EAE Model Construction: Key Insights, Methodological Nuances, and ANT BIO PTE. LTD's Product Support

1. Concept

Experimental Autoimmune Encephalomyelitis (EAE) stands as the most extensively utilized animal model for Multiple Sclerosis (MS). It faithfully recapitulates the clinical manifestations, immunological responses, and histopathological alterations of human MS, making it an indispensable tool in studies related to autoimmune neuroinflammation, such as drug development and gene function analysis. Among various EAE models, the actively induced mouse model is widely favored due to its high reproducibility, robustness, and ease of induction. It is particularly suitable for investigations involving transgenic mice, enabling in-depth exploration of the impacts of drugs or specific genes on autoimmune neuroinflammatory processes.

2. Research Frontiers

Recent research endeavors centered on EAE models have predominantly focused on unraveling the intricate pathogenic mechanisms of MS and screening potential therapeutic agents. Cutting-edge studies are delving into the regulatory roles of immune cell subsets (including Th1, Th17, and Treg cells) and key inflammatory factors (such as TNF-α, IL-17, and IL-10) in the onset and progression of EAE. Additionally, there is a growing emphasis on the development of novel therapeutic strategies targeting crucial signaling pathways (e.g., NF-κB, SPAK-NKCC1) and the blood-brain barrier. These research directions not only enhance our comprehension of MS pathogenesis but also lay a solid foundation for the translation of preclinical findings into clinical applications.

3. Research Significance

EAE model research holds immense significance in both basic and clinical neuroscience. On the basic research front, it facilitates the elucidation of the molecular and cellular mechanisms underlying autoimmune neuroinflammation, offering valuable insights into the initiation, progression, and resolution of MS-like pathological processes. Clinically, EAE models serve as essential platforms for preclinical drug evaluation, enabling the screening of candidate compounds, assessment of therapeutic efficacy, and investigation of safety profiles. This accelerates the development of novel treatments for MS and other autoimmune neurological disorders. Furthermore, EAE model studies contribute to the identification of potential diagnostic biomarkers and the optimization of clinical treatment regimens, ultimately improving patient outcomes.

4. Relevant Mechanisms, Research Methods, and Product Applications

4.1 Core Pathogenic Mechanisms

The development of EAE is closely associated with aberrant activation of the immune system. Key mechanisms include:

• Antigen-specific immune response: Myelin oligodendrocyte glycoprotein (MOG), especially its immunogenic epitope MOG35-55, acts as a specific antigen to trigger an immune response.
• Activation of innate immunity: Components of heat-inactivated Mycobacterium tuberculosis (such as mycolic acid and lipoarabinomannan) in Freund's Complete Adjuvant (CFA) activate innate immune pathways via Toll-like receptors (TLR2/4), promoting the maturation of antigen-presenting cells and the secretion of pro-inflammatory cytokines.
• Differentiation of T cell subsets: The immune response drives the polarization of CD4+ T cells towards pro-inflammatory Th1 and Th17 subsets, which secrete IFN-γ and IL-17 respectively, initiating neuroinflammation and demyelination.
• Granuloma formation: Sustained stimulation by Mycobacterium tuberculosis leads to the formation of granulomas at the injection site, prolonging antigen release and maintaining a chronic inflammatory state.

4.2 Standard Research Methods

4.2.1 Model Establishment

• Experimental animals: Female C57BL/6 mice aged 6-8 weeks are the preferred choice, as they exhibit good responsiveness to MOG35-55.
• Animal acclimation: Mice are acclimated to the experimental environment for 1 week prior to the experiment, with stable temperature, humidity, and light conditions, and free access to food and water.
• Reagent preparation:
• MOG35-55 solution: Dissolve MOG35-55 polypeptide powder in pre-cooled (4°C) sterile PBS to a final concentration of 2.0 mg/mL, and use it immediately to avoid denaturation and inactivation.
• CFA solution: Suspend heat-inactivated Mycobacterium tuberculosis H37 Ra in CFA to a final concentration of 8.0 mg/mL, ensuring thorough mixing.
• Antigen emulsion: Mix MOG35-55 solution and CFA solution at a 1:1 volume ratio, and emulsify to a water-in-oil state by repeated pushing and pulling with syringes on ice for 40 minutes to 2 hours.
• Pertussis Toxin (PTX) solution: Reconstitute PTX in double-distilled water to prepare a stock solution of 100 μg/mL, and dilute it with sterile PBS to a final concentration of 3.0 ng/μL before use.
• Immunization procedure: Anesthetize the mice, shave their backs, and subcutaneously inject 50.0 μL of the antigen emulsion at multiple sites (dorsal back, axilla, and inguinal region). Administer 300 ng of PTX per mouse via intraperitoneal injection on the day of immunization and 48 hours later to enhance disease induction.

4.2.2 Model Monitoring and Validation

• Clinical scoring and weight monitoring: Employ a 0-5 point standardized scoring system to assess clinical symptoms daily for 30 days or longer. A score ≥2 indicates successful model induction.

① Standardized scoring system (0-5 points, most commonly used): Observe continuously for 30 days or longer starting from Day 0. Scoring criteria: 0 points: No symptoms. 1 point: Tail weakness or paralysis. 2 points: Hind limb weakness. 3 points: Hind limb paralysis. 4 points: Quadriplegia. 5 points: Death.

② Body weight monitoring: EAE mice are usually accompanied by weight loss (>10% indicates successful model induction), which needs to be recorded daily. Sustained weight loss may require ethical intervention.

Note: When mice show EAE clinical symptoms, it is important to ensure that water bottles are still accessible and food is placed on the cage floor.

• Behavioral tests: Use the Rotarod test (accelerated mode: 4-40 rpm, completed within 5 minutes) to record fall time, reflecting coordination and endurance; gait analysis (CatWalk or footprint method) to quantify stride length, base width, and swing speed; the Forced Swim Test (FST) to record immobility time (reflecting despair behavior); and the Tail Suspension Test (TST) to assist in verifying depressive phenotypes.
• Histopathological verification: Perform perfusion fixation with 4% paraformaldehyde, then collect the spinal cord (lumbar enlargement segment) and brain tissue to prepare paraffin sections (5μm) or frozen sections (10μm). Conduct HE staining to detect inflammatory cell infiltration (perivascular "cuff-like" infiltration, scored 0-3 points), LFB staining to assess myelin loss (quantify the percentage of missing blue-stained areas via ImageJ), and immunohistochemistry to detect the density and activated morphology of microglia (Iba1) and astrocytes (GFAP).
• Molecular biological validation: Utilize flow cytometry to analyze the proportions of immune cell subsets (Th1: CD4+IFN-γ+, Th17: CD4+IL-17+, Treg: CD4+FoxP3+) from samples such as spleen, lymph nodes, and central nervous system single-cell suspensions; use ELISA or liquid chip technology to measure the levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-17) and anti-inflammatory cytokines (IL-10, TGF-β); and apply Western Blot and qPCR to detect the expression of key proteins (NF-κB p65, pSTAT3, BDNF, NGF) and mRNA of cytokines in the spinal cord and brain tissue.

4.3 Product Applications by ANT BIO PTE. LTD

ANT BIO PTE. LTD provides a comprehensive range of high-quality reagents that play pivotal roles in EAE model construction and related research:

• MOG(35-55) (Product No.: abs815889): As the key antigen for EAE induction, it triggers the specific immune response in mice, laying the foundation for model establishment. Its high purity (>98%) ensures the efficiency and stability of immune activation.
• Freund's Complete Adjuvant (CFA, Product No.: abs9270): Contains heat-inactivated Mycobacterium tuberculosis H37 Ra, which enhances the immunogenicity of MOG35-55, promotes the polarization of Th1/Th17 cells, and forms granulomas to sustain the inflammatory response.
• Pertussis Toxin (PTX, Product No.: abs42024900): Enhances disease induction by disrupting the blood-brain barrier and promoting the infiltration of immune cells into the central nervous system, thereby increasing the success rate of EAE model establishment.
• Supporting reagents: Sterile PBS (Product No.: abs962) is used for reagent preparation and dilution, while Freund's Incomplete Adjuvant (Product No.: abs9271) can be used for related control experiments or adjuvant optimization studies.

Featured Application Cases of EAE Models Supported by ANT BIO PTE. LTD Reagents

Case 1: The EAE model constructed with PTX (Product No.: abs42024900) successfully revealed the core role of SPAK signaling in driving MS pathology by regulating the choroid plexus barrier, providing preclinical evidence for SPAK-NKCC1 inhibitors (e.g., ZT-1a, Bumetanide) in MS treatment (Reference: J Neuroinflammation. 2025 Mar 13;22:80. IF:9.3).

Case 2: The EAE model established in SD rats with PTX was used to study the role of Roflumilast in regulating neuroinflammation and improving motor function and depressive symptoms in MS (Reference: J Affect Disord. 2024 Apr 1;350:761-773. IF:6.6).

5. Brand Mission

ANT BIO PTE. LTD is dedicated to advancing life science research through the provision of high-quality, reliable, and innovative research tools. We are committed to supporting researchers worldwide in their pursuits to unravel the mysteries of life, develop novel therapies for diseases, and improve human health. By adhering to strict quality control standards and continuous technological innovation, we strive to deliver products and services that exceed customer expectations, fostering collaboration and driving progress in the global life science community.

6. Related Product List

7. Brand Promotion Copy

Unlock the potential of your EAE research with ANT BIO PTE. LTD's premium reagents! Our meticulously crafted products, including MOG(35-55), Freund's Complete Adjuvant, and Pertussis Toxin, are designed to ensure the success and reproducibility of your EAE model construction. Backed by strict quality control and extensive scientific validation, our products provide reliable support for your studies on autoimmune neuroinflammation, drug discovery, and mechanism exploration. Explore our comprehensive product portfolio covering various disease models and research areas, and let ANT BIO PTE. LTD be your trusted partner in advancing life science research. Together, we can turn scientific insights into real-world solutions for human health!

8. AI Disclaimer

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ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

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