Adult (8–9 weeks old and 18–20 g) female C57BL/6 mice were purchased from North China University of Science and Technology. All mice were housed in a temperature-controlled room under a 12-h light/dark cycle for 4 weeks with food and water ad libitum. The mice were randomly divided into three groups; control group (n=12), experimental autoimmune encephalomyelitis group (EAE group, n=12) and Tuftsin group (n=12). All protocols were approved by the Animal Ethics Committee of the North China University of Science and Technology.

EAE mice were induced with MOG35-55 (200 µg), and the mice were intraperitoneally injected with pertussis toxin (500 ng, List Biological Laboratories, Inc.) at 0 and 48 h following immunization. At least two investigators weighed and evaluated the animals for clinical scores in a blinded manner. There were two animals which appeared to be in intolerable distress and self-mutilated limbs; these were sacrificed using pentobarbital sodium (150 mg/kg).

Clinical scores (10) were determined in accordance with the following criteria: 0, healthy; 1, limp tail; 2, ataxia and/or paresis of the hind limbs; 3, paralysis of the hind limbs; 4, paresis and/or paralysis of the forelimbs; 5, moribund or dead.

The present study used ALZET mini-osmotic pumps to control drug delivery over time. The mice were injected with either PBS or 500 mM tuftsin [Gen Script (Nanjing) Co., Ltd.] at a rate of 0.25 ml/h (total volume was 100 ml). Pumps were implanted subcutaneously in the backs of anaesthetized mice on day 1 following immunization. On day 15, the pumps were replaced with fresh pumps and maintained thus until day 28.

Spinal cords were obtained from anaesthetized mice, which were perfused intracardially with 4% paraformaldehyde. The samples underwent a dehydration in graded ethanol (70% ethanol 3–5 min; 80% ethanol 3–5 min; 90% ethanol 3–5 min; 95% ethanol 3–5 min). Paraffin-embedded tissue sections were cut in the coronal plane at a thickness of 5 µm. Histological staining, including LFB staining, was performed to identify demyelination. The sections were left in LFB solution (Beijing Solarbio Science & Technology Co., Ltd.) at 56°C overnight, excess stain rinsed off with 95% ethyl alcohol and distilled water, differentiated in lithium carbonate solution for 30 sec and 70% ethyl alcohol for 30 sec, counterstained in cresyl violet solution (Guidechem) for 30–40 sec, rinsed in distilled water, differentiated in 95% ethyl alcohol for 5 min then placed in 100% alcohol for 5 min (twice) and finally two baths in xylene for 5 min each. Immunohistochemistry was performed with anti-myelin basic protein (MBP) antibodies to identify MBP (1:100; sc-271524, Santa Cruz Biotechnology, Inc.). Hematoxylin was used to stain cell morphology. The sections were observed under light microscope (magnification, ×40) (11) and analyzed by Image 2 Pro plus 5.0 (Media Cybernetics, Inc.).

Total RNA was extracted from the brain and spinal cord in all groups using the RNAeasy Micro kit purchased from OMEGA Company following the manufacturer's instructions. Reverse transcription was performed on 1 µg of total RNA with an RT-PCR kit (Life Technologies; Thermo Fisher Scientific, Inc.), Purity quantification, cDNA synthesis and qPCR were performed according to the manufacturer's protocols. Reaction procedures were as follows: An initial step at 95°C for 5 min, 40 cycles of 94°C for 15 sec, and 60°C for 34 sec; reaction volume 20 µl. The primer sequences were: GAPDH: Forward: 5′-TTCACCACCATGGAGAAGGC-3′, Reverse: 5′-GGCATGGACTGTGGTCATGA-3′; tumor necrosis factor (TNF)-α: Forward: 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, Reverse: 5′-TGGG-AGTAGACAAGGTACAACCC-3′; IL-10: Forward: 5′-TGGCCACACTTGA-GAGCTGC-3′, Reverse: 5′-TTCAGGGATGAAGCGGCTGG-3′; TGF-β: Forward: 5′-CCGCAACAACGCAATCTATG-3′, Reverse: 5′-AGCCCTGTATTCCGTCTCCTT-3′.

qPCR was carried out using SYBR green mix (Roche, USA) and analyzed using the 2^−ΔΔCq method (11). The relative expression levels of the mRNAs were reported as fold changes vs. control.

Protein was extracted from the brain and spinal cord. The samples were lysed in Tissue Protein Lysis Solution (Thermo Fisher Scientific, Inc.). The protein concentration was determined using a BCA protein assay kit (OriGene Technologies, Inc.) and 25 µg of protein was loaded per lane. The primary antibodies used were specific for β-actin (1:1,000; cat. no. AB8227, Abcam), for the detection of ionized calcium binding adaptor molecule 1 (iba-1; 1:1,000; no. AB5076, Abcam), and for the detection of MBP (1:500, no. sc-271524, Santa Cruz Biotechnology, Inc.). Protein extracts were separated by electrophoresis on 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% non-fat milk for 2 h at room temperature and washed three times in PBS with Tween-20 (0.05%). The membranes were sequentially incubated at 4°C with primary antibodies (2 h) and secondary antibodies (1 h; 1:1,000; no. AB205718, Abcam) and enhanced chemiluminescence (ECL) solution. Finally, the images were captured and analyzed by using ImageJ v1.41 software (National Institutes of Health).

Data from experiments performed in triplicate were analyzed by One-way ANOVA, and multiple comparisons between groups were performed using the Student-Newman-Keuls method. The results are shown as the mean ± standard error of mean. P<0.05, **P<0.01 or ***P<0.001 was considered to indicate a statistically significant difference.

To explore the function of microglia in EAE, the expression of microglia in mice induced with tuftsin or PBS was first examined. EAE mice were induced with the MOG35-55 peptide. The expression levels of all groups were compared using western blotting at different time points of EAE. As shown in Fig. 1, western blots indicated increases in iba-1 and MBP expression in the spinal cords of tuftsin-treated mice compared with those of the EAE mice on day 7 (P>0.05), day 14 (P<0.01), day 21 (P<0.01), and day 28 (P<0.01). In a comparison of the EAE group and the control group, expression of iba-1 in EAE group demonstrated notably higher expression than the control group on day 14 (P<0.01), day 21 (P<0.001), and day 28 (P<0.001); the EAE group demonstrated significantly higher expression of MBP compared with the control group on day 14 (P<0.001), day 21 (P<0.001) and day 28 (P<0.001).

Clinical scores are one of the validated behavioral methods used to evaluate the symptoms of EAE in mice. Following induction of EAE with MOG35-55 or PBS, the symptoms of EAE mice with and without tuftsin treatment were compared by using clinical score testing. In Fig. 2A, we observed that in the acute phase of EAE, all groups had similar clinical scores. The clinical scores of the EAE and tuftsin groups changed from day 11; however, the EAE and tuftsin groups achieved a higher clinical score than the control group on day 14, day 21 and day 28 (P<0.05). However, during the progression of EAE, the control group had no mice without the disease. Notably, EAE mice treated with tuftsin had a significantly lower clinical score than did mice treated with PBS on day 14 (P<0.05), day 21 (P<0.05) and day 28 (P<0.05). These results demonstrated that tuftsin treatment can ameliorate the behavior impairment induced by MOG35-55.

A typical pathological change during EAE development is demyelination, which can be evaluated utilizing LFB staining. To investigate this aspect comprehensively, a histological examination of spinal cord tissue isolated from mice in the control, EAE and tuftsin groups was performed at different time points of the EAE process. The spinal cord tissue sections were subjected to LFB staining in which myelin is stained blue to green, immunohistochemical examination. As shown in Figs. 2B and C, and 3, significant demyelination was not observed in any group on day 7. At day 14, the clinical onset point, LFB staining of the EAE group demonstrated slight demyelination in the white matter. However, in the tuftsin group, the demyelination was less intense at the same time point. The structure of the myelin sheath in the control group, however, exhibited integrity. At the peak of the EAE process, day 21, a gradual but obvious change in the myelin sheath revealed severe deficiency in the EAE group. In contrast, demyelination was milder in the tuftsin group. On day 28, during the recovery stage of EAE, the loss of myelin did not continue but remyelination began, particularly in the tuftsin group, in which remyelination was more obvious than in the EAE group, showing that the structure of the myelin sheath was relatively intact. It should be noted that the structure of myelin in the control group remained unaltered.

Microglia were demonstrated to have M1 and M2 subsets, which represent pro- and anti-inflammatory populations, respectively (12). TNF-α and nitric oxide were secreted by M1 microglia, while neuroprotective M2 microglia produced protective cytokines, including IL-10 and TGF-β. M2 microglia have been reported to serve a protective role in EAE by ameliorating experimental autoimmune encephalomyelitis (13). To test whether M1 or M2 microglia contributed to the severity of EAE during development, the concentrations of cytokines produced by M1 or M2 microglia were detected. Total RNA was extracted the from the brains from each group and RT-qPCR performed to determine the microglial phenotype based on TNF-α levels for M1, and IL-10 and TGF-β levels for M2. Fig. 4 shows that the amounts of TNF-α and TGF-β were reduced in the tuftsin group and that the level of IL-10 was increased in the EAE group. The levels of TNF-α, IL-10 and TGF-β were increased at day 14 in the tuftsin group and EAE group, and the levels of IL-10 and TGF-β in the tuftsin-treatment group exceeded those in the EAE group (P<0.01 and P<0.05, respectively), but the level of TNF-α was less than that in the EAE group (P<0.01). At days 21 and 28, a comparison of the levels of IL-10 and TGF-β in the tuftsin treatment group with those in the EAE group revealed that the former increased more than the latter (P<0.001, P<0.001); however, the level of TNF-α demonstrated the opposite pattern. The control group generated little TNF-α, IL-10 and TGF-β. Collectively, these data indicated that microglial activation did not contribute to the development of EAE but rather the secretion of protective cytokines, which ameliorated the progression of clinical EAE. Thus, these experiments indicated that microglia released anti-inflammatory cytokines, improving remyelination.