Purinergic control of vascular tone in the retina.

Purinergic control of vascular tone in the CNS has been largely unexplored. This study examines the contribution of endogenous extracellular ATP, acting on vascular smooth muscle cells, in controlling vascular tone in the in vivo rat retina. Retinal vessels were labelled by i.v. injection of a fluorescent dye and imaged with scanning laser confocal microscopy. The diameters of primary arterioles were monitored under control conditions and following intravitreal injection of pharmacological agents. Apyrase (500 units ml(-1)), an ATP hydrolysing enzyme, dilated retinal arterioles by 40.4 ± 2.8%, while AOPCP (12.5 mm), an ecto-5'-nucleotidase inhibitor that increases extracellular ATP levels, constricted arterioles by 58.0 ± 3.8% (P < 0.001 for both), demonstrating the importance of ATP in the control of basal vascular tone. Suramin (500 µm), a broad-spectrum P2 receptor antagonist, dilated retinal arterioles by 50.9 ± 3.7% (P < 0.001). IsoPPADS (300 µm) and TNP-ATP (50 µm), more selective P2X antagonists, dilated arterioles by 41.0 ± 5.3% and 55.2 ± 6.1% respectively (P < 0.001 for both). NF023 (50 µm), a potent antagonist of P2X1 receptors, dilated retinal arterioles by 32.1 ± 2.6% (P < 0.001). A438079 (500 µm) and AZ10606120 (50 µm), P2X7 antagonists, had no effect on basal vascular tone (P = 0.99 and P = 1.00 respectively). In the ex vivo retina, the P2X1 receptor agonist α,ß-methylene ATP (300 nm) evoked sustained vasoconstrictions of 18.7 ± 3.2% (P < 0.05). In vivo vitreal injection of the gliotoxin fluorocitrate (150 µm) dilated retinal vessels by 52.3 ± 1.1% (P < 0.001) and inhibited the vasodilatory response to NF023 (50 µm, 7.9 ± 2.0%; P < 0.01). These findings suggest that vascular tone in rat retinal arterioles is maintained by tonic release of ATP from the retina. ATP acts on P2X1 receptors, although contributions from other P2X and P2Y receptors cannot be ruled out. Retinal glial cells are a possible source of the vasoconstricting ATP.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Light adaptation does not prevent early retinal abnormalities in diabetic rats.

The aetiology of diabetic retinopathy (DR), the leading cause of blindness in the developed world, remains controversial. One hypothesis holds that retinal hypoxia, exacerbated by the high O2 consumption of rod photoreceptors in the dark, is a primary cause of DR. Based on this prediction we investigated whether early retinal abnormalities in streptozotocin-induced diabetic rats are alleviated by preventing the rods from dark adapting. Diabetic rats and their non-diabetic littermates were housed in a 12:12 hour light-dim light photocycle (30 lux during the day and 3 lux at night). Progression of early retinal abnormalities in diabetic rats was assessed by monitoring the ERG b-wave and oscillatory potentials, Müller cell reactive gliosis, and neuronal cell death, as assayed by TUNEL staining and retinal thickness at 6 and 12 weeks after diabetes induction. Maintaining diabetic animals in a dim-adapting light did not slow the progression of these neuronal and glial changes when compared to diabetic rats maintained in a standard 12:12 hour light-dark photocycle (30 lux during the day and 0 lux at night). Our results indicate that neuronal and glial abnormalities in early stages of diabetes are not exacerbated by rod photoreceptor O2 consumption in the dark.

Ischemia-induced spreading depolarization in the retina.

Cortical spreading depolarization is a metabolically costly phenomenon that affects the brain in both health and disease. Following severe stroke, subarachnoid hemorrhage, or traumatic brain injury, cortical spreading depolarization exacerbates tissue damage and enlarges infarct volumes. It is not known, however, whether spreading depolarization also occurs in the retina in vivo. We report now that spreading depolarization episodes are generated in the in vivo rat retina following retinal vessel occlusion produced by photothrombosis. The properties of retinal spreading depolarization are similar to those of cortical spreading depolarization. Retinal spreading depolarization waves propagate at a velocity of 3.0 ± 0.1 mm/min and are associated with a negative shift in direct current potential, a transient cessation of neuronal spiking, arteriole constriction, and a decrease in tissue O2 tension. The frequency of retinal spreading depolarization generation in vivo is reduced by administration of the NMDA antagonist MK-801 and the 5-HT(1D) agonist sumatriptan. Branch retinal vein occlusion is a leading cause of vision loss from vascular disease. Our results suggest that retinal spreading depolarization could contribute to retinal damage in acute retinal ischemia and demonstrate that pharmacological agents can reduce retinal spreading depolarization frequency after retinal vessel occlusion. Blocking retinal spreading depolarization generation may represent a therapeutic strategy for preserving vision in branch retinal vein occlusion patients.

Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.

PURPOSE: To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). METHODS: Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacologic agents inhibiting Ca(2+) sparks and oscillations and K(+) channels. Subcellular Ca(2+) signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. RESULTS: Arachidonic acid dilated pressurized retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. Arachidonic acid-induced dilation was associated with an inhibition of subcellular Ca(2+) signals. Interventions known to block Ca(2+) sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. Arachidonic acid accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous large-conductance calcium-activated K(+) (BK) currents, but only at positive membrane potentials. Pharmacologic inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. Arachidonic acid suppressed L-type Ca(2+) currents. CONCLUSIONS: These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca(2+)-signaling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca(2+)-channel activity. Arachidonic acid actions on K(+) currents are inconsistent with a model in which K(+) channels contribute to the vasodilator effects of AA.

Cellular and physiological mechanisms underlying blood flow regulation in the retina and choroid in health and disease.

We review the cellular and physiological mechanisms responsible for the regulation of blood flow in the retina and choroid in health and disease. Due to the intrinsic light sensitivity of the retina and the direct visual accessibility of fundus blood vessels, the eye offers unique opportunities for the non-invasive investigation of mechanisms of blood flow regulation. The ability of the retinal vasculature to regulate its blood flow is contrasted with the far more restricted ability of the choroidal circulation to regulate its blood flow by virtue of the absence of glial cells, the markedly reduced pericyte ensheathment of the choroidal vasculature, and the lack of intermediate filaments in choroidal pericytes. We review the cellular and molecular components of the neurovascular unit in the retina and choroid, techniques for monitoring retinal and choroidal blood flow, responses of the retinal and choroidal circulation to light stimulation, the role of capillaries, astrocytes and pericytes in regulating blood flow, putative signaling mechanisms mediating neurovascular coupling in the retina, and changes that occur in the retinal and choroidal circulation during diabetic retinopathy, age-related macular degeneration, glaucoma, and Alzheimer's disease. We close by discussing issues that remain to be explored.