A preferential loss of GABAergic, symmetric synapses in epileptic foci: a quantitative ultrastructural analysis of monkey neocortex.

Previous immunocytochemical results from five monkeys with cortical focal epilepsy produced by alumina gel showed a severe decrease at seizure foci of axon terminals that contained glutamic acid decarboxylase (GAD), the synthesizing enzyme for the inhibitory neurotransmitter, GABA. These data indicated a functional loss of GABAergic terminals but did not show: whether this loss was caused by GABAergic nerve terminal degeneration or by a lack of GAD immunoreactivity within these terminals and if this loss of GABAergic terminals was selective for only this terminal type. To resolve these issues, cortical tissue from three of the five monkeys used in the previous study was reexamined using electron microscopy, and a quantitative morphological analysis of cortical structures was made to compare profiles of terminals and glia in the nonepileptic cortex with those in the focus and parafocus. The following statistically significant changes were observed: the number of axosomatic symmetric synapses with layer V pyramidal cells was decreased 80% at the focus and 50% at the parafocus; in the neuropil adjacent to these pyramidal somata, the number of terminals forming symmetric synapses was reduced 50% at the epileptic focus but was unchanged at the parafocus, while the number of asymmetric synapses was reduced 25% at the focus and 15% at the parafocus; and a 50% increase of glial profiles occurred at epileptic foci both in the neuropil and at sites apposed to pyramidal cell somata. The quantitative results also showed that terminals which form symmetric synapses had twice the number of mitochondria per terminal as those that form asymmetric synapses. Axon terminals which form symmetric synapses with somata and dendrites in the neocortex have been shown previously to contain GAD. Therefore, the large reduction in the number of symmetric synapses at epileptic foci and the increased gliosis indicate that the previously observed loss of GABAergic terminals at sites of focal epilepsy is caused by terminal degeneration. Since such terminals are reduced more severely at epileptic foci than other terminals, their loss could be the basis for seizure activity due to a preferential decrease of inhibitory function at epileptic foci. Hypoxia has been shown to cause a selective degeneration of terminals with the same morphology as GABAergic terminals in the cortex, and the basis for this loss could be related to higher physiological and/or metabolic activities of GABAergic cortical cells which may inhibit other cells tonically. The fact that increased numbers of mitochondria occur in GABAergic terminals supports this idea

Water-pipe smoke condensate increases the internalization of Mycobacterium Bovis of type II alveolar epithelial cells (A549)

Background: Tuberculosis (TB) is a major global health problem, and there is an association between tobacco smoke and TB. Water pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally because users consider it as safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be a predisposing factor that enhances the incidence of pulmonary disorders. For example, uncontrolled macropinocytosis in alveolar epithelial cells following exposure to water-pipe smoke may predispose subjects to pulmonary infection. Here, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium Bovis BCG by macropinocytosis in the alveolar epithelial cell line A549. Methods: A549 cells were exposed to WPC (4 mg/ml) for 24, 48, 72 and 96 h. Cell viability was studied using the methyl thiazolyldipenyl-tetrazolium bromide (MTT) reduction assay and proliferation by bromodeoxyUridine (BrdU) incorporation. Cells were exposed to FITC-Dextran (1 mg/ml) (as a control) and FITC-BCG (MOI = 10) for 20 min at 37 ° Cbeforecellswere collected and the uptake of BCG-FITC determined by flow cytometry. Similar experiments were performed at 4 ° Casacontrol . The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (1 μ M) was used to assess the mechanism by which WPC enhanced BCG uptake. Results: WPC (4 mg/ml) increased the uptake of BCG-FITC after 72 (1.3 ± 0.1 fold, p < 0.05) and 96 (1.4 ± 0.05 fold, p < 0.05) hours. No effect on BCG-FITC uptake was observed at 24 or 48 h. WPC also significantly increased the uptake of FITC-Dextran (2.9 ± 0.3 fold, p < 0.05) after 24 h. WPC significantly decreased cell viability after 24 (84 ± 2%, p < 0.05), 48 (78±, 3%, p < 0.05), 72 (64 ± 2%, p < 0.05) and 96 h (45 ± 2%, p < 0.05). Y-27632 completely attenuated the increased uptake of BCG by WPC. Cell proliferation showed a decreasing trend in a time-dependent manner with WPC exposure. Conclusion: WPC exposure increased epithelial cell endocytosis activity and death as well as enhancing their capacity for macropinocytosis. Our in vitro data indicates possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacterium

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements

Abstract Background When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. Results The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. Conclusions This study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs