Preparation of Cultured Smooth Muscle Cells from Human Myometrium for X-Ray Microanalysis

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer\u27s solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer\u27s solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer\u27s solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer\u27s solution

Use of In Vitro Systems for X-Ray Microanalysis

The use of X-ray microanalysis in human pathology may require the use of cryoprepared tissue. Often it is impossible to carry out freezing of the tissue in an optimal way, and in addition, it is difficult to carry out experiments in living patients. The use of in vitro systems and cell cultures allows separation of the process of tissue removal and the freezing procedure, and also makes testing of pharmacological or toxic substances possible. In experiments with animal tissue it was shown that incubation in a physiological buffer induced significant changes in the concentrations of Na, K, and Cl. In general, the concentrations of Na and Cl increased, those of K decreased. Prolonged incubation of brain tissue (cortex and hippocampus) and of liver resulted in further changes of the cellular ion contents in the same direction. Incubation of pancreas and submandibular gland resulted in a limited reversal of the changes induced by dissection. The submandibular gland in vitro showed the same response to cholinergic stimulation as the gland in situ. The use of cell cultures for X-ray microanalysis is briefly reviewed and illustrated by an example of analysis of an immortalized sweat gland cell line. It was shown that these cells respond to stimulation by cAMP with loss of Cl and that this response was unaffected by the type of substrate the cells were grown on

In Vitro Systems and Cultured Cells as Specimens for X-Ray Microanalysis

In vitro systems and cultured cells are recognized as useful systems in many areas of biomedical research, including X-ray microanalysis. To be reliable, in an vitro system should have an elemental composition close to that of the tissue in situ, react in the same way to stimuli, and retain the in situ regulation of ion transport. In the present paper, four of the most commonly used in vitro systems will be reviewed: incubated tissue slices (liver and pancreas), isolated glands (submandibular gland acini, sweat glands), primary cell cultures (sweat glands, endometrium), and cell lines (the colon cancer cell line T84, immortalized sweat gland cells). Incubation of tissue slices of liver in Krebs-Ringers buffer caused a significant increase in Na and Cl and a decrease in K. Initially, these changes were also observed in the pancreas, but here the values gradually returned to normal. Isolated submandibular gland acini, and isolated sweat gland ducts and coils react in a similar way to stimulation as their in situ counterparts. In primary cultures of coil cells, however, part of the cell population acquires different ion transport characteristics. Technically simplest is the use of cell lines originating from cancer cells (e.g., the T84 cell line) and immortalized cell lines. X-ray microanalysis not only confirms data on ion transport obtained with other techniques, but adds the possibility to investigate the presence of subpopulations within a culture

PKCε Activator Protects Hippocampal Microvascular Disruption and Memory Defect in 3×Tg-Alzheimer\u27s Disease Mice with Cerebral Microinfarcts

BACKGROUND: Current evidence suggests that microvessel disease is involved in Alzheimer\u27s disease (AD). Cerebrovascular disease correlates with cardiovascular disease and is complicated in ≈40% of AD patients. The protein kinase C (PKC) ε activator DCPLA can stimulate human antigen (Hu) R that prevents degradation and promotes the translation of mitochondrial Mn-superoxide dismutase (MnSOD) and vascular endothelial growth factor-A (VEGF) mRNAs. METHODS: To induce brain microinfarcts, we injected triple transgenic (3×Tg) and wild-type (WT) control mice with microbeads (20 μm caliber) into common carotid arteries, with or without the DCPLA-ME (methyl-ester) for 2 weeks. After water maze training, mice at 16 months old were examined for confocal immunohistochemistry at a single cell or microvessel level in the hippocampal CA1 area, important for spatial memory storage, and in the dorsal hippocampus by western blots. RESULTS: In 3×Tg mice without cerebral microinfarcts, an accelerating age-related increase in (mild) oxidative stress and hypoxia inducible factor (HIF)-1α, but a reduction in VEGF, mitochondrial transcription factor A (TFAM), and MnSOD were associated with capillary loss. The change was less pronounced in arterioles. However, in 3×Tg mice with cerebral microinfarcts, increasing arteriolar diameter and their wall cells were related with the strong oxidative DNA damage 8-hydroxy-2\u27-deoxyguanosine (8-OHdG), apoptosis (cleaved caspase 3), and sustained hypoxia (increased HIF-1α and VEGF/PKCε/extracellular signal regulated kinase or ERK pathway). Microocclusion enhanced the loss of the synaptic marker spinophilin, astrocytic number, and astrocyte-vascular coupling areas and demyelination of axons. DCPLA-ME prevented spatial memory defect; strong oxidative stress-related apoptosis; sustained hypoxia (by reducing HIF-1α and VEGF); and exaggerated cell repair in arteriolar walls, pericapillary space dilation, neuro-glial-vascular disruption, and demyelination. CONCLUSION: In conclusion, in 3×Tg mice with cerebral microinfarcts, sustained hypoxia (increased HIF-1α and VEGF signals) is dominant with arteriolar wall thickening, and DCPLA has a protective effect on sustained hypoxia

PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer\u27s Disease Hippocampus

Vascular endothelial dysfunction and capillary loss are currently considered to be a primary phenotype of normal human aging and Alzheimer\u27s disease (AD). Activation of protein kinase C (PKCε) improves several molecular, cellular, physiological, and behavioral endpoints, yet it is not known whether a loss of PKCε activity occurs in the microvascular endothelium in aged and AD hippocampi, whether this loss contributes to microvascular change, or whether activation of PKCε protects against microvascular damage, an early change that induces age-associated memory defect and AD. We investigated the effect of the PKCε activation on microvascular loss in the hippocampus, important for memory storage. In cultured human brain microvascular endothelial cells, tert-butyl hydroperoxide induced oxidative stress and a decrease in manganese superoxide dismutase (MnSOD) mRNA and protein expression that were blocked by the antioxidant drugs. The PKCε activators bryostatin and DCPLA methyl ester increased PKCε, associated with an increase in MnSOD mRNA and its protein as well as vascular endothelial growth factor (VEGF), which was inhibited by the mRNA-stabilizing HuR inhibitors. In rats (\u3e24 months old) and AD transgenic mice Tg2576 (5 months old), bryostatin or DCP-LA prevented a decrease in vascular PKCε, MnSOD, and VEGF and prevented microvascular loss and age-related memory impairment. An autopsy-confirmed AD hippocampus showed a decrease in PKCε and MnSOD mRNAs and their proteins and VEGF as well as in microvascular density compared to non-AD controls. In conclusion, the PKCε activation can rescue a decrease in PKCε, MnSOD, and VEGF via posttranscription regulation and alleviate oxidative stress, and in doing so, prevent microvascular loss during aging and AD

Adaptive regulation of the brain's antioxidant defences by neurons and astrocytes

AbstractThe human brain generally remains structurally and functionally sound for many decades, despite the post-mitotic and non-regenerative nature of neurons. This is testament to the brain’s profound capacity for homeostasis: both neurons and glia have in-built mechanisms that enable them to mount adaptive or protective responses to potentially challenging situations, ensuring that cellular viability and functionality is maintained. The high and variable metabolic and mitochondrial activity of neurons places several demands on the brain, including the task of neutralizing the associated reactive oxygen species (ROS) produced, to limit the accumulation of oxidative damage. Astrocytes play a key role in providing antioxidant support to nearby neurons, and redox regulation of the astrocytic Nrf2 pathway represents a powerful homeostatic regulator of the large cohort of Nrf2-regulated antioxidant genes that they express. In contrast, the Nrf2 pathway is weak in neurons, robbing them of this particular homeostatic device. However, many neuronal antioxidant genes are controlled by synaptic activity, enabling activity-dependent increases in ROS production to be offset by enhanced antioxidant capacity of both glutathione and thioredoxin-peroxiredoxin systems. These distinct homeostatic mechanisms in neurons and astrocytes together combine to promote neuronal resistance to oxidative insults. Future investigations into signaling between distinct cell types within the neuro-glial unit are likely to uncover further mechanisms underlying redox homeostasis in the brain

Regulation of neuronal development and function by ROS.

Reactive oxygen species (ROS) have long been studied as destructive agents in the context of nervous system ageing, disease and degeneration. Their roles as signalling molecules under normal physiological conditions is less well understood. Recent studies have provided ample evidence of ROS-regulating neuronal development and function, from the establishment of neuronal polarity to growth cone pathfinding; from the regulation of connectivity and synaptic transmission to the tuning of neuronal networks. Appreciation of the varied processes that are subject to regulation by ROS might help us understand how changes in ROS metabolism and buffering could progressively impact on neuronal networks with age and disease