Contributions
Abstract: P779
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Neurodegeneration
Background: Recent studies have demonstrated the prognostic role of the cerebrospinal fluid (CSF) biomarker chitinase 3-like 1 (CHI3L1) in the development of neurological disability, particularly in patients with clinically isolated syndrome (CIS). In this study, we investigated the potential neurotoxic effect of CHI3L1 in primary cultures of mouse embryonic cortical neurons.
Methods: A preliminary time and dose-response study was conducted to determine the optimal exposure conditions of CHI3L1 in cortical neurons isolated from E16 C57BL6/J mouse embryos. We tested CHI3L1 concentrations below and above 170 ng/ml, a cut-off value that was demonstrated to have prognostic implications in CIS patients (Cantó et al., Brain 2015). After testing 100, 170, 300 and 600 ng/ml exposure conditions for 12, 24 and 48 h, neurons at 11 days in vitro were treated with vehicle (PBS) or mouse recombinant CHI3L1 at 300 ng/ml during 24 and 48 h. Then, cells were fixed with 4% paraformaldehyde and inmunostained with mouse monoclonal primary antibody MAP2 (1:500) and goat anti-mouse alexa fluor 594 (1:500). Five microscope fields (20x) of each replicate were randomly chosen, imaged and counted. We assessed the effect of CHI3L1 on cell viability by quantifying the total number of neurons (MAP2+ cells) and neuronal functionality by neurite length retraction study with NeuriteTracer Image J plugin. A total of 5 independent experiments (vehicle: n=3) with 4 replicates per condition were performed.
Results: In agreement with the CSF CHI3L1 associated with the development of neurological disability in CIS patients, CHI3L1 concentrations of 100 ng/ml and 170 ng/ml in the preliminary time-dose-response study had no neurotoxic effect. In addition, no significant changes in neuronal death were observed at the 12 h time point. A CHI3L1 concentration of 300 ng/ml was finally selected as the lowest effective dose following 24-48 h of exposure. After 48 h of treatment, CHI3L1 reduced neuronal survival, insomuch as a concentration of 300 ng/ml was associated with a significant decrease in the total number of cortical neurons (p=0.009 vs. vehicle), and significantly induced neurite length retraction (p=0.034 vs. vehicle).
Conclusions: These results point to a neurotoxic effect of CHI3L1 in vitro and suggest a potential role of CHI3L1 as therapeutic target to prevent disability progression in MS patients.
Disclosure: C. Matute-Blanch, I. Carballo-Carbajal and L. Navarro report no disclosures.
X Montalban has received speaking honoraria and travel expenses for scientific meetings, has been a steering committee member of clinical trials or participated in advisory boards of clinical trials in the past years with Bayer Schering Pharma, Biogen Idec, EMD Merck Serono, Genentech, Genzyme, Novartis, Sanofi-Aventis, Teva Phramaceuticals, Almirall and Roche.
M Comabella has received compensation for consulting services and speaking honoraria from Bayer Schering Pharma, Merk Serono, Biogen-Idec, Teva Pharmaceuticals, Sanofi-Aventis, Genzyme, and Novartis.
Abstract: P779
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Neurodegeneration
Background: Recent studies have demonstrated the prognostic role of the cerebrospinal fluid (CSF) biomarker chitinase 3-like 1 (CHI3L1) in the development of neurological disability, particularly in patients with clinically isolated syndrome (CIS). In this study, we investigated the potential neurotoxic effect of CHI3L1 in primary cultures of mouse embryonic cortical neurons.
Methods: A preliminary time and dose-response study was conducted to determine the optimal exposure conditions of CHI3L1 in cortical neurons isolated from E16 C57BL6/J mouse embryos. We tested CHI3L1 concentrations below and above 170 ng/ml, a cut-off value that was demonstrated to have prognostic implications in CIS patients (Cantó et al., Brain 2015). After testing 100, 170, 300 and 600 ng/ml exposure conditions for 12, 24 and 48 h, neurons at 11 days in vitro were treated with vehicle (PBS) or mouse recombinant CHI3L1 at 300 ng/ml during 24 and 48 h. Then, cells were fixed with 4% paraformaldehyde and inmunostained with mouse monoclonal primary antibody MAP2 (1:500) and goat anti-mouse alexa fluor 594 (1:500). Five microscope fields (20x) of each replicate were randomly chosen, imaged and counted. We assessed the effect of CHI3L1 on cell viability by quantifying the total number of neurons (MAP2+ cells) and neuronal functionality by neurite length retraction study with NeuriteTracer Image J plugin. A total of 5 independent experiments (vehicle: n=3) with 4 replicates per condition were performed.
Results: In agreement with the CSF CHI3L1 associated with the development of neurological disability in CIS patients, CHI3L1 concentrations of 100 ng/ml and 170 ng/ml in the preliminary time-dose-response study had no neurotoxic effect. In addition, no significant changes in neuronal death were observed at the 12 h time point. A CHI3L1 concentration of 300 ng/ml was finally selected as the lowest effective dose following 24-48 h of exposure. After 48 h of treatment, CHI3L1 reduced neuronal survival, insomuch as a concentration of 300 ng/ml was associated with a significant decrease in the total number of cortical neurons (p=0.009 vs. vehicle), and significantly induced neurite length retraction (p=0.034 vs. vehicle).
Conclusions: These results point to a neurotoxic effect of CHI3L1 in vitro and suggest a potential role of CHI3L1 as therapeutic target to prevent disability progression in MS patients.
Disclosure: C. Matute-Blanch, I. Carballo-Carbajal and L. Navarro report no disclosures.
X Montalban has received speaking honoraria and travel expenses for scientific meetings, has been a steering committee member of clinical trials or participated in advisory boards of clinical trials in the past years with Bayer Schering Pharma, Biogen Idec, EMD Merck Serono, Genentech, Genzyme, Novartis, Sanofi-Aventis, Teva Phramaceuticals, Almirall and Roche.
M Comabella has received compensation for consulting services and speaking honoraria from Bayer Schering Pharma, Merk Serono, Biogen-Idec, Teva Pharmaceuticals, Sanofi-Aventis, Genzyme, and Novartis.