EAE Modeling Solutions

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June 16, 2025

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Experimental Autoimmune Encephalomyelitis (EAE) is the gold standard animal model for studying Multiple Sclerosis (MS). It simulates the attack of autoimmune T cells on the Central Nervous System (CNS), reproducing the core pathological features of MS, such as neuroinflammation, demyelination, and neurological dysfunction. The EAE model is widely used in the research fields of MS pathogenesis, screening of neuroimmunomodulatory drugs (such as Roflumilast), and the development of myelin repair and neuroprotective therapies.

With professional R&D capabilities and a strict quality control system, Absin has meticulously crafted a complete set of high-stability and high-reproducibility reagent combinations for EAE model research. The advantages of the core reagents for model construction are as follows:

✅ Fast delivery from stock: Whooping cough toxin is available in domestic stock, with extremely high purity (>99%), high quality, and cost-effectiveness.

✅ High incidence rate: C57BL/6 mice stably develop the disease (clinical score of 2-4) 10-14 days after immunization.

✅ Good pathological recurrence: Clear myelin loss (as demonstrated by LFB staining) & Th17 cell infiltration (verified by flow cytometry).

✅ Multidimensional evaluation system: Supports comprehensive validation across clinical scoring, behavioral studies, histopathology, and molecular biology.

1. Core Reagents for EAE Model

Item No.	Product Name	Specification
abs42024900	Pertussis Toxin	50μg
abs815889	MOG_35-55	1mg
abs9270	Complete Freund's Adjuvant (CFA)	10mL

(1) Immunogen (Antigen)

MOG₃₅₋₅₅ (Myelin Oligodendrocyte Glycoprotein 35-55 peptide)

Source: Synthetic peptide, mimicking the autoantigen in MS

Concentration: Typically used at 150-300 μg per mouse (dissolved in PBS, saline, or CFA)

(2) Adjuvant

Complete Freund’s Adjuvant (CFA)

Composition: Mineral oil + inactivated Mycobacterium tuberculosis (e.g., Mycobacterium tuberculosis H37Ra, concentration 4-5 mg/mL)

Function: Enhances the immune response, used after emulsification with MOG₃₅₋₅₅

(3) Immuno-Enhancer

Pertussis Toxin (PTX)

Dosage: 200-500 ng per mouse (i. p. injection)

Injection Time: On the day of immunization (Day 0) and the second day (Day 2)

Function: Disrupts the blood-brain barrier, facilitating T cell infiltration into the central nervous system

2. EAE Model Induction Method

（1）Experimental Preparation

Experimental Animals: Typically, female C57BL/6 mice or Sprague-Dawley rats aged 6-8 weeks are chosen.

Number of Animals: At least 10 animals per group are required to ensure statistical significance, based on experimental design.

Animal Acclimation: Allow animals to acclimate to the experimental environment for one week, maintaining stable temperature, humidity, and lighting conditions.

Reagent Preparation: Dissolve MOG35-55 peptide in saline and mix with an equal volume of CFA to form a uniform emulsion.

（2）EAE Induction

Day 0:

Emulsion Preparation: Mix 300 µg of MOG35-55 peptide with 300 µL of CFA to emulsify.

Injection: Administer the emulsion in two divided doses (100 µL per side) subcutaneously into both axillary, inguinal, and dorsal regions of the mouse.

PTX Injection: Intravenously inject 200 ng of PTX.

Day 2:

Repeat PTX Injection: Intravenously inject 200 ng of PTX.

3. EAE Model Validation

Validation of the EAE model requires a multidimensional approach, including clinical assessment, behavioral testing, histopathology, and molecular biology. The following are systematic validation methods and operational key points.

（1）Clinical Scoring Validation

①Standardized 0-5 point scale (most commonly used): Observe continuously for 30 days or longer starting from Day 0.

Scoring criteria: 0 points: No symptoms. 1 point: Weakness or paralysis of the tail. 2 points: Weakness in the hind limbs. 3 points: Paralysis of the hind limbs. 4 points: Paralysis of all four limbs. 5 points: Death.

②Weight Monitoring: EAE mice usually experience weight loss (>10% indicates successful modeling), which needs to be recorded daily. Continuous weight loss may require ethical intervention.

（2）Behavioral Testing

① Motor Function Assessment

Rotarod: Accelerating mode (4-40 rpm, completed within 5 minutes) records the time to fall, reflecting coordination and endurance.

Gait Analysis (CatWalk or footprint method): Quantifies stride length, base width, and swing speed.

② Detection of Depression-like Behavior

Forced Swimming Test (FST): Records immobility time (reflecting despair behavior).

Tail Suspension Test (TST): Assists in verifying depressive phenotypes.

（3）Histopathological Validation

① Sampling and Processing

Perfusion fixation: After perfusion fixation with 4% paraformaldehyde, collect spinal cord (lumbar enlargement) and brain tissue.

Section preparation: Paraffin sections (5µm) or cryosections (10µm).

② Staining Methods

Staining Type	Detection Target	Result Interpretation
HE Staining	Inflammatory Cell Infiltration	"Sleeve-like" infiltration around blood vessels (score 0-3)
LFB Staining	Myelin Loss	Percentage of blue-stained area loss (quantified by ImageJ)
Immunohistochemistry	Oligodendrocytes (Iba1), Astrocytes (GFAP)	Positive cell density/morphology

（4）Molecular Biology Validation

① Flow Cytometry

Cell subset analysis: Th1 (CD4+IFN-γ+), Th17 (CD4+IL-17+), Treg (CD4+FoxP3+).

Sample source: Spleen, lymph nodes, single-cell suspension from the central nervous system.

② Cytokine Detection

ELISA/liquid phase chip: Pro-inflammatory factors: TNF-α, IL-6, IL-17.

Anti-inflammatory factors: IL-10, TGF-β.

qPCR: mRNA expression of cytokines in spinal cord/brain tissue.

③ Protein Expression Analysis

Western Blot：

Inflammatory pathway proteins: NF-κB p65, pSTAT3.

Neuroprotective markers: BDNF, NGF.

4. Model Success Criteria

Basic criteria: Incidence rate >80% (clinical score ≥2); pathological confirmation with HE showing inflammatory infiltration + LFB confirming myelin loss.

Advanced criteria: Behavioral abnormalities (Rotarod time decreased by 50%); molecular markers: Th17 proportion increased by 2 times, significantly elevated IL-17 levels in the CNS.

5. Common Issues and Solutions

Issue	Possible Cause	Solution
No clinical symptoms	Ineffective CFA/PTX	Change the batch of reagents, increase PTX dosage
High mortality rate	Excessive PTX/disease	Decrease PTX dosage, sterile operation
Negative pathological results	Too early sampling time	Delay until the peak of disease (days 18-21 post-immunization)

6. Featured Application Case

^{Professor Zhang Zhijun's team from the Department of Neurology, The Affiliated Zhongda Hospital of the Medical School of Southeast University, used an Experimental Autoimmune Encephalomyelitis (EAE) rat model to study the effects of Roflumilast. Their research indicates that Roflumilast has the potential to become a drug for improving depressive symptoms and acute phase motor disorders in Multiple Sclerosis (MS). Its anti-inflammatory, inflammation-reducing, and microglia activation-inhibiting properties suggest its potential in the treatment of MS. IL6, IL1B, and TNF are key genes shared by MS and depression, and the cytokines they encode have protein interactions. IL-6 may play an important role in depression related to multiple sclerosis, and IL-6 antagonists may become a potential therapeutic strategy for improving depression in multiple sclerosis. The research results provide important clues for further clarifying the pathogenesis of MS and depression [1]}。

References：

[1] Wang Z, Zhang Y, Chai J, Wu Y, Zhang W, Zhang Z. Roflumilast: Modulating neuroinflammation and improving motor function and depressive symptoms in multiple sclerosis[J]. J Affect Disord. 2024;350:761-773. doi:10.1016/j.jad.2023.12.074

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Email: worldwide@absin.net