[Fungal keratitis: A 5-year monocentric retrospective study on Reunion Island]. / Kératites fongiques : étude rétrospective monocentrique sur 5ans à l'île de la Réunion.

OBJECTIVES: Fungal keratitis is rare in France, but could be a severe sight-threatening condition. Here, we aimed to describe the epidemiology of fungal keratitis in Réunion Island. METHODS: In a retrospective study, we analyzed 13 culture-proven keratitis episodes, occurred between January 2013 and July 2017 in the ophthalmology ward of a University Hospital, Saint-Pierre. Twelve isolates were genotyped and antifungal susceptibility testing was performed. RESULTS: Corneal abrasion caused by vegetable matter was the main predisposing factor. Stromal infiltration was observed in 12 patients. Six patients did not response to medical treatment, requiring surgical care, including two enucleations surgery. Fusarium solani (n = 6) and Fusarium dimerum (n = 4) were the main fungal species involved in fungal keratitis. Clinical failures were more prevalent with F. solani infections. The lowest minimal inhibitory concentrations for Fusarium sp. were observed with voriconazole and amphotericin B. CONCLUSION: In Reunion Island, the epidemiology of fungal keratitis is characterized by the predominance of Fusarium species, potentially involved in visual loss. This pattern is consistent with the epidemiology usually observed in tropical areas.

Cavernous sinus fistula treated through the transvenous approach: report of four cases.

PURPOSE: To describe transvenous embolization in four patients with indirect dural carotid cavernous fistulas (CCFs) via the inferior petrosal sinus (IPS) or superior ophthalmic vein (SOV), and their clinical outcomes. METHODS: The CCF approach was performed after retrograde venous catheterization from the femoral vein to the cavernous sinus via the IPS (n=1) or SOV (n=3). SOV catheterization was possible without surgical intervention. All patients presented initially with typical clinical signs of CCF. Patients treated via the SOV presented with thrombosis of the IPS. RESULTS: Catheterization and embolization were successful in all patients, with complete angiographic occlusion of the fistula. No early or late complications occurred. All patients presented with favorable clinical outcomes and complete recovery of ocular symptoms. CONCLUSION: Retrograde transvenous embolization of CCF via the IPS, or SOV if the IPS is thrombosed, is a safe procedure with a good clinical outcome.

Na+ channel-mediated Ca2+ entry leads to glutamate secretion in mouse neocortical preplate.

Before synaptogenesis, early excitability implicating voltage-dependent and transmitter-activated channels is known to be crucial for neuronal development. We previously showed that preplate (PP) neurons of the mouse neocortex express functional Na(+) channels as early as embryonic day 12. In this study, we investigated the role of these Na(+) channels in signaling during early development. In the neocortex of embryonic-day-13 mice, activation of Na(+) channels with veratridine induced a large Ca(2+) response throughout the neocortex, even in cell populations that lack the Na(+) channel. This Na(+)-dependent Ca(2+) activity requires external Ca(2+) and is completely blocked by inhibitors of Na(+)/Ca(2+) exchangers. Moreover, veratridine-induced Ca(2+) increase coincides with a burst of exocytosis in the PP. In parallel, we show that Na(+) channel stimulation enhances glutamate secretion in the neocortical wall. Released glutamate triggers further Ca(2+) response in PP and ventricular zone, as indicated by the decreased response to veratridine in the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and NMDA-receptor inhibitors. Therefore, the combined activation of the Na(+) channel and the Na(+)/Ca(2+) exchanger triggers Ca(2+) signaling in the PP neurons, leading to glutamate secretion, which amplifies the signal and serves as an autocrine/paracrine transmitter before functional synapses are formed in the neocortex. Membrane depolarization induced by glycine receptors activation could be one physiological activator of this Na(+) channel-dependent pathway.

Developmental expression of the calcium release channels during early neurogenesis of the mouse cerebral cortex.

The developmental changes of intracellular calcium release channels of mouse neocortex were studied at the onset of neurogenesis, which occurs between embryonic days E11 and E17. The three main isoforms of the two families of intracellular calcium release channels, namely the inositol trisphosphate receptors (IP3R) and the ryanodine receptors (RyR), were detected by their transcripts in the cerebral hemispheres, as early as stage E11. The major isoforms of each family, IP3R-1 and RyR-2, were found at the protein level by Western blot analysis. Expression of these proteins increases progressively throughout brain development. Their localization in coronal sections of cortex has been observed by immunodetection from E12, and compared to the TuJ1 (anti-class III beta-tubulin antibody) neuronal specific labelling. The expression of both channels is greatly enhanced after E12, and both were seen to be present in most of the proliferative and neuronal cells of the slice. Between E12 and E13, there is a striking transition in the pattern of calcium release elicited by specific agonists of these channels, thimerosal for IP3R and caffeine for RyR. The signals induced by thimerosal were not zone-specific, while the observed calcium release signals induced by caffeine were predominantly restricted out of the ventricular zone. This zone-specific caffeine sensitivity is consistent with the main RyR localization immunodetected at E13. Our results indicate that there is a time lag of several days between the molecular detection of calcium release channels and their functional expression, around the time of neuronal differentiation. Altogether, they provide a molecular basis for analyzing the developmental modulation of calcium signals useful for neurogenesis progression.

Bilateral asymmetry of the inositol trisphosphate-mediated calcium signaling in two-cell ascidian embryos.

In ascidian oocytes, numerous calcium signaling events occur at fertilization which contribute to resume and complete meiosis, and determine the three embryonic axes. The main ooplasmic and intracellular calcium channels at work in the calcium signaling of the one-cell embryo have different roles and fates when the first mitosis begins. By whole-cell patch-clamp recording, we observed different families of these calcium channels in the blastomeres of Phallusia mammillata two-cell ascidian embryos. Membrane capacitance has been measured to evaluate the oocyte and blastomere surface area, allowing certification of the exact time of cell division. At the two-cell stage, no difference was observed in the density of voltage-dependent calcium channels in each blastomere, or in the ryanodine-sensitive calcium stores. In contrast, a bilateral asymmetry was recorded for the ooplasmic channels responsible for calcium entry after calcium store depletion: they could be activated only in the blastomere not wearing the polar bodies. The same laterality was observed in the InsP3-induced internal calcium release. Moreover, this asymmetry included a one-way communication in the InsP3-dependent calcium signaling between the two blastomeres. These results enhance the understanding of the early steps of development, and underscore the interest for ascidians in studies of polarity patterning.

Calmodulin and immunophilin are required as functional partners of a ryanodine receptor in ascidian oocytes at fertilization.

Fertilization of oocytes incites numerous changes relying on Ca(2+) signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, we show here that this calcium channel is asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FKBP-like protein and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca(2+) release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself.

Calcium signaling by cyclic ADP-ribose, NAADP, and inositol trisphosphate are involved in distinct functions in ascidian oocytes.

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADPR) from beta-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from beta-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were characterized in connection with that induced by inositol 1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is accompanied by a decrease in the oocyte Ca2+ current and an increase in membrane capacitance due to the addition of membrane to the cell surface. Both of these electrical changes could be induced by perfusion, through a patch pipette, of nanomolar concentrations of cADPR or its precursor, beta-NAD, into unfertilized oocytes. The changes induced by beta-NAD showed a distinctive delay consistent with its enzymatic conversion to cADPR. The cADPR-induced changes were inhibited by preloading the oocytes with a Ca2+ chelator, indicating the effects were due to Ca2+ release induced by cADPR. Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a specific antagonist of cADPR, but not heparin, inhibited the cADPR-induced changes. Both inhibitors likewise blocked the membrane insertion that normally occurred at fertilization consistent with it being mediated by a ryanodine receptor. The effects of NAADP were different from those of cADPR. Although NAADP induced a similar decrease in the Ca2+ current, no membrane insertion occurred. Moreover, pretreatment of the oocytes with NAADP inhibited the post-fertilization Ca2+ oscillation while cADPR did not. A similar Ca2+ oscillation could be artificially induced by perfusing into the oocytes a high concentration of InsP3 and NAADP could likewise inhibit such an InsP3-induced oscillation. This work shows that three independent Ca2+ signaling pathways are present in the oocytes and that each is involved in mediating distinct changes associated with fertilization. The results are consistent with a hierarchical organization of Ca2+ stores in the oocyte.

The two intracellular Ca2+ release channels, ryanodine receptor and inositol 1,4,5-trisphosphate receptor, play different roles during fertilization in ascidians.

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs of Phallusia mammillata using whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3 receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3 (10 microM perfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3 perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3 causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15-20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.

A ryanodine-sensitive calcium store in ascidian eggs monitored by whole-cell patch-clamp recordings.

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.