The Toxic Effects And Mechanisms Of Dimethyl Sulfoxide On Astrocytes

Background:Dimethyl sulfoxide (DMSO) is a polar organic solvent with a variety of biological functions and has been widely applied in the medical field. In neuroscience research, DMSO is used to dissolve the neuroprotective or neurotoxic drugs, but it has been shown the toxic effects to neurons. Astrocytes (Ast) play an important role in maintaining the homeostasis of brain microenvironment, the toxic effects and mechanisms of DMSO on Ast remain to be determined.Objective:The aim of the present study was to observe the effects of different concentrations of DMSO on the Ast morphology, cell vitality and mitochondria! functions, and further explore its toxic mechanisms, which may provide guidelines for selecting a safe dose of DMSO in the medical research and clinical utilization.Methods:Cortical Ast were isolated and cultured from 1-3 day old mouse cortex tissue, then incubated with different DMSO concentrations (1%,5% and 10%) for 24 h. Then experiments were performed as following:1) Cell morphology and number changes were observed under an optical microscope; cells vitality and apoptosis were determined by MTT and TUNEL assays; 2) Changes in immunofluorescence density of glial fibrillary acidic protein (GFAP) expression, mitochondrial membrane potential (TMRE) and reactive oxygen species (ROS) were captured and quantified. The levels of mitochondrial membrane potential (JC-1) and ROS were also detected by the flow cytometry; 3) The mitochondrial ultrastructures were observed by the transmission electron microscopy; 4) The release of cytochrome c was detected by the flow cytometry; 5) The active-caspase 3 and Bcl-2 expression levels were detected by Western-blot; 6) The immunocytochemistry was performed to observe glutamate transporter-1 (GLT-1), glutamate/aspartate transporter protein (GLAST) and glutamine synthetase (GS) expression levels. In vivo experiments, mice received microinjection of 50%DMSO 1 ?l into the hippocampus, and perfusion 24 h later, then the hippocampal sections were peroformed immunohistochemical stains to observe 7) the neuronal apoptosis, reactivity of GFAP positive Ast and expression levels of the GLAST and GLT-1 and GS.Results:The mouse Ast showed no differences in growth and the following indicators from the control group when cultured at the medium containing 1% DMSO. However, when treated by 5% and 10% DMSO, Ast showed 1) abnormal cell growth with significant reductions in the cell vitality and significant increases in the apoptotic rate; 2) decreases in mitochondrial membrane potential but increases in intracellular ROS levels; 3) mitochondrial matrix swelling, crest shorten and reduced in the number, or vacuolar changes even; 4) down-regulation in glutamate metabolism markers including GLT-1, GLAST and GS; 5) up-regulation of the apoptotic protein active-caspase 3 expression, while down-regulation of the anti-apoptotic protein Bcl-2 expression; 6) increases in the cytochrome c release at 5% DMSO, but no significant difference at 10%, when compared with the baseline control.Conclusions:The results confirm that the low concentration of DMSO (< 1%) is safe to Ast. However, higher concentrations (> 5%) exhibit its toxic effects on Ast with dose-dependent manner. Its underlying mechanisms may as following:DMSO damages Ast mitochondrial membrane integrity and membrane potential level, which not only leads to the release of cytochrome c and subsequent activation of caspase 3 apoptotic pathway, but also leads to an increase in ROS production and down-regulation in ROS-sensitive glutamate metabolism proteins, affecting uptake of glutamate within synaptic space, and thus exacerbating its toxic effects to neurons.