The relationship between changes in retinal vessel morphology and the onset and progression of diseases such as diabetes, hypertension and retinopathy of prematurity (ROP) has been the subject of several large scale clinical studies. However, the difficulty of quantifying changes in retinal vessels in a sufficiently fast, accurate and repeatable manner has restricted the application of the insights gleaned from these studies to clinical practice. This paper presents a novel algorithm for the efficient detection and measurement of retinal vessels, which is general enough that it can be applied to both low and high resolution fundus photographs and fluorescein angiograms upon the adjustment of only a few intuitive parameters. Firstly, we describe the simple vessel segmentation strategy, formulated in the language of wavelets, that is used for fast vessel detection. When validated using a publicly available database of retinal images, this segmentation achieves a true positive rate of 70.27%, false positive rate of 2.83%, and accuracy score of 0.9371. Vessel edges are then more precisely localised using image profiles computed perpendicularly across a spline fit of each detected vessel centreline, so that both local and global changes in vessel diameter can be readily quantified. Using a second image database, we show that the diameters output by our algorithm display good agreement with the manual measurements made by three independent observers. We conclude that the improved speed and generality offered by our algorithm are achieved without sacrificing accuracy. The algorithm is implemented in MATLAB along with a graphical user interface, and we have made the source code freely available.

Abstract-Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca 2ϩ release events (Ca 2ϩ -sparks), trigger the activation of large-conductance Ca 2ϩ -activated K ϩ (BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca 2ϩ ] i release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca 2ϩ -sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BK␣ subunit was unchanged. The Ca 2ϩ -sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BK␤1 subunit confers Ca 2ϩ -sensitivity to BK channel complexes and both transcript and protein levels for BK␤1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BK␤1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension. (Circ Res. 2007;100:703-711.)

Summary1. Potential changes in isolated rat superior cervical ganglia following addition and removal of depolarizing agents were recorded using a moving-fluid extracellular electrode system. 2. Ganglionic negativity produced by carbachol was followed by a pronounced ganglionic positivity on washing. This after-positivity was attributed to hyperpolarization of the ganglion cells since it was unaffected by crushing the postganglionic trunk.3. The after-hyperpolarization was selectively depressed by (a) cooling (Qlo Na+. This suggested a close dependence on active Na+ transport. 4. When K+ was restored to K +-free solution, or the preparation was warmed rapidly, or when metabolic inhibitors were washed away, the hyperpolarization was rapidly regenerated. The effect of restoring K+ indicated that the hyperpolarization was generated directly by the Na+ pump.5. The hyperpolarization was not altered by replacing Cl-with isethionate, indicating that the voltage change produced by the Na+ current was not modified by passive Cl-movements. 6. Hexamethonium added to the washout fluid augmented the after-hyperpolarization, suggesting that there was a high (cationic) leak current due to continued receptor-activation on washing with normal Krebs solution. 7. The hyperpolarization was reduced by omission of Ca2+ and restored by addition of Mg2+. This was considered to result from changes in passive membrane permeability. 8. The time-course of post-carbachol hyperpolarization accorded with a Na+ extrusion process whose rate was directly proportional to [Na+]i with a rate constant of 038 +0-02 min-' at 23-270 C. 9. With increasing concentrations of carbachol, the amplitude of the hyperpolarization increased in proportion to the preceding depolarization, but the rate constant of the hyperpolarization was unchanged. 11. A hyperpolarization also followed depolarization by acetylcholine or by EDTA in Ca2+-free solution. 12. Nicotine-depolarization was not followed by a hyperpolarization unless hexamethonium was added to the washout fluid. This was attributed to prolonged receptor-stimulation by nicotine. 13. It was concluded that the after-hyperpolarization was due to the electrogenic extrusion of Na+ accumulated in the ganglion cells during the preceding depolarization, with no involvement of specific muscarinic receptors. The relationship of this process to post-tetanic hyperpolarization and to other forms of drug-induced ganglionic hyperpolarization is discussed.

This study assessed the contribution of L‐type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage‐clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing < 10 cells) using the whole‐cell perforated patch‐clamp technique. Cell Ca2+ was estimated by fura‐2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mm), refilling was slower in cells held at ‐20 mV compared to ‐80 mV (refilling half‐time was 38 ± 10 and 20 ± 6 s, respectively). To attempt faster refilling via L‐type Ca2+ channels, depolarising steps from ‐60 to ‐20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L‐type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At ‐80 mV and in 20 mm TEA, prolonged caffeine exposure produced a transient Ca2+‐activated Cl− current (ICl(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene‐9‐carboxylic acid (1 mm; an ICl(Ca) blocker) and to BAPTA AM, but was abolished by 1 μm nifedipine. This nifedipine‐sensitive current reversed at +29 ± 2 mV, which shifted to +7 ± 5 mV in Ca2+‐free solution. Cyclopiazonic acid (20 μm; an inhibitor of sarcoplasmic reticulum Ca2+‐ATPase) also activated the nifedipine‐sensitive sustained current. At ‐80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient ICl(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 μm) during refilling reduced the caffeine‐activated ICl(Ca) by 38 ± 5 %. The effect was concentration dependent (1‐3000 nm, EC50 64 nm). In Ca2+‐free solution, store refilling was similarly depressed (by 46 ± 6 %). Endothelin‐1 (10 nm) applied at ‐80 mV increased [Ca2+]i, which subsided to a sustained 198 ± 28 nm above basal. Cell Ca2+ was then lowered by 1 μm nifedipine (to 135 ± 22 nm), which reversed on washout. These results show that L‐type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non‐selective store‐operated cation conductance that is blocked by nifedipine.

This study showed moderate associations between depression and poor treatment adherence. Targeting behaviour and social environments, however, may ultimately provide more cost-effective health gains than targeting depressive symptoms.