Justin Wanek

Three-dimensional mapping of chorioretinal vascular oxygen tension in the rat.

PURPOSE An optical section phosphorescence lifetime imaging system was developed for three-dimensional mapping of oxygen tension (P(O2)) in chorioretinal vasculatures. METHODS A laser line was projected at an oblique angle and scanned on the retina after intravenous injection of an oxygen-sensitive molecular probe to generate phosphorescence optical section images. An automated software algorithm segmented and combined images from spatially adjacent locations to construct depth-displaced en face retinal images. Intravascular P(O2) was measured by determining the phosphorescence lifetime. Three-dimensional chorioretinal P(O2) maps were generated in rat eyes under varying fractions of inspired oxygen. RESULTS Under an air-breathing condition, mean P(O2) in the choroid, retinal arteries, capillaries, and veins were 58+/-2 mm Hg, 47+/-2 mm Hg, 44+/-2 mm Hg, and 35+/-2 mm Hg, respectively. The mean arteriovenous P(O2) difference was 12+/-2 mm Hg. With a lower fraction of inspired oxygen, chorioretinal vascular P(O2) and mean arteriovenous P(O2) differences decreased compared with measurements under an air-breathing condition. Retinal venous P(O2) was statistically lower than P(O2) measured in the retinal artery, capillaries, and choroid (P<0.004). CONCLUSIONS Three-dimensional mapping of chorioretinal oxygen tension allowed quantitative P(O2) measurements in large retinal blood vessels and in retinal capillaries. This method has the potential to facilitate better understanding of retinal oxygenation in health and disease.

Quantitative assessment of conjunctival microvascular circulation of the human eye

Accessibility to the bulbar conjunctival microvasculature provides a means to assess blood supply to the cerebral cortex and thus optimize therapeutic interventions designed to prevent or reduce the risk of cerebral vascular disease and stroke. The feasibility of a method for quantitative measurements of conjunctiva blood vessel diameter, blood velocity, and flow in the human eye is reported. The method is based on slit lamp biomicroscope digital imaging coupled with a space time image analysis technique. A sequence of conjunctiva microvasculature images was captured at a rate of 50 Hz. The images were analyzed to determine blood vessel diameter, velocity and flow. Blood vessel diameter measurements ranged between 8.7 and 24.3 microns, with a mean value of 15.5 microns. Blood flow rate ranged between 27.3 and 296.9 pl/s, with a mean value of 111.8 pl/s. The relationship between blood flow and vessel diameter was fit with a power law curve (R=0.87). The application of this technique for in vivo quantitative assessment of blood flow dynamics has potential to impact diagnosis and monitoring of various cardiovascular and blood disorders.

Inner retinal oxygen delivery and metabolism under normoxia and hypoxia in rat.

PURPOSE Retinal hypoxia is a common pathological condition usually caused by ischemia that may result in alterations in oxidative energy metabolism. We report measurements of oxygen delivery by the retinal circulation (DO2_IR) and inner retinal oxygen metabolism (MO2_IR) under systemic normoxia and hypoxia in rat. METHODS Rats were ventilated with fractions of inspired oxygen (FiO2) to induce either normoxia (n = 10), moderate hypoxia (n = 14), or severe hypoxia (n = 10). Oxygen tension was measured in retinal vessels using phosphorescence lifetime imaging and converted to arterial (O2A) and venous (O2V) oxygen contents. Total retinal blood flow (F) was assessed by red-free and fluorescent microsphere imaging. DO2_IR and MO2_IR were calculated as the products of F and O2A, and F and the arteriovenous oxygen content difference (O2A-V), respectively. RESULTS Measurements of O2A, O2V, and O2A-V were significantly reduced with decreased FiO2 (P < 0.001). In response to reduced oxygen availability, F increased under moderate hypoxia (P < 0.001) but did not increase further under severe hypoxia (P = 0.5). DO2_IR was similar under normoxia and moderate hypoxia (P = 0.7), but significantly lower under severe hypoxia (P < 0.001). Likewise, MO2_IR under normoxia and moderate hypoxia was similar (P = 0.1), but significantly reduced under severe hypoxia (P ≤ 0.02). CONCLUSIONS DO2_IR and MO2_IR were maintained during moderate hypoxia, but reduced under severe hypoxia, indicating blood flow compensation became insufficient for the reduced oxygen availability. Future studies may aid our understanding of retinal metabolic function in ischemic conditions.

Feasibility of a Method for En Face Imaging of Photoreceptor Cell Integrity

PURPOSE To report a method for en face imaging of the photoreceptor inner and outer segment junction by spectral-domain optical coherence tomography (SD OCT) and to describe findings in normal subjects and patients with various retinal diseases. DESIGN Observational case series. METHODS SD OCT images were acquired from 6 normal subjects (mean age, 44 ± 11 years) and from 5 subjects with retinal diseases (mean age, 66 ± 22 years). A customized high-density SD OCT volume scan was acquired on the retina. SD OCT B-scan images were segmented automatically to extract intensity data along the inner and outer segment junction. Data obtained from the raster B-scans were combined to generate an inner and outer segment en face image in a 4.4 × 4.4-mm retinal area centered on the fovea. The foveal-to-parafoveal mean intensity ratio was measured, and repeatability was determined. An infrared scanning laser ophthalmoscope image was acquired and was cropped to provide a field of view similar to the inner and outer segment en face image. RESULTS Inner and outer segment en face images generated in normal subjects provided clear visualization of the retinal vasculature, matching the vascular network observed in the infrared scanning laser ophthalmoscope image. In normal subjects, the foveal-to-parafoveal mean intensity ratio was 0.88 ± 0.06, and repeatability of measurements was, on average, 7%. In macular hole, a dark circular region was observed in the inner and outer segment en face image, indicative of photoreceptor cell loss. In age-related macular degeneration, the en face image displayed nonuniform texture corresponding to topographic variations in the inner and outer segment junction. In central serous retinopathy, areas of lower intensity were visible on the en face image corresponding to regions of prior neurosensory elevation. In cystoid macular edema, reduced intensity was present in the inner and outer segment en face image in areas with increased retinal thickness. In diabetic retinopathy, the inner and outer segment en face image displayed regions of reduced intensity resulting from edema, laser scars, or both. CONCLUSIONS Detection of intensity abnormalities in the inner and outer segment en face image is useful for monitoring the integrity of photoreceptor cells in the course of disease progression and therapeutic intervention.

Retinal Tissue Oxygen Tension Imaging in the Rat

PURPOSE To report an imaging technique for measurement of oxygen tension (PO2) in retinal tissue and establish its feasibility for measuring retinal PO2 variations in rat eyes by adjusting the fraction of inspired oxygen (FiO2). METHODS A narrow laser line was projected at an angle on the retina, and phosphorescence emission was imaged after intravitreal injection of an oxygen-sensitive molecular probe. A frequency-domain approach was used for phosphorescence lifetime measurements. Retinal PO2 maps were computed from phosphorescence lifetime images, and oxygen profiles through the retinal depth were derived in rats in conditions of 10%, 21%, and 50% FiO2. RESULTS Retinal PO2 measurements were repeatable, and variations in outer and inner retina PO2 at different locations along the image were not significant (P>or=0.3). Maximum outer retinal PO2 obtained in 10%, 21%, and 50% FiO2 were significantly different (P<0.0001). Maximum outer retinal PO2 correlated with systemic arterial PO2 (R=0.70; P<0.0001). The slope of the outer retina PO2 profile correlated with maximum outer retinal PO2 (R=0.84; P<0.0001). Mean inner retina PO2 correlated with maximum outer retinal PO2 (R=0.88; P<0.0001). CONCLUSIONS A technique has been developed for quantitative mapping of retinal tissue oxygen tension with the potential to enable sequential monitoring of retinal oxygenation in health and disease.

Conjunctival microvascular haemodynamics in sickle cell retinopathy.

To determine alterations in bulbar conjunctival microvascular haemodynamics in sickle cell retinopathy (SCR) subjects with focal macular thinning (FMT).

Inner Retinal Oxygen Extraction Fraction in Rat

PURPOSE Oxygen extraction fraction (OEF), defined by the ratio of oxygen consumption to delivery, may be a useful parameter for assessing the retinal tissue status under impaired circulation. We report a method for measurement of inner retinal OEF in rats under normoxia and hypoxia based on vascular oxygen tension (PO(2)) imaging. METHODS Retinal vascular PO(2) measurements were obtained in 10 rats, using our previously developed optical section phosphorescence lifetime imaging system. Inner retinal OEF was derived from retinal vascular PO(2) measurements based on Ficks principle. Measurements of inner retinal OEF obtained under normoxia were compared between nasal and temporal retinal sectors and repeatability was determined. Inner retinal OEF measurements obtained under normoxia and hypoxia were compared. RESULTS Retinal vascular PO(2) and inner retinal OEF measurements were repeatable (ICC ≥ 0.83). Inner retinal OEF measurements at nasal and temporal retinal sectors were correlated (R = 0.71; P = 0.02; n = 10). Under hypoxia, both retinal arterial and venous PO(2) decreased significantly as compared with normoxia (P < 0.001; n = 10). Inner retinal OEF was 0.46 ± 0.13 under normoxia and increased significantly to 0.67 ± 0.16 under hypoxia (mean ± SD; P < 0.001; n = 10). CONCLUSIONS Inner retinal OEF is a promising quantitative biomarker for the adequacy of oxygen supply for metabolism under physiologically and pathologically altered conditions.

Human bulbar conjunctival hemodynamics in hemoglobin SS and SC disease

The known biophysical variations of hemoglobin (Hb) S and Hb C may result in hemodynamic differences between subjects with SS and SC disease. The purpose of this study was to measure and compare conjunctival hemodynamics between subjects with Hb SS and SC hemoglobinopathies. Image sequences of the conjunctival microcirculation were acquired in 9 healthy control subjects (Hb AA), 24 subjects with SC disease, and 18 subjects with SS disease, using a prototype imaging system. Diameter (D) and blood velocity (V) measurements were obtained in multiple venules of each subject. Data were categorized according to venule caliber by averaging V and D for venules with diameters less than (vessel size 1) or greater than (vessel size 2) 15 µm. V in vessel size 2 was significantly greater than V in vessel size 1 in the AA and SS groups (P ≥ 0.009), but not in the SC group (P = 0.1). V was significantly lower in the SC group as compared to the SS group (P = 0.03). In AA and SS groups, V correlated with D (P ≤ 0.005), but the correlation was not statistically significant in the SC group (P = 0.08). V was inversely correlated with hematocrit in the SS group for large vessels (P = 0.03); however, no significant correlation was found in the SC group (P ≥ 0.2). Quantitative assessment of conjunctival microvascular hemodynamics in SS and SC disease may advance understanding of sickle cell disease pathophysiology and thereby improve therapeutic interventions. Am. J. Hematol. 88:661–664, 2013.

Retinal vessel diameter assessment in papilledema by semi-automated analysis of SLO images: feasibility and reliability.

PURPOSE To report feasibility and reliability of a semi-automated image analysis method for retinal vessel diameter measurements in subjects with papilledema before and after treatment. METHODS Scanning laser ophthalmoscopy (SLO) was performed in seven normal, five pseudopapilledema, and seven papilledema subjects. In four papilledema subjects, SLO was performed both before and following treatment. Two observers measured diameters of superior and inferior retinal arteries and veins from SLO images using two methods: manual analysis and semi-automated customized analysis. Vessel measurements were compared between observers and between image analysis methods. Retinal vein and artery diameters for each subject were compared between papilledema, pseudopapilledema, and normal subjects, and before and following treatment for papilledema subjects. RESULTS Interobserver reliability was 0.97 (Pearsons correlation, r) and 0.90 for semi-automated and manual measurements, respectively. Correlation coefficient of manual and semi-automated measurements was 0.85. Retinal vein diameter in papilledema subjects was larger than in pseudopapilledema and normal subjects (P = 0.03, 0.04, Mann-Whitney). Papilledema subjects had a decrease in retinal vein diameter following treatment for and resolution of papilledema (P = 0.04, Wilcoxon signed rank). Retinal artery diameters were not significantly different between papilledema and pseudopapilledema or normal groups, and did not significantly change following papilledema treatment. CONCLUSIONS A feasible and reliable semi-automated image analysis method for measurement of retinal artery and vein diameters from SLO images of elevated optic nerves is reported. Further studies are needed to determine the clinical utility of retinal vein diameter measurements as a marker for diagnosis and treatment of papilledema.

Inner retinal metabolic rate of oxygen by oxygen tension and blood flow imaging in rat

The metabolic function of inner retinal cells relies on the availability of nutrients and oxygen that are supplied by the retinal circulation. Assessment of retinal tissue vitality and function requires knowledge of both the rate of oxygen delivery and consumption. The purpose of the current study is to report a novel technique for assessment of the inner retinal metabolic rate of oxygen (MO2) by combined measurements of retinal blood flow and vascular oxygen tension (PO2) in rat. The application of this technology has the potential to broaden knowledge of retinal oxygen dynamics and advance understanding of disease pathophysiology.