Name: scoring central nervous system inflammation, demyelination, and axon injury in experimental autoimmune encephalomyelitis
Uploaded: 2024-02-23T14:35:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Unveiling the Pathway Linking Obesity to Autoimmune Inflammation in Multiple Sclerosis at JoVE.com

We thank Dr. Raymond Sobel (Stanford University) for showing us his method of grossing and fix brain and spinal cord sections. We thank Kyle Roberton and Milan Ganguly from the Toronto Centre for Phenogenomics for learning the embedding method and for cutting so many of our brain and spinal cord sections. We thank Dr. Matthew Cussick and Dr. Robert Fujinami (University of Utah) for sharing their protocols for scoring submeningeal and perivascular inflammation in the spinal cord. We thank Shalina Ousman for sharing the clone of the CD45 antibody. We thank Xiofang Lu for training on the tissue processor and tissue embedding station and maintaining this equipment at the Keenan Research Centre of Biomedical Research at St. Michael&#8217;s Hospital. This work was supported by a Biomedical grant from MS Canada (to SED). Carmen Ucciferri is supported by a studentship from the Government of Canada. Nuria Alvarez-Sanchez is supported by a Keenan post-doctoral fellowship.&#160; &#160;&#160;

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

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Shannon Dunn consults for FSD Lucid Psycheceuticals. &#160;&#160;&#160;&#160;&#160;

Histological staining of the spinal cord is an important tool in assessing EAE disease severity, particularly in instances where there are differences between treatment groups in the extent of disease recovery in the post-acute phase of disease. Staining for immune cell infiltration (CD45), myelin (LFB) and axonal injury (SMI-32) helps characterize the underlying cause of the altered clinical scores in mice. The histological staining protocol described here provides a perspective of inflammation as well as the extent of myelin and axonal injury. Furthermore, the results shown validate sNF-L measurement as a method to assess the extent of overall neuronal damage in EAE.
The critical parameters for this analysis are to ensure that investigators are blinded to the identity of the sections and that there is equivalent sampling at each level of the spinal cord across the different mice. This is because the severity of inflammation can be greater at lower levels of the cord. Another critical parameter is the size of the experimental groups. Spinal cords and brains are typically harvested from 6&#8211;8 mice per group at endpoint to see significant differences between groups with treatments or genotypes having modest effect sizes. It is also important to ensure that selected mice, when averaged, have representative mean scores of the entire group. Regarding trouble shooting, a common problem encountered by those who are inexperienced with the protocol is that the spinal cord is fixed for an insufficient length of time and it is not easily extruded from the spinal column. If this is the case, the spinal cord can be manually dissected from the column by clipping along the spinous processes using fine scissors and opening the column to reveal the spinal cord. Alternatively, tissues can be fixed for a few additional days without interfering with the success of antibody staining. The antibody clones that are described here work in tissue fixed up to 2 weeks in formalin.
Embedding the spinal cord pieces requires skill and practice. It is recommended that eye loupes be worn and a lamp be directed over the embedding station to better visualize whether the sections are falling in cross-section or in longitudinal section. Keeping the lengths of the spinal cord pieces at less than 2 mm during grossing will help them fall in cross-section. Another common problem encountered for less-experienced users is that the LFB evaporates during the overnight incubation, leaving half of the slide stained and half unstained. To avoid evaporation, the glass staining dish should be sealed with thermoplastic film and then plastic wrap. If evaporation occurs and sections are unevenly stained, it is recommended to completely de-blue the slides with lithium carbonate and re-stain them again in LFB overnight. Another common issue is that users do not fully de-blue the grey matter after LFB. It is critical to examine individual sections under the microscope to ensure that a sufficient amount of de-bluing has been reached before proceeding with other steps in the protocol. In addition, though the CD45 and SMI-32 IHC stains perform robustly, it is still important to trouble shoot antibody concentrations in preliminary experiments for each new antibody lot received. This can be done by testing a variety of concentrations of the antibody on a positive control section (EAE spinal cord). First-time staining should also include a negative control that consists of secondary antibody alone without primary antibody added. Finally, it is critical in the image analysis to threshold individual images as staining can be uneven across slides or sections.
This protocol uses freely available software. If one does not have access to a processor, an embedder, or microtome, these steps can be sourced to a hospital-based pathology core that offers these services. Also, if one does not have access to a slide scanner, one can use a light microscope that is fitted with a video camera to save TIFF images of the spinal cord or brain regions. For a microscope-based workflow, capture LFB or LFB/H&#38;E sections at low power (40x magnification) and for CD45 and SMI-32 staining, image at least four windows that are centered in the ventral, dorsal, and lateral parts of the spinal cord (200x magnification for CD45 and 400x magnification for SMI-32). Image analysis can be performed on these images to quantify staining in a similar manner as described.
The decision of what histological approach to take to score EAE is dependent on how much the clinical scores differ between groups. For example, if there are drastic differences in EAE clinical score (one group got EAE and one did not), this usually relates to differences in peripheral-mediated inflammation. In this case, scoring for the presence of demyelinating lesions on LFB/H&#38;E-stained sections is sufficient and will reveal differences between groups. If groups are more similar in clinical score at onset and there are instead differences in the extent of clinical recovery (e.g. experiment in Figure 5A), it is best to apply the full histological workflow that is outlined here, including scoring of brain inflammation in the brain stem and cerebellum, to distinguish whether the differences the disease chronicity relate to differences in inflammation or tissue damage.&#160; If differences in inflammation are found as assessed by CD45 counting, further IHC studies can be done to stain for T cells (anti-CD3), infiltrating monocyte/macrophages (Mac3) and microglia (Iba-1/TMEM119) (recommended antibody clones are in Supplementary Table 3). Microglia activation is reflected by an increase in the intensity of Iba-1 staining on double-labeled Iba-1+TMEM-19+ microglia and an increased retraction of microglia processes that can be assessed by Sholl analysis on sections32. Furthermore, techniques like flow cytometry or single cell RNA sequencing can be applied to conduct a deeper characterization of the frequency and phenotype of immune populations in the brain and spinal cord.
The counting of SMI-32+ axons is a sensitive method to detect axon injury in EAE32,33 and in MS34. SMI-32, which detects the non-phosphorylated form of neurofilament heavy or medium accumulates in end-bulbs of transected neurons. An alternative to detect injured axons is to stain with amyloid precursor protein (APP) that can accumulate in axons as a result of disrupted axon transport33. The pattern of staining for SMI-32 and APP though both both reflective of axon injury, do not typically overlap, indicating they they are detecting different pathologies33. One can also complement histological measures of axon injury by measuring sNF-L, which is a rapid and sensitive measure of ongoing axonal injury in both the spinal cord and the brain. It offers the advantage that it can be done in half a day in living mice. A drawback of this method is that the kits are expensive and the machine is highly specialized. The company that sells the sNF-L kit does offer a fee for service for those who do not have access to a SIMOA machine. An alternative to assessing axon injury is to score for axon loss by either counting axons in toluidine blue-stained sections of the spinal cord12 or counting neurofilament bundles detected by SMI-31 in areas of the spinal cord white matter32. Both of these are more laborious approaches than SMI-32 or sNF-L measurement.
If EAE clinical scores differ between groups, but scoring for inflammation, demyelination and axonal injury does not reveal differences between groups, it may be useful to stain for astrocyte activation using GFAP (see Supplementary Table 3 for recommended antibody clone). Astrocyte activation is associated with an increase in GFAP staining and this has been shown to correlate with EAE progression in some EAE models including chronic EAE in the DA rat35.
In conclusion, this protocol describes methods and provides an analysis workflow to conduct histological scoring of EAE.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for the human demyelinating disease, Multiple Sclerosis (MS)1. Classic MS inflammatory pathology, including the infiltration of IFN-&#947; (gamma) and IL-17-producing T helper cells2, the infiltration of inflammatory monocytes3, the formation of perivascular and sub-meningeal inflammatory demyelinating lesions4, and the occurrence of axon injury4 in the central nervous system (CNS), is also observed in EAE5,6,7,8,9. The similarity in immune mechanisms between EAE and MS has made EAE a suitable pre-clinical model for testing the efficacy and mechanisms of action of a number of approved immune-based therapies for MS, including natalizumab, fingolimod, dimethyl fumarate, and glatiramer acetate (reviewed in1,5). Certain EAE regimens model other aspects of progressive MS pathology beyond axonal injury, including the development of sub-meningeal inflammation in the brain, chronic demyelination, spinal cord atrophy, synapse, and neuron loss6,10,11,12. Thus, EAE has utility for screening the efficacy of neuroprotective therapies for MS.
EAE is induced in rodents in a number of ways. Active immunization is the most common induction method and involves immunization of rodents with myelin antigens (either whole proteins or peptides) emulsified in CFA supplemented with heat-killed Mycobacterium tuberculosis13. Depending on the strain of mouse, pertussis toxin (PTX) is also administered on day 0 and day 2 of immunization to increase the penetrance of disease13. EAE can also be induced by adoptively transferring myelin-specific T cells obtained from myelin/CFA-primed mice into healthy mice14&#160;or can develop spontaneously in mice that overexpress T cell receptors specific for the major myelin antigens5.
EAE disease severity and progression are commonly scored using a discrete 5-point clinical scale: 1 &#8211; tail limpness, 2 &#8211; hindlimb or foot weakness, 3 &#8211; complete paralysis in one or both hindlimbs, 4 &#8211; forelimb weakness, 5 &#8211; moribund or dead13. This clinical scoring system is sound in documenting the progression of ascending paralysis that occurs at disease onset but is less sensitive at capturing the extent of recovery from CNS inflammatory attacks. For example, both mice that ambulate with difficulty and mice that ambulate easily but exhibit foot-grasping weakness are assigned a score of 2 on the EAE scale. Scores can remain high in the post-acute phase of EAE due to the presence of permanent axon injury or loss, even despite the resolution of the inflammatory response9. There have been a variety of attempts at developing more refined scoring systems, behavioral tests, measures of hindlimb and grip strength, and infrared monitoring systems&#160;to better capture differences in clinical deficits in EAE9,16,17,18; however, these more intricate scoring measures do not distinguish the contribution of inflammation versus tissue injury to the underlying neurological deficits. Thus, the gold standard approach to score the severity of EAE is to conduct both clinical and histological scoring.
Here, a protocol is described for how to dissect and embed mouse spinal cord and brain specimens in paraffin in a way that captures the stochastic process of lesion formation that occurs in EAE. A protocol is also presented of how to stain sections with Luxol fast blue (LFB), originally created by Kluver and Barrera19, which detects myelin in the CNS. Sections are either stained with LFB alone (for demyelination analysis) or are counter-stained with hematoxylin and eosin (H&#38;E) to help visualize and score inflammatory lesions. Protocols are also provided to quantify the presence of total leukocytes (CD45), the loss of myelin, and the number of injured axons (SMI-32) in the spinal cord using commercially available antibodies, immunohistochemical (IHC) techniques, and publicly accessible software. The protocol used to quantify leukocytes in the spinal cord can also be applied to quantify leukocytes in the brain.
Histological evaluation of axonal loss and injury in the brain is comparatively more difficult than in the spinal cord since brain white matter tracts do not run in parallel to one another. The measurement of serum neurofilament light (sNF-L) has emerged as a promising biomarker for&#160;neuronal injury in MS20,21. Recent studies have extended this technology to EAE22,23,24. Here, a method is presented to measure serum neurofilament light (sNF-L) in living mice using a Small Molecule Assay (SIMOA) assay. This method requires only a small quantity of serum and can be done in live mice in just half a day, providing rapid feedback on how a tested therapy is affecting overall CNS injury. All of the methods described here can be applied to mice of any sex or strain.

<table><tbody><tr><td>10% Neutral Buffered Formalin</td><td>Sigma Aldrich</td><td>HT501128-4L</td><td>Used to fix spinal cord and brain specimens</td></tr><tr><td>1000 mL Glass Beaker</td><td>Pyrex</td><td>1000</td><td></td></tr><tr><td>15 mL Falcon Tube</td><td>Starstedt</td><td>62.554.100</td><td>Fixing and storing spinal cord and brain</td></tr><tr><td>250 mL Erlenmeyer Flask</td><td>Pyrex</td><td>4980</td><td></td></tr><tr><td>500 mL Glass Beaker</td><td>Pyrex</td><td>1003</td><td></td></tr><tr><td>92 mm x 16 mm Petri Dishes</td><td>Starstedt</td><td>82-1473-001</td><td>Used in the tissue grossing procedure</td></tr><tr><td>95% Ethyl Alcohol</td><td>Commercial Alcohols</td><td>P016EA95</td><td>Dehydration and rehydration steps</td></tr><tr><td>ABC Elite Kit</td><td>Vector Labratories</td><td>PK6100</td><td>Used for immunohistochemistry labeling</td></tr><tr><td>Aqua Hold 2 PAP Pen</td><td>Cole Parmer</td><td>UZ-75955-53</td><td>Used for drawing around tissue sections in Immunohistochemical Staining</td></tr><tr><td>Avidin/Biotin Blocking Kit</td><td>Vector Labratories</td><td>SP-2001</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Any</td><td></td><td></td></tr><tr><td>Biotinylated rabbit anti-rat IgG</td><td>Vector Labratories</td><td>BA-4000</td><td>Used for CD45 staining</td></tr><tr><td>C57BL6/J Mice</td><td>Jackson Laboratory</td><td>Stock # 664</td><td>These mice were used in experiments shown in paper.</td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific</td><td>Sorvall ST Plus</td><td></td></tr><tr><td>CitriSolv</td><td>Fisher Scientific</td><td>04-355-121</td><td>Used for de-waxing. Is an alternative to xylene</td></tr><tr><td>DAB Kit</td><td>Vector Labratories</td><td>SK-4100</td><td>Used for developing in immunohistochemistry</td></tr><tr><td>ddH2O</td><td>-</td><td>-</td><td></td></tr><tr><td>Disposable Scalpel</td><td>Magna</td><td>M92-10</td><td>Used for grossing spinal cord and brain</td></tr><tr><td>DWK Life Sciences (Wheaton) glass staining dish</td><td>Cole Parmer</td><td>UZ-48585-60</td><td>Used for histochemical staining and washes</td></tr><tr><td>DWK Life Sciences (Wheaton) glass staining rack</td><td>Cole Parmer</td><td>10061392</td><td>Used for immunohistochemistry and histochemistry</td></tr><tr><td>Eosin Y</td><td>Bioshop</td><td>173772-87-1</td><td>Stains cytoplasm</td></tr><tr><td>Feather Microtome Blades</td><td>Fisher Scientific</td><td>12-634-1C</td><td>Used for sectioning paraffin</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>1001110</td><td>Used to filter the formalin (during grossing) and the luxol fast blue</td></tr><tr><td>Fine Surgical Scissors</td><td>Fine Science Tools</td><td>14160-10</td><td>Used to snip brain and the skull</td></tr><tr><td>Fumehood</td><td>Any</td><td></td><td></td></tr><tr><td>Gibco DPBS</td><td>Fisher Scientific</td><td>14190944</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>BioShop</td><td>ACE333.4</td><td>Used in the luxol fast blue staining procedure</td></tr><tr><td>Histoplex Histology Containers</td><td>Starplex Scientific</td><td>565-060-26</td><td>Fixing spinal cord and brain</td></tr><tr><td>Hydrogen Peroxide</td><td>Fisher Chemicals</td><td>H325-500</td><td>Used to remove endogenous peroxidase in the tissue</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>https://imagej.nih.gov/ij/download.html</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Kimberly Clark Professional</td><td>34155</td><td>Used for immunohistochemistry</td></tr><tr><td>Lens paper</td><td>VWR</td><td>52846-001</td><td>Used for trapping spinal cord species in cassette during processing</td></tr><tr><td>Light microscope</td><td>Any</td><td></td><td></td></tr><tr><td>Lithium carbonate</td><td>Sigma Aldrich</td><td>554-13-3</td><td>De-blueing after luxol fast blue staining</td></tr><tr><td>Luxol blue</td><td>Sigma Aldrich</td><td>1328-51-4</td><td>Stains CNS myelin</td></tr><tr><td>M.O.M Immunodetection Kit</td><td>Vector Labratories</td><td>BMK-2202</td><td>Used to stain SMI-32</td></tr><tr><td>Methanol</td><td>Fisher Chemicals</td><td>A454.2</td><td>Used for fixation</td></tr><tr><td>Mayer&#039;s Hematoxylin</td><td>Electron Microscopy Sciences</td><td>26381-02</td><td>Stains nuclei</td></tr><tr><td>Micro-Adson Forceps with Teeth</td><td>Fine Science Tools</td><td>11027-12</td><td>Used for reflecting the skull during dissections</td></tr><tr><td>Microcentrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Model 5417R</td><td></td></tr><tr><td>Microvette Capillary Tubes CB 300 Z</td><td>Starstedt</td><td>16.440.100</td><td>Used for blood collection</td></tr><tr><td>Micrscope Cover Glass</td><td>Fisher Scientific</td><td>12545A</td><td>Used for coverslipping</td></tr><tr><td>Mini Shaker</td><td>VWR</td><td>12620-938</td><td>Used for making buffers</td></tr><tr><td>NF light kit</td><td>Quanterix</td><td>103186</td><td>This kit can be used for detection of mouse or human soluble neurofilament in serum</td></tr><tr><td>Nitrile Gloves</td><td>VWR</td><td>76307-462</td><td>Safety</td></tr><tr><td>Normal Goat Serum</td><td>Vector Labratories</td><td>S-1000</td><td>Blocking reagent</td></tr><tr><td>Normal Rabbit Serum</td><td>Vector Labratories</td><td>S-5000</td><td>Blocking reagent</td></tr><tr><td>OmniSette Tissue Cassettes</td><td>Fisher Scientific</td><td>M4935FS</td><td>Used for embedding spinal cord and brain</td></tr><tr><td>p1000 Pipette and Tips</td><td>various</td><td></td><td></td></tr><tr><td>p200 Pipette and Tips</td><td>various</td><td></td><td></td></tr><tr><td>Paraffin Embedding station</td><td>Leica Biosystems</td><td>Model EG1160</td><td></td></tr><tr><td>Paraplast Tissue Infiltration/Embedding Medium</td><td>Leica Biosystems</td><td>39601006</td><td>Used for embedding spinal cord and brain</td></tr><tr><td>Permount Mounting Medium</td><td>Fisher Chemicals</td><td>SP15-100</td><td>Used for mounting coverslips on slides</td></tr><tr><td>pH meter</td><td>Fisher Scientific</td><td>13636AB315B</td><td>Used for pHing buffers</td></tr><tr><td>Plastic Transfer Pipettes</td><td>Fisher Scientific</td><td>13-711-20</td><td>Used for pHing buffers</td></tr><tr><td>Potassium Chloride</td><td>BioShop</td><td>7447-40-7</td><td>Used for making PBS</td></tr><tr><td>Potassium Phosphate Monobasic</td><td>BioShop</td><td>7778-77-0</td><td>Used for making PBS</td></tr><tr><td>Pressure Cooker</td><td>Nordic Ware</td><td>Tender Cooker</td><td></td></tr><tr><td>Purified rat anti-mouse CD45</td><td>Vector Labratories</td><td>553076</td><td>Detects leukocytes</td></tr><tr><td>Reagent grade alcohol 100%</td><td>VWR</td><td>89370-084</td><td>Dehydration and rehydration steps</td></tr><tr><td>Reagent grade alcohol 70%</td><td>VWR</td><td>64-17-5</td><td>Dehydration and rehydration steps</td></tr><tr><td>Rotary Microtome</td><td>Leica Biosystems</td><td>Model RM2235</td><td></td></tr><tr><td>Simoa Machine</td><td>Quanterix</td><td>HD-X</td><td></td></tr><tr><td>Slide Scanner</td><td>Zeiss</td><td>AxioScan.Z1</td><td></td></tr><tr><td>SMI-32 mouse IgG1 antibody</td><td>Biolegend</td><td>801701</td><td>Detects damaged axons</td></tr><tr><td>Sodium Chloride</td><td>BioShop</td><td>7647-14-5</td><td>Used for making PBS</td></tr><tr><td>Sodium Phosphate Dibasic&amp;nbsp;</td><td>Bioshop</td><td>7558-79-4</td><td>Used for making PBS</td></tr><tr><td>Standard Adson Forceps</td><td>Fine Science Tools</td><td>11150-10</td><td>Used for dissection steps</td></tr><tr><td>Superfrost Plus Microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td>Used to collect sections</td></tr><tr><td>Surgical Tough Cuts</td><td>Fine Science Tools</td><td>14110-15</td><td>Used to cut through the spine, body wall, and skin</td></tr><tr><td>Tissue Processor</td><td>Leica Biosystems</td><td>Model TP1020</td><td></td></tr><tr><td>Tri-soldium citrate</td><td>Thermo Fisher Scientific</td><td>03-04-6132</td><td>Used for antigen retrieval</td></tr><tr><td>Tween-20</td><td>BioBasic</td><td>9005-64-5</td><td>Used for washing sections</td></tr><tr><td>X-P Pierce XP-100 plate seal</td><td>Excel Scientific</td><td>12-140</td><td>Used for the sNF-L Assay</td></tr><tr><td>Xylene</td><td>Fisher Chemicals</td><td>1330-20-7</td><td>Used for de-waxing and clearing sections</td></tr><tr><td>Funnel</td><td>Cole Parmer</td><td>RK-63100-64</td><td>Used to filter formalin before grossing tissue</td></tr><tr><td>Stir Plate</td><td>Any</td><td></td><td>Used to make solutions</td></tr><tr><td>Oven</td><td>Any</td><td></td><td>Used to bake tissue sections after cutting</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>13-374-10</td><td>Used to seal LFB staining dish</td></tr><tr><td>Microwave</td><td>Any</td><td></td><td>Timing may vary depending on the microwave model</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich</td><td>9048-46-8</td><td>Used to make blocking buffer</td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fisher Scientific</td><td>05-408--129</td><td>Used to store mouse serum samples</td></tr><tr><td>Vortex</td><td>Any</td><td></td><td>Used to prepare samples for sNF-L assay</td></tr><tr><td>Waterbath</td><td>Any</td><td></td><td>Used to warm enzyme substrate for sNF-L assay</td></tr></tbody></table>

scoring central nervous system inflammation, demyelination, and axon injury in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund&#39;s adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into na&#239;ve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS).
Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides&#160;feedback on the extent of overall CNS injury in live mice.

Figure 3 shows representative IHC and histochemical staining, with examples of both acute (left) and older EAE lesions (right). Representative CD45 staining with hematoxylin counterstaining is shown in Figure 3A,B.&#160; Figure 3C&#8211;F show examples of LFB staining with (Figure 3C,D) or without (Figure 3E,F)&#160;the H&#38;E counterstain. Though hematoxylin is not specific to immune cells, the nuclei of the immune cells stain more darkly and can be distinguished from CNS resident cells. Figure 3G,H show representative staining of SMI-32+ axons, counterstained with hematoxylin. Notice the increased appearance of this stain in older EAE lesions.
Damage of myelinated tracks is most prevalent in the spinal cord in active murine EAE and this is the main driver of paralysis in this disease7,9. Thus, scoring for the presence of inflammation and tissue damage in the spinal cord is prioritized in histological analyses. EAE lesions occur sporadically at different regions (anterior, lateral or dorsal) (Figure 2A,B) and at different levels (sacral, lumbar, thoracic, cervical) of the spinal cord. The described embedding method ensures good sampling of lesions throughout the cord. More sections are embedded than analyzed, since some sections can become damaged in the processing or sectioning process. To ensure representative sampling, a minimum of 3 representative sections are analyzed at cervical, thoracic, and lumbar levels of the spinal cord for each mouse. The identity of each specimen is blinded in order that the person conducting the analysis is not biased when selecting representative sections for analysis.
To gain quick insights into differences in the histological severity of EAE, one can score for the presence of sub-meningeal demyelinating lesions in spinal cord quadrants in selected sections (Figure 2A,B). This is a rapid method that can be performed on scanned images or using a light microscope. This analysis is sensitive enough to detect differences in histological EAE severity between groups when EAE is severe in one group and mild in another. For example, in the experiment in Figure 4, EAE was induced in female wild type (WT) and mice with a deletion in OGR1 (OGR1 KO) using MOG p35-55/CFA plus PTX. Mice in the WT group developed severe EAE with complete paralysis, whereas the OGR1 knockout group developed mild disease. This difference in clinical score corresponded to a difference in the fraction of quadrants that had sub-meningeal lesions (Figure 4C).
It is important to complement the scoring of demyelinating lesions with percent area fraction of myelin staining to capture the extent of loss of myelin and/or myelinated axons during the autoimmune attack. In the example in Figure 4, the percent myelin fraction also differed signficiantly between the OGR1 and WT mice (Figure 4D). The percent myelin fraction also significantly correlates with the cumulative EAE score in mice with EAE (Figure 4E) and therefore serves as a good measure of overall tissue damage in this disease. Note that this protocol does not distinguish the intensity of myelin stain. If this is the desired outcome, one should conduct immunofluorescence staining for myelin proteins such as proteolipid protein or myelin basic protein and measure the intensity of this staining.
In the case where EAE is severe in both comparator groups, a higher fraction of spinal cord quadrants will contain inflammatory/demyelinating lesions. In this case, a more sensitive approach to score inflammation is to count the number of CD45+ leukocytes per mm2 white matter (see representative staining in Figure 3A). The CD45 antibody clone described here detects all infiltrating leukocytes, and only stains occasional microglia that upregulate CD45 expression in EAE (see open arrow in Figure 3B) and therefore is useful at capturing peripheral immune cell infiltration.
In longer-term EAE studies (&#62;20 days), it is recommended that one also carry out an analysis of axon injury. SMI-32 staining in spinal cord sections is a sensitive method to detect damaged axons. Though inflammation in the spinal cord subsides with time and spared axons can re-myelinate, surviving axons exhibit a differential extent of residual injury9 (Figure 3G,H). For example, in the MOG p35-55-induced model of EAE in C57BL6/J mice, the extent of axonal injury and loss is a driver of clinical scores after the inflammatory process has subsided9. Figure 5 shows an example of this in an EAE experiment in male and female mice WT mice and mice that are deficient in a gene called peroxisome proliferator-activated receptor-delta (PPAR-delta) in the myeloid compartment (LysMCre: Ppardfl/fl). In the males, the WT mice regained hindlimb function, yet the clinical scores remained high in male LysMCre: Ppardfl/fl group. By contrast, in the experiment in the females, both experimental groups had high scores throughout. At first glance, this result suggested that PPAR-delta had a sex-specific effect in EAE; however, pathological scoring of the spinal cord revealed that mice of both sexes in the LysMCre: Ppardfl/fl group had increased axonal injury compared to WT counterparts (Figure 5B). A genotype effect on clinical scores was likely not observed in females because WT female mice tended to exhibit increased axonal injury, which manifested into chronic neurological deficits.
In this same experiment, female LysMCre: Ppardfl/fl female mice were found to have more extensive T cell infiltration in the cerebellum, providing an example of how scoring brain inflammation may be useful in EAE. In EAE, inflammation in the brain is predominantly found in the cerebellum and brain stem (Figure 6A,D,G), but can also be found in the meninges (seen under hippocampus in Figure 6C), near the ventricles (Figure 6F), and other white matter tracts including the optic nerve and corpus collosum (Figure 6B,E). Scoring brain inflammation is done in a specific brain region (e.g., cerebellar white matter) by counting the number of CD45 cells per mm2 tissue region using the same methodology as outlined for the spinal cord protocol. In the grossing method outlined here, a cut is in the middle of the cerebellum, which provides the perspective of the cerebellum and brain stem as shown in Figure 6A.
Measuring sNF-L using a SIMOA assay has become a useful biomarker for assessing ongoing axonal injury and responses to therapy in relapsing-remitting MS20,21,27,28. The same SIMOA assay kit used to measure of sNF-L humans can be applied to measure mouse sNFL22,23,24. To explore how well this assay performs is detecting axonal injury in EAE, sNF-L was measured in female C57BL6/J mice at the end-point of an EAE experiment and levels were compared to those in sex-matched healthy control mice that did not have EAE. It was found that mice with EAE had much higher levels of sNF-L than in healthy mice (Figure 7A) and these levels correlated with the density of SMI-32+ axons in the spinal cord &#160;(Figure 7B). Compared to histological scoring of axonal injury, the SIMOA assay is faster (from bleeding mice to results can be achieved in just over half a day) and therefore provides rapid feedback of how a treatment is working in living mice. This assay also has the advantage that it reflects axonal injury in both the spinal cord and the brain.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65738/65738fig01.jpg" /> 
Figure 1: Representative paraffin block of brain and spinal cord sections. The 5 coronal brain sections and spinal cord cross sections (1.5&#8211;2 mm thick) are embedded in the same block in order that they can be cut in one section. At least 15 sections of spinal cord should be embedded, allowing for adequate selection of sections for analysis. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65738/65738fig02.jpg" /> 
Figure 2: Scoring meningeal inflammation and percent myelin area at the level of the thoracic spinal cord. (A,B) show images of the thoracic spinal cord from a female C57BL6/J mouse with MOG p35-55-induced EAE stained with LFB/H&#38;E. Shown is the approach used to visualize quadrants and examples of demyelinating lesions (traced in dotted line). The mouse in A has 4 of 4 quadrants with confluent demyelinating lesions, while the mouse in B has 1 of 4 quadrants affected. The mouse in B does have some inflammation in other quadrants, but this has not manifested into a confluent lesion and therefore is not scored. (C&#8211;E) Example of LFB image, and the greyscale and thresholded image in imageJ. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65738/65738fig03.jpg" /> 
Figure 3: Spinal cord sections stained with CD45, LFB H&#38;E, LFB and SMI-32. Examples of an early (A,C,E,G) and late (B,D,F,H) sub-meningeal lesion in the spinal cord stained for CD45 antibody (A,B), LFB/H&#38;E (C,D), LFB alone (E,F), and SMI-32 antibody (G,H). Black arrows show examples of cells stained with each respective antibody. White arrows show putative microglia that have been stained as CD45+. Scale bar = 50 &#181;m. This figure shows representative staining of lesions in the spinal cord of a female C57BL6/J mouse during EAE and highlights how pathology can be different across different spinal cord sections. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65738/65738fig04v2.jpg" /> 
Figure 4: Application of scoring for lesions and percent demyelination in EAE. Shown is an example of an EAE experiment where female mice deficient in Ovarian cancer G-protein coupled receptor 1 (OGR1) gene on the C57BL6/J background developed less severe clinical EAE than wildtype (WT) female C57BL6/J mice. EAE was induced by immunization with MOG p35-55/CFA plus PTX and mice were scored according to the following clinical scale: 1 = tail paralysis. 2 = hindlimb and foot weakness, 3 = hindlimb paralysis, 4 = forelimb weakness, 5 = moribund. (A) Mean + SEM clinical scores of mice over time. (B) Shown is an example of LFB/H&#38;E staining in the ventral spinal cord. Scale bar = 50 &#181;m. (C)&#160; Mean + SEM percent quadrants that contained demyelinating lesions. (D) Mean + SEM percent demyelination in each group. (E) shows result from another experiment in MOG p35-55-induced EAE in C57BL6/J mice where EAE scores of individual mice were summed over the 30 days of observation and were correlated with the percent demyelination in the spinal cord. Correlations were performed using a Spearman test. Panels in (A&#8211;D) are adapted from Souza C et al.29. Data in (E) are original data. *P&#60;0.05, **P&#60;0.01, ***P&#60;0.001. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65738/65738fig05.jpg" /> 
Figure 5: Application of SMI-32 staining to understand the effect of a genotype on clinical EAE phenotype. This figure shows an example of an EAE experiment where male and female wildtype (carry floxed allele of Ppard) and myeloid specific Ppard mutant mice (LysMCre: Ppardfl/fl) on the C57BL6/J background were immunized with MOG p35-55/CFA and PTX and were followed for 45 days. (A) shows the mean + SEM clinical scores of mice. (B) shows mean + SEM results of histological scoring of the number of SMI-32+ axons in the spinal cord, %quadrants with submeningeal lesions, percent quadrants with perivascular cuffs, and #CD3 lesions in the cerebellum per mm2 tissue. This experiment showed a genotype effect on SMI-32 staining. This figure is adapted from Drohomyrecky. et al.15. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65738/65738fig06.jpg" /> 
Figure 6: Examples of CD45+/hematoxylin staining in brain coronal sections in MOG p35-55-induced EAE in female C57BL6/J mice. CD45+ lesions are shown in brown. (A) CD45+ lesions in the brain stem of coronal sections. Scale bar = 150 &#956;m. (B&#8211;G) Examples of CD45+ &#160;lesions in the optic nerves (B), meningeal extensions under the hippocampus (C), the brain stem (D), the corpus collosum (E), the medial habenula near the ventricle (F), and the cerebellum (G).&#160; Scale bar: (B&#8211;G) = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65738/65738fig07v2.jpg" /> 
Figure 7: sNF-L levels in serum in MOG p35-55-induced EAE. (A) Serum NFL levels collected from female control and EAE mice at end-point of one experiment. Data was analysed using a two-tailed Mann Whitney test. (****p value &#60; 0.0001). (B) Spinal cord sections were harvested at end-point and stained with SMI-32. The number of positive cells per white matter tissue area was determined and correlated with serum NF-L at endpoint using a Spearman test. <a href="https://www.jove.com/files/ftp_upload/65738/65738fig07largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Description of baths used in tissue processing. Cassettes are automatically moved through these series of baths using an automated processor. <a href="https://www.jove.com/files/ftp_upload/65738/JOVESupplementaryTable1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Steps in Luxol Fast Blue and Hematoxylin and Eosin staining. This table outlines the order of steps in the Luxol Fast Blue and Hematoxylin and Eosin staining protocol. <a href="https://www.jove.com/files/ftp_upload/65738/JOVESupplementaryTable2.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 3: Antibodies used for immunohistochemical staining. Described are the antibodies that are used in this protocol as well as those that can be used to further explore inflammation, microgliosis, and astrogliosis. <a href="https://www.jove.com/files/ftp_upload/65738/SupplementaryJOVETable3_Revised(1).zip" target="_blank">Please click here to download this File.</a>
Supplementary Table 4: How to convert .czi to TIFF files. Note that it is optimal to use a high-resolution image, but medium-resolution images can be saved instead if the working memory of the computer is limiting. It is imperative to use images of the same resolution across analyses. Also, note that the last image of the series is the slide label. Avoid reading the label to ensure that the analysis is blinded.30,31 <a href="https://www.jove.com/files/ftp_upload/65738/SupplementaryJOVETable4.xlsx" target="_blank">Please click here to download this File.</a>

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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

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...

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Research

JoVE Journal

Immunology and Infection

3.9K Views.  University of Toronto. We zijn geïnteresseerd in het begrijpen van hoe auto-immuniteit en multiple sclerose of MS ontstaan. Dit onderzoek maakt gebruik van verschillende benaderingen om te bestuderen hoe risicofactoren geassocieerd zijn met MS. Leid tot de ontwikkeling van auto-immuniteit. Experimentele auto-immuun encefalomyelitis of EAE is een muismodel van MS dat kan worden gebruikt om het effect van verschillende risicofactoren op auto-immuniteit te onderzoeken.We hebben onlangs de biologische route ont...

Video: Auteur in de schijnwerpers: onthulling van de route die obesitas koppelt aan auto-immuunontsteking bij multiple sclerose 

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).
Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.
The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.

This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

Biology

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

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

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

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.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

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

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

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 limitans.&#160;

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Isolating All CNS Resident Cell Types Simultaneously from Autoimmune Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Author Spotlight: Studying Neuroinflammatory Pathways Through Simultaneous Isolation of All Main CNS-Resident Cell Types 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...

Neuroscience

Role of CD4 T Cell in Relapsing-Remitting Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) serves as a prototypic animal model for studying MS. In this study, we aimed to investigate the role of CD4 T cells in the initiation and relapse of EAE, focusing on the activation phase and immune response. To create the EAE mice model, female mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 emulsified with complete Freund&#39;s adjuvant (CFA). Clinical scores were assessed daily, and results demonstrated that mice in the EAE group exhibited a classic relapsing-remitting pattern. Hematoxylin-eosin (H&#38;E) and luxol fast blue (LFB) staining analysis revealed significant infiltration of inflammatory cells in the CNS and demyelination in EAE mice. Regarding the activation phase, both CD4+CD69+ effector T (Teff) cells and CD4+CD44+CD62L- effector memory T (Tem) cells may contribute to the initiation&#160;of EAE, however, the relapse stage was probably dominated by CD4+CD44+CD62L- Tem cells. Additionally, in terms of immune function, helper T (Th)1 cells are primarily involved in initiating the EAE. However, both Th1 and Th17 cells contribute to the relapse stage, and the immunosuppressive function of regulatory T (Treg) cells was inhibited during the EAE pathological process.

An experimental autoimmune encephalomyelitis (EAE) model in mice was employed to investigate the role of CD4 T cells in the initial and relapse of EAE from the perspective of the activation phase and immune effect function.

Multiple sclerosis (MS) is an autoimmune disease characterized by the infiltration of immune cells and demyelination in the central nervous system (CNS). ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

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.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Tietz, S. M. et al. <a href="https://app.jove.com/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EAE ...

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

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