Molecular Vision 1999; 5:6 <http://www.molvis.org/molvis/v5/p6/> Received 15 January 1999 | Accepted 22 March 1999 | Published 6 May 1999

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The Internalization of Posterior Subcapsular Cataracts (PSCs) in Royal College of Surgeons (RCS) Rats. I. Morphological Characterization

Kristin J. Al-Ghoul,¹ Kurt L. Peterson,¹ Jer. R. Kuszak^1,2
 
 

Departments of ¹Pathology and ²Ophthalmology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL

Correspondence to: K. J. Al-Ghoul, Ph.D., Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL, 60612; Phone: (312) 942-6871; FAX: (312) 942-2371; email: kalghoul@rush.edu

Abstract

Purpose: To document lens ultrastructure during and after internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats, a model for human autosomal retinal degenerative disease.

Methods: RCS rat lenses at 2, 2.5, 3, 4, 6, 9, 12, and 15 months old were enucleated and fixed. For light and transmission electron microscopy (TEM), lenses were embedded in epoxy and sectioned along the visual axis. For scanning electron microscopy, lenses were dissected to expose the posterior fibers in concentric growth shells down to the internalized PSC plaques.

Results: Overgrowth of the plaque began between 8 and 9 weeks postnatal and proceeded from the periphery to the posterior pole. This is in contrast to PSC formation which begins centrally and enlarges radially between 4-6 weeks postnatal. Peripheral-to-central overgrowth resulted in the formation of a convexo-concave, disk-shaped suture plane oriented parallel to the capsule. The initial fibers overlying the plaque were extremely flattened at their posterior ends. However, by 3 months postnatal, fiber ultrastructure was relatively normal and displayed only minor morphological irregularities. These temporal and structural changes were used to create 3-dimensional computer assisted-drawing (3D-CAD) reconstructions and animations. TEM examination of plaques revealed scattered fiber defects such as membrane whorls, globular aggregates and intracellular voids in both the internalized plaques and the initial overgrowth. The internalized PSC plaques had comparable morphology in all animals, regardless of age. Specifically, the posterior segments of fibers were enlarged and curved abnormally toward the capsule.

Conclusions: PSC plaques are not internalized and broken down in the classical cell biological sense (i. e. via lysosomal degradation). Rather the plaques retain their structure indefinitely as lens growth proceeds (albeit not entirely normally). This demonstrates that the lens has a restricted ability to respond to growth defects and effect a limited recovery after PSC formation.

Introduction

The Royal College Of Surgeons (RCS) rat is an animal model for autosomal recessive retinitis pigmentosa (ARRP) wherein the retinas of RCS rats spontaneously degenerate between 2 and 6 weeks postnatal [1,2]. These animals also develop bilateral posterior subcapsular cataracts (PSCs) which can be detected by slit lamp examination at 7-8 weeks of age [3]. Hence, the RCS rat is also a useful model for PSC associated with ARRP.

Our recent investigation of RCS rat PSCs [4] demonstrated that cataract formation occurs from four to six weeks postnatal and results from a growth malformation affecting the posterior segments of elongating fibers. Specifically, the posterior fiber ends curve away from the polar axis toward the vitreous and enlarge into ovate globules under the capsule, forming a PSC plaque. Consequently, plaque formation precludes normal posterior suture formation during posterior subcapsular cataractogenesis.

It has been reported that subsequent to PSC formation, 75% of animals "recover" by internalizing the PSC plaque via new growth, while the remaining 25% of animals develop mature cataracts by 9-12 months of age [3]. In lenses with internalized PSCs, new fiber growth over the PSC plaque is transparent and thus has been characterized as healthy [5]. However, transparency alone does not ensure good optical quality. Recent structural analyses in various types of lenses has shown that excessive disorder due to abnormal suture formation is correlated with a decrease in optical quality [6-8]. The aim of this investigation was to characterize the ultrastructure of internalized PSCs and the posterior portions of overlying lens fibers as a function of time after internalization.

Methods

Lenses

RCS rat lenses at 2, 2.5, 3, 4, 6, 9, 12, and 15 months (n = 6-8 animals per group) were utilized in this investigation. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were euthanized by intra peritoneal injection of sodium pentobarbital and the eyes were enucleated. The tissue posterior to the ora serrata was removed and the remaining anterior portion of the eye was immediately placed into fixative. Some RCS rat lenses were obtained from breeding colonies maintained at Alcon Laboratories Inc., Ft. Worth, Texas and at Columbia University, NY (kindly provided by Dr. K. Bhuyan). In these cases, lenses were placed in fixative after enucleation and dissection from the orbit, then shipped to the laboratory by overnight express.

Fixation

Lenses were fixed by one of two methods: (1) 24 hours in 10% buffered formalin, then 2 days in 2.5% glutaraldehyde in 0.07 M sodium cacodylate buffer with fresh fixative daily, or (2) 3 days in 2.5% glutaraldehyde in 0.07 M sodium cacodylate buffer with fresh fixative daily. All lenses were washed overnight in 0.2 M sodium cacodylate buffer, then examined under a dissecting microscope (Zeiss, New York, NY) and photographed. Color slides of whole lenses were digitized using a Polaroid Sprint Scan 35 (Polaroid Corp., Bedford, MA) operated with Adobe Photoshop version 5 (Adobe Systems Inc., San Jose, CA) on a pentium pc platform.

Scanning Electron Microscopy

To expose the PSCs, lenses were either split along the optic axis or dissected as previously described [4]. Briefly, superficial fibers were peeled away from the lens around its entire diameter, resulting in crescent-shaped groups of fibers and the remaining lens core. Subsequent layers of fibers were removed in the same manner until the internalized PSC was visible on the lens core. Both the superficial fiber "peels" and the remaining lens mass were collected and processed for SEM.

Specimens were post-fixed in 1% aqueous osmium tetroxide at 4 °C overnight, washed in cacodylate buffer, and dehydrated through a graded ethanol series. After overnight dehydration in absolute ethanol, the alcohol was replaced through a graded series of Freon 113. Specimens were critical point dried in Freon 13 in a Balzers CPD 020 (Balzers, Hudson, NH), secured on aluminum stubs with silver paste, sputter coated with gold and examined in a JEOL JSM 35c scanning electron microscope (JEOL USA, Peabody, MA) at 15 kV.

Light Microscopy/Transmission Electron Microscopy

Lenses were post-fixed overnight in 1% aqueous osmium tetroxide at 4 °C, then washed in cacodylate buffer and dehydrated through a graded ethanol series to propylene oxide. Lenses were infiltrated and flat-embedded in epoxy resin. For light microscopy, embedded lenses were bisected along the optic axis with a jeweler's saw and 1-2 µm thick sections were cut using a glass knife. Sections were stained with a 1:1 mixture of methylene blue and azure II, then mounted on glass slides with permount. Light micrographs were taken on an Olympus Vanox AHBS3 photographic microscope (Olympus America Inc., Melville, NY).

For TEM, mesas were positioned to include portions of both the PSC plaque and the initial overgrowth. Thin sections were cut at 60 nm with a diamond knife (Diatome, Ft. Washington, PA) on a Sorvall Ultratome V 2088 microtome (LKB, Washington, DC). Sections were mounted on uncoated 200 mesh Hex grids (Electron Microscopy Sciences, Ft. Washington, PA), stained with uranyl acetate and lead citrate, then examined on a JEOL JEM-1200EX transmission electron microscope (JEOL USA, Peabody, MA) at 60 kV.

Results

Whole lenses from 2 to 3 month old RCS rats displayed superficially located speckled posterior opacities (Figure 1A). When viewed along the equatorial axis (Figure 1B), the PSCs were located progressively deeper within the lens as a function of time. Although fibers overgrowing the PSC were transparent, posterior suture formation was abnormal as indicated by the lack of a typical inverted Y pattern (Figure 1C).

Light microscopy of thick axial sections demonstrated that at 2 months postnatal, new fiber growth had accumulated adjacent to the plaque (Figure 2A, arrows). The posterior ends of upturned fibers comprising the PSC plaque were separated from the capsule. At 3 months postnatal, the plaque was completely internalized by growth of new fibers (Figure 2B). Overgrowth of the PSC began at the perimeter of the plaque and progressed centrally to confluence at the posterior pole. During overgrowth of the plaque, the posterior ends of new fibers abutted with the enlarged fiber ends in the plaque (Figure 2C), forming an abnormal, convexo-concave, disk-shaped suture plane oriented parallel to the capsule (with the convex surface facing the posterior). After the PSC plaque was completely overgrown, new fibers abutted with each other in the conventional manner [9,10] to form suture planes perpendicular to the capsule (Figure 2B, arrows). However, as demonstrated in Figure 1C, posterior suture patterns were not normal. The functional implications of abnormal suture formation during PSC internalization have been explored in a concurrent investigation [11].

Scanning electron microscopic examination of the initial fibers overlying the plaque showed that their posterior ends were extremely flattened (Figure 3A,B, asterisks) and had irregular, branched, terminal extensions (Figure 3C, inset). This is in contrast to the slightly-flared fiber ends characteristically seen at sutures [12]. Inspection of fiber peels obtained during lens dissections revealed that fiber morphology was normal except over the plaque (Figure 3C and Figure 4A-C). In some lenses, the posterior ends of fibers directly over the plaque displayed concavities which were complementary to the enlarged ends of fibers comprising the plaque. The concave ends of fibers also lacked fiber-fiber interdigitations (Figure 4C, arrowheads) which are typically present both at fiber ends and along the rest of the fiber length in rat lenses [4,12]. In addition, it was apparent that during internalization of PSCs, each successive growth shell of fibers terminated closer to the posterior pole (Figure 3C and Figure 4A,B).

Transmission electron microscopic observations of the plaque and initial overgrowth were completely consistent with features noted by light and scanning electron microscopy. The ovate profiles of fiber ends in the plaque were overlain by attenuated, irregularly-shaped fiber ends which, in turn, were overlain by flattened, hexagonal profiles typical of cross-sectioned fibers (Figure 5). Scattered defects such as membrane whorls, intracellular voids, and small globules were located in both the plaque and initial overgrowth (Figure 6). However, there was no evidence of wide-spread breakdown of the internalized PSC.

Precise dissection of RCS rat lenses to a diameter of 2.5 mm (equivalent to 6 weeks postnatal) exposed the internalized plaques for SEM evaluation. In animals 3 to 15 months old, internalized plaques were structurally similar regardless of the length of time since internalization occurred (Figure 7). Specifically, the plaques were composed of enlarged, globulized fiber ends abnormally curved towards the capsule at all ages examined.

Discussion

In this study, correlative LM, SEM and TEM have elucidated the structure of RCS rat lenses during and after PSC internalization. Precise characterization of fiber morphology throughout PSC formation [4] and internalization was utilized to create 3-D CAD reconstructions (Figure 8 and Figure 9) and an animation (Figure 10) depicting these events. Figure 8 summarizes PSC formation from 2 to 6 weeks. Figure 9 outlines the initial internalization of PSC plaques from 8 to 12 weeks.

The 3D-CADs in Figure 8 demonstrate that the PSC was due to a growth malformation which affects the posterior segments of each successive growth shell of fibers, resulting in gradual plaque formation. In a similar manner, as shown in Figure 9, plaque internalization was accomplished progressively as each new growth shell of fibers extends closer to the posterior pole. The rate of PSC formation and internalization was therefore dependent on the growth rate of the affected lens (and would be expected to be more rapid in juvenile rat lenses as compared to mature human lenses).

The initial internalization of the PSC created an abnormal suture plane parallel to the capsule and essentially perpendicular to the optic axis. Since sutures are regions of naturally occurring disorder which have a measurable negative effect on lens optics [6-8], the addition of an abnormal suture plane directly across the optic axis would be expected to adversely affect lens function. In fact, laser scan analysis of recovered RCS rat lenses implied that the light path was obscured in central locations [11]. Conversely, there are two factors which may exert a positive influence on the optics of recovering lenses. First, the posterior ends of new fibers formed concavities which covered the irregular surface of the plaque, effectively minimizing extracellular space between the two disparate regions. Second, relatively normal fiber morphology was reestablished rapidly (within only a few growth shells), restoring the ordered array of radial cell columns and suture patterns which are optimal for lens optics. Once again, analysis of the optical quality of RCS rat lenses after recovery from PSC confirms the importance of correctly-formed suture patterns in minimizing light diffraction [11].

It is generally presumed that human PSCs, regardless of etiology, are the result of dysplastic cells which migrate posteriorly and aggregate at the posterior pole. However, recent evidence indicates that this may be incorrect for PSC associated with retinal degenerative disease and for post-vitrectomy PSC. In both the present model (RCS rat) and in a transgenic mouse model for autosomal dominant retinitis pigmentosa, there was no evidence of posterior migration of nucleated cells [4,13]. Rather, the PSCs were formed by abnormal posterior fiber growth. Similarly, in a rabbit model for post-vitrectomy PSC, cataracts were due to abnormal posterior suture development rather than posterior migration of cells from the bow region (unpublished data, Kuszak et al.). In addition, it has long been recognized that some PSCs noted clinically have a stellate appearance, indicating involvement of the sutures in posterior opacification [14,15]. This suggests that there may be two types of PSC: one caused by posterior migration of dysplastic cells, and one caused by abnormal fiber growth resulting in posterior sutural malformations.

SEM examination of 3 to 15 month old lenses indicated that the structure of internalized plaques remained stable throughout the life span of animals. Similarly, TEM examination of internalized plaques revealed only scattered ultrastructural defects. It is clear that the fibers comprising PSC plaques were neither internalized nor broken down via lysosomal degradation, as occurs during internalization of necrotic and senescent cells in other tissues [16-18]. The sparse ultrastructural damage located within the plaque and initial overgrowth in RCS rats is dissimilar to previous observations of human PSC associated with retinal degenerative disease [19-21] which described widespread fiber degeneration in the posterior region. Human lens observations in those studies are more consistent with observations from RCS rat lenses which do not recover, but instead progress to mature cataracts [22].

The results of this investigation demonstrate that the lens has the ability, although restricted, to respond to growth defects and effect a limited recovery after PSC formation associated with retinal degeneration. This is not entirely unexpected since spontaneous recovery from PSCs have been reported in diabetic patients [23-25]. Evaluation of the optical quality of internalized RCS rat PSCs has provided a quantitative measure of the extent and limitations of the recovery of lens function and is described in the concurrent investigation [11].

Acknowledgements

The authors thank Layne A. Novak for his expert technical assistance. We are also grateful to Alcon Laboratories Inc. (Ft. Worth TX.) And Dr. K. Bhuyan (Columbia University, NY) for providing RCS rat lenses. This work was supported by NIH-NEI grant EY-06642 to JRK and by the Louise C. Norton Trust (Chicago, IL).

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