Retinal Neuropathy in IGT Stage of OLETF Rats: Another Characteristic Change of Diabetic Retinopathy.

AIMS: We investigated the changes of retinal structure in normal glucose tolerance (NGT), impaired glucose tolerance (IGT), diabetes mellitus (DM), and diabetic kidney disease (DKD) stages in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: We assigned OLETF rats to four groups based on their OGTT results and 24 h urinary microalbumin (24 h UMA) levels: NGT, IGT, DM, and DKD groups. We observed the structural and the corresponding pathological changes and quantified the expression of HIF-1α, iNOS, NF-κB, VEGF, ICAM-1, and occludin in the retina. RESULTS: Significant damage to the retinal structure, especially in retinal ganglion cells (RGCs), was observed in the IGT stage. The expression of HIF-1α, iNOS, NF-κB, VEGF, and ICAM-1 was significantly upregulated, while that of occludin was downregulated. CONCLUSION: Significant retinal neuropathy occurs in the IGT stage. Inflammation and hypoxia may damage the blood retina barrier (BRB), leading to diabetic retinopathy.

Early (5-Day) Onset of Diabetes Mellitus Causes Degeneration of Photoreceptor Cells, Overexpression of Incretins, and Increased Cellular Bioenergetics in Rat Retina.

The effects of early (5-day) onset of diabetes mellitus (DM) on retina ultrastructure and cellular bioenergetics were examined. The retinas of streptozotocin-induced diabetic rats were compared to those of non-diabetic rats using light and transmission electron microscopy. Tissue localization of glucagon-like-peptide-1 (GLP-1), exendin-4 (EXE-4), and catalase (CAT) in non-diabetic and diabetic rat retinas was conducted using immunohistochemistry, while the retinal and plasma concentration of GLP-1, EXE-4, and CAT were measured with ELISA. Lipid profiles and kidney and liver function markers were measured from the blood of non-diabetic and diabetic rats with an automated biochemical analyzer. Oxygen consumption was monitored using a phosphorescence analyzer, and the adenosine triphosphate (ATP) level was determined using the Enliten ATP assay kit. Blood glucose and cholesterol levels were significantly higher in diabetic rats compared to control. The number of degenerated photoreceptor cells was significantly higher in the diabetic rat retina. Tissue levels of EXE-4, GLP-1 and CAT were significantly (p = 0.002) higher in diabetic rat retina compared to non-diabetic controls. Retinal cellular respiration was 50% higher (p = 0.004) in diabetic (0.53 ± 0.16 µM O2 min-1 mg-1, n = 10) than in non-diabetic rats (0.35 ± 0.07 µM O2 min-1 mg-1, n = 11). Retinal cellular ATP was 76% higher (p = 0.077) in diabetic (205 ± 113 pmol mg-1, n = 10) than in non-diabetic rats (116 ± 99 pmol mg-1, n = 12). Thus, acute (5-day) or early onslaught of diabetes-induced hyperglycemia increased incretins and antioxidant levels and oxidative phosphorylation. All of these events could transiently preserve retinal function during the early phase of the progression of diabetes.

Livelayer: a semi-automatic software program for segmentation of layers and diabetic macular edema in optical coherence tomography images.

Given the capacity of Optical Coherence Tomography (OCT) imaging to display structural changes in a wide variety of eye diseases and neurological disorders, the need for OCT image segmentation and the corresponding data interpretation is latterly felt more than ever before. In this paper, we wish to address this need by designing a semi-automatic software program for applying reliable segmentation of 8 different macular layers as well as outlining retinal pathologies such as diabetic macular edema. The software accommodates a novel graph-based semi-automatic method, called "Livelayer" which is designed for straightforward segmentation of retinal layers and fluids. This method is chiefly based on Dijkstra's Shortest Path First (SPF) algorithm and the Live-wire function together with some preprocessing operations on the to-be-segmented images. The software is indeed suitable for obtaining detailed segmentation of layers, exact localization of clear or unclear fluid objects and the ground truth, demanding far less endeavor in comparison to a common manual segmentation method. It is also valuable as a tool for calculating the irregularity index in deformed OCT images. The amount of time (seconds) that Livelayer required for segmentation of Inner Limiting Membrane, Inner Plexiform Layer-Inner Nuclear Layer, Outer Plexiform Layer-Outer Nuclear Layer was much less than that for the manual segmentation, 5 s for the ILM (minimum) and 15.57 s for the OPL-ONL (maximum). The unsigned errors (pixels) between the semi-automatically labeled and gold standard data was on average 2.7, 1.9, 2.1 for ILM, IPL-INL, OPL-ONL, respectively. The Bland-Altman plots indicated perfect concordance between the Livelayer and the manual algorithm and that they could be used interchangeably. The repeatability error was around one pixel for the OPL-ONL and < 1 for the other two. The unsigned errors between the Livelayer and the manual algorithm was 1.33 for ILM and 1.53 for Nerve Fiber Layer-Ganglion Cell Layer in peripapillary B-Scans. The Dice scores for comparing the two algorithms and for obtaining the repeatability on segmentation of fluid objects were at acceptable levels.

Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.

Ferrostatin-1 attenuates ferroptosis and protects the retina against light-induced retinal degeneration.

Degenerative retinal diseases, including age-related macular degeneration, are serious diseases that may lead to irreversible retinal neuron damage and permanent vision impairment. There are currently no effective treatments for these diseases due to our incomplete understanding of the underlying pathological mechanisms. Ferroptosis, a newly identified iron-dependent mode of cell death, is implicated in various diseases. However, it is unknown whether ferroptosis is involved in light-induced retinal degeneration. In this study, we found that light exposure significantly reduced the viability of photoreceptor cells in vitro and induced pro-ferroptotic changes, including iron accumulation, mitochondrial shrinkage, glutathione depletion, increased malondialdehyde (MDA), and decreased protein expression of SLC7A11 and GPX4. The effects of light exposure on ferroptosis were attenuated by ferrostatin-1. Consistently, the results of in vivo studies demonstrated that ferrostatin-1 protected against light-induced ferroptosis. And it exerted therapeutic effects by inhibiting neuroinflammation and prevented the effects of light exposure on the structure and function of the retina. The findings reveal an important role of ferroptosis in the pathogenesis of light-induced retinal degeneration and suggest that ferroptosis may be a novel treatment target for preventing retinal degeneration.

Renin-angiotensin system impairs macrophage lipid metabolism to promote age-related macular degeneration in mouse models.

Metabolic syndrome, a condition involving obesity and hypertension, increases the risk of aging-associated diseases such as age-related macular degeneration (AMD). Here, we demonstrated that high-fat diet (HFD)-fed mice accumulated oxidized low-density lipoprotein (ox-LDL) in macrophages through the renin-angiotensin system (RAS). The ox-LDL-loaded macrophages were responsible for visual impairment in HFD mice along with a disorder of the retinal pigment epithelium (RPE), which is required for photoreceptor outer segment renewal. RAS repressed ELAVL1, which reduced PPARγ, impeding ABCA1 induction to levels that are sufficient to excrete overloaded cholesterol within the macrophages. The ox-LDL-loaded macrophages expressed inflammatory cytokines and attacked the RPE. An antihypertensive drug, angiotensin II type 1 receptor (AT1R) blocker, resolved the decompensation of lipid metabolism in the macrophages and reversed the RPE condition and visual function in HFD mice. AT1R signaling could be a future therapeutic target for macrophage-associated aging diseases, such as AMD.

Stereopsis and retinal microstructures following macular hole surgery.

The aim of this prospective study was to evaluate the changes in stereopsis in patients who underwent vitrectomy for macular hole (MH) and assess the relationship between stereopsis and retinal microstructures. Fifty-two patients who underwent successful vitrectomy for unilateral MH and 20 control participants were recruited. We examined stereopsis using the Titmus Stereo Test (TST) and TNO stereotest (TNO), optical coherence tomography, and best-corrected visual acuity measurements, preoperatively, and at 3, 6, and 12 months postoperatively. As a result, preoperative and postoperative 3, 6, and 12-month values of stereopsis assessed by TST (log) were 2.7, 2.2, 2.2, and 2.2, respectively. TNO (log) were 2.8, 2.5, 2.4, and 2.4, respectively. Stereopsis in MH after surgery was significantly worse than that in normal participants (p < 0.001). Preoperative TST significantly correlated with MH size and defect length of external limiting membrane (ELM). Postoperative TST demonstrated significant correlation with the preoperative ELM defect length, and postoperative TNO was associated with the preoperative interdigitation zone defect length. Vitrectomy for MH significantly improved stereopsis, although not to normal levels. The ELM defect lengths, which approximately correspond to TST circles, are prognostic factors for postoperative stereopsis by TST. The interdigitation zone defect length, similar in size to the TNO index, is a prognostic factor for postoperative stereopsis by TNO.

The Impact of the Timing of Dosing on the Severity of UNC569-Induced Ultrastructural Changes in the Mouse Retina.

Mer proto-oncogene tyrosine kinase (MerTK), expressed in the retinal pigment epithelium (RPE), regulates the phagocytosis of shed photoreceptor outer segments. To investigate the influence of dosing time on MerTK inhibitor UNC569-induced retinal toxicity, UNC569 at 100 mg/kg was orally administered to male mice at 2 different Zeitgeber times (ZT5.5 or ZT22) for 28 days. Electron microscopy was conducted at ZT2 after the final dosing. Additionally, the visual cycle components (11-cis-retinal, all-trans-retinal, all-trans-retinol, and 11-cis-retinol), which play an important role in maintaining retinal homeostasis, were quantified by liquid chromatography/mass spectrometry/mass spectrometry. Under electron microscopic examination, the number of phagosomes and phagolysosomes in the RPE increased in both the ZT5.5 and ZT22 administered groups, while endoplasmic reticulum dilatation in the RPE and chromatin aggregation of photoreceptor nuclei were observed only in the ZT22 administered group. No change was observed in any of the visual cycle components. These results suggest that the timing of the dosing in relation to the physiological MerTK phosphorylation affected the severity of changes in the RPE, leading to the apoptosis of the photoreceptor cells.

Loss of ß-catenin via activated GSK3ß causes diabetic retinal neurodegeneration by instigating a vicious cycle of oxidative stress-driven mitochondrial impairment.

Synaptic neurodegeneration of retinal ganglion cells (RGCs) is the earliest event in the pathogenesis of diabetic retinopathy. Our previous study proposed that impairment of mitochondrial trafficking by hyperphosphorylated tau is a potential contributor to RGCs synapse degeneration. However, other molecular mechanisms underlying mitochondrial defect in diabetic retinal neurodegeneration remain to be elucidated. Here, using a high-fat diet (HFD)-induced diabetic mouse model, we showed for the first time that downregulation of active ß-catenin due to abnormal GSK3ß activation caused synaptic neurodegeneration of RGCs by inhibiting ROS scavenging enzymes, thus triggering oxidative stress-driven mitochondrial impairment in HFD-induced diabetes. Rescue of ß-catenin via ectopic expression of ß-catenin with a recombinant adenoviral vector, or via GSK3ß inhibition by a targeted si-GSK3ß, through intravitreal administration, abrogated the oxidative stress-derived mitochondrial defect and synaptic neurodegeneration in diabetic RGCs. By contrast, ablation of ß-catenin by si-ß-catenin abolished the protective effect of GSK3ß inhibition on diabetic RGCs by suppression of antioxidant scavengers and augmentation of oxidative stress-driven mitochondrial lesion. Thus, our data identify ß-catenin as a part of an endogenous protective system in diabetic RGCs and a promising target to develop intervention strategies that protect RGCs from neurodegeneration at early onset of diabetic retinopathy.

Modeling and Rescue of RP2 Retinitis Pigmentosa Using iPSC-Derived Retinal Organoids.

RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death.

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization.

Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging.

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

A Common Outer Retinal Change in Retinal Degeneration by Optical Coherence Tomography Can Be Used to Assess Outcomes of Gene Therapy.

Identifying early disease hallmarks in animal models with slow disease progression may expedite disease detection and assessment of treatment outcomes. Using optical coherence tomography, a widely applied noninvasive method for monitoring retinal structure changes, we analyzed retinal optical sections from six mouse lines with retinal degeneration caused by mutations in different disease-causing genes. While images from wild-type mice revealed four well-separated hyper-reflective bands in the outer retina (designated as outer retina reflective bands, ORRBs) at all ages, the second band (ORRB2) and the third band (ORRB3) were merged in retinas of five mutant mouse lines at early ages, suggesting the pathological nature of this alteration. This ORRB change appeared to be degenerating photoreceptor related, and occurred before obvious morphological changes that can be identified on both hematoxylin and eosin-stained sections and electron microscopic sections. Importantly, the merging of ORRB2 and ORRB3 was reversed by treatment with adeno-associated viral vector-mediated gene replacement therapies, and this restoration occurred much earlier than measurable functional or structural improvement. Our data suggest that the ORRB change could be a common hallmark of early retinal degeneration and its restoration could be used for rapid and noninvasive assessment of therapeutic effects following gene therapy or other treatment interventions.

Astragalus root extract inhibits retinal cell apoptosis and repairs damaged retinal neovascularization in retinopathy of prematurity.

Since the functions of Astragalus root extract in retinopathy remain to be unraveled, this study is performed to elucidate whether Astragalus root extract functions in retinal cell apoptosis and angiogenesis in retinopathy of prematurity (ROP). Newborn mice were selected for establishing mice models of oxygen-induced retinopathy (OIR), which were treated with high-, medium- or low-Astragalus root extract. Evans Blue (EB) was perfused to detect the blood retinal barrier. Additionally, the vascular morphology, number of endothelial cell nuclei of neovascularization, proliferation of blood vessels, ultrastructural changes were determined via a series of assays. Moreover, levels of reactive oxygen species (ROS), expression of other factors such as VEGF, PEDF, IGF-1, HIF-1α, Bax, Bcl-2, eNOS, nNOS, and iNOS were detected. Astragalus root extract was found to protect blood-retinal barrier in the OIR model mice through repairing the structure and morphology of retina, inhibiting ROS production, retinal cell apoptosis, as well as improving retinal vascular angiogenesis. Astragalus root extract was also found to decrease VEGF and HIF-1α expression, but enhance PEDF and IGF-1 expression in the OIR model mice, thereby protecting retinas in ROP. This study highlights that Astragalus root extract is able to suppress retinal cell apoptosis and repair damaged retinal neovascularization in ROP, which provides basis for ROP therapy.

CLINICAL FINDINGS IN TRIAMCINOLONE-ASSOCIATED MACULOPATHY.

PURPOSE: To describe a crystalline retinopathy observed in patients greater than 1 year after intravitreal injection of triamcinolone acetonide (IVTA). METHODS: A retrospective, interventional, noncomparative, single-center case series of patients who received IVTA and developed subsequent crystalline retinopathy lasting greater than 1 year after injection. RESULTS: Eighteen eyes of 16 patients in which preretinal crystals were observed >1 year after IVTA were included in the study, with a mean follow-up (range) of 5.8 years (1.1-9.2) after IVTA. The crystals were refractile, not visible on fluorescein nor indocyanine green angiography, exhibited slow dissolution and movement, and were occasionally distributed in a circular fashion. Optical coherence tomography confirmed the preretinal and/or subhyaloid location of crystals. CONCLUSION: Macular crystals can persist for years after IVTA. The crystals localize to the preretinal or subhyaloid space, are angiographically silent, can exhibit slow dissolution and movement, may be distributed in a circular fashion reflecting the bursa premacularis, and appear nonpathologic.

Neuroprotective effect of insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA hydrogel on diabetic retinopathy in rats.

BACKGROUND: To pursuit effective sustained release systems for insulin to treat diabetic retinopathy (DR), a novel insulin delivering system was developed via loading onto chitosan nanoparticles/poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) hydrogel (ICNPH). METHODS AND MATERIALS: Examinations including electroretinography, HE staining, transmission electron microscopy, terminal deoxynucleotidyl transferased UTP nick-end labeling, immunofluorescence, Western blot, and real-time polymerase chain reaction were performed to evaluate the neuroprotective efficacy of ICNPH on DR by a single subconjunctival injection. RESULTS: Compared with the insulin, blank, and sham treatment groups, subconjunctival injection of ICNPH significantly reduced the decrease of scotopic B-wave amplitude, alleviated retinal micro- and ultrastructural changes, and reduced retinal cell apoptosis caused in DR rats. Meanwhile, a significant reduction of vascular endothelial growth factor and glial fibrillary acidic protein expression as well as a remarkable increase in Occludin expression was also found in retinas in ICNPH group compared with the sham treatment group. CONCLUSION: The results indicate that ICNPH has sufficient neuroprotective effect on retinas through subconjunctival injection in DR rats and facilitates controlled insulin delivery. It might be one of the therapeutic strategies for DR in the near future.

Typhae pollen polysaccharides ameliorate diabetic retinal injury in a streptozotocin-induced diabetic rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient traditional Chinese medicine, Typhae Pollen (TP) is commonly used to treat fundus haemorrhage because it improves blood circulation. AIMS OF THE STUDY: This study evaluated the role of the main TP component, polysaccharides (TPP), on diabetic retinopathy (DR) and its possible mechanisms of inhibiting inflammation and improving blood circulation. MATERIALS AND METHODS: After successful establishment of a diabetic rat model, TPP was administered to diabetic rats for treatment, and the rats were sacrificed at 12 weeks. Retinal electrophysiology and ultrastructures were observed, and serum interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) levels were also measured. Changes in the retinal expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were examined by immunofluorescence. A mouse model of acute blood stasis was then established, and the effects of TPP on haemorheology were observed. The anti-inflammatory effect of TPP was analysed based on the changes in abdominal capillary permeability and the degree of auricle swelling in the mice. RESULTS: In streptozotocin (STZ)-induced DR rats, TPP (0.4 g/kg) treatment restored electrophysiology indexes and retinal ultrastructures, reduced serum IL-6 and TNF-α levels, decreased VEGF and bFGF expression in retinal tissues, and improved haemorheology indexes. Moreover, TPP reduced abdominal capillary permeability and relieved auricle swelling in a dose-dependent manner. CONCLUSIONS: TPP treatment ameliorated DR by inhibiting inflammation and improving blood circulation.

Acute and long-term effects of trophic exposure to silver nanospheres in the central nervous system of a neotropical fish Hoplias intermedius.

Nanotechnologies are at the center of societal interest, due to their broad spectrum of application in different industrial products. The current concern about nanomaterials (NMs) is the potential risks they carry for human health and the environment. Considering that NMs can reach bodies of water, there is a need for studying the toxic effects of NMs on aquatic organisms. Among the NMs' toxic effects on fish, the interactions between NMs and the nervous system are yet to be understood. For this reason, our goal was to assess the neurotoxicity of polyvinylpyrrolidone coated silver nanospheres [AgNS (PVP coated)] and compare their effects in relation to silver ions (Ag+) in carnivorous Hoplias intermedius fish after acute and subchronic trophic exposure through the analysis of morphological (retina), biochemical (brain) and genetic biomarkers (brain and blood). For morphological biomarkers, damage by AgNS (PVP coated) in retina was found, including morphological changes in rods, cones, hemorrhage and epithelium rupture, and also deposition of AgNS (PVP coated) in retina and sclera. In the brain biomarkers, AgNS (PVP coated) did not disturb acetylcholinesterase activity. However, lowered migration of the DNA tail in the Comet Assay of blood and brain cells was observed for all doses of AgNS (PVP coated), for both acute and subchronic bioassays, and in a dose-dependent manner in acute exposure. Ag+ also reduced the level of DNA damage only under subchronic conditions in the brain cells. In general, the results demonstrated that AgNS (PVP coated) do not cause similar effects in relation to Ag+. Moreover, the lowered level of DNA damage detected by Comet Assay suggests that AgNS (PVP coated) directly interacts with DNA of brain and blood cells, inducing DNA-DNA or DNA-protein crosslinks. Therefore, the AgNS (PVP coated) accumulating, particularly in the retina, can lead to a competitive disadvantage for fish, compromising their survival.

Immunohistochemical Procedures for Characterizing the Retinal Expression Patterns of Cre Driver Mouse Lines.

The retina is a thin neural tissue sitting on the backside of the eye, composed of light-sensing cells, interneurons, and output ganglion neurons. The latter send electrical signals to higher visual centers in the brain. Transgenic mouse lines are becoming one of the most valuable mammalian animal models for the study of visual signal processing within the retina. Especially, the generation of Cre recombinase transgenic mouse lines provides a powerful tool for genetic manipulation. A key step for the utilization of transgenic lines is the characterization of their transgene expression patterns in the retina. Here we describe a standard protocol for characterizing the expression pattern of the Cre recombinase or fluorescent proteins in the retina with an immunohistochemical approach.

Preliminary study of the safety and efficacy of medium-chain triglycerides for use as an intraocular tamponading agent in minipigs.

PURPOSE: To date, only silicone oils and gases have the appropriate characteristics for use in vitreo-retinal surgery as vitreous substitutes with intraocular tamponading properties. This preliminary study evaluated the safety and efficacy of medium-chain triglycerides (MCTs) for use as a tamponading agent in minipigs. METHODS: In 15 minipigs, 15 right eyes underwent vitrectomies followed by injection of MCT tamponade (day 1). Two groups were defined. In Group A (ten eyes), the surgical procedure before MCT injection included induced rhegmatogenous retinal detachment (RRD), retina flattening, and retinopexy. In Group B (five eyes), MCT was injected without inducing RRD; in these eyes, MCT was removed on day 90. Pigs were sacrificed on day 45 (Group A) or 120 (Group B). Eyes were examined on days 1, 5, 15, and 45 in both groups and on days 90 and 120 in Group B. In Group B only, we performed bilateral electroretinography examinations on days 1 and 120, and histological examinations of MCTs and controlateral eyes were performed after sacrifice. RESULTS: In Group A eyes (n = 9; one eye was non-assessable), on day 45, the retina was flat in seven eyes and two RRD detachments were observed in insufficiently MCT-filled eyes. In Group B, electroretinography showed no significant differences between MCT eyes and controls on days 1 or 120. Histological analyses revealed no signs of retinal toxicity. CONCLUSIONS: This study showed that MCT tamponade seems to be effective and safe; however, additional studies are needed before it becomes commonly used as a tamponading agent in humans.