In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.
Based on proteomic analyses we investigated the differences of released molecules in the conditioned media (CM) from the spinal cord central lesion and adjacent rostral and caudal segments at 3, 7, and 10 days after spinal cord injury (SCI), in order to specify the molecular environment within greater extent of tissue damage. Proteins found in CM were analyzed by shot-gun MS using nanoLC coupled to an orbitrap. The results showed some specific proteins at each site of the lesion at 3days. Among the proteins from rostral and lesion segments, some are related to chemokines, cytokines or to neurogenesis factors. In contrast, proteins from caudal segments are more related to necrosis factors. The CM from each spinal segment were used in vitro, on microglial BV2 cell lines and DRGs explants, showing a lesion site-dependent impact on microglia activation and DRGs neurite outgrowth. In addition, while naive BV2 cells exhibited insignificant staining for CX3CR1 receptor, the level of CX3CR1 was strongly enhanced in some BV2 cells after their stimulation by CM collected from SCI. The molecular data might correlate with different polarization of activated microglia and macrophages along the rostro-caudal axis following acute injury. This was partially confirmed in vivo with CX3CR1 receptor, revealing higher expression in the rostral segment, with potential neuroprotective action. In addition, the neurotrophic factors released from rostral and lesion segments enhanced outgrowth of DRGs explants. Taken together these data suggest that regionalization in terms of inflammatory and neurotrophic responses may occur between rostral and caudal segments in acute SCI.
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