No Impact of Corticosteroid Use During the Acute Phase on Persistent Symptoms Post-COVID-19.

Persistent COVID-19 symptoms may be related to residual inflammation, but no preventive treatment has been evaluated. This study aimed to analyze, in a prospective cohort, whether corticosteroid use in the acute phase of COVID-19 in hospitalized patients may reduce the risk of persistent COVID-19 symptoms. A total of 306 discharged patients, including 112 (36.6%) from the ICU, completed a structured face-to-face assessment 4 months after admission. Of these, 193 patients (63.1%) had at least one persistent symptom, mostly dyspnea (38.9%) and asthenia (37.6%). One-hundred and four patients have received corticosteroids. In multivariable adjusted regression analysis, corticosteroid use was not associated with the presence of at least one symptom (OR=1.00, 95% CI: 0.58-1.71, p=0.99) or with the number of persistent symptoms (p=0.74). Corticosteroid use remained ineffective when analyzing the ICU subpopulation separately. Our study suggests that corticosteroid use had no impact on persistent symptoms after COVID-19 in discharged patients.

3D MALDI mass spectrometry imaging reveals specific localization of long-chain acylcarnitines within a 10-day time window of spinal cord injury.

We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.

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

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.

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.

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.

Comparison of dynamic behavior and maturation of neural multipotent cells derived from different spinal cord developmental stages: an in vitro study.

Neural progenitor cells (NPCs) are characterized as undifferentiated cells with the ability of self-renewal and multipotency to give rise to other cells of the nervous system. In our in vitro study we demonstrate the proliferative and differentiative potential of NPCs isolated from the spinal cord at different developmental stages (embryonal, early postnatal, adult), maintained and expanded within neurospheres (NSs). Using the NSs culture system, we examined the size, number of NSs and their fate when exposed to differentiation conditions. Based on immunocytochemical analyses for cell markers (MAP 2, GFAP, RIP) we evaluated the occurrence of various cell types: neurons, astrocytes and oligodendrocytes. The results show that NSs increased in size during cultivation time via NPC proliferation, but proliferation potential decreased Turing maturation stages. In addition, NPCs derived from spinal cord developmentally different stages gave rise to a consistent ratio of glial and neuronal progeny (3:1), and adult tissues represent a comparable source of NPCs compared to embryonal and early postnatal tissues. These data provide useful information for large-scale in vitro expansion of NPCs required for potential cell therapy after spinal cord injury.

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

In the present paper we develop a new non-cell based (cell-free) therapeutic approach applied to BV2 microglial cells and spinal cord derived primary microglia (PM) using conditioned media from rat bone marrow stromal cells (BMSCs-CM). First we collected conditioned media (CM) from either naive or injured rat spinal cord tissue (SCI-CM, inflammatory stimulation agent) and from rat bone marrow stromal cells (BMSCs-CM, therapeutic immunomodulation agent). They were both subsequently checked for the presence of chemokines and growth, neurotrophic and neural migration factors using proteomics analysis. The data clearly showed that rat BMSCs-CM contain in vitro growth factors, neural migration factors, osteogenic factors, differentiating factors and immunomodulators, whereas SCI-CM contain chemokines, chemoattractant factors and neurotrophic factors. Afterwards we determined whether the BMSCs-CM affect chemotactic activity, NO production, morphological and pro-apoptotic changes of either BV2 or PM cells once activated with SCI-CM. Our results confirm the anti-migratory and NO-inhibitory effects of BMSCs-CM on SCI-CM-activated microglia with higher impact on primary microglia. The cytotoxic effect of BMSCs-CM occurred only on SCI-CM-stimulated BV2 cells and PM, not on naive BV2 cells, nor on PM. Taken together, the molecular cocktail found in BMSCs-CM is favorable for immunomodulatory properties.

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

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.