Retinal tissue oxygen tension and consumption during light flicker stimulation in rat.

Light flicker stimulation has been shown to increase inner retinal oxygen metabolism and supply. The purpose of the study was to test the hypothesis that sustained light flicker stimulation of various durations alters the depth profile metrics of oxygen partial pressure in the retinal tissue (tPO2) but not the outer retinal oxygen consumption rate (QO2). In 17 rats, tPO2 depth profiles were derived by phosphorescence lifetime imaging after intravitreal injection of an oxyphor. tPO2 profile metrics, including mean inner retinal tPO2, maximum outer retinal tPO2 and minimum outer retinal tPO2 were determined. QO2 was calculated using a one-dimensional oxygen diffusion model. Data were acquired at baseline (constant light illumination) and during light flicker stimulation at 10 Hz under the same mean illumination levels, and differences between values obtained during flicker and baseline were calculated. None of the tPO2 profile metrics or QO2 differences depended on the duration of light flicker stimulation (R2 ≤ 0.03). No significant change in any of the tPO2 profile metrics was detected with light flicker compared with constant light (P ≥ 0.08). Light flicker decreased QO2 from 0.53 ±â€¯0.29 to 0.38 ±â€¯0.30 mL O2/(min*100 gm), a reduction of 28% (P = 0.02). The retinal compensatory responses to the physiologic challenge of light flicker stimulation were effective in maintaining the levels of oxygen at or near baseline in the inner retina. Oxygen availability to the inner retina during light flicker may also have been enhanced by the decrease in QO2.

The effect of intravitreal vascular endothelial growth factor on inner retinal oxygen delivery and metabolism in rats.

Vascular endothelial growth factor (VEGF) is stimulated by hypoxia and plays an important role in pathologic vascular leakage and neovascularization. Increased VEGF may affect inner retinal oxygen delivery (DO2) and oxygen metabolism (MO2), however, quantitative information is lacking. We tested the hypotheses that VEGF increases DO2, but does not alter MO2. In 10 rats, VEGF was injected intravitreally into one eye, whereas balanced salt solution (BSS) was injected into the fellow eye, 24 h prior to imaging. Vessel diameters and blood velocities were determined by red-free and fluorescent microsphere imaging, respectively. Vascular PO2 values were derived by phosphorescence lifetime imaging of an intravascular oxyphor. Retinal blood flow, vascular oxygen content, DO2 and MO2 were calculated. Retinal arterial and venous diameters were larger in VEGF-injected eyes compared to control eyes (P < 0.03), however no significant difference was observed in blood velocity (P = 0.21). Thus, retinal blood flow was greater in VEGF-injected eyes (P = 0.007). Retinal vascular PO2 and oxygen content were similar between control and VEGF-injected eyes (P > 0.11), while the arteriovenous oxygen content difference was marginally lower in VEGF-injected eyes (P = 0.05). DO2 was 950 ± 340 and 1380 ± 650 nL O2/min in control and VEGF-injected eyes, respectively (P = 0.005). MO2 was 440 ± 150 and 490 ± 190 nL O2/min in control and VEGF-injected eyes, respectively (P = 0.31). Intravitreally administered VEGF did not alter MO2 but increased DO2, suggesting VEGF may play an offsetting role in conditions characterized by retinal hypoxia.

Feasibility of assessment of conjunctival microvascular hemodynamics in unilateral ischemic stroke.

Since the internal carotid artery supplies blood to both the eye and the brain, ocular microvascular hemodynamics can be altered due to ischemic stroke. The purpose of the current study was to establish the feasibility of conjunctival microcirculation imaging for detection of inter-ocular differences in microvascular hemodynamics in subjects with unilateral ischemic stroke. Conjunctival microcirculation imaging was performed in both eyes of 15 healthy control subjects and 12 subjects following unilateral ischemic stroke. Diameter and axial blood velocity were measured in multiple conjunctival venules of each eye. A two-way repeated measures analysis of variance was performed to determine the effects of stroke (control vs. stroke) and side of stroke (ipsilateral vs. contralateral) on conjunctival diameter and axial blood velocity. There was no significant main effect of stroke on conjunctival diameter (P=0.7) or conjunctival axial blood velocity (P=0.9). There was no significant main effect of side of stroke on conjunctival diameter (P=0.8), but there was a significant main effect of side of stroke on conjunctival axial blood velocity (P=0.02). There was a significant interaction effect between stroke and side of stroke (P=0.04), indicating that conjunctival axial blood velocity was lower in ipsilateral eyes than in contralateral eyes of stroke subjects. Conjunctival axial blood velocity and internal carotid artery blood velocity were correlated in stroke subjects (r=0.75, P=0.01, N=10). Conjunctival microcirculation imaging is a feasible method to detect inter-ocular differences in microvascular hemodynamics in subjects with unilateral ischemic stroke.

Changes in Conjunctival Hemodynamics Predict Albuminuria in Sickle Cell Nephropathy.

BACKGROUND: Albuminuria is an early manifestation of deterioration in renal function in subjects with sickle cell disease (SCD). Hyperfiltration may be an early mechanism for kidney damage in SCD. The purpose of the current study was to determine the association between conjunctival hemodynamics and albuminuria in SCD subjects with preserved glomerular filtration rate. METHODS: Conjunctival microcirculation imaging was performed to measure conjunctival diameter and axial blood velocity (V) in 35 SCD and 10 healthy control subjects. Albuminuria, defined as albumin excretion ratio (AER), was obtained from the medical charts. Based on the 95% CI of conjunctival V in control subjects (0.40-0.60 mm/s), SCD subjects were allocated to 3 groups: V1 <0.40 mm/s (n = 7), V2 of 0.40-0.60 mm/s (n = 18) and V3 ≥0.60 mm/s (n = 10). RESULTS: Mean log(AER) measurements in the V1, V2 and V3 groups were 1.08 ± 0.67, 1.39 ± 0.59 and 2.00 ± 0.91 mg/g creatinine, respectively, and followed a positive linear trend from the V1 to V3 groups (p = 0.01). By multivariate linear regression analysis, conjunctival V significantly correlated with albuminuria (p = 0.01) independent of age, blood pressure, α-thalassemia, hematocrit, white blood cell count and lactate dehydrogenase concentration. CONCLUSIONS: Increased conjunctival V is associated with albuminuria in SCD subjects. Assessment of conjunctival microvascular hemodynamics may improve our understanding of the pathophysiology and clinical management of sickle cell nephropathy.

Effects of optical blur reduction on equivalent intrinsic blur.

PURPOSE: To determine the effect of optical blur reduction on equivalent intrinsic blur, an estimate of the blur within the visual system, by comparing optical and equivalent intrinsic blur before and after adaptive optics (AO) correction of wavefront error. METHODS: Twelve visually normal subjects (mean [±SD] age, 31 [±12] years) participated in this study. Equivalent intrinsic blur (σint) was derived using a previously described model. Optical blur (σopt) caused by high-order aberrations was quantified by Shack-Hartmann aberrometry and minimized using AO correction of wavefront error. RESULTS: σopt and σint were significantly reduced and visual acuity was significantly improved after AO correction (p ≤ 0.004). Reductions in σopt and σint were linearly dependent on the values before AO correction (r ≥ 0.94, p ≤ 0.002). The reduction in σint was greater than the reduction in σopt, although it was marginally significant (p = 0.05). σint after AO correlated significantly with σint before AO (r = 0.92, p < 0.001), and the two parameters were related linearly with a slope of 0.46. CONCLUSIONS: Reduction in equivalent intrinsic blur was greater than the reduction in optical blur after AO correction of wavefront error. This finding implies that visual acuity in subjects with high equivalent intrinsic blur can be improved beyond that expected from the reduction in optical blur alone.

Correspondence of retinal thinning and vasculopathy in mice with oxygen-induced retinopathy.

The aberrantly vascularized peripheral retina in retinopathy of prematurity (ROP) may be associated with visual field constriction, retinal dysfunction, and abnormalities in retinal thickness which is commonly assessed by spectral domain optical coherence tomography (SDOCT). However, due to the limitation of SDOCT for peripheral retinal imaging, retinal thickness in avascular peripheral retina in ROP has not been evaluated. Oxygen induced retinopathy (OIR) in mice has features of vasculopathy similar to those in human ROP. These features occur in the posterior retina and thereby are accessible by standard imaging methods. The purpose of the current study was to determine the correspondence between abnormalities in retinal thickness and vasculopathy in neonatal OIR mice by simultaneous SDOCT imaging and fluorescein angiography (FA). Newborn mice (N = 19; C57BL/6J strain) were exposed to 77% oxygen from postnatal day 7 (P7) to P12. Age-matched control mice (N = 12) were raised in room air. FA and SDOCT were performed in mice between P17 and P19 to visualize retinal vasculature and measure retinal thickness, respectively. Retinal thickness measurements in vascular regions of interest (ROIs) of control mice, and in hypovascular and avascular ROIs of OIR mice were compared. In control mice, FA showed uniformly dense retinal capillary networks between major retinal vessels and retinal thickness of vascular ROIs was 260 ± 7 µm (N = 12). In OIR mice, FA displayed hypovascular regions with less dense and fewer capillaries and avascular regions devoid of visible capillaries. Retinal thickness measurements of hypovascular and avascular ROIs were 243 ± 21 µm and 209 ± 11 µm (N = 19), respectively. Retinal thickness in hypovascular and avascular ROIs of OIR mice was significantly lower than in vascular ROIs of control mice (p ≤ 0.01). Likewise, retinal thickness in avascular ROIs was significantly lower than in hypovascular ROIs (p < 0.001). Retinal thinning in hypovascular and avascular regions may be due to arrested retinal development and/or ischemia induced apoptosis.

Neural constraints on visual acuity in proliferative diabetic retinopathy.

PURPOSE: Visual acuity (VA) in normally sighted individuals is highly correlated with equivalent intrinsic blur, a measure of the amount of blur within the visual system that is generated by optical and neural sources. This study assessed the extent to which VA, equivalent intrinsic blur, optical blur, and neural blur are abnormal in subjects with proliferative diabetic retinopathy (PDR) and characterized the relationships among these parameters. METHODS: Best-corrected VA of 10 subjects with PDR (ages 25 to 68) and 10 normally sighted individuals (ages 46 to 63) was measured for tumbling E optotypes. The Es were either unblurred or blurred through convolution with Gaussian functions of different widths. Values of equivalent intrinsic blur (σ(int)) and unblurred VA (MAR0) were derived using a standard model. Optical blur (σ(opt)), a measure of blur generated by higher-order aberrations, was quantified using Shack-Hartmann aberrometry. An index of neural blur (η) was defined as 1--σ(opt)/σ(int), which represents the remaining blur once the contributions of σ(opt) to σ(int) have been accounted for. RESULTS: Log MAR0 and log σ(int) were correlated significantly (r = 0.98, p < 0.05) for the PDR subjects and the values of these parameters ranged from normal to more than a factor of 2 above the upper limit of normal. In comparison, log MAR measured for the most blurred E was elevated by a relatively small amount for all PDR subjects and was not correlated significantly with log σ(int) (r = 0.40, p = 0.25). MAR0, σ(int), and η differed significantly between the PDR subjects and the controls (all p < 0.05) but σ(opt) did not (p = 0.50). CONCLUSIONS: Subjects with PDR and VA loss had higher than normal equivalent intrinsic blur that resulted primarily from neural blur elevations, suggesting that neural blur is an important factor that limits VA in these patients.

Wavefront error correction with adaptive optics in diabetic retinopathy.

PURPOSE: To determine the effects of diabetic retinopathy (DR), increased foveal thickness (FT), and adaptive optics (AO) on wavefront aberrations and Shack-Hartmann (SH) image quality. METHODS: Shack-Hartmann aberrometry and wavefront error correction were performed with a bench-top AO retinal imaging system in 10 healthy control and 19 DR subjects. Spectral domain optical coherence tomography was performed and central FT was measured. Based on the FT data in the control group, subjects in the DR group were categorized into two subgroups: those with normal FT and those with increased FT. Shack-Hartmann image quality was assessed based on spot areas, and high-order (HO) root mean square (RMS) and total RMS were calculated. RESULTS: There was a significant effect of DR on HO and total RMS (p = 0.01), and RMS decreased significantly after AO correction (p < 0.001). Shack-Hartmann spot area was significantly affected by DR (p < 0.001), but it did not change after AO correction (p = 0.6). High-order RMS, total RMS, and SH spot area were higher in DR subjects both before and after AO correction. In DR subgroups, HO and total RMS decreased significantly after AO correction (p < 0.001), whereas the effect of increased FT on HO and total RMS was not significant (p ≥ 0.7). There were no significant effects of increased FT and AO on SH spot area (p = 0.9). CONCLUSIONS: Diabetic retinopathy subjects had higher wavefront aberrations and less compact SH spots, likely attributable to pathological changes in the ocular optics. Wavefront aberrations were significantly reduced by AO, although AO performance was suboptimal in DR subjects as compared with control subjects.

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.

Feasibility of conjunctival hemodynamic measurements in rabbits: reproducibility, validity, and response to acute hypotension.

OBJECTIVE: To evaluate the feasibility of conjunctival hemodynamic measurements based on assessment of reproducibility, validity, and response to acute hypotension. METHODS: Image sequences of the conjunctival microvasculature of rabbits were captured using a slit lamp biomicroscope under a steady-state condition, after topical administration of phenylephrine, and after intravenous administration of esmolol. Venous hemodynamic parameters (diameter, blood velocity, blood flow, and wall shear stress) were derived. RESULTS: Conjunctival venous diameters ranged from 9 to 34 µm and blood velocities ranged from 0.08 to 0.95 mm/s. Coefficients of variation of venous diameter and blood velocity measurements were, on average, 6% and 14%, respectively. Automated and manual measurements of venous diameter and velocity were highly correlated (R = 0.97; p < 0.001; n = 16). With phenylephrine administration, diameter and velocity were reduced by 21% and 69%, respectively. Following esmolol administration, blood pressure was reduced with a concomitant decrease in velocity, followed by recovery to baseline. Venous blood velocity, flow, and WSS were correlated with blood pressure (R ≥ 0.52; p ≤ 0.01). CONCLUSIONS: The feasibility of quantifying alterations in microvascular hemodynamics in the bulbar conjunctiva was established. The method is of potential value in evaluating microcirculatory hemodynamics related to cardiovascular function.

Oxygen tension and gradient measurements in the retinal microvasculature of rats.

BACKGROUND: Oxygen delivery from the retinal vasculature plays a crucial role in maintaining normal retinal metabolic function. Therefore, measurements of retinal vascular oxygen tension (PO(2)) and PO(2) longitudinal gradients (gPO(2)) along retinal blood vessels may help gain fundamental knowledge of retinal physiology and pathological processes. METHODS: Three-dimensional retinal vascular PO(2) maps were generated in rats by optical section phosphorescence lifetime imaging. A major retinal artery and vein pair, and a smaller blood vessel (microvessel) between them were segmented, and PO(2) along each blood vessel was measured. In each blood vessel, an average PO(2) (mPO(2)) was calculated, and gPO(2) was determined by linear regression analysis. Reproducibility of measurements was assessed by calculating intraclass correlation coefficient (ICC) of repeated measurements. The correlations of mPO(2) and gPO(2) measurements with systemic arterial oxygen tension (P(a)O(2)) and carbon dioxide tension (P(a)CO(2)) was determined. RESULTS: Measurements of mPO(2) and gPO(2) in retinal arteries, microvessels and veins were reproducible (ICC > 0.86; p < 0.01; N = 8), except for retinal arterial gPO(2). Retinal arterial, microvessel and venous mPO(2) were 41 ± 8, 32 ± 8 and 25 ± 7 mmHg, respectively (mean ± SD; N = 27). Retinal arterial mPO(2) was correlated with P(a)O(2) and P(a)CO(2) (R > 0.44; p < 0.03), while retinal microvessel and venous mPO(2) were only correlated with P(a)CO(2) (R > 0.68; p < 0.01). Retinal microvessel gPO(2) (-3.8 ± 1.5 mmHg/100 µm) was significantly steeper (more negative) than venous gPO(2) (0.02 ± 0.43 mmHg/100 µm) (p < 0.01; N = 27), and neither were significantly correlated with P(a)O(2) or P(a)CO(2). CONCLUSIONS: Quantitative measurement of mPO(2) and gPO(2) in the retinal microvasculature was demonstrated. A significant decrease in PO(2) was observed along most retinal microvessels, indicative of substantial oxygen extraction by the retinal tissue. This method has the potential to help elucidate retinal microvascular oxygen transport in health and disease.

Representing the retinal line spread shape with mathematical functions.

OBJECTIVE: To report a mathematical function that characterizes the double-pass line spread function (LSF) of the human eye. Determining analytical functions that represent the double-pass LSF is important because it allows modeling the optical performance of the eye. METHODS: Optical section retinal images, generated in normal human eyes using a modified slit-lamp biomicroscope, were analyzed to derive the double-pass LSF by plotting the intensity distribution of laser light reflected/ scattered from the vitreoretinal interface. Three mathematical functions (Lorentzian, Gaussian, exponential) were fitted to the double-pass LSF and the root mean square error (RMSE) was calculated to provide a measure of the goodness of fit. RESULTS: The Lorentzian function provided the best representation of the double-pass LSF of normal human eyes. The full width at half maximum (FWHM) of the Lorentzian fitted curve was positively correlated with age, indicating that the double-pass LSF broadens with age. Furthermore, the goodness of fit of the Lorentzian function was significantly better in younger subjects as compared with older subjects, suggesting that the fitted function to the double-pass LSF may vary according to age. CONCLUSION: The results demonstrate an age-related change in the double-pass LSF width and the goodness of fit of the Lorentzian function.

Assessment of Global and Local Alterations in Retinal Layer Thickness in Ins2 (Akita) Diabetic Mice by Spectral Domain Optical Coherence Tomography.

PURPOSE/AIM: The Ins2 (Akita) mouse is a spontaneous diabetic mouse model with a heterozygous mutation in the insulin 2 gene that results in sustained hyperglycemia. The purpose of the study was to assess global and local retinal layer thickness alterations in Akita mice by analysis of spectral domain optical coherence tomography (SD-OCT) images. MATERIALS AND METHODS: SD-OCT imaging was performed in Akita and wild-type mice at 12 and 24 weeks of age. Inner retinal thickness (IRT), outer retinal thickness (ORT), total retinal thickness (TRT), and photoreceptor outer segment length (OSL) were measured. Mean global thickness values were compared between Akita and wild-type mice. Local thickness variations in Akita mice were assessed based on normative values in wild-type mice. RESULTS: Akita mice had higher blood glucose levels and lower body weights (p < 0.001). On average, IRT, ORT, and TRT were approximately 2% lower in Akita mice than in wild-type mice (p ≤ 0.02). In Akita mice, the percent difference between retinal areas with thickness below and above normative values for IRT, ORT, and TRT was 22%, 32%, and 38%, respectively. CONCLUSIONS: These findings support the use of the Akita mouse model to study the retinal neurodegenerative effects of hyperglycemia.

A method for volumetric retinal tissue oxygen tension imaging.

PURPOSE: Inadequate retinal oxygenation occurs in many vision-threatening retinal diseases, including diabetic retinopathy, retinal vascular occlusions, and age-related macular degeneration. Therefore, techniques that assess retinal oxygenation are necessary to understand retinal physiology in health and disease. The purpose of the current study is to report a method for the three-dimensional (3D) imaging of retinal tissue oxygen tension (tPO2) in rats. METHODS: Imaging was performed in Long Evans pigmented rats under systemic normoxia (N = 6) or hypoxia (N = 3). A vertical laser line was horizontally scanned on the retina and a series of optical section phase-delayed phosphorescence images were acquired. From these images, phosphorescence volumes at each phase delay were constructed and a 3D retinal tPO2 volume was generated. Retinal tPO2 volumes were quantitatively analyzed by generating retinal depth profiles of mean tPO2 (MtPO2) and the spatial variation of tPO2 (SVtPO2). The effects of systemic condition (normoxia/hypoxia) and retinal depth on MtPO2 and SVtPO2 were determined by mixed linear model. RESULTS: Each 3D retinal tPO2 volume was approximately 500 × 750 × 200 µm (horizontal × vertical × depth) and consisted of 45 en face tPO2 images through the retinal depth. MtPO2 at the chorioretinal interface was significantly correlated with systemic arterial oxygen tension (P = 0.007; N = 9). There were significant effects of both systemic condition and retinal depth on MtPO2 and SVtPO2, such that both were lower under hypoxia than normoxia and higher in the outer retina than inner retina (P < 0.001). CONCLUSION: For the first time, 3D imaging of retinal tPO2 was demonstrated, with potential future application for assessment of physiological alterations in animal models of retinal diseases.

Association between Visual Acuity and Retinal Layer Metrics in Diabetics with and without Macular Edema.

PURPOSE: Diabetes is known to cause alterations in retinal microvasculature and tissue that progressively lead to visual impairment. Optical coherence tomography (OCT) is useful for assessment of total retinal thickening due to diabetic macular edema (DME). In the current study, we determined associations between visual acuity (VA) and retinal layer thickness, reflectance, and interface disruption derived from enface OCT images in subjects with and without DME. MATERIALS AND METHODS: Best corrected VA was measured and high-density OCT volume scans were acquired in 149 diabetic subjects. A previously established image segmentation method identified retinal layer interfaces and locations of visually indiscernible (disrupted) interfaces. Enface thickness maps and reflectance images of the nerve fiber layer (NFL), combined ganglion cell and inner plexiform layer (GCLIPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptor outer segment layer (OSL), and retinal pigment epithelium (RPE) were generated in the central macular subfield. The associations among VA and retinal layer metrics were determined by multivariate linear regressions after adjusting for covariates (age, sex, race, HbA1c, diabetes type, and duration) and correcting for multiple comparisons. RESULTS: In DME subjects, increased GCLIPL and OPL thickness and decreased OSL thickness were associated with reduced VA. Furthermore, increased NFL reflectance and decreased OSL reflectance were associated with reduced VA. Additionally, increased areas of INL and ONL interface disruptions were associated with reduced VA. In subjects without DME, increased INL thickness was associated with reduced VA, whereas in subjects without DME but with previous antivascular endothelium growth factor treatment, thickening of OPL was associated with reduced VA. CONCLUSIONS: Alterations in retinal layer thickness and reflectance metrics derived from enface OCT images were associated with reduced VA with and without presence of DME, suggestive of their potential for monitoring development, progression, and treatment of DME.

Retinal Vascular and Oxygen Temporal Dynamic Responses to Light Flicker in Humans.

Purpose: To mathematically model the temporal dynamic responses of retinal vessel diameter (D), oxygen saturation (SO2), and inner retinal oxygen extraction fraction (OEF) to light flicker and to describe their responses to its cessation in humans. Methods: In 16 healthy subjects (age: 60 ± 12 years), retinal oximetry was performed before, during, and after light flicker stimulation. At each time point, five metrics were measured: retinal arterial and venous D (DA, DV) and SO2 (SO2A, SO2V), and OEF. Intra- and intersubject variability of metrics was assessed by coefficient of variation of measurements before flicker within and among subjects, respectively. Metrics during flicker were modeled by exponential functions to determine the flicker-induced steady state metric values and the time constants of changes. Metrics after the cessation of flicker were compared to those before flicker. Results: Intra- and intersubject variability for all metrics were less than 6% and 16%, respectively. At the flicker-induced steady state, DA and DV increased by 5%, SO2V increased by 7%, and OEF decreased by 13%. The time constants of DA and DV (14, 15 seconds) were twofold smaller than those of SO2V and OEF (39, 34 seconds). Within 26 seconds after the cessation of flicker, all metrics were not significantly different from before flicker values (P ≥ 0.07). Conclusions: Mathematical modeling revealed considerable differences in the time courses of changes among metrics during flicker, indicating flicker duration should be considered separately for each metric. Future application of this method may be useful to elucidate alterations in temporal dynamic responses to light flicker due to retinal diseases.

A Method for Combined Retinal Vascular and Tissue Oxygen Tension Imaging.

The retina requires adequate oxygenation to maintain cellular metabolism and visual function. Inner retinal oxygen metabolism is directly related to retinal vascular oxygen tension (PO2) and inner retinal oxygen extraction fraction (OEF), whereas outer retinal oxygen consumption (QO2) relies on oxygen availability by the choroid and is contingent upon retinal tissue oxygen tension (tPO2) gradients across the retinal depth. Thus far, these oxygenation and metabolic parameters have been measured independently by different techniques in separate animals, precluding a comprehensive and correlative assessment of retinal oxygenation and metabolism dynamics. The purpose of the current study is to report an innovative optical system for dual oxyphor phosphorescence lifetime imaging to near-simultaneously measure retinal vascular PO2 and tPO2 in rats. The use of a new oxyphor with different spectral characteristics allowed differentiation of phosphorescence signals from the retinal vasculature and tissue. Concurrent measurements of retinal arterial and venous PO 2 , tPO2 through the retinal depth, inner retinal OEF, and outer retinal QO 2 were demonstrated, permitting a correlative assessment of retinal oxygenation and metabolism. Future application of this method can be used to investigate the relations among retinal oxygen content, extraction and metabolism under pathologic conditions and thus advance knowledge of retinal hypoxia pathophysiology.

Retinal Oximetry and Vessel Diameter Measurements With a Commercially Available Scanning Laser Ophthalmoscope in Diabetic Retinopathy.

Purpose: To test the hypothesis that retinal vascular diameter and hemoglobin oxygen saturation alterations, according to stages of diabetic retinopathy (DR), are discernible with a commercially available scanning laser ophthalmoscope (SLO). Methods: One hundred eighty-one subjects with no diabetes (No DM), diabetes with no DR (No DR), nonproliferative DR (NPDR), or proliferative DR (PDR, all had photocoagulation) underwent imaging with an SLO with dual lasers (532 nm and 633 nm). Customized image analysis software determined the diameters of retinal arteries and veins (DA and DV) and central retinal artery and vein equivalents (CRAE and CRVE). Oxygen saturations of hemoglobin in arteries and veins (SO2A and SO2V) were estimated from optical densities of vessels on images at the two wavelengths. Statistical models were generated by adjusting for effects of sex, race, age, eye, and fundus pigmentation. Results: DA, CRAE, and CRVE were reduced in PDR compared to No DM (P ≤ 0.03). DV and CRVE were similar between No DM and No DR, but they were higher in NPDR than No DR (P ≤ 0.01). Effect of stage of disease on SO2A differed by race, being increased relative to No DM in NPDR and PDR in Hispanic participants only (P ≤ 0.02). Relative to No DM, SO2V was increased in NPDR and PDR (P ≤ 0.05). Conclusions: Alterations in retinal vascular diameters and SO2 by diabetic retinopathy stage can be detected with a widely available SLO, and covariates such as race can influence the results.

Assessment of Conjunctival Microvascular Hemodynamics in Stages of Diabetic Microvasculopathy.

Diabetes impairs the microcirculation and function of various vital tissues throughout the body. The conjunctival microcirculation can be non-invasively imaged and thus enables assessment of microvascular hemodynamics. In this study, alterations in conjunctival microvascular hemodynamics were quantitatively assessed at stages of increasing diabetic microvasculopathy based on diabetic retinopathy (DR). Subjects were categorized into non-diabetic control (C, N = 34), no clinically visible DR (NDR, N = 47), non-proliferative DR (NPDR, N = 45), and proliferative DR (PDR, N = 35). Conjunctival hemodynamic descriptors, namely vessel diameter (D), blood velocity (V), blood flow (Q), wall shear rate (WSR), and wall shear stress (WSS) were measured in arterioles and venules, and compared between DR and C subjects using generalized linear mixed models. In arterioles, V, WSR, and WSS were lower in NDR (P ≤ 0.01). V was lower in NDR than NPDR and PDR subjects (P ≤ 0.02). In venules, D was higher in NDR and NPDR (P ≤ 0.03), while V was lower in PDR (P = 0.04). Venular V and Q were higher in NPDR than PDR subjects (P ≤ 0.04). WSR and WSS were lower in all stages of DR (P ≤ 0.05), suggestive of the potential of WSS as a marker of diabetic microvasculopathy. Quantitative assessment of conjunctival hemodynamics can potentially be useful for evaluation of diabetic microvasculopathy.

Automated Assessment of Hemodynamics in the Conjunctival Microvasculature Network.

The conjunctival microcirculation is accessible for direct visualization and quantitative assessment of microvascular hemodynamic properties. Currently available methods to assess hemodynamics in the conjunctival microvasculature use manual or semi-automated algorithms, which can be inefficient for application to a large number of microvessels within the microvascular network. We present an automated image analysis method for measurements of diameter and blood velocity in microvessels. The method was applied to conjunctival microcirculation images acquired in 15 healthy human subjects. Frangi filtering, thresholding, and morphological closing were applied to automatically segment microvessels, while variance filtering was used to detect blood flow. Diameter and blood velocity were measured in arterioles and venules within the conjunctival microvascular network, and blood flow and wall shear rate were calculated. Repeatability and validity of hemodynamic measurements were established. The automated image analysis method allows reliable, rapid and quantitative assessment of hemodynamics in the conjunctival microvascular network and can be potentially applied to microcirculation images of other tissues.