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, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.
Highlights
The presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration
Each of these analyses were performed on spinal cord segments obtained on days 3, 7, or 10 post-Spinal cord injury (SCI) giving rise to a set of 80 proteomic data covering an average of 1500 proteins identified par sample with at least 2 peptides per protein recognized and percentage of false positive (FDR) Ͻ 1%
We have previously investigated the spectrum of released molecules in the conditioned media (CM) from the spinal cord central lesion and adjacent rostral and caudal segments at 3 days after spinal cord injury (SCI) in order to specify the molecular environment within the proximity of the injured tissue
Summary
Reagents—Dulbecco’s modified Eagle’s medium (DMEM) media, phosphate buffer saline (PBS), fetal calf serum (FCS) were purchased. IgG Purification with Dynabeads Protein A—CM (150 l) obtained from lesion site(ϳ1.5 mg of protein in 2 ml) after SCI3, 7 or days after SCI and from the corresponding segment of control spinal cord were bound with 50 l of Dynabeads Protein-A 200 l of PBS-Tween 20 and incubated 10 min with rotation at room temperature. Immunohistochemical Analysis of IgG Deposition—Tissue sections of 40 m thickness from rostral and caudal segment adjacent to the lesion at the 3, 7 and 10 days post-injury and corresponding cuts for controls (embedded in gelatin-egg albumin protein matrix, same procedure as for IHC for immune response) were cut. MALDI Imaging Data Analyses—Twelve micrometer tissue sections from the R1, lesion and C1 segments were obtained using a cryostat (Leica Microsystems, Nanterre, France) These were mounted on indium tin oxide (ITO)-coated slides and placed under vacuum in a dessicator for 15 min. The PCA was performed with no scaling, a threshold of 29.0588, maximum interval processing mode, lower m/z range threshold mode and individual spectrum mode [30]
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