Name: bioluminescence and near-infrared imaging of optic neuritis and brain inflammation in the eae model of multiple sclerosis in mice
Uploaded: 2017-03-01T10:10:00
Description: Watch this Scientific Journal Video about Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice at JoVE.com

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 co...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="1287">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">AngioSpark-680</td>
			<td style="width:267px;">Perkin Elmer, Inc., Waltham, USA</td>
			<td style="width:119px;">NEV10149</td>
			<td style="width:636px;">Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">PLP139-151/CFA emulsion&#160;</td>
			<td>Hooke Labs, St Lawrence, MA</td>
			<td>EK-0123</td>
			<td>EAE induction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pertussis Toxin</td>
			<td>Hooke Labs, St Lawrence, MA</td>
			<td>EK-0123</td>
			<td>EAE induction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS Lumina Spectrum</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td style="width:119px;"></td>
			<td>Bioluminescence and Infrared Imaging System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LivingImage 4.5 software&#160;</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td>CLS136334</td>
			<td>IVIS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Isoflurane</td>
			<td style="width:267px;">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

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...

Watch this Scientific Journal Video about Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice at JoVE.com

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

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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The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool

The Multiple Sclerosis Performance Test MSPT: An iPad-Based Disability Assessment Tool

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation (tDCS) in Multiple Sclerosis (MS)

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation tDCS in Multiple Sclerosis MS

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...