Localization, dynamics, and function of survivin revealed by expression of functional survivinDsRed fusion proteins in the living cell.

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.

Inhibition of apoptosis and reduction of intracellular pH decrease in retinal neural cell cultures by a blocker of carbonic anhydrase.

PURPOSE: Methylglyoxal and glyoxal are intermediates of advanced glycation end products (AGEs). These substances, as well as hydrogen peroxide, induce retinal neurons to reduce their intracellular pH and augment their production of reactive oxygen species, leading to apoptosis. Because these processes may play a role in diabetic retinopathy, the authors undertook this study to investigate the protective action of dorzolamide, an inhibitor of carbonic anhydrase, on retinal neural cells. METHODS: E1A-NR3 cells were incubated with varying concentrations of glyoxal, methylglyoxal, and H2O2 for different periods of time in the presence or absence of dorzolamide. Apoptotic changes were determined by cytofluorometry after the cells were incubated with appropriate dyes and antibodies. The parameters studied were DNA strand breaks (TUNEL assay), subdiploid DNA content (sub-G1 assay), annexin V binding, reactive oxygen species intermediates production, active caspase-3, N(epsilon)-(carboxymethyl)lysine (a glycation product), and intracellular pH. RESULTS: Optimal conditions for detection of the cell-protecting effect of dorzolamide were incubation with 0.6 to 0.8 mM glyoxal or methylglyoxal for 5 hours or with 0.1 mM H2O2 for 30 minutes, respectively, followed by 20-hour incubation with fresh medium. All apoptotic changes were reduced in the assays in which dorzolamide was included. CONCLUSIONS: Dorzolamide reduced the damage inflicted on retinal neural cells by agents that induced apoptosis and, therefore, can be considered a neuroprotectant.

Endoscopic exploration of the brachial plexus: technique and topographic anatomy--a study in fresh human cadavers.

OBJECTIVE: The indications for and timing of brachial plexus exploration in closed injuries are controversial. The time-consuming surgery proves its worth in some cases, whereas spontaneous regeneration might have been possible in others. The differentiation is difficult, because no investigational method reveals the exact morphological correlates of the nerve lesions. Minimally invasive, direct observation of the structures is a possible solution. Here we describe our surgical technique and the anatomic features of the normal brachial plexus appreciated with the endoscope. METHODS: Twenty-one brachial plexus in 11 fresh cadavers were investigated. Endoscopic exploration was performed at the supraclavicular and infraclavicular levels. The method involves insertion of an optic shaft-integrated retractor through a stab wound; retraction of landmark muscles produces a working space, into which other instruments are introduced for dissection. After completion of endoscopic surgery, open dissection was performed to verify the endoscopically identified structures and to assess iatrogenic injuries. RESULTS: The omohyoid muscle is a reliable landmark in the supraclavicular region, beneath which the suprascapular nerve can be observed. Following the suprascapular nerve proximally leads to the plexus trunks. Infraclavicular exploration first reveals the axillary artery. The plexus and its nerves are traced around this artery. The anatomic features were constant in all cases, with variations in fat accumulation depending on the corporeal constitution. We detected iatrogenic injuries to the medial circumflex humeral vessels in two cases. No nerve injuries were observed. CONCLUSION: The endoscopic technique combined with intraoperative nerve stimulation studies might provide important information on the type of morphological damage in closed brachial plexus injuries and thus might become an important tool for determination of the surgical treatment strategy. Clinical work is under way.

Cross-linking of human amniotic membrane by glutaraldehyde.

PURPOSE: The transplantation of cryopreserved human amniotic membrane has been introduced recently for the reconstruction of the ocular surface. However, in some diseases the transplant usually dissolves rather quickly and early detachment may occur. Therefore, we tried to stabilize the amniotic transplant by applying glutaraldehyde for collagen cross-linking of the membrane. METHODS: 18 human amnions were prepared. 4 x 4 cm pieces of amnion were treated with 0.1% glutaraldehyde solution for 30 min. Biomechanical force-elongation measurements were performed and resistance to enzymatic digestion by 0.1% collagenase solution was tested and compared to cryopreserved and untreated fresh amnion. 8 patients with various ocular surface defects were treated with cross-linked amnion and compared to 5 patients with cryopreserved amnion. RESULTS: The force of the amnion cross-linked with glutaraldehyde at 2.5 mm elongation was increased statistically significant by 175% versus fresh amnion and 76.8% versus the cryopreserved amnion. Glutaraldehyde-treated membranes were virtually completely resistant to enzymatic digestion, while fresh and cryopreserved amnions were dissolved completely by day 7. In patients, the cross-linked membrane was preserved for up to 90 days without any signs of dissolution of the membrane and good transparency. CONCLUSIONS: Collagen cross-linking using glutaraldehyde leads to a significant increase in the biomechanical strength and enzymatic resistance of amnion, better transparency and less wrinkling. The cross-linked membrane does not dissolve for months and is well suited for the surface reconstruction of the cornea.

Blockers of carbonic anhydrase can cause increase of retinal capillary diameter, decrease of extracellular and increase of intracellular pH in rat retinal organ culture.

BACKGROUND: At least in normal-pressure glaucoma a vascular genesis with hypoperfusion and regulation impairment is discussed. This may lead to malnutrition of retinal ganglion cells and apoptosis. The retinal microvasculature has a small functional reserve. In addition, the retinal microvessels lack the autonomic nerves that are normally found in other tissues. Thus, no systemic influences reach the retinal capillaries apart from circulating hormones or transmitters. Blockers of carbonic anhydrase (CA) may modulate regional blood flow by mediating changes in extra- and intracellular pH. However, it is still unclear (1) whether blockers of CA really change the pH near the retinal capillaries and (2) how changes in the local pH affect the capillary tone in situ. Therefore, we tested dorzolamide and acetazolamide in our model of the freshly enucleated rat retina. METHODS: Adult Sprague-Dawley rats (of both sexes, 250-350 g) were killed and retinae were prepared. The retinae were gently separated from the retinal pigmented epithelium and were observed in a chamber for electronic light microscopy or were fixed for immunohistochemistry. Electronic light microscopy of the retinal cells was performed with a Zeiss Axiovert microscope equipped with differential interference contrast (DIC) optics. Changes in capillary diameter were measured using an Openlab acquisition system and analyzed statistically using ANOVA. In addition to light microscopy the intracellular pH was analyzed in the whole mounts by ratio imaging of the pH using the special dye BCECF-AM (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluoresceinacetoxymethyl ester) and the extracellular pH using BCECF (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein). RESULTS: Pericytes of most segments of retinal capillaries are immunoreactive for alpha-smooth muscle actin (SMA). The SMA immunostaining is strong around the nucleus; the endothelial tube is visible by virtue of the slight immunoreactivity of the surrounding pericyte processes. Acetazolamide and dorzolamide showed statistically significant vasoactive effects in retinal capillaries. Vasodilation increased by up to 105% of that in control capillaries after 5, 10 and 15 min. CA inhibitors were found to be able to induce intracellular alkalization in retinal cells. After addition of dorzolamide or acetazolamide the extracellular pH decreased from 7.4 to 7.2 concomitant with diameter changes. CONCLUSIONS: The tube-like pattern of SMA immunoreactivity demonstrates the presence of contractile elements within the pericyte processes of the rat retina. Thus, pericytes may act as a regulation element within the retinal microcirculation. Our results further suggest that CA inhibitors are able to decrease pH in the extracellular space; however, the pH within the cells increases. The increase in capillary diameter is concomitant with these pH changes. Thus, we may conclude that CA inhibitors can relax pericytes and might improve the retinal blood supply.

Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro.

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 microM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 microm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

Alteration of the intracellular pH and apoptosis induction in a retinal cell line by the AGE-inducing agent glyoxal.

BACKGROUND: Methylglyoxal and glyoxal, intermediate products of glycation, are known to accelerate glycation and the formation of advanced glycation endproducts (AGEs). These mechanisms may play a role in the degenerative progression of diabetic retinopathy and macular degeneration. The present study was undertaken to elucidate the retinal neurotoxicity of the Maillard reaction intermediate alpha-oxoaldehyde glyoxal. METHODS: E1A-NR3 is an immortalized retinal cell line that manifests specific phenotypes of retinal neurons. These cells were incubated with 0 microM (control), 200 microM, 400 microM, and 800 microM glyoxal for different lengths of time. For intracellular pH measurements, cells were incubated for 30 min with BCECF-AM prior to measurement of the emission ratio at two wavelengths. For semiquantitative analysis of the mitochondrial potential, cells were incubated for 15 min with 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). For detection of apoptotic cells, cultures were incubated for 15 min with YO-PRO-1 iodide. Immunohistochemical labeling of cell components, which are indicative for apoptosis like active caspase-3 and the caspase cleavage product fractin, was performed after fixation. DNA gel electrophoresis was carried out to detect apoptotic cell death at the DNA level. RESULTS: Morphological changes in living cells after glyoxal incubation showed signs of cell damage, including cell membrane blebbing and aggregation of intracellular organelles. Glyoxal produced a dose- and time-dependent acidification from physiological pH to pH 7.2. The semiquantitative analysis of mitochondrial membrane potential in living control cells showed nearly all mitochondria with intact hyperpolarized membranes. One hour after the beginning of incubation with 200 microM glyoxal we found most mitochondria depolarized but still with an elongated shape and only regional swelling. With higher glyoxal concentrations more depolarized mitochondria developed blebs and were located around the nucleus. The membrane permeability assay with the nucleic acid stain YO-PRO-1 showed increasing cell membrane permeability with time and glyoxal dose and, finally, nuclear fragmentation. By means of immunohistochemistry we found a glyoxal-induced accumulation of the AGE N(epsilon)-(carboxymethyl) lysine in the cytoplasm and nuclei. The cells were immunoreactive for active caspase-3 after glyoxal in a dose-dependent manner. Neurofilament protein was strongly expressed in controls and disappeared gradually with increasing glyoxal concentration. The immunoreactivity for the caspase cleavage product fractin was found in most of the cells after 800 microM glyoxal. DNA gel electrophoresis showed DNA ladder formation. CONCLUSIONS: The ability of glyoxal to induce apoptosis was confirmed by our findings demonstrating the time- and dose-dependent acidification of retinal cells. The onset of acidification is a hallmark because acidification is a measurable cytosolic event that follows the mitochondrial change but precedes caspase activation. Therefore, monitoring of pHi can allow one to assess cell stresses such as hypoxia, and metabolic stress (AGEs), and to test whether these stresses have a cumulative effect on apoptosis induction. Our study showed that intracellular pH and mitochondrial potential in living retinal cells are useful parameters for monitoring metabolic status in retinal tissue. These findings may be relevant in the study of retinal cell death mechanisms associated with age-related diabetic retinopathy and macular degeneration.

Graded sensitiveness of the various retinal neuron populations on the glyoxal-mediated formation of advanced glycation end products and ways of protection.

BACKGROUND: The accumulation of advanced glycation end products (AGEs) in retinal cells is known to be associated with the risk of diabetic retinopathy. To develop a model of AGE-related metabolic stress in retinal organ cultures, we investigated the accumulation of a typical glycoxidation product (N(epsilon)-[carboxymethyl] lysine [CML]) and its possible pro-apoptotic effects on different retinal cell populations. METHODS: Retinal organ cultures (rat) were kept for 9 h in the Ames medium containing 0 (control), 5, 25, 50, 150, 300 and 800 micro M glyoxal. The expression of bax, active caspase-3, and the accumulation of CML were studied by using immunohistochemistry after the paraffin embedding of retinal explants. Apoptosis was studied using the terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling (TUNEL) test and electron microscopy. Alpha lipoic acid (alpha-LA), sodium metavanadate (NaVO(3)), N-acetylcysteine (NAC), aminoguanidine (AG), and nicotinamide (NA) were used to influence glyoxal effects in organ cultures. RESULTS: In cultured normal non-diabetic retinae, small amounts of CML and the apoptosis-promoting factors bax and active caspase-3 were present. CML, bax and active caspase-3 increased after incubation with glyoxal. Incubation with glyoxal (<300 micro M, 9 h) increased apoptotic events in all layers. At low glyoxal concentrations, we found a graded sensitiveness of the different layers: at 25 micro M 39.4% in GCL, 28.2% in INL, 11.9% in ONL. After 800 micro M glyoxal, approximately 50% of the cells in all layers of the retina were apoptotic. In the ONL, this ratio was reduced by NaVO(3) (17%), by AG (27%), by NA (24.8%), by NAC (25.2%), and by alpha-LA (33.5%). In the INL, AG (25.9%) produced the best result. In the GCL, NAC, NaVO(3) and AG reduced apoptosis. A-LA had no significant protective effect. CONCLUSION: The glyoxal-induced rapid formation of CML shows the ability of our retina model to simulate AGE-related effects in vitro. The dose-dependent expression of apoptosis-promotor molecules indicates that the apoptosis-inducing machinery starts in most retinal cells within 9 h. The neurotoxicity of glyoxal-induced AGE formation was shown by the significantly increased rate of cell death in the retina. The significant decrease of apoptotic events (P<0.01) indicates that antioxidants and AGE formation blocker can exert a differentiated cytoprotection for each of the retinal cell layers.