &#181;L) plus PTX without PLP.</li>
	</ol>
	</li>
	<li>Mouse handling after immunization
	<ol>
		<li>Weigh the mice every other day up to day 7, and then weigh them daily. 
		NOTE: Mice lose about 1 - 2 g of body weight during EAE. The decline marks the onset of EAE.</li>
		<li>Assess clinical symptoms daily from day 7 according to the standard scoring systems (i.e., Score 0: no obvious changes in motor functions; score 0.5: distal paralysis of the tail; score 1: complete tail paralysis; score 1.5: mild paresis of one or both hind legs; score 2: severe paresis of hind legs; score 2.5: complete paralysis of one hind leg; score 3: complete paralysis of both hind legs; score 3.5: complete paralysis of hind legs and paresis of one front leg. Euthanize mice with scores of 3.5 or higher for &#62; 12 h.</li>
	</ol>
	</li>
	<li>EAE course and time of imaging
	<ol>
		<li>Perform the first imaging at the onset of the disease, when the mice reach scores &#62; 1, which will occur around day 10 - 12 after immunization.</li>
		<li>Perform the second imaging at the peak, which will be reached 1 or 2 days after the initial symptoms develop and will last for 1 - 3 days. 
		NOTE: Subsequently, the mice will fully recover within 7 to 10 days. Imaging during the intervals may still show vascular leaks, but inflammation indicators should be negative.</li>
	</ol>
	</li>
</ol>
2. Bioluminescent and Near-infrared Imaging of Optic Neuritis and Brain Inflammation
<ol>
	<li>Setup of the imaging system
	<ol>
		<li>Perform in vivo imaging with any equipment that allows for the analysis of bioluminescence and near-infrared signals.</li>
		<li>Keep mice under 2 - 2.5% isoflurane anesthesia during all imaging procedures.</li>
		<li>Position one or two mice beside each other in the apparatus using the middle gas supplies. Position the upper spine in the center.</li>
		<li>Use two mice simultaneously, one per group, for evaluation of medication effects to compare pairs. This is important for bioluminescent imaging.</li>
		<li>Shield the site of immunization with black cloth and take a photograph and baseline image to assess the correct positioning of the mouse/mice. Use the B-focus with a 6.5-cm distance to the camera for all images.</li>
	</ol>
	</li>
	<li>Injection and imaging of bioluminescent inflammation probe
	<ol>
		<li>Use the in vivo imaging system settings: Epi-BLI, Em filter open, Ex filter block, fstop 1, binning 8, focus B = 6.5 cm, ad exposure 120 s; take a baseline image.</li>
		<li>Inject 100 &#181;L i.p. of the ready-to-use chemiluminescent reagent (40 mg/mL). Mix well before filling the syringe.</li>
		<li>Capture bioluminescence images 5, 10, and 15 min after injection. The time course of the bioluminescent peak differs between animals. 
		NOTE: The peak will occur 5 - 10 min after injection; a decline at 15 min indicates that no further images are required. Use mouse pairs of control and treatment groups to eliminate minor biases due to different time courses.</li>
		<li>Fill in descriptions relevant to the experiment. Observe a dialog box pop up automatically; it includes information, such as mouse strain, sex, time point, time of probe injection, group, etc. Save the files all in one folder; they will have time tags and all descriptions.</li>
	</ol>
	</li>
	<li>Injection and imaging of near-infrared fluorescent nanoparticles for BBB integrity
	<ol>
		<li>Use near-infrared epifluorescence imaging in the B-focus (distance: 6.5 cm). The excitation/emission maxima of the pegylated fluorescent nanoparticles are 675/690 nm. Capture two images at different wavelengths, at Ex640/Em700 and Ex675/Em720; use a 2-s exposure, binning 8, and fstop 2. Take a baseline image.</li>
		<li>Inject 70 &#181;L of pegylated fluorescent infrared nanoparticles i.v. through the tail vein and imagine the mice 3 h and 24 h after injection using the above settings. Mix the solution well before filling the syringe.</li>
		<li>Inject 0.9% sodium chloride in control mice, which will be needed to assess the specificity of the signal.</li>
	</ol>
	</li>
	<li>Injection and imaging of the near-infrared fluorescent probe for MMP activity
	<ol>
		<li>Shave or depilate the head and upper spine region cautiously one day before taking the baseline image. The skin must not be injured.</li>
		<li>Use near-infrared epifluorescence imaging in the B-focus (distance: 6.5 cm). The excitation/emission maxima of the MMP activatable probe are 680/700 nm. Capture two images at different wavelengths, at Ex640/Em700 and Ex675/Em720; use a 1-s exposure, binning 8, and fstop 2. Take a baseline image.</li>
		<li>Add 200 &#181;L of 1x PBS to the 1.5-mL tube of the ready-to-use solution (20 nmol/1.5 mL in 1x PBS) so that it will be sufficient for 10 mice; mix well before filling the syringe. 
		NOTE: The provided volume does not take into account that some volume is lost during syringe filling and injection.</li>
		<li>Inject 150 &#181;L of the probe i.v. through the tail vein 24 h before imaging. Inject PBS only in control animals to assess the specificity of the signal.</li>
		<li>At 24 h after injection, take Epi-FL images at least at two wavelengths, Ex640/Em700 and Ex 675/Em720, with the settings explained above (1-s exposure, focus B, binning 8, and fstop 2). 
		NOTE: The use of two wavelengths allows for spectral unmixing by subtracting the unspecific signal.</li>
	</ol>
	</li>
</ol>
3. Image Analyses
<ol>
	<li>Bioluminescence analysis (BLI)
	<ol>
		<li>Double-click the software to open it.</li>
		<li>In the upper menu bar, click on the file browser icon, go to the directory of the folder of the experiment, and select it; this will open all files in the folder in a table.</li>
		<li>Configure the columns showing the descriptions relevant to the experiment.</li>
		<li>For quality control, double-click on a file of a non-responder mouse without symptoms of EAE and/or a na&#239;ve mouse to check the specificity of the EAE signals. 
		NOTE: There should be no signal in na&#239;ve mice and minimal signal in non-responder mice.
		<ol>
			<li>Check the baseline image before the injection of the probe for each mouse as further control of the specificity of the signal; it should be negative.</li>
		</ol>
		</li>
		<li>To select an image, double-click on the first file of the first EAE mouse and check the bioluminescent intensity (LUT bar range) and localization. Check all images one by one. Close the file with the lowest intensity for each mouse (i.e., keep 2 out of 3 images for each mouse).</li>
		<li>Image adjustment and exportation
		<ol>
			<li>Double-click the first file to be quantified. Observe a new window pop up. Under &#34;options&#34; (upper menu), customize the labels to display in each image.</li>
			<li>In the right tool palette, go to &#34;image adjustment.&#34; By default, minimum and maximum intensities are set to &#34;auto&#34; and displayed in rainbow pseudocolor. Select &#34;manual&#34; to change the settings if necessary. 
			NOTE: For example, all exported images may have identical LUT bars (identical minima and maxima) to be easily comparable. The adjustment of the minima and maxima has no impact on the quantitative results.</li>
			<li>Click on image export, select png, the directory, and an image name</li>
		</ol>
		</li>
		<li>Quantification of Regions of Interest (ROI)
		<ol>
			<li>Go to the &#34;ROI&#34; tool in the tool palette. Select the ROI method (circle, rectangular, auto, or free-hand) and the number of ROIs. Observe the ROI window pop up in the image window.</li>
			<li>Using the mouse, adjust the size and position. Use identical ROI thresholds for all images if the auto-ROI tool is used. Use identical areas for all images if ROI sizes and positions are defined manually (e.g., circular ROIs).</li>
			<li>Click on &#34;measure RO.I. Observe a new window pop up. Customize the columns (e.g., file name, animal number, group, experiment, area, total counts, average counts, SD counts, min and max counts, area, time point, time of probe injection, etc.). Save the customized settings. When ready, select all (Ctrl + A), and copy and paste the table into a spreadsheet.</li>
			<li>Export the image as a png with the ROIs in place. Save and close the image file.</li>
		</ol>
		</li>
		<li>Repeat the procedure (steps 3.1.6 - 3.1.7) for all image files that need to be quantified. Copy all ROI quantifications into the spreadsheet. 
		NOTE: Here, results can be sorted by group, time point, etc. and statistically analyzed. Use the total counts of bioluminescence signals in the ROIs for statistical analyses.</li>
	</ol>
	</li>
	<li>Near-Infrared (NIR) analysis of fluorescent nanoparticles
	<ol>
		<li>Using the controls in the software, adjust the threshold of the image. 
		NOTE: This has no impact on the quantitative result.</li>
		<li>Visually compare the images captured at Ex/Em 675/720 and Ex/Em 640/700 to assess the specificity of the signal.</li>
		<li>Use the images captured at Ex/Em 675/720 for the quantitative analysis (excitation maximum: 680 nm). Define ROIs, for which the auto-ROI tool may be used. Adjust the auto-ROI threshold and use it for all images. Quantify the total radiant efficiency in ROIs (see step 3.1).</li>
	</ol>
	</li>
	<li>Near-Infrared (NIR) analysis of protease-sensitive probe
	<ol>
		<li>Using software controls, adjust the threshold of the image. 
		NOTE: This has no impact on the quantitative result. Perform spectral unmixing of the auto-fluorescence. The unmixing tool is implemented in Living Image. The auto-unmixing uses the Ex/Em 640/700 as the specific and 675/720 as the auto-fluorescent image.</li>
		<li>Select the unmixed image and define the ROIs, as described above. Use the total radiant efficiency in ROIs for quantitative and statistical analyses.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The present video shows techniques for bioluminescence and near-infrared fluorescence in vivo imaging of EAE in SJL/J mice. We show that bioluminescence imaging using an inflammation-sensitive probe mainly shows optic neuritis, and the quantification agrees with the clinical evaluation of EAE severity and the effects of medication. However, the bioluminescence imaging method was not able to detect inflammation of the lumbar spinal cord, which is a primary site of EAE manifestation17, like...

Multiple sclerosis is caused by the autoimmune-mediated attack and destruction of the myelin sheath in the brain and the spinal cord1. With an overall incidence of about 3.6 cases per 100,000 people a year in women and about 2.0 in men, MS is the second most common cause of neurological disability in young adults, after traumatic injuries2,3. The disease pathology is contributed to by genetic and environmental factors4 but is still not completely understood. Autoreactive T lymphocytes enter the central nervous system and trigger an inflammatory cascade that causes focal infiltrates in the white matter of the brain, spinal cord, and optic nerve. In most cases, these infiltrates are initially reversible, but persistence increases with the number of relapses. A number of rodent models have been developed to study the pathology of the disease. The relapsing-remitting EAE in SJL/J mice and the primary-progressive EAE in C57BL6 mice are the most popular models.
The clinical EAE scores, which describe the extent of the motor function deficits, and body weight are the gold standards to assess EAE severity. These clinical signs agree with the extent of immune cell infiltration and myelin destruction in the spinal cord and moderately predict drug treatment efficacy in humans5. However, these signs mainly reflect the destruction of the ventral fiber tracts in the spinal cord. Presently, there is no easy, non-invasive, reliable, and reproducible method to assess in vivo brain infiltration and optic neuritis in living mice. 
The in vivo imaging agrees with the 3 "R" principles of Russel and Burch (1959), which claim a Replacement, Reduction, and Refinement of animal experiments6, because imaging increases the readouts of one animal at several time points and allows for a reduction of the overall numbers. Presently, inflammation or myelin status is mainly assessed ex vivo via immunohistochemistry, FACS-analysis, or different molecular biological methods7, all requiring euthanized mice at specific time points. 
A number of in vivo imaging system probes have been developed to assess inflammation in the skin, joints, and vascular system. The techniques rely on the activation of bioluminescent or near-infrared fluorescent substrates by tissue peroxidases, including myeloperoxidase (MPO), matrix metalloproteinases (MMPs)8, and cathepsins9 or cyclooxygenase2. These probes have been mainly validated in models of arthritis or atherosclerosis9,10. A cathepsin-sensitive probe has also been used for fluorescence molecular tomographic imaging of EAE11. MMPs, particularly MMP2 and MMP9, contribute to the protease-mediated BBB disruption in EAE and are upregulated at sites of immune cell infiltration12, suggesting that these probes may be useful for EAE imaging. The same holds true for peroxidase or cathepsin-based probes. Technically, imaging of inflammation in the brain or spinal cord is substantially more challenging because the skull or spine absorb bioluminescent and near-infrared signals. 
In addition to inflammation indicators, fluorescent chemicals have been described, which specifically bind to myelin and may allow for quantification of myelination13. A near-infrared fluorescent probe, 3,3'-diethylthiatricarbocyanine iodide (DBT), was found to specifically bind to myelinated fibers and was validated as a quantitative tool in mouse models of primary myelination defects and in cuprizone-evoked demyelination14. In EAE, the DBT signal was rather increased, reflecting the inflammation of the myelin fibers5. 
An additional hallmark of EAE and MS is the BBB breakdown, resulting in increased vascular permeability and the extravasation of blood cells, extracellular fluid, and macromolecules into the CNS parenchyma. This can lead to edema, inflammation, oligodendrocyte damage, and, eventually, demyelination15,16. Hence, visualization of the BBB leak using fluorescent probes, such as fluorochrome-labeled bovine serum albumin5, which normally distribute very slowly from blood to tissue, may be useful to assess EAE.
In the present study, we have assessed the usefulness of different probes in EAE and show the procedure for the most reliable and robust bioluminescent technique. In addition, we discuss the pros and cons of near-infrared probes for MMP activity and BBB integrity.

<table><tbody><tr><td>AngioSpark-680</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>NEV10149</td><td>Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity</td></tr><tr><td>MMP-sense 680</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>NEV10126</td><td>Imaging probe, activatable by matrix metalloproteinases, useful for imaging of inflammation</td></tr><tr><td>XenoLight RediJect Inflammation Probe</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>760535</td><td>Imaging probe, activatable by oxidases, useful for imaging of inflammation</td></tr><tr><td>PLP139-151/CFA emulsion&amp;nbsp;</td><td>Hooke Labs, St Lawrence, MA</td><td>EK-0123</td><td>EAE induction kit</td></tr><tr><td>Pertussis Toxin</td><td>Hooke Labs, St Lawrence, MA</td><td>EK-0123</td><td>EAE induction kit</td></tr><tr><td>IVIS Lumina Spectrum</td><td>Perkin Elmer, Inc., Waltham, USA</td><td></td><td>Bioluminescence and Infrared Imaging System</td></tr><tr><td>LivingImage 4.5 software&amp;nbsp;</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>CLS136334</td><td>IVIS analysis software</td></tr><tr><td>Isoflurane</td><td>Abbott Labs, Illinois, USA</td><td>26675-46-7</td><td>Anaesthetic</td></tr></tbody></table>

bioluminescence and near-infrared imaging of optic neuritis and brain inflammation in the eae model of multiple sclerosis in mice

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

Time Course of Bioluminescence of Optic Neuritis
The bioluminescence signal of the inflammation probe was the strongest around the eyes and occurred exclusively in EAE mice with optic neuritis. A signal occurred in neither the non-EAE mice nor the mice not injected with the inflammation probe. The signal disappeared when the mice recovered. Hence, the signal is specific for optic neuritis, and the peak of the si...

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

The overall goal of this imaging technique to to visualize and quantify optic neuritis and brain inflammation in the EAE model of multiple sclerosis in living mice. The method can help answer key questions in the neuroimmunology field, such as novel mechanisms which impact the course of EAE and multiple sclerosis. The main advantage of this technique is that EAE evoked optic neuritis and brain inflammation can be observed and quantified in living mice throughout the course of the disease.The implications of this technique extend toward therapy because we can observe effect of medication, which do not reveal from the EAE scores. In this procedure dissolve PTX at two micrograms per milliliter in 1x PBS and mix well. Then inject 100 microliters of PLP/CFA in the mice subcutaneously twice at the base of the tail during short isoflurane anesthesia.Do not inject into the back of the neck, because any immune reactions in the skin in the upper back or neck will disturb imaging of the head and spinal cord. One to two hours after immunization, inject 100 microliters of PTX in the mice intraperitoneally. Then 24 hours after immunization, inject the mice with 100 microliters of PTX again.For the control mice, inject 100 microliters of CFA without PLP twice at the base of the tail. From day seven post immunization, assess the clinical symptoms daily according to the standard scoring systems. When the mice reach a score larger than one, which will occur around day 10 to 12 after immunization, perform the first imaging at the onset of the disease.Then perform the second imaging at the peak of the disease, which is about one to two days after the initial symptoms develop, and will last for one to three days. Subsequently, the mice will fully recover within seven to 10 days. Imaging during the intervals may still show vascular leaks, but inflammation indicators should be negative.To perform in vivo imaging keep the mice under 2%to 2.5%isoflurane anesthesia during all imaging procedures. Position two mice next to each other in the apparatus, and position the upper spines of the mice in the center. Next, shield the site of immunization with black cloth.Open the software for image capture and initialize the camera. Then take a baseline image to assess the correct positioning of the mice. Subsequently use B focus with a 6.5 centimeter distance to the camera for capturing all the images.Afterward mix the ready-to-use chemiluminescent reagent at 40 milligrams per milliliter before filling the syringe. Then, inject 100 microliters of it in the mice intraperitoneally and capture the bioluminescence images five, 10, and 15 minutes after injection. In this procedure open the bioluminescence analysis software.In the upper menu bar click on the File Browser icon. Go to the directory of the experiment folder and open the files, and then configure the columns showing the descriptions relevant to the experiment. For quality control click on a file of a non-responder mouse without the symptoms of EAE, or a naive mouse to check the specificity of the EAE signals.As further control of the specificity of the signal, check the baseline image before the probe injection for each mouse, which should be negative. To select an image click on the first file of the first EAE mouse and check the bioluminescent intensity and localization. Check all images one by one.For quantification, use the image with the strongest signal for each mouse. To adjust and export the image, double click the first file to be quantified. In a new popped up window under the Options button, customize the labels to display in each screen.In the right tool palette, go to Image Adjustment. By default minimum and maximum intensities are set to auto and displayed in rainbow pseudocolor. Select Manual to change the settings if necessary.Next, click in Image Export, select PNG, the directory, and an image name. For quantification of the regions of interest go to the ROI Tool in the tool palette. Select the ROI method and the number of ROIs.In the ROI window within the image window, adjust the size and position of the ROI. Use identical ROI thresholds for all the images when the auto ROI tool is used. Use identical areas for all the images if the ROI sizes and positions are defined manually.After that, click on Measure ROI. Customize the columns on the experimental details in a new pop-up window and save them. When ready, select all and copy and paste the table into a spreadsheet.After that, export the image as a PNG with the ROIs in place. Then, save and close the image file. Here are the examples of two SJL/J mice that were imaged at day 10 and 14 after EAE induction.The bioluminescent signal around the eyes was highest at day 10, and disappeared when the mice started to recover. The bar charts here show the quantification of the bioluminescent signals at day 10 and 14, whereas the clinical EAE scores are shown here. In this figure the bioluminscent images of five mice in each group show the effect of the medication in which optic neuritis was reduced in mice treated with R-flurbiprofen.The quantification of the total bioluminescent counts in ROIs confirmed the significant treatment efficacy. Once mastered, this technique can be done in two to three hours, depending on the number of mice to be imaged. While attempted this procedure, it's important to remember to use white mice and to closely observe the clinical EAE course.Following this procedure other methods like FACS analysis or immunohistochemistry can be performed in order to answer additional questions like immune cell infiltration of the spinal cord. After watching video, you should have a good understanding of how to asses optic neuritis and brain inflammation in the EAE model of multiple sclerosis using bioluminescent imaging.

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JoVE Journal

Medicine

This video demonstrates a procedure for assessing blood-brain barrier disruption in an EAE mouse model. Fluorescent dyes of different sizes are injected retro-orbitally and circulate through the brain's blood vessels. The smaller dye leaks through gaps into the brain tissue, while the larger dye enters only if the disruption is severe, correlating with the extent of barrier damage.

Determining Blood-Brain Barrier Disruption Using Fluorescent Dyes in an EAE Mouse Model

In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that...

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Breid, S., et. al. Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils. J. Vis. Exp. (2017)This video demonstrates the bioluminescence imaging of neuroinflammation in transgenic mice, showcasing luciferase-luciferin reactions in activated astrocytes. The protocol enables non-invasive quantification of astrocytic activation, aiding studies on CNS inflammation and therapeutic efficacy.

Bioluminescence Imaging of Neuroinflammation in a Transgenic Mouse

Source: Schmitz, K., et al., Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice. J. Vis. Exp. (2017).This video demonstrates the method for visualizing and monitoring Experimental Autoimmune Encephalomyelitis or EAE-induced inflammation in the optic nerve using bioluminescence imaging. It includes positioning the mouse, reagent injection, and capturing sequential images to monitor changes in bioluminescence over time.

Bioluminescence Imaging in an EAE Mouse Model

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Learn bioluminescence imaging to visualize and quantify optic neuritis and brain inflammation in the EAE model of multiple sclerosis in living mice. This method enables real-time monitoring of disease progression and therapeutic effects.

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice (Video) | JoVE Journal

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

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