Calpains are activated by light but their inhibition has no neuroprotective effect against light-damage.

Calpain had been shown to be highly activated at one day after exposure to the damaging light (Perche et al. (2007)Caspase-dependent apoptosis in light-induced retinal degeneration. Invest Ophthalmol Vis Sci 48:2753-2759.), suggesting that they might play a critical role in photoreceptor apoptosis induced by light. Therefore in the present study we investigate the role of calpain in light-induced photoreceptor cell death. In a first set of experiments, untreated albino Wistar rats were sacrificed at 0, 2, 4, 6, 12, 24 h of light exposure and at one day after the light was turned off (D1) to measure retinal calpain activity and to study calpain expression. In a second set of experiments, after control electroretinograms (ERGs), rats were uninjected or injected intravitreally with DMSO or the calpain inhibitor Mu-Phe-hPhe-FMK, before being exposed to the damaging light for 24 h. ERGs were then recorded at one day (D1) and fifteen days (D15) after the end of light exposure. Rats were sacrificed at D1 for apoptotic cell detection or D15 for histological analysis (ONL thickness). Calpain activity and expression significantly increased in Untreated retinas, from 0 h to D1. DMSO has no effect on calpain activity. Mu-Phe-hPhe-FMK significantly inhibited retinal calpain activity by 85% at 2 h of light exposure and still 48% at D1. However, Mu-Phe-hPhe-FMK has no effect on light-induced retinal degeneration as evidence by equivalent loss of function, equivalent loss of photoreceptor cells and an equivalent number of apoptotic cells in Mu-Phe-hPhe-FMK and DMSO retinas. Therefore, calpains are up-regulated by light stress but they do not have a pivotal role in photoreceptor apoptosis.

Structural, conformational and vibrational properties of 1,1,1-Trifluoro-N-(1,1,2,2,2-pentafluoroethyl) methanesulfinimidoyl chloride, CF3CF2-N=S(Cl)CF3.

The structural, conformational, and configurational properties of 1,1,1-Trifluoro-N-(1,1,2,2,2-pentafluoroethyl) methanesulfinimidoyl chloride, CF3CF2NS(Cl)CF3 have been studied by vibrational spectroscopy [IR (vapor) and Raman (liquid)] and quantum chemical calculations [B3LYP, MP2 and B3PW91 levels of theory using the 6-311+G(d), 6-311+G(df) and 6-311+G(2df) basis sets]. According to these theoretical approximations, CF3CF2-N=S(Cl)CF3 exists in the gas phase as a mixture of a favored anticlinal form (CN bond anticlinal with respect to the CSCl bisector) with C1 symmetry and a less abundant syn conformer showing C1 symmetry as well (ΔG° ≈ 1.20 kcal mol(-1)). Due to the small contribution only a few corresponding vibrational modes of the syn conformer could be assigned confidently in the experimental spectra. Compared to CF3CF2-N=S(F)CF3, the replacement of F by Cl produces a clear change in NS bond length and the corresponding stretching frequency, without affecting the conformational properties.

Modification of reperfusion-induced ionic imbalance by free radical scavengers in spontaneously hypertensive rat retina.

We studied the effects of free radical scavengers, superoxide dismutase (SOD), vitamin E, and EGB 761, on ion shifts (Na+, K+, and Ca2+) induced by ischemia reperfusion in rat retina obtained from spontaneously hypertensive rats. Eyes were subjected to 90 min of retinal ischemia followed by 24 h of reperfusion. Two basic protocols were used: (1) chronic application, in which rats received SOD (7500, 15,000, and 30,000 U/kg, i.v.), vitamin E (50, 100, and 200 mg/kg, i.v.), and EGB 671 (50, 100, and 200 mg/kg, orally) for 10 d, respectively; and (2) acute administration, in which 7500, 15,000, and 30,000 U/kg of SOD, 50, 100, and 200 mg/kg of vitamin E, and 50, 100, and 200 mg/kg of EGB 761 were administered after an ischemic episode, at the onset of reperfusion, respectively. In the drug-free control group, 90 min ischemia followed by 24 h of reperfusion resulted in an accumulation of retinal sodium and calcium from their nonischemic control values of 76 +/- 4 and 3.2 +/- 0.1 mumol/g dry weight to 112 +/- 6 (p < .001) and 6.2 (p < .001) mumol/g dry weight, respectively. Tissue potassium loss was also observed in this model of retinal ischemia reperfusion, and after 90 min ischemia followed by 24 h of reperfusion potassium content was significantly reduced from its nonischemic control value of 266 +/- 5 to 207 +/- 6 (p < .001) mumol/g dry weight. The chronic administration of SOD, vitamin E, and EGB 761 dose dependently reduced the reperfusion-induced ionic imbalance and improved the recovery of retinal ion contents.(ABSTRACT TRUNCATED AT 250 WORDS)

Ischemia and reperfusion-induced histologic changes in the rat retina. Demonstration of a free radical-mediated mechanism.

Histologic alterations of ischemia- and reperfusion-induced retinal damage are critically dependent on the duration of the period of ischemia. Male Sprague Dawley rats were anesthetized, and a suture was placed behind the globe including the central retinal artery. Because it was desirable that untreated eyes show a great histologic change due to reperfusion-induced damage (in order that maximum scope would exist for demonstration of any protective effect of a drug treatment), a preliminary series of studies established the time-induced characteristics for the retina with transient regional ischemia. Eyes (n = 6-12 in each group) were subjected to 30, 60, or 90 min of ischemia followed by 0.5, 1, 2, 4, and 24 hr of reperfusion, respectively. The 30-min ischemia followed by reperfusion did not result in any histologic changes; 60-min ischemia followed by reperfusion induced a moderate retinal edema which returned to the preischemic value after 24 hr of reperfusion. The 90-min ischemia followed by reperfusion further aggravated retinal edema and increased the migration of neutrophil leukocytes. Even after 24 hr of reperfusion, the retinal edema had not disappeared although an attenuation was observed. In this study, the rats were treated with superoxide dismutase (SOD-PEG, 15 x 10(3) U/kg) or EGB 761 (100 mg/kg) for 10 days (chronic treatment). The SOD and EGB 761 significantly reduced the development of reperfusion-induced retinal edema and significantly prevented the neutrophil leukocyte infiltration. Both also had a protective effect against reperfusion-induced injury when these agents were administered just before reperfusion ("late" administration).(ABSTRACT TRUNCATED AT 250 WORDS)

Functional protection of photoreceptors from light-induced damage by dimethylthiourea and Ginkgo biloba extract.

PURPOSE: To investigate the functional protective effect of a synthetic (dimethylthiourea, DMTU) and a natural antioxidant (Ginkgo biloba extract, EGb 761) against light-induced retinal degeneration. METHODS: Wistar rats were exposed for 24 hours to 1700-lux light after treatment with DMTU or EGb 761. Electroretinograms were recorded before and on day (D)1, D3, D8, D15, D22, and D29 after light exposure. The b-wave amplitude was plotted against log L (ganzfeld luminance), providing the b-wave sensitivity curve. The Naka-Rushton function fitted to the sensitivity curve enabled derivation of the parameters Bmax (saturated amplitude) and K (luminance-inducing Bmax/2). In addition, rats from each group were killed for retinal morphometric analyses. RESULTS: In the untreated group, light exposure caused collapse of the b-wave sensitivity curves. Bmax was reduced by 51% at D1 without subsequent recovery. K increased temporarily, reverting to normal values 8 days later. The outer nuclear layer thicknesses decreased markedly in the superior retina. In the treated groups, light exposure had a weaker effect on sensitivity curves. The values of Bmax were not significantly different from those in the unexposed-untreated group, although K increased temporarily. Retinal morphometry was preserved. CONCLUSIONS: Dimethylthiourea and EGb 761 afford functional protection against light-induced retinal damage.

Oxidative stress in diabetic retina.

The authors describe the alterations usually associated with diabetic retinopathy. They concern the classical thickening of the basal membrane of retinal capillaries and the associated modification of retinal vessel permeability. These alterations correspond to the blood-retinal barrier disruption. The authors then discuss the participation of oxygenated free radicals in the pathogenesis of diabetic retinopathy. They report several experimental studies establishing such a participation and finally describe their own results obtained on a model of retinas isolated from alloxan-induced diabetic rats. After one month of evolution, the electroretinograms (ERG) recorded on isolated retinas from diabetic rats had an amplitude about 20% lower than the controls, whereas after two months of diabetes, this decrease was about 60%. Under these conditions, the authors tested the protective properties of Ginkgo biloba extract (EGb 761) on their model. They observed that in EGb-treated animals (100 mg/kg/day), the ERG had a significantly (p less than 0.001) greater amplitude than untreated animals after two months of diabetes evolution. In conclusion, the authors discuss the possible utilization of a free radical scavenger, such as EGb 761, in the prevention of the retinal impairment in diabetes.

A program for automatic acquisition and processing of electroretinograms obtained in vivo in albino rats.

A program sequence has been developed on a microcomputer that affords automatic acquisition and processing of electroretinogram (ERG) obtained in vivo in dark-adapted albino rats. A 50-Hz digital filter and an averaging summation remove the background noise.

Presence of specific platelet-activating factor binding sites in the rat retina.

The specificity of the effects of platelet-activating factor (PAF) on the electrophysiology of the retina suggests the existence of specific sites for PAF in this tissue. In this study, we report the presence of tritiated PAF ([3H]PAF) specific binding sites in membrane preparations of the retina of albino rats. The binding of [3H]PAF was saturable, specific, time-dependent and reversible. Scatchard analysis of the data revealed that the high-affinity retinal binding site possessed a Kd of 2.9 +/- 0.4 nM and Bmax of 0.85 +/- 0.16 pmol/mg protein. These values are comparable with those found for the membranous PAF receptor sites in platelets, neutrophils, lung tissue and brain. We have recently reported that PAF dose dependently modulates the b-wave of the electroretinogram (ERG) obtained from the isolated rat retina. The results of the present study suggest that such PAF-induced disturbances of the ERG may be mediated via specific receptors located in the retina.

Effect of a cGMP-specific phosphodiesterase inhibitor on retinal function.

Multiple forms of phosphodiesterase have been reported in many tissues. Phosphodiesterase 6, a cGMP-specific phosphodiesterase, is described as a photoreceptor cell-specific phosphodiesterase. Phosphodiesterase 6 is known to play a crucial role in visual function. A novel phosphodiesterase inhibitor, GF248 (5["(propoxy),7'(4-morpholino)-phenacyl],[1-methyl-3 propyl]pyrazolo[4,3d]pyrimidin-7-one), has been described to be a very potent cGMP-specific phosphodiesterase inhibitor. In the present study, we compared the potency of GF248 and other known cGMP-specific phosphodiesterase inhibitors on phosphodiesterase 5 and phosphodiesterase 6. GF248 displayed an IC50 of 2 and 5 nM for phosphodiesterase 5 and phosphodiesterase 6, respectively. Thereafter, we assessed the effect of GF248 on retinal function, using an ex vivo model of isolated retina electroretinogram recording. Exposure of retina to GF248 resulted in a dose-dependent decrease in electroretinogram amplitude (PIII and b-waves), with no marked modification of PIII and b-wave implicit time. Among other phosphodiesterase inhibitors, DMPPO (1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazol ol[3,4d]-pyrimidin-4-(5H)-one) and dipyridamole, cGMP-specific phosphodiesterase inhibitors, and IBMQ (1-isobutyl-3-methylimidazol[1,5a]quinoxalin-4-(5H)one), a nonselective phosphodiesterase inhibitor, altered retinal function but less potently than GF248, consistent with their in vitro phosphodiesterase 6 inhibition. Phosphodiesterase 3- and phosphodiesterase 4-selective inhibitors, cilostamide and rolipram, respectively, did not affect retinal function at 10 micromol l(-1). Our conclusion from these data is that GF248, a potent phosphodiesterase 6 inhibitor, could interfere with visual transduction by cGMP accumulation.

A reversed-phase high-performance liquid chromatographic method to analyze retinal isomers.

A high-performance liquid chromatographic (HPLC) procedure was developed to separate all-trans-, 13-cis-, 11-cis- and 9-cis-retinal isomers. Two reversed-phase Vydac C18 columns in series were used with an isocratic solvent system of 0.1 M ammonium acetate-acetonitrile (40:60, v/v) as mobile phase and all-trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-no natetraene-1-ol (TMMP) as internal standard. Prior to HPLC, the retinal isomers were efficiently extracted in their original isomeric conformation using dichloromethane-n-hexane in the presence of formaldehyde. This technique is suitable for the assay of 11-cis- and all-trans-retinal isomers in retina.

Transduction of the light message: from the photon to the optic nerve.

Retinal transduction consists of the conversion of a physical stimulus, light, into an electrophysiological signal. This conversion takes place in several stages. First of all, at the photoreceptor level, via a sequence of molecular activations and deactivations, the detection of light results in an hyperpolarization of the cell membrane. This initial electrical signal is then relayed onto the functional cells of the retina. The bipolar cells are the first associated neurons, responding to the light stimulus by either hyperpolarization (OFF), or depolarization (ON). The second associated neurons are the ganglion cells where the ON-OFF duality also operates and whose fibers make up the optic nerve. In coloured photopic vision, the photoreceptor--bipolar cell--ganglion cell circuit is direct. For the cone-bipolar cell transmission, horizontal cells delimit excitatory (center) and inhibitory (surround) zones at the origin of the receptive field. In scotopic vision, however, i) there is only a single class of bipolar cells, that depolarize in response to light, and ii) the bipolar-ganglion cell connection is not direct. Here, the AII amacrine cells are responsible for the inhibition of the OFF ganglion cells directly connected to them or for the excitation of the ON ganglion cells via ON bipolar cells of the cone circuit. Finally, in mesopic vision, the sensory message originates in rods, and is subsequently relayed by the cone circuit via gap junctions between photoreceptors.

Prospective longitudinal study of urinary eosinophil protein X in children with asthma and chronic cough.

Airway inflammation is the principal abnormality in asthma and many other respiratory diseases. Eosinophils are the cells primarily involved in this process. The aim of this study was to analyze sequential changes in urinary eosinophil protein X (EPX) a biological marker of eosinophil activation in asthmatic children and chronic coughers, and to confirm the importance of such changes in evaluating the inflammatory process once regular treatment was initiated. Eighty-eight asthmatic children (AC), 33 children with chronic cough (CC), and 34 control children were included in the study. All those with respiratory disease underwent allergy tests (serum total IgE, serum-specific IgE for common allergens, peripheral blood eosinophil (PBE), and skin prick tests) and a pulmonary function test (PFT), and had chest X-ray and serum eosinophil cationic protein (s-ECP) and urinary EPX assays. All subjects attended the outpatient clinic every 3 months, irrespective of the treatment prescribed following inclusion in this investigation. At baseline, urinary EPX concentrations were higher in children with asthma and those with chronic cough than in controls (mean 171.1 and 131.3, respectively, vs. 60.2 microg/mmol creatinine, P < 0.001). CC children had lower eosinophil counts (0.25 vs. 0.39 x 10(9)/L, P < 0.02) than those with asthma. There was no significant difference between the AC and CC groups in urinary EPX and s-ECP levels. s-ECP concentrations were significantly higher (P < 0.01) in atopic vs. nonatopic patients (44 vs. 29.9 ng/mL), but no significant difference was observed for urinary EPX. Concentrations of urinary EPX were significantly correlated with s-ECP levels (r = 0.24, P < 0.025) and with PBE (r = 0.38, P < 0.01). No correlation was found between urinary EPX values and PFT results. In AC receiving inhaled steroids after the start of the study, there was a significant reduction after 3 months in urinary EPX (-54, P < 0.02). In contrast, there was no significant change in PBE levels. Urinary EPX concentrations are sensitive, noninvasive technique that could be useful to the clinician in the evaluation of manifestations of airway inflammation.

Light-induced variations of retinal sensitivity in rats.

PURPOSE: ERG responses were measured as a function of Ganzfeld luminance to evaluate functional damage induced by light on rat retinas. METHODS: Wistar rats were exposed to a fluorescent light of 1700 lux for 12 h, 24 h, 48 h and 72 h. We recorded ERGs before and one night after exposure, then 3, 8, 15, 22 and 29 days later. The b- and PIII-wave amplitudes were plotted against luminance for each group at each recovery time. RESULTS: The retinal damage induced by a pupillary illuminance of 1700 lux ranged from low to severe as exposure duration increased from 12 h to 72 h, respectively. We observed an effect immediately after light exposure but no improvement during the recovery period. The b-wave amplitude was reduced by 40, 60, 80 and 90 percent after 12, 24, 48 and 72 h of light exposure, respectively; the PIII-wave amplitude was reduced by 30, 40, 70 and 90 percent after these respective exposures. The Ganzfeld luminance eliciting a 50 microV b-wave amplitude increased significantly with exposure duration, but the luminance eliciting the maximal b-wave amplitude was not dependent on this duration. Hence we suggest that the ERG decrease is due to a reduction in photoreceptor number. CONCLUSIONS: We present a full analysis of the electrophysiological parameters recorded from light-exposed or non-exposed rats. This model is a useful tool to study in vivo retinal degeneration.

Ginkgo biloba extract (EGb 761) and a platelet-activating factor antagonist protect the retina in experimental autoimmune uveoretinitis.

Oxygen-free radical toxicity is an important factor of tissue necrosis in the eye, especially in the retina. Activation of synthesis and release of platelet-activating factor (PAF) by ocular inflammatory cells and resident cells initiates cascades of mediators and cytokines which contribute to tissue damage in several ocular pathologies. The authors studied the therapeutic effectiveness of Ginkgo biloba extract (EGb 761), a potent free radical scavenger with anti-PAF activity, and of BN 50730, a specific PAF antagonist, on acute experimental autoimmune uveoretinitis induced in rats by S-antigen immunization. These treatments slightly delayed disease onset but had little effect on the severity of uveal inflammation. However, they significantly reduced inflammatory cell infiltration of the retina and the damage of the outer retinal layers. These drugs should become useful adjuvants in the therapy of posterior uveitis and other disorders that might damage the retina.

Protective effect of Ginkgo biloba extract (EGB 761) on free radical-induced changes in the electroretinogram of isolated rat retina.

The retina is a tissue particularly rich in polyunsaturated fatty acids and thus highly sensitive to lipid peroxidation initiated by oxygenated free radicals. By recording the electroretinogram (ERG) b wave amplitude on isolated rat retina, the authors have investigated the anti-oxidant properties of Ginkgo biloba extract (EGB 761). Two groups of rats were used: one group was treated with EGB 761 at a dose of 100 mg/kg/day per os for 10 days; the other one of untreated animals served as a control. At the end of the treatment (10 days), rats were sacrificed, one retina isolated and perfused in order to record ERG. Lipid peroxidation was induced by adding a mixture of (FeSO4 + Na ascorbate) to the perfusion solution. In the untreated rats a 50% decrease in ERG was observed after only 55 min. Such a delay in the decrease and subsequent maintenance of ERG b wave amplitude confirm that the anti-oxidant properties of EGB 761 can protect the retina against lipoperoxidation.

Experimental electroretinographic exploration of retinal ischemia: preventive use of free radical scavengers and anti-PAF agents.

Electroretinographic exploration is an effective approach to evaluate retinal function. In order to investigate physiopathological mechanisms and evaluate potentially protective therapies for retinal ischemia, we developed three experimental models: the first two on isolated retina, with ischemia induced by either stopping perfusion or clamping the ophthalmic artery, and the third, in vivo, with ischemia induced by ocular hypertonia. Since free radicals are implicated in the formation of post-ischemic lesions, we evaluated the protective effects of drugs known to be free radical scavengers and of an immunomediator antagonist, an anti-PAF (platelet activating factor) agent.

Comparison of intraocular treatment of DMTU and SOD following retinal ischemia in rats.

The effect of intravitreal injections of DMTU (dimethylthiourea) and SOD (superoxide dismutase), two free radical scavengers, was evaluated in a rat model of retinal ischemia induced by elevated intraocular pressure. The drugs were administered just before or just after a 60 min ischemia. At days 2 and 7 after reperfusion, retinal recovery was evaluated by electroretinography. At day 7, layer thicknesses and cell rows were measured from histologic sections of paraffin-embedded retinas. In the vehicle-treated control group, we observed a decrease in the inner retinal layers and b-wave amplitude impairment. SOD injection (6 units/eye) protected the retina from ischemia/reperfusion injury. At day 2 after reperfusion, electroretinographic recovery was more efficient when SOD was administered just after ischemia (99%) than after pretreatment with SOD (81%) (p<0.03). In the DMTU-treated group (75 microg/eye), only the pretreatment induced significant electrophysiologic (40%) (p<0.001) and morphologic recovery.

[Analysis of hematopoietic growth factor prescriptions in 19 french cancer centers]. / Analyse de la prescription de facteurs de croissance hématopoïétique dans 19 centres anticancéreux français. Groupe de pharmaciens de la Fédération nationale des centres de lutte contre le cancer.

Medical prescription of hematopoietic growth factors (HGF) was analysed in 19 anticancer french centers during 2 months. About 4% of anticancer chemotherapeutic cycles prescribed during this period were supported by HGF prescription. The mean duration of treatment was 8 days. Among the 755 collected prescriptions, two tumor localizations represented about 50% of the prescriptions: malignant non Hodgkin lymphomas and breast cancer. The other main localizations concerned adult or pediatric soft tissue sarcomas (18%), testicular cancer (7%) and gynecologic tumors (6%). The prescription for primary prophylaxis for febrile neutropenia remains the main use of HGF (44%). The respect of the guidelines established by the F|d|ration nationale des centres de lutte contre le cancer was analyzed. Overall, 66% of the prescriptions were in adequation with these guidelines. Whereas the consommation of HGF decreased in the 19 considered institutions, it did not reach a plateau and could decrease in institutions which are awaked to the international and national recommendations.

Effects of PAF-acether on electrophysiological response of isolated retina.

Results of experiments performed on rat isolated retina indicate that platelet-activating factor (PAF) is able to inhibit the functional response of the retina electroretinogram (ERG) recorded in response to a brief light flash. In the presence of PAF, the ERG b-wave amplitude decreases according to a dose-dependent (2.10(-11) M; 2.10(-9) M; 2.10(-7) M) process. This effect is partially inhibited by the simultaneous administration of a Ginkgo biloba extract (GBE, 10 mg/l) or Ginkgolide B (BN 52021, 2.10(-5)M). The authors interpret these results with reference to the main mechanism of the membrane signal triggered by PAF, namely the activation of phosphatidylinositol cycle with the formation of inositol-triphosphate, the inhibition of the light-induced response of the retina by administration of inositol-triphosphate, and the antagonistic effect of GBE and BN 52021 on specific PAF-receptors demonstrated on other models. Thus specific PAF-receptors may exist at the level of the retina, which suggests that they are also present in the brain.

Ischaemia and reperfusion-induced injury in rat retina obtained from normotensive and spontaneously hypertensive rats: effects of free radical scavengers.

The authors have studied the effects of free radical scavengers, superoxide dismutase (SOD) and extract of Ginkgo biloba (EGb 761, flavone-rich extract) on ion shifts (Na, K and Ca) induced by ischaemia and reperfusion in rat retina obtained from normotensive and spontaneously hypertensive rats. Eyes were subjected to 90 min of ischaemia by occlusion of the retinal artery, followed by 4 and 24 hours of reperfusion. SOD (15,000 U/kg, i.v.) or EGb 761 (50 mg/kg, per os) was administered in a daily dose for 10 days. In the drug-free control groups, 90 min of ischaemia significantly increased tissue Na gains from their pre-ischaemic control values of 63 +/- 7 microM/g dry weight (in retina obtained from normotensive rats) and 76 microM/g dry weight (in retina obtained from hypertensive rats) to 89 +/- 9 microM/g dry weight and 101 +/- 7 microM/g dry weight, respectively. During reperfusion, a further elevation was found in retinal Na in both the normotensive and hypertensive groups. Probably, because of the ischaemia-induced inhibition of Na-K-ATPase, retinal K loss was detected after ischaemia and reperfusion, respectively. An accumulation of retinal Ca was measured after ischaemia and reperfusion in the normotensive and spontaneously hypertensive groups. Both free radical scavengers significantly reduced the maldistribution of ions induced by ischaemia and reperfusion, but the effectiveness of drugs was more evident in normotensive than hypertensive groups.(ABSTRACT TRUNCATED AT 250 WORDS)