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.

Representative 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 the disease worsens, it gradually develops into the weakness of the forelimbs, paralysis, and in severe cases, causes difficulty in moving the mice and even near death. As shown in Figure 1A, the state diagram of the mice with different degrees of EAE pathology depicts an exemplary picture of a group of mice changing from asymptomatic to high-scoring EAE symptoms (score 4). It has also been mentioned earlier that the bodyweight of EAE mice is correlated with clinical symptoms. Compared with WT mice, weight loss in EAE mice may start to occur in the first few days after immunization, while the clinical symptoms of EAE mice usually begin on day 6-9 after immunization and will reach a peak on day 14-16. After this, the symptoms of EAE mice generally partially recover, and at the same time, the weight loss of the mice will be alleviated (Figure 1B,C). Thus, the course of EAE onset is usually divided into early-onset, peak, and remission periods, and the prediction of these time points is important in assessing outcome parameters. In general, to analyze the production of immune cells and cytokines at the site of EAE lesions, immune cells in the brain and spinal cord of EAE mice at the peak of the disease can be isolated and further processed, which can be analyzed by flow cytometry^14,15. The spinal cord tissue at peak onset is also most suitable for preparing hematoxylin and eosin (H&E) staining and Luxol fast blue staining to further investigate the inflammatory cell infiltration and demyelination of the spinal cord^14,16. For monitoring changes in the immune system at different onset times of EAE, spleen and lymph nodes at early onset are also essential options^17,18. In addition, cells from the spleen or lymph nodes of MOG-immunized EAE mice are commonly used to construct transfer models, which are transferred to recipient mice after restimulation of MOG in vitro to induce passive immunization of EAE mice¹⁸.

MS is an autoimmune inflammatory induced demyelinating lesion of the central nervous system characterized by inflammatory demyelination and neuronal loss^2,3. This disease is usually accompanied by psychiatric comorbidities, such as affective disorders, of which anxiety disorder is very common in MS patients, with up to 30% of MS patients suffering from anxiety^9,19. Anxiety disorder is a psychiatric abnormality characterized by excessive emotional stress and worry. Open field test is often used to analyze behaviors for anxiety in rodents^20,21. With the help of analyzing the exploration behaviors of EAE mice in open field tests during early onset, peak, and remission periods, it was found that EAE mice also had anxiety-like behaviors similar to those of MS patients (Figure 2A). In the open field test, anxious rodents tend to have reduced activity and increased stereotypic behavior, including a preference for being closer to corners, a bias toward the peripheral domain, and a lack of desire to explore the central area. Compared to WT mice, EAE mice had significantly lower walking distance and movement time in all three periods of the disease, even in the early onset of the disease when EAE mice did not yet have a motor impairment (Figure 2B,C). In addition, EAE mice pass significantly less distance and stay in the central area less than normal mice, and even move only in the peripheral area, showing obvious anxiety-like mood (Figure 2D,E).EAE mice still exhibit a strong anxiety-like mood when the onset is mild, that is, motor coordination. Some studies suggest that this may be attributed to mild neuroinflammation, which further affects neurotransmitter secretion^22,23. Open field tests monitoring anxiety-like mood triggers in EAE mice could help researchers understand and treat MS psychiatric comorbidities.

With the progress of the disease, MS essentially eventually manifests as dyskinesia. Studies have found that MS patients have a higher susceptibility to osteoporosis and fracture, mainly due to bone mass loss, and that the severity of dyskinesia is strongly correlated with the patient's bone density^24,25. A similar phenomenon can be observed by Micro-CT analysis with the help of the EAE animal model (Figure 3A,H). From the trabecular analysis data of femur in mice, EAE mice underwent a significant decrease in bone mineral density (BMD) compared to WT mice, which is an important indicator of the response to bone strength and an important basis for the diagnosis of osteoporosis (Figure 3B). Further analysis showed that trabecular bone loss occurred significantly in EAE mice compared with healthy WT mice and was accompanied by a reduction in trabecular connection density, trabecular numbers, and trabecular thickness. These are all characteristic of reduced bone mass, suggesting that EAE also causes trabecular bone loss in the femur of mice (Figure 3C-F).At the same time, the structural morphology of the bone trabeculae changed, and the spacing of the trabeculae increased significantly; the greater the spacing, the more osteoporotic the bone (Figure 3G). This is consistent with the notion that MS patients are prone to osteoporosis. In cortical bones of the femur diaphysis, the thickness of cortical bone in the EAE model was significantly less than that in normal mice (Figure 3I). In MS, decreased motility and increased muscular dystrophy are strongly associated with osteoporosis, fracture, and increased bone resorption due to reduced mechanical forces, gradually decreasing bone integrity, thereby increasing the risk of osteoporosis and fracture²⁶. Micro-CT analysis of the femur in EAE mice can monitor bone health well, and the intervention is beneficial in controlling the condition of EAE.

Figure 1: Monitoring of clinical symptoms of EAE. (A) The exemplary picture of the mice with different degrees of EAE pathology. (B) Weight change in WT and EAE mice. (C) Clinical score in WT and EAE mice. Data are given as mean ± SEM (n = 5), ***p < 0.001 versus WT mice,two-way ANOVA test. Please click here to view a larger version of this figure.

Figure 2: Anxiety-like behavior of EAE mice in the open field test. (A) Representative track plots of the open field test. Travel distance (B), duration of activity (C), travel distance in the center (D), and time spent in the center (E) of WT mice and EAE mice in early-onset, peak, and remission periods. Data are mean ± SEM (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001 versus WT mice, Student's t-test. Please click here to view a larger version of this figure.

Figure 3: Micro-CT analysis of bone health in EAE mice. (A) Representative 3D images of femoral trabecular architecture (scale bars, 100 µm). Bone mineral density (B), bone volume/tissue volume ratio (C), connection density (D), numbers (E), thickness (F), and separation (G) of femoral trabecular were determined by Micro-CT analysis. (H) Representative 3D images of cortical bone (scale bars, 100 µm). (I) Cortical thickness obtained from microcomputed tomography data. Data are mean ± SEM (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001 versus WT mice, Student's t-test. Please click here to view a larger version of this figure.

Score	Clinical sign
0	No clinical signs
0.5	Tail weakness, front of tail dropping
1	Tail completely paralyzed
2	Mild paralysis of hind limbs (weakness of both hind limbs or unilateral paralysis, uncoordinated walking, response to pinch)
3	Complete paralysis of hind limbs, hind limbs dragging and walking, hind limbs not responding to pinch
4	Paralysis of the hind limbs and weakness of the forelimbs
5	Near death or dying

Table 1: Clinical scoring system.

Antigen	Strain	Characteristic	Application		Limitation
PLP	SJL/J34	Relapsing-remitting EAE	Study of the cellular and molecular events involved in clinical relapse		Limited to T-cell specific responses; Studying specific genes and pathways in the SJL/J strain is challenging
MBP	SJL/J35	Relapsing paralysis followed in partial or total recipients after recovery from acute paralysis	Study of neuroinflammation and immune system activation		Limited to T-cell specific responses; Studying specific genes and pathways in the SJL/J strain is challenging
MOG35-55	C57BL/631	Primary progressive EAE; Chronic progressive EAE	Study of axonal damage mechanisms; Study of molecular disease mechanisms in transgenic and knockout models		Primary demyelination and myelin regeneration studies are of limited value
Spinal cord homogenate	Biozzi ABH36	Relapsing-remitting EAE	Study of myelin regeneration and neuroprotective therapy for MS		Not clinically progressive, with accumulation of neurological deficits over time; Limited to CD4 T-cell specific responses
Theiler’s murine encephalomyelitis virus	Multiple strains of mice are highly susceptible, including SJL/J, SWR, PL/J12,31	Viral models of inflammatory demyelination	Study of axonal injury and inflammation-induced demyelination		No MS-specific viral infections have been identified; Myelin regeneration is difficult to assess, demyelination and myelin regeneration occur simultaneously
Cuprizone	Widely used in C57BL/6 mice, while other strains, such as the CD1 strain, are less susceptible to copper cuprizone-induced damage37,38	Toxic model of demyelination and remyelination	Study of T cells-independent demyelination, especially myelin regeneration and basic myelin repair processes		Significant spontaneous myelin regeneration occurs after cessation of toxic injury; Demyelination is brain region-specific and not useful for studying spinal cord demyelination
Lysolecithin	SJL/J12, C57BL/639, etc.	Toxic model of demyelination and remyelination	Study of T cells-independent demyelination, especially myelin regeneration and basic myelin repair processes		Lack of immune response observed during MS
Ethidium bromide	C57BL/640, etc.	Toxic model of demyelination and remyelination	Study of focal demyelinating lesions and prediction of the kinetics of demyelination and myelin regeneration		Ethidium bromide damages all nucleolus containing cells; Limited correlation with MS

Table 2: Different mouse models of MS.

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 impairment, motor disorders, cognitive and emotional disturbances, etc^6,7. MS is a devastating disease that will progressively worsen if not properly treated, leading to severe blindness and paralysis²⁸. The pathogenesis of MS is not yet fully understood, as its clinical manifestations and pathological progression are heterogeneous, and therefore animal models are essential for the research and development of treatment of MS.

The most frequently studied animal model for MS is EAE, which mainly has two modeling methods: active immunization and passive immunization²⁹. The former is widely adopted because of its simplicity, lack of mouse irradiation, and short cycle time. The EAE active immune model induces autoimmune responses in the CNS by immunizing rodents with self-myelin antigens or their subsequent peptides, usually manifesting as tail weakness and limb paralysis¹³. Specifically, the EAE model induced by MOG_35-55 in C57BL/6J mice is characterized by alternating remission-relapse cycles, accompanied by CNS inflammation, demyelination, and axonal injury, which makes the MOG_35-55-induced EAE the preferred model^11,12. EAE can be well induced in mice sensitized by subcutaneous injection of MOG_35-55 peptide, emulsified with immunopotentiator CFA enriched with MTB, and given an intraperitoneal injection of PTX with blood-brain barrier disrupting effect on days 0-2³⁰. The biggest challenge to the EAE model is the low incidence of weak symptoms, and several influencing factors are critical to the course of the experiment. (1) The age and environment of mice will affect the susceptibility to EAE. Therefore, mice of the same age need to be selected before the experiment. The mice should be weighted and randomly grouped before cage allocation, and the weight of each group needs to be kept close. Also, ensure that environmental conditions between independent experiments are comparable. (2) The concentration of PTX: The main role of PTX is to increase the permeability of the blood-brain barrier, which allows pathogenic T cells to enter the CNS to secrete relevant cytokines and promote the inflammatory response, ultimately leading to the hydrolysis of myelin proteins wrapped in the outer layer of nerve axons. The onset of disease in EAE mice was delayed with decreasing PTX administration concentration, and the severity of the disease would be weakened if PTX decreased by more than 60%. (3) The dissolution of MOG_35-55 antigen: To avoid freezing and thawing, it is recommended to procure the lyophilized MOG_35-55 antigen in separate packages, 4 mg/tube, to avoid freezing and thawing. Also, PTX is used for dissolution instead of sterile water because it is better to emulsify with CFA using PTX. (4) MOG_35-55 antigen and CFA must be sufficiently emulsified by oscillating to form a water-in-oil structure, and the emulsion droplets do not disperse on the water surface for a long time, indicating good emulsification. (5) Two-point injection was used for mouse EAE modeling. The injection site is preferably located above the waist and below the neck, close to the spine, to avoid mice licking and biting the skin, resulting in the spillage of MOG_35-55 emulsion.

However, the EAE model induced by the MOG_35-55 antigen still has some limitations. As an inflammatory encephalopathy model of primary axonal injury mediated by CD4 T cells, the main lesion is massive axonal degeneration with secondary demyelination and less primary demyelination, which is different from the MS pathology^31,32,33.The EAE model induced by MOG_35-55 mainly reflects the immune response driven by CD4 T cells, while other immune cells such as CD8 T and B cells do not have a strong sense of participation in this model³¹. Another limiting factor is that EAE induced by MOG_35-55 has a certain bias towards the immunological component of MS pathophysiology,and other animal models can be considered when considering studies focused on CNS pathologies, such as the study of demyelination and myelin regeneration processes^{34,35,36,37,38,39,40}, as described in Table 2.

EAE is an effective predictor in imitating the autoimmune properties of MS, as well as its inflammation and demyelination mechanism¹², which makes many scholars keen to study its immunomodulatory mechanisms, ignoring other clinical symptoms of MS. Studies have shown that MS-associated anxiety is attributed to pathophysiological processes such as immune dysregulation and brain inflammation^22,23,41. Mental health issues, such as anxiety, are particularly common in people with MS, and anxiety disorder can impede the performance of basic daily tasks and significantly affect the quality of life^42,43. Alterations in the locomotor ability of EAE mice may predict alterations in neural processes. In open-field experiments, anxiety-like behaviors occurred in EAE mice,including a decrease in exploratory behaviors that started early onset of the disease (essentially, test subjects with clinical scores <0.5 and locomotor ability almost as good as normal mice), suggesting that the onset of anxiety-like behaviors precedes motor impairment. In addition, EAE mice in the remission period did not improve anxiety-like behavior with increased motor ability. The emergence of the inflammatory response may be responsible for the mental impairment in EAE mice through neuronal activity, leading to changes in mood, and upregulation of inflammatory cytokine levels interferes with striatal synaptic function prior to the onset of motor impairment^23,41. However, the open field test requires attention to the fact that the test room must be a quiet area, and the test mice need to acclimatize to the room for 1 h in advance to avoid excessive pressure caused by the new environment. Additional cleaning with ethanol is required between tests to remove odors and other substances left behind by the last test subject. The main limitation of the open field test is the interference caused by motor dysfunction in EAE mice, and the test for evaluating rodent anxiety-like behavior is based on the distance, time, and range of rodent activity in the reaction chamber, and cannot rule out the effect of motor dysfunction on anxiety-like behavior. Therefore, future studies need to take locomotor impairment into account.

Traditional methods to analyze bone tissue-related studies often use two-dimensional bone section microscopy, which can have greater difficulties in analyzing structural morphology, density distribution, orientation distribution, and other characteristics due to the influence of two-dimensional planes and inhomogeneity of biological specimens. Many studies have confirmed the accuracy and effectiveness of the Micro-CT system to describe bone morphology, and many studies^44,45,46 have confirmed microstructure detection results. The three-dimensional imaging of bone tissue by Micro-CT allows for more intuitive and accurate bone histomorphometry results. It has been achieved to scan small animals and specimens without executing the animal or damaging the tissue specimen, allowing for detailed three-dimensional spatial structure information inside the tissue⁴⁴. Studies have shown that MS patients are prone to bone mass loss and osteoporosis^24,25; By scanning and imaging the femur of EAE mice with a Micro-CT system, an obvious deterioration of bone microarchitecture and decreased bone strength could be observed in the femur of EAE mice compared to WT mice that had developed osteoporosis. Micro-CT analysis is relatively simple to perform and relatively inexpensive, and Micro-CT live imaging can be used to monitor bone health in the future when administering treatment to EAE mice. However, the drawback is the absence of bone turnover markers assessed through blood samples or bone histomorphometry to observe fundamental changes in bone metabolic homeostasis in EAE mice⁴⁷. Regulation of osteoblasts and osteoclasts could be considered within the study in the future.

MS is a multi-symptom disease, and the sole aim of disease management is to minimize disease symptoms. In the protocol mentioned above, the clinical symptoms of MS can be simulated by constructing the MOG_35-55 induced EAE model. EAE mice showed clinical symptoms of motor dysfunction and anxiety-like behavior, and osteoporosis. The 5-point scoring system, open field test, and Micro-CT analysis described in this protocol can monitor the pathological symptoms of EAE mice from multiple perspectives and provide a reference scheme for the treatment of EAE. In the absence of effective disease-modifying drugs, a potential combination of synergistic therapy can provide the best opportunity to relieve symptoms and improve function.

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