Membrane Permeability Is Required for the Vasodilatory Effect of Carbonic Anhydrase Inhibitors in Porcine Retinal Arteries.

It has been demonstrated previously that a variety of carbonic anhydrase inhibitors (CAIs) can induce vasodilation in pre-contracted retinal arteriolar segments although with different efficacy and potency. Since the CAIs tested so far are able to permeate cell membranes and inhibit both intracellular and extracellular isoforms of the enzyme, it is not clear whether extra- or intracellular isoforms or mechanisms are mediating their vasodilatory effects. By means of small wire myography, we have tested the effects of four new CAIs on wall tension in pre-contracted retinal arteriolar segments that demonstrably do not enter cell membranes but have high affinity to both cytosolic and membrane-bound isoforms of CA. At concentrations between 10-6 M to 10-3 M, none of the four membrane impermeant CAIs had any significant effect on arteriolar wall tension, while the membrane permeant CAI benzolamide (10-3 M) fully dilated all arteriolar segments tested. This suggests that CAI act as vasodilators through cellular mechanisms located in the cytoplasm of vascular cells.

Molecular genetics of inherited retinal degenerations in Icelandic patients.

The study objective was to delineate the genetics of inherited retinal degenerations (IRDs) in Iceland, a small nation of 364.000 and a genetic isolate. Benefits include delineating novel pathogenic genetic variants and defining genetically homogenous patients as potential investigative molecular therapy candidates. The study sample comprised patients with IRD in Iceland ascertained through national centralized genetic and ophthalmological services at Landspitali, a national social support institute, and the Icelandic patient association. Information on patients' disease, syndrome, and genetic testing was collected in a clinical registry. Variants were reevaluated according to ACMG/AMP guidelines. Overall, 140 IRD patients were identified (point prevalence of 1/2.600), of which 70 patients had a genetic evaluation where two-thirds had an identified genetic cause. Thirteen disease genes were found in patients with retinitis pigmentosa, with the RLBP1 gene most common (n = 4). The c.1073 + 5G > A variant in the PRPF31 gene was homozygous in two RP patients. All tested patients with X-linked retinoschisis (XLRS) had the same possibly unique RS1 pathogenic variant, c.441G > A (p.Trp147X). Pathologic variants and genes for IRDs in Iceland did not resemble those described in ancestral North-Western European nations. Four variants were reclassified as likely pathogenic. One novel pathogenic variant defined a genetically homogenous XLRS patient group.

Carbonic Anhydrase Inhibitors of Different Structures Dilate Pre-Contracted Porcine Retinal Arteries.

Carbonic anhydrase inhibitors (CAIs), such as dorzolamide (DZA), are used as anti-glaucoma drugs to lower intraocular pressure, but it has been found that some of these drugs act as vasodilators of retinal arteries. The exact mechanism behind the vasodilatory effect is not yet clear. Here we have addressed the issue by using small vessel myography to examine the effect of CAIs of the sulfonamide and coumarin type on the wall tension in isolated segments of porcine retinal arteries. Vessels were pre-contracted by the prostaglandin analog U-46619, and CAIs with varying affinity for five different carbonic anhydrase (CA) isoenzymes found in human tissue tested. We found that all compounds tested cause a vasodilation of pre-contracted retinal arteries, but with varying efficacy, as indicated by the calculated mean EC50 of each compound, ranging from 4.12 µM to 0.86 mM. All compounds had a lower mean EC50 compared to DZA. The dilation induced by benzolamide (BZA) and DZA was additive, suggesting that they may act on separate mechanisms. No clear pattern in efficacy and affinity for CA isoenzymes could be discerned from the results, although Compound 5, with a low affinity for all isoenzymes except the human (h) CA isoform IV, had the greatest potency, with the lowest EC50 and inducing the most rapid and profound dilation of the vessels. The results suggest that more than one isozyme of CA is involved in mediating its role in controlling vascular tone in retinal arteries, with a probable crucial role played by the membrane-bound isoform CA IV.

Retinal A2A and A3 adenosine receptors modulate the components of the rat electroretinogram.

Adenosine is a neuromodulator present in various areas of the central nervous system, including the retina. Adenosine may serve a neuroprotective role in the retina, based on electroretinogram (ERG) recordings from the rat retina. Our purpose was to assess the role of A2A and A3 adenosine receptors in the generation and modulation of the rat ERG. The flash ERG was recorded with corneal electrodes from Sprague Dawley rats. Agonists and antagonists for A2A and A3 receptors, and adenosine were injected (5 µl) into the vitreous. The effects on the components of the single flash scotopic and photopic ERGs were examined, and ERG flicker. Adenosine (0.5 mM) increased the mean amplitudes of the scotopic ERG a-waves (68 ± 8 to 97 ± 14 µV, P = 0.042), and b-waves (236 ± 38 µV to 305 ± 42 µV). A2A agonist CGS21680 (2 mM) reduced the mean amplitude of the ERG b-wave, from 298 ± 21 µV in response to the brightest stimulus to 212 ± 19 µV (P = 0.005), and mean scotopic oscillatory potentials (OPs) from 100 ± 9 µV to 47 ± 11 µV (P = 0.023). ZM241385 [4 mM], an A2A antagonist, decreased the scotopic b-wave of the ERG. A3 agonist 2-CI-IB-MECA (0.5 mM) increased the a-wave, while decreasing the scotopic and photopic ERG b-waves, and the scotopic OPs. A3 antagonist VUF5574 (1 mM) increased the mean amplitude of the scotopic a-wave (66 ± 8 to 140 ± 29 µV, P = 0.046) and b-wave (224 ± 20 to 312 ± 39 µV, P = 0.0037). No significant effects on ERG flicker were found. We conclude that retinal neurons containing A2A and/or A3 adenosine receptors contribute to the generation of the ERG a- and b-waves and OPs.

Lysosomal alkalinization, lipid oxidation, and reduced phagosome clearance triggered by activation of the P2X7 receptor.

Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca(2+); Ca(2+) influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4(-/-) mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.

Carbonic anhydrase, its inhibitors and vascular function.

It has been known for some time that Carbonic Anhydrase (CA, EC 4.2.1.1) plays a complex role in vascular function, and in the regulation of vascular tone. Clinically employed CA inhibitors (CAIs) are used primarily to lower intraocular pressure in glaucoma, and also to affect retinal blood flow and oxygen saturation. CAIs have been shown to dilate vessels and increase blood flow in both the cerebral and ocular vasculature. Similar effects of CAIs on vascular function have been observed in the liver, brain and kidney, while vessels in abdominal muscle and the stomach are unaffected. Most of the studies on the vascular effects of CAIs have been focused on the cerebral and ocular vasculatures, and in particular the retinal vasculature, where vasodilation of its vessels, after intravenous infusion of sulfonamide-based CAIs can be easily observed and measured from the fundus of the eye. The mechanism by which CAIs exert their effects on the vasculature is still unclear, but the classic sulfonamide-based inhibitors have been found to directly dilate isolated vessel segments when applied to the extracellular fluid. Modification of the structure of CAI compounds affects their efficacy and potency as vasodilators. CAIs of the coumarin type, which generally are less effective in inhibiting the catalytically dominant isoform hCA II and unable to accept NO, have comparable vasodilatory effects as the primary sulfonamides on pre-contracted retinal arteriolar vessel segments, providing insights into which CA isoforms are involved. Alterations of the lipophilicity of CAI compounds affect their potency as vasodilators, and CAIs that are membrane impermeant do not act as vasodilators of isolated vessel segments. Experiments with CAIs, that shed light on the role of CA in the regulation of vascular tone of vessels, will be discussed in this review. The role of CA in vascular function will be discussed, with specific emphasis on findings with the effects of CA inhibitors (CAI).

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Progressive Cone-Rod Dystrophy and RPE Dysfunction in Mitfmi/+ Mice.

Mutations in the mouse microphthalmia-associated transcription factor (Mitf) gene affect retinal pigment epithelium (RPE) differentiation and development and can lead to hypopigmentation, microphthalmia, deafness, and blindness. For instance, an association has been established between loss-of-function mutations in the mouse Mitf gene and a variety of human retinal diseases, including Waardenburg type 2 and Tietz syndromes. Although there is evidence showing that mice with the homozygous Mitfmi mutation manifest microphthalmia and osteopetrosis, there are limited or no data on the effects of the heterozygous condition in the eye. Mitf mice can therefore be regarded as an important model system for the study of human disease. Thus, we characterized Mitfmi/+ mice at 1, 3, 12, and 18 months old in comparison with age-matched wild-type mice. The light- and dark-adapted electroretinogram (ERG) recordings showed progressive cone-rod dystrophy in Mitfmi/+ mice. The RPE response was reduced in the mutant in all age groups studied. Progressive loss of pigmentation was found in Mitfmi/+ mice. Histological retinal sections revealed evidence of retinal degeneration in Mitfmi/+ mice at older ages. For the first time, we report a mouse model of progressive cone-rod dystrophy and RPE dysfunction with a mutation in the Mitf gene.

Mouse microphthalmia-associated transcription factor (Mitf) mutations affect the structure of the retinal vasculature.

PURPOSE: Mice carrying pathogenic variants in the microphthalmia transcription factor (Mitf) gene show structural and functional changes in the retina and retinal pigment epithelium. The purpose of this study was to assess the vascular changes in Mitf mice carrying pathogenic variants by determining their retinal vessel diameter. METHODS: Mice examined in this study were: B6-Mitfmi-vga9/+ (n = 6), B6-Mitfmi-enu22(398) /Mitfmi-enu22(398) (n = 6) and C57BL/6J wild type mice (n = 6), all 3 months old. Fundus images were taken with a Micron IV camera after intraperitoneal injection of fluorescein salt. Images were adjusted to enhance contrast and a custom written MATLAB program used to extract the mean vascular diameter at a pre-defined distance from the optic disc. The number of vessels, mean diameter and mean total diameter were examined. RESULTS: The mean diameter of retinal veins in Mitfmi-enu22(398) /Mitfmi-enu22(398) mice was 18.8% larger than in wild type (p = 0.026). No differences in the mean diameter of the retinal arteries were found between the genotypes. Mitfmi-enu22(398) /Mitfmi-enu22(398) mice have 17.2% more retinal arteries (p = 0.026), and 15.6% more retinal veins (p = 0.041) than wild type. A 24.8% increase was observed in the mean combined arterial diameter in mice with the Mitfmi-enu22(398)/ Mitfmi-enu22(398) compared to wild type mice (p = 0.024). A 38.6% increase was found in the mean combined venular diameter in mice with the Mitfmi-enu22(398) /Mitfmi-enu22(398) pathogenic variation as compared to wild type (p = 0.004). The mean combined retinal venular diameter in the Mitfmi-vga9/+ mice was 17.8% larger than in wild type (p = 0.03). CONCLUSION: An increase in vascularization of the retina in Mitfmi-enu22(398) /Mitfmi-enu22(398) mice was found, indicating an increased demand for blood flow to the retina.

Vasodilation of Pre-contracted Porcine Retinal Arteries by Carbonic Anhydrase Inhibitors with Enhanced Lipophilicity.

PURPOSE: In this study, we investigated the vasodilation properties on pre-contracted retinal arteries of a restricted series of carbonic anhydrase inhibitors (CAIs) of the sulfonamide type with enhanced lipophilicity, to assess if it affects the potency of CAIs as vasodilators. METHODS: Carbonic anhydrase (CA) inhibition and in vitro kinetics of the compounds designed and synthesized for testing in this study were assessed by extracting human CA isoform proteins (hCA) from human cells expressing the isoforms of interest, and then measure the affinity of the novel compound for the hCAs by stopped-flow CO2 hydrase spectroscopy. Lipophilicity of compounds was measured by obtaining their octanol-water partition coefficient, expressed as calculated logP. Porcine eyes were obtained from a local abattoir, and the wall tension of porcine retinal arteriole segments dissected from the eyes was measured with small wire vessel myography. The effects of the CA compounds on wall tension were assessed by adding them to the myography bath, after pre-contracting the vessel by prostaglandin analog U-46619. RESULTS: All compounds induced vasodilation but at different concentrations. Among the tested compounds the most potent vasodilators were found to be the seleno-compound 4 and sulfur-ether compound 8 with EC50 values of 7.13 × 10-5 and 7.93 × 10-5 M, respectively, whereas the remaining ones induced complete vasodilation at EC50 comprised within the sub millimolar range. CONCLUSIONS: All the data reported in this study (i.e. results from myography, in vitro kinetics and LogPs) confirm the important role played by several CA isoforms in vasodilation, although the precise mechanism of action still remains to be elucidated.

Mechanisms of Ion Transport Across the Mouse Retinal Pigment Epithelium Measured In Vitro.

Purpose: To examine ion transport across the mouse retinal pigment epithelium (RPE), measured by the short-circuit current (ISC) and transepithelial resistance (TER). Methods: Sheets of RPE from mice (C57BL6/J) with retina, choroid, and sclera attached were mounted in Ussing chambers (0.031-cm2 aperture) and Krebs solution. The ISC and TER were recorded with voltage clamps. Receptors implicated in ion transport were blocked or stimulated by ligands applied to both sides. Results: The mean initial ISC was -12.0 ± 3.9 µA/cm2 (basolateral negative), and mean TER was 67.1 ± 8.0 ohm·cm2. RPE preparations remained stable for 3 hours, with ISC decreasing by 0.078 ± 0,033 µA/cm2/hr. Adenosine triphosphate (100 µM) increased ISC by 2.22 ± 0.41 µA/cm2 (P = 0.003). Epinephrine (100 µM) increased ISC by 1.14 ± 0.19 µA/cm2 (P = 0.011). Bumetanide (100 µM) reduced ISC by 1.72 ± 0.73 µA/cm2 (P = 0.027). Ouabain (1 mM) induced a biphasic response: an ISC increase from -7.9 ± 2.4 to -15.49 ± 2.12 µA/cm2 and then a decrease to -3.7 ± 2.2 µA/cm2. Ouabain increased TER by 15.3 ± 4.8 ohm·cm2. These compounds were added sequentially. Apical [K+]o at zero mM transiently increased ISC by 3.36 ± 1.06 µA/cm2. Ba++ decreased ISC from -10.4 ± 3.1 to -6.6 ± 1.8 µA/cm2 (P = 0.01). Ba++ reversed the K+-free response, with Isc decreasing further from -5.65 ± 1.24 to -3.37 ± 0.79 µA/cm2 (P = 0.029). Conclusions: The ISC and TER can be recorded from the mouse RPE for 3 hours. Adrenergic and purinergic receptors affect murine RPE ion transport. Sodium-potassium adenosine triphosphatase plays a role in net ion transport across mouse RPE, and Na-K-2Cl cotransporter activity partly accounts for transepithelial ion transport. Mimicking light-induced changes, low subretinal [K+]o increases ion transport transiently, dependent on K+ channels.

Mitf Links Neuronal Activity and Long-Term Homeostatic Intrinsic Plasticity.

Neuroplasticity forms the basis for neuronal circuit complexity and differences between otherwise similar circuits. We show that the microphthalmia-associated transcription factor (Mitf) plays a central role in intrinsic plasticity of olfactory bulb (OB) projection neurons. Mitral and tufted (M/T) neurons from Mitf mutant mice are hyperexcitable, have a reduced A-type potassium current (IA) and exhibit reduced expression of Kcnd3, which encodes a potassium voltage-gated channel subunit (Kv4.3) important for generating the IA Furthermore, expression of the Mitf and Kcnd3 genes is activity dependent in OB projection neurons and the MITF protein activates expression from Kcnd3 regulatory elements. Moreover, Mitf mutant mice have changes in olfactory habituation and have increased habituation for an odorant following long-term exposure, indicating that regulation of Kcnd3 is pivotal for long-term olfactory adaptation. Our findings show that Mitf acts as a direct regulator of intrinsic homeostatic feedback and links neuronal activity, transcriptional changes and neuronal function.

GABA agonists fail to protect the retina from ischemia-reperfusion injury.

The purpose of this study was to test the hypothesis that ischemia/reperfusion injury in the rat retina may be ameliorated by reducing retinal metabolism with either hypothermia or inhibitory GABA agonists. The intraocular pressure of each right eye in rats was raised to 130 mm Hg for 60 min with the left eye serving as normal control. The rats were divided into four groups in terms of drug and hypothermia treatment: (1) Untreated ischemia, (2) Hypothermia, (3) Baclofen/midazolam and (4) Baclofen/muscimol. Electroretinogram was recorded before ischemia and again after 10-day reperfusion. Histological analysis with H&E staining and cell counts was performed. Untreated ischemia/reperfusion resulted in severely reduced ERG responses. The ERG b-wave was reduced from 423+/-144 microV to 130+/-91 microV (mean+/-SD, n=5). With hypothermia the ERG b-wave was reduced from 499+/-80 microV to 237+/-111 microV (n=4). With combinations of baclofen and midazolam the ERG b-wave was reduced from 432+/-96 microV to 104+/-67 microV (n=7). In baclofen/muscimol treated eyes the ERG b-wave went from 426+/-101 microV to 148+/-118 microV (n=6). The histological tissue damage was severe in untreated ischemia and the baclofen/midazolam and baclofen/muscimol groups, but less severe in the hypothermia group. The GABA agonists do not provide any protection in our ischemia/reperfusion model. Our results are consistent with earlier reports that hypothermia may be helpful in ischemic conditions in the retina.

The microphthalmia-associated transcription factor (Mitf) gene and its role in regulating eye function.

Mutations in the microphthalmia-associated transcription factor (Mitf) gene can cause retinal pigment epithelium (RPE) and retinal dysfunction and degeneration. We examined retinal and RPE structure and function in 3 month old mice homo- or heterozygous or compound heterozygous for different Mitf mutations (Mitfmi-vga9/+, Mitfmi-enu22(398)/Mitfmi-enu22(398), MitfMi-Wh/+ and MitfMi-Wh/Mitfmi) which all have normal eye size with apparently normal eye pigmentation. Here we show that their vision and retinal structures are differentially affected. Hypopigmentation was evident in all the mutants while bright-field fundus images showed yellow spots with non-pigmented areas in the Mitfmi-vga9/+ mice. MitfMi-Wh/+ and MitfMi-Wh/Mitfmi mice showed large non-pigmented areas. Fluorescent angiography (FA) of all mutants except Mitfmi-vga9/+ mice showed hyperfluorescent areas, whereas FA from both Mitf-Mi-Wh/+ and MitfMi-Wh/Mitfmi mice showed reduced capillary network as well as hyperfluorescent areas. Electroretinogram (ERG) recordings show that MitfMi-Wh/+ and MitfMi-Wh/Mitfmi mice are severely impaired functionally whereas the scotopic and photopic ERG responses of Mitfmi-vga9/+ and Mitfmi-enu22(398)/Mitfmi-enu22(398) mice were not significantly different from wild type mice. Histological sections demonstrated that the outer retinal layers were absent from the MitfMi-Wh/+ and MitfMi-Wh/Mitfmi blind mutants. Our results show that Mitf mutations affect eye function, even in the heterozygous condition and that the alleles studied can be arranged in an allelic series in this respect.

Retinal oximetry: Metabolic imaging for diseases of the retina and brain.

Retinal oximetry imaging of retinal blood vessels measures oxygen saturation of hemoglobin. The imaging technology is non-invasive and reproducible with remarkably low variability on test-retest studies and in healthy cohorts. Pathophysiological principles and novel biomarkers in several retinal diseases have been discovered, as well as possible applications for systemic and brain disease. In diabetic retinopathy, retinal venous oxygen saturation is elevated and arteriovenous difference progressively reduced in advanced stages of retinopathy compared with healthy persons. This correlates with pathophysiology of diabetic retinopathy where hypoxia stimulates VEGF production. Laser treatment and vitrectomy both improve retinal oximetry values, which correlate with clinical outcome. The oximetry biomarker may allow automatic measurement of severity of diabetic retinopathy and predict its response to treatment. Central retinal vein occlusion is characterized by retinal hypoxia, which is evident in retinal oximetry. The retinal hypoxia seen on oximetry correlates with the extent of peripheral ischemia, visual acuity and thickness of macular edema. This biomarker may help diagnose and measure severity of vein occlusion and degree of retinal ischemia. Glaucomatous retinal atrophy is associated with reduced oxygen consumption resulting in reduced arteriovenous difference and higher retinal venous saturation. The oximetry findings correlate with worse visual field, thinner nerve fiber layer and smaller optic disc rim. This provides an objective biomarker for glaucomatous damage. In retinitis pigmentosa, an association exists between advanced atrophy, worse visual field and higher retinal venous oxygen saturation, lower arteriovenous difference. This biomarker may allow measurement of severity and progression of retinitis pigmentosa and other atrophic retinal diseases. Retinal oximetry offers visible light imaging of systemic and central nervous system vessels. It senses hypoxia in cardiac and pulmonary diseases. Oximetry biomarkers have been discovered in Alzheimer's disease and multiple sclerosis and oxygen levels in the retina correspond well with brain.

Sveinsson chorioretinal atrophy: the mildest changes are located in the photoreceptor outer segment/retinal pigment epithelium junction.

PURPOSE: To locate the mildest and/or earliest changes in the retina and/or choroid in Sveinsson chorioretinal atrophy (SCRA), using more advanced techniques than previous studies. METHODS: We used fundus photography, intravenous fluorescein angiography (IVFA) enhanced ocular coherence tomography (OCT) scans, microperimetry and multifocal electroretinography (mfERG) in an attempt to locate the mildest changes in SCRA. Eight patients with SCRA were examined. To improve the resolution of OCT scans, several consecutive recorded B-scans were retrieved for each location of interest. The scans were processed off-line with an averaging algorithm developed to maximally reduce laser speckle (noise). Static microperimetry was performed using the Rodenstock scanning laser ophthalmoscope (SLO). RESULTS: Biomicroscopy and fundus photographs disclosed an apparent thinning of the retinal pigment epithelium (RPE) in the areas minimally affected, where possible changes in the transparent sensory retina were not visible. In minimally affected areas a choriocapillaris filling defect was evident on IVFA, but some choroidal blood vessels remained open. High-resolution OCT scans in normal eyes showed three highly reflective outer layers, probably representing the junction of the inner and outer photoreceptor segments in the case of the innermost layer, the interdigitizing outer photoreceptors and RPE villi in the case of the middle layer, and the outer RPE in the case of the outermost layer. The middle layer was absent in the transition between affected and unaffected areas in all eyes with SCRA. In the more severely affected areas, the innermost layer was discontinuous and associated with increasing thinning of the outermost layer. Microperimetry of the transition areas sometimes showed clearly defined lesions that were non-responsive to stimuli. It also revealed elevated thresholds (10-15 dB) at the margins and normal thresholds in apparently unaffected areas. CONCLUSIONS: The mildest changes seen in SCRA on OCT are found at the outer photoreceptor/RPE junction; the changes in the outer RPE, choriocapillaris and inner photoreceptor segments may be secondary. Corresponding functional deficits are confirmed on microperimetry and mfERG.

Retinal Oximetry Discovers Novel Biomarkers in Retinal and Brain Diseases.

Purpose: Biomarkers for several eye and brain diseases are reviewed, where retinal oximetry may help confirm diagnosis or measure severity of disease. These include diabetic retinopathy, central retinal vein occlusion (CRVO), retinitis pigmentosa, glaucoma, and Alzheimer's disease. Methods: Retinal oximetry is based on spectrophotometric fundus imaging and measures oxygen saturation in retinal arterioles and venules in a noninvasive, quick, safe manner. Retinal oximetry detects changes in oxygen metabolism, including those that result from ischemia or atrophy. Results: In diabetic retinopathy, venous oxygen saturation increases and arteriovenous difference decreases. Both correlate with diabetic retinopathy severity as conventionally classified on fundus photographs. In CRVO, vein occlusion causes hypoxia, which is measured directly by retinal oximetry to confirm the diagnosis and measure severity. In both diseases, the change in oxygen levels is a consequence of disturbed blood flow with resulting tissue hypoxia and vascular endothelial growth factor (VEGF) production. In atrophic diseases, such as retinitis pigmentosa and glaucoma, retinal oxygen consumption is reduced and this is detected by retinal oximetry. Retinal oximetry correlates with visual field damage and retinal atrophy. It is an objective metabolic measure of the degree of retinal atrophy. Finally, the retina is part of the central nervous system tissue and reflects central nervous system diseases. In Alzheimer's disease, a change in retinal oxygen metabolism has been discovered. Conclusions: Retinal oximetry is a novel, noninvasive technology that opens the field of metabolic imaging of the retina. Biomarkers in metabolic, ischemic, and atrophic diseases of the retina and central nervous system have been discovered.

Automatic retinal oximetry.

PURPOSE: To measure hemoglobin oxygen saturation (SO(2)) in retinal vessels and to test the reproducibility and sensitivity of an automatic spectrophotometric oximeter. METHODS: Specialized software automatically identifies the retinal blood vessels on fundus images, which are obtained with four different wavelengths of light. The software calculates optical density ratios (ODRs) for each vessel. The reproducibility was evaluated by analyzing five repeated measurements of the same vessels. A linear relationship between SO(2) and ODR was assumed and a linear model derived. After calibration, reproducibility and sensitivity were calculated in terms of SO(2). Systemic hyperoxia (n = 16) was induced in healthy volunteers by changing the O(2) concentration in inhaled air from 21% to 100%. RESULTS: The automatic software enhanced reproducibility, and the mean SD for repeated measurements was 3.7% for arterioles and 5.3% venules, in terms of percentage of SO(2) (five repeats, 10 individuals). The model derived for calibration was SO(2) = 125 - 142 . ODR. The arterial SO(2) measured 96% +/- 9% (mean +/- SD) during normoxia and 101% +/- 8% during hyperoxia (n = 16). The difference between normoxia and hyperoxia was significant (P = 0.0027, paired t-test). Corresponding numbers for venules were 55% +/- 14% and 78% +/- 15% (P < 0.0001). SO(2) is displayed as a pseudocolor map drawn on fundus images. CONCLUSIONS: The retinal oximeter is reliable, easy to use, and sensitive to changes in SO(2) when concentration of O(2) in inhaled air is changed.

Optic nerve oxygenation.

The oxygen tension of the optic nerve is regulated by the intraocular pressure and systemic blood pressure, the resistance in the blood vessels and oxygen consumption of the tissue. The oxygen tension is autoregulated and moderate changes in intraocular pressure or blood pressure do not affect the optic nerve oxygen tension. If the intraocular pressure is increased above 40 mmHg or the ocular perfusion pressure decreased below 50 mmHg the autoregulation is overwhelmed and the optic nerve becomes hypoxic. A disturbance in oxidative metabolism in the cytochromes of the optic nerve can be seen at similar levels of perfusion pressure. The levels of perfusion pressure that lead to optic nerve hypoxia in the laboratory correspond remarkably well to the levels that increase the risk of glaucomatous optic nerve atrophy in human glaucoma patients. The risk for progressive optic nerve atrophy in human glaucoma patients is six times higher at a perfusion pressure of 30 mmHg, which corresponds to a level where the optic nerve is hypoxic in experimental animals, as compared to perfusion pressure levels above 50 mmHg where the optic nerve is normoxic. Medical intervention can affect optic nerve oxygen tension. Lowering the intraocular pressure tends to increase the optic nerve oxygen tension, even though this effect may be masked by the autoregulation when the optic nerve oxygen tension and perfusion pressure is in the normal range. Carbonic anhydrase inhibitors increase the optic nerve oxygen tension through a mechanism of vasodilatation and lowering of the intraocular pressure. Carbonic anhydrase inhibition reduces the removal of CO2 from the tissue and the CO2 accumulation induces vasodilatation resulting in increased blood flow and improved oxygen supply. This effect is inhibited by the cyclo-oxygenase inhibitor, indomethacin, which indicates that prostaglandin metabolism plays a role. Laboratory studies suggest that carbonic anhydrase inhibitors might be useful for medical treatment of optic nerve and retinal ischemia, potentially in diseases such as glaucoma and diabetic retinopathy. However, clinical trials and needed to test this hypotheses.

Cyclodextrin formulation of dorzolamide and its distribution in the eye after topical administration.

Due to limited aqueous solubility of dorzolamide at physiologic pH, the pH of Trusopt eye drops (cont. 2% dorzolamide) has to be kept at about 5.65, and to increase the topical bioavailability of the drug from Trusopt the contact time of the drug with the eye surface is increased by increasing the viscosity of the eye drops to 100 cps. This low pH and high viscosity can lead to local irritation. In this study, dorzolamide hydrochloride was formulated as 2% and 4% low viscosity solutions (viscosity 3 to 5 cps) containing randomly methylated beta-cyclodextrin at pH 7.45. These formulations were evaluated in rabbits. The animals were sacrificed at various time points after topical administration of the drug and the dorzolamide concentration determined in the different parts of the eye. Trusopt was used as a reference standard. The topical availability of dorzolamide from the cyclodextrin-containing eye drops appeared to be comparable to that from Trusopt and the drug reached retina and optic nerve to give measurable concentrations for at least 8 h after administration of the eye drops.