Induction of EAE and histological Analysis

Encephalomyelitis was induced in mice (n = 5 per condition) using the MOG35-55 peptide in complete Freund's adjuvant (CFA) with pertussis toxin (PTx) as described previously ³⁶. Following the appearance of EAE symptoms, the mice were scored daily by two independent investigators (0, no sign of disease; 1, limp tail; 2, limp tail and partial hind limb weakness; 3, complete hind limb paralysis; 4, complete hind limb and partial front limb paralysis; 5, death), stratified, and assigned to separate test groups in order to obtain equally weighted average disease scores prior to experimental interventions. EVs were injected at 0 and 7 days (pre-onset stage of EAE) or every alternate day for 10 days (post-onset stage of EAE). EAE mice receiving PKH67-EVs were euthanized 24h after injection. Brain and spleen sections were observed under a Lionheart imaging system (BioTek Instruments, Winooski, VT). Images were processed using Gen5 software (BioTek Instruments). For histological analysis, routine histology methods were carried out to obtain morphological details of the brain tissue in EAE mice. Paraformaldehyde fixed tissues were embedded in paraffin, and serial sections (8 μm) were prepared. Sections were stained with the conventional hematoxylin and eosin (H and E) staining method. Digital images were taken using a ×20 objective. Brain sections were observed under a Lionheart imaging system (BioTek Instruments, Winooski, VT), and images were processed to measure infiltrated cell numbers and the inflammation area using Gen5 software (BioTek Instruments).