Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Invest Ophthalmol Vis Sci ; 55(11): 7535-44, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25316724

ABSTRACT

PURPOSE: Advances in retinal imaging have led to the discovery of long-lasting retinal changes caused by light exposures below published safety limits, including disruption of the RPE. To investigate the functional consequences of RPE disruption, we combined adaptive optics ophthalmoscopy with retinal densitometry. METHODS: A modified adaptive optics scanning light ophthalmoscope (AOSLO) measured the apparent density and regeneration rate of rhodopsin in two macaques before and after four different 568-nm retinal radiant exposures (RREs; 400-3200 J/cm(2)). Optical coherence tomography (OCT) was used to measure the optical path length through the photoreceptor outer segments before and after RPE disruption. RESULTS: All tested RREs caused visible RPE disruption. Apparent rhodopsin density was significantly reduced following 1600 (P = 0.01) and 3200 J/cm(2) (P = 0.007) exposures. No significant change in apparent density was observed in response to 800 J/cm(2). Surprisingly, exposure to 400 J/cm(2) showed a significant increase in apparent density (P = 0.047). Rhodopsin recovery rate was not significantly affected by these RREs. Optical coherence tomography measurements showed a significant decrease in the optical path length through the photoreceptor outer segments for RREs above 800 J/cm(2) (P < 0.001). CONCLUSIONS: At higher RREs, optical path length through the outer segments was reduced. However, the rate of photopigment regeneration was unchanged. While some ambiguity remains as to the correlation between measured reflectivity and absolute rhodopsin density; at the lowest RREs, RPE disruption appears not to be accompanied by a loss of apparent rhodopsin density, which would have been indicative of functional loss.


Subject(s)
Dark Adaptation/physiology , Densitometry/methods , Retinal Pigment Epithelium/metabolism , Rhodopsin/metabolism , Animals , Macaca , Retinal Photoreceptor Cell Outer Segment/metabolism , Retinal Pigment Epithelium/cytology , Tomography, Optical Coherence/methods
2.
Invest Ophthalmol Vis Sci ; 55(11): 7525-34, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25316726

ABSTRACT

PURPOSE: Retinal densitometry provides objective information about retinal function. But, a number of factors, including retinal reflectance changes that are not directly related to photopigment depletion, complicate its interpretation. We explore these factors and suggest a method to minimize their impact. METHODS: An adaptive optics scanning light ophthalmoscope (AOSLO) was used to measure changes in photoreceptor reflectance in monkeys before and after photopigment bleaching with 514-nm light. Reflectance measurements at 514 nm and 794 nm were recorded simultaneously. Several methods of normalization to extract the apparent optical density of the photopigment were compared. RESULTS: We identified stimulus-related fluctuations in 794-nm reflectance that are not associated with photopigment absorptance and occur in both rods and cones. These changes had a magnitude approaching those associated directly with pigment depletion, precluding the use of infrared reflectance for normalization. We used a spatial normalization method instead, which avoided the fluctuations in the near infrared, as well as a confocal AOSLO designed to minimize light from layers other than the receptors. However, these methods produced a surprisingly low estimate of the apparent rhodopsin density (animal 1: 0.073 ± 0.006, animal 2: 0.032 ± 0.003). CONCLUSIONS: These results confirm earlier observations that changes in photopigment absorption are not the only source of retinal reflectance change during dark adaptation. It appears that the stray light that has historically reduced the apparent density of cone photopigment in retinal densitometry arises predominantly from layers near the photoreceptors themselves. Despite these complications, this method provides a valuable, objective measure of retinal function.


Subject(s)
Dark Adaptation/physiology , Densitometry/methods , Retina/metabolism , Rhodopsin/metabolism , Animals , Female , Macaca , Male , Ophthalmoscopy , Retina/cytology
3.
J Neurosci ; 34(19): 6596-605, 2014 May 07.
Article in English | MEDLINE | ID: mdl-24806684

ABSTRACT

The fovea dominates primate vision, and its anatomy and perceptual abilities are well studied, but its physiology has been little explored because of limitations of current physiological methods. In this study, we adapted a novel in vivo imaging method, originally developed in mouse retina, to explore foveal physiology in the macaque, which permits the repeated imaging of the functional response of many retinal ganglion cells (RGCs) simultaneously. A genetically encoded calcium indicator, G-CaMP5, was inserted into foveal RGCs, followed by calcium imaging of the displacement of foveal RGCs from their receptive fields, and their intensity-response functions. The spatial offset of foveal RGCs from their cone inputs makes this method especially appropriate for fovea by permitting imaging of RGC responses without excessive light adaptation of cones. This new method will permit the tracking of visual development, progression of retinal disease, or therapeutic interventions, such as insertion of visual prostheses.


Subject(s)
Eye/radiation effects , Fovea Centralis/radiation effects , Light , Retinal Ganglion Cells/radiation effects , Anesthesia , Animals , Dependovirus/genetics , Electrophysiological Phenomena , Eye/anatomy & histology , Eye Movements/physiology , Female , Fluorescent Dyes , Fovea Centralis/cytology , Gene Transfer Techniques , Light Signal Transduction/physiology , Macaca fascicularis , Microscopy, Confocal , Neuroimaging/methods , Photic Stimulation , Photoreceptor Cells, Vertebrate/physiology , Retina/growth & development , Retinal Degeneration/pathology , Signal-To-Noise Ratio
4.
Invest Ophthalmol Vis Sci ; 55(6): 3929-38, 2014 May 20.
Article in English | MEDLINE | ID: mdl-24845640

ABSTRACT

PURPOSE: Many retinal imaging instruments use infrared wavelengths to reduce the risk of light damage. However, we have discovered that exposure to infrared illumination causes a long-lasting reduction in infrared autofluorescence (IRAF). We have characterized the dependence of this effect on radiant exposure and investigated its origin. METHODS: A scanning laser ophthalmoscope was used to obtain IRAF images from two macaques before and after exposure to 790-nm light (15-450 J/cm(2)). Exposures were performed with either raster-scanning or uniform illumination. Infrared autofluorescence images also were obtained in two humans exposed to 790-nm light in a separate study. Humans were assessed with direct ophthalmoscopy, Goldmann visual fields, multifocal ERG, and photopic microperimetry to determine whether these measures revealed any effects in the exposed locations. RESULTS: A significant decrease in IRAF after exposure to infrared light was seen in both monkeys and humans. In monkeys, the magnitude of this reduction increased with retinal radiant exposure. Partial recovery was seen at 1 month, with full recovery within 21 months. Consistent with a photochemical origin, IRAF decreases caused by either raster-scanning or uniform illumination were not significantly different. We were unable to detect any effect of the light exposure with any measure other than IRAF imaging. We cannot exclude the possibility that changes could be detected with more sensitive tests or longer follow-up. CONCLUSIONS: This long-lasting effect of infrared illumination in both humans and monkeys occurs at exposure levels four to five times below current safety limits. The photochemical basis for this phenomenon remains unknown.


Subject(s)
Fluorescein Angiography , Infrared Rays/adverse effects , Optical Imaging , Radiation Injuries, Experimental/etiology , Retina/radiation effects , Retinal Diseases/etiology , Animals , Electroretinography , Female , Lipofuscin/metabolism , Macaca fascicularis , Macaca mulatta , Male , Maximum Allowable Concentration , Ophthalmoscopy , Radiation Injuries, Experimental/diagnosis , Radiation Injuries, Experimental/metabolism , Retinal Diseases/diagnosis , Retinal Diseases/metabolism , Retinal Pigment Epithelium/metabolism , Visual Field Tests , Visual Fields
5.
Exp Eye Res ; 119: 88-96, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24316158

ABSTRACT

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.


Subject(s)
Photoreceptor Cells/pathology , Retinal Degeneration/pathology , Retinal Ganglion Cells/pathology , Vision, Low/etiology , Visual Acuity , Animals , Disease Progression , Fluorescein Angiography , Fundus Oculi , Macaca , Retinal Degeneration/complications , Retinal Degeneration/physiopathology , Tomography, Optical Coherence , Vision, Low/pathology , Vision, Low/physiopathology
6.
Invest Ophthalmol Vis Sci ; 52(5): 2775-83, 2011 Apr 25.
Article in English | MEDLINE | ID: mdl-21310920

ABSTRACT

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.


Subject(s)
Dependovirus/genetics , Genetic Vectors , Green Fluorescent Proteins/genetics , Microglia/metabolism , Retinal Ganglion Cells/metabolism , Transduction, Genetic , Actins/genetics , Animals , Axons/metabolism , Connexins/genetics , Gene Expression , Genes, Reporter , Intravitreal Injections , Macaca , Microscopy, Confocal , Synapsins/genetics , Gap Junction delta-2 Protein
7.
Biomed Opt Express ; 2(1): 139-48, 2010 Dec 17.
Article in English | MEDLINE | ID: mdl-21326644

ABSTRACT

In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.

8.
BMC Ophthalmol ; 9: 9, 2009 Aug 23.
Article in English | MEDLINE | ID: mdl-19698151

ABSTRACT

BACKGROUND: Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed. METHODS: We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue. RESULTS: Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections. CONCLUSION: This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.


Subject(s)
Retina/cytology , Retinal Vessels/cytology , Animals , Fluorescein , Glaucoma/pathology , Humans , In Vitro Techniques , Macaca mulatta , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Nerve Fibers/ultrastructure , Ophthalmoscopy/methods , Retinal Ganglion Cells/cytology
9.
Invest Ophthalmol Vis Sci ; 49(8): 3715-29, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18408191

ABSTRACT

PURPOSE: Autofluorescence fundus imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) allows for imaging of individual retinal pigment epithelial (RPE) cells in vivo. In this study, the potential of retinal damage was investigated by using radiant exposure levels that are 2 to 150 times those used for routine imaging. METHODS: Macaque retinas were imaged in vivo with a fluorescence AOSLO. The retina was exposed to 568- or 830-nm light for 15 minutes at various intensities over a square (1/2) degrees per side. Pre- and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2 degrees field. Long-term AOSLO imaging was performed intermittently from 5 to 165 days after exposure. Exposures delivered over a uniform field were also investigated. RESULTS: Exposures to 568-nm light caused an immediate decrease in autofluorescence of RPE cells. Follow-up imaging revealed either full recovery of autofluorescence or long-term damage in the RPE cells at the exposure. The outcomes of AOSLO exposures and uniform field exposures of equal average power were not significantly different. No effects from 830-nm exposures were observed. CONCLUSIONS: The study revealed a novel change in RPE autofluorescence induced by 568-nm light exposure. Retinal damage occurred as a direct result of total average power, independent of the light-delivery METHOD: Because the exposures were near or below permissible levels in laser safety standards, these results suggest that caution should be used with exposure of the retina to visible light and that the safety standards should be re-evaluated for these exposure conditions.


Subject(s)
Fluorescence , Light/adverse effects , Lipofuscin/metabolism , Pigment Epithelium of Eye/metabolism , Radiation Injuries, Experimental/metabolism , Retina/radiation effects , Retinal Degeneration/metabolism , Animals , Fluorescein Angiography , Macaca fascicularis , Macaca nemestrina , Male , Ophthalmoscopy/methods , Pigment Epithelium of Eye/pathology , Radiation Injuries, Experimental/diagnosis , Radiation Injuries, Experimental/etiology , Retina/pathology , Retinal Degeneration/diagnosis , Retinal Degeneration/etiology
10.
Invest Ophthalmol Vis Sci ; 49(1): 467-73, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18172127

ABSTRACT

PURPOSE: The extent to which the fine structure of single ganglion cells, such as dendrites and axons, can be resolved in retinal images obtained from the living primate eye was investigated. METHODS: Macaque retinal ganglion cells were labeled with retrograde transport of rhodamine dextran injected into the lateral geniculate nucleus. Fluorescence images of the ganglion cells were obtained in vivo with an adaptive optics scanning laser ophthalmoscope. RESULTS: Axons and dendritic arborization could be resolved in primate retinal ganglion cells in vivo, comparing favorably in detail with ex vivo confocal images of the same cells. The full width at half maximum of the transverse line spread function (LSF) was 1.6 microm, and that of the axial point spread function (PSF) was 115 microm. The axial positional accuracy of fluorescence-labeled objects was approximately 4 microm. CONCLUSIONS: This in vivo method applied to ganglion cells demonstrates that structures smaller than the somas of typical retinal cells can be accessible in living eyes. Similar approaches may be applied to image other relatively transparent retinal structures, providing a potentially valuable tool for microscopic examination of the normal and diseased living retina.


Subject(s)
Axons , Dendrites , Macaca mulatta/anatomy & histology , Retinal Ganglion Cells/cytology , Animals , Dextrans , Fluorescent Dyes , Microscopy, Confocal , Ophthalmoscopy , Rhodamines
SELECTION OF CITATIONS
SEARCH DETAIL
...