Experimental Autoimmune Encephalomyelitis: C57BL/6 Mice Show Gender-based Differences in Neuronal and Immune Injury

Research Article

Austin J Mult Scler & Neuroimmunol. 2014;1(1): 7.

Experimental Autoimmune Encephalomyelitis: C57BL/6 Mice Show Gender-based Differences in Neuronal and Immune Injury

Tao Yang1, Qi Zheng2, Hui Zhao2, ZiJing Hu3, Lei Wang2* and Yongping Fan1*

1Department of Traditional Chinese Medicine, Capital Medical University, China

2School of Traditional Chinese Medicine, Capital Medical University, China

3School of Basic Medical Sciences, Capital Medical University, China

*Corresponding author: Lei Wang, School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China, Tel: +86-010-83911626; Fax: +86-010-83911627; E-mail: tmwangl@ccmu.edu.cn

*Corresponding author: Yongping Fan, Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University, 100050, China

Received: October 14, 2014; Accepted: December 01, 2014; Published: December 04, 2014


Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system (CNS). We investigated the role of gender in neuronal and immune injury in experimental autoimmune encephalomyelitis (EAE), using C57BL/6 mice induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 and complete Freund's adjuvant (CFA) supplemented with pertussis toxin (PTX). The body weights and the neurological function scores of mice in different genders were assessed once a day for 40 days. The histopathologic changes in the brains of mice were observed by light microscope with hematoxylin-eosin (H&E) staining. The protein expression of myelin basic protein (MBP) and microtubule-associated protein (MAP)-2 in the brain of mice were measured by Western-blotting (WB) on day 22 post-immunization (PI) and by immunohistochemistry (IHC) on day 40 PI. The Interleukin (IL)-17A and forkhead box P3 (Foxp3) were assessed by IHC on day 22 and 40 PI. In addition to neurological dysfunction and weight loss, decreased protein expression of MBP and MAP-2 was seen. The ratio of IL-17A/Foxp3 was dramatically enhanced in female and male EAE mice, more so in males than females, compared with normal mice, respectively, The mortality and the neurological function scores were significantly increased, In conclusion, EAE results from an imbalance between Th17 and Treg cells, along with the associated demyelination and axonal damage, affecting males more severely.

Keywords: Experimental autoimmune encephalomyelitis; Gender; Myelin basic protein; Microtubule-associated protein; Interleukin-17A; Forkhead box P3


Multiple sclerosis (MS) is a common inflammatory demyelinating disease of the central nervous system (CNS), which is characterized by multifocal inflammation, demyelination, and neuronal damage [1- 3]. With different lesions in the brain, the clinical symptoms of MS are complex and varied [4]. Most patients showed impaired vision, numbness, movement disorders and other symptoms. Symptoms accumulate with relapse of MS, severely reducing the patient's quality of life. Increased rate of relapse and severe disability are a major challenge [5]. MS is the most common chronic disabling disease in young adults [2,6-8], with a female-to-male preponderance approaching 2:1 to 3:1 [9-11], and widening gender gap [11]. Our research explores the underlying differences between genders.

Experimental autoimmune encephalomyelitis (EAE) has been proven to be the ideal animal model presenting with similar pathological and clinical manifestations as human MS [12]. Due to the limited evidence available, related to differential incidence between genders, we studied the distinguishing features in EAE [13]. In this study, we compared body weights, neurological function scores, incidence, mortality and latency between the genders. We observed the expression of myelin basic protein (MBP), the microtubule-associated protein (MAP)-2, Interleukin (IL)-17A, and forkhead box P3 (Foxp3) in the brain of mice. A study of the gender-based differences in EAE using a reliable animal model to investigate MS is clinically significant.

Materials and Methods


Specific pathogen-free grade, 6-8 weeks-old C57BL/6 mice of were purchased from Beijing Weitong Lihua Experimental Animal Tech. Co., Ltd., China [certification NO. SCXK (JING) 2006-0009]. The animals were housed in the Center of Laboratory Animals at Capital Medical University [certification number SYXK (JING) 2010- 0020]. The experiments were approved by the Ethics Committee of Capital Medical University.


Myelin oligodendrocyte glycoprotein peptides (MOG) 35-55 (MEVGWYRSPFSRVVHLYR NGK) were synthesized by Beijing SciLight Biotechnology Co. Ltd. (China) at a purity of >95%. Complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were purchased from Sigma (USA). Rabbit anti-MBP mouse, rabbit anti-MAP-2 mouse, rabbit anti-IL-17A mouse, rabbit anti-Foxp3 mouse were purchased from Abcam (UK). β-actin was purchased from Epitomics (USA). Sheep anti-rabbit IgG and DAB were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (China). Western-blot kit was purchased from Beijing CoWin Bioscience Co., Ltd. (China).

Induction of EAE animal model

The mice were randomly assigned to four groups: female normal control (F NC) group (n = 12), male normal control (M NC) group (n = 12), female EAE (F EAE) group (n = 15), and male EAE (M EAE) group (n = 15). Each of the EAE mice was injected subcutaneously with 0.2 ml emulsion, containing 50 μg MOG 35-55 in 100 μl of normal saline (NS) and 100 μl of CFA (H37Ra, 4 mg/ml), followed by peritoneal injections of 500 ng of PTX on day 0 and 2 post-immunization (PI) [14]. The mice in NC groups were given NS instead.

Animal observations

Individual animals were examined daily. The body weights, neurological function scores, incidence, mortality and latency data of mice were recorded at the end of experiment. The neurological function scores were defined as reported previously [14]: 0 for no signs, 1 for flaccid tail, 2 for moderate hind-limp paralysis, 3 for complete hind-limp paralysis, 4 for fore-limb paralysis, and 5 deaths.

Sample collection

The mice were sacrificed on day 22 (acute stage) and 40 (remission stage) PI. The four mice out of 15 were anesthetized with an intraperitoneal injection of 10% chloral hydrate (5 ml/kg). The mice were perfused with ice-cold NS (50 ml), followed by slow perfusion with 4% paraformaldehyde through the left ventricle. The brains of mice were removed, fixed, and embedded in paraffin. The tissues were sectioned (thickness, 4μm) for hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining and detection. The brains of four mice from each group were quickly removed, and immediately frozen in liquid nitrogen, and then kept at -80oC for Western-blotting (WB) detection.


The sections were dewaxed in xylene, dehydrated with gradient alcohol for 5 min each, and stained with Harris hematoxylin for 1 min, followed by eosin for 10 min. The sections were dehydrated in gradient alcohol, permeabilized with xylene, mounted on neutral gum, and observed with light microscopy (Nikon eclipse 80i, Tokyo, Japan).

Immunohistochemical analysis

The paraffin slices were dewaxed in xylene for 15 min, dehydrated in gradient alcohol for 5 min each. The slices were treated with 3% H2O2 for 10 min at room temperature, washed 3 times with PBS (pH7.2) for 5 min each. The slices were pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) for 20 min, washed 3 times with PBS. The slices were incubated with primary antibody [rabbit anti-mouse MBP (1:100), MAP-2 (1:200), IL-17A (1:100) and Foxp3 (1:200)] at 4oC overnight. The slices were later washed 3 times with PBS and incubated with biotin-labeled secondary antibody (sheep anti-rabbit IgG) at 37°C for 30 min, and then washed 3 times with PBS and the colored reaction product was developed using 3,3'-diaminobenzidine tetrahydrochloride (DAB). Finally, the slices were dehydrated and mounted. Each slice was observed microscopically with six high-power fields (x400) randomly selected. The quantitative analysis of immunohistochemical images was carried out with a NIS-Elements BR 3.0 system (Nikon, Japan). Positive results were expressed by integral optical density (IOD).

Western-Blotting analysis

Protein extraction and quantification were performed according to the procedures of specification. Protein bands were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred onto nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% defatted dry milk for 60 min, followed by incubation with primarily anti- MBP antibody (1:1000), anti-MAP-2 antibody (1:2000) and anti β-acting rabbit polyclonal antibody (1:3000) in blocking solution at 4oC overnight. The membranes were washed 4 times (10 min each), followed by incubation with goat anti-rabbit IgG (1:10000) for 60 min. The membranes were then washed 4 times (10 min each) and incubated in electrochemiluminescence (ECL) for 5 min, followed by exposure to Kodak film (Comwin, China) and development with an enhanced chemiluminescent kit (Amersham, UK). The image analysis software ImageQuant TL 2005 (Amersham, Biosciences, Piscataway, NJ) was used to analyze IOD value of each band. The relative expression of the target protein was represented by the IOD ratio, IODMBP/ IODβ-actin, IODMAP-2/ IODβ-actin.

Statistical analysis

Data were expressed as mean ± standard error and analyzed with SPSS software (Version 17.0, Chicago, IL, USA,). The test of normality was conducted by one-way ANOVA with a post-hoc LSD test, or otherwise with a rank-sum test. P values less than 0.05 were considered statistically significant.


Incidence, mortality and latency of EAE mice

The experimental groups developed typical symptoms (limp tail, limb weakness or even death) of F EAE mice after 12.7±3.2 days and M EAE mice after 10.0±1.5 days following immunization, with 100% incidence and a chronic monophasic course. The mortality was 6.3% in F EAE mice, and was significantly higher, up to 20.0% in M EAE mice (p < 0.01). The latency in M EAE group was significantly shorter compared with the F EAE group (p < 0.01, Table 1).