The internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats. II. The inter-relationship of optical quality and structure as a function of age.

PURPOSE: The Royal College of Surgeons (RCS) rat is an animal model for human retinal degenerative disease and posterior subcapsular cataracts (PSCs). The purpose of this study was to correlate the structure and optical quality of RCS lenses with PSCs as a function of their internalization, with normal, non-cataractous, age-matched control lenses. METHODS: Correlative light (LM), scanning electron microscopic (SEM), three-dimensional computer assisted drawings (3D-CADs) and low power helium-neon laser scan analysis were used to examine the structure and function of lenses. RESULTS: The optical properties (average focal length variability; sharpness of focus) of RCS rat lenses are quantitatively compromised by PSCs. Correlative LM and SEM analysis of RCS lenses at various stages of PSC internalization (1.5, 3, 6, 9, 12 and 15 months of age), revealed that the sutures formed by additional fiber growth were progressively more abnormal. During PSC internalization, two to nine small suture branches were formed and arranged in modified line to multiple y configurations rather than the normal three branch y sutures. These temporal changes were also chronicled in animated 3D-CAD videos derived from lens reconstructions based on LM and SEM micrographs from the selected time points stated above. However, laser scan analysis also revealed that as the PSCs of RCS rat lenses were progressively internalized, there was a steady improvement in total sharpness of focus that reached normal levels by 12 months of age. The correlation of laser scan and structural data from specific regions of lenses revealed the following: 1. The abnormal posterior sutures of RCS rats with internalized PSCs effect a greater reduction in optical quality than normal posterior sutures of age-matched controls; 2. However, the resulting abnormal suture plane area was cumulatively similar to that of age-matched controls; 3. Thus, total optical quality was similar between RCS lenses with internalized PSCs and age-matched controls by 12 months of age. CONCLUSIONS: The results of this study show that RCS lenses with internalized PSCs can appear grossly, and indeed optically perform, at levels comparable to aged lenses. These findings are consistent with clinical observations of spontaneous recovery from PSC. The results suggest that human PSCs that occur as a consequence of retinal degenerative disease could also be the result of abnormal posterior suture growth. If this is proven to be the case, such PSCs may have some capacity for repair or recovery thereby obviating their surgical removal.

Effect of diabetes and fructose/non-fructose diet on the optical quality (cataracts) of the rat lens.

PURPOSE: This study measures the effect of moderate and severe levels of diabetes on the optical performance of the rat lens, and evaluates the effect of dietary fructose on diabetic lens damage. METHODS: Moderate and severe diabetes were induced in 250 g rats (Harlan Sprague Dawley) with streptozotocin (35 and 55 mg/kg body weight iv). Animals were fed either a control (glucose/cornstarch) or a 40% (wt:wt) fructose and cornstarch diet and then sacrificed after 7 or 12.5 weeks. These two diets were also fed to two groups in which diabetes was not induced and these rats were also sacrificed at 7 or 12.5 weeks. Lenses removed from animals were analyzed in terms of average focal length (mm), focal length variability (spherical aberration, mm) and relative transmission of light (scatter or number of pixels excited by a refracted laser beam) using an automated laser scanning system. RESULTS: Diabetes disrupted rat lens optical function, both at 7 and 12.5 weeks, as indicated by an increase in focal length variability (FLV). This was true for control and fructose diets. For example, after 12.5 weeks on the diets average FLV values of 0.10 +/- 0.00 mm [n = 9], 0.11 +/- 0.01 mm [n = 9],(control and fructose diets, no diabetes) and 0. 48 +/- 0.04 mm [n = 10], 0.95 +/- 0.11 mm [n = 9], (control and fructose diets, severe diabetes), were measured. The difference between control and fructose diets was significant at 12.5 weeks in the severe diabetes group (p

Mitochondria of rat lenses: distribution near and at the sutures.

PURPOSE: This study is part of an effort to clarify mitochondrial distribution in the lens in order to better understand lens metabolic function. This study of the rat lens involves: 1) Using confocal microscopy, Rhodamine-123 and Calcium Green fluorescent dyes, to characterise the distribution of mitochondria and calcium in whole rat lenses of different ages in epithelial and superficial cortical fibre cells approaching sutures and 2) Using a scanning laser system to measure the optical quality at the sutures. METHODS: Lenses of rats from age 1 week to 22 months were pre-incubated for 24 hrs in 1.5 ml medium 199 (M199). Those exhibiting damage, as evaluated by protein leakage or visual opacities, were discarded. Lenses were labelled with 50 microg/ml Calcium Green for 45 min and/or 14 microM Rhodamine-123 for 25 min and embedded in 1% agarose in M199 for inverted laser scanning confocal microscopy with a 40 x water immersion lens. The lens optical properties were determined with a scanning laser system. RESULTS: Lens focal length variability significantly increased at the sutures of 13 month-old lenses, the only age investigated. An absence of both mitochondria and calcium was observed at the sutures in rat lenses of all ages. Elongated (up to 108 mm) mitochondria were present in superficial cortical fibre cells approaching the sutures of 16 month-old lenses. Calcium Green fluorescent staining was seen closer to the border of the suture, where mitochondria were absent. Along the axis, 1 week-old lenses showed a mitochondria free zone (MFZ) starting 177 microm below the lens surface, whereas in 22 month-old lenses the MFZ started only 29 microm below the surface. In the equatorial fibre cells, mitochondria were seen to a depth of 220 microm. CONCLUSIONS: Optical quality near and at the suture decreased in 13 month-old lenses despite the reduction in light scattering that should be associated with absence of mitochondria at the sutures. This suggests that mitochondrial loss in superficial cortical fibre cells may originate at the sutures and may compensate for loss of optical quality at the sutures.

In vitro ocular irritancy measure of four contact lens solutions: damage and recovery.

We measured the potential toxicity of four contact lens solutions using an in vitro approach in which the optical quality of the cultured bovine lens was measured as a function of exposure to each substance tested. This approach uses an automated scanning laser to measure the focal variability of lenses contained in special culture cells and maintained under long-term culture conditions. The products tested included three rigid gas permeable contact lens conditioning solutions (Boston Conditioning Solution, Boston Advance Conditioning Solution, and a new formulation of Boston Advance Conditioning Solution [Polymer Technology]) and one soft contact lens disinfecting system (OptimEyes; Core Technologies). The results indicate a wide range of toxicologic potential that corresponds, on a relative basis, with published in vivo evaluation of the same substances. Moreover, the results demonstrate that this in vitro system can be used to evaluate the potential for recovery from damage caused by the four solutions tested.

The interrelationship of lens anatomy and optical quality. I. Non-primate lenses.

We have quantified the influence of 'Y' sutures on lens optical quality (spherical aberration, i.e. focal length variability) as a function of age. Young (n = 6) and old (group 1, n = 5; and group 2, n = 4) bovine lenses were initially scanned by a low-power (2 mW) helium-neon laser beam passed either through or at a series of acute angles to suture branches. In all lenses, focal length variability was least when the beam was passed through areas of the lens devoid of sutures and greatest when passed through sutures. In older lenses, variability was also significantly increased in all locations though to a greater degree at sutures. Correlative morphological analysis by scanning electron microscopic (SEM) and three dimensional (3-D) computer-assisted drawings (CADs) revealed the following: (1) young lenses had uniformly hexagonal fibers arranged in parallel radial cell columns (RCCs), while old lenses had nonuniformly hexagonal fibers arranged in variably parallel RCCs; (2) the irregularly-sized and -shaped ends of young fibers overlapped within growth shells to form complementary anterior and posterior symmetrical 'Y' suture patterns, while larger and more irregularly-shaped ends of older lenses overlapped to form asymmetrical 'Y' patterns; and (3) the identical suture patterns in successive shells of young lenses resulted in inverted triangular suture planes extending from the embryonic nucleus to the lens periphery, while the progressively wider and more serpentine suture branches of old lenses resulted in inverted pyramidal suture planes with narrow apices oriented towards the embryonic nucleus and broad irregular bases oriented toward the lens periphery. Thus, there is a significant interrelationship between lens optical quality and structure that varies as a function of age. These results extend and confirm the results of our earlier studies on lenses with simpler 'line' sutures and preface our studies of primate lenses with more complex 'star' sutures. All of these studies show that lens sutural anatomy should be considered when evaluating the optical quality of normal and pathological lenses.

The interrelationship of lens anatomy and optical quality. II. Primate lenses.

We have quantified the influence of lens sutural anatomy on optical quality (focal length variability, i.e. spherical aberration) in adult monkeys (Macaque nemestrina). Adult lenses (n = 6) were initially scanned by a low-power helium-neon laser beam that was passed at a series of acute angles to, and/or directly through, lens sutures. Optical analysis showed that while the 'star' sutures of primate lenses exerted a quantifiable negative effect on focal length variability, this detrimental effect was far less significant than that attributable to 'line' and 'Y' sutures in non-primate lenses. Correlative morphological and 3-D computer-assisted drawing (CAD) analysis of the laser-scanned lenses areas, as well as of variably aged lenses (n = 30), revealed that primates have a more complex lens architecture than non-primates. Non-primate lenses feature suture planes, aligned along the visual axis that are responsible for a significant quantifiable increase in spherical aberration. Primate lenses are characterized by an absence of continuous suture planes aligned along the visual axis. Rather, 3-D-CADs of primate lenses demonstrate that distinct generations of progressively more complex sutures are produced as a function of development, growth, and age. In succession, 'Y' sutures (three branches) are formed throughout embryonic development, 'simple star' sutures (three-six branches) evolve after birth and through infancy, 'star' sutures (six-nine branches) are made in young adult lenses and, finally, 'complex star' sutures (nine-15 branches) are laid down from middle through old age. In view of the fact that slit-lamp evaluation of cataractous lenses often reveals abnormally thin zones of discontinuity, it is significant to note that the temporal development of the zones of discontinuity in normal human lenses is essentially identical to the progressive iteration of offset monkey lens sutures. In conclusion, these studies describe a specific structural aspect of lenses that adversely influences optical quality, and relates it to the most commonly employed clinical technique to identify and monitor the progress of cataracts.

Effects of continuous light on experimental refractive errors in chicks.

It is possible to induce ametropias in young chicks either by depriving the developing eye of clear form vision with a translucent goggle or by defocusing the retinal image with convex or concave lenses. The refractive properties of the developing chick eye are also altered by raising young birds in a continuous light environment. The effects of superimposing form deprivation or defocus treatments on chicks raised in continuous light are unclear. Newly hatched (n = 31) chicks were raised for 2 weeks under continuous light while wearing either translucent goggles or + 10 or -10 diopter (D) lenses over one eye. Refractive states, corneal curvature and intraocular dimensions were measured periodically by retinoscopy, keratometry and A-scan ultrasound. The birds were sacrificed after 2 weeks and the eyes removed and measured with calipers. Under continuous light, all eyes treated with translucent goggle and -10 D lens developed moderate myopia (-2.6 +/- 0.5 D and -1.4 +/- 0.3 D, respectively) by day 4. The eyes treated with a + 10 D lens developed moderate hyperopia (+ 4.8 +/- 0.5 D) at day 4. Corneal curvatures of all treated eyes were slightly, but significantly, larger than contralateral control eyes by day 4. After 2 weeks of goggle or lens application, all the treated eyes were hyperopic due to corneal flattening. But the eyes treated with a goggle or a -10 D lens still showed relative myopia compared to the fellow eyes (treated minus untreated = -3.8 +/- 0.4 D and -2.8 +/- 0.4 D, respectively), and the eyes treated with a + 10 D lens showed more hyperopia than fellow eyes (treated minus untreated = + 5.1 +/- 0.6 D). Compared with the control eyes, the axial length (mainly vitreous chamber depth) was slightly, but significantly, increased in the eyes treated with a goggle or a -10 D lens, and the axial length decreased slightly in the eyes treated with + 10 D lens. The results suggest that form deprivation and retinal defocus (induced by +/- 10 D lenses) could still induce experimental refractive errors (myopia and hyperopia) in chicks kept under continuous light, but the effects of form deprivation and retinal defocus were partially suppressed by continuous light.

The relationship between rabbit lens optical quality and sutural anatomy after vitrectomy.

This study was conducted first, to characterize structural changes in rabbit lenses after vitrectomy; and second, to assess whether such changes correlate with a quantifiable compromise in optical function. Unilateral, partial vitrectomies were performed on 2.5 month old Dutch Belted rabbits (n = 64). Age matched non-operated rabbits (n = 32) were used as controls. Lenses were analysed by correlative structural (light, scanning electron microscopic and three-dimensional computer-assisted drawings) and optical (low power helium-neon laser scan) analysis at 1.5, 3, 6 and 12 months post-surgery (n = 16 lenses from operated animals and n = 8 lenses from non-operated controls at each time point). Results demonstrate that in rabbits lens growth, or fiber formation, is compromised after vitrectomy. From 1.5 to 12 months after surgery, lenses had progressively more crooked posterior line sutures with sub-branches of increasing size and number in successive growth shells. Quantification of lens optical quality specifically along and/or between these atypical suture branches and sub-branches revealed a significant increase in focal length variability (sharpness of focus) after vitrectomy. A peripheral zone of fibers with abnormal posterior ends was produced surrounding the pre-surgical lens mass. This additional zone of aberrant fibers was associated with a quantifiable degradation in lens optics. Studies on the prevention of post-vitrectomy lens changes in this rabbit model may yield useful information applicable to the human condition.

Effect of maintaining rainbow trout in creosote microcosms on lens optical properties and liver 7-ethoxyresorufin-O-deethylase (EROD) activity.

Previously, exposure of fish to polycyclic aromatic hydrocarbons (PAHs) in both field and laboratory settings has been associated with eye damage, but this has only been expressed qualitatively. In this study, an automated scanning laser system has been employed to quantitatively evaluate changes in lens optical quality in rainbow trout (Oncorhynchus mykiss) following their in vivo exposure to creosote, which is a complex mixture with many PAHs. Rainbow trout were placed in 12,000-L outdoor microcosms dosed with 0, 3, or 10 microl/L liquid creosote for a 28-day period. Collected fish were examined for changes in focal length variability (FLV), lens size, and weight. These measurements were compared with induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity and hepatic and water concentrations of priority pollutant PAHs. The optical quality of rainbow trout lenses was significantly reduced following creosote exposure, as indicated by increased FLV. Lens damage was significantly related to hepatic EROD activity, and both effects rose with creosote dose. Analytical measurements of microcosm water indicated elevated concentrations of PAHs in creosote-dosed ponds, including compounds capable of inducing rainbow trout EROD activity in vitro. Hepatic concentrations of PAHs were low and not related to creosote dose, likely due to rapid metabolism and elimination. This study demonstrates for the first time employment of a highly sensitive and quantitative technique to measure lens damage in fish exposed to contaminants in situ. The relationship between this effect and hepatic CYP1A activity may suggest a mechanistic linkage, which could lead to the use of EROD activity as an indicator of toxic effect rather than just chemical exposure.