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Fabrication of conductive fibrous scaffold for photoreceptor differentiation of mesenchymal stem cell
摘要： Conductive nanofibrous scaffolds with that can conduct electrical current have a great potential in neural tissue engineering. The purpose of this study was to survey effects of electrical stimulation and polycaprolactone/polypyrrole/multiwall carbon nanotube (PCL/PPY/MWCNTs) fibrous scaffold on photoreceptor differentiation of trabecular meshwork mesenchymal stem cells (TM‐MSCs). PCL/PPY/MWCNTs scaffold was made by electrospinning method. TM‐MSCs were seeded on PCL/PPY/MWCNTs scaffold and stimulated with a potential of 115 V/m. Scanning electron microscopy, transmission electron microscopy, and FT‐IR were used to evaluate the fabricated scaffold. Immunofluorescence and quantitative real‐time polymerase chain reaction were used to examine differentiated cells. Scanning electron microscopy, transmitting electron microscopy, and FT‐IR confirmed the creation of the composite structure of fibers. RT‐qPCR analysis showed that the expression of rhodopsin and peripherin genes in electrically stimulated cells were significantly higher (5.7‐ and 6.23‐fold, respectively; p ≤ 0.05) than those with no electrical stimulation. Collectively, it seems that the combination of PCL/PPY/MWCNTs scaffold, as a suitable conductive scaffold, and electrical stimulation could be an effective approach in the differentiation of stem cells in retinal tissue engineering.
关键词： electrical conductive,trabecular meshwork mesenchymal stem cells,photoreceptor‐like cells,nanostructure
更新于2025-11-14 15:18:02
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Assessment of Different Sampling Methods for Measuring and Representing Macular Cone Density Using Flood-Illuminated Adaptive Optics
摘要： PURPOSE. To describe a standardized flood-illuminated adaptive optics (AO) imaging protocol suitable for the clinical setting and to assess sampling methods for measuring cone density. METHODS. Cone density was calculated following three measurement protocols: 50 × 50-μm sampling window values every 0.5° along the horizontal and vertical meridians (fixed-interval method), the mean density of expanding 0.5°-wide arcuate areas in the nasal, temporal, superior, and inferior quadrants (arcuate mean method), and the peak cone density of a 50 × 50-μm sampling window within expanding arcuate areas near the meridian (peak density method). Repeated imaging was performed in nine subjects to determine intersession repeatability of cone density. RESULTS. Cone density montages could be created for 67 of the 74 subjects. Image quality was determined to be adequate for automated cone counting for 35 (52%) of the 67 subjects. We found that cone density varied with different sampling methods and regions tested. In the nasal and temporal quadrants, peak density most closely resembled histological data, whereas the arcuate mean and fixed-interval methods tended to underestimate the density compared with histological data. However, in the inferior and superior quadrants, arcuate mean and fixed-interval methods most closely matched histological data, whereas the peak density method overestimated cone density compared with histological data. Intersession repeatability testing showed that repeatability was greatest when sampling by arcuate mean and lowest when sampling by fixed interval. CONCLUSIONS. We show that different methods of sampling can significantly affect cone density measurements. Therefore, care must be taken when interpreting cone density results, even in a normal population.
关键词： cone photoreceptor density,retina,adaptive optics
更新于2025-09-23 15:23:52
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Electron microscopic observation of photoreceptor cells in directly inserted anesthetized <i>Drosophila</i> into a high-pressure freezing unit
摘要： The high-pressure freezing (HPF) technique is known to cryofix water-containing materials with little ice-crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light-exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight-exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.
关键词： photoreceptor cell,high-pressure freezing,Drosophila
更新于2025-09-23 15:23:52
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Choroidal thickness in healthy eyes using enhanced depth imaging optical coherence tomography and comparison with cases of retinitis pigmentosa
摘要： Introduction. — The goal of this study was to measure by spectral domain optical coherence tomography (SD-OCT) with EDI the choroidal thickness in healthy subjects and to compare these parameters with those of patients with retinitis pigmentosa (RP). Methods. — Data were obtained from 60 healthy patients without history or family history of retinal or choroidal disease or glaucoma. A case-control study was also conducted on 40 eyes of 20 patients with RP and 40 eyes of 20 healthy refraction- and age-matched controls, selected from among the 60 healthy patients. OCT was used with the EDI protocol. The primary outcome measure was choroidal thickness. Results. — Among healthy patients, the overall choroidal thickness was 287.7 μm. Mean choroidal thickness was lower on the nasal side (236.6 μm from the fovea) compared with the temporal side (262.3 μm, P = 0.002). It also varied according to age, being highest among 20—29-year-old patients and decreasing thereafter with increasing age. Choroidal thickness was significantly higher in healthy patients than in RP patients, regardless of the location (P < 0.001). Conclusion. — This observational study confirms that choroidal thickness varies with age and location. It decreases in subjects with RP and is related to worsening of retinal damage, independently of age-related thinning. Further studies are needed to understand whether choroidal vascular alteration is a cause or a consequence of the degenerative pathology.
关键词： Photoreceptor degeneration,Choroidal thickness,Case-control study,Retinitis pigmentosa,Enhanced depth imaging optical coherence tomography
更新于2025-09-23 15:23:52
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Retinal photoreceptor apoptosis is associated with impaired serum ionized calcium homeostasis in diabetic retinopathy: An in-vivo analysis
摘要： Purpose: The aim of this work was to study the association of serum ionized calcium with retinal photoreceptor apoptosis on spectral domain optical coherence tomography (SD-OCT) in diabetic retinopathy (DR). Methods: Sixty consecutive cases with Type 2 diabetes mellitus were categorized into three groups: no diabetic retinopathy; non-proliferative DR; proliferative DR. The eye with more severe form of the disease was considered. Twenty healthy controls were also included. Best corrected visual acuity (BCVA) was measured on logMAR scale. Retinal photoreceptor apoptosis was defined as disruption of retinal photoreceptor ellipsoid zone (EZ). Ellipsoid zone disruption was assessed using SD-OCT. Serum levels of total and ionized calcium were measured using standard protocol. Results: EZ disruption was found to be positively associated with serum total calcium and ionized calcium. Also, EZ disruption was found to be positively associated with logMAR BCVA. Conclusion: Increased serum ionized calcium induces retinal photoreceptor apoptosis resulting in increased EZ disruption in DR.
关键词： Ionized calcium,Retinal photoreceptor apoptosis,Diabetic retinopathy,Autophagy,Ellipsoid zone,Spectral-domain optical coherence tomography
更新于2025-09-23 15:23:52
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Pharmacological interference of adrenergic receptor signaling preserve photoreceptors after retinal detachment through inhibition of oxidative stress and inflammation
摘要： Background and Purpose: The current strategy is not sufficient to halt progression of photoreceptor death and subsequent visual impairment related to retinal detachment (RD) which is observed in various retinal disorders. This study investigated the neuroprotective effects of adrenergic receptor (AR)-targeting pharmaceuticals, the α1-AR antagonist doxazosin or the α2-AR agonist guanabenz, against photoreceptor cell death in RD. Experimental Approach: Brown-Norway rats were created with experimental RD by subretinal injection of sodium hyaluronate. Oxidative stress biomarkers and cytokine production were quantified with ELISA. Protein expression levels and immunofluorescent labelling were determined in rats with RD and controls for mechanistic elucidation. The effects of systemic administration of doxazosin, guanabenz on photoreceptor apoptosis, retinal histology and electroretinography were evaluated in rats with RD compared to vehicle controls. Key Results: Photoreceptors were the major source of RD-induced ROS overproduction in the rat retina through the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Systemic administration of doxazosin or guanabenz significantly alleviated the RD-induced production of ROS and proinflammatory cytokines, including IL-1β and MCP-1, and suppressed retinal gliosis, resulting in attenuation of photoreceptor death and preservation of retinal structures and functions in RD. Conclusions and Implications: Our findings point to adrenergic receptors as novel therapeutic targets for photoreceptor protection and suggest that both doxazosin and guanabenz, two FDA-approved drugs, could be further explored to treat retinal diseases.
关键词： G protein-coupled receptors,adrenergic receptors,retinal detachment,photoreceptor neuroprotection,NADPH oxidase
更新于2025-09-23 15:23:52
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Early Onset Ultrastructural and Functional Defects in RPE and Photoreceptors of a Stargardt-Like Macular Dystrophy (STGD3) Transgenic Mouse Model
摘要： PURPOSE. We investigated the interplay between photoreceptors expressing mutant ELOVL4 (responsible for Stargardt-like disease, STGD3) and RPE in the initial stages of retinal degeneration. METHODS. Using electron microscopy and electroretinogram (ERG), we assessed RPE and photoreceptor ultrastructure and function in transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates. Experiments were done at P30, 1 month before photoreceptor loss in TG and at P90, a time point with approximately 30% rod loss. To further elucidate the mechanism underlying our ultrastructural and functional results, we undertook Western blotting and immunohistochemistry of key proteins involved in phagocytosis of outer segments by RPE cells. RESULTS. Firstly, we showed that in TG mouse photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of the mutated ELOVL4 protein. Secondly, we found evidence of RPE toxicity at P30, preceding any photoreceptor loss. Pathology in RPE cells was exacerbated at P90. Furthermore, higher proportions of phagosomes remained at the apical side of RPE cells. Subretinal lysosomal deposits were immunopositive for phagocytic proteins. Ultrastructural analysis of photoreceptor (rod) outer segments showed disrupted surface morphology consisting of disc spacing irregularities. Finally, rods and RPE exhibited signs of dysfunction as measured by the ERG a-wave leading edge (P30) and c-wave (P90), respectively. CONCLUSIONS. The presence of human mutant ELOVL4 in transgenic mouse photoreceptors leads to early outer segment disc pathology and RPE cytotoxicity. Defective processing of these abnormal discs by RPE cells ultimately may be responsible for outer segment truncation, photoreceptor death, and vision loss.
关键词： mouse,outer segment,Stargardt-like dystrophy,photoreceptor,STGD3,phagocytosis,ELOVL4,RPE
更新于2025-09-23 15:23:52
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DWARF4 accumulation in root tips is enhanced via blue light perception by cryptochromes
摘要： Brassinosteroid (BR) signalling is known to be coordinated with light signalling in aboveground tissue. Many studies focusing on the shade avoidance response in aboveground tissue or hypocotyl elongation in darkness have revealed the contribution of the BR signalling pathway to these processes. We previously analysed the expression of DWARF 4 (DWF4), a key BR biosynthesis enzyme, and revealed that light perception in aboveground tissues triggered DWF4 accumulation in root tips. To determine the required wavelength of light and photoreceptors responsible for this regulation, we studied DWF4-GUS marker plants grown in several monochromatic light conditions. We revealed that monochromatic blue LED light could induce DWF4 accumulation in primary root tips and root growth as much as white light, while monochromatic red LED could not. Consistent with this, a cryptochrome1/2 double mutant showed retarded root growth under white light while a phytochromeA/B double mutant did not. Taken together, our data strongly indicated that blue light signalling was important for DWF4 accumulation in root tips and root growth. Furthermore, DWF4 accumulation patterns in primary root tips were not altered by auxin or sugar treatment. Therefore, we hypothesize that blue light signalling from the shoot tissue is different from auxin and sugar signalling.
关键词： light signalling,brassinosteroid,DWF4,phytochromes,cryptochromes,hypocotyl elongation,photoreceptor,root growth,BR biosynthesis
更新于2025-09-23 15:22:29
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Characterization and engineering of photoactivated adenylyl cyclases
摘要： Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3′, 5′-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.
关键词： synthetic biology,phytochrome,sensory photoreceptor,adenylyl cyclase,BLUF,optogenetics
更新于2025-09-23 15:22:29
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Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors
摘要： Centrins (CETN1–4) are ubiquitous and conserved EF–hand family Ca2+-binding proteins associated with the centrosome, basal body, and transition zone. Deletion of CETN1 or CETN2 in mice causes male infertility or dysosmia, respectively, without affecting photoreceptor function. However, it remains unclear to what extent centrins are redundant with each other in photoreceptors. Here, to explore centrin redundancy, we generated Cetn3GT/GT single-knockout and Cetn2-/-; Cetn3GT/GT double-knockout mice. Whereas the Cetn3 deletion alone did not affect function, simultaneous ablation of Cetn2 and Cetn3 resulted in attenuated scotopic and photopic electroretinography (ERG) responses in mice at three months of age, with nearly complete retina degeneration at one year. Removal of CETN2 and CETN3 activity from the lumen of the connecting cilium (CC) destabilized the photoreceptor axoneme and reduced the CC length as early as postnatal day 22 (P22). In Cetn2-/-; Cetn3GT/GT double-knockout mice, spermatogenesis-associated 7 (SPATA7), a key organizer of the photoreceptor-specific distal CC, was depleted gradually and CETN1 was condensed to the mid-segment of the CC. Ultrastructural analysis revealed that in this double knockout, the axoneme of the CC expanded radially at the distal end, with vertically misaligned outer segment discs and membrane whorls. These observations suggest that CETN2 and CETN3 cooperate in stabilizing the CC/axoneme structure.
关键词： centriole,connecting cilium,retina degeneration,cilium,retina,electroretinography,Ca2+-binding proteins,photoreceptor,centrin 2/centrin 3 double knockout,centrin
更新于2025-09-23 15:22:29