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

这项研究是由德意志研究联合会（CRC1039 A3）和研究资助计划黑森州，研究中心转化医学和药理学TMP的国家“Landesoffensive祖尔发展协会wissenschaftlich-ökonomischerExzellenz”（LOEWE）和否则克朗，费森尤斯基金会支持（EKFS），研究培训组转化研究创新 - 制药（TRIP）。

1. EAE诱导SJL / J小鼠<ol><li> 老鼠 <ol><li>使用11周龄的雌性SJL / J小鼠，并允许他们使其习惯的实验室进行约7天。使用每组N = 10只。 </li><li>对于药物影响的评估，通过饮用水或通过启动免疫后3或5天（每组n = 10）的食物颗粒连续地管理为对照组的药物和安慰剂。在疾病的高峰期，管理药物或安慰剂用牛奶或3％的糖水渍玉米片。 </li></ol></li><li> 免疫物质 <ol><li>在用完全弗氏佐剂（CFA，热?...

<ol>
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H., 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=Suppression+of+EAE+and+prevention+of+blood-brain+barrier+breakdown+after+vaccination+with+novel+bifunctional+peptide+inhibitor.">Suppression of EAE and prevention of blood-brain barrier breakdown after vaccination with novel bifunctional peptide inhibitor.</a> Neuropharmacology. 62 (4), 1874-1881 (2012).</li><li>Simmons, S. B., Pierson, E. R., Lee, S. Y., Goverman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+heterogeneity+of+multiple+sclerosis+in+animals.">Modeling the heterogeneity of multiple sclerosis in animals.</a> Trends in immunology. 34 (8), 410-422 (2013).</li><li>Lin, T. H., 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=Diffusion+fMRI+detects+white-matter+dysfunction+in+mice+with+acute+optic+neuritis.">Diffusion fMRI detects white-matter dysfunction in mice with acute optic neuritis.</a> Neurobiol Dis. 67, 1-8 (2014).</li><li>Knier, B., 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=Neutralizing+IL-17+protects+the+optic+nerve+from+autoimmune+pathology+and+prevents+retinal+nerve+fiber+layer+atrophy+during+experimental+autoimmune+encephalomyelitis.">Neutralizing IL-17 protects the optic nerve from autoimmune pathology and prevents retinal nerve fiber layer atrophy during experimental autoimmune encephalomyelitis.</a> J Autoimmun. 56, 34-44 (2015).</li><li>Schellenberg, A. E., Buist, R., Yong, V. W., Del Bigio, M. R., Peeling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+blood-spinal+cord+barrier+disruption+in+mice+with+experimental+autoimmune+encephalomyelitis.">Magnetic resonance imaging of blood-spinal cord barrier disruption in mice with experimental autoimmune encephalomyelitis.</a> Magn Reson Med. 58 (2), 298-305 (2007).</li><li>Mori, Y., 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=Early+pathological+alterations+of+lower+lumbar+cords+detected+by+ultrahigh-field+MRI+in+a+mouse+multiple+sclerosis+model.">Early pathological alterations of lower lumbar cords detected by ultrahigh-field MRI in a mouse multiple sclerosis model.</a> Int Immunol. 26 (2), 93-101 (2014).</li><li>Bittner, S., 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=Endothelial+TWIK-related+potassium+channel-1+(TREK1)+regulates+immune-cell+trafficking+into+the+CNS.">Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS.</a> Nat Med. 19 (9), 1161-1165 (2013).</li><li>Theien, B. 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本视频显示了在 SJL / J小鼠EAE的体内成像生物发光和近红外荧光的技术。我们表明，使用炎症敏感探针生物发光成像主要表示视神经炎，以及量化与EAE严重程度的临床评估和药物的效果一致。但是，由于该信号被脊柱所吸收的生物发光成像方法是不能检测的腰脊髓，这是EAE表现17的原发部位的炎症，可能的。 近红外成像是更敏感，但在与自体荧?...

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

在SJL / J小鼠实验性自身免疫性脑脊髓炎（EAE）是复发缓解型多发性硬化症（RRMS）的模型。描述运动功能障碍的临床EAE分数是脊髓的免疫介导的炎症的基本读数。但是，得分和体重不允许大脑炎症和视神经炎的体内评估。后者是在MS患者的大约2/3的早期和频繁表现。这里，我们表明使用体内成像系统在活小鼠生物发光和近红外实时成像以评估EAE诱发视神经炎，脑炎症，和血-脑屏障（BBB）的破坏的方法。通过氧化酶激活的生物发光底物，主要表现为视神经炎。该信号是具体和允许的药物治疗效果和疾病时程可视化，从而平行临床得分。聚乙二醇化荧光纳米粒子仍然是vasculatur内e表示延长的时间段被用来评估血脑屏障完整性。近红外成像显示在疾病的峰的血脑屏障渗漏。该信号是眼睛周围的最强的。为基质金属蛋白酶近红外基材是用来评估EAE诱发炎症。自动荧光干扰信号，要求对定量光谱分离。总体而言，生物发光成像是评估EAE相关视神经炎和药物作用的可靠的方法，并在信号的特异性，鲁棒性，易于量化的，和成本方面优于近红外技术。

视神经炎的生物发光的时间进程 炎症探针的生物发光信号是眼睛周围的最强和只在EAE小鼠视神经炎的发生。发生在没有非EAE小鼠也不不与炎症探针注入的小鼠的信号。当小白鼠恢复的信号消失了。因此，该信号是特异于视神经炎，信号的峰值平行于临床EAE分数的峰。 图1示出了在EAE诱导后第10天14成像...

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

我们表明用于体内实时生物发光和近红外中在SJL / J小鼠多发性硬化的实验性自身免疫性脑脊髓炎（EAE）模型视神经炎和脑炎的成像技术。

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

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.

Research

JoVE Journal

Medicine

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

动态视觉测试，以确定和量化视觉损伤与修复继脱髓鞘视神经炎患者

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

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

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

多发性硬化症性能测试（MSPT）：一个iPad为基础的残疾评估工具

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

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

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

季节变化的三角建模的方法证明与多发性硬化症复发数据

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

一个协议，用于远程监督经颅直流电刺激（TDCS）在多发性硬化症的使用（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

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

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

解剖性脑 MRI 的全脑分割及变化点分析--Premanifest 亨廷顿病的应用

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