Objective. Glaucoma is a leading cause of severe visual impairment and blindness. Although high intraocular pressure (IOP) is an established risk factor for the disease, the role of abnormal ocular vessel function in the pathophysiology of glaucoma gains more and more attention. We tested the hypothesis that elevated intraocular pressure (IOP) causes vascular dysfunction in the retina. Methods. High IOP was induced in one group of mice by unilateral cauterization of three episcleral veins. The other group received sham surgery only. Two weeks later, retinal vascular preparations were studied by video microscopy in vitro. Reactive oxygen species (ROS) levels and expression of hypoxia markers and of prooxidant and antioxidant redox genes as well as of inflammatory cytokines were determined. Results. Strikingly, responses of retinal arterioles to stepwise elevation of perfusion pressure were impaired in the high-IOP group. Moreover, vasodilation responses to the endothelium-dependent vasodilator, acetylcholine, were markedly reduced in mice with elevated IOP, while no differences were seen in response to the endothelium-independent nitric oxide donor, sodium nitroprusside. Remarkably, ROS levels were increased in the retinal ganglion cell layer including blood vessels. Expression of the NADPH oxidase isoform, NOX2, and of the inflammatory cytokine, TNF-α, was increased at the mRNA level in retinal explants. Expression of NOX2, but not of the hypoxic markers, HIF-1α and VEGF-A, was increased in the retinal ganglion cell layer and in retinal blood vessels at the protein level. Conclusion. Our data provide first-time evidence that IOP elevation impairs autoregulation and induces endothelial dysfunction in mouse retinal arterioles. Oxidative stress and inflammation, but not hypoxia, appear to be involved in this process.

Angiotensin II (Ang II) has been implicated in the pathophysiology of various age-dependent ocular diseases. The purpose of this study was to test the hypothesis that Ang II induces endothelial dysfunction in mouse ophthalmic arteries and to identify the underlying mechanisms. Ophthalmic arteries were exposed to Ang II in vivo and in vitro to determine vascular function by video microscopy. Moreover, the formation of reactive oxygen species (ROS) was quantified and the expression of prooxidant redox genes and proteins was determined. The endothelium-dependent artery responses were blunted after both in vivo and in vitro exposure to Ang II. The Ang II type 1 receptor (AT1R) blocker, candesartan, and the ROS scavenger, Tiron, prevented Ang II-induced endothelial dysfunction. ROS levels and NOX2 expression were increased following Ang II incubation. Remarkably, Ang II failed to induce endothelial dysfunction in ophthalmic arteries from NOX2-deficient mice. Following Ang II incubation, endothelium-dependent vasodilation was mainly mediated by cytochrome P450 oxygenase (CYP450) metabolites, while the contribution of nitric oxide synthase (NOS) and 12/15-lipoxygenase (12/15-LOX) pathways became negligible. These findings provide evidence that Ang II induces endothelial dysfunction in mouse ophthalmic arteries via AT1R activation and NOX2-dependent ROS formation. From a clinical point of view, the blockade of AT1R signaling and/or NOX2 may be helpful to retain or restore endothelial function in ocular blood vessels in certain ocular diseases.

Visual impairment and blindness are often caused by retinal ischemia-reperfusion (I/R) injury. We aimed to characterize a new model of I/R in pigs, in which the intraocular pathways were not manipulated by invasive methods on the ocular system. After 12 min of ischemia followed by 20 h of reperfusion, reactivity of retinal arterioles was measured in vitro by video microscopy. Dihydroethidium (DHE) staining, qPCR, immunohistochemistry, quantification of neurons in the retinal ganglion cell layer, and histological examination was performed. Retinal arterioles of I/R-treated pigs displayed marked attenuation in response to the endothelium-dependent vasodilator, bradykinin, compared to sham-treated pigs. DHE staining intensity and messenger RNA levels for HIF-1α, VEGF-A, NOX2, and iNOS were elevated in retinal arterioles following I/R. Immunoreactivity to HIF-1α, VEGF-A, NOX2, and iNOS was enhanced in retinal arteriole endothelium after I/R. Moreover, I/R evoked a substantial decrease in Brn3a-positive retinal ganglion cells and noticeable retinal thickening. In conclusion, the results of the present study demonstrate that short-time ocular ischemia impairs endothelial function and integrity of retinal blood vessels and induces structural changes in the retina. HIF-1α, VEGF-A, iNOS, and NOX2-derived reactive oxygen species appear to be involved in the pathophysiology.

Ischemia-reperfusion (I/R) events are involved in the development of various ocular pathologies, e.g., retinal artery or vein occlusion. We tested the hypothesis that resveratrol is protective against I/R injury in the murine retina. Intraocular pressure (IOP) was elevated in anaesthetized mice to 110 mm Hg for 45 min via a micropipette placed in the anterior chamber to induce ocular ischemia. In the fellow eye, which served as control, IOP was kept at a physiological level. One group received resveratrol (30 mg/kg/day p.o. once daily) starting one day before the I/R event, whereas the other group of mice received vehicle solution only. On day eight after the I/R event, mice were sacrificed and retinal wholemounts were prepared and immuno-stained using a Brn3a antibody to quantify retinal ganglion cells. Reactivity of retinal arterioles was measured in retinal vascular preparations using video microscopy. Reactive oxygen species (ROS) and nitrogen species (RNS) were quantified in ocular cryosections by dihydroethidium and anti-3-nitrotyrosine staining, respectively. Moreover, hypoxic, redox and nitric oxide synthase gene expression was quantified in retinal explants by PCR. I/R significantly diminished retinal ganglion cell number in vehicle-treated mice. Conversely, only a negligible reduction in retinal ganglion cell number was observed in resveratrol-treated mice following I/R. Endothelial function and autoregulation were markedly reduced, which was accompanied by increased ROS and RNS in retinal blood vessels of vehicle-exposed mice following I/R, whereas resveratrol preserved vascular endothelial function and autoregulation and blunted ROS and RNS formation. Moreover, resveratrol reduced I/R-induced mRNA expression for the prooxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2). Our data provide evidence that resveratrol protects from I/R-induced retinal ganglion cell loss and endothelial dysfunction in the murine retina by reducing nitro-oxidative stress possibly via suppression of NOX2 upregulation.

The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M₁–M₅) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M₃ receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M₃ receptor (M3R^−/−). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmental scanning electron microscopy, assessment of proliferating cells in the corneal epithelium and of goblet cells in the conjunctiva, quantification of mRNA for inflammatory cytokines and prooxidant redox enzymes and quantification of reactive oxygen species. Tear volume was reduced in M3R^−/− mice compared to age-matched controls at the age of 3 months and 15 months, respectively. This was associated with mild corneal epitheliopathy in the 15-month-old but not in the 3-month-old M3R^−/− mice. M3R^−/− mice at the age of 15 months also displayed changes in corneal epithelial cell texture, reduced conjunctival goblet cell density, oxidative stress and elevated mRNA expression levels for inflammatory cytokines and prooxidant redox enzymes. The findings suggest that the M₃ receptor plays a pivotal role in tear production and its absence leads to ocular surface changes typical for DED at advanced age.

Objective. Atherogenic lipoproteins may impair vascular reactivity consecutively causing tissue damage in multiple organs via abnormal perfusion and excessive reactive oxygen species generation. We tested the hypothesis that chronic hypercholesterolemia causes endothelial dysfunction and cell loss in the retina. Methods. Twelve-month-old apolipoprotein E-deficient (ApoE-/-) mice and age-matched wild-type controls were used in this study (n=8 per genotype for each experiment). Intraocular pressure, blood pressure, and ocular perfusion pressure were determined. Retinal arteriole responses were studied in vitro, and reactive oxygen and nitrogen species were quantified in the retinal and optic nerve cryosections. The expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the NADPH oxidase isoforms, NOX1, NOX2, and NOX4, were determined in retinal cryosections by immunofluorescence microscopy. Pro- and antioxidant redox genes were quantified in retinal explants by PCR. Moreover, cell number in the retinal ganglion cell layer and axon number in the optic nerve was calculated. Results. Responses to the endothelium-dependent vasodilator, acetylcholine, were markedly impaired in retinal arterioles of ApoE-/- mice (P<0.01). LOX-1 (P=0.0007) and NOX2 (P=0.0027) expressions as well as levels of reactive oxygen species (P=0.0022) were increased in blood vessels but not in other retinal structures. In contrast, reactive nitrogen species were barely detectable in both mouse genotypes. Messenger RNA for HIF-1α, VEGF-A, NOX1, and NOX2, but also for various antioxidant redox genes was elevated in the retina of ApoE-/- mice. Total cell number in the retinal ganglion cell layer did not differ between ApoE-/- and wild-type mice (P=0.2171). Also, axon number in the optic nerve did not differ between ApoE-/- and wild-type mice (P=0.6435). Conclusion. Apolipoprotein E deficiency induces oxidative stress and endothelial dysfunction in retinal arterioles, which may trigger hypoxia in the retinal tissue. Oxidative stress in nonvascular retinal tissue appears to be prevented by the upregulation of antioxidant redox enzymes, resulting in neuron preservation.