Axonal damage induced by cerebrospinal fluid from patients with relapsing-remitting multiple sclerosis

The importance of axonal damage in multiple sclerosis (MS) has been recently stressed in proton magnetic resonance spectroscopy and pathological studies, but the exact mechanism producing this damage is unknown. The aim of our study was to ascertain whether soluble mediators present in the cerebrospinal fluid (CSF) of patients with relapsing-remitting MS could induce neuron injury in culture. Different biochemical and cytochemical parameters were determined in primary embryonal rat neuron cultures following 8 days of exposure to CSF. Cytotoxic activity was evaluated with a blue formazan production colorimetric assay. Morphological and immunocytochemical studies performed with antibodies against beta-tubulin revealed neuritic fragmentation, axonal damage and cellular shrinkage indicating apoptosis. Detection of apoptosis was carried out using the fluorescent DNA-binding dye Hoechst 33342, as well as by a Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay. We observed that soluble factors in CSF from patients with "aggressive" MS i.e, those with poor recovery after relapses, induced neurite breakdown and neuronal apoptosis in cultures. Neuron injury is not related with blood-brain barrier dysfunction nor with IgG index. Interestingly, CSF from patients with "non-aggressive" MS i.e., relapsing-remitting patients with a good recovery after relapses, did not induce any damage. In conclusion, we report that CSF from patients with aggressive MS bears soluble mediators that induce axonal damage and apoptosis of neurons in culture. These mediators can be present during the first attack of the disease, and the neuronal damage caused could be related to the functional deficit of these MS patients.