A retinal imaging system for combined measurement of optic nerve head vascular pulsation and stimulated vasodilation in humans.

Vascular pulsation at the optic nerve head (ONH) reflects vessel properties. Reduction in the stimulated retinal vasodilatory capacity has been reported in diabetes, but its relation with vascular pulsation is unknown. Here we report a new retinal imaging system for correlative assessment of ONH vascular pulsation and stimulated retinal vasodilation. Retinal reflectance images were acquired before and during light flicker stimulation to quantify arterial and venous vasodilation (DAR, DVR) in subjects with and without diabetic retinopathy (N = 25). ONH vascular pulsation amplitude and frequency (PA, PF), were quantified by curve fitting of periodic intensity waveforms acquired in retinal vasculature (RV) and ONH tissue (ONHT) regions. The relationships between pulsation metrics, heart rate (HR), intraocular pressure (IOP), and vasodilatory responses were evaluated. Pulsation metrics were not significantly different between regions (p ≥ 0.70). In RV, inter-image variabilities of PA and PF were 10% and 6%, whereas inter-observer variabilities were 7% and 2% respectively. In both regions, PF was correlated with HR (p ≤ 0.001). PA was associated with DAR in both regions (p ≤ 0.03), but only with DVR in RV (p ≤ 0.05). Overall, ONH vascular pulsation was associated with stimulated retinal vasodilation, suggesting diabetes may have concomitant effects on retinal vasculature compliance and neurovascular coupling.

Alterations in retinal pulse wave velocity under experimental ocular hypertension.

Impairments of blood flow and autoregulation have been implicated in diabetic retinopathy and glaucoma. Thus, identifying biomarkers of retinal vascular compliance and regulatory capacity is of potential value for understanding the pathophysiology and evaluating onset or progression of disease. Pulse wave velocity (PWV) represents the speed of the pulse-propagated pressure wave within blood vessels and has shown promise as a marker of vascular compliance. The purpose of the current study was to report a method for comprehensive assessment of retinal PWV based on spectral analysis of pulsatile intravascular intensity waveforms and determine alterations due to experimental ocular hypertension. Retinal PWV was linearly related to vessel diameter. Increased retinal PWV was associated with elevated intraocular pressure. Retinal PWV has the potential to serve as a vasoregulation biomarker for investigating vascular factors that contribute to the development of retinal diseases in animal models.

Development of a programmable magnetic agitation device to maintain colloidal suspension of cells during microfluidic syringe pump perfusion.

Droplet-based microfluidic devices have been used to achieve homogeneous cell encapsulation, but cells sediment in a solution, leading to heterogeneous products. In this technical note, we describe automated and programmable agitation device to maintain colloidal suspensions of cells. We demonstrate that the agitation device can be interfaced with a syringe pump for microfluidic applications. Agitation profiles of the device were predictable and corresponded to device settings. The device maintains the concentration of cells in an alginate solution over time without implicating cell viability. This device replaces manual agitation, and hence is suitable for applications that require slow perfusion for a longer period of time in a scalable manner.

Design of a navigational catheter system for the targeted delivery of therapeutics within the suprachoroidal space.

Diseases of the posterior segment of the eye are common causes of blindness and can be difficult to treat due to their location. Recently, there has been increased interest in the use of the suprachoroidal space to deliver therapeutics to the posterior segment. This space is accessible through a trans-scleral approach and blunt dissection of the adjacent scleral and choroidal tissues. However, despite recent commercial interest, there are few tools designed specifically to provide targeted delivery of therapeutics to a localised region within the suprachoroidal space. Therefore, we designed and prototyped a novel navigational catheter system for the targeted delivery of payloads within the suprachoroidal space. The system consists of a customised catheter tip designed to minimise blunt dissection stresses on neighbouring tissues, a mechanism for controlled catheter navigation, and a method for targeted delivery of large payloads. A customised in vitro model of the eye was also designed to visually demonstrate the capability of the catheter system to controllably navigate within the suprachoroidal space and deliver a targeted payload. This system can enable the delivery of large therapeutic payloads to the eye for the treatment of posterior eye diseases, thereby impacting the development and availability of vision-saving treatments.

Perspectives on Bioengineering Clinical Immersion: History, Innovation, and Impact.

Opportunities to provide clinical immersion experiences to bioengineering undergraduate students have expanded over the last several years. These programs allow students to observe the clinical environment in order to better understand workflow processes, the context in which medical equipment is used, and identify unmet needs firsthand. While each program focuses on identifying unmet needs, these experiences vary in content and implementation. Here we discuss features of clinical immersion programs, share details of our program after six years, and present data regarding post-graduation employment of our participants. Students who participated in the University of Illinois at Chicago Clinical Immersion Program are not more likely to pursue careers in industry as compared to non-participants, nor do they demonstrate an ability to find a job more quickly than non-participants. However, participants who did enter into industry self-reported that the program was impactful to both their career interests and ability to find their first employment position.

Retinal Oxygen Delivery, Metabolism and Extraction Fraction and Retinal Thickness Immediately Following an Interval of Ophthalmic Vessel Occlusion in Rats.

Limited knowledge is currently available about alterations of retinal blood flow (F), oxygen delivery (DO2), oxygen metabolism (MO2), oxygen extraction fraction (OEF), or thickness after the ophthalmic blood vessels have been closed for a substantial interval and then reopened. We ligated the ophthalmic vessels for 120 minutes in one eye of 17 rats, and measured these variables within 20 minutes after release of the ligature in the 10 rats which had immediate reflow. F, DO2 and MO2 were 5.2 ± 3.1 µL/min, 428 ± 271 nL O2/min, and 234 ± 133 nL O2/min, respectively, that is, to 58%, 46% and 60% of values obtained from normal fellow eyes (P < 0.004). OEF was 0.65 ± 0.23, 148% of normal (P = 0.03). Inner and total retinal thicknesses were 195 ± 24 and 293 ± 20 µm, respectively, 117% and 114% of normal, and inversely related to MO2 (P ≤ 0.02). These results reflect how much energy is available to the retina immediately after an interval of nonperfusion for 120 minutes. Thus, they elucidate aspects of the pathophysiology of nonperfusion retinal injury and may improve therapy in patients with retinal artery or ophthalmic artery obstructions.

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.

A Model for Graded Retinal Ischemia in Rats.

PURPOSE: Retinal ischemic injury depends on grade and duration of an ischemic insult. We developed a method to induce ischemic injury in rats permitting: (1) Variable grades of retinal blood flow (F) reduction, (2) controllable duration of F reduction, (3) injury without collateral neural damage, and (4) optical measurements of F and O2-related factors: O2 delivery (DO2), O2 extraction fraction (OEF), and metabolic rate of O2 (MO2). METHODS: In five anesthetized rats the left common carotid artery (CA) was ligated and the right CA was exposed. A variable clamp having a backstop and a rod mounted on a micromanipulator straddled the right CA. Advancing the rod with the micromanipulator produced graded compressions of the CA. F and O2-related factors were measured with established optical techniques. RESULTS: Four to seven grades of F for at least 10 minutes were achieved per rat. F decreased only with compressions of over 60%. DO2 changed in proportion to F, particularly at low F. As F decreased, OEF initially changed little, but then rose steeply to its maximum of 1 when F was approximately 4 µL/min. MO2 was stable with reduced F until OEF maximized, after which it decreased progressively. CONCLUSIONS: This model in rats permits acute, graded inner retinal ischemia that is reversible after prescribed durations, does not otherwise injure the eye and allows optical measurement of important physiologic factors during ischemia. TRANSLATIONAL RELEVANCE: This model will allow improved understanding of retinal ischemic injury and enable better management of this common, sight-threatening affliction.

Retinal Oxygen Delivery and Metabolism in Healthy and Sickle Cell Retinopathy Subjects.

Purpose: Reduction in inner retinal oxygen delivery (DO2) can cause retinal hypoxia and impair inner retinal oxygen metabolism (MO2), leading to vision loss. The purpose of the current study was to establish measurements of DO2 and MO2 in healthy subjects and test the hypothesis that DO2 and MO2 are reduced in sickle cell retinopathy (SCR) subjects. Methods: Dual wavelength retinal oximetry and Doppler optical coherence tomography were performed in 12 healthy control and 12 SCR subjects. Images were analyzed to measure retinal arterial and venous oxygen content (O2A and O2V), venous diameter (DV), and total retinal blood flow (TRBF). Retinal arteriovenous oxygen content difference (O2AV), DO2, MO2, and oxygen extraction fraction (OEF) were calculated according to the following equations: O2AV = O2A - O2V; DO2 = TRBF * O2A; MO2 = TRBF * O2AV; OEF = MO2/DO2. Results: Retinal DV and TRBF were higher in the SCR group as compared to the control group, whereas, O2A, O2V, and O2AV were lower in SCR group as compared to the control group. DO2, MO2, and OEF were not significantly different between control and SCR groups. MO2 and DO2 were linearly related, such that higher MO2 was associated with higher DO2. There was an inverse relationship between TRBF and OEF, such that lower TRBF was associated with higher OEF. Conclusions: Increased blood flow compensated for decreased oxygen content, thereby maintaining DO2, MO2, and OEF at predominately lower stages of SCR. Quantitative assessment of these parameters has the potential to advance knowledge and improve diagnostic evaluation of retinal ischemic conditions.

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.

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.

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.

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.

Inner Retinal Oxygen Delivery, Metabolism, and Extraction Fraction in Ins2Akita Diabetic Mice.

PURPOSE: Retinal nonperfusion and hypoxia are important factors in human diabetic retinopathy, and these presumably inhibit energy production and lead to cell death. The purpose of this study was to elucidate the effect of diabetes on inner retinal oxygen delivery and metabolism in a mouse model of diabetes. METHODS: Phosphorescence lifetime and blood flow imaging were performed in spontaneously diabetic Ins2Akita (n = 22) and nondiabetic (n = 22) mice at 12 and 24 weeks of age to measure retinal arterial (O2A) and venous (O2V) oxygen contents and total retinal blood flow (F). Inner retinal oxygen delivery (DO2) and metabolism (MO2) were calculated as F ∗ O2A and F ∗ (O2A - O2V), respectively. Oxygen extraction fraction (OEF), which equals MO2/DO2, was calculated. RESULTS: DO2 at 12 weeks were 112 ± 40 and 97 ± 29 nL O2/min in nondiabetic and diabetic mice, respectively (NS), and 148 ± 31 and 85 ± 37 nL O2/min at 24 weeks, respectively (P < 0.001). MO2 were 65 ± 31 and 66 ± 27 nL O2/min in nondiabetic and diabetic mice at 12 weeks, respectively, and 79 ± 14 and 54 ± 28 nL O2/min at 24 weeks, respectively (main effects = NS). At 12 weeks OEF were 0.57 ± 0.17 and 0.67 ± 0.09 in nondiabetic and diabetic mice, respectively, and 0.54 ± 0.07 and 0.63 ± 0.08 at 24 weeks, respectively (main effect of diabetes: P < 0.01). CONCLUSIONS: Inner retinal MO2 was maintained in diabetic Akita mice indicating that elevation of the OEF adequately compensated for reduced DO2 and prevented oxidative metabolism from being limited by hypoxia.

The Effects of Diabetic Retinopathy Stage and Light Flicker on Inner Retinal Oxygen Extraction Fraction.

PURPOSE: We determined the effects of light flicker and diabetic retinopathy (DR) stage on retinal vascular diameter (D), oxygen saturation (SO2), and inner retinal oxygen extraction fraction (OEF). METHODS: Subjects were categorized as nondiabetic control (NC, n = 42), diabetic with no clinical DR (NDR; n = 32), nonproliferative DR (NPDR; n = 42), or proliferative DR (PDR; n = 14). Our customized optical imaging system simultaneously measured arterial and venous D (DA, DV) and SO2 (SO2A, SO2V) before and during light flicker. Inner retinal OEF was derived from SO2 values. Light flicker-induced ratios of metrics (DAR, DVR, SO2AR, SO2VR, OEFR) were calculated. RESULTS: Arterial D was larger in NPDR compared to NC (P = 0.01) and PDR (P = 0.002), whereas DV was similar among groups (P ≥ 0.16). Light flicker increased DA and DV (P ≤ 0.004), but DAR and DVR were similar among groups (P ≥ 0.09). Arterial SO2 was higher in all groups compared to NC (P ≤ 0.02) and higher in PDR compared to NDR and NPDR (P<0.001). Arterial SO2 did not change with light flicker (P ≥ 0.1). Venous SO2 was higher in NPDR and PDR compared to NC and NDR (P ≤ 0.02). Light flicker increased SO2V in NC, NDR, and PDR (P ≤ 0.003), and SO2VR was lower in NPDR compared to NC and NDR (P ≤ 0.05). Inner retinal OEF was lower in NPDR compared to NDR and PDR (P ≤ 0.02). Light flicker decreased OEF (P ≤ 0.03), but OEFR was greater in NPDR compared to NC and NDR (P ≤ 0.03). CONCLUSIONS: The findings of alterations in retinal D, SO2, OEF, and their light flicker-induced responses at stages of DR may be useful to elucidate the pathophysiology of DR.

Automated Real-Time Conjunctival Microvasculature Image Stabilization.

The bulbar conjunctiva is a thin, vascularized membrane covering the sclera of the eye. Non-invasive imaging techniques have been utilized to assess the conjunctival vasculature as a means of studying microcirculatory hemodynamics. However, eye motion often confounds quantification of these hemodynamic properties. In the current study, we present a novel optical imaging system for automated stabilization of conjunctival microvasculature images by real-time eye motion tracking and realignment of the optical path. The ability of the system to stabilize conjunctival images acquired over time by reducing image displacements and maintaining the imaging area was demonstrated.

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.

Inner Retinal Oxygen Extraction Fraction in Response to Light Flicker Stimulation in Humans.

PURPOSE: Light flicker has been shown to stimulate retinal neural activity, increase blood flow, and alter inner retinal oxygen metabolism (MO2) and delivery (DO2). The purpose of the study was to determine the change in MO2 relative to DO2 due to light flicker stimulation in humans, as assessed by the inner retinal oxygen extraction fraction (OEF). METHODS: An optical imaging system, based on a modified slit lamp biomicroscope, was developed for simultaneous measurements of retinal vascular diameter (D) and oxygen saturation (SO2). Retinal images were acquired in 20 healthy subjects before and during light flicker stimulation. Arterial and venous D (DA and DV) and SO2 (SO2A and SO2V) were quantified within a circumpapillary region. Oxygen extraction fraction was defined as the ratio of MO2 to DO2 and was calculated as (SO2A - SO2V)/SO2A. Reproducibility of measurements was assessed. RESULTS: Coefficients of variation and intraclass correlation coefficients of repeated measurements were <5% and ≥0.83, respectively. During light flicker stimulation, DA, DV , and SO2V significantly increased (P ≤ 0.004). Oxygen extraction fraction was 0.37 ± 0.08 before light flicker and significantly decreased to 0.31 ± 0.07 during light flicker (P = 0.001). CONCLUSIONS: Oxygen extraction fraction before and during light flicker stimulation is reported in human subjects for the first time. Oxygen extraction fraction decreased during light flicker stimulation, indicating the change in DO2 exceeded that of MO2. This technology is potentially useful for the detection of changes in OEF response to light flicker in physiological and pathological retinal conditions.