Fluorescence Lifetime Imaging of Human Retinal Pigment Epithelium in Pentosan Polysulfate Toxicity Using Adaptive Optics Scanning Light Ophthalmoscopy.

Purpose: Fluorescence lifetime ophthalmoscopy (FLIO) is an emerging clinical modality that could provide biomarkers of retinal health beyond fluorescence intensity. Adaptive optics (AO) ophthalmoscopy provides the confocality to measure fluorescence lifetime (FL) primarily from the retinal pigment epithelium (RPE) whereas clinical FLIO has greater influence from fluorophores in the inner retina and lens. Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) measures of FL in vivo could provide insight into RPE health at different stages of disease. In this study, we assess changes in pentosan polysulfate sodium (PPS) toxicity, a recently described toxicity that has clinical findings similar to advanced age-related macular degeneration. Methods: AOFLIO was performed on three subjects with PPS toxicity (57-67 years old) and six age-matched controls (50-64 years old). FL was analyzed with a double exponential decay curve fit and with phasor analysis. Regions of interest (ROIs) were subcategorized based on retinal features on optical coherence tomography (OCT) and compared to age-matched controls. Results: Twelve ROIs from PPS toxicity subjects met the threshold for analysis by curve fitting and 15 ROIs met the threshold for phasor analysis. Subjects with PPS toxicity had prolonged FL compared to age-matched controls. ROIs of RPE degeneration had the longest FLs, with individual pixels extending longer than 900 ps. Conclusions: Our study shows evidence that AOFLIO can provide meaningful information in outer retinal disease beyond what is obtainable from fluorescence intensity alone. More studies are needed to determine the prognostic value of AOFLIO.

Imaging Transplanted Photoreceptors in Living Nonhuman Primates with Single-Cell Resolution.

Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX+/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo. Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.

Endogenous fluorophores enable two-photon imaging of the primate eye.

PURPOSE: Noninvasive two-photon imaging of a living mammalian eye can reveal details of molecular processes in the retina and RPE. Retinyl esters and all-trans-retinal condensation products are two types of retinoid fluorophores present in these tissues. We measured the content of these two types of retinoids in monkey and human eyes to validate the potential of two-photon imaging for monitoring retinoid changes in human eyes. METHODS: Two-photon microscopy (TPM) was used to visualize excised retina from monkey eyes. Retinoid composition and content in human and monkey eyes were quantified by HPLC and mass spectrometry (MS). RESULTS: Clear images of inner and outer segments of rods and cones were obtained in primate eyes at different eccentricities. Fluorescence spectra from outer segments revealed a maximum emission at 480 nm indicative of retinols and their esters. In cynomolgus monkey and human retinal extracts, retinyl esters existed predominantly in the 11-cis configuration along with notable levels of 11-cis-retinol, a characteristic of cone-enriched retinas. Average amounts of di-retinoid-pyridinium-ethanolamine (A2E) in primate and human eyes were 160 and 225 pmol/eye, respectively. CONCLUSIONS: These data show that human retina contains sufficient amounts of retinoids for two-photon excitation imaging. Greater amounts of 11-cis-retinyl esters relative to rodent retinas contribute to the fluorescence signal from both monkey and human eyes. These observations indicate that TPM imaging found effective in mice could detect early age- and disease-related changes in human retina.

Focal damage to macaque photoreceptors produces persistent visual loss.

Insertion of light-gated channels into inner retina neurons restores neural light responses, light evoked potentials, visual optomotor responses and visually-guided maze behavior in mice blinded by retinal degeneration. This method of vision restoration bypasses damaged outer retina, providing stimulation directly to retinal ganglion cells in inner retina. The approach is similar to that of electronic visual protheses, but may offer some advantages, such as avoidance of complex surgery and direct targeting of many thousands of neurons. However, the promise of this technique for restoring human vision remains uncertain because rodent animal models, in which it has been largely developed, are not ideal for evaluating visual perception. On the other hand, psychophysical vision studies in macaque can be used to evaluate different approaches to vision restoration in humans. Furthermore, it has not been possible to test vision restoration in macaques, the optimal model for human-like vision, because there has been no macaque model of outer retina degeneration. In this study, we describe development of a macaque model of photoreceptor degeneration that can in future studies be used to test restoration of perception by visual prostheses. Our results show that perceptual deficits caused by focal light damage are restricted to locations at which photoreceptors are damaged, that optical coherence tomography (OCT) can be used to track such lesions, and that adaptive optics retinal imaging, which we recently used for in vivo recording of ganglion cell function, can be used in future studies to examine these lesions.

Toward an understanding of bisretinoid autofluorescence bleaching and recovery.

PURPOSE: To understand molecular mechanisms underlying photobleaching of the RPE fluorophores responsible for fundus autofluorescence. METHODS: ARPE-19 cells were allowed to accumulate the bisretinoid, A2E, and were irradiated at 430 nm. For some experiments, the cells were pretreated with vitamin E or sulforaphane and N-acetylcysteine; samples included A2E-free cells. The cells were analyzed by fluorescence microscopy and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. A2E free cells were also irradiated and analyzed. Cell death was quantified by double labeling with a membrane impermeable dye and 4',6'-diamino-2-phenylindole (DAPI). RESULTS: A2E that had accumulated in ARPE-19 cells exhibited irradiation-associated autofluorescence bleaching despite the absence of appreciable cell death. Chromatographic analysis with absorbance, fluorescence, and mass spectrometry detection revealed that irradiation of A2E was associated with A2E photoisomerization, photooxidation, and photodegradation. Pretreatment with vitamin E favored fluorescence recovery; this finding was consistent with a process involving photooxidation. A2E that was not cell-associated underwent irradiation-induced bleaching, but fluorescence recovery was not observed. CONCLUSIONS: Using cell-associated A2E as a model of RPE bisretinoid behavior, photobleaching and autofluorescence recovery was observed; these changes were similar to RPE autofluorescence reduction in vivo. The potential for autofluorescence recovery is dependent on light dose and antioxidant status. Fluorescence bleaching of bisretinoid involves photooxidative and photodegradative processes.

Intravitreal injection of AAV2 transduces macaque inner retina.

PURPOSE: Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. METHODS: In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. RESULTS: AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-ß-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters--human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)--AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. CONCLUSIONS: The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.

In vivo imaging of microscopic structures in the rat retina.

PURPOSE: The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. The authors have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo. METHODS: Enhanced green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague-Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy. RESULTS: Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics. Adaptive optics correction reduced the total root mean square wavefront error, on average, from 0.30 microm to 0.05 microm (measured at 904 nm, 1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was approximately 1.84 microm, approximately 82% greater than the FWHM of the diffraction-limited LSF. CONCLUSIONS: With perfect aberration compensation, the in vivo resolution in the rat eye could be approximately 2x greater than that in the human eye because of its large numerical aperture (approximately 0.43). Although the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using adaptive optics to investigate the rodent eye in vivo with high resolution.

Potential effect on the retinoscopic reflex of scleral expansion surgery for presbyopia.

PURPOSE: In this initial study, we model the impact of crystalline lens movement and tilt, which we postulate are a potential consequence of scleral expansion surgery (SES). We demonstrate the possibility that these lead to an improvement in near vision with no concurrent restoration of lens accommodative function. With the implantation of scleral expansion bands (SEB), Schachar predicts an increase in the amplitude of accommodation not observed objectively. We postulate that the SEB implants cause the crystalline lens to be shifted forward and become misaligned. Using mathematical models of the optics of the human eye, we assess the effects of our postulate on the power of the eye, the depth of field, optical aberrations, and the appearance of the horizontal retinoscopic reflex. METHODS: Using anatomically based models of the human eye, the postulated effects of SES are theoretically modeled and optically analyzed in Code V for unintentional lens anterior movement, tilts, and decentrations of up to 1 mm, +/- 3 degrees , and +/- 0.3mm, respectively. The transverse aberrations are calculated before and after SES. Because it has been reported that the appearance of the retinoscopic reflex is consistent with the presence of excess aberrations, we also predict the appearance of the one-dimensional retinoscopic reflex for our models. The change in refractive error is also determined. RESULTS.: Unintentional lens shift, tilt, and decentration, as might occur as a result of SES surgery, would result in an increase in the total power of the eye along with an increase in asymmetric aberrations and little change in symmetric aberrations. The calculated appearances of the retinoscopic reflexes pre- and postsurgery are consistent with observations in the literature. CONCLUSIONS: Scleral expansion surgery could produce an improvement in near vision through an unintentional anterior displacement of the crystalline lens in combination with excess tilts and decentrations rather than as a result of a restoration of lens accommodation. There is a need for measurements and further analysis of the optical and visual properties of SES patients postsurgery.

Optical analysis of an accommodating intraocular lens.

PURPOSE: To model the feasibility of an accommodating intraocular lens (IOL) that allows near vision by means of an anterior translation within the capsular bag. SETTING: University of Waterloo, Waterloo, Ontario, Canada. METHODS: Model eyes were constructed and analyzed based on experimental data using Code V, a computerized optical design tool. The potential near vision of IOLs of different powers was calculated as they were moved anteriorly within the capsular bag. The conditions under which a spherical lens performs well and when an aspheric design should be considered were determined. RESULTS: Accommodation (the dioptric change from the far to the near point) varies linearly with lens movement and is sensitive to corneal and IOL powers. Simple equations were derived and accurately predicted induced accommodation. Retinal image quality varies significantly with pupil size and IOL power. However, image quality is minimally affected by the amount of induced accommodation. CONCLUSIONS: From an optical standpoint accommodation with adequate image quality can be achieved by anterior movement of a spherical IOL within the capsular bag.