3.1. Induction of EAE
We applied mostly the same procedures as other studies stated. Twenty female mice, 5–8 weeks old, (C57BL/6, 19.4 g) were obtained from Pasteur Institute of Iran. The animals were divided randomly into two groups (n = 10). The animals were immunized subcutaneously with 200 μg of MOG 35–55 peptide (Alexis, Switzerland), emulsified in Complete Freund’s adjuvant (CFA), and supplemented with 5 mg/mL of Mycobacterium tuberculosis (Sigma-Aldrich, USA). Then, they received intraperitoneal injections of 400 ng pertussis toxin (Sigma-Aldrich, USA) at the time of immunization, and 48 hours later (9, 10). Following induction, animals of group one were placed in separated room with the least local transfer, and animals of the second group were placed in the room with other experimental animals having normal local transfer and conditions.
3.2. Clinical EAE Score
To approve the onset and stage of progression of the disease, the standard scoring system cited elsewhere was used (6). Based on this method, clinical signs of no symptoms, distal weakness or spastic tail and completely limp tail, bilateral partial hind-limb paralysis, complete hind-limb and unilateral partial forelimb paralysis, moribund, and dead were scored 0, 1, 2, 3, 4, and 5, respectively. We confirmed the EAE model by Luxol Fast Blue staining in optic nerve (Figures 1 and 2).
Figure 1.
LFB in Optic nerve to Confirm of Model
Luxol Fast Blue staining used for rate of demyelination showed significant differences among groups including group 1 (A) and group 2 (B).
Figure 2.
Histogram of Demyelination in Optic Nerve
Significant difference between EAE (group 1) and EAE (group 2).
3.3. Tissue Preparation
Perfusion and fixation was done transcardially via left ventricle by aldehyde solutions. Optic nerve was removed and postfixed in the same solution overnight. After that, the optic nerve was exposed to tissue processing and in turn paraffin embedding. By using rotatory microtome, coronal sections of 4 µ thickness, were prepared. To study the myelination, Luxol Fast Blue staining with Nissl contra staining was used. The total surface of demyelinated regions was calculated by Infinity Software (Version 4.4.0).
3.4. Statistical Analysis
By using SPSS V 21(SPSS Inc., Chicago, USA), data were analyzed and presented as mean ± SEM. All mean differences were considered significant if P < 0.05.
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