**2. Material and methods**

### **2.1 Material**

Sixty adult Lewis rats (AgB1) of both sexes (30 male and 30 female animals), of 150–200 mean body weight, were immunized by slow injection in their hind footpads of 50 μm of guinea pig myelin basic protein, emulsified in Freund's complete adjuvant.

All the rats were clinically examined and scored daily following the immunization. The first clinical findings appeared between the 10th and 12th day following the injection. On the 18th day after the immunization, all the clinical manifestations of the animals were quantified and scored based on a 0–5 disease severity scale, where 0 means no clinical findings, 1 means loss of the tone of the nail, 2 means weakness of the hind limb, 3 means paralysis of the hind limb, 4 means paralysis of the hind limb and severe weakness or paralysis of the forelimb, and 5 means expiring condition or death [36, 37].

From the 60 immunized animals according to the final clinical evaluation, 2 of them were scored 0, 12 were scored 1, 10 were scored 2, 22 were scored 3, and 14 were scored 4. No one died.

Then, under ether anesthesia, all the rats were sacrificed, by perfusion with 200 ml buffer solution (buffered physiologic saline) followed by 250 ml οf Sotelo fixing solution [38] containing 2.5% glutaraldehyde, 1% paraformaldehyde in 0.2 cacodylate buffer, adjusted at pΗ 7.35. For the perfusion, a Holter pump (flow 25 ΗΡΜ) was used.

After the fixation, the skull of each animal was opened as well as the spinal canal, and the brain and the spinal cord were quickly removed and immersed in newly prepared Sotelo solution at 4°C.

#### **2.2 Method**

Coronal sections of the brain hemispheres were performed. The brain stem, the cerebellum, and the spinal cord were cut into sections of 2 mm thickness. Samples were taken under a dissecting microscope and immediately processed for electron microscopy.

#### *Demyelination Disorders*

All the specimens were immersed in newly prepared Sotelo fixing solution, for 3 h, then they were postfixed in 1% of osmium oxide for 30 min at room temperature and dehydrated in graded alcohol solutions and propylene oxide. After the dehydration, the specimens were embedded in Araldite mixture.

Semi-thin sections were performed on a Porter-Blum microtome and stained with 1% toluidine blue. Thin sections of silver-gray inference color were cut in a Reichert ultratome, mounted on bare 400 mesh grids, contrasted, with uranyl acetate and lead citrate, and studied in an electron microscope Zeiss 9As.

Besides, multiple sections of the brain hemispheres, the brain stem, the cerebellum, and the spinal cord, were prepared for histological examination. Thus paraffin-embedded sections were stained with hematoxylin and eosin and were serially studied in the light microscope at a magnification of 25× and 100×.

The histological diagnosis of the experimental allergic encephalomyelitis was based on the identification and quantitation of the perivascular infiltrates of mononuclear cells and lymphocytes in the brain, the cerebellum, and the spinal cord.
