Presumed SARS-CoV-2 Viral Particles in the Human Retina of Patients With COVID-19.

Importance: The presence of the SARS-CoV-2 virus in the retina of deceased patients with COVID-19 has been suggested through real-time reverse polymerase chain reaction and immunological methods to detect its main proteins. The eye has shown abnormalities associated with COVID-19 infection, and retinal changes were presumed to be associated with secondary microvascular and immunological changes. Objective: To demonstrate the presence of presumed SARS-CoV-2 viral particles and its relevant proteins in the eyes of patients with COVID-19. Design, Setting, and Participants: The retina from enucleated eyes of patients with confirmed COVID-19 infection were submitted to immunofluorescence and transmission electron microscopy processing at a hospital in São Paulo, Brazil, from June 23 to July 2, 2020. After obtaining written consent from the patients' families, enucleation was performed in patients deceased with confirmed SARS-CoV-2 infection. All patients were in the intensive care unit, received mechanical ventilation, and had severe pulmonary involvement by COVID-19. Main Outcomes and Measures: Presence of presumed SARS-CoV-2 viral particles by immunofluorescence and transmission electron microscopy processing. Results: Three patients who died of COVID-19 were analyzed. Two patients were men, and 1 was a woman. The age at death ranged from 69 to 78 years. Presumed S and N COVID-19 proteins were seen by immunofluorescence microscopy within endothelial cells close to the capillary flame and cells of the inner and the outer nuclear layers. At the perinuclear region of these cells, it was possible to observe by transmission electron microscopy double-membrane vacuoles that are consistent with the virus, presumably containing COVID-19 viral particles. Conclusions and Relevance: The present observations show presumed SARS-CoV-2 viral particles in various layers of the human retina, suggesting that they may be involved in some of the infection's ocular clinical manifestations.

Localization of Tfap2ß, Casq2, Penk, Zic1, and Zic3 Expression in the Developing Retina, Muscle, and Sclera of the Embryonic Mouse Eye.

Optic development involves sequential interactions between several different tissue types, including the overlying ectoderm, adjacent mesoderm, and neural crest mesenchyme and the neuroectoderm. In an ongoing expression screen, we identified that Tfap2ß, Casq2, Penk, Zic1, and Zic3 are expressed in unique cell types in and around the developing eye. Tfap2ß, Zic1, and Zic3 are transcription factors, Casq2 is a calcium binding protein and Penk is a neurotransmitter. Tfap2ß, Zic1, and Zic3 have reported roles in brain and craniofacial development, while Casq2 and Penk have unknown roles. These five genes are expressed in the major tissue types in the eye, including the muscles, nerves, cornea, and sclera. Penk expression is found in the sclera and perichondrium. At E12.5 and E15.5, the extra-ocular muscles express Casq2, the entire neural retina expresses Zic1, and Zic3 is expressed in the optic disk and lip of the optic cup. The expression of Tfap2ß expanded from corneal epithelium to the neural retina between E12.5 to E15.5. These genes are expressed in similar domains as Hedgehog (Gli1, and Ptch1) and the Wnt (Lef1) pathways. The expression patterns of these five genes warrant further study to determine their role in eye morphogenesis.

Repulsive Environment Attenuation during Adult Mouse Optic Nerve Regeneration.

The regenerative capacity of CNS tracts has ever been a great hurdle to regenerative medicine. Although recent studies have described strategies to stimulate retinal ganglion cells (RGCs) to regenerate axons through the optic nerve, it still remains to be elucidated how these therapies modulate the inhibitory environment of CNS. Thus, the present work investigated the environmental content of the repulsive axon guidance cues, such as Sema3D and its receptors, myelin debris, and astrogliosis, within the regenerating optic nerve of mice submitted to intraocular inflammation + cAMP combined to conditional deletion of PTEN in RGC after optic nerve crush. We show here that treatment was able to promote axonal regeneration through the optic nerve and reach visual targets at twelve weeks after injury. The Regenerating group presented reduced MBP levels, increased microglia/macrophage number, and reduced astrocyte reactivity and CSPG content following optic nerve injury. In addition, Sema3D content and its receptors are reduced in the Regenerating group. Together, our results provide, for the first time, evidence that several regenerative repulsive signals are reduced in regenerating optic nerve fibers following a combined therapy. Therefore, the treatment used made the CNS microenvironment more permissive to regeneration.

A crustacean lobula plate: Morphology, connections, and retinotopic organization.

The lobula plate is part of the lobula complex, the third optic neuropil, in the optic lobes of insects. It has been extensively studied in dipterous insects, where its role in processing flow-field motion information used for controlling optomotor responses was discovered early. Recently, a lobula plate was also found in malacostracan crustaceans. Here, we provide the first detailed description of the neuroarchitecture, the input and output connections and the retinotopic organization of the lobula plate in a crustacean, the crab Neohelice granulata using a variety of histological methods that include silver reduced staining and mass staining with dextran-conjugated dyes. The lobula plate of this crab is a small elongated neuropil. It receives separated retinotopic inputs from columnar neurons of the medulla and the lobula. In the anteroposterior plane, the neuropil possesses four layers defined by the arborizations of such columnar inputs. Medulla projecting neurons arborize mainly in two of these layers, one on each side, while input neurons arriving from the lobula branch only in one. The neuropil contains at least two classes of tangential elements, one connecting with the lateral protocerebrum and the other that exits the optic lobes toward the supraesophageal ganglion. The number of layers in the crab's lobula plate, the retinotopic connections received from the medulla and from the lobula, and the presence of large tangential neurons exiting the neuropil, reflect the general structure of the insect lobula plate and, hence, provide support to the notion of an evolutionary conserved function for this neuropil.

Retinal Toxicity of Acai Fruit (Euterpe Oleracea) Dye Concentrations in Rabbits: Basic Principles of a New Dye for Chromovitrectomy in Humans.

PURPOSE: Evaluate toxicity of acai fruit (Euterpe oleracea) dye concentrations in a rabbit model. METHODS: Rabbits were injected intravitreously with 10%, 25%, and 35% acai dye concentrations. Control eyes received balanced salt solution (BSS). Electroretinogram (ERG), fundus imaging, fluorescein angiography (FA), optical coherence tomography (OCT), and light and transmission electron microscopy (LM/TEM) were performed. RESULTS: Fundus imaging showed increased vitreous opacity with increased dye concentrations. FA and OCT showed normality with all concentrations. Comparisons between BSS and dye concentrations were analyzed using Kruskal-Wallis and Mood's median test (p < 0.05). At 24 h, ERGs showed reduced amplitudes from baseline in all eyes. Median b-wave amplitudes nonsignificantly decreased and latency increased with 10% and 25%; findings were significant (p < 0.05) for 35%. LM and TEM showed no abnormalities for 10% and 25%. With 35%, TEM showed ganglion cell edema at 24 h that resolved after 7 days. Vacuolization, multilamellar bodies, and nerve bundle damage occurred at 24 h/7 days in the inner nuclear layer. Mitochondrial cristae disruption occurred in the inner photoreceptor segment at 24 h that decreased by 7 days. CONCLUSION: Ten and twenty-five percent concentrations were safe and may improve identification of the posterior hyaloid and internal limiting membrane during chromovitrectomy in humans.

Characterization of primary cilia during the differentiation of retinal ganglion cells in the zebrafish.

BACKGROUND: Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. The primary cilium, as a signaling hub in the cell, may have a role during this process but its presence and localization during RGC generation, and its contribution to the process of cell differentiation, have not been previously assessed in vivo. METHODS: In this work we analyzed the distribution of primary cilia in vivo using laser scanning confocal microscopy, as well as their main ultrastructural features by transmission electron microscopy, in the early stages of retinal histogenesis in the zebrafish, around the time of RGC generation and initial differentiation. In addition, we knocked-down ift88 and elipsa, two genes with an essential role in cilia generation and maintenance, a treatment that caused a general reduction in organelle size. The effect on retinal development and RGC differentiation was assessed by confocal microscopy of transgenic or immunolabeled embryos. RESULTS: Our results show that retinal neuroepithelial cells have an apically-localized primary cilium usually protruding from the apical membrane. We also found a small proportion of sub-apical cilia, before and during the neurogenic period. This organelle was also present in an apical position in neuroblasts during apical process retraction and dendritogenesis, although between these stages cilia appeared highly dynamic regarding both presence and position. Disruption of cilia caused a decrease in the proliferation of retinal progenitors and a reduction of neural retina volume. In addition, retinal histogenesis was globally delayed albeit RGC layer formation was preferentially reduced with respect to the amacrine and photoreceptor cell layers. CONCLUSIONS: These results indicate that primary cilia exhibit a highly dynamic behavior during early retinal differentiation, and that they are required for the proliferation and survival of retinal progenitors, as well as for neuronal generation, particularly of RGCs.

Apoptosis durante el desarrollo embrionario de la retina de tortuga (Trachemys scripta elegans) / Apoptosis in turtle embryonic retina (Trachemys scripta elegans)

La apoptosis o muerte celular programada es un proceso que ocurre durante el desarrollo del sistema nervioso. El objetivo de este estudio fue observar los patrones de apoptosis que se producen durante el desarrollo embrionario de la retina, desde el estadio S16 al momento del nacimiento, mediante miscoscopía óptica y electrónica. Se utilizaron retinas de embriones de tortuga. Nuestros datos muestran que los primeros signos de apoptosis comienzan en el estadio S16, en la capa nuclear interna y alcanzan su máxima densidad tanto en la capa nuclear interna como en la capa de células ganglionares en S20, para extinguirse, prácticamente, en el momento del nacimiento. Por otra parte, la apoptosis sigue un gradiente centro-periferia.

Apoptosis or programmed cell death is a process that occurs during development of the nervous system. The aim of this study was to observe the patterns of apoptosis that occur during embryonic development of the retina from the stage S16 at birth, by light and electron miscoscopia. Turtle embryonic retinas were used for the study. Our data show that the first signs of apoptosis begins at stage S16 in the inner nuclear layer and reaches maximum density both in the inner nuclear layer and the ganglion cell layer in S20 until they practically disappear at the time of birth. Furthermore, apoptosis follows a gradient center-periphery.

Dye solutions based on lutein and zeaxanthin: in vitro and in vivo analysis of ocular toxicity profiles.

PURPOSE: To study the safety profile of Lutein/Zeaxanthin(L/Z)-based natural dye solutions in in vitro and in vivo models. MATERIAL AND METHODS: In vitro cytotoxicity and cellular growth experiments were carried out on ARPE-19 and human corneal epithelial (HCE) cell lines using different L/Z-based dye solutions, either alone or in association with brilliant blue (BB) or trypan blue (TB). Light and transmission electron microscopy studies were performed seven days after intravitreal injection of dye solutions in rabbits. Electroretinogram (ERG) recordings were taken at baseline and before histopathology. RESULTS: In vitro cytotoxicity assays demonstrated that the different L/Z-based solutions (from 0.3 to 2%), either alone or in association with BB (0.025%) or TB (0.04%), did not significantly alter mitochondrial activity (≤15%) in the cell lines tested. In addition, in vitro cell growth was inhibited by up to 60% depending on the dye solution, and in direct proportion to the concentration assayed. There was no evidence of structural alterations in the neurosensory retina, retinal pigment epithelium (RPE), or choriocapillaris-choroidal complex. b-Wave ERG records showed no significant differences (±15.2%) in comparison with baseline. CONCLUSIONS: L/Z-based dye solutions demonstrated a safe profile in in vitro and in vivo models, and may be a useful tool for staining intraocular structures.

Características morfológicas de la retina del calderón común (Globicephala melas; traill, 1809) y su relación con el hábitat / Morphological characteristics of pilot whales retina (Globicephala melas; traill, 1809) and their relationship to habitat

Existe una estrecha relación entre las características morfológicas de los sistemas sensoriales, su funcionamiento y el hábitat al que están adaptados los organismos. En este sentido, de todos los mamíferos marinos estudiados, los cetáceos son los que más profundamente han modificado su estructura y fisiología ocular por su estrecha adaptación a una vida exclusivamente acuática. Para aportar más datos a la literatura, el objetivo de este trabajo es describir morfológicamente la retina de la ballena piloto a través de técnicas de microscopia óptica, con el fin de relacionarla con su adaptación al medio acuático. Nuestros datos muestran que la retina de Globicephala melas se organiza de acuerdo al mismo plan básico de los vertebrados. Tiene un grosor medio alrededor de 330±23 µm en las zonas de alta densidad de células ganglionares y 175±2 µm en la zona periférica. La capa de los fotorreceptores se corresponde con el 45% del grosor de la retina total. Presenta largos segmentos externos. La capa más característica de cetáceos en general y de Globicephala melas en particular, es la capa de células ganglionares. Su grosor, de 77,76±37,26 siendo la más variable de toda la retina. Esta capa presenta baja densidad celular pero tamaños excepcionalmente grandes, de 10 a 75 µm (promedio de 33,5 µm), denominadas células ganglionares gigantes.

There is a close relationship between morphological features of sensory systems, their function and habitat to which these organisms are adapting. In this sense, of all marine mammals that have been studied, cetaceans are the ones that have profoundly changed structure and ocular physiology in their adaptation to an exclusively aquatic life. To add further data to the literature, the aim of this paper is to describe morphologically the retina of the pilot whale through optical microscopy and relate their adaptation to the aquatic environment. Our data show that the retina of the long-finned pilot whale is organized according to the same basic plan of vertebrates. It has an average thickness of about 330±23 microns in areas of high ganglion cell density of 175±2 microns in the peripheral zone. Photoreceptor layer corresponds to 45% of total thickness of the retina and has long outer segments. The most significant characteristic of cetaceans in general and long-finned pilot whale in particular, is the ganglion cell layer. Thickness of 77.76±37.26 being the most variable of the entire retina. This layer has a low density but exceptionally large cell size of 10 to 75 microns (average of 33.5 microns), known as giant ganglion cells.

Morphology of the eyeball from the Humpback whale (Megaptera novaeangliae).

Aquatic mammals underwent morphological and physiological adaptations due to the transition from terrestrial to aquatic environment. One of the morphological changes regards their vision since cetaceans' eyes are able to withstand mechanical, chemical, osmotic, and optical water conditions. Due to insufficient information about these animals, especially regarding their sense organs, this study aimed to describe the morphology of the Humpback whale (Megaptera novaeangliae) eyeball. Three newborn females, stranded dead on the coast of Sergipe and Bahia, Brazil, were used. Samples were fixed in a 10% formalin solution, dissected, photographed, collected, and evaluated through light and electron microscopy techniques. The Humpback whale sclera was thick and had an irregular surface with mechanoreceptors in its lamina propria. Lens was dense, transparent, and ellipsoidal, consisting of three layers, and the vascularized choroid contains melanocytes, mechanoreceptors, and a fibrous tapetum lucidum. The Humpback whale eyeball is similar to other cetaceans and suggests an adaptation to diving and migration, contributing to the perception of differences in temperature, pressure, and lighting.

Anaglyph of retinal stem cells and developing axons: selective volume enhancement in microscopy images.

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high-quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three-dimensional (3-D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high-quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3-D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells.

Morphological evidence of neurotoxicity in retina after methylmercury exposure.

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 µgg(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure.

[Characteristics associated with visual improvement after focal photocoagulation in diabetic macular edema]. / Características asociadas con la mejoría visual después de la fotocoagulación en pacientes con edema macular diabético.

BACKGROUND: Photocoagulation reduces the incidence of moderate visual loss in patients with clinically significant macular edema (CSME). However, the incidence of visual improvement after treatment is low. Diffuse retinal thickening identified with optical coherence tomography (OCT) and visual loss before treatment have been associated with visual improvement. OBJECTIVE: To identify features associated with visual improvement after focal photocoagulation for CSME. METHODS: Observational, analytical, longitudinal, retrospective study. Diabetic patients with CSME treated with focal photocoagulation were evaluated. Study variables were: visual improvement (defined as a gain of one or more lines in a vision chart) and ocular and systemic features before treatment. Clinical and ancillary test variables were compared between eyes with and without visual improvement. Mann-Whitney's U was used to compare quantitative variables; χ(2) was used for qualitative variables. RESULTS: 99 eyes of 72 patients mean age 60.6 years, 59.6% females. 38.4% had visual improvement after treatment. The incidence of visual improvement was higher in eyes with vision < 20/40 before treatment (58.8 vs. 16.7%, p < 0.001, relative risk 3.53). In eyes with vision = 20/40 before treatment, the probability of improving was 12 times higher when field 7 of the OCT map had no thickening (p < 0.001). The remaining variables showed no significant associations. CONCLUSIONS: Up to 48% of treated eyes could achieve visual improvement. Consistently with previous reports, the probability increased in eyes with vision < 20/40 before photocoagulation; when vision before treatment was = 20/40, the incidence of visual improvement increased significantly in eyes without thickening in the temporal parafoveal retina.

Novel vitreous modulators for pharmacologic vitreolysis in the treatment of diabetic retinopathy.

Vitreous constitutes about 80% of the volume of the human eye. It is an extended extracellular matrix that is composed of collagen, hyaluronan, and other extracellular matrix molecules, but mostly water. In both health as well as disease, especially diabetic retinopathy (DR), special attention should be drawn to the posterior vitreous cortex and its relation to the retinal surface. The important role of vitreous in the pathogenesis of proliferative DR has already been demonstrated by several experimental and clinical studies. Thus, vitreo-retinal separation by pharmacologic vitreolysis and/or removal by surgical means are appropriate approaches to interrupt the pathogenic contribution of vitreous and prevent progression of diabetic retinopathy to more advanced stages. This review describes various aspects of the molecular morphology and structural anatomy of vitreous and the vitreo-retinal interface, as well as the role of vitreous in the pathophysiology of DR. Lastly, this treatise provides a critical analysis of novel vitreous modulators for pharmacologic vitreolysis in the treatment of DR. Microplasmin is currently the most promising approach to treat vitreoretinal traction by pharmacologic vitreolysis.

A six-rhabdomere, open rhabdom arrangement in the eye of the chrysanthemum beetle Phytoecia rufiventris: some ecophysiological predictions based on eye anatomy

We are describing a rhabdom organization of the eye of the chrysanthemum beetle Phytoecia rufiventris that to date has not been described from any other insect. In cerambycid beetles free rhabdomeres, forming a circular, open rhabdom, surround a central rhabdom made up of the rhabdomeres of one or two cells. In Phytoecia rufiventris the central rhabdomeres are missing throughout the eye and the microvilli of the outer 6 rhabdomeres are regularly oriented in three directions. Following the classification of rhabdom types suggested by Wachmann (1979), we suggest to name the rhabdom arrangement seen in the retina of Phytoecia rufiventris [quot ]Grundmuster 3[quot ]. This pattern ought to facilitate polarization sensitivity and movement perception, features that agree with the behavioural repertoire of Phytoecia rufiventris.

A six-rhabdomere, open rhabdom arrangement in the eye of the chrysanthemum beetle Phytoecia rufiventris: some ecophysiological predictions based on eye anatomy

We are describing a rhabdom organization of the eye of the chrysanthemum beetle Phytoecia rufiventris that to date has not been described from any other insect. In cerambycid beetles free rhabdomeres, forming a circular, open rhabdom, surround a central rhabdom made up of the rhabdomeres of one or two cells. In Phytoecia rufiventris the central rhabdomeres are missing throughout the eye and the microvilli of the outer 6 rhabdomeres are regularly oriented in three directions. Following the classification of rhabdom types suggested by Wachmann (1979), we suggest to name the rhabdom arrangement seen in the retina of Phytoecia rufiventris [quot ]Grundmuster 3[quot ]. This pattern ought to facilitate polarization sensitivity and movement perception, features that agree with the behavioural repertoire of Phytoecia rufiventris.(AU)

Immunoelectron microscopy reveals the presence of neurofilament proteins in retinal terminals undergoing dark degeneration.

After nerve crushing or section, the distal stump undergoes morphological changes described as Wallerian degeneration (WD). Immediately after nerve injury, early ultrastructural alterations occur in the terminal boutons, a process known as terminal degeneration (TD), which occurs before degeneration of the axon and leads to electrophysiological impairment. In this study we investigated the presence of neurofilament (NF) proteins in TD and compared the results with degeneration in the optic nerve. Young adult Wistar rats were submitted to bilateral enucleation and perfused after 24 h, 48 h and 1 week. Optic nerves (ON) and superior colliculus (SC) segments were processed for electron microscopy (EM) and immunoelectron microscopy (IEM) for NF subunits. Analysis of ultrathin sections of SC, at 24 h, revealed terminals undergoing TD. At 48 h and 1 week after enucleation, there was a clear increase in the number of degenerating terminals. The cytoarchitecture of the optic nerve did not change considerably at 24 h, but it was progressively altered at 48 h and 1 week after enucleation, when we observed intense astrogliosis, and most fibers exhibited dark degeneration (DD). The IEM for the NF subunits of normal ON showed gold particles located along the filaments, but we did not observe labeling for neurofilament proteins in normal retinal terminals. However, 48 h after lesion, we observed immunogold particles for the NF proteins in fibers undergoing DD and on terminals undergoing TD. Therefore, we can conclude that NF proteins participate in the process of TD, and this event occurs before complete axonal degeneration, suggesting different mechanisms for TD and DD.

Differential expression of connexins during histogenesis of the chick retina.

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species.

Subretinal injection of preservative-free triamcinolone acetonide and supernatant vehicle in rabbits: an electron microscopy study.

BACKGROUND: To evaluate the effect of injections of benzyl alcohol (BA)-free triamcinolone acetonide (TA) solution (MTA-PF) and the supernatant vehicle of TA (STA) containing BA into the subretinal space of rabbit eyes. METHODS: Sixteen rabbits underwent vitrectomy and subretinal injection of 0.02 ml of either 40 mg/ml MTA-PF, 40 mg/ml STA, or balanced salt solution (BSS). The animals were examined 6, 12, and 24 hours and 14 days after the procedure by fundus examination and fluorescein angiography (FA), as well as histological studies by light and transmission electron microscopy. The histological injury was classified in four stages: (1) stage 1, photoreceptor outer segment injury, (2) stage 2, stage 1 + photoreceptor inner segment injury, (3) stage 3, stage 2 + outer nuclear layer damage, and (4) stage 4, stage 3 + retinal pigment epithelium (RPE) damage. RESULTS: FA showed no window defects in areas where MTA-PF, STA, or BSS have been injected. Histological examination revealed that subretinal BSS-injection resulted in stage 1 damage during entire follow-up. Subretinal injection of MTA-PF resulted in damage stage 2 at 24 h and 14 days after surgery. However, at the STA position, stage 3 damage was noted 24 h and 14 days postoperatively. No RPE or choroidal damage was observed. CONCLUSIONS: The histological lesions induced by subretinal STA were more relevant than the damage induced by MTA-PF. The vehicle BA may be involved in these abnormalities. The data indicate that care must be taken when using TA during internal limiting membrane peeling in macular hole surgery, due to the possibility of unintentional subretinal migration and for retinal pharmacotherapy.

Intercellular junctions in rabbit eye ora serrata.

Summary The aim of this study was to describe and localize the intercellular junctions in the ora serrata region of albino and pigmented rabbit eyes. Eyes of albino and pigmented rabbits were fixed and processed for transmission electron microscopy. Light and electron microscope examination was carried out on semithin and ultrathin sections. The ora serrata region showed adherens, gap and tight junctions in the retinal and ciliary margins of albino and pigmented rabbit eyes. In the retinal margin, zonulae adherens between Müller cells and photoreceptors are associated with tight junctions. In the ciliary margin, epithelial cells are joined by adherens, gap and tight junctions localized between apical and apicolateral cell membranes. Tight junctions appear as zonulae occludens in the non-pigmented apicolateral cell membranes and as tight focal junctions between pigmented and non-pigmented apical cell membranes. Between the ciliary and retinal margins there are adherens and tight focal junctions which attach pigmented apical cell membranes to adjacent cells. There were no differences in the distribution of intercellular junctions between albino and pigmented rabbits.