**4.1 Experimental autoimmune encephalomyelitis (EAE)**

EAE is a spectrum of neurological disorders. EAE can be induced in laboratory animals such as rats and mice after immunization with CNS antigens emulated in an adjuvant to enhance immune response [20].

Usually, in these models, purified myelin, recombinant proteins, or peptides related to encephalitogenic myelin protein are applied. Notably, myelin lesions and inflammation in MS are common features; however, neurodegeneration is a major characteristic as well. Therefore, the secondary progressive EAE was developed with main characteristics such as cortical demyelination, experimental inflammatory neurodegenerative, and spastic diseases [22, 65].

The clinical course of EAE depends on several factors, including the immunization protocol, the antigen, and age, gender species, and strain of the animals. With the active immunization protocol, the first signs of neurological illness are usually weight and activity loss and can be observed between 10 and 17 days. When the adoptive transfer method is used, signs are seen a little earlier, starting 5–7 days after cell transfer [21, 24].

The EAE clinical signs are typically rated based on a muscle force scale (0 to 5) that reflects increasing degrees of paresis, with grade zero being normal and graded 5 being moribund. There are other scoring systems depending on the species used and focus on other clinical signs than just paresis (muscle weakness). Neurological disease can also be monitored using a rotor rod to measure dexterity, and as well as by monitoring behavioral changes [66].

## **4.2 Toxin models**

A further possibility of investigating demyelination with subsequent remediation is the use of toxin models [47]. In these models, demyelination is induced after focal application or systemic administration of the toxin. Copper-chelating cuprizone is the most common toxin used to induce demyelination in the CNS using systemic administration [28].

Cuprizone or oxalic acid bis cyclohexylidene hydrazide is a selective and sensitive copper-chelating agent. Shortly after its discovery, it was used to detect copper in serum. W. Carlton was one of the first ones to systematically study and describes disabling changes in cuprizone-fed animals, including sponginess, edema formation, hydrocephalus, demyelination of the central nervous system, and liver damage [46]. Based on Carlton's findings, we can find several studies using 6–9-week-old mice with a diet containing 0.2–0.3% cuprizone. In older animals, the concentration of comparison must be high in the feeding to guarantee adequate demyelination. In addition, vulnerability to cuprizone that depends on the strain has been reported. For example, there is a difference in the anatomical distribution of demyelinated foci between SJL and C57BL/6 mice. Based on previous studies, we cannot observe demyelination at the brain midline within the corpus callosum in SJL mice; however, demyelination can be seen immediately from the lateral part of mice brain [47]. Therefore, focusing on these variables can be very important before starting any studies.
