Dietary Alcohol Consumption Elicits Corneal Toxicity Through the Generation of Cellular Oxidative Stress.

Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity.

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Compatibility of intravitreally applied epidermal growth factor and amphiregulin.

INTRODUCTION: To examine the compatibility of intravitreally injected epidermal growth factor (EGF) and amphiregulin as EGF family member. METHODS: Four rabbits (age: 4 months; body weight: 2.5 kg) received three intravitreal injections of EGF (100 ng) uniocularly in monthly intervals and underwent ocular photography, tonometry, biometry, and optical coherence tomography. After sacrificing the rabbits, the globes were histomorphometrically examined. In a second study part, eyes of 22 guinea pigs (age: 2-3 weeks) received two intravitreal administrations of amphiregulin (10 ng) or phosphate buffered solution (PBS) in 10-day interval, or were left untouched. Ten days after the second injection, the guinea pigs were sacrificed, the enucleated eyes underwent histological and immune-histological examinations. RESULTS: The rabbit eyes with EGF injections versus the contralateral untouched eyes did not show significant differences in intraocular pressure (7.5 ± 2.4 mmHg vs. 6.8 ± 2.2 mmHg; P = 0.66), retinal thickness (158 ± 5 µm vs. 158 ± 3 µm; P = 1.0), cell counts in the retinal ganglion cell layer (3.3 ± 1.7 cells/150 µm vs. 3.0 ± 1.4 cells/150 µm; P = 0.83), inner nuclear layer (46.4 ± 23.2 cells/150 µm vs. 39.6 ± 6.4 cells/150 µm; P = 0.61), and outer nuclear layer (215 ± 108 cells/150 µm vs. 202 ± 47 cells/150 µm; P = 0.83), or any apoptotic retinal cells. The guinea pig eyes injected with amphiregulin versus eyes with PBS injections did not differ (P = 0.72) in the degree of microglial activation, and both groups did not differ from untouched eyes in number of apoptotic retinal cells and retinal gliosis. CONCLUSIONS: Intravitreal applications of EGF (100 ng) in rabbits nor intravitreal applications of amphiregulin (10 ng) in guinea pigs led to intraocular specific inflammation or any observed intraocular destructive effect. The findings support the notion of a compatibility of intraocular applied EGF and amphiregulin.

Viability of Mouse Retinal Explant Cultures Assessed by Preservation of Functionality and Morphology.

Purpose: Retinal explant cultures provide simplified systems where the functions of the retina and the effects of ocular therapies can be studied in an isolated environment. The purpose of this study was to provide insight into long-term preservation of retinal tissue in culture conditions, enable a deeper understanding of the interdependence of retinal morphology and function, and ensure the reliability of the explant technique for prolonged experiments. Methods: Retinal explants from adult mice were cultured as organotypic culture at the air-medium interface for 14 days in vitro (DIV). Retinal functionality was assessed by multielectrode array technique and morphology by immunohistochemical methods at several time points during culture. Results: Retinal explants retained viability for 14 DIV, although with diminishing neuronal activity, progressing neuronal loss, and increasing reactive gliosis. We recorded spontaneous retinal ganglion cell (RGC) activity up to 14 DIV with temporally changing distribution of RGC firing rates. Light responsiveness was measurable from RGCs for 7 DIV and from photoreceptors for 2 DIV. Apoptotic cells were detected beginning at 3 DIV with their density peaking at 7 DIV. The number of RGCs gradually decreased by 70% during 14 DIV. The change was accompanied by the loss of RGC functionality, resulting in 84% loss of electrically active RGCs. Conclusions: Retinal explants provide a valuable tool for studies of retinal functions and development of ocular therapies. However, critical for long-term use, retinal functionality was lost before structural loss, emphasizing a need for both functional and morphologic readouts to determine the overall state of the cultured retina.

Manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin, a superoxide dismutase mimetic, reduces disease severity in in vitro and in vivo models for dry-eye disease.

PURPOSE: To determine the efficacy of the superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), in vitro in human corneal epithelial (HCE-T) cells and in vivo in a preclinical mouse model for dry-eye disease (DED). METHODS: In vitro, HCE-T cultures were exposed either to tert-butylhydroperoxide (tBHP) to generate oxidative stress or to hyperosmolar conditions modeling cellular stress during DED. Cells were pre-treated with Mn-TM-2-PyP or vehicle. Mn-TM-2-PyP permeability across stratified HCE-T cells was assayed. In vivo, Mn-TM-2-PyP (0.1% w/v in saline) was delivered topically as eye drops in a desiccating stress/scopolamine model for DED. Preclinical efficacy was compared to untreated, vehicle- and ophthalmic cyclosporine emulsion-treated mice. RESULTS: Mn-TM-2-PyP protected HCE-T cells in a dose-dependent manner against tBHP-induced oxidative stress as determined by calculating the IC50 for tBHP in the resazurin, MTT and lactate dehydrogenase release cell viability assays. Mn-TM-2-PyP did not protect HCE-T cells from hyperosmolar insult. Its permeability coefficient across a barrier of HCE-T cells was 1.1 ±â€¯0.05 × 10-6 cm/s and the mass balance was 62 ±â€¯0.6%. In vivo, topical dosing with Mn-TM-2-PyP resulted in a statistically significant reduction of corneal fluorescein staining, similar to ophthalmic cyclosporine emulsion. Furthermore, Mn-TM-2-PyP significantly reduced leukocyte infiltration into lacrimal glands and prevented degeneration of parenchymal tissue. No protective effect against loss of conjunctival goblet cells was observed. Notably, Mn-TM-2-PyP did not produce ocular toxicity when administered topically. DISCUSSION: Our data suggest that Mn-TM-2-PyP, a prototypic synthetic metalloporphyrin compound with potent catalytic antioxidant activity, can improve signs of DED in vivo by reducing oxidative stress in corneal epithelial cells.

Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina.

The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found Angpt4 expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression. Correlating with its spatiotemporal expression, deletion of Angpt4 resulted in defective venous development causing impaired venous drainage and defects in neuronal cells. In vitro characterization of angiopoietin-4 proteins revealed both ligand-specific and redundant functions among the angiopoietins. Our study identifies Angpt4 as the first growth factor for venous-specific development and its importance in venous remodeling, retinal fluid clearance and neuronal function.

Efficacy of Trabodenoson in a Mouse Keratoconjunctivitis Sicca (KCS) Model for Dry-Eye Syndrome.

Purpose: To determine the efficacy of trabodenoson, an adenosine mimetic with highly selective adenosine A1 receptor binding properties, in a preclinical mouse model for dry-eye disease. Methods: Dry-eye disease was induced in adult male C57BL/6 mice using a combination of desiccating environment and transdermal administration of scopolamine. Mice were treated concurrently and twice daily with either vehicle, 6% trabodenoson, or 0.05% cyclosporine (Restasis). Efficacy (P < 0.05 versus vehicle) was determined by clinical assessment of dry-eye symptoms using corneal fluorescein staining and tear volumes and histopathologically by quantifying lacrimal gland pathology and conjunctival goblet cells. Results: Twice-daily topical (ocular) administration of trabodenoson increased tear levels and reduced corneal fluorescein staining (P < 0.05) as compared with vehicle-treated eyes in a mouse model of dry-eye disease. Furthermore, significant infiltration of immune cells in the lacrimal gland and reduced number of mucin-producing conjunctival goblet cells were noted in both untreated and vehicle-treated eyes. Comparatively, trabodenoson treatment significantly reduced lacrimal gland infiltration and increased the number of goblet cells (P < 0.05 for both versus vehicle). These trabodenoson-related effects on lacrimal gland pathology and goblet cells were similar to or better than the effects observed with cyclosporine treatment. Conclusions: Topical ocular delivery of trabodenoson significantly improves the clinical and histopathological signs associated with dry-eye disease in mice. This improvement appears to be related to anti-inflammatory effects from targeting adenosine signaling and represents a novel therapeutic approach to develop for the management of dry-eye disease.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period. The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch's membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch's membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ. This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

Retinal Degeneration In A Mouse Model Of CLN5 Disease Is Associated With Compromised Autophagy.

The Finnish variant of late infantile neuronal ceroid lipofuscinosis (CLN5 disease) belongs to a family of neuronal ceroid lipofuscinosis (NCLs) diseases. Vision loss is among the first clinical signs in childhood forms of NCLs. Mutations in CLN5 underlie CLN5 disease. The aim of this study was to characterize how the lack of normal functionality of the CLN5 protein affects the mouse retina. Scotopic electroretinography (ERG) showed a diminished c-wave amplitude in the CLN5 deficient mice already at 1 month of age, indicative of pathological events in the retinal pigmented epithelium. A- and b-waves showed progressive impairment later from 2 and 3 months of age onwards, respectively. Structural and immunohistochemical (IHC) analyses showed preferential damage of photoreceptors, accumulation of autofluorescent storage material, apoptosis of photoreceptors, and strong inflammation in the CLN5 deficient mice retinas. Increased levels of autophagy-associated proteins Beclin-1 and P62, and increased LC3b-II/LC3b-I ratio, were detected by Western blotting from whole retinal extracts. Photopic ERG, visual evoked potentials, IHC and cell counting indicated relatively long surviving cone photoreceptors compared to rods. In conclusion, CLN5 deficient mice develop early vision loss that reflects the condition reported in clinical childhood forms of NCLs. The vision loss in CLN5 deficient mice is primarily caused by photoreceptor degeneration.

Acute cytotoxic effects of marketed ophthalmic formulations on human corneal epithelial cells.

The purpose of the study was to devise a fast, reliable and sensitive cell viability assay for assessment of acute cytotoxicity on human corneal epithelial cells by using a clinically relevant exposure time. Acute cytotoxic effects of the pharmaceutical excipients benzalkonium chloride (BAC), macrogolglycerol hydroxystearate (MGHS40), polysorbate 80 (PS80) and marketed ophthalmic formulations (Lumigan(®), Monoprost(®), Taflotan(®), Travatan(®), Xalatan(®)) containing these excipients were tested. Human corneal epithelial cell (HCE-T) viability was assessed by measuring the reduction of resazurin to highly fluorescent resorufin. Expression of the tight junction proteins in HCE-T cells were characterized by immunofluorescence staining. Presence of tight junction proteins in HCE-T cells was demonstrated. BAC preserved ophthalmic formulations showed concentration-dependent and time-dependent cytotoxicity to human corneal epithelium. In contrast, no acute cytotoxicity of non-ionic stabilizing/solubilizing excipients (MGSH40 and PS80) or ophthalmic formulation containing these excipients was observed. Marketed ophthalmic formulations used for glaucoma medication show differential toxicity on human corneal epithelial cells. The present study revealed that BAC-preserved ophthalmic formulations were able to induce acute cytotoxic effects even during a clinically relevant exposure time, which was not observed with MGSH40 and PS80 excipients or ophthalmic formulations containing these excipients.

Early retinal function deficit without prominent morphological changes in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder that primarily affects the medium-size GABAergic neurons of striatum. The R6/2 mouse line is one of the most widely used animal models of HD. Previously the hallmarks of HD-related pathology have been detected in photoreceptors and interneurons of R6/2 mouse retina. Here we aimed to explore the survival of retinal ganglion cells (RGCs) and functional integrity of distinct retinal cell populations in R6/2 mice. The pattern electroretinography (PERG) signal was lost at the age of 8 weeks in R6/2 mice in contrast to the situation in wild-type (WT) littermates. This defect may be attributable to a major reduction in photopic ERG responses in R6/2 mice which was more evident in b- than a-wave amplitudes. At the age of 4 weeks R6/2 mice had predominantly the soluble form of mutant huntingtin protein (mHtt) in the RGC layer cells, whereas the aggregated form of mHtt was found in the majority of those cells from the 12-week-old R6/2 mice and onwards. Retinal astrocytes did not contain mHtt deposits. The total numbers of RGC layer cells, retinal astrocytes as well as optic nerve axons did not differ between 18-week-old R6/2 mice and their WT controls. Our data indicate that mHtt deposition does not cause RGC degeneration or retinal astrocyte loss in R6/2 mice even at a late stage of HD-related pathology. However, due to functional deficits in the rod- and cone-pathways, the R6/2 mice suffer progressive deficits in visual capabilities starting as early as 4 weeks; at 8 weeks there is severe impairment. This should be taken into account in any behavioral testing conducted in R6/2 mice.