Posterior Capsule Opacification in Mice FREE

Posterior capsule opacification (PCO) remains the most common complication of cataract surgery. Despite recent advances in surgical techniques and intraocular lens designs, it has been estimated that between 14.1% and 18.8% of patients will require Nd:YAG posterior capsulotomy following cataract surgery.¹ Complications of this latter procedure are well known to ophthalmologists and include retinal detachment, cystoid macular edema, and increased intraocular pressure. Furthermore, the procedure represents a cost burden to health care systems, and it is not available in underdeveloped countries.²

Several in vitro and in vivo models of PCO have been described. These models have been used to study the pathogenic mechanisms that lead to PCO and to search for new therapies to prevent or treat this common complication of cataract surgery. In vitro models have included the use of cultures of lens epithelial cells (LECs)^{3 - 4} and intact capsular bags.^{5 - 6} Although cell cultures are adequate to evaluate LEC growth, the matrix and the culture medium used to grow these cells would be expected to affect the growth rate and the molecular characteristics of these cells. Capsular bag models seem to mimic more closely the events that take place during the development of PCO, such as LEC proliferation and capsular wrinkling. However, in these models, lens fiber regeneration has not been observed. Furthermore, cultures of human capsular bags are restricted by the unavailability of this tissue.

The most widely used in vivo animal models of PCO include those in the monkey,⁷ dog,⁸ cat,⁷ and rabbit.^{9 - 10} These animals are difficult to handle and expensive to purchase and maintain. This may limit the number of animals that can be used in any given study and, thus, the statistical evaluation of the data. Furthermore, given the large size of the eye, it may be more difficult to study in great detail all tissue available. Moreover, and unlike what occurs in humans, a marked inflammatory reaction is usually seen after lens extraction in dogs, cats, and rabbits.^{7 - 10} Ethical considerations may also arise when using these species.

A new model of PCO in rats has been introduced recently.¹¹ In the rat, marked LEC proliferation and capsular wrinkling occurred only 3 days following extracapsular lens extraction (ECLE), and new lens fibers and Soemmerring ring formation were observed only 14 days following this surgical procedure. The disadvantages of using higher species, such as those already listed, can be overcome now by using this new PCO model.

In the present report, we have adapted this model for use in mice. Despite the smaller size of the mouse eye compared with the rat eye, we have found that a reproducible PCO model in mice is possible. Certain differences in ocular wound healing compared with the rat were observed, particularly in relation to iris cell behavior. In addition to the advantages of the rat model, the mouse model opens up the possibility of developing new research strategies by using genetically modified mice to study selectively the effect of different molecules in vivo in the development of PCO.

Twenty-eight BALB/c male mice (weight, 20-25 g) were used. All animal procedures were performed in accord with the United Kingdom Home Office regulations and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Institutional guidelines regarding animal experimentation were followed.

An ECLE was performed in the right eye of all 28 animals. All surgical procedures were done consecutively. Animals were anesthetized using an intraperitoneal injection of ketamine hydrochloride (Vetalar; Pharmacia & Upjohn Ltd, St Albans, Herts, England) at a dose of 60 mg/kg of body weight and xylazine (Rompum; Bayer AG, Wuppertal, Germany) at a dose of 5 mg/kg. Pupils were dilated using 1% tropicamide (Chauvin, Essex, England) and 2.5% phenylephrine hydrochloride (Chauvin). The surgery was performed as previously described.¹¹ In brief, a corneal incision was made, followed by injection of 1% sodium hyaluronate in the anterior chamber. Once the corneal incision was extended, an anterior curvilinear continuous capsulorrhexis was done, followed by hydrodissection and lens removal. To facilitate the removal of the lens in toto, including the cortex, the hydrodissection was performed as a single rapid maneuver. The anterior chamber was then filled with sodium hyaluronate, and the surgery was completed by suturing the corneal wound with 11-0 nylon interrupted sutures. Topical 2.5% phenylephrine, 1% tropicamide, and 1% atropine (Chauvin, for all) were administered at the end of the surgery.

Animals were humanely killed at 0 hours (after the surgery was completed) and 24 hours, 3 days, and 14 days after surgery by using a lethal dose of carbon dioxide. Seven animals were killed at each time point. The right eye was enucleated and processed for light microscopy (n = 1) or immunohistochemistry (n = 6) studies.

Before death, animals were anesthetized and the anterior segment was evaluated using the operating microscope. The status of the posterior capsule (presence or absence of opacification), cornea (clear or opaque), corneal wound (presence or absence of neovascularization and presence or absence of iris synechiae into the wound), iris (presence or absence of posterior synechiae), and pupil (good or poor dilation) was recorded.

For light microscopy studies, eyes were fixed in 2.5% glutaraldehyde and processed to glycol methacrylate resin (Histocryl; London Resin Company, Reading, England). Then, 2.5-μm sections were cut at 3 levels through the midpoint of the eye and stained with hematoxylin-eosin. For immunohistochemistry, eyes were embedded in optimal cutting temperature compound, snap frozen, and stored at −80°C. Cryostat sections (8-10 μm) of tissues were placed on poly-L-lysine–coated slides at −20°C, air dried, and fixed in acetone. They were then rehydrated in Tris-buffered saline and incubated in the primary antibody. The following primary antibodies were used: MOMA1, MOMA2, CD4, CD8, CD11b, CD45, CD3e, CD11c, GR1, and α smooth muscle actin (α-SMA) (Table 1). After two 5-minute washes, a secondary biotinylated rabbit antimouse antibody (DAKO, Glostrup, Denmark) was added for 30 minutes, followed by further washes. For CD3e and CD11c primary antibodies, a biotin antihamster IgG cocktail (BD Biosciences, United Kingdom) was used. Sections were then incubated with streptavidin-biotin complex for 30 minutes at room temperature, washed in Tris-buffered saline, and rinsed briefly in distilled water. This was followed by the addition of the substrate and further rinsing in distilled water. Sections were then counterstained with hematoxylin-eosin. Washed sections were mounted and viewed under the microscope. For α-SMA, the Vector M.O.O immunodetection kit (Vector Laboratories, Ltd, Orton Southgate, Peterborough, England) was used, and the antibody to α-SMA was used at dilutions of 1:200 and 1:400 for all specimens.

Positive-stained cells were counted in 3 fields per eye studied using ×40 objective magnification. One section per eye was counted. The mean number of cells per eye was recorded. Cell counts were done in a masked fashion.

t Test was used to evaluate differences in cell numbers at all time points. For the statistical analysis, the number of cells at 0 hours was compared with that at 24 hours, 3 days, and 14 days. Differences were considered to be statistically significant at P<.05.

The surgical procedure was uncomplicated in 22 animals (79%). In 5 animals (18%), some bleeding from the iris occurred immediately after lens removal (n = 1) or at the time of the wound closure (n = 4). In all these cases, the bleeding was limited and was successfully controlled during the surgery. In only 1 of these cases, a small hyphema, which did not obscure the view of the anterior segment structures, was seen at the end of the surgery. In 1 animal (4%), a choroidal detachment was observed at the end of the surgical procedure.

In 4 animals, a small amount of cortex was left behind after ECLE in the superior aspect of the capsular bag. There were no occurrences of posterior capsule rupture, vitreous loss, or corneal edema during surgery. The eye appeared well formed at the end of the procedure in all animals.

In 20 animals (71%), the eye appeared well healed, with a clear cornea and a deep anterior chamber before death (Table 2). The wound appeared variably opaque, with mild neovascularization or none. In 2 of these animals, a small granuloma was seen at the site of a corneal suture, and in 4 there were anterior synechiae into the wound. In 2 animals, small amounts of fibrin were present in the anterior chamber, which did not obscure the view of the iris or capsular bag. In 4 animals, posterior synechiae were present at the pupillary margin, which prevented full dilation of the pupil.

In 8 cases (29%), postoperative complications were noted, including a small hyphema that did not obscure the view of anterior segment structures (n = 3), corneal opacification that obscured the view of anterior segment structures (n = 2), and poor dilation of the pupil (n = 3). In 2 of these latter cases, poor pupillary dilation seemed to be related to the presence of synechiae at the pupillary margin, and in 1 the cause was undetermined (Table 2).

Dusting (small pinpoint areas of opacification) at the posterior capsule was detected in the first 3 days after ECLE. All animals in which a clear view of the posterior capsule was achieved before death had developed PCO 2 weeks after surgery (Table 2).

Immediately after ECLE, a monolayer of LECs was observed in the inner surface of the anterior capsule and at the lens bow. The capsular bag appeared open, and there was no contact between the inner surface of the anterior and posterior lens capsule.

Twenty-four hours after surgery, the anterior and posterior leaves of the capsular bag were in contact, and the space between the anterior and posterior lens capsule was filled with LECs. No LECs were seen in the center of the capsular bag, where only the posterior capsule was present. Cells from the posterior iris root and ciliary body were seen forming membranelike structures, which appeared to extend toward the center of the capsular bag (Figure 1A). Some inflammatory cells were also present.

Three days following lens extraction, multiple layers of LECs, some with a spindle-shaped appearance, were filling the capsular bag, including its center. Wrinkling of the posterior capsule was evident.

At 2 weeks, there were well-differentiated lens fibers and Soemmerring ring formation in the periphery of the lens. In the center of the capsular bag, where there was no anterior lens capsule present, several layers of elongated LECs and marked wrinkling in the posterior capsule were observed (Figure 1B).

Inflammatory cells were evaluated by immunohistochemistry using a wide range of cell markers (Table 1). CD11b⁺ cells were found in greater numbers 24 hours and 3 days after surgery (P = .01 and P = .004, respectively) (Figure 2 and Figure 3A). Similarly, the number of CD45⁺ cells was statistically significantly higher 24 hours and 3 days after ECLE (P = .04 and P<.001, respectively) and remained statistically significantly higher (P = .04) 14 days after ECLE (Figure 2 and Figure 3B). In addition, a statistically significant increase in MOMA1⁺ cells (P = .04) compared with baseline was found 3 days following ECLE. Few GR1⁺ cells were seen at all time points (Figure 2 and Figure 3C).

Marked immunoreactivity to α-SMA was found in the blood vessels (Figure 4A) and in the iris (Figure 4B; and C). Weak immunoreactivity to α-SMA was observed in the center of the lens capsule only in those specimens obtained 14 days following ECLE (Figure 4D). No immunoreactivity to α-SMA was detected in the lens capsule at other time points or in any other areas of the capsular bag. In some specimens, marked immunoreactivity to α-SMA was also observed in layers of cells present between the capsular bag and the iris (Figure 4C).

In some specimens, cellular sheets that appeared to arise from the iris root and ciliary body were seen forming membranelike structures (see the “Light Microscopy” subsection of this section). These appeared to extend centripetally to the central aspect of the lens capsule, and, in some cases, they seemed to contribute to PCO formation.

This study demonstrates that, despite the small size of the eye, ECLE is a feasible surgical procedure in mice. As in rats,¹¹ PCO was observed clinically and histopathologically in all animals 14 days following ECLE. Similarly, ECLE in mice was associated with a low-grade inflammatory response.

Difficulties encountered during and after ECLE in mice compared with rats included the performance of an adequate cleaning of the capsular bag after lens extraction and the maintenance, after surgery, of a mobile iris that would allow an adequate dilation of the pupil. To achieve a complete cleaning of the capsular bag, including the removal of all lens cortex, a rapid injection of fluid during capsulorrhexis, in a single maneuver, was required. Posterior synechiae that limited pupillary dilation were observed often in mice (Table 2). It is not clear whether these iris adhesions to the lens capsule were real posterior synechiae or whether they may represent, at least in some cases, newly developed sheets of cells, such as those described as “pigment membranes” by Cobo and colleagues⁷ in cats. As in cats, these cellular sheets appeared to extend centripetally from the iris root to the central aspect of the lens capsule, and, in some cases, they appeared to contribute to PCO formation.

As in other animal models of PCO,^{7 - 11} proliferation of LECs, capsular wrinkling, and new lens fiber formation occurred in mice. However, the time required for these changes to develop was shorter in the mice and rat models of PCO. Therefore, confluent proliferation of LECs over the posterior capsule and capsular wrinkling were evident in rodent models only 3 days following ECLE, whereas these changes were observed after 1 week in cats⁷ and after 4 weeks in dogs⁸ and rabbits.⁹ As in mice, proliferation of spindle-shaped cells at the edge of the incised anterior capsule in primates caused capsular opacification at that site. However, opacification at the center of the lens capsule was not observed even after 6 months following surgery.⁷ Fiber differentiation was noted only 2 weeks after surgery in rodents but was detected over 1 month following surgery in dogs⁸ and rabbits.¹⁰

In mice, a moderate degree of inflammation was found following ECLE. CD45⁺, CD11b⁺, and MOMA1⁺ cells were detected in greater numbers 3 days after lens extraction, when maximal LEC proliferation and capsular wrinkling were evident. The number of CD45⁺ cells remained high 14 days following ECLE, when well-differentiated lens fibers were present. CD11b and MOMA1 are expressed by macrophages. CD45 is the common leukocyte marker, expressed by macrophages and other leukocytes. Given that few GR1⁺ cells and few lymphocytes (CD4⁺, CD8⁺, and CD3e⁺ cells) were seen at all time points, it is likely that most CD45⁺ cells found were macrophages rather than neutrophils. Macrophages are highly phagocytic cells that are known to play a role in the clearance of diverse tissues of damaged, infected, or senescent cells^{12 - 14} and are involved in the process of wound healing.¹⁵ In this regard, opening the anterior lens capsule and removing the lens fibers during ECLE would be expected to initiate a wound-healing response; thus, the infiltration of macrophages observed after ECLE would not be unexpected. Macrophages produce growth factors, including transforming growth factor β¹⁶ and fibroblast growth factor,^{17 - 19} which have been associated with capsular wrinkling²⁰ and lens fiber differentiation,²¹ respectively, and could play a role in the development of PCO. Although CD11c⁺ cells were seen in greater numbers at 3 and 14 days, their numbers did not reach statistical significance. CD11c is expressed by murine dendritic cells.²² CD11c⁺ cells seem to play an important role in the iris, taking up foreign proteins.²³ It is possible, therefore, that exposure of normally sequestered lens proteins after opening of the lens capsule would allow uptake and presentation of lens antigens by iris-resident dendritic cells, thus initiating an autoimmune response. It is interesting that there is an increase in antilens crystallin antibodies with age and with development of cataract.^{24 - 25}

In the mice model of PCO, only weak immunoreactivity to α-SMA was found in the lens capsule 14 days after ECLE. This contrasts with findings reported in aphakic rabbits, in which α-SMA–positive LECs were observed in the adhesive region (where the incised edge of the anterior capsule adheres to the posterior capsule) 5, 7, and 14 days following ECLE.²⁶ It is unlikely that this represents a technical problem with the antibody used, because marked immunoreactivity to α-SMA was found in blood vessels and the iris. Tissue sections were used in this study; thus, it would be possible that no α-SMA–positive cells may have been included within these sections. Further studies in flat whole mounts and at other time points are under way to try to elucidate whether α-SMA–positive cells are implicated in the process of PCO in rodents.

In conclusion, we believe that this newly described model of PCO in the mouse allows the possibility of using genetically modified mice to (1) study the sequence of events that lead to PCO, including the role of different molecules involved in its development; (2) evaluate the signals that lead to cell differentiation; and (3) search for new therapeutic modalities to prevent or treat PCO.

Correspondence: Noemi Lois, MD, PhD, Retina Service, Department of Ophthalmology, Aberdeen Royal Infirmary, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZN, Scotland (noemilois@aol.com).

Submitted for Publication: September 4, 2003; final revision received April 21, 2004; accepted June 1, 2004.

Financial Disclosure: Drs Lois and Forrester have a patent on the mouse model of posterior capsule opacification.

Funding/Support: This research was supported by the Royal Blind Asylum and School, Edinburgh, Scotland, and by the Scottish National Institution for the War Blinded, Edinburgh.

Acknowledgment: We thank Marlene Arthur, Lynne Doverty, and Bruce Mireylees for their technical assistance.