Thirty female Wistar rats, 8–10 weeks of age, were purchased from the Beijing Vital-River Experimental Animal Company, China, and housed in specific pathogen-free conditions at the Henan Province Chinese Medicine Research Institute. Every effort was made to ensure minimal animal suffering, and the guidelines of the Animal Care and Use Committee of the Henan Province Chinese Medicine Research Institute were followed for all the procedures in this study.
2.2. Induction of rat EAE model
EAE was induced in 20 rats as described previously (20). Briefly, guinea pig spinal cord homogenate (GPSCH) was made from an equal amount of guinea pig (Beijing Vital River Experimental Animal Company) spinal cord and pre-chilled saline, and then emulsified with the same volume of complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MI, USA) containing 6 mg/ml Bacillus Calmette–Guérin vaccine (Solarbio Bio-Technology Co., Shanghai, China). Each rat was subcutaneously injected on the same day at four separate sites with 0.5 ml of antigen emulsion. All the experiments were approved by the Bioethics Committee of Zhengzhou University.
2.3. MAT treatment and disease assessment
Immunized rats were randomly divided into two groups (n = 10 each group). The EAE incidence was 80%, and each EAE group contained two rats that did not show clinical signs of EAE. Treatment groups include: (1) MAT (MW: 264.36, a small molecule that was purchased from Chia-Tai Tianqing Pharmaceutical Co.), was injected intraperitoneally (i.p.) at 250 mg/kg in 1 ml normal saline daily, starting from day 11 post immunization (p.i.) until the end of the experiment (day 18 p.i.); (2) immunized rats that received the same amount of saline via i.p. served as control; (3) non-immunized naïve rats that received the same amount of saline i.p. served as naïve control. All rats were monitored and scored daily from day 0 to 18 p.i. by two independent observers in a blinded manner following the standard 0–5 EAE grading scale as previously published (6, 8): 0, natural; 0.5, partial tail paralysis; 1, tail limpness or waddling gait; 1.5, loss of tail tonicity or waddling gait; 2, hind limb weakness; 2.5, partial limb paralysis; 3, paralysis of one limb; 3.5, paralysis of one limb and partial paralysis of another; 4, paralysis of two limbs; 4.5, moribund state, and 5, death.
2.4. Histopathological evaluation of optic nerves
To assess the extent of CNS inflammation and demyelination, the rats were sacrificed on day 18 p.i. Rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and extensive perfusion with 0.9% normal saline. Both sides of the optic nerves were quickly harvested and post-fixed with 4% paraformaldehyde, embedded in paraffin, and cut into paraffin sections (2–5 µm). Cross-sectional optic nerve sections (the anterior part of the optic nerve) and longitudinal optic nerve sections (the posterior part of the optic nerve) were examined from both sides of eyes for HE staining and LFB staining. The histological examination was performed and scored by light microscopy by two investigators in a blinded manner using a grading scale as previously published criteria (8): 0, no inflammatory infiltration; 1, a few scattered inflammatory cells of the optic nerve or optic nerve sheath; 2, moderate inflammatory infiltrates; 3, severe inflammatory infiltrates; 4, massive inflammatory infiltrates. Given that ON can occur either bilaterally or unilaterally, all subsequent analyses were performed using both eyes from each rat as individual data points and both cross-sectional and longitudinal optic nerve sections. An Olympus BX53 microscope (Japan Olympus Corporation) was used for the histological examination. Scores of demyelination and inflammation were calculated by Image-Pro Plus 5.0 (IPP5.0) software.
2.5. Immunofluorescence analysis of optic nerves and retina cross-sections
Both optic nerves and retinas from each rat were paraffin-embedded and cut into 5-μm-thick sections for immunofluorescence. CD4, Iba1 and neurofilaments (NFs) were assessed in the anterior part of the optic nerve and the retina, and Brn3a and TUNEL were done in the temporal retina (close to the optic nerve head). First, non-specific binding was blocked with 3% bovine serum albumin (BSA) (Serotec, UK), and permeabilized with 0.3% Triton X-100 in 1% BSA-phosphate buffer saline (PBS) for 30 min. The optic nerve sections were incubated at 4°C overnight with mouse anti-rat CD4 (1:100, Bioss, Beijing, China), mouse anti-rat Iba1 (1:100, Abcam, London, UK), rabbit anti-rat NFs (1:100, Bioss), followed by incubation with corresponding secondary antibodies (goat anti-mouse Cy3 conjugate, 1:200; goat anti-rabbit Alexa Fluor 488, 1:200; Invitrogen, Carlsbad, CA, USA) at room temperature for 2 h.
For double staining of RGCs and TUNEL (for apoptotic cells), comparable areas of the temporal retina were analyzed in all animals of all three groups. TUNEL reaction mixture was added before the primary antibody (Abcam) following the manufacture’s instructions. RGCs-positive were detected by rabbit anti-Brn3a (1:100, both from Abcam), followed by incubation with corresponding secondary antibodies - goat anti-rabbit Cy3 conjugate (1:200, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. To assess the number of cells, a nuclear stain 4′,6-diamidino-2-phenylindole (DAPI, Roche, Shanghai, China), was added to tissue sections for 15 min prior to final washes after adding secondary antibodies. Finally, slides were visualized with confocal microscope (Olympus Fluoview FV1000).
Numbers of CD4+ and Iba1+ cells that had been co-stained for DAPI were counted as positive cells. Brn3a+TUNEL+ cells that were co-stained for DAPI were counted as apoptotic RGCs. Image J (National Institutes of Health, USA) was used for the cell counting. Image J was also used to assess average optical density (AOD) of NFs. The anterior part of the optic nerve was used to quantify NF intensity. Exposure time was fixed for all microscope images. All these studies were performed by two investigators in a blinded manner.
2.6. Statistical analysis
All the animal groups were coded and analyses were conducted by two researchers blind to experimental conditions. Multiple comparisons were performed using one-way ANOVA, followed by Student-Newman-Keuls test. Clinical EAE scores were compared at individual time points between vehicle- and MAT-treated rats. Statistical software (GraphPad Prism 5.0; GraphPad Software) was used for statistical analyses; P<0.05 was considered significant. For all histological experiments, each eye was used as an independent data point, based on previous reports showing that optic neuritis can occur bilaterally, or unilaterally in either eye, and thus can occur as an independent event (21). Generalized estimating equation (GEE) models were used as previously described (22) for within-subject inter-eye correlations between numbers of Brn3a+TUNEL+ cells and scores of optic nerve inflammation in EAE+Vehicle and EAE+MAT groups.