Alexander Pinhas

In vivo imaging of human retinal microvasculature using adaptive optics scanning light ophthalmoscope fluorescein angiography

The adaptive optics scanning light ophthalmoscope (AOSLO) allows visualization of microscopic structures of the human retina in vivo. In this work, we demonstrate its application in combination with oral and intravenous (IV) fluorescein angiography (FA) to the in vivo visualization of the human retinal microvasculature. Ten healthy subjects ages 20 to 38 years were imaged using oral (7 and/or 20 mg/kg) and/or IV (500 mg) fluorescein. In agreement with current literature, there were no adverse effects among the patients receiving oral fluorescein while one patient receiving IV fluorescein experienced some nausea and heaving. We determined that all retinal capillary beds can be imaged using clinically accepted fluorescein dosages and safe light levels according to the ANSI Z136.1-2000 maximum permissible exposure. As expected, the 20 mg/kg oral dose showed higher image intensity for a longer period of time than did the 7 mg/kg oral and the 500 mg IV doses. The increased resolution of AOSLO FA, compared to conventional FA, offers great opportunity for studying physiological and pathological vascular processes.

Assessment of Perfused Foveal Microvascular Density and Identification of Nonperfused Capillaries in Healthy and Vasculopathic Eyes

PURPOSE To analyze the foveal microvasculature of young healthy eyes and older vasculopathic eyes, imaged using in vivo adaptive optics scanning light ophthalmoscope fluorescein angiography (AOSLO FA). METHODS AOSLO FA imaging of the superficial retinal microvasculature within an 800-μm radius from the foveal center was performed using simultaneous confocal infrared (IR) reflectance (790 nm) and fluorescence (488 nm) channels. Corresponding IR structural and FA perfusion maps were compared with each other to identify nonperfused capillaries adjacent to the foveal avascular zone. Microvascular densities were calculated from skeletonized FA perfusion maps. RESULTS Sixteen healthy adults (26 eyes; mean age 25 years, range, 21-29) and six patients with a retinal vasculopathy (six eyes; mean age 55 years, range, 44-70) were imaged. At least one nonperfused capillary was observed in five of the 16 healthy nonfellow eyes and in four of the six vasculopathic eyes. Compared with healthy eyes, capillary nonperfusion in the vasculopathic eyes was more extensive. Microvascular density of the 16 healthy nonfellow eyes was 42.0 ± 4.2 mm(-1) (range, 33-50 mm(-1)). All six vasculopathic eyes had decreased microvascular densities. CONCLUSIONS AOSLO FA provides an in vivo method for estimating foveal microvascular density and reveals occult nonperfused retinal capillaries. Nonperfused capillaries in healthy young adults may represent a normal variation and/or an early sign of pathology. Although limited, the normative data presented here is a step toward developing clinically useful microvascular parameters for ocular and/or systemic diseases.

Classification of Human Retinal Microaneurysms Using Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography

PURPOSE Microaneurysms (MAs) are considered a hallmark of retinal vascular disease, yet what little is known about them is mostly based upon histology, not clinical observation. Here, we use the recently developed adaptive optics scanning light ophthalmoscope (AOSLO) fluorescein angiography (FA) to image human MAs in vivo and to expand on previously described MA morphologic classification schemes. METHODS Patients with vascular retinopathies (diabetic, hypertensive, and branch and central retinal vein occlusion) were imaged with reflectance AOSLO and AOSLO FA. Ninety-three MAs, from 14 eyes, were imaged and classified according to appearance into six morphologic groups: focal bulge, saccular, fusiform, mixed, pedunculated, and irregular. The MA perimeter, area, and feret maximum and minimum were correlated to morphology and retinal pathology. Select MAs were imaged longitudinally in two eyes. RESULTS Adaptive optics scanning light ophthalmoscope fluorescein angiography imaging revealed microscopic features of MAs not appreciated on conventional images. Saccular MAs were most prevalent (47%). No association was found between the type of retinal pathology and MA morphology (P = 0.44). Pedunculated and irregular MAs were among the largest MAs with average areas of 4188 and 4116 μm(2), respectively. Focal hypofluorescent regions were noted in 30% of MAs and were more likely to be associated with larger MAs (3086 vs. 1448 μm(2), P = 0.0001). CONCLUSIONS Retinal MAs can be classified in vivo into six different morphologic types, according to the geometry of their two-dimensional (2D) en face view. Adaptive optics scanning light ophthalmoscope fluorescein angiography imaging of MAs offers the possibility of studying microvascular change on a histologic scale, which may help our understanding of disease progression and treatment response.

Comparison of adaptive optics scanning light ophthalmoscopic fluorescein angiography and offset pinhole imaging

Recent advances to the adaptive optics scanning light ophthalmoscope (AOSLO) have enabled finer in vivo assessment of the human retinal microvasculature. AOSLO confocal reflectance imaging has been coupled with oral fluorescein angiography (FA), enabling simultaneous acquisition of structural and perfusion images. AOSLO offset pinhole (OP) imaging combined with motion contrast post-processing techniques, are able to create a similar set of structural and perfusion images without the use of exogenous contrast agent. In this study, we evaluate the similarities and differences of the structural and perfusion images obtained by either method, in healthy control subjects and in patients with retinal vasculopathy including hypertensive retinopathy, diabetic retinopathy, and retinal vein occlusion. Our results show that AOSLO OP motion contrast provides perfusion maps comparable to those obtained with AOSLO FA, while AOSLO OP reflectance images provide additional information such as vessel wall fine structure not as readily visible in AOSLO confocal reflectance images. AOSLO OP offers a non-invasive alternative to AOSLO FA without the need for any exogenous contrast agent.

Outer retinal structure after closed-globe blunt ocular trauma.

Purpose: To evaluate outer retinal structural abnormalities in patients with visual deficits after closed-globe blunt ocular trauma. Methods: Nine subjects with visual complaints after closed-globe blunt ocular trauma were examined between 1 month after trauma and 6 years after trauma. Spectral domain optical coherence tomography was used to assess the outer retinal architecture, whereas adaptive optics scanning light ophthalmoscopy was used to analyze the photoreceptor mosaic integrity. Results: Visual deficits ranged from central scotomas to decreased visual acuity. Spectral domain optical coherence tomography defects included focal foveal photoreceptor lesions, variable attenuation of the interdigitation zone, and mottling of the outer segment band, with one subject having normal outer retinal structure. Adaptive optics scanning light ophthalmoscopy revealed disruption of the photoreceptor mosaic in all subjects, variably manifesting as foveal focal discontinuities, perifoveal hyporeflective cones, and paracentral regions of selective cone loss. Conclusion: We observe persistent outer retinal disruption in subjects with visual complaints after closed-globe blunt ocular trauma, albeit to a variable degree. Adaptive optics scanning light ophthalmoscopy imaging allows the assessment of photoreceptor structure at a level of detail not resolvable using spectral domain optical coherence tomography or other current clinical imaging tools. Multimodal imaging seems to be useful in revealing the cause of visual complaints in patients after closed-globe blunt ocular trauma. Future studies are needed to better understand how photoreceptor structure changes longitudinally in response to various traumas.

Fellow Eye Changes In Patients With Nonischemic Central Retinal Vein Occlusion: Assessment of Perfused Foveal Microvascular Density and Identification of Nonperfused Capillaries

Purpose: Eyes fellow to nonischemic central retinal vein occlusion (CRVO) were examined for abnormalities, which might explain their increased risk for future occlusion, using adaptive optics scanning light ophthalmoscope fluorescein angiography. Methods: Adaptive optics scanning light ophthalmoscope fluorescein angiography foveal microvascular densities were calculated. Nonperfused capillaries adjacent to the foveal avascular zone were identified. Spectral domain optical coherence tomography, ultrawide field fluorescein angiographies, and microperimetry were also performed. Results: Ten fellow eyes of nine nonischemic CRVO and 1 nonischemic hemi-CRVO subjects and four affected eyes of three nonischemic CRVO and one nonischemic hemi-CRVO subjects were imaged. Ninety percent of fellow eyes and 100% of affected eyes demonstrated at least 1 nonperfused capillary compared with 31% of healthy eyes. Fellow eye microvascular density (35 ± 3.6 mm−1) was significantly higher than that of affected eyes (25 ± 5.2 mm−1) and significantly lower than that of healthy eyes (42 ± 4.2 mm−1). Compared with healthy controls, spectral domain optical coherence tomography thicknesses showed no significant difference, whereas microperimetry and 2/9 ultrawide field fluorescein angiography revealed abnormalities in fellow eyes. Conclusion: Fellow eye changes detectable on adaptive optics scanning light ophthalmoscope fluorescein angiography reflect subclinical pathology difficult to detect using conventional imaging technologies. These changes may help elucidate the pathogenesis of nonischemic CRVO and help identify eyes at increased risk of future occlusion.

Longitudinal imaging of microvascular remodelling in proliferative diabetic retinopathy using adaptive optics scanning light ophthalmoscopy

To characterise longitudinal changes in the retinal microvasculature of type 2 diabetes mellitus (T2DM) as exemplified in a patient with proliferative diabetic retinopathy (PDR) using an adaptive optics scanning light ophthalmoscope (AOSLO).

Isoforms of Secretory Group Two Phospholipase A (sPLA2) in Mouse Ocular Surface Epithelia and Lacrimal Glands

PURPOSE To compare and contrast the distribution patterns of select secretory group two phospholipase A (sPLA2) isoforms in corneal epithelia (CN), conjunctival epithelia (CNJ), and lacrimal glands (LG) of BALB/c and C57BL/6 mice. METHODS Gene expression of select sPLA2 isoforms was quantified via real-time reverse-transcription PCR (qRT(2)-PCR). Immunofluorescence assay (IFA) of the sPLA2-IIa, -V, and -X isoforms were used to confirm qRT(2)-PCR results. sPLA2-IIa function was confirmed via in vitro CN and CNJ culturing. RESULTS qRT(2)-PCR revealed that sPLA2 isoforms (pla2g5, 12a, and 12b), cPLA2 isoform (pla2g4a), iPLA2 isoform (pla2g6), and PLA2-receptor (pla2r1) were present in all tissues of both strains, whereas sPLA2 isoforms (pla2g1b, 2e, and 3) were absent. sPLA2 isoforms (pla2g2a, 2d, 2f, and 10) showed tissue- and strain-specific expression: 2a in BALB/c CNJ only; 2d at higher levels in CNJ than LG; and 2f and 10 in CN and CNJ, but absent in LG. Upon dry eye (DE) induction, pla2g2a, 2d, and 2f were upregulated in BALB/c CNJ, and 10 was absent from CN. Furthermore, BALB/c DE mice showed upregulation of pla2r1 in CN and CNJ and downregulation of 12a and 12b in LG. IFA of sPLA2-IIa, -V, and -X in DE CNJ confirmed the upregulation of pla2g2a, 5, and 10. Last, in vitro CN and CNJ culturing confirmed that sPLA2-IIa amplifies ocular surface inflammation in CNJ but not in CN. CONCLUSIONS sPLA2 isoforms exhibit differential expression patterns when comparing BALB/c with C57BL/6 mice; and DE with control BALB/c mice. These findings suggest that at least some sPLA2 isoforms must have significant roles in ocular surface physiology and inflammation.

Human retinal microvascular imaging using adaptive optics scanning light ophthalmoscopy

BackgroundRetinal microvascular imaging is an especially promising application of high resolution imaging since there are increasing options for therapeutic intervention and need for better structural and functional biomarkers to characterize ocular and systemic vascular diseases.Main bodyAdaptive optics scanning light ophthalmoscopy (AOSLO) is an emerging technology for improving in vivo imaging of the human retinal microvasculature, allowing unprecedented visualization of retinal microvascular structure, measurements of blood flow velocity, and microvascular network mapping. This high resolution imaging technique shows significant potential for studying physiological and pathological conditions of the retinal microvasculature noninvasively.ConclusionThis review will briefly summarize the abilities of in vivo human retinal microvasculature imaging in healthy controls, as well as patients with diabetic retinopathy, retinal vein occlusion, and sickle cell retinopathy using AOSLO and discuss its potential contribution to scientific research and clinical applications.

A method for age-matched OCT angiography deviation mapping in the assessment of disease- related changes to the radial peripapillary capillaries

Purpose To present a method for age-matched deviation mapping in the assessment of disease-related changes to the radial peripapillary capillaries (RPCs). Methods We reviewed 4.5x4.5mm en face peripapillary OCT-A scans of 133 healthy control eyes (133 subjects, mean 41.5 yrs, range 11–82 yrs) and 4 eyes with distinct retinal pathologies, obtained using spectral-domain optical coherence tomography angiography. Statistical analysis was performed to evaluate the impact of age on RPC perfusion densities. RPC density group mean and standard deviation maps were generated for each decade of life. Deviation maps were created for the diseased eyes based on these maps. Large peripapillary vessel (LPV; noncapillary vessel) perfusion density was also studied for impact of age. Results Average healthy RPC density was 42.5±1.47%. ANOVA and pairwise Tukey-Kramer tests showed that RPC density in the ≥60yr group was significantly lower compared to RPC density in all younger decades of life (p<0.01). Average healthy LPV density was 21.5±3.07%. Linear regression models indicated that LPV density decreased with age, however ANOVA and pairwise Tukey-Kramer tests did not reach statistical significance. Deviation mapping enabled us to quantitatively and visually elucidate the significance of RPC density changes in disease. Conclusions It is important to consider changes that occur with aging when analyzing RPC and LPV density changes in disease. RPC density, coupled with age-matched deviation mapping techniques, represents a potentially clinically useful method in detecting changes to peripapillary perfusion in disease.