In the course of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), the infiltration of lymphocytes and other inflammatory cells across the blood–brain barrier is associated with interactions between adhesion molecules expressed by infiltrating cells and vascular endothelium. Monoclonal antibodies (mAb) against the α4 subunit of α4-β1 integrin (VLA-4) show beneficial effects in both MS and EAE. (1) Background: The aim of this study was to examine the expression of selected adhesion molecules: VLA-4, VCAM-1, LFA-1, ICAM-1 and PECAM-1 in the successive phases of EAE and the effect of anti-VLA-4 mAb treatment on that expression. (2) Methods: EAE was induced in C57BL/6 mice by immunization with MOG_35–55 peptide. The animals were killed in three successive phases of the disease: onset (day 13), peak (day 18) and chronic (day 28). Frozen sections of the lumbar spinal cord were examined by quantitative immunofluorescence microscopy. The expression of the studied molecules was quantified as the percentage of the cross-sectioned spinal cord lesion area occupied by immunopositive structures. (3) Results: The expression of the studied molecules showed two temporal patterns: (1) an increase in the onset phase, a maximum in the peak phase and a decrease in the chronic phase, which corresponded to the temporal pattern of the clinical score, the number of lesions and the inflammation level (ICAM-1, LFA-1 and PECAM-1), and (2) an increase in the peak phase and no significant change or further increase in the chronic phase (VCAM-1, VLA-4). Among the molecules studied, ICAM-1 and LFA-1 exhibited the highest expression levels in the peak phase of EAE. Anti-VLA-4 mAb inhibited the expression of not only VLA-4 but also other adhesion molecules. (4) Conclusions: The interactions of adhesion molecules governing the migration of leukocytes across the blood–brain barrier change in the successive phases of EAE. The therapeutic mechanism of anti-VLA-4 mAb treatment seems to include a complex influence on a variety of adhesion molecules expressed by infiltrating cells and vascular endothelium.

Introduction. Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for studying immunopathology of multiple sclerosis (MS) because it repeats the hallmarks of the human disease, such as focal inflammation and demyelination of the central nervous system, subsequently leading to axonal and neuronal loss. The interrelationships, timing and sequence of different pathological processes that lead to histologically observed lesions in SM are still incompletely understood. Material and methods. EAE was induced in female C57Bl/6 mice by active immunization with MOG35-55 antigen. Development of the neurological symptoms in the animals was monitored and on that basis spinal cords were collected in three successive phases of the disease (onset, peak, chronic). Total leukocytes, T cells, macrophages/microglia, oligodendrocytes, damaged axons and surviving neuronal cell bodies were visualized using appropriate immunohistochemical markers and their density was quantitatively assessed using image analysis software. Results. The density of all studied cells except neurons was significantly higher in EAE mice than in the control mice. The density of total leukocytes, T cells, and damaged axons increased from the onset to the peak phase and decreased in the chronic phase to reach values lower than those in the peak phase. The density of macrophages/microglia increased in the peak phase and remained at the elevated level in the chronic phase. Oligodendrocytes showed the highest density in the onset phase and gradually decreased afterwards. The density of neuronal cell bodies decreased only in the chronic phase of the disease. Conclusions. In mouse model of EAE, inflammatory cells predominate in the early phases of the disease. This study shows for the first time that inflammation precedes oligodendrocyte death and neuronal loss and that macrophages/ microglia are the only cells persisting in large numbers in the chronic phase of the disease, probably because of the switch from proinflammatory to anti-inflammatory phenotype. Introduction. Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for studying immunopathology of multiple sclerosis (MS) because it repeats the hallmarks of the human disease, such as focal inflammation and demyelination of the central nervous system, subsequently leading to axonal and neuronal loss. The interrelationships, timing and sequence of different pathological processes that lead to histologically observed lesions in SM are still incompletely understood. Material and methods. EAE was induced in female C57Bl/6 mice by active immunization with MOG35-55 antigen. Development of the neurological symptoms in the animals was monitored and on that basis spinal cords were collected in three successive phases of the disease (onset, peak, chronic). Total leukocytes, T cells, macrophages/microglia, oligodendrocytes, damaged axons and surviving neuronal cell bodies were visualized using appropriate immunohistochemical markers and their density was quantitatively assessed using image analysis software. Results. The density of all studied cells except neurons was significantly higher in EAE mice than in the control mice. The density of total leukocytes, T cells, and damaged axons increased from the onset to the peak phase and decreased in the chronic phase to reach values lower than those in the peak phase. The density of macrophages/microglia increased in the peak phase and remained at the elevated level in the chronic phase. Oligodendrocytes showed the highest density in the onset phase and gradually decreased afterwards. The density of neuronal cell bodies decreased only in the chronic phase of the disease. Conclusions. In mouse model of EAE, inflammatory cells predominate in the early phases of the disease. This study shows for the first time that inflammation precedes oligodendrocyte death and neuronal loss and that macrophages/ microglia are the only cells persisting in large numbers in the chronic phase of the disease, probably because of the switch from proinflammatory to anti-inflammatory phenotype.

Matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs) play a significant role in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), as they degrade extracellular matrix including vascular basal laminae and by damaging blood-brain barrier (BBB) facilitate transmigration of immune cells into the central nervous system. MMPs are also involved in destruction of myelin sheaths, leading to axonal and neuronal loss. The aim of the present study was to assess whether natalizumab, a transmigration-inhibiting monoclonal antibody against alpha 4 beta 1 integrin, influences expression of MMPs and TIMPs in the central nervous system of mice with EAE. MMP-2 and MMP-9, their respective inhibitors TIMP-2 and TIMP-1 and laminin were assessed by quantitative immunohistochemistry in the spinal cord cryosections of C57BL/6 mice with EAE in the successive phases of the disease (onset, peak and chronic). The percentage of immunopositive areas were calculated in sections encompassing the whole spinal cord cross-sectional area occupied by the gray and white matter. Results obtained in animals administered with 5 mg/kg natalizumab were compared with those collected from control mice receiving 5 mg/kg IgG. Both studied MMPs and both TIMPs were upregulated in control EAE mice. Natalizumab treatment significantly reduced expression of MMPs and increased expression of TIMPs in the peak and chronic phases of the disease. This effect was accompanied by inhibition of laminin degradation in the vascular basal laminae and reduction of inflammatory infiltration. Results of this study demonstrate that in addition to its well known anti-integrin activity counteracting transmigration of immune cells into the central nervous system, natalizumab strengthens this effect by its probably indirect influence on MMPs and TIMPs leading to protection of blood-brain barrier integrity.

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are often accompanied by optic neuritis associated with neurofilament disruption. In this study, the stiffness of the optic nerve was investigated by atomic force microscopy (AFM) in mice with induced EAE in the successive phases of the disease: onset, peak, and chronic. AFM results were compared with the intensity of the main pathological processes in the optic nerve: inflammation, demyelination, and axonal loss, as well as with the density of astrocytes, assessed by quantitative histology and immunohistochemistry. Optic nerve tissue and serum levels of neurofilament light chain protein (NEFL) were also examined by immunostaining and ELISA, respectively. The stiffness of the optic nerve in EAE mice was lower than that in control and naïve animals. It increased in the onset and peak phases and sharply decreased in the chronic phase. Serum NEFL level showed similar dynamics, while tissue NEFL level decreased in the onset and peak phases, indicating a leak of NEFL from the optic nerve to body fluids. Inflammation and demyelination gradually increased to reach the maximum in the peak phase of EAE, and inflammation slightly declined in the chronic phase, while demyelination did not. The axonal loss also gradually increased and had the highest level in the chronic phase. Among these processes, demyelination and especially axonal loss most effectively decrease the stiffness of the optic nerve. NEFL level in serum can be regarded as an early indicator of EAE, as it rapidly grows in the onset phase of the disease.