This research was supported by National Natural Science Foundation of China grant (31570921 to ZJ, 81571533 to LS), Shanghai Municipal Commission of Health, and Family Planning (201540206 to ZJ), Ruijin Hospital North research grant (2017ZX01 to ZJ).

All methods described here have been approved by the animal committee of the School of Basic Medical Sciences, Shanghai Jiao Tong University.
1. Preparation of the materials
<ol>
	<li>Use the MEVGWYRSPFSRVVHLYRNGK sequence of MOG35&#8211;55 to obtain the lyophilized peptide from commercial sources. Ensure that the purity of the peptide is &#62;95%. Prepare 10 mg/mL MOG stock solution in phosphate-buffered saline (PBS) and store at -20 &#176;C.</li>
	<li>Prepare a 4 mg/mL stock solution of M. tuberculosis H37 Ra by putting one 100 mg tube of M. tuberculosis H37 Ra into 25 mL of Complete Freund&#39;s Adjuvant (CFA) and mixing. Store the stock solution at -20 &#176;C.</li>
	<li>Prepare 1 ng/&#956;L Pertussis Toxin Working Solution (PTX) by adding 50 &#956;g of PTX into 50 mL of PBS. Store the working solution at -20 &#176;C.</li>
	<li>Store all antibodies (i.e., FITC anti-mouse CD3, PE/Cy7 anti-mouse CD4, PerCP/Cy5.5 anti-mouse CD11b, Alexa Fluor700 anti-mouse CD45.2, PE anti-mouse IL-17A, and APC anti-mouse IFN-&#947;) at 4 &#176;C.</li>
	<li>Make the Flow Cytometry Staining (FCS) Buffer by adding 2 mM ethylene diamine tetraacetic acid (EDTA) and 1% fetal bovine serum (FBS) into 500 mL of PBS.</li>
</ol>
2. Housing of C57BL/6 mice
<ol>
	<li>Use female C57BL/6 mice at 8&#8211;12 weeks of age.</li>
	<li>Acclimate C57BL/6J mice for at least 7 days prior to the injection.</li>
	<li>House mice in an animal facility under pathogen-free conditions at constant temperature and humidity in a 12 h light/dark cycle and provide free access to water and standard pellet food.</li>
</ol>
3. Immunization of C57BL/6 mice
<ol>
	<li>Leave all stock solutions for 15 min at room temperature (RT) to ensure complete rehydration.</li>
	<li>Dilute 300 &#956;L of MOG-peptide stock solution with 700 &#956;L of PBS for preparing 3 mg/mL work solution.</li>
	<li>Put 1 mL of M. tuberculosis H37 Ra stock solution and 1 mL of MOG35&#8211;55 peptides working solution into separate 10 mL syringes, then use a four-way stop cock to emulsify for at least 10 min. Ensure complete emulsification before injection.</li>
	<li>Anesthetize mice at the peak of EAE with an intraperitonial injection of 1% sodium pentobarbital (50 mg/kg).</li>
	<li>With a 1 mL syringe, subcutaneously inject mice with 100 &#956;L of a MOG 35&#8211;55/CFA emulsion (300 &#181;g/200 &#956;L) at two sites, both at the back near the neck. Subcutaneously inject control mice with 200 &#956;L of PBS.</li>
	<li>On the same day (day 0) and on day 2 post immunization (PI), Intraperitoneally inject mice with 200 &#956;L of 1 ng/&#956;L PTX working solution. Intraperitoneally&#160;inject control mice with 200 &#956;L of PBS.</li>
	<li>Transfer the mice to their home cage with a warming pad.</li>
	<li>Examine and grade all mice every day after the injection in a blinded manner for the neurological signs shown in Table 111,13. Euthanize the animals if the scores are worse than grade 4.</li>
	<li>Record the weight changes during the disease course. This is a valuable additional measure for disease activity in the EAE model11,13.</li>
	<li>Add the first day of clinical signs for individual mice and divide by the number of mice in the group; the result is the onset. Add the first day of the maximum EAE score for individual mice and divide by the number of mice in the group; the result is the peak.</li>
</ol>
4. Single-cell suspension preparation from brain
<ol>
	<li>Dilute density gradient medium in 9:1 ratio with PBS in a 15 mL conical tube to yield a final 100% solution.</li>
	<li>Anesthetize mice at the peak of EAE with an intraperitonial injection of 1% sodium pentobarbital (50 mg/kg) and perfuse intracardially with 20 mL of sterile ice-cold PBS. Achieve this by slowly and steadily injecting PBS into the left ventricle of the heart using a 20 mL syringe and opening the right atrium.</li>
	<li>Cut the cranium carefully from the nose to the neck, then remove the brain from the cranial box into 10 mL of RPMI in 50 mL conical tubes. Mix well to remove adherent red blood cells. Then remove the medium by aspiration and add 10 mL of RPMI.</li>
	<li>Place the brains and medium in a 100 mm dish. Finely chop with a razor blade.</li>
	<li>Transfer 6 mL from the dish to an ice-cold 7 mL sintered glass homogenizer with a clean pipette. Avoid leaving large quantities of tissue in the pipette. A small amount is unavoidable.</li>
	<li>Grind the brain using the &#34;loose&#34; plunger of the pestle first, then use the &#34;tight&#34; plunger until the suspension is homogeneous, and pour into a prechilled 15 mL conical tube and keep on ice.</li>
	<li>After all the samples are homogenized, estimate the volume. Adjust the volume with RPMI to 7 mL. Then place 3 mL of ice-cold 100% basement membrane matrix in a chilled 15 mL conical centrifuge tube and add 7 mL of the brain homogenate to yield a final 30% density gradient medium. Mix by inversion a couple of times. Do not vortex.</li>
	<li>To ensure a sharp interface, carefully and slowly add 1 mL of 70% underlay density gradient medium in RPMI with a 3 mL pipette.</li>
	<li>Centrifuge at 800 x g for only 20 min at 4 &#176;C. Set the acceleration to 1 and deceleration to 0. After centrifugation, aspirate almost all of the top phase, being careful to completely remove the myelin at the top (Figure 1).</li>
	<li>Remove the interface into a new 15 centrifuge tube. Adjust the volume to 10 mL with RPMI.</li>
	<li>Centrifuge at 500 x g for 10 min. After centrifugation, aspirate the supernatant. Resuspend the pellet in ~200 &#956;L of flow cytometry staining (FSC) buffer. The pellets are then ready to stain for FACS.</li>
</ol>
5. Flow cytometric analysis of single cells from brain
<ol>
	<li>Use a hemocytometer and microscope to count the cells. Add 10 &#956;L of the cells to 10 &#956;L of trypan blue, mix well, and place 10 &#956;L on a hemocytometer to count the cells. Then calculate the number of live cells per microliter under an inverted microscope (e.g., Olympus Inverted microscope).</li>
	<li>Aliquot approximately 2 x 106 of cells in RPMI into a single well of a 96 well plate.</li>
	<li>Add 500x cell stimulation cocktail plus protein transport inhibitors to the wells.</li>
	<li>Incubate the plate in the incubator at 37 &#176;C for 4 h.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at RT. Discard the supernatant and resuspend the cells in 100 &#956;L of FCS Buffer.</li>
	<li>Preincubate the cells with anti-mouse CD16/CD32 Fc block (1:33) for 10 min at 4 &#176;C before staining to block nonspecific Fc-mediated interactions.</li>
	<li>Stain cell surface markers without washing. Add anti-mouse CD45.2 (1:200), anti-mouse CD11b (1:200), anti-mouse CD3 (1:200), and anti-mouse CD4 (1:200) antibodies. 
	NOTE: To determine positive and negative gates, a fluorescence minus one (FMO) for each color and an isotype control antibody should be stained.</li>
	<li>Incubate the plate for at least 30 min at 4 &#176;C or on ice. Protect from light.</li>
	<li>Wash the cells by adding FCS Buffer. Use 200 &#956;L/well for microtiter plates. Centrifuge at 400 x g for 5 min at RT. Discard the supernatant.</li>
	<li>Add 200 &#956;L of intracellular (IC) fixation buffer to each well to fix the cells. Ensure the cells are fully resuspended in the solution.</li>
	<li>Incubate 30&#8211;60 min at RT. Protect from light.</li>
	<li>Centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Add 200 &#956;L of 1x permeabilization buffer to each well and centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in residual volume and adjust volume to about 100 &#956;L with 1x permeabilization buffer.</li>
	<li>Add anti-mouse IL-17A (1:200) and anti-mouse IFN-g (1:200) antibodies for detection of intracellular antigens to cells.</li>
	<li>Incubate for at least 30 min at 4 &#176;C. Protect from light.</li>
	<li>Add 100 &#956;L of 1x permeabilization buffer to each well and centrifuge the samples at 400 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the stained cells in 100 &#956;L of flow cytometry staining buffer.</li>
	<li>Analyze by flow cytometry. 
	NOTE: The laser and compensation settings on the flow cytometer are adjusted, the samples are placed onto the cytometer, and all events are recorded as per the manufacturer&#8217;s recommendations.</li>
</ol>
6. Data analysis
<ol>
	<li>Gate singlets using FSC-A vs. FSC-H and SSC-A vs. SSC-H.</li>
	<li>Gate live cells using FSC-A and SSC-A based on size.</li>
	<li>Next, identify the leukocytes, excluding the monocytes, by gating on CD45+ CD11b- cells.</li>
	<li>Then, identify the CD4 T lymphocytes by gating on CD3+CD4+ cells.</li>
	<li>Lastly, identify the Th1 and Th17 subsets by gating on IFN-&#947;+ cells and IL-17+ cells separately, and determine the positive and negative populations using isotype controls and FMO.</li>
</ol>

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The authors have no conflicts of interest to declare.

This study presents a protocol to induce and monitor EAE using MOG35-55 in C57BL/6 mice, which are considered a typical neuroimmunological experimental animal model of MS. EAE can be induced varying the mice strains or the type of protein used for induction according to the purpose of the study. For example, using PLP139&#8211;151 peptide in SJL mice can induce a relapsing-remitting EAE disease course that is especially well-suited for assessing therapeutic effects on relapses15. The experimental ...

As a chronic demyelinating disease of the CNS, MS affects about 2.5 million people worldwide and lacks curative treatments1. It is also considered an autoimmune disease, in which myelin antigen specific T lymphocytes initiate an inflammatory reaction and lead to demyelination and axonal injury in the CNS2. Experimental autoimmune encephalomyelitis (EAE) has been widely used to investigate pathogenic mechanisms of MS as a classic autoimmune demyelination disease model in CNS3. There are two ways to induce EAE: one is to induce EAE actively by immunizing animals with myelin components, another is adoptive transfer by transferring encephalitogenic T cells into receptor2,4,5. The susceptibilities to EAE are different in different animal strains6. In C57BL/6 mice, myelin oligodendrocyte glycoprotein (MOG) 35&#8211;55 challenge induces a monophasic disease with extensive demyelination and inflammation in the CNS, which is frequently used in experiments with gene-targeted mice7.
The generation of myelin-specific reactive T cells is required for the occurrence and development of disease in EAE and is an immunological sign of both EAE and MS. Activated autoreactive T lymphocytes cross the blood brain barrier (BBB) into the healthy CNS and initiate EAE disease. When MOG 35&#8211;55 Ag is encountered, these T lymphocytes induce inflammation and the recruitment of effector cells into the CNS, resulting in demyelination and axon destruction8,9. In the EAE model, there is ample evidence that neuroantigen-specific CD4+ T cells can initiate and sustain neuroinflammation and pathology3,10. Depending on the major cytokines produced, CD4+ T lymphocytes have been classified into different subsets: Th1 (characterized by the production of interferon-&#947;), Th2 (characterized by the production of interleukin 4), and Th17 (characterized by the production of interleukin 17). It is believed that activation of Th1 and Th17 cells contribute to the induction, maintenance, and regulation of inflammatory demyelination in EAE and MS by secreting effector cytokines IFN-&#947; and IL-17, which are capable of activating macrophages and recruiting neutrophils to the inflammatory sites to accelerate the lesions11.
Because autoreactive T cells cross the BBB into the CNS and induce the development of disease in MS and EAE, it is very important to analyze T cells in the CNS. However, there are very few established protocols for the isolation of lymphocytes from the CNS12. Therefore, a method optimized for isolating mononuclear cells from the brain and analyzing T lymphocytes with markers CD45, CD11b, CD3, CD4, INF-g, and IL-17 for flow cytometry was developed. The method uses MOG35&#8211;55 adjuvant Mycobacterium tuberculosis H37 Ra and Pertussis Toxin Working Solution (PTX) to induce an active immunization model of EAE in mice. Then, mechanical separation and density gradient centrifugation methods are used for the isolation of CNS mononuclear cells. Finally, an optimized flow cytometry gating strategy is used to identify T lymphocytes and subsets from the brain by staining multiple markers.

<table><tbody><tr><td>Alexa Fluor700 anti-mouse CD45.2</td><td>eBioscience</td><td>56-0454-82</td><td></td></tr><tr><td>Anti-Mouse CD16/CD32 Fc block</td><td>BioLegend</td><td>101302</td><td></td></tr><tr><td>APC anti-mouse IFN-g</td><td>eBioscience</td><td>17-7311-82</td><td></td></tr><tr><td>BD LSRFortessa X-20</td><td>BD</td><td></td><td></td></tr><tr><td>Dounce homogenizer</td><td>Wheaton</td><td>353107542</td><td></td></tr><tr><td>eBioscience Cell Stimulation Cocktail (plus protein transport inhibitors) (500X)</td><td>eBioscience</td><td>00-4975-03</td><td></td></tr><tr><td>eBioscience Intracellular Fixation &amp;amp; Permeabilization Buffer Set</td><td>eBioscience</td><td>88-8824-00</td><td></td></tr><tr><td>FITC anti-mouse CD3</td><td>BioLegend</td><td>100203</td><td></td></tr><tr><td>FITC Rat IgG2b, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400605</td><td></td></tr><tr><td>Freund&#039;s Adjuvant Complete (CFA)</td><td>Sigma-Aldrich</td><td>F5881</td><td></td></tr><tr><td>Mouse IgG2a kappa Isotype Control (eBM2a), Alexa Fluor 700, eBioscience</td><td>eBioscience</td><td>56-4724-80</td><td></td></tr><tr><td>Mycobacterium tuberculosis H37 Ra</td><td>Difco Laboratories</td><td>231141</td><td></td></tr><tr><td>PE anti-mouse IL-17A</td><td>eBioscience</td><td>12-7177-81</td><td></td></tr><tr><td>PE/Cy7 anti-mouse CD4</td><td>BioLegend</td><td>100422</td><td></td></tr><tr><td>PE/Cy7 Rat IgG2b, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400617</td><td></td></tr><tr><td>Percoll</td><td>GE</td><td>17-0891-01</td><td></td></tr><tr><td>PerCP/Cy5.5 anti-mouse CD11b</td><td>BioLegend</td><td>101228</td><td></td></tr><tr><td>PerCP/Cy5.5 Rat IgG2b, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400631</td><td></td></tr><tr><td>pertussis toxin (PTX)</td><td>Sigma-Aldrich</td><td>P-2980</td><td></td></tr><tr><td>Rat IgG1 kappa Isotype Control (eBRG1), APC, eBioscience</td><td>eBioscience</td><td>17-4301-82</td><td></td></tr><tr><td>Rat IgG2a kappa Isotype Control (eBR2a), PE, eBioscience</td><td>eBioscience</td><td>12-4321-80</td><td></td></tr><tr><td>Rat MOG35&amp;ndash;55 peptides</td><td>Biosynth International</td><td></td><td>MEVGWYRSPFSRVVHLYRNGK</td></tr></tbody></table>

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.

After immunization of C57BL/6 mice, all mice were weighed, examined, and graded daily for neurological signs. The representative clinical course of EAE should result in a disease curve as presented in Figure 2A and a change of body weight in the mouse as presented in Figure 2B. C57BL/6 mice immunized with MOG35-55 usually started to develop disease symptoms around day 10&#8211;12 and achieved the peak of disease around day 15&#8211;21 after active immunization (...

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

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 ...

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7.0K Views.  Shanghai Jiao Tong University School of Medicine. Het protocol dat we hier presenteren zal nuttig zijn voor ons om te begrijpen hoe lymfocyten betrokken zijn bij de ontwikkeling van het centrale zenuwstelsel auto-immuunziekte. Met deze methode kunnen lymfocyten in de hersenen per cel worden bestudeerd. Dit protocol kan ook worden toegepast op andere modellen en de ziekte van het centrale zenuwstelsel die nodig zijn om de kenmerken en functies van immuuncellen te analyseren.In deze video kan het protocol gemakkelijk aantonen hoe u mode...

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

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

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) ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

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 ...

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 ...

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

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.

Author Spotlight: Unveiling the Pathway Linking Obesity to Autoimmune Inflammation in Multiple Sclerosis 

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.

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

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Neuroscience

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 ...

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 ...

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

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