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

Summary

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.

Abstract

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-1β, IL-6, IL-23, TNFα, IFNγ) 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.

Introduction

Multiple sclerosis (MS) and its corresponding animal model, experimental autoimmune encephalomyelitis (EAE), show autoimmune neuroinflammation changes in the central nervous system (CNS). Early active MS and EAE lesions are characterized by the presence of infiltrated immune cells. The etiology of MS remains unknown, but is widely considered to involve the destruction of myelin mediated by autoreactive T cells. These autoreactive T cells secrete pro-inflammatory cytokines and chemokines which attract other immune cells such as B cells, monocytes and neutrophils from the circulation. Monocytes differentiate into macrophages. Interferon gamma (IFNγ) secreted by aut....

Protocol

ETHICS STATEMENT: Our experimental procedures are approved by the Ethics Committee of the Regierungspräsidium Darmstadt (Germany) and confirm to National and European regulations. All efforts were made to minimize animal suffering and reduce the number of animals used.

1. EAE Model

1. Induction of the EAE model
   1. Use 10- to 13-week-old female 129S4/SvJae×C57BL/6 mice for the induction of EAE.
   2. Give the mice a subcutaneous injection, into the upper and lower back, of the encephalitogenic MOG_{35 - 55} (myelin oligodendrocyte glycoprotein) peptide (200 µg), emulsified in 200 µl complete....

Results

Figure 1 gives a schematic overview of the different methods described in this article. 1) Mice receive an injection of MOG_35-55 antigen and develop initial clinical symptoms after 10.7 ± 0.3 days²⁸. A representative disease course of EAE mice is shown in Figure 1. 2) Various tissues (spleen, lymph nodes, lumbar spinal cord) and blood are extracted from control and EAE mice at different time points d.......

Discussion

The EAE model described here has received the most attention as a model of MS and is routinely used in testing therapeutic strategies for MS³². The mouse disease exhibits many clinical and histological features of MS and is caused by the induction of autoimmunity to neuronal antigens. The sensitization to myelin antigens is associated with blood brain barrier dysfunction and thereby, immune cell infiltration into the CNS. Our findings show that immune cells increase transiently in the lymph nodes in the acute .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ABI Prism 7500 Sequence Detection System	Applied Biosystems, Austin, USA		quantitative PCR system
Accutase	Sigma Aldrich Munich, Germany	A6964	cell detachment solution
CD3-PE-CF594	BD, Heidelberg, Germany	562286
CD4-V500	BD, Heidelberg, Germany	560782
CD8-eFluor650	eBioscience, Frankfurt, Germany	95-0081-42
CD11b-eFluor605	eBioscience, Frankfurt, Germany	93-0112-42
CD11c-AlexaFluor700	BD, Heidelberg, Germany	560583
CD19-APC-H7	BD, Heidelberg, Germany	560143
CD45-Vioblue	Miltenyi Biotec, Bergisch Gladbach, Germany	130-092-910
CompBeads	BD, Heidelberg, Germany	552843	compensation beads
Collagenase A	Sigma Aldrich Munich, Germany	C0130
Cytometric absolute count standard	Polyscience, Eppelheim, Germany	BLI-580-10
Cytometer Setup and Tracking beads	BD, Heidelberg, Germany	642412
DNase I	Sigma Aldrich Munich, Germany	D5025
EAE Kit	Hooke Laboratories, Lawrence, USA	EK2110
F4/80-PE-Cy7	BioLegend, Fell, Germany	123114
First Strand cDNA-Synthesis kit	Thermo Scientific, Schwerte, Germany	K1612
Fc receptor-1 blocking buffer	Miltenyi Biotec, Bergisch Gladbach, Germany	130-092-575
Flow cytometric absolute count standard	Polyscience, Eppelheim, Germany	580
FlowJo software v10	Treestar, Ashland, USA		flow cytometry software
LSRII/Fortessa	BD, Heidelberg, Germany		flow cytometer
Ly6G-APC-Cy7	BD, Heidelberg, Germany	560600
Lysing solution	BD, Heidelberg, Germany	349202
Maxima SYBR Green	Thermo Scientific, Schwerte, Germany	K0221	fluorescent DNA binding dye
RNeasy Mini Kit	Qiagen, Hilden, Germany	74104	RNA extraction kit