Mark Wendt

Monitoring of glucose permeability in monkey skin in vivo using Optical Coherence Tomography.

Topical trans-dermal delivery of drugs has proven to be a promising route for treatment of many dermatological diseases. The aim of this study is to monitor and quantify the permeability rate of glucose solutions in rhesus monkey skin noninvasively in vivo as a primate model for drug diffusion. A time-domain Optical Coherence Tomography (OCT) system was used to image the diffusion of glucose in the skin of anesthetized monkeys for which the permeability rate was calculated. From 5 experiments on 4 different monkeys, the permeability for glucose-20% was found to be (4.41 +/- 0.28) 10(-6) cm/sec. The results suggest that OCT might be utilized for the noninvasive study of molecular diffusion in the multilayered biological tissues in vivo.

Changes in crystalline lens radii of curvature and lens tilt and decentration during dynamic accommodation in rhesus monkeys

Dynamic changes in crystalline lens radii of curvature and lens tilt and decentration were measured during centrally stimulated accommodation in four iridectomized eyes of two adolescent rhesus monkeys. Phakometry measurements were performed dynamically using a custom-built, video-based, Purkinje-image instrument. Lens anterior and posterior radii were calculated from reflections of paired light sources from the ocular surfaces (Purkinje images PI, PIII, and PIV). Lens tilt and decentration were calculated assuming linearity between Purkinje image positions, eye rotation, lens tilt, and decentration. Because the monkey eyes were iridectomized, Purkinje images were referred to the mid-point of the double first Purkinje image (PI). Mean unaccommodated values of anterior and posterior lens radii of curvature were 11.11 +/- 1.58 mm and -6.64 +/- 0.62 mm, respectively, and these decreased relatively linearly with accommodation in all eyes, at a rate of 0.48 +/- 0.14 mm/D and 0.17 +/- 0.03 mm/D for anterior and posterior lens surfaces, respectively. Tilt and decentration did not change significantly with accommodation except for tilt around the horizontal axis, which changed at a rate of 0.147 +/- 0.25 deg/D. These results are important to fully characterize accommodation in rhesus monkeys.

Lens diameter and thickness as a function of age and pharmacologically stimulated accommodation in rhesus monkeys

Uncertainty exists regarding accommodative and age changes in lens diameter and thickness in humans and monkeys. In this study, unaccommodated and accommodated refraction, lens diameter, and lens thickness were measured in rhesus monkeys across a range of ages. Iridectomized eyes were studied in 33 anesthetized monkeys aged 4-23 years. Refraction was measured using a Hartinger coincidence refractometer and lens thickness was measured with A-scan ultrasound. Lens diameters were measured with image analysis from slit-lamp images captured via a video camera while a saline filled, plano perfusion lens was placed on the cornea. Accommodation was pharmacologically stimulated with 2% pilocarpine via the perfusion lens in 21 of the monkeys and lens diameters were measured until a stable minimum was achieved. Refraction and lens thickness were measured again after the eye was accommodated. Unaccommodated lens thickness increased linearly with age by 0.029 mm/year while unaccommodated lens diameter showed no systematic change with age. Accommodative amplitude decreased by 0.462 D/year in response to pilocarpine. The accommodative increase in lens thickness decreased with age by 0.022 mm/year. The accommodative decrease in lens diameter declined linearly with age by 0.021 mm/year. Rhesus monkeys undergo the expected presbyopic changes including increasing lens thickness and a decreasing ability of the lens to undergo changes in thickness and diameter with accommodation, however without an age-related change in unaccommodated lens diameter. As in humans, the age-related decrease in accommodative amplitude in rhesus monkeys cannot be attributed to an age-related increase in lens diameter.

Topical and intravenous pilocarpine stimulated accommodation in anesthetized rhesus monkeys

Many studies have used pilocarpine to stimulate accommodation in both humans and monkeys. However, the concentrations of pilocarpine used and the methods of administration vary. In this study, three different methods of pilocarpine administration are evaluated for their effectiveness in stimulating accommodation in rhesus monkeys. Experiments were performed in 17 iridectomized, anesthetized rhesus monkeys aged 4-16 years. Maximum accommodation was stimulated in all these monkeys with a 2% pilocarpine solution maintained on the cornea for at least 30 min in a specially designed perfusion lens. In subsequent topical pilocarpine experiments, baseline refraction was measured with a Hartinger coincidence refractometer and then while the monkeys were upright and facing forward, commercially available pilocarpine (2, 4, or 6%) was applied topically to the cornea as 2 or 4 drops in two applications or 6 drops in three applications over a five minute period with the eyelids closed between applications. Alternatively, while supine, 10-12 drops of pilocarpine were maintained on the cornea in a scleral cup for 5 min. Refraction measurements were begun 5 min after the second application of pilocarpine and continued for at least 30 min after initial administration until no further change in refraction occurred. In intravenous experiments, pilocarpine was given either as boluses ranging from 0.1mg/kg to 2mg/kg or boluses followed by a constant infusion at rates between 3.06 mg/kg/h and 11.6 mg/kg/h. Constant 2% pilocarpine solution on the eye in the perfusion lens produced 10.88+/-2.73 D (mean+/-SD) of accommodation. Topically applied pilocarpine produced 3.81 D+/-2.41, 5.49 D+/-4.08, and 5.55 D+/-3.27 using 2%, 4%, and 6% solutions respectively. When expressed as a percentage of the accommodative response amplitude obtained in the same monkey with constant 2% pilocarpine solution on the eye, the responses were 34.7% for 2% pilocarpine, 48.4% for 4% pilocarpine, and 44.6% for 6% pilocarpine. Topical 4% and 6% pilocarpine achieved similar, variable accommodative responses, but neither achieved maximum accommodation. IV boluses of pilocarpine achieved near maximal levels of accommodation at least ten times faster than topical methods. Doses effective for producing maximum accommodation ranged from 0.25mg/kg to 1.0mg/kg. IV pilocarpine boluses caused an anterior movement of the anterior lens surface, a posterior movement of the posterior lens surface, and a slight net anterior movement of the entire lens. Considerable variability in response amplitude occurred and maximum accommodative amplitude was rarely achieved with topical application of a variety of concentrations of commercially available pilocarpine. Intravenous infusion of pilocarpine was a rapid and reliable method of producing a nearly maximal accommodative response and maintaining accommodation when desired.

Full-field accommodation in rhesus monkeys measured using infrared photorefraction.

PURPOSE Full-field photorefraction was measured during accommodation in anesthetized monkeys to better understand the monkey as a model of human accommodation and how accommodation affects off-axis refraction. METHODS A photorefraction camera was rotated on a 30-cm-long rod in a horizontal arc, with the eye at the center of curvature of the arc so that the measurement distance remained constant. The resistance of a potentiometer attached to the rotation center of the rod changed proportionally with the rotation angle. Photorefraction and rotation angle were simultaneously measured at 30 Hz. Trial-lens calibrations were performed on-axis and across the full field in each eye. Full-field refraction measurements were compared using on-axis and full-field calibrations. In five iridectomized monkeys (mean age in years ± SD: 12.8 ± 0.9), full-field refraction was measured before and during carbachol iontophoresis stimulated accommodation, a total of seven times (with one repeat each in two monkeys). RESULTS Measurements over approximately 20 seconds had <0.1 D of variance and an angular resolution of 0.1°, from at least -30° to 30°. Photorefraction calibrations performed over the full field had a maximum variation in the calibration slopes within one eye of 90%. Applying full-field calibrations versus on-axis calibrations resulted in a decrease in the maximum SDs of the calculated refractions from 1.99 to 0.89 D for relative peripheral refractive error and from 4.68 to 1.99 D for relative accommodation. CONCLUSIONS By applying full-field calibrations, relative accommodation in pharmacologically stimulated monkeys was found to be similar to that reported with voluntary accommodation in humans.

Influence of Amplitude, Starting Point, and Age on First- and Second-Order Dynamics of Edinger-Westphal–Stimulated Accommodation in Rhesus Monkeys

PURPOSE In humans, accommodative and disaccommodative dynamics depend on response amplitude and starting point. The purpose of this study was to determine the influence of amplitude and starting point on open-loop accommodative dynamics in Edinger-Westphal (EW)-stimulated, anesthetized rhesus monkeys of different ages. METHODS One eye each of two younger and two older iridectomized rhesus monkeys, (aged 6.8, 8.9, 15.0, and 16.3 years) were studied. The experiment was repeated in one eye of one younger monkey. Lens thickness changes were recorded by dynamic ultrasound biometry at 100 Hz. Stimuli used produced accommodative responses: (1) starting from baseline with increasing amplitudes; (2) from increasing starting points to maximum accommodation; and (3) from increasing starting points with a constant amplitude of 1 D. The lens thickness measurements were converted into accommodation and velocities and accelerations of the responses were determined by using a two-point difference algorithm. RESULTS Maximum accommodative amplitudes ranged from 4.68 to 6.37 D in the older monkeys and 9.33 to 11.59 D in the younger monkeys. The peak velocity of accommodation and disaccommodation increased linearly with response amplitude. Peak velocity and peak acceleration of accommodation and disaccommodation were independent of the response starting point. Subtle variations in disaccommodative response peak velocities were found to vary with age. CONCLUSIONS The results suggest that, in anesthetized rhesus monkeys, disaccommodative rather than accommodative dynamics may be more sensitive to age-related changes and that, unlike in conscious human subjects, the starting configuration of the accommodative plant has little influence on accommodative dynamics.

Reproducibility of carbachol stimulated accommodation in rhesus monkeys

Approaches are being explored to restore accommodation to the presbyopic eye. Some of these approaches can be tested in monkeys by stimulating accommodation in various ways including using carbachol iontophoresis. Knowledge of the repeatability of carbachol iontophoresis stimulated accommodation in the monkey phakic eye is necessary to understand the variability of this method of evaluating accommodation. Data from 9 to 10 separate carbachol iontophoresis experiments performed on phakic eyes from 8 monkeys were retrospectively analyzed. For each experiment, carbachol was applied iontophoretically to the eyes of anesthetized monkeys and refraction generally measured every two minutes until accommodation reached a plateau. Repeated experiments were performed in each monkey over periods ranging from 10 to 18 months. Maximum accommodation measured for each monkey ranged from 11.1 D to 18.3 D with standard deviations from 0.8 D to 2.1 D and differences in accommodative amplitude varying from 2.2 D to 7.5 D. Time to reach maximum accommodation ranged from 18 to 64 min in individual experiments. Averaged time-courses indicate that maximum accommodation is generally achieved between 10 and 20 min after carbachol administration. Although carbachol iontophoresis is considered a reliable method to stimulate maximum accommodation in anesthetized monkeys, the amplitude achieved typically varies by more than 2 D. Presbyopia treatments evaluated in this way in phakic monkeys would need to show an increase in accommodation of over 2 D to clearly demonstrate that the treatments work when being tested with carbachol iontophoresis stimulation.

Accuracy and resolution of in vitro imaging based porcine lens volumetric measurements.

There is considerable interest in determining lens volume in the living eye. Lens volume is of interest to understand accommodative changes in the lens and to size accommodative IOLs (A-IOLs) to fit the capsular bag. Some studies have suggested lens volume change during accommodation. Magnetic Resonance Imaging (MRI) is the only method available to determine lens volume in vivo. MRI is, by its nature, relatively low in temporal and spatial resolution. Therefore analysis often requires determining lens volume from single image slices with relatively low resolution on which only simple image analysis methods can be used and without repeated measures. In this study, 7 T MRI scans encompassing the full lens volume were performed on 19 enucleated pig eyes. The eyes were then dissected to isolate and photograph the lens in profile and the lens volumes were measured empirically using a fluid displacement method. Lens volumes were calculated from two- and three-dimensional (2D and 3D) MR and 2D photographic profile images of the isolated lenses using several different analysis methods. Image based and actual measured lens volumes were compared. The average image-based volume of all lenses varied from the average measured volume of all lenses by 0.6%-6.4% depending on the image analysis method. Image analysis methods that use gradient based edge detection showed higher precision with actual volumes (r(2): 0.957-0.990), while threshold based segmentation had poorer correlations (r(2): 0.759-0.828). The root-mean-square (RMS) difference between image analysis based volumes and fluid displacement measured volumes ranged from 8.51 μl to 25.79 μl. This provides an estimate of the error of previously published methods used to calculate lens volume. Immobilized, enucleated porcine eyes permit improved MR image resolution relative to living eyes and therefore improved image analysis methods to calculate lens volume. The results show that some of the accommodative changes in lens volume reported in the literature are likely below the resolution limits of imaging methods used. MRI, even with detailed image analysis methods used here, is unlikely to achieve the resolution required to accurately size an A-IOL to the capsular bag.

Long-term reproducibility of Edinger-Westphal stimulated accommodation in rhesus monkeys.

If longitudinal studies of accommodation or accommodation restoration procedures are undertaken in rhesus monkeys, the methods used to induce and measure accommodation must remain reproducible over the study period. Stimulation of the Edinger-Westphal (EW) nucleus in anesthetized rhesus monkeys is a valuable method to understand various aspects of accommodation. A prior study showed reproducibility of EW-stimulated accommodation over 14 months after chronic electrode implantation. However, reproducibility over a period longer than this has not been investigated and therefore remains unknown. To address this, accommodation stimulation experiments in four eyes of two rhesus monkeys (13.7 and 13.8 years old) were evaluated over a period of 68 months. Carbachol iontophoresis stimulated accommodation was first measured with a Hartinger coincidence refractometer (HCR) two weeks before electrode implantation to determine maximum accommodative amplitudes. EW stimulus-response curves were initially measured with the HCR one month after electrode implantation and then repeated at least six times for each eye in the following 60 months. At 64 months, carbachol iontophoresis induced accommodation was measured again. At 68 months, EW stimulus-response curves were measured with an HCR and photorefraction every week over four consecutive weeks to evaluate the short-term reproducibility over one month. In the four eyes studied, long-term EW-stimulated accommodation decreased by 7.00 D, 3.33 D, 4.63 D, and 2.03 D, whereas carbachol stimulated accommodation increased by 0.18 D-0.49 D over the same time period. The short-term reproducibility of maximum EW-stimulated accommodation (standard deviations) over a period of four weeks at 68 months after electrode implantation was 0.48 D, 0.79 D, 0.55 D and 0.39 D in the four eyes. Since the long-term decrease in EW-stimulated accommodation is not matched by similar decreases in carbachol iontophoresis stimulated accommodation, the decline in accommodation cannot be due to the progression of presbyopia but is likely to result from variability in EW electrode position. Therefore, EW-stimulated accommodation in anesthetized monkeys is not appropriate for long-term longitudinal studies of age-related loss of accommodation or accommodation restoration procedures.

Segmentation of crystalline lens in photorefraction video

With an increase in the percentage of people over the age of 65 years or older, there is a growing interest in finding ways to slow or reverse some of the effects of aging on the human body. One of these effects is the loss in the ability of the human eye to visually focus on near objects. Understanding the properties of the natural lens and the associated mechanisms of accommodation will greatly increase our knowledge to identify alternate solutions to reverse this phenomenon. Towards that end, a technique called photorefraction is currently being used in laboratory studies involving monkeys. To quantitate the lens properties, there is a need to accurately measure monkey lenses in images produced by this technique. In this paper, we present two probabilistic methods for segmenting the lens from photorefraction video sequences. Results of the developed methods are compared and evaluated against ground-truth segmentations. In addition, the results obtained are also compared to those obtained by a level set segmentation method.