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

Abstract

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover

RhoA Inhibitor Treatment At Acute Phase of Spinal Cord Injury May Induce Neurite Outgrowth and Synaptogenesis

“…Our data suggest that in acute SCI regionalization in terms of inflammatory and neurotrophic responses may occur because of alterations in protein dynamics between rostral and caudal segments (13). In addition, the proteomic profile in caudal segments was characterized by the neuronal expression of IgG2a and by a signature of axonal regrowth inhibition associating CSPG and proteins of the MEMO1-RHOA-DIAPH1 signaling pathway (14). The MEMO1-RHOA-DIAPH1 signaling pathway plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex and inhibits neurite outgrowth.…”

“…The cells were starved overnight with DMEM medium supplemented with 2% Fetal bovine serum (FBS) ϩ 1% antibiotics ϩ 1% L-glutamine. Afterward, cells were stimulated with 1/3 R1 or C1 CM and 2/3 DMEM ϩ 1% L-1% L-Glutamine ϩ 1% penicillin-streptomy-cin (serum free medium) (14). The cells were treated or not with 1 g/ml RhoAi in combination with 1/3 of CM with 2/3 DMEM supplemented medium 24 h after C1 or R1 stimulation in order to reproduce the injured environment.…”

“…The cells were treated or not with 1 g/ml RhoAi in combination with 1/3 of CM with 2/3 DMEM supplemented medium 24 h after C1 or R1 stimulation in order to reproduce the injured environment. The optimum split ratio 1/3 (CM, RhAi): 2/3 culture DMEM was set to perform sustained culture conditions for cells, according to our previous studies (14). Live images of cells not stimulated with RhoAi were captured 48 h after R1 or C1 CM stimulation and the images of ND7/23 cells stimulated with CM and RhoAi were captured 24 h after RhoAi stimulation (corresponding to 48 h after CM stimulation) with a camera mounted on a phase-contrast microscope (Nikon Eclipse TS100).…”

“…To confirm the proteomic data outlining the role of the RhoAi treatment at early stage of the lesion, we decided to design in vivo experiments consisting in the local intraspinal delivery of RhoAi and by an intraperitoneal injection of an immunosuppressant, calcineurin inhibitor (FK506) to diminish inflammation (14). Here were administered RhoAi in an alginate scaffold (with no growth factors) which biocompatibility and its intrinsic beneficial impact on neurite outgrowth was previously showed (16).…”

Neuroproteomics and Systems Biology Approach to Identify Temporal Biomarker Changes Post Experimental Traumatic Brain Injury in Rats

“…The potential of neuroproteomics platforms have been explored using acute paradigms of TBI (26, 27, 29–35), spinal cord injury (36–44), and cerebral ischemia or stroke (34, 45–50). Our group has previously reported TBI effects upon the global proteome where we combined cyanine labeling with SDS PAGE–capillary LC–MS/MS to study hippocampal tissue (30).…”