Androgen influence on lacrimal gland apoptosis, necrosis, and lymphocytic infiltration.
(9/6081)
PURPOSE: Previous studies have shown that ovariectomy and hypophysectomy cause regression of the lacrimal gland and have implicated androgens as trophic hormones that support the gland. The purposes of this study were to test the hypothesis that glandular regression after ovariectomy is due to apoptosis, to identify the cell type or types that undergo apoptosis, to survey the time course of the apoptosis, and to determine whether ovariectomy-induced apoptosis could be prevented by dihydrotestosterone (DHT) treatment. METHODS: Groups of sexually mature female New Zealand White rabbits were ovariectomized and killed at various time periods up to 9 days. Additional groups of ovariectomized rabbits were treated with 4 mg/kg DHT per day. At each time period, sham-operated rabbits were used as controls. Lacrimal glands were removed and processed for analysis of apoptosis as assessed by DNA fragmentation and for morphologic examination. DNA fragmentation was determined using the TdT-dUTP terminal nick-end labeling assay and by agarose gel electrophoresis. Labeled nuclei were quantified by automated densitometry. Sections were also stained for RTLA (rabbit thymic lymphocyte antigen), rabbit CD18, and La antigen. Morphology was evaluated by both light and electron microscopy. RESULTS: The time course of apoptosis exhibited two phases, a rapid and transient phase and a second prolonged phase. A transient phase peaked at approximately 4 to 6 hours after ovariectomy. The values for degraded DNA as a percentage of total nuclear area were 4.29%+/-0.79% and 4.26%+/-0.54%, respectively. The values for sham-operated controls examined at the same time periods were 1.77%+/-0.08% and 0.82%+/-0.21%, respectively. The percentage of degraded DNA at 24 hours after ovariectomy was not different from controls examined at the same interval after sham operation. The percentage of degraded DNA 6 days after ovariectomy was significantly increased (8.5%+/-2.4%), compared with sham-operated animals at the same time period (0.68%+/-0.03%). DNA laddering was more pronounced after ovariectomy. Dihydrotestosterone treatment in ovariectomized rabbits suppressed the increase in DNA degradation. Morphologic examination of lacrimal gland sections indicated that ovariectomy caused apoptosis of interstitial cells rather than acinar or ductal epithelial cells. Tissue taken 4 hours and 6 days after ovariectomy showed nuclear chromatin condensation principally in plasma cells. Increased numbers of macrophages were also evident. Significant levels of cell degeneration and cell debris, characteristic of necrosis, were observed in acinar regions 6 days after ovariectomy. Dihydrotestosterone prevented this necrosis. Increased numbers of RTLA+, CD18+, and La+ interstitial cells were also evident 6 days after ovariectomy. In addition, ovariectomy increased La expression in ductal cells. Dihydrotestosterone treatment prevented the increase in numbers of lymphoid cells and La expression. Dihydrotestosterone also promoted the appearance of mitotic figures in acinar cells and increased the sizes of acini by 43% (P < 0.05). CONCLUSIONS: Glandular atrophy observed after ovariectomy is likely to proceed by necrosis of acinar cells rather than apoptosis. This process begins with an apparent time lag after a rapid phase of interstitial cell apoptosis. These processes are accompanied by increased lymphocytic infiltration. These results suggest that a critical level of androgen is necessary to maintain lacrimal gland structure and function and that a decrease in available androgen below this level could trigger lacrimal gland apoptosis and necrosis, and an autoimmune response. Because apoptotic and necrotic cell fragments may be sources of autoantigens that can be processed and presented to initiate an autoimmune reaction, we surmise that cell death triggered by androgen withdrawal may trigger an autoimmune response such as that encountered in Sjogren's syndrome. (ABSTRACT TRUNCATED) (+info)
Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells.
(10/6081)
PURPOSE: The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. METHODS: A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. RESULTS: Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. CONCLUSIONS: These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs. (+info)
Necrosis and apoptosis after retinal ischemia: involvement of NMDA-mediated excitotoxicity and p53.
(11/6081)
PURPOSE: Accumulated evidence has shown that apoptosis and necrosis contribute to neuronal death after ischemia. The present study was performed to study the temporal and spatial patterns of neuronal necrosis and apoptosis after ischemia in retina and to outline mechanisms underlying necrosis and apoptosis. METHODS: Retinal ischemia was induced by increasing intraocular pressure to a range of 160 mm Hg to 180 mm Hg for 90 minutes in adult rats. The patterns of neuronal cell death were determined using light and electron microscopy and were visualized by TdT-dUTP nick-end labeling (TUNEL). The mRNA expression profile of p53 was examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. Immunohistochemistry was performed using anti-p53, anti-microtubule associated protein-2, and anti-glial fibrillary acidic protein antibodies. RESULTS: Within 4 hours after ischemia, neurons in the inner nuclear cell layer (INL) and ganglion cell layer (GCL) underwent marked necrosis, made apparent by swelling of the cell body and mitochondria, early fenestration of the plasma membrane, and irregularly scattered condensation of nuclear chromatin. After 3 days, the INL and GCL neurons showed further degeneration through apoptosis marked by cell body shrinkage, aggregation, and condensation of nuclear chromatin. Apoptotic neurons were also observed sparsely in the outer nuclear cell layer. Intravitreal injections of MK-801 prevented early neuronal degeneration after ischemia. Of note, mRNA and protein levels of p53, the tumor suppressor gene known to induce apoptosis, were increased in the retinal areas undergoing apoptosis 1 to 3 days after ischemic injury. CONCLUSIONS: Ischemia produces the N-methyl-D-aspartate-mediated necrosis and slowly evolving apoptosis of neurons in the retina. The latter may depend on the expression of the p53 proapoptosis gene. (+info)
Kleine-Levin and Munchausen syndromes in a patient with recurrent acromegaly.
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Hypothalamic disease often affects the patients' personality and this also applies to pituitary tumors with suprasellar extension. We report on a patient with a 12-year history of recurrent acromegaly, treated with three transphenoidal operations, single field radiation therapy and bromocriptine/octreotide administration. During the course of follow-up she presented with self-inflicted anemia and Kleine-Levin syndrome (hypersomnia, hyperphagia and hypersexuality). Furthermore, she developed post-radiation necrosis within the right temporal lobe. Whether her neurological and personality disorders result - at least partially - from the acromegaly or the temporal lobe necrosis remains unclear. (+info)
Evaluation of lidocaine as an analgesic when added to hypertonic saline for sclerotherapy.
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PURPOSE: The efficacy of sclerosing agents for the treatment of telangiectasias and reticular veins is well established. The injection of these agents is often associated with pain, and it is not uncommon for sclerotherapists to include lidocaine with the sclerosants in an attempt to reduce the pain associated with treatment. However, there are concerns that this may reduce the overall efficacy of the treatment because of dilution of the sclerosant. Patient comfort and overall outcome associated with treatment using HS with lidocaine (LIDO) versus that using HS alone was compared. METHODS: Forty-two patients were prospectively entered into the study and randomized blindly to sclerotherapy with 23.4% HS or 19% LIDO. Study subjects and treating physicians were blinded to the injection solution used. Injection sites were chosen for veins ranging in size from 0.1 to 3 mm. Photographs of the area to be treated were taken, and the patients rated their pain. They were then observed at regular intervals for four months, and clinical data was collected. Thirty-five subjects completed the full follow-up period, and photographs of the injected area were taken again. Three investigators blinded to the treatment assignment then evaluated the photographs and scored the treatment efficacy according to a standardized system. RESULTS: In the HS group, 61.9% (13 of 21) patients rated their pain as none or mild, whereas 90.5% (19 of 21) of patients in the LIDO group had no or mild discomfort. This difference is significant, with a P value of.034. There was no difference in the overall efficacy of treatment between the two groups. The groups had similar rates of vein thrombosis and skin necrosis. CONCLUSION: Although lidocaine is often used with sclerosing agents, there are no previous reports in the literature to evaluate its effectiveness in reducing the pain experienced by the patient. In this study, patients receiving LIDO experienced significantly less discomfort at the time of injection than patients who received HS alone. There were no differences in the effectiveness of treatment or in the incidence of complications between the two groups. (+info)
Monocytic cell necrosis is mediated by potassium depletion and caspase-like proteases.
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Apoptosis is a physiological cell death that culminates in mitochondrial permeability transition and the activation of caspases, a family of cysteine proteases. Necrosis, in contrast, is a pathological cell death characterized by swelling of the cytoplasm and mitochondria and rapid plasma membrane disruption. Necrotic cell death has long been opposed to apoptosis, but it now appears that both pathways involve mitochondrial permeability transition, raising the question of what mediates necrotic cell death. In this study, we investigated mechanisms that promote necrosis induced by various stimuli (Clostridium difficile toxins, Staphylococcus aureus alpha toxin, ouabain, nigericin) in THP-1 cells, a human monocytic cell line, and in monocytes. All stimuli induced typical features of necrosis and triggered protease-mediated release of interleukin-1beta (IL-1beta) and CD14 in both cell types. K+ depletion was actively implicated in necrosis because substituting K+ for Na+ in the extracellular medium prevented morphological features of necrosis and IL-1beta release. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum caspase inhibitor, prevented morphological features of necrosis, plasma membrane destruction, loss of mitochondrial membrane potential, IL-1beta release, and CD14 shedding induced by all stimuli. Thus, in monocytic cells, necrosis is a cell death pathway mediated by passive K+ efflux and activation of caspase-like proteases. (+info)
Tumor necrosis factor-alpha and ceramide induce cell death through different mechanisms in rat mesangial cells.
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It has been proposed that ceramide acts as a cellular messenger to mediate tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Based on this hypothesis, it was postulated that resistance of some cells to TNF-alpha cytotoxicity was due to an insufficient production of ceramide on stimulation by TNF-alpha. The present study was initiated to investigate whether this was the case in mesangial cells, which normally are insensitive to TNF-alpha-induced apoptosis. Our results indicate that although C2 ceramide was toxic to mesangial cells, the cell death it induced differed both morphologically and biochemically from that induced by TNF-alpha in the presence of cycloheximide (CHX). The most apparent effect of C2 ceramide was to cause cells to swell, followed by disruption of the cell membrane. It is evident that C2 ceramide caused cell death by necrosis, whereas TNF-alpha in the presence of CHX killed the cells by apoptosis. C2 ceramide did not mimic the effects of TNF-alpha on the activation of c-Jun NH2-terminal protein kinase and nuclear factor-kappaB transcription factor. Although mitogen-activated protein kinase [extracellular signal-related kinase (ERK)] was activated by both C2 ceramide and TNF-alpha, such activation appeared to be mediated by different mechanisms as judged from the kinetics of ERK activation. Furthermore, the cleavage of cytosolic phospholipase A2 during cell death induced by C2 ceramide and by TNF-alpha in the presence of CHX showed distinctive patterns. The present study provides evidence that apoptosis and necrosis use distinctive signaling machinery to cause cell death. (+info)
Myocardial temperature reduction attenuates necrosis after prolonged ischemia in rabbits.
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OBJECTIVE: Previously we observed that a large reduction in infarct size was attained by cooling the risk region of the heart, either before or early after the onset of a 30-min coronary artery occlusion. While this is a standard duration of ischemia used in the rabbit model of infarction, it may not reflect the situation of patients who are reperfused late. The effects of regional hypothermia with a longer duration of ischemia, and when the intervention is applied later, are unknown. This study tests the hypothesis that a local reduction in cardiac temperature protects myocardium during prolonged ischemia (2 h) even if begun well after coronary artery occlusion. METHODS: Anesthetized rabbits received 2 h of coronary artery occlusion and 3 h of reperfusion. Rabbits were randomly assigned to a treated group: topical myocardial cooling starting 30 min after coronary occlusion (n = 14), or control group, no intervention (n = 12). Myocardial temperature in the risk zone, hemodynamics and regional myocardial blood flow were measured. RESULTS: Ischemic zone temperature was similar in both groups at 30 min post occlusion, but the cooling maneuver produced a reduction in temperature in the risk region of the treated group such that myocardial temperature was reduced an average of 10 degrees C between 30 and 60 min of coronary artery occlusion. Myocardial temperature in the control group remained within 0.3 degree C of baseline during coronary artery occlusion and into reperfusion. Core temperatures were similar in both groups. Hemodynamic parameters and collateral blood flow during occlusion were also equivalent in both groups. After 120 min of coronary occlusion, necrosis in the control group comprised 72 +/- 3% of the ischemic risk region. However, in cooled hearts, infarct size, expressed as a fraction of the risk region was significantly lower. Infarct size in this group averaged 59 +/- 3% of the risk region (p < 0.004 vs. controls), and thus cooling resulted in a salvage of approximately 18% of the risk region. CONCLUSION: These results show that reducing myocardial temperature protects ischemic myocardium during a long duration of ischemia even if initiated after coronary artery occlusion. (+info)