Neurotoxicity Study with Titanium Dioxide Nanoparticles on Rat

Numerous studies have shown titanium dioxide nanoparticles (TiO2 NPs) could present a risk or potential risk to humans and other living organisms in certain conditions via inhalation and skin contact. Dermal exposure has limited adverse effects and the possible risks for exogenous inhaled nanoparticles migrating to the brain through the olfactory nerve is still under research. To study the in vivo and in vitro neurotoxicity of brain tissue in rats induced by TiO2 NPs. For in vitro study, rat astrocytes were exposed to TiO2 NPs with three different diameters (10, 50 and 200 nm) at five concentrations levels. Cellular morphology and sulfur rhodamine B (SRB) were carried out to evaluate the viability of particle-treated cells after 72 hours exposure. For in vivo study, suspensions of test material above mentioned were injected into tracheas of Wistar rats at dose of 0.1, 1.0 and 10.0 mg·kg-1 in three groups, respectively. After 72 hours of exposure, the concentration of TiO2 NPs in brain tissue and the levels of IL-1β, TNF-α and IL-10 in brain homogenate were measured, while the cell morphology induced by TiO2 NPs was observed by light microscopy and transmission electron microscopy. TiO2 NPs can significantly affect the growth and morphology of rat astrocytes and inhibit the proliferation of astrocytes, which was positively related to dose-effect and size-dependent response. Pathological observations indicated that TiO2 NPs could penetrate the blood-brain barrier, leading to blood-brain barrier damage in rats, brain tissue necrosis, mitochondrial swelling and apoptosis while the non-nanoscale TiO2 particles showed no significant toxicity in the central nervous system cells.

Feasibility of Photodynamic Therapy for Glioblastoma with the Mitochondria-Targeted Photosensitizer Tetramethylrhodamine Methyl Ester (TMRM)

One of the most challenging problems in the treatment of glioblastoma (GBM) is the highly infiltrative nature of the disease. Infiltrating cells that are non-resectable are left behind after debulking surgeries and become a source of regrowth and recurrence. To prevent tumor recurrence and increase patient survival, it is necessary to cleanse the adjacent tissue from GBM infiltrates. This requires an innovative local approach. One such approach is that of photodynamic therapy (PDT) which uses specific light-sensitizing agents called photosensitizers. Here, we show that tetramethylrhodamine methyl ester (TMRM), which has been used to asses mitochondrial potential, can be used as a photosensitizer to target GBM cells. Primary patient-derived GBM cell lines were used, including those specifically isolated from the infiltrative edge. PDT with TMRM using low-intensity green light induced mitochondrial damage, an irreversible drop in mitochondrial membrane potential and led to GBM cell death. Moreover, delayed photoactivation after TMRM loading selectively killed GBM cells but not cultured rat astrocytes. The efficacy of TMRM-PDT in certain GBM cell lines may be potentiated by adenylate cyclase activator NKH477. Together, these findings identify TMRM as a prototypical mitochondrially targeted photosensitizer with beneficial features which may be suitable for preclinical and clinical translation.

Overexpression of miR-221-3p effecting cell proliferation, apoptosis and inflammation by targeting Toll-like receptors 4 in propofol-induced rat astrocytes

IntroductionGrowing evidence indicated that propofol has neurotoxic effects on the brains of developing rodents, leading to neuronal cell death, neurodegeneration, and brain injury.Material and methodsEctopic miR-221-3p was transfected into rat astrocytes, and Cell Counting Kit-8 assay and flow cytometry were performed to evaluate cell growth and apoptosis. The mRNA levels of Toll-like receptors 4 (TLR4), nuclear factor kappa B, interleukin-6, interleukin-1β, myeloid differentiation primary response 88 (MyD88), caspase-3, caspase-12, STAT3, and GRP78 were detected using quantitative real-time polymerase chain reaction. The proteins of TLR4 and MyD88 were determined using Western blotting. The association between miR-221-3p and TLR4 was measured using Dual-Luciferase Reporter Assay (Promega Corporation, Wisconsin, USA). Then, siTLR4 was transfected with 293T cells to study the role of TLR4 in astrocytes with propofol treatment.ResultsThe miR-221-3p expression in rat astrocytes was markedly suppressed by propofol treatment. The miR-221-3p mimics transfection in propofol-treated astrocytes effectively reduced the suppressive effect of propofol on astrocyte growth, repressed the propofol-induced apoptosis in rat astrocytes, and decreased the cell number during the G2–M phase. The expression of MyD88 and TLR4 was induced by propofol, whereas the transfection of miR-221-3p mimics dramatically reduced these genes expression at the mRNA and protein expression. After that, TLR4 was found to be target of miR-221-3p using Dual-Luciferase Reporter Assay. Furthermore, knockdown of TLR4 could suppress the apoptosis rate in propofol-treated astrocytes.ConclusionsThis study revealed that miR-221-3p might prevent astrocytes from propofol-induced damage by targeting TLR4.

High Glucose Shifts the Oxylipin Profiles in the Astrocytes towards Pro-Inflammatory States

Hyperglycemia is associated with several complications in the brain, which are also characterized by inflammatory conditions. Astrocytes are responsible for glucose metabolism in the brain and are also important participants of inflammatory responses. Oxylipins are lipid mediators, derived from the metabolism of polyunsaturated fatty acids (PUFAs) and are generally considered to be a link between metabolic and inflammatory processes. High glucose exposure causes astrocyte dysregulation, but its effects on the metabolism of oxylipins are relatively unknown and therefore, constituted the focus of our work. We used normal glucose (NG, 5.5 mM) vs. high glucose (HG, 25 mM) feeding media in primary rat astrocytes-enriched cultures and measured the extracellular release of oxylipins (UPLC-MS/MS) in response to lipopolysaccharide (LPS). The sensitivity of HG and NG growing astrocytes in oxylipin synthesis for various serum concentrations was also tested. Our data reveal shifts towards pro-inflammatory states in HG non-stimulated cells: an increase in the amounts of free PUFAs, including arachidonic (AA), docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, and cyclooxygenase (COX) mediated metabolites. Astrocytes cultivated in HG showed a tolerance to the LPS, and an imbalance between inflammatory cytokine (IL-6) and oxylipins release. These results suggest a regulation of COX-mediated oxylipin synthesis in astrocytes as a potential new target in treating brain impairment associated with hyperglycemia.

A1 Reactive Astrocytes Activated by 2-chloroethanol Modulate M1 Microglia Polarization through Upregulating IL-1β and TNF-α

Background1,2-Dichloroethane (1,2-DCE) is a synthetic organic chemical that causes brain edema under subacute poisoning. Our previous studies indicated that the neuroinflammation could be induced due to activation of both astrocytes and microglia during the course of brain edema in 1,2-DCE intoxicated mice. However, the crosstalk between the two glial cells in 1,2-DCE-induced neuroinflammation is unclear. In the current studies, we hypothesized that astrocytes are the first responder to the effects of 1,2-DCE in the brain, as they adhere to the cerebral capillaries, and they are an essential component of the blood-brain barrier (BBB).MethodsWe used primary cultured rat astrocytes and microglia, as well as a highly aggressively proliferating immortalized (HAPI) microglia cell line to study the effects of astrocytes on microglia polarization following exposure to 2-CE. ResultsFindings from the present studies demonstrated that treatment of primary rat astrocytes with 2-chloroethanol (2-CE), the intermediate metabolite of 1,2-DCE in vivo, can stimulate the activation of A1 reactive astrocytes (A1s) through p38 mitogen-activated protein kinase (p38 MAPK)/ nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling pathways by the reactive oxygen species (ROS) produced during 2-CE metabolism. A1s activated by 2-CE can upregulate the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS), and stimulate the M1 polarization of microglia through IL-1β and TNF-α released by 2-CE activated A1s. Microglia are less sensitive to 2-CE than astrocytes, since treatment of primary rat microglia with 30 mM 2-CE alone failed to activate them, though this dose of 2-CE can activate A1s and in turn stimulate M1 polarization of microglia through the factors released by A1s. ConclusionThe neuroinflammation induced by 1,2-DCE in the brain of mice is most probably triggered by the activation of astrocytes. The understanding of the multidimensional roles of reactive astrocytes may further the development of new treatment strategies in reducing neuroinflammation and brain edema following 1,2-DCE-induced toxic encephalopathy.

A1 astrocytes activated by 2-Chloroethanol via the ROS-induced p38 MAPK/NF-κB and AP-1 pathways modulate microglia polarization

Background1,2-Dichloroethane (1,2-DCE) is a synthetic organic chemical that causes brain edema under subacute poisoning. Our previous studies indicated that the neuroinflammation could be induced due to activation of both astrocytes and microglia during the course of brain edema in 1,2-DCE intoxicated mice. However, the crosstalk between the two glial cells in 1,2-DCE-induced neuroinflammation is unclear. In the current studies, we hypothesized that astrocytes are the first responder to the effects of 1,2-DCE in the brain, as they adhere to the cerebral capillaries, and they are an essential component of the blood-brain barrier (BBB).MethodsWe used primary cultured rat astrocytes and microglia, as well as a highly aggressively proliferating immortalized (HAPI) microglia cell line to study the effects of astrocytes on microglia polarization following exposure to 2-CE.ResultsFindings from the present studies demonstrated that treatment of primary rat astrocytes with 2-chloroethanol (2-CE), the intermediate metabolite of 1,2-DCE in vivo, can stimulate the activation of A1 reactive astrocytes (A1s) through p38 mitogen-activated protein kinase (p38 MAPK)/ nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling pathways by the reactive oxygen species (ROS) produced during 2-CE metabolism. A1s activated by 2-CE can upregulate the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS), and stimulate the M1 polarization of microglia through IL-1β and TNF-α released by 2-CE activated A1s. Microglia are less sensitive to 2-CE than astrocytes, since treatment of primary rat microglia with 30 mM 2-CE alone failed to activate them, though this dose of 2-CE can activate A1s and in turn stimulate M1 polarization of microglia through the factors released by A1s.ConclusionThe neuroinflammation induced by 1,2-DCE in the brain of mice is most probably triggered by the activation of astrocytes. The understanding of the multidimensional roles of reactive astrocytes may further the development of new treatment strategies in reducing neuroinflammation and brain edema following 1,2-DCE-induced toxic encephalopathy.