Dark-adapted State Research Articles

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Comparative analysis of leaf photosynthetic characteristics and fruit sugar content in trees of Citrus cultivar 'Huangguogan' of different age

Citrus trees of cv. Huangguogan, 8-, 30-, and 280-year-old (8s, 30s, and 280s, respectively), were used to explore the photosynthesis and related enzyme activity. In spring shoot leaves, net photosynthetic rate (PN) of the 280s was significantly lower than that of the 30s and 8s, but it was opposite in summer shoot leaves. PN of the summer shoots was higher than that of the spring shoots. PN was significantly positively correlated with stomatal conductance, intercellular CO2 concentration, and transpiration rate, but was negatively correlated with maximum photochemical quantum yield of PSII photochemistry. The effect of age on variable fluorescence/minimal fluorescence was similar to that of minimal fluorescence, variable fluorescence, maximal fluorescence yield of the dark-adapted state. Fruits of the 280s had significantly lower sugar components than those of the 8s and 30s. Results showed that fruits of the 8s and 30s actively accumulated photosynthesis products, which was verified by the higher PN in the spring shoots, lower acidic invertase and neutral invertase activities, and higher sucrose phosphate synthase than that of the 280s.

Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

The photoactive Orange Carotenoid Protein (OCP) plays a key role in cyanobacterial photoprotection. In OCP, a single non-covalently bound keto-carotenoid molecule acts as a light intensity sensor, while the protein is responsible for forming molecular contacts with the light-harvesting antenna, the fluorescence of which is quenched by OCP. Activation of this physiological interaction requires signal transduction from the photoexcited carotenoid to the protein matrix. Recent works revealed an asynchrony between conformational transitions of the carotenoid and the protein. Intrinsic tryptophan (Trp) fluorescence has provided valuable information about the protein part of OCP during its photocycle. However, wild-type OCP contains five Trp residues, which makes extraction of site-specific information impossible. In this work, we overcame this problem by characterizing the photocycle of a fully photoactive OCP variant (OCP-3FH) with only the most critical tryptophan residue (Trp-288) in place. Trp-288 is of special interest because it forms a hydrogen bond to the carotenoid’s keto-oxygen to keep OCP in its dark-adapted state. Using femtosecond pump-probe fluorescence spectroscopy we analyzed the photocycle of OCP-3FH and determined the formation rate of the very first intermediate suggesting that generation of the recently discovered S* state of the carotenoid in OCP precedes the breakage of the hydrogen bonds. Therefore, following Trp fluorescence of the unique photoactive OCP-3FH variant, we identified the rate of the H-bond breakage and provided novel insights into early events accompanying photoactivation of wild-type OCP.

Assessment of the safety of intravitreal injection of metoprolol tartrate in rabbits.

To verify the safety of different doses of intravitreal metoprolol tartrate (MT) after intravitreal injection in rabbit eyes. Animals were randomly assigned into 2 groups: group I received 50µg of MT and group II 100µg of MT. A volume of 0.05mL of the drug solution was administered through an intravitreal injection, while the control eyes received an equal volume of saline solution. Safety was assessed by clinical observation, electroretinography (ERG) and histological evaluation. No evidence of clinical toxicity was observed. ERG waveforms from the MT treated eyes were similar to those recorded from the control eyes in dark-adapted state, amplitude and the implicit time are similar between the groups in light-adapted state, and their retinas had no signs of toxicity by histological evaluation 7days after intravitreal injection. The intravitreal use of metoprolol at 50 and 100µg dosages does not cause short-term retinal toxicity in rabbits.

Light-harvesting complex II is an antenna of photosystem I in dark-adapted plants.

Photosystem I (PSI) is a major player in the light reactions of photosynthesis. In higher plants, it consists of a core complex and four external antennae, Lhca1-4 forming the PSI-light-harvesting complex I (LHCI) supercomplex. The protein and pigment composition as well as the spectroscopic properties of this complex are considered to be identical in different higher plant species. In addition to the four Lhca, a pool of mobile LHCII increases the antenna size of PSI under most light conditions. In this work, we have first investigated purified PSI complexes and then PSI in vivo upon long-term dark-adaptation of four well-studied plant species: Arabidopsis thaliana, Zea mays, Nicotiana tabacum and Hordeum vulgare. By performing time-resolved fluorescence measurements, we show that LHCII is associated with PSI also in a dark-adapted state in all the plant species investigated. The number of LHCII subunits per PSI is plant-dependent, varying between one and three. Furthermore, we show that the spectroscopic properties of PSI-LHCI supercomplexes differ in different plants.

Transient pupillary light reflex in CEP290- or NPHP5-associated Leber congenital amaurosis: Latency as a potential outcome measure of cone function

Mutations in photoreceptor cilium genes CEP290 and NPHP5 cause a form of Leber congenital amaurosis (LCA) which typically lacks rods but retains central cones. The current study evaluated the transient pupillary light reflex (TPLR) as an objective outcome measure to assess efficacy of ongoing and future therapies. Eleven eyes of six patients selected for retained cone function were tested with TPLR using full-field stimuli in the dark-adapted state. Stimuli were red or blue with 1 s duration and spanned a 6-log unit dynamic range. TPLR response amplitude was quantified at fixed times of 0.9 and 2 s after stimulus onset and TPLR latency was defined as the time to reach 0.3 mm constriction. Full-field stimulus testing (FST) and static perimetry were used to correlate subjective perception with objective TPLR parameters. TPLR and FST thresholds with both red and blue stimuli were abnormally elevated in patients to near −1.25 log phot-cd·m−2 consistent with the lack of rods. TPLR latencies were delayed on average but showed some differences among patients. Remnant extrafoveal vision was correlated with faster TPLR latencies. Our results support the use of a short TPLR protocol with full-field red stimuli of 0.7 log phot-cd·m−2 or brighter as an objective and convenient outcome measure of cone function in CEP290- and NPHP5-LCA. The latency parameter of the TPLR would be expected to show a detectable change when an intervention modifies cone sensitivity in the extrafoveal region.

Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function

Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCPO) to the red (OCPR) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCPR accumulation implicates restructuring of a compact dark-adapted OCPO state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCPR-like absorption spectrum and an OCPO-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCPCC). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCPCC, FRP binds reduced OCPCC nearly as efficiently as OCPO devoid of the NTE, suggesting that the low-affinity FRP binding to OCPO is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCPCC indicates that OCPCC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCPR-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Structural elements regulating the photochromicity in a cyanobacteriochrome

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore's peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation.

ISCEV extended protocol for derivation and analysis of the strong flash rod-isolated ERG a-wave

The International Society for the Clinical Electrophysiology of Vision (ISCEV) standard for full-field electroretinography (ERG) describes a minimum set of tests, but encourages the use of additional protocols for clinical ERG testing. This extended protocol describes recording methods and derivations that will allow analysis of rod-driven components of the dark-adapted (DA) strong flash ERG a-wave, more closely related to rod phototransduction than ISCEV standard DA ERGs. The method involves recording ERGs to a flash strength equivalent to 30 cd s m2 under conditions of dark adaptation and additionally to the same stimulus following light adaptation (LA) and in the presence of a standard photopic background luminance of 30 cd m−2. The isolated rod-driven ERG a-wave is derived by subtracting the LA response from the DA ERG. The method is likely to be of value in the characterization of retinal disorders which affect rod quantal catch, diseases that affect the dynamics of any component of the activation phase of rod phototransduction, or those affecting total numbers of rod photoreceptors.

Circadian clock regulation of cone to horizontal cell synaptic transfer in the goldfish retina.

Although it is well established that the vertebrate retina contains endogenous circadian clocks that regulate retinal physiology and function during day and night, the processes that the clocks affect and the means by which the clocks control these processes remain unresolved. We previously demonstrated that a circadian clock in the goldfish retina regulates rod-cone electrical coupling so that coupling is weak during the day and robust at night. The increase in rod-cone coupling at night introduces rod signals into cones so that the light responses of both cones and cone horizontal cells, which are post-synaptic to cones, become dominated by rod input. By comparing the light responses of cones, cone horizontal cells and rod horizontal cells, which are post-synaptic to rods, under dark-adapted conditions during day and night, we determined whether the daily changes in the strength of rod-cone coupling could account entirely for rhythmic changes in the light response properties of cones and cone horizontal cells. We report that although some aspects of the day/night changes in cone and cone horizontal cell light responses, such as response threshold and spectral tuning, are consistent with modulation of rod-cone coupling, other properties cannot be solely explained by this phenomenon. Specifically, we found that at night compared to the day the time course of spectrally-isolated cone photoresponses was slower, cone-to-cone horizontal cell synaptic transfer was highly non-linear and of lower gain, and the delay in cone-to-cone horizontal cell synaptic transmission was longer. However, under bright light-adapted conditions in both day and night, cone-to-cone horizontal cell synaptic transfer was linear and of high gain, and no additional delay was observed at the cone-to-cone horizontal cell synapse. These findings suggest that in addition to controlling rod-cone coupling, retinal clocks shape the light responses of cone horizontal cells by modulating cone-to-cone horizontal cell synaptic transmission.

Co-expression of opsins in the eye photoreceptor cells of the terrestrial slug Limax valentianus.

Visual opsins coupled with Gq -type G protein have been considered to be responsible for the vision in mollusks. Recent transcriptomic studies, however, revealed the presence of opsin mRNA belonging to different groups of opsin subfamilies in the eyes of mollusks. In the present study, we found that at least three different opsins, Gq -coupled rhodopsin, opsin5A, and xenopsin, are co-expressed in the rhabdomeric photoreceptor cell in the eyes of the terrestrial slug Limax valentianus. These opsins were all localized to the microvilli of the rhabdomere. Co-expression of rhodopsin and opsin5A mRNA was also demonstrated by dual fluorescence in situ hybridization. Co-expression of multiple opsins in the rhabdomeric photoreceptors cells may explain the previously reported shift in the action spectra of the electroretinogram of eyes of Limax flavus between the light- and dark-adapted states, which was also reproduced in the present study in L. valentianus.

THE INFLUENCE OF BIO-ALGEEN S90 ON THE GROWTHOF MULTIFLORA ROSE SEEDLINGS (Rosa multiflora Thunb.)

Striving to intensify horticultural production, new and more effective bio-preparations are being sought to stimulate plant growth and development. Bio-algeen S90 is a natural agent based on sea algae, the high bi- ological activity of which results from the high content of natural growth regulators. The aim of the study was to verify the influence of Bio-algeen S90 on the growth, morphological characteristics and chlorophyll fluorescence of Rosa multiflora seedlings. The bio-preparation was applied one, two and three times at con- centrations: 0.1, 0.2, 0.4 and 0.6 mg.dm−3. Following parameters were measured to evaluate the response of plants to the bio-preparation: F0 – initial fluorescence, Fm – maximal fluorescence in the dark-adapted state, Fv/Fm – maximum photochemical efficiency of PSII. All concentrations of the bio-preparation and frequency of its application stimulated the number of shoots in a bush, the length of shoots and the diameter of the root crown of plants intended for budding. The most beneficial was the two-fold bio-preparation application at a concentration of 0.4 mg.dm–3. Bio-algeen also positively influenced the chlorophyll fluorescence parame- ters. The highest mean F0 and Fm values were recorded with the two-fold preparation treatment. There was no significant effect of the bio-preparation on the Fv/Fm index, which was within the range of 0.75–0.66.

Effect of Ecklonia maxima extract on photosynthesis activity and chlorophyll content of Medicago × varia Martyn leaves

Recently, there is a big interest in science to use seaweed extract in agricultural practice. Therefore, the aim of this paper was to determine the effect of foliar application of Ecklonia maxima extract on photosynthetic activity and chlorophyll content of Medicago ×varia Martyn leaves. The experimental factor was a commercial product containing E. maxima extract (Em). The following parameters were determined: maximum photosystem II efficiency (Fv/Fm) in the dark-adapted state, actual photosystem II efficiency in the light-adapted state (∆F/Fm’), the photochemical quenching coefficient (qP), the non-photochemical quenching coefficient (qN), chlorophyll content determination. The Fisher-Snedecor test was used to determine whether the impact of experimental factor was significant, while the value of the HSD0.05 was calculated with Tukey’s test. As a reaction to the particular conditions during the experiment alfalfa treated with Em had a better efficiency of the photosynthetic apparatus in relation to the control. Algae extract used on hybrid alfalfa leaves caused a 12.4% increase of Fv/Fm parameter, from 0.565 on the control to 0.635. The value of the actual efficiency of photosystem II (∆F/Fm) determined under the same conditions turned out to be about 20% higher for plants treated with the extract. Chlorophyll a and b concentration, as a response to the extract, increased by 12% and 16.7%, respectively. The experiment showed that periods of rainfall shortages did not reverse the effect of the extract on plants, with an increase in chlorophyll a and b content during the growing seasons with dry and extremely dry months.

Water Bridges Conduct Sequential Proton Transfer in Photosynthetic Oxygen Evolution.

Proton transfer using water bridges has been observed in bulk water, acid-base reactions, and several proton-translocating biological systems. In the photosynthetic water-oxidizing enzyme, photosystem II (PSII), protons from substrate water are transferred 35 Å from the Mn4CaO5 catalytic site to the chloroplast lumen. This process leads to acidification of the lumen and ATP synthesis. Water oxidation occurs in a flash-induced, five-step S n state cycle; acetate is a chloride-dependent inhibitor of the S2 to S3 step of this cycle. Here, we study the effect of acetate on a previous step of the cycle, the S1 to S2 transition, using reaction-induced infrared spectroscopy. PSII was isolated from spinach, and experiments were conducted at pH 7.5, using 532 nm laser flashes to advance the cycle from the dark-adapted state S1 to the S2 state. Isotope-editing of acetate reveals direct contributions to the S2-minus-S1 infrared spectrum consistent with protonation of bound acetate in PSII. In the acetate-derived S2-minus-S1 PSII spectra, an accompanying decrease in the intensity of a 2830 cm-1 band is observed when compared to the chloride control. The 2830 cm-1 band has been assigned previously to a stretching vibration of an internal, hydrated hydronium ion, W n+. Density functional studies of a catalytic site model predict the spontaneous transfer of a proton from this internal hydronium ion to acetate, when acetate is substituted at a chloride-binding site. Taken together, the results show that the mechanism of PSII proton transfer at pH 7.5 involves proton hopping through an internal, water-containing network.

Two-color pupillometry in KCNV2 retinopathy.

To investigate receptor and post-receptor function in KCNV2 retinopathy [cone dystrophy with supernormal rod electroretinogram (ERG)], using the pupillary light reflex (PLR) and the ERG. Two unrelated patients (1 male and 1 female) with molecularly confirmed KCNV2 retinopathy underwent full-field two-color pupillometry testing in one eye, with monitoring of the stimulated eye by an infrared digital camera. Pupillometry stimuli consisted of 1-s duration, short-wavelength (465-nm, blue) and long-wavelength (642-nm, red) stimuli. Pupillometry intensity series were performed under both a dark-adapted condition and a light-adapted condition (on a 0.76-logcdm-2 blue background). The transient PLR, defined as the maximum constriction following flash onset, was measured under all conditions. The melanopsin-mediated sustained constriction was measured 5-7s following flash offset for the highest flash luminance presented in the dark. Both patients were also tested in one eye with the full-field ERG, including a dark-adapted intensity series and ISCEV standard stimuli. Dark-adapted PLRs were markedly attenuated or extinguished for low-luminance stimuli, but the responses to higher-luminance blue stimuli were within normal limits. Light-adapted PLRs to blue stimuli were generally within normal limits, exceeding the responses to photopically matched red stimuli. Thus, light-adapted responses were consistent with either rod or S-cone mediation of the PLR. Melanopsin-mediated sustained PLRs were within normal limits. ERG showed the characteristic findings previously reported in this condition. Cone-mediated ERG responses were markedly decreased in amplitude. Rod-mediated ERG responses were absent for low-luminance stimuli (- 3 logcdsm-2), but had normal amplitude for stimuli of - 2 log cdsm-2 and above (although none were "supernormal"). The b-wave for the dark-adapted ISCEV standard - 2 log cd s m-2 stimulus was markedly delayed, whereas the b-wave timing was generally normal for higher flash luminances. The abnormalities measured by pupillometry have a similar pattern to the outer-retinal abnormalities measured by ERG in KCNV2 retinopathy. These findings as well as the normal sustained PLR suggest that inner-retinal function may be preserved in KCNV2 retinopathy and highlight the potential for therapies designed to restore outer-retinal function in these individuals.

Pathway-specific light adaptation in human electroretinograms.

The cellular origins of slow ERG changes during light adaptation following a dark-adapted state are still unclear. To study light adaptation, six healthy, normal trichromats were dark-adapted for 30 min prior to full-field ERG recordings to sinusoidal stimuli that isolate responses of the L- or M-cones or that stimulate luminance and chromatic mechanisms at 12 or 36 Hz. Recordings were performed for 16 min with 2-min intervals after onset of a constant background. Generally, the responses were sine-wave-like, and the first harmonic (fundamental) component dominated the Fourier spectrum except for the 12-Hz luminance stimulus in which two components, a sine-wave-like component and a transient component, determined the response profiles, leading to large second harmonic components. The amplitude of the first harmonic component (F) increased as a function of the light-adaptation time except for the 12-Hz luminance stimulus at which the F component decreased as a function of the light-adaptation period. The phase of the first harmonic component changed only slightly (less than 30°) during the light-adaptation period for all stimuli conditions. The L/M ratio in luminance reflecting ERGs decreased with increasing adaptation time. Our present data suggest that the light-adaptation process mainly reflects changes in the luminance pathway. The responses to 12-Hz luminance stimuli are determined by two different luminance driven pathways with different adaptation characteristics.

Bleaching effects and fluorescence lifetime imaging ophthalmoscopy.

This study investigates the influence of photopigment bleaching on autofluorescence lifetimes in the fundus in 21 young healthy volunteers. Three measurements of 30° retinal fields in two spectral channels (SSC: 498-560 nm, LSC: 560-720 nm) were obtained for each volunteer using fluorescence lifetime imaging ophthalmoscopy (FLIO). After dark-adaptation by wearing a custom-made lightproof mask for 30 minutes, the first FLIO-measurement was recorded (dark-adapted state). Subsequently, the eye was bleached for 1 minute (luminance: 3200 cd/m2), followed by a second FLIO-measurement (bleached state). Following an additional 10 minute dark adaptation using the mask, a final FLIO-measurement was recorded (recovered state). Average values of the fluorescence lifetimes were calculated from within different areas of a standardized early treatment diabetic retinopathy study (ETDRS) grid (central area, inner and outer rings). The acquisition time in the bleached state was significantly shortened by approximately 20%. The SSC did not show any significant changes in fluorescence lifetimes with photopigment bleaching, only the LSC showed small but significant bleaching-related changes in the fluorescence lifetimes τ1 and τ2 from all regions, as well as the mean fluorescence lifetime in the central area. The fluorescence lifetime differences caused by bleaching were by far less significant than pathological changes caused by eye diseases. The magnitudes of fluorescence lifetime changes are <10% and do not interfere with healthy or disease related FLIO patterns. Thus, we conclude that bleaching is not a relevant confounder in current clinical applications of FLIO.

Hemodynamic Response of the Three Macular Capillary Plexuses in Dark Adaptation and Flicker Stimulation Using Optical Coherence Tomography Angiography.

PurposeTo assess retinal microvascular reactivity during dark adaptation and the transition to ambient light and after flicker stimulation using optical coherence tomography angiography (OCTA).MethodsFifteen eyes of 15 healthy participants were dark adapted for 45 minutes followed by OCTA imaging in the dark-adapted state. After 5 minutes of normal lighting, subjects underwent OCTA imaging. Participants were then subjected to a flashing light-emitting diode (LED) light and repeat OCTA. Parafoveal vessel density and adjusted flow index (AFI) were calculated for superficial (SCP), middle (MCP), and deep capillary plexuses (DCP), and then compared between conditions after adjusting for age, refractive error, and scan quality. SCP vessel length density (VLD) was also evaluated. Between-condition capillary images were aligned and subtracted to identify differences. We then analyzed images from 10 healthy subjects during the transition from dark adaptation to ambient light.ResultsSCP vessel density was significantly higher while SCP VLD was significantly lower during ambient light and flicker compared to dark adaptation. There was a significant positive mean value for DCP “flicker minus dark or light,” suggesting more visible vessels during flicker due to changes in flow, dilation, or vessel recruitment. We found a significant, transient increase in SCP and decrease in both MCP and DCP vessel density during the transition from dark to light.ConclusionsWe show evidence suggesting constriction of deeper vessels and dilation of large SCP vessels during the transition from dark to light. This contrasts to redistribution of blood flow to deeper layers during dark adaptation and flicker stimulation.

Effect of Ocriplasmin on objectively assessed retinal function after treatment of vitreomacular diseases.

The scope of the study was the functional assessment of possible effects of intravitreally injected ocriplasmin using electroretinographical (ERG) examinations. Seven subjects suffered from pathologies within the label of ocriplasmin (Jetrea® ) were examined in the study. An extended international society for clinical electrophysiology of vision ERG protocol was used for baseline recordings (before treatment) and was repeated for comparison 3months after treatment with intravitreally injected ocriplasmin. All subjects were examined using 6 steps and a 9Hz flicker protocol as scotopic flicker response under dark-adapted conditions. Under light-adapted conditions, two single-flash responses (standard flash and On/Off protocol) and a 31Hz flicker response was used for functional assessment. A significant reduction of amplitudes was found for the lowest stimulation intensity (0.0001cd.s/m²) under dark-adapted conditions after treatment (p=0.027). All other parameters were not significantly different after treatment including the scotopic flicker response. Under light-adapted state, the a- and b-wave amplitudes were not significantly altered after treatment for single-flash and flicker responses. The On- and Off-responses were not significantly different between baseline and after the treatment. One single significant difference of all examined parameters mirrors a difference due to chance, even if a negative effect of proteolytic enzymes on laminin and fibronectin of retinal cells cannot be entirely excluded. Nevertheless, this result should be respected in order to ensure a safe use of ocriplasmin for patients with vitreoretinal disorders.