Alterations of protein composition along the rostro-caudal axis after spinal cord injury: proteomic, in vitro and in vivo analyses

Abstract

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.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair

“…Indeed, the microenvironment in which microglia and/or macrophages are activated influences their neurotrophic or neurotoxic effector potential (52,53) that correspond to M1 and M2 phenotypes. The predominance of microglia activation in caudal segments is in line with our previous and present studies confirming the occurrence of severe inflammationassociated tissue damage taking place in this segment and possibility to switch/or not from M1 to M2 phenotype (29).…”

“…Tregs can reduce inflammation and enhance CNS repair (57). Presence of such cells predominantly in rostral segment is in line with the cytokine medium which favor expression of macrophages expressing an M2 phenotype and production of neurotrophic factors (29). Moreover, the time difference of recruitment between rostral and caudal segments of these cells can also contribute to the less enhancement of neurites outgrowth in C1 compared with R1.…”

“…Silver destaining was performed using 30 mM of potassium ferricyanide and 100 mM sodium thiosulfate (1:1) 200 l per band for 20 min in the dark. Bands were washed 2 times for 15 min with Milli-Q water followed by an in-gel digestion as previously described (29). The samples were reconstituted with 10 l of 5% AcN/0.1% FA and injected on a UPLC MS instrument.…”

Modulation properties of factors released by bone marrow stromal cells on activated microglia: an in vitro study

“…Thus, based on the secretion profile, BMSCs-CM. may contribute to neuroprotection in a direct manner by rescuing neural cells and switching the microglial cells/macrophages to a M2 polarization33.…”

“…Moreover, BMSCs have been transplanted into rodent models of SCI by various research groups33343536. Despite considerable variation, a clear functional beneficial effects can be expected after transplantation of BMSCs into the injured spinal cord37383940.…”