Wistar rats (weighing 200±18 g, male) were purchased from and fed at the Jiangsu Laboratory Animal Center. The rats were fed in a specific pathogen-free environment at a room temperature of 25±2°C, with free intake of food and water. The study was approved by the Ethics Committee of Daqing Longnan Hospital (Daqing, China).

The EAE models were established according to foreign literature (10): Myelin oligodendrocyte glycoprotein 35–55 (MOG35-55) (article number: 2568, R&D Systems Europe, Ltd., Abingdon, UK, 400 µg), mycobacterium tuberculosis H37Ra (400 µg), phosphate-buffered saline (PBS) 200 µl; and complete Freund's adjuvant (CFA) solution 100 µl were mixed; and the mixture was made into milky-white suspension through ultrasonic emulsification. The rats in the EAE group were intraperitoneally injected with immunopotentiator pertussis toxin (500 ng/rat) at 0 and 2 days, followed by 0.1 ml MOG35-55 emulsion. Rats in the control group were injected with an equivalent volume of normal saline. The clinical symptoms of the rats were observed for 20 days, and scores were recorded in accordance with the Wear neurological function scale as: 0 point, no signs; 1, limp tail and ataxia; 3, paralysis of one hind limb; 4, paralysis of both hind limbs; and 5, dying or death.

The rats were anaesthetised with pentobarbital (2.5 mg/100 g) then sacrificed by decapitation. The brains of the two groups of rats were fixed in an appropriate amount of formalin overnight, which were then dehydrated in graded alcohol, cleared, wax-impregnated and embedded in paraffin, after which the paraffin tissues were sliced to 0.4 µm. The sections were baked in an oven at 65°C for 3–5 h, followed by dewaxing in xylene reagent and rehydration with graded ethanol. H&E staining, rehydration with graded ethanol and drying of sections were conducted in sequence, and the sections were mounted with neutral balsam. The histomorphology of the sections was observed and photographed under a microscope (DM-5000B, Leica Microsystems, Wetzlar, Germany).

The brain tissue sections of the rats were permeabilized with 0.1% Triton X-100 for 10 min after dewaxing and rehydration with ethanol, which were then blocked in 5% standard protein bovine serum albumin for 30 min. The rabbit anti-rat primary antibodies to glial fibrillary acidic protein (GFAP) polyclonal antibody were added to the sections in drops and blocked at 4°C overnight (1:200; cat. no. ab7260; Abcam, Cambridge, UK). The sections were washed in PBS with Tween-20 three times the next day, followed by incubation with Alexa Fluor-labeled fluorescent goat anti-rabbit secondary polyclonal antibody (1:1,000; cat. no. A-11034; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at room temperature for 1 h. The sections were mounted after 4′, 6-diamidino-2-phenylindole was added for visualization of cell nuclei for 3 min, which were observed and photographed under an inverted fluorescence microscope (DM-6000B, Leica Microsystems).

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was applied to extract the total RNA in the brain tissue of the rats. The RNA concentration was detected using a spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNA (1 µg) was withdrawn for reverse transcription (Takara Bio, Inc., Otsu, Japan), and the obtained complementary DNA was stored at −20°C. The messenger RNA (mRNA) levels of various indexes were measured in accordance with the instructions of an All-in-One™ qPCR Mix (GeneCopoeia, Inc., Rockville, MD, USA) kit. Reaction conditions of RT-qPCR were: Step 1, at 90°C for 10 min; 2, at 95°C for 10 sec; 3, at 60°C for 20 sec and 4, at 70°C for 20 sec; 45 cycles in total. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as the internal reference. The formula for the mRNA relative expression levels of the indexes was: 2^−ΔΔCq [ΔCq = Cq (target gene) - Cq (GAPDH)]. The corresponding primer sequences are shown in Table I.

Brain tissues (5 g) of the rats were cut up and placed into a 2.5 ml Eppendorf tube, to which 150 µl mixture of radio immtmoprecipitation assay lysis buffer and protease inhibitor Cocktail (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added, followed by homogenizing in a tissue homogenizer for 10 min. After centrifugation at 12,000 × g for 15 min (at 4°C), the upper liquid, namely, tissue protein, was absorbed. The concentration of the protein was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). After being denatured, the total protein was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was transferred to the nitrocellulose membrane (Merck KGaA). The bands were blocked in 5% skim milk for 1 h and then incubated in monoclonal antibodies overnight. Interleukin-10 (IL-10; Abcam, 1:1,000), tumor necrosis factor-α (TNF-α; Abcam, 1:2,000) and interferon-γ (IFN-γ; Abcam, 1:1,000) were added. The bands were then incubated in secondary antibodies of anti-rabbit immunoglobulin G for 1 h after the membrane was washed, and enhanced chemiluminescence system (Bio-Rad Laboratories, Inc.) was used to reveal the bands of target proteins.

GraphPad Prism software (Version 5.01; GraphPad Software, Inc., La Jolla, CA, USA) was utilized for analysis. Independent-sample t-test was applied to compare the differences in the indexes between the two groups of samples. P<0.05 was considered to indicate a statistically significant difference.

There was no onset of disease in rats of the control group from beginning to end. However, in the EAE group, the disease occurred in the 10 rats at 9 days after immunization, with an average onset time of 5.4±1.5 days. Eight rats were attacked by the disease, 1 rat was not attacked and 1 rat was dead; the incidence rate was 80%. During the onset of disease, the Wear score of the rats in EAE group was markedly increased (P<0.05) (Fig. 1). In addition, the weight of the rats in EAE group at 9 days after immunity was markedly lower than that in the control group (P<0.05) (Fig. 2).

As shown in Fig. 3, the brain tissue sections of the control and EAE groups were compared, and it was indicated that the inflammatory lesion was mainly located in the region of brain parenchyma in rats of the EAE group, which was presented as local infiltration of inflammatory cells dominated by lymphocytes and monocytes. It confirmed that the EAE model had been successfully established.

The activation degree of the astrocytes in the rat CNS was detected using GFAP specificity. In Fig. 4, the green fluorescence intensity represented the expression level of GFAP. The GFAP level in the brain sections of rats in EAE group was notably elevated compared with that in control group, suggesting that the activation degree of the astrocytes was significantly increased in the EAE rat models.

As shown in Fig. 5, compared with those in control group, the mRNA expression level of IL-10 in the brains of the rats in EAE group was lowered obviously (P<0.05), while the mRNA expression levels of IFN-γ and TNF-α were elevated notably (P<0.05).

In order to further investigate the expression of inflammatory factors in the EAE models, western blot analysis was performed to measure the levels of inflammatory factors in the brain tissues of the two groups of rats. Fig. 6 shows that, the protein level of IL-10 in the brains of rats in the EAE group was decreased significantly (P<0.05), while those of IFN-γ and TNF-α were markedly increased (P<0.05).