Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Summary

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.

Abstract

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 (MOG_35-55) 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.

Introduction

Autoimmune diseases are a spectrum of disorders caused by the immune system's immune response to its own antigens resulting in tissue damage or dysfunction¹. Multiple sclerosis (MS) is a chronic autoimmune disease of polyneuropathy in the central nervous system (CNS), characterized by inflammatory infiltration, demyelination, and neuronal axonal degeneration^2,3. At present, MS has affected as many as 2.5 million people around the world, mostly young and middle-aged people aged 20-40, who are often the backbone of their families and society. This has caused considerable impact and harm to families and society^2,4.

MS is a multifactorial disease with diverse and complex clinical manifestations. In addition to classic neurological disorders characterized by inflammatory infiltration and demyelination, MS often shows visual impairment, limb dyskinesia, and cognitive and emotional disorders^5,6,7. If MS patients do not get the proper and correct treatment, half of them will live in wheelchairs after 20 years, and nearly half of them will experience depressive and anxiety symptoms, leading to much higher levels of suicidal ideation than the general population^8,9.

Despite a long research period, the etiology of MS remains elusive, and the pathogenesis of MS has not yet been elucidated. Animal models of MS have allowed serving as testing tools to explore disease development and new therapeutic approaches, despite the significant differences between the rodent and human immune systems, while at the same time sharing some basic principles. Experimental autoimmune encephalomyelitis (EAE) is currently the ideal animal model for studying MS, which uses autoantigen immunity from myelin proteins to induce autoimmunity to CNS components in susceptible mice, with the addition of complete Freund's adjuvant (CFA) and pertussis toxin (PTX) to enhance the humoral immune response. Depending on the genetic background and immune antigens, different disease processes, including acute, relapsing-remitting, or chronic, are obtained to mimic various clinical forms of MS^10,11,12. The relevant immunogens commonly used in the construction of EAE models come from self-CNS proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG). MBP- or PLP-immunized SJL/L mice develop a relapsing-remitting course, and MOG triggers chronic progressive EAE in C57BL/6 mice^11,12,13.

The main purpose of disease-modifying therapy (DMT) is to minimize disease symptoms and improve function⁶. Several drugs are used clinically to alleviate MS, but no drug has yet been used to completely cure it, revealing the necessity of synergistic treatment. C57BL/6 mice are currently the most commonly used to construct transgenic mice, and in this work, an EAE model induced by MOG_35-55 in C57BL/6J mice with a 5-point scale was used to monitor the disease progression. EAE models also suffer from anxiety-like moods and bone loss, and the widely known demyelinating lesions. Here, the method to assess the symptoms of EAE from multiple perspectives using open-field test and micro-computed tomography (Micro-CT) analysis is also described.

Protocol

The Animal Care Committee of Tongji University approved the present work, and all animal care guidelines were followed. Male or female C57BL/6J mice between 8-12 weeks of age were used for the experiments. It was ensured that the age and sex were the same in the experimental groups; otherwise, the susceptibility to the disease was affected. Mice were housed in a specific pathogen-free environment with alternating 12 h light and dark cycles under constant conditions (room temperature 23 ± 1 °C, humidity 50% ± 10%), with free access to mouse food and water.

1. Preparation of MOG_35-55 emulsion

1. Add heat-inactivated lyophilized Mycobacterium tuberculosis (MTB, H37Ra) to complete Freund's adjuvant (itself containing 1 mg/mL of heat-inactivated MTB, H37Ra), resulting in a final MTB concentration of 5 mg/mL (see Table of Materials).
   NOTE: The entire operation must be completed in the biosafety cabinet; do not open the blowing air.
2. Dissolve the lyophilized MOG_35-55 peptide (see Table of Materials) with sterile pre-cooled Phosphate Buffered Saline (PBS) (without calcium and magnesium ions, pH 7.4) to prepare the antigen solution at the concentration of 2 mg/mL.
3. Take a clean 2 mL microcentrifuge tube and add one sterilized 5 mm steel ball (see Table of Materials) to each tube.
4. Add 500 µL of complete Freund's adjuvant containing 5 mg/mL of MTB and 500 µL of MOG_35-55 antigen solution to the above microcentrifuge tube containing one steel ball.
5. Oscillate the above tube on a TissueLyser (see Table of Materials) for 10 min, cool on ice for 10 min, and repeat four times to mix it well and finally form a white viscous solution.
   NOTE: Good emulsification is a key step in preparing MOG_35-55 emulsion, so thorough mixing is required. The TissueLyser is set to a speed of 28 Hz.

2. Preparation of pertussis toxin (PTX)

1. Prepare PTX with ddH₂O into a 100 µg/mL concentration and store at 4 °C.
2. Dilute the PTX stock solution 50 times with sterile 1x PBS (without calcium and magnesium ions, pH 7.4) to make a 200 ng/100 µL solution for use.

3. Establishment of EAE animal model

1. Construct the EAE model using the 8-12-week-old male or female C57BL/6J mice. Ensure that the mice are adequately acclimatized to the feeding environment prior to immunization.
2. Centrifuge the prepared MOG_35-55 emulsion (step 1) at 4 °C for 2-3 s by pressing the Pulse button of the equipment (see Table of Materials) to precipitate all the emulsions at the bottom of the tube.
   NOTE: MOG_35-55 emulsion can be stored at -20 °C for several days. To avoid drug failure, it is recommended to use it as soon as possible.
3. Attach a 22 G needle to a 1 mL syringe barrel, aspirate the MOG_35-55 emulsion, and transfer the MOG_35-55 emulsion into a new 1 mL syringe barrel. Secure the connection between the 1 mL syringe barrel and a 26 G needle with sealing film (see Table of Materials).
   NOTE: Avoid air bubbles when loading MOG_35-55 emulsion into 1 mL syringe barrels.
4. Wipe and disinfect the injection site with 70% ethanol.
5. Inject the MOG_35-55 emulsion subcutaneously on each side of the dorsal spine of the mice, 100 µL on each side. Observe the automatic formation of bulbous masses under the skin of the mice's dorsum after the injection operation is completed.
   NOTE: Ensure that experienced experimenters perform the immunization process and that the injection is done gently and slowly to minimize the pressure on mice.
6. Inject the above mice intraperitoneally with 100 µL of PTX (step 2).
   NOTE: The day of immunization is day 0. Also, ensure that the mice can be accurately identified for subsequent daily evaluation, such as using a color marker on the tail of the mice.
7. Inject the same dose of PTX on day 2 after immunization.
8. Prepare a group of unimmunized mice as wild-type (WT) mice.

4. Clinical monitoring of mice

1. Record the bodyweight of EAE and WT mice daily.
   NOTE: The severity of EAE is positively correlated with the weight loss of mice, so bodyweight is also a very important monitoring index.
2. Monitor the status of mice from 0-21 days after immunization using the 0-5 scoring system listed in Table 1.
   NOTE: Symptoms in between are counted as plus or minus 0.5 points.

5. Open field test

NOTE: The experimental animals selected for this step are EAE mice in the early onset, peak, and remission periods. In addition, WT mice were used as control. It is to be noted that all mice were tested for anxiety-like behavior prior to modeling to exclude mice with anxiety disorders for EAE modeling. In addition, EAE mice in peak and remission periods with complete motor incapacity were excluded from the test.

1. Prepare a 40 × 40 × 40 cm³ open field reaction chamber and a locomotion activity (open field) video analysis system (see Table of Materials).
   NOTE: The camera is installed in a position that completely covers the box, the reaction room is evenly lit, and the test room is required to be a quiet area.
2. Place the test mice in the test room for habituation 1 h before starting the experiment.
3. Spray the entire area with 70% ethanol and wipe with a clean paper towel to ensure the reaction chamber is clean before starting the test.
4. Remove each mouse individually from its cage and place it in the same corner of the arena before starting to explore.
   NOTE: The bottom of the box is divided into 16 grids, of which the middle four grids area is the central area and the surrounding area is the peripheral area.
5. Click on the Start Capture button in the menu bar of the video analysis system, record time, and begin shooting.
6. Keep quiet in the test room.
7. Let the mouse move freely for 5 min during the recording process.
8. Stop the acquisition system and save the video.
9. Take the mouse out of the arena, put it back in the cage, and proceed to the next mouse.
   NOTE: Clean the test area with 70% ethanol between runs to remove odors and other substances.
10. Analyze the results using the video analysis system.

6. Analysis of bone phenotype

1. Euthanize the EAE and WT mice by cervical dislocation on the 21st day.
   NOTE: Personnel performing cervical dislocation operations must be well trained to minimize the pain endured during the animal's death.
2. Make the mouse lie flat in a dissecting tray and fix the extremities.
3. Hold the mouse hind limb skin with forceps and open the mouse skin and muscle tissue with scissors.
4. Separate the femur from the tibia and hip bone carefully with scissors.
5. Remove the muscle adhering to the femur with scissors and place the femur in 70% ethanol at room temperature.
6. Scan the distal femur using a micro-CT system (see Table of Materials) with an isotropic voxel size of 10 µm, with a peak X-ray tube voltage of 70 kV and an X-ray intensity of 0.114 mA.
   NOTE: A 3D Gaussian filter allows the denoising of 2D threshold images.
7. Analyze the 100 slices scanned from the middle femoral shaft to measure femur parameters, including bone volume, tissue volume, bone mineral density, trabecular separation, trabecular number, trabecular connection density, and trabecular and cortical thickness.
   NOTE: Starting from the proximal end of the distal femur growth plate in mice, sections completely devoid of epiphyseal cap structures were found and continued to extend 100 slices towards the proximal femur, which were manually outlined contours at several voxels away from the inner cortical surface to identify epiphyseal trabeculae.
8. Create the 3D reconstructions by stacking threshold 2D images from contour regions in the micro-CT system.

Results

After immunization of the mice, the bodyweight of the mice is recorded daily, and their clinical symptoms are evaluated according to the protocol described above (step 4). In C57BL/6J mice immunized with MOG peptide, because the location of the lesion is mainly confined to the spinal cord,the pathogenesis of EAE mice spreads from the tail end to the head. At the beginning of the disease, EAE mice exhibit weakness and drooping of the tail, followed by weakness of the hind limbs, uncoordinated movement, and paralysis. As t...

Discussion

MS is a demyelinating inflammatory disease of the CNS and is one of the most common neurological disorders causing chronic disability in young people, imposing a huge burden on families and society^3,4. MS has always been classified as an organ-specific T cell-mediated autoimmune disease, inducing the autoimmune system to slowly erode CNS, which will involve multiple systems throughout the body²⁷. Typical clinical symptoms include visual im...

Materials

Name	Company	Catalog Number	Comments
1 mL syringe(with 26 G needle)	Shanghai Kindly Medical Instruments Co., Ltd	60017031
2 mL microcentrifuge tube	HAIKELASI	KY-LXG2A
22 G needle	Shanghai Kindly Medical Instruments Co., Ltd	60017208
Complete Freund’s Adjuvant	Sigma	F5881	Stored at 4 °C, 1 mg of heat-inactivated MTB (H37Ra) per mL
Conditioned place preference system	Shanghai Jiliang Software Technology Co., Ltd		Animal behavior
Ethanol	Sinopharm Chemical Reagent Co., Ltd	10009218	Stored at RT
Locomotion activity (open field) video analysis system	Shanghai Jiliang Software Technology Co., Ltd	DigBehv-002	Animal behavior
MOG35-55 peptide	Gill Biochemical Co., Ltd	GLS-Y-M-03590	Stored at -20 °C
Mycobacterium tuberculosis H37Ra	BD	231141	Stored at 4 °C
Open field reaction chamber	Shanghai Jiliang Software Technology Co., Ltd		Animal behavior
Pertussis toxin	Calbiochem	516560	Stored at 4 °C
Phosphate Buffered Saline			Made in our laboratory
Scissor	Shanghai Medical Instrument (group) Co., Ltd	J21010
Sealing film	Heathrow Scientific	HS 234526B
Sorvall Legend Micro 21R Microcentrifuge	Thermo Scientific	75002447
Steel ball	QIAGEN	69975
TissueLyser II	QIAGEN	85300
Tweezer	Shanghai Medical Instrument (group) Co., Ltd	JD1060
μCT 35 desktop microCT scanner	Scanco Medical AG, Bassersdorf, Switzerland