Mechanisms of bFGF and NT-4 potentiation of necrotic neuronal death

The effects of neurotrophic factors on necrotic neuronal death are controversial. In this study we found that both neurotrophin-4 (NT-4) and basic fibroblast growth factor (bFGF) potentiated necrotic neuronal death caused by exposure to oxygen-glucose deprivation or iron-citrate (Fe) in cortical cultures. However, there were significant differences in the actions of the two neurotrophic factors. Neurotrophin-4 protected against apoptotic neuronal death, while bFGF had no effect on apoptotic death in these cultures. Furthermore, potentiation of oxygen-glucose deprivation induced necrotic death by NT-4 required pretreatment (24 h), while pretreatment with bFGF had no effect. However, acute treatment with bFGF during oxygen-glucose deprivation did potentiate neuronal death. Both neurotrophic factors potentiated free radical mediated necrotic neuronal death induced by exposure to Fe. However, the RNA synthesis inhibitor, actinomycin-D, blocked the injury potentiation by NT-4, but not that caused by bFGF. Also, NT-4, but not bFGF, potentiated Fe induced necrotic death in pure neuronal cultures. Expression of mRNA for FGF receptors FGFR1 and FGFR2 was observed at high levels in astrocytes. The results indicate that the injury enhancing effects of bFGF are acute, while those of NT-4 require prolonged exposure and new protein synthesis. Furthermore, the effects of bFGF appear to be mediated through actions on astrocytes, while NT-4 appears to act directly on neurons. The fact that neurotrophic factors from two distinct families can potentiate neuronal death by two different mechanisms suggests that such injury potentiation may be a common concern regarding the use of neurotrophic factors.     

Beta-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms

The idea that the environmental toxin beta-N-methylamino-l-alanine (BMAA) is involved in neurodegenerative diseases on Guam has risen and fallen over the years. The theory has gained greater interest with recent reports that BMAA is biomagnified, is widely distributed around the planet, and is present in the brains of Alzheimer's patients in Canada. We provide two important new findings. First, we show that BMAA at concentrations as low as 10 muM can potentiate neuronal injury induced by other insults. This is the first evidence that BMAA at concentrations below the mM range can enhance death of cortical neurons and illustrates potential synergistic effects of environmental toxins with underlying neurological conditions. Second, we show that the mechanism of BMAA toxicity is threefold: it is an agonist for NMDA and mGluR5 receptors, and induces oxidative stress. The results provide further support for the hypothesis that BMAA plays a role in neurodegenerative diseases.     

Collagenous matrix supported by a 3D-printed scaffold for osteogenic differentiation of dental pulp cells

Objective

A systematic characterization of hybrid scaffolds, fabricated based on combinatorial additive manufacturing technique and freeze-drying method, is presented as a new platform for osteoblastic differentiation of dental pulp cells (DPCs).

In vitro cytotoxicity of orthodontic archwires in cortical cell cultures

There have been a number of studies regarding the toxicity of orthodontic archwires, but little is known concerning the mechanism of their toxicity. This investigation used murine cortical cell cultures to examine the in vitro neurotoxicity of commonly used orthodontic metallic archwire alloys. The materials examined included 0.016 inch nickel-titanium (NiTi), copper-nickel-titanium, titanium-molybdenum, Elgiloy, and stainless steel archwire alloys. Standard sized samples of each material were placed on tissue culture inserts suspended above the cell cultures. Neuronal death was determined using the lactate dehydrogenase release assay 24 hours after exposure to the archwires. The results indicated that NiTi, copper-nickel-titanium and titanium-molybdenum alloys were not neurotoxic, while stainless steel and Elgiloy were significantly toxic. Washing the archwires for 7 days in a saline solution did not alter the toxicity. However, the free radical scavenger, trolox, blocked the toxicity of both stainless steel and Elgiloy, indicating that the death was free radical mediated. The caspase inhibitor, Z-VAl-Ala-Asp-fluoromethylketone (zVAD-FMK), blocked the toxicity of stainless steel, but not Elgiloy, suggesting that stainless steel induced apoptosis. Further evidence that stainless steel induced apoptosis was provided by propidium staining which showed nuclear chromatin condensation and fragmentation into discrete spherical or irregular shapes, characteristic of apoptosis. The specific metal responsible for the toxicity was not determined; the metals common to each of the toxic archwires were nickel, iron, and chromium.     

Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release

Central neurons exposed to several types of sublethal stress, including ischemia, acquire resistance to injury induced by subsequent ischemic insults, a phenomenon called ischemic preconditioning. We modeled this phenomenon in vitro, utilizing exposure to 45 mM KCl to reduce the vulnerability of cultured murine cortical neurons to subsequent oxygen-glucose deprivation. Twenty-four hours after preconditioning, cultures exhibited enhanced depolarization-induced, tetanus toxin-sensitive GABA release and a modest decrease in glutamate release. Total cellular GABA levels were unaltered. Inhibition of GABA degradation with the GABA transaminase inhibitor (+/-)-gamma-vinyl GABA, or addition of low levels of GABA, muscimol, or chlormethiazole to the bathing medium, mimicked the neuroprotective effect of preconditioning against oxygen-glucose deprivation-induced death. However, neuronal death was enhanced by higher levels of these manipulations, as well as by prior selective destruction of GABAergic neurons by kainate. Finally, selective blockade of GABA(A) receptors during oxygen-glucose deprivation or removal of GABAergic neurons eliminated the neuroprotective effects of prior preconditioning.Taken together, these data predict that presynaptic alterations, specifically enhanced GABA release together with reduced glutamate release, may be important mediators of ischemic preconditioning, but suggest caution in regard to interventions aimed at increasing GABA(A) receptor activation.     

In vitro neurotoxic evaluation of root-end-filling materials

Root-end-filling materials have been tested for toxicity on several cell types, but their toxicity has not been tested on neurons. In this study we evaluated the neurotoxicity in murine cerebral cortical cell cultures of four commonly used root-end-filling materials: mineral trioxide aggregate, amalgam, Super EBA, and Diaket. Standardized amounts of each material were placed on culture-well inserts, allowing the material to be exposed to the culture bathing media without causing physical disruption of the cells. Cell death was quantified by assaying release of the cytosolic enzyme lactate dehydrogenase. Exposure of cortical cultures to freshly mixed or 7-day-old MTA did not cause significant neuronal death, whereas exposure to freshly mixed or 7-day-old amalgam, Super EBA, and Diaket resulted in significant neuronal death (p < .05). Thus, each material, except for mineral trioxide aggregate, can induce neurotoxicity, even when allowed to set thoroughly.     

Glutathione-mediated neuroprotection against methylmercury neurotoxicity in cortical culture is dependent on MRP1

Methylmercury (MeHg) exposure at high concentrations poses significant neurotoxic threat to humans worldwide. The present study investigated the mechanisms of glutathione-mediated attenuation of MeHg neurotoxicity in primary cortical culture. MeHg (5 μM) caused depletion of mono- and disulfide glutathione in neuronal, glial and mixed cultures. Supplementation with exogenous glutathione, specifically glutathione monoethyl ester (GSHME) protected against the MeHg induced neuronal death. MeHg caused increased reactive oxygen species (ROS) formation measured by dichlorodihydrofluorescein (DCF) fluorescence with an early increase at 30 min and a late increase at 6h. This oxidative stress was prevented by the presence of either GSHME or the free radical scavenger, trolox. While trolox was capable of quenching the ROS, it showed no neuroprotection. Exposure to MeHg at subtoxic concentrations (3 μM) caused an increase in system x(c)(-) mediated (14)C-cystine uptake that was blocked by the protein synthesis inhibitor, cycloheximide (CHX). Interestingly, blockade of the early ROS burst prevented the functional upregulation of system x(c)(-). Inhibition of multidrug resistance protein-1 (MRP1) potentiated MeHg neurotoxicity and increased cellular MeHg. Taken together, these data suggest glutathione offers neuroprotection against MeHg toxicity in a manner dependent on MRP1-mediated efflux.     

Insulin-like growth factor 1 and transforming growth factor-β stimulate cystine/glutamate exchange activity in dental pulp cells

Introduction

The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-β (TGF-β) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through the enhancement of an endogenous antioxidant mechanism.

Methods

We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-).

Results

We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc-mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line.

Conclusions

The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc-mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells.     

Melatonin Use in Hospitalized Children for Non-Anesthetic Indications: A Systematic Review

Melatonin, a potent free radical scavenger, plays an important role in homeostasis of cell and organ physiology. The increased demand and synthesis from the pineal gland during times of oxidative stress suggests a potential benefit of melatonin supplementation during hospitalization for acute illness. Yet, the paucity of clinical studies for non-anesthetic–associated indications in pediatric populations hampers the safe, effective, and consistent use of melatonin. The objective of this study was to systematically review published studies of melatonin use for non-sedative and non-analgesic indications in hospitalized pediatric patients. We conducted a search of PubMed, EMBASE, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Web of Science, and Scopus databases for articles on the use of melatonin for pediatric patients in a hospital setting. Thirteen eligible studies, all in neonates, were identified. Data elements extracted included study design, number of study subjects, indication for melatonin therapy, and melatonin regimen (formulation, dosage, and duration). Because study methodologies were very heterogeneous, a quantitative synthesis of the published findings was not possible. The identified studies were therefore categorized by the indication of melatonin (adjuvant-antioxidant or anti-inflammatory therapy) in the following specific disease states: (i) acute infections, (ii) respiratory distress syndrome, (iii) neurologic injury, and (iv) jaundice. The current data suggest that melatonin is safe for use in hospitalized neonates. Melatonin may be beneficial for reducing inflammatory markers in neonatal patients with disease states and clinical sequelae that are associated with increased inflammation and oxidative stress. Melatonin, in conjunction with phototherapy, is not superior to use of vitamin D with phototherapy for treatment of neonatal jaundice. However, studies in other pediatric populations are needed given widespread use across clinical inpatient settings.