Whole-eye electrical stimulation therapy preserves visual function and structure in P23H-1 rats.

Low-level electrical stimulation to the eye has been shown to be neuroprotective against retinal degeneration in both human and animal subjects, using approaches such as subretinal implants and transcorneal electrical stimulation. In this study, we investigated the benefits of whole-eye electrical stimulation (WES) in a rodent model of retinitis pigmentosa. Transgenic rats with a P23H-1 rhodopsin mutation were treated with 30 min of low-level electrical stimulation (4 µA at 5 Hz; n = 10) or sham stimulation (Sham group; n = 15), twice per week, from 4 to 24 weeks of age. Retinal and visual functions were assessed every 4 weeks using electroretinography and optokinetic tracking, respectively. At the final time point, eyes were enucleated and processed for histology. Separate cohorts were stimulated once for 30 min, and retinal tissue harvested at 1 h and 24 h post-stimulation for real-time PCR detection of growth factors and inflammatory and apoptotic markers. At all time-points after treatment, WES-treated rat eyes exhibited significantly higher spatial frequency thresholds than untreated eyes. Inner retinal function, as measured by ERG oscillatory potentials (OPs), showed significantly improved OP amplitudes at 8 and 12 weeks post-WES compared to Sham eyes. Additionally, while photoreceptor segment and nuclei thicknesses in P23H-1 rats did not change between treatment groups, WES-treated eyes had significantly greater numbers of retinal ganglion cell nuclei than Sham eyes at 20 weeks post-WES. Gene expression levels of brain-derived neurotrophic factor (BDNF), caspase 3, fibroblast growth factor 2 (FGF2), and glutamine synthetase (GS) were significantly higher at 1 h, but not 24 h after WES treatment. Our findings suggest that WES has a beneficial effect on visual function in a rat model of retinal degeneration and that post-receptoral neurons may be particularly responsive to electrical stimulation therapy.

Reduction of amyloid-beta levels in mouse eye tissues by intra-vitreally delivered neprilysin.

Amyloid-beta (Aß) is a group of aggregation-prone, 38- to 43-amino acid peptides generated in the eye and other organs. Numerous studies suggest that the excessive build-up of low-molecular-weight soluble oligomers of Aß plays a role in the progression of Alzheimer's disease and other brain degenerative diseases. Recent studies raise the hypothesis that excessive Aß levels may contribute also to certain retinal degenerative diseases. These findings, together with evidence that a major portion of Aß is released as monomer into the extracellular space, raise the possibility that a technology enabling the enzymatic break-down of monomeric Aß in the living eye under physiological conditions could prove useful for research on ocular Aß physiology and, perhaps ultimately, for therapeutic applications. Neprilysin (NEP), an endopeptidase known to cleave Aß monomer into inactive products, is a membrane-associated protein. However, sNEP, a recombinant form of the NEP catalytic domain, is soluble in aqueous medium. With the aim of determining the Aß-cleaving activity of exogenous sNEP in the microenvironment of the intact eye, we analyzed the effect of intra-vitreally delivered sNEP on ocular Aß levels in mice that exhibit readily measurable, aqueous buffer-extractable Aß40 and Aß42, two principal forms of Aß. Anesthetized 10-month wild-type (C57BL/6J) and 2-3-month 5XFAD transgenic mice received intra-vitreal injections of sNEP (0.004-10 µg) in one eye and were sacrificed at defined post-treatment times (30 min - 12 weeks). Eye tissues (combined lens, vitreous, retina, RPE and choroid) were homogenized in phosphate-buffered saline, and analyzed for Aß40 and Aß42 (ELISA) and for total protein (Bradford assay). The fellow, untreated eye of each mouse served as control, and concentrations of Aß (pmol/g protein) in the treated eye were normalized to that of the untreated control eye. In C57BL/6J mice, as measured at 2 h after sNEP treatment, increasing amounts of injected sNEP yielded progressively greater reductions of Aß40, ranging from 12% ± 3% (mean ± SEM; n = 3) with 4 ng sNEP to 85% ± 13% (n = 5) with 10 µg sNEP. At 4 ng sNEP the average Aß40 reduction reached >70% by 24 h following treatment and remained near this level for about 8 weeks. In 5XFAD mice, 10 µg sNEP produced an Aß40 decrease of 99% ± 1% (n = 4) and a substantial although smaller decrease in Aß42 (42% ± 36%; n = 4) within 24 h. Electroretinograms (ERGs) were recorded from eyes of C57BL/6J and 5XFAD mice at 9 days following treatment with 4 ng or 10 µg sNEP, conditions that on average led, respectively, to an 82% and 91% Aß40 reduction in C57BL/6J eyes, an 87% and 92% Aß40 reduction in 5XFAD eyes, and a 23% and 52% Aß42 reduction in 5XFAD eyes. In all cases, sNEP-treated eyes exhibited robust ERG responses, consistent with a general tolerance of the posterior eye tissues to the investigated conditions of sNEP treatment. The sNEP-mediated decrease of ocular Aß levels reported here represents a possible approach for determining effects of Aß reduction in normally functioning eyes and in models of retinal degenerative disease.

Spatial differences in corneal electroretinogram potentials measured in rat with a contact lens electrode array.

PURPOSE: It has been known for several decades that the magnitude of the corneal electroretinogram (ERG) varies with position on the eye surface, especially in the presence of focal or asymmetric stimuli or retinal lesions. However, this phenomenon has not been well-characterized using simultaneous measurements at multiple locations on the cornea. This work provides the first characterization of spatial differences in the ERG across the rat cornea. METHODS: A contact lens electrode array was employed to record ERG potentials at 25 corneal locations simultaneously following brief full-field flash stimuli in normally sighted Long-Evans rats. These multi-electrode electroretinogram (meERG) responses were analyzed for spatial differences in a-wave and b-wave amplitudes and implicit times. RESULTS: Spatially distinct ERG potentials could be recorded reliably. Comparing relative amplitudes across the corneal locations suggested a slight non-uniform distribution when using full-field, near-saturating stimuli. Amplitudes of a- and b-waves were approximately 3 % lower in the inferior quadrant than in the superior quadrant of the cornea. CONCLUSIONS: The present results comprise the start of the first normative meERG database for rat eyes and provide a basis for comparison of results from eyes with functional deficit. Robust measures of spatial differences in corneal potentials will also support optimization and validation of computational source models of the ERG. To fully utilize the information contained in the meERG data, a detailed understanding of the roles of the many determinants of local corneal potentials will eventually be required.

Neuroprotective effects of low level electrical stimulation therapy on retinal degeneration.

Low-level electrical stimulation applied to the eye has been shown to have neuroprotective effects on photoreceptors and retinal ganglion cells. In this review, we compare the effects of Subretinal Electrical Stimulation (SES), Transcorneal Electrical Stimulation (TES), and Whole Eye Stimulation (WES) on preserving retinal structure and function, and visual acuity, in retinal degeneration. Similarities and differences in stimulus parameters, targeted cells and growth factor expression will be discussed with emphasis on studies that have translated laboratory findings into clinical trials.

Effects of normal aging on the mouse retina assessed by full-field flash and flicker electroretinography.

Changes in the full-field flash and flicker electroretinogram (ERG) that accompany normal aging were evaluated in mice. ERGs were recorded from a single cohort of C57BL/6J mice from 5 to 70 weeks of age using conventional techniques. Dark-adapted ERGs were recorded for flash luminances of - 3.0 to 1.5 log cd-s-m-2; a- and b-wave amplitude and implicit time (IT) were calculated from these responses. In addition, light-adapted flicker ERGs elicited by sinusoidally modulated light were measured for temporal frequencies of 2 to 31 Hz. Amplitudes and phases were extracted from the flicker responses using Fourier analysis. Linear quantile mixed models were used for statistical comparisons of the effects of age on amplitude and timing. There was a significant decrease in a-wave amplitude (p < 0.001) and b-wave amplitude (p < 0.001) over the 65 week study. From 5 to 70 weeks, the a- and b-wave amplitudes decreased by a factor of approximately 2. There was a small (2-14 ms), but significant (p < 0.001), delay in a- and b-wave IT over the 65 week study. There was also a significant decrease in fundamental amplitude (factor of 1.8, p < 0.001) and second harmonic amplitude (factor of 1.5, p < 0.001) over time. There were no significant age-related effects on the phase of these components (both p > 0.06). These results indicate that age scales the single flash and flicker ERG similarly, reducing response amplitude by a factor of approximately 2, from 5 to 70 weeks, with small or no effect on response timing. These data may be useful for guiding future longitudinal pre-clinical therapeutic studies.

Visualizing spatial differences in corneal electroretinogram potentials using a three-dimensional surface spline.

Objective. The spatial distribution of activity at the retina determines the spatial distribution of electroretinogram potentials at the cornea. Here a three-dimensional surface spline method is evaluated for interpolating corneal potentials between measurement points in multi-electrode electroretinography (meERG) data sets.Approach. 25-channel meERG responses were obtained from rat eyes before and after treatment to create local lesions. A 3rd order surface spline was used to interpolate meERG values resulting in smooth color-coded maps of corneal potentials. Potential maps were normalized using standard score values. Pre- and post-treatment responses were characterized by spatial standard deviation and by difference-from-normal plots.Main results. The spatial standard deviation for eyes with local lesions were significantly higher than for healthy eyes. The 3rd order spline resulted in well-behaved corneal potential maps that maintained low error rate when up to 30% of recording channels were excluded from analysis. Post-normalization, responses could be combined within experimental groups, and individual eyes with lesions were clearly distinguished from the healthy-eye mean response. A 3rd order surface spline is an acceptable means of interpolating meERG potentials to create corneal potential maps. The spatial standard deviation is more sensitive to local dysfunction than absolute amplitudes.Significance. This work demonstrates solutions to key challenges in the recording and analysis of meERG responses: visualization, normalization, channel loss, and identification of abnormal responses. Continued development of the meERG technique is relevant to research and clinical applications, especially where local dysfunction (early progressive disease) or local therapeutic effect (subretinal injection) is of interest.

Detection and discrimination of mixed odor strands in overlapping plumes using an insect-antenna-based chemosensor system.

Olfactory signals, a major means of communication in insects, travel in the form of turbulent odor plumes. In terrestrial environments, an odor blend emitted from a single point source exists in every strand of the plume, whereas, in confluent plumes from two different odor sources, the strands have some chance of being coincident and comprising a new third odor in those strands. Insects have the ability to detect and interpret necessary olfactory information from individual filamentous odor strands in complex multifilament odor plumes. However, behaviorists have had no way to measure the stimulus situations they are presenting to their temporally acute insect subjects when performing Y-tube olfactometer or confluent pheromone plume wind tunnel assays. We have successfully measured the degree of plume-strand mixing in confluent plumes in a wind tunnel by using a multichannel insect-antenna-based chemosensor. A PC-based computer algorithm to analyze antennal signals from the probe portion of the system performed real-time signal processing and, following a short training session, classified individual odorant/mixture strands at sub-second temporal resolution and a few tens of millimeters of spatial resolution. In our studies, the chemosensor classified a higher frequency of strands of two different odorants emitted from two closely spaced filter papers as being "mixed" when the sources were located only 1 or 2 cm apart than when the sources were 5 or 10 cm apart. These experiments demonstrate the chemosensor's potential to be used for measuring odor stimulus situations in more complex multiple-plume environments.

Three-Dimensional Model of Electroretinogram Field Potentials in the Rat Eye.

OBJECTIVE: The information derived from the electroretinogram (ERG), especially with regard to local areas of retinal dysfunction or therapeutic rescue, can be enhanced by an increased understanding of the relationship between local retinal current sources and local ERG potentials measured at the cornea. A critical step in this direction is the development of a robust bioelectric field model of the ERG. METHODS: A finite-element model was created to simulate ERG potentials at the cornea resulting from physiologically relevant transretinal currents. A magnetic resonance image of a rat eye was segmented to define all major ocular structures, tissues were assigned conductivity values from the literature. The model was optimized to multi-electrode ERG (meERG) data recorded in healthy rat eyes, and validated with meERG data from eyes with experimental lesions in peripheral retina. RESULTS: Following optimization, the simulated distribution of corneal potentials was in good agreement with measured values; residual error was comparable to the average difference of individual eyes from the measured mean. The model predicted the corneal potential distribution for eight eyes with experimental lesions with similar accuracy, and a measure of pre- to post-lesion changes in corneal potential distribution was well correlated with the location of the lesion. CONCLUSION: An eye model with high anatomical accuracy was successfully validated against a robust dataset. SIGNIFICANCE: This model can now be used for optimization of ERG electrode design, and to support functional mapping of the retina from meERG data via solving the inverse bioelectric source problem.

Three Dimensional Stimulus Source for Pattern Electroretinography in Mid- and Far-peripheral Retina.

PURPOSE: The pattern electroretinogram (pERG) response reflects, in part, ganglion cell function. However, probing retinal ganglion cell (RGC) function in the mid- and far peripheral retina is difficult with conventional flat-panel pERG stimulus sources. A pattern stimulus source is presented for probing the peripheral retina. Peripheral pERG (ppERG) responses were evaluated versus luminance, reversal rate, and field subtended, and were compared with conventional pERG in healthy eyes. METHODS: Eleven normally-sighted subjects were recruited. A hemispherical surface was used to present a reversing checkerboard pattern to the peripheral retina, from approximately 35° to 85° of visual field, in all directions. Responses to stimuli presented to peripheral field sectors (superior, nasal, inferior, temporal) were also recorded. Conventional pERG responses were recorded on the same day. Amplitudes and implicit times of waveform peaks were evaluated. RESULTS: Robust pERG responses from peripheral retina resemble conventional pERG responses but with shorter implicit times and reduced positive component. Responses to high-luminance patterns include high-frequency components resembling flash ERG oscillatory potentials. Negative response component amplitudes increased with increasing pattern luminance, and decreased with increasing reversal rate. CONCLUSIONS: Peripheral-field pERG responses are robust and repeatable; the unique response properties reflect differences between central and peripheral retina. Field-sector response ratios can be used to probe for sectoral dysfunction associated with disease. TRANSLATIONAL RELEVANCE: The ppERG approach provides direct measurement of proximal retinal function beyond the fields probed by conventional perimetry and pERG, providing access to a relatively under studied part of the retina relevant to early stage glaucoma.

Corneal Potential Maps Measured With Multi-Electrode Electroretinography in Rat Eyes With Experimental Lesions.

Purpose: Conventional full-field flash electroretinography (ERG) yields a single response waveform that can be useful in the early detection and diagnosis of many diseases affecting the retina. It is an objective measurement that probes the entire retina. However, localized areas of dysfunction have relatively small influence on ERG amplitudes compared to normal ranges. Here we evaluate the use of corneal potential maps obtained in response to full-field flash stimuli for sensitivity to local areas of retinal damage. Methods: A contact lens electrode array was used to record 25 ERG waveforms simultaneously following saturating full-field flash stimuli (multi-electrode electroretinography, meERG) in rats. Waveforms were evaluated for a-wave and b-wave amplitudes; these values were normalized and further evaluated for spatial differences across the corneal surface. Cluster analysis and a support vector machine approach were used to classify meERG responses from healthy eyes and eyes with central (photocoagulation) or peripheral (cryocoagulation) experimental lesions. Results: A normative normalized corneal potential map was obtained from healthy eyes (n = 26). Corneal potential maps from eyes with experimental lesions (n = 13) could be classified with sensitivity and specificity of approximately 80% based solely on the normalized spatial distribution of corneal potentials, that is, with no knowledge of absolute amplitudes. Conclusions: Corneal potential maps obtained in response to full-field flash stimuli are altered in eyes with scotomas in the central and far-peripheral retina. The meERG approach yields useful spatial information following a single brief flash, analogous to body-surface potential maps used to evaluate heart and brain.

Dark adaptation of rod photoreceptors in normal subjects, and in patients with Stargardt disease and an ABCA4 mutation.

PURPOSE: Psychophysical and electroretinographic (ERG) studies indicate that patients with Stargardt disease exhibit abnormally slow rod dark adaptation after illumination that bleaches a substantial fraction of rhodopsin. However, relatively little information is available concerning rod recovery in this disease after weaker adapting (i.e., conditioning) light. With the use of a paired-flash ERG method, properties of the derived rod response to a low-bleach (<1%) but rod-saturating conditioning flash were investigated in seven normal subjects and in five Stargardt patients with identified sequence variations in the ABCA4 gene. METHODS: In the first of two experiments, the interval between a fixed conditioning flash (67 or 670 scotopic cd s m(-2)) and a bright probe flash of fixed strength was varied to determine the falling-phase kinetics of the derived rod response to the conditioning flash. In the second, the instantaneous amplitude-intensity function for the rod response at an intermediate stage of recovery from the conditioning flash was determined by presenting a test flash of various strengths at a fixed time after the conditioning flash, and a probe flash at 200 ms after the test flash. RESULTS: The maximum peak amplitude of the dark-adapted, rod-mediated a-wave determined in Stargardt patients (211 +/- 87 microV) was on average lower than that determined in normal subjects (325 +/- 91 microV; P = 0.06). The derived rod response to the 670 scotopic cd s m(-2) conditioning flash determined in normal subjects and Stargardt patients exhibited a biphasic recovery, and the kinetics of the early stage of this recovery were similar in the two subject groups. For both normal subjects and patients, normalized amplitude-intensity functions describing the dark-adapted derived rod response exhibited half-saturation at approximately 1.5 log scotopic troland second. In both groups, the normalized amplitude-intensity function determined at approximately 2 seconds after the 67 scotopic cd s m(-2) conditioning flash and at approximately 9 seconds after the 670 scotopic cd s m(-2) conditioning flash exhibited an average desensitization (i.e., an increase of test flash strength at half-saturation) of approximately 0.5 to 0.6 log unit relative to that determined under dark-adapted conditions. CONCLUSIONS: The results indicate that, despite a reduction in the average dark-adapted maximum a-wave amplitude in the Stargardt/ABCA4 patients, the early-stage recovery kinetics of the derived rod response to a low-bleaching conditioning flash as well as the lingering rod desensitization produced by such a flash are similar to those determined in normal subjects.

Neural prostheses for vision: designing a functional interface with retinal neurons.

A number of prevalent eye diseases exist which may lead to partial or total blindness, and for which there are currently no cures or means by which to restore lost sight. Based on recent progress, it has become apparent that artificial prosthetic devices, which would use electrical stimulation of neurons in the visual pathway to elicit visual percepts, are likely to some day become a viable treatment for patients blinded by these diseases. A number of recent scientific reviews have summarized general functional electrical stimulation (FES) approaches related to the visual system, and many of the technical considerations regarding fabrication, biocompatibility, stimulation thresholds and electrotoxicity. This review will address a principal outstanding question in retinal prosthesis development: the design and implementation of a functional interface with the retina. A functional interface between electrodes and retinal neurons will be stable, biocompatible, and will convey useful information to the visual system. Several parameters related to both the artificial and biological aspects of the interface must be considered; this paper will emphasize electrode design. Additional issues central to the development of prosthesis interface design, including retinal physiology, eye diseases, and existing animal models of retinal degeneration, are also summarized.

Subretinal implantation of semiconductor-based photodiodes: durability of novel implant designs.

Selective degeneration of the retinal photoreceptor layers underlies blindness in retinitis pigmentosa (RP) and other inherited retinal disorders. Because there are no therapies for these patients, we are evaluating the possibility that electrical stimulation delivered to the subretinal space by a microphotodiode array (MPA) could replace, in some aspect, the function of diseased photoreceptors. Early MPA prototypes utilized gold as the electrode material, which gradually dissolved during the postoperative period following subretinal implantation. Here we present the results obtained when different MPA materials were used. Semiconductor-based silicon MPAs (2 mm in diameter; 50 microm in thickness), incorporating iridium/iridium oxide (IrOx) or platinum (Pt) electrodes, were implanted into the subretinal space of the right eye of normal cats with the use of vitreoretinal surgical techniques. Indirect ophthalmoscopy, fundus photography, ganzfeld electroretinography, and histology were used for the evaluation of the implanted retinas postoperatively. Infrared (IR) stimulation was used to isolate electrical responses generated by the MPA. The unimplanted left eyes were used for control purposes. After the implantation surgery, subretinal MPAs retained a stable position in the subretinal space. Up to 12 months after surgery, there was little change in the magnitude of the electrical response of IrOx- and Pt-based MPAs to a standard IR light stimulus. Overlying the implant, there was a near-complete loss of the outer retinal layer, which is likely to reflect obstruction of choroidal nourishment to these layers by the solid disk implant. In addition, the inner retinal layers showed variable disorganization. Away from the implant, the retina displayed a normal appearance. In comparison to electroretinograms (ERGs) obtained from unimplanted eyes, responses recorded from implanted eyes had a normal waveform but were slightly smaller in amplitude. These results indicate that IrOx and Pt improve implant electrode durability and that implants incorporating these materials into the electrode layer do not induce panretinal abnormalities.

Chronic delivery of low-level exogenous current preserves retinal function in pigmented P23H rat.

Diffuse electrical currents delivered to the eye were investigated in a rat model of retinitis pigmentosa for potentially therapeutic effects. Low-level currents were passed between electrodes placed on the cornea and in the mouth during 30-min sessions two times per week from 4 to 16 weeks of age. Single-flash electroretinograms (ERG) were recorded and analyzed for amplitude and measures of sensitivity, and basic histology was performed. ERG a-wave amplitudes were slightly greater in treated vs. age-matched controls at 16 weeks of age, but the combined thicknesses of the outer nuclear layer and outer segment layer were similar at this age. Treated animals exhibited a significant preservation of b-wave amplitudes, and a striking preservation of rod sensitivity, measured as the stimulus strength required to reach half-saturation of the a-wave. Analysis of the leading edge of the a-wave using a delayed Gaussian function revealed a decrease in the parameter reflecting gain of the phototransduction cascade over the 12-week course of treatment, and no significant change in control animals over the same period. These results suggest that while the exogenous currents failed to preserve the number or gross structure of rods, the responsivity of individual photoreceptors was relatively preserved, perhaps via an increase in efficiency of photon capture (R/photon). This preserved functionality may delay the retraction of bipolar cell dendrites from degenerating photoreceptors.

Features of visual function in the naked mole-rat Heterocephalus glaber.

The eyes and visual capacity of the naked mole-rat, Heterocephalus glaber, a subterranean rodent, were evaluated using anatomical, biochemical, and functional assays, and compared to other rodents of similar body size (mouse and gerbil). The eye is small compared to mouse, yet possesses cornea, lens, and retina with typical mammalian organization. The optic nerve cross-sectional area and fiber density are approximately 10% and approximately 50% that of gerbil, respectively. Levels per unit retinal area of 11-cis and all-trans retinal, derivatives of vitamin A associated with the visual cycle, are comparable to mouse. The corneal electroretinogram (ERG) exhibits early and late negative components that scale with flash strength; raising the body temperature of this poikilothermic animal from 30 degrees C (normal for H. glaber ) to 37 degrees C (normal for mouse) revealed an ERG response with typically mammalian features, but greatly attenuated and with slower kinetics. Leaving the nest chamber was a behavior correlated with light onset displayed preferentially by breeding females. Optical models of five mole-rat eyes suggest reasonable, but variable, image formation at the retina, possibly related to age. Results are consistent with amorphous light detection, possibly useful for circadian entrainment or escape behavior in the event of tunnel breeches.