Ультраструктурные изменения хориоретинального комплекса кроликов после воздействия лазерного излучения с длиной волны 577 нм

Background: Diode lasers have been used in ophthalmic practice for several years. They are useful in the management of vasoproliferative retinopathies and have been shown to be comparable in efficacy to the argon laser. Aim: To report on the outcome of transpupillary diode laser treatment for retinal diseases. Patients and Methods: A retrospective study carried out using the case files of patients that had retinal diode laser photocoagulation between June 2011 and May 2012 at the Jos University Teaching Hospital, Jos, Nigeria. Information retrieved included patients' age, sex, diagnosis, indication for retinal laser photocoagulation, prelaser and postlaser visual acuity (VA), best corrected VA, and operative complications. Results: A total of 31 eyes of 21 patients had transpupillary retinal laser photocoagulation during the study period. The commonest indication for retinal laser photocoagulation was proliferative diabetic retinopathy (PDR), and this was seen in 16 eyes (52%). Six eyes (19%) had both PDR and clinically significant macular edema (CSMO), while 3 eyes had CSMO only. Other indications were retinal vein occlusion (RVO) with secondary neovascularization (four eyes), proliferative sickle cell retinopathy (one eye), and traumatic retinal tear (one eye). Following laser therapy in those with diabetic retinopathy, VA improved by at least a line in 6 eyes, remained the same in 13 eyes, and deteriorated in 2 eyes. Neovascular regression was observed in 13 eyes and resolution of macular edema in 3 eyes. In the eyes with RVO, neovascular regression was observed in two eyes. Conclusion: Transpupillary diode laser photocoagulation was beneficial in the treatment of retinal diseases. Fibrovascular proliferation with significant fibrous tissue component was, however, associated with poor response to laser treatment in proliferative retinopathies.

To determine the specific location of the initial lesion in acute retinal pigment epitheliitis. Four patients diagnosed with acute retinal pigment epitheliitis were studied. Fundus photographs, fluorescein angiography and indocyanine green angiography, and spectral-domain optical coherence tomography findings were reviewed. Four healthy young patients presented with acute onset of unilateral decreased vision. Ophthalmoscopy showed macular pigment mottling with surrounding yellow hypopigmented areas at the level of the retinal pigment epithelium (RPE). Fluorescein angiography revealed transmission hyperfluorescence. Early-phase and midphase indocyanine green angiography images showed a patchy macular hyperfluorescence. At late phase of indocyanine green angiography, a hyperfluorescent halo with a cockadelike appearance of the macular area was observed. Spectral-domain optical coherence tomography showed a disruption of the photoreceptors' inner segment and outer segment interface associated with a wider disruption of the RPE inner band. These disrupted lines were replaced by a dome-shaped highly reflective lesion involving the RPE inner layer, the photoreceptors' inner segment and outer segment layers, and, in two cases, the outer nuclear layer. With time, indocyanine green angiography showed resolution of the observed lesions. Spectral-domain optical coherence tomography showed restored and continuous inner segment and outer segment layers and RPE inner band. Spectral-domain optical coherence tomography findings suggest that the initial lesion in acute retinal pigment epitheliitis is located at the junction between the photoreceptor outer segments and the apical side of the RPE cells. Indocyanine green angiography and spectral-domain optical coherence tomography show that the RPE appears to be more widely involved than the neurosensory retina.

Purpose Age‐related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface. Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT‐PCR. Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch’s membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF‐alpha and IL‐6, whereas the expression of CFB was positively regulated by TNF‐alpha and IFN‐gamma. Short‐term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long‐term incubation of RPE cells with POS significantly down‐regulated CFH expression but up‐regulated CFB expression. Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS.

We have documented the ultrastructural changes that occur within the photoreceptor outer segment and the retinal pigment epithelium (RPE) during photosensitive membrane turnover. We employed an in vitro eyecup preparation from Xenopus laevis in which a large shedding event was induced by adding the excitatory amino acid L-aspartate (Greenberger & Besharse 1985; J. comp. Neurol. 239, 361-372). We found that during L-aspartate-induced shedding the RPE cells formed, on their apical domains, previously undescribed processes that were directly involved in disc phagocytosis. These processes are structurally similar to processes formed by macrophages during phagocytosis and are accordingly referred to as pseudopodia. Pseudopodia were distinguishable from the apical villous process normally extended from the RPE in that they were closely applied to the surface of the outer segment, had a cytoplasmic matrix of low electron density that was devoid of most cellular organelles and were enriched in thin (7 nm diameter) filaments. Filament size, specific pseudopodial staining with the actin-specific probe rhodamine phalloidin and inhibition of pseudopod formation by cytochalasin D suggested that the thin filaments were composed of actin. Pseudopodial formation also occurs during a normal light-initiated shedding event. However, the low frequency of shedding, the asynchrony of the individual shedding events and the transient appearance of the pseudopodia prevented a full appreciation of their role during normal disc shedding. Associated with massive shedding and pseudopodial formation, there was an increased adherence between retina and RPE. During L-aspartate treatment, the apical portions of the RPE cells partitioned with the distal outer segment during retinal isolation. This effect was directly related to the development of pseudopodia and may reflect alteration of surface features of the rod outer segment (ROS)-RPE interface related to phagocytosis. Our observations show that transiently forming pseudopodia are the organelles of phagocytosis and that they may play a role in disc detachment as well.

Macular telangiectasia Type 2 (MacTel 2) is a bilateral disease characterized by neurodegenerative and vascular changes affecting the fovea.1 The occurrence of subretinal neovascularization in MacTel 2 is rather rare but constitutes a vision-threatening complication.2 We describe longitudinal multimodal imaging features of a novel variant of bacillary layer detachment (BLD, outer retinal split at the level of the photoreceptor inner segment myoid)3 and termed “hemorrhagic BLD” in a case of subretinal neovascularization associated with MacTel 2. This report followed the tenets of the Declaration of Helsinki. A 55-year-old woman presented with proliferative MacTel 2. Fundus photography showed active yellowish neovascular membrane with subretinal hemorrhages (Figure 1A). Optical coherence tomography (OCT) angiography segmented at the level of the outer retina confirmed subretinal neovascularization (Figure 1B). Optical coherence tomography disclosed a parafoveal outer retinal cyst consistent with BLD and adjacent subretinal fluid (Figure 1, C–E). The higher optical intensity in the BLD compared with that of subretinal fluid and its colocalization with subretinal hemorrhage on fundus photography suggested heme accumulation in the BLD. Eye-tracked follow-up OCT performed one month after a single intravitreal injection of ranibizumab (0.5 mg/0.05 mL) demonstrated significant regression of the BLD (Figure 1, F and G). Persistent focal attenuations of the ellipsoid and interdigitation zones were noted.Fig. 1.: Multimodal imaging features of hemorrhagic bacillary layer detachment. A. Color fundus photography shows typical features of macular telangiectasia Type 2 including retinal graying, crystalline deposits, ectatic capillaries, and hyperpigmentation foci. There is an active yellowish neovascular membrane (white arrowhead) with subretinal hemorrhages. B. Optical coherence tomography angiography segmented from the outer retina to the choriocapillaris shows a subretinal neovascular membrane (white arrowhead). The inset shows segmentation lines of the en face optical coherence tomography angiography image with red flow signal overlay. C–E. The (C) is a magnified view of the BLD on OCT as illustrated in (E). The intraretinal split occurs at the level of the hyporeflective myoid zone (green arrowheads). The ellipsoid zone (orange arrowhead) of the attached retina is continuous with a faint hyperreflective line (orange arrowhead) that likely represents split photoreceptor inner segments and outer segments remaining adherent to the retinal pigment epithelium/Bruch membrane complex (yellow arrowhead). The external limiting membrane (blue arrowheads) is visible over the top of the BLD (withe arrowhead). Note the dense hyperreflective material within the BLD that corresponds to heme accumulation and may interfere with accurate delineation of the outer retinal layers. The (D) is the near infrared reflectance image with the green line indicating the position of the OCT scan. Vertical OCT scan (E) shows parafoveal outer retinal cyst consistent with BLD (white arrowhead) and adjacent subretinal fluid (asterisk). F and G. Eye-tracked follow-up near infrared reflectance image (F) and OCT scan (G) performed 1 month after an intravitreal injection of ranibizumab shows dramatic resolution of the BLD. The external limiting membrane is intact. Note the persistent focal attenuations of the ellipsoid and interdigitation zones (between red arrowheads).To the best of our knowledge, hemorrhagic BLD has not been associated with proliferative MacTel 2, although similar findings coined “atypical outer retinal fluid accumulation” have been reported in neovascular age-related macular degeneration.4 Intense leakage of fluid from the subretinal neovascularization and modulation of the adherence between photoreceptor outer segments and retinal pigment epithelium by the subretinal hemorrhage may have induced BLD development.5 In fact, fibrin interdigitation between photoreceptor outer segments has been documented after experimental subretinal hemorrhages and may explain the presumed photoreceptor fracture under hydrostatic pressure effect.

To determine the nature of the cellular infiltrate, alterations in cell adhesion molecules, and MHC II antigen expression in the rat retina following diode laser retinal photocoagulation. 20 normal Lister rats underwent diode laser photocoagulation of the retina. Frozen sections from eyes enucleated at 0, 1, 5, 13, and 33 days post laser were examined for T cells (R7.3), CD4 T cells (W3/25), activated CD4 T cells (OX-40), CD8 T cells (OX-8), B cells (OX-33), and macrophages (OX-42), MHC II antigen (OX-6), and E-Selectin-1, VCAM-1, and ICAM-1. Retinal diode laser photocoagulation stimulated a wound healing response in the outer retina and choroid. The cellular infiltrate included macrophages and activated CD4 T cells at 13 and 33 days post laser. Glial cells in the inner plexiform and inner nuclear layers expressed MHC II antigen at 24 hours only. ICAM-1 antigen was induced in RPE cells and in Muller cells in the inner retina at all time intervals post laser and intense staining for ICAM-1 was present around intraretinal migrated cells at 13 and 33 days post laser. VCAM-1 antigen expression was induced in the choroidal vascular endothelium and RPE at 13 and 33 days after laser as was E-Selectin-1 antigen expression which was also evident focally at the external limiting membrane in association with migrated cells adjacent to the burn. These results suggest that alterations in cell adhesion molecules may regulate the migration and activation of retinal pigment epithelium, macrophages and CD4 T cells at the outer blood-retinal barrier and choroid following diode laser photocoagulation of the normal Lister rat retina.

During embryonic development, the rudimentary layers that will become the pigment epithelium (PE) and the neural retina are brought into close proximity upon the collapse of the optic vesicles. At this developmental stage, the retina is morphologically undifferentiated and photoreceptor outer segments have not yet begun to form. The close apposition of the PE and photoreceptors prior to the time when outer segments first appear raises the possibility that the PE could be a source of signals that induce or regulate photoreceptor development and outer segment elaboration. Also, the observation that outer segment development is limited in the absence of the PE in most species suggests that interactions between these two cell types may be of fundamental importance for the structural and functional maturation of photoreceptors [1, 2, 3, 5, 13, 16, 19, 20, 26].

Disruption of the blood-aqueous barrier (BAB) induced by retinal photocoagulation and cryopexy in pigmented rabbits was evaluated by laser flare photometry. A significant increase in flare values after retinal photocoagulation was measured from the 1st postoperative day, with values returning to baseline levels by day 7. Cryopexy induced consistently high flare values for 14 days. Intravitreal injection of interleukin (IL) 1, IL-6 and prostaglandin (PG) E₂ induced a significant increase in flare values. Following these treatments, introduction of a PG synthetase inhibitor can partially ameliorate BAB disruption. IL-1, IL-6 and PGE₂ may be involved in BAB disruption following retinal photocoagulation and cryopexy.

Reduced birth weight is associated with an increased risk of visual impairments. This study was undertaken to determine whether prenatal exposure to a chronic compromise sufficient to cause fetal growth restriction (FGR) results in long-term alterations to the retina and optic nerve. FGR was induced by umbilicoplacental embolization (UPE) in two cohorts of pregnant ewes from (1) 120 days of gestation (dg) until 140 dg and (2) 120 dg until term ( approximately 147 dg). Control fetuses were not subjected to UPE. The structure and neurochemistry of the retina and number and structure of ganglion cell axons were assessed in near-term (140 dg) and adult animals (2.3 years). In near-term FGR fetuses compared with control fetuses there were significant reductions (P < 0.05) in the outer plexiform layer (OPL), the photoreceptor inner and outer segment layers, the inner nuclear layer (INL) in the central retina and the outer nuclear layer (ONL) in the peripheral retina, and the diameter of ganglion cell axons in the optic nerve, with a proportional reduction in the thickness of myelin sheaths. In FGR animals compared with the control at 2.3 years, there were significant reductions (P < 0.05) in the total thickness of the retina, the thickness of the photoreceptor outer segment layer and the INL and the number of tyrosine hydroxylase-immunoreactive (TH-IR) dopaminergic amacrine cells. Axonal diameter and myelin sheath thickness in the optic nerve were not different (P > 0.05) between groups. Chronic placental insufficiency in late gestation results in long-lasting effects on specific retinal components, including photoreceptor outer segments and TH-IR amacrine cells. Other alterations observed at term, including reductions in growth and myelination of optic nerve axons, do not persist, suggesting delayed rather than permanently compromised development. Alterations persisting into adulthood could affect visual function.

PurposeThe determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon.MethodsThe endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS) length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin.ResultsThe post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induction of inflammatory cytokines, such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3), which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-κB activation, mTOR inhibition suppressed NF-κB activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-κB in the retina. In addition, the inhibition of NF-κB suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-κB during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin.ConclusionsOur results propose the potential use of rapamycin as a neuroprotective therapy to suppress local activated mTOR levels, related inflammatory molecules, and the subsequent visual dysfunction during retinal inflammation.

Protein kinase C (PKC) has been implicated in regulating several proteins involved in phototransduction. This contribution characterizes the biochemical and immunological properties of PKC isozyme(s) in the photoreceptor outer segment. Activity measurements revealed that at least 85% of the PKC in this specialized compartment belongs to the subfamily of Ca2+-regulated (conventional) PKCs. Of the known Ca2+-dependent PKCs, only PKC alpha was immunodetected by western blot analysis of rod outer segment proteins. However, the ratio of immunoreactivity to enzyme activity for rod outer segment PKC was no more than 40% of that for brain PKC, using antibodies against conventional PKCs. Therefore, at least half the Ca2+/lipid-stimulated activity in rod outer segment preparations cannot be accounted for by the known isozymes, suggesting the presence of a previously uncharacterized isozyme. Despite extensive tests using a variety of antibodies against different domains of PKC alpha, PKC alpha could not be detected in rod outer segments by immunofluorescence of retinal sections. In summary, our data reveal that most of the PKC in photoreceptor outer segments is of the conventional type and that most, if not all, of this conventional PKC activity comes from a novel isozyme(s).

In humans, inositol polyphosphate-5-phosphatase E (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi, mislocalized rhodopsin and an accumulation of extracellular vesicles. In addition, knockout cells showed disruption of the actin network. Together, these data demonstrate that Inpp5e plays a crucial role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

Diabetic retinopathy (DR) is a prevalent chronic ocular complication of diabetes, which ranks as the leading cause of blindness in individuals aged 40 and above. Recent studies have demonstrated that neuroglial vascular unit (NGVU) injury leads to distinct fundus changes in DR, including exudates, cotton‐fluff spots, microangiomas, hemorrhages, and neovascularization. Currently, the primary clinical treatment options primarily target retinal microvascular degeneration during the middle and late stages of DR through techniques such as retinal laser photocoagulation, antivascular endothelial growth factor (VEGF) therapy, and vitrectomy. However, progression to these stages often results in irreversible damage to visual acuity with limited treatment efficacy. In recent years, relevant research has confirmed that NGVU injury occurs prior to retinal microangiopathy in patients with DR, and it is closely associated with impaired visual function. Therefore, targeting NGVU holds potential for future therapeutic interventions aimed at preventing and treating early‐stage DR. This review identifies six key molecular targets (P2X7 receptor, NLRP3 inflammasome, retinal microglial cell necroptosis pathway, spermine oxidase, and AQP4‐AS1 lncRNA) that mitigate NGVU dysfunction in preclinical models. Literature search was conducted in PubMed/Embase using keywords “diabetic retinopathy,” “neuroglial vascular unit,” and “targeted therapy” (2018–2024), focusing on preclinical studies with in vivo efficacy data.

The photoreceptor outer segment (OS) is a unique modification of the primary cilium, specialized for light perception. Being homologous organelles, the primary cilium and the OS share common building blocks and molecular machinery to construct and maintain them. The OS, however, has several unique structural features that are not seen in primary cilia. Although these unique features of the OS have been well documented, their implications in protein localization have been under-appreciated. In this review, we compare the structural properties of the primary cilium and the OS, and propose a hypothesis that the OS can act as a sink for membrane proteins. We further discuss the implications of this hypothesis in polarized protein localization in photoreceptors and mechanisms of photoreceptor degeneration in retinal ciliopathies.

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.