Glutamate excitotoxicity is implicated in the pathogenesis of numerous diseases, such as stroke, traumatic brain injury, and Alzheimer’s disease, for which insulin resistance is a concomitant condition, and intranasal insulin treatment is believed to be a promising therapy. Excitotoxicity is initiated primarily by the sustained stimulation of ionotropic glutamate receptors and leads to a rise in intracellular Ca2+ ([Ca2+]i), followed by a cascade of intracellular events, such as delayed calcium deregulation (DCD), mitochondrial depolarization, adenosine triphosphate (ATP) depletion that collectively end in cell death. Therefore, cross-talk between insulin and glutamate signaling in excitotoxicity is of particular interest for research. In the present study, we investigated the effects of short-term insulin exposure on the dynamics of [Ca2+]i and mitochondrial potential in cultured rat cortical neurons during glutamate excitotoxicity. We found that insulin ameliorated the glutamate-evoked rise of [Ca2+]i and prevented the onset of DCD, the postulated point-of-no-return in excitotoxicity. Additionally, insulin significantly improved the glutamate-induced drop in mitochondrial potential, ATP depletion, and depletion of brain-derived neurotrophic factor (BDNF), which is a critical neuroprotector in excitotoxicity. Also, insulin improved oxygen consumption rates, maximal respiration, and spare respiratory capacity in neurons exposed to glutamate, as well as the viability of cells in the MTT assay. In conclusion, the short-term insulin exposure in our experiments was evidently a protective treatment against excitotoxicity, in a sharp contrast to chronic insulin exposure causal to neuronal insulin resistance, the adverse factor in excitotoxicity.

AimAn impaired biological response to insulin in the brain, known as central insulin resistance, was identified during stroke and traumatic brain injury, for which glutamate excitotoxicity is a common pathogenic factor. The exact molecular link between excitotoxicity and central insulin resistance remains unclear. To explore this issue, the present study aimed to investigate the effects of glutamate-evoked increases in intracellular free Ca2+ concentrations [Ca2+]i and mitochondrial depolarisations, two key factors associated with excitotoxicity, on the insulin-induced activation of the insulin receptor (IR) and components of the Akt/ mammalian target of rapamycin (mTOR) pathway in primary cultures of rat cortical neurons.MethodsChanges in [Ca2+]i and mitochondrial inner membrane potentials (ΔΨm) were monitored in rat cultured cortical neurons, using the fluorescent indicators Fura-FF and Rhodamine 123, respectively. The levels of active, phosphorylated signalling molecules associated with the IR/Akt/mTOR pathway were measured with the multiplex fluorescent immunoassay.ResultsWhen significant mitochondrial depolarisations occurred due to glutamate-evoked massive influxes of Ca2+ into the cells, insulin induced 48% less activation of the IR (assessed by IR tyrosine phosphorylation, pY1150/1151), 72% less activation of Akt (assessed by Akt serine phosphorylation, pS473), 44% less activation of mTOR (assessed by mTOR pS2448), and 38% less inhibition of glycogen synthase kinase β (GSK3β) (assessed by GSK3β pS9) compared with respective controls. These results suggested that excitotoxic glutamate inhibits signalling via the IR/Akt/mTOR pathway at multiple levels, including the IR, resulting in the development of acute neuronal insulin resistance within minutes, as an early pathological event associated with excitotoxicity.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

No abstract

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 μM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

No abstract

No abstract

No abstract