Posterior Capsule Opacification: A Review of Experimental Studies

Posterior capsule opacification (PCO) is the most common complication of cataract surgery. It causes a gradual deterioration of visual acuity, which would otherwise improve after a successful procedure. Despite recent advances in ophthalmology, this complication has not been eradicated, and the incidence of PCO can be as high as 10%. This article reviews the literature concerning the pathomechanism of PCO and examines the biochemical pathways involved in its formation and methods to prevent this complication. We also review the reported tests performed in cell cultures under laboratory conditions and in experimental animal models and in ex vivo human lens capsules. Finally, we describe research involving human eyes in the clinical setting and pharmacological methods that may reduce the frequency of PCO. Due to the multifactorial etiology of PCO, in vitro studies make it possible to assess the factors contributing to its complications and search for new therapeutic targets. Not all pathways involved in cell proliferation, migration, and contraction of the lens capsule are reproducible in laboratory conditions; moreover, PCO in humans and laboratory animals may be additionally stimulated by various degrees of postoperative reactions depending on the course of surgery. Therefore, further studies are necessary.


Background
Cataract is the most common cause of blindness worldwide, and surgical removal is the most common method used to treat it (1). Cataract surgery has improved in recent years, but postoperative complications still occur. The most common complication of cataract surgery is postoperative capsular opacification (PCO), which has a multifactorial etiology (2). PCO not only causes quantitative visual disturbances but also reduces the quality of vision, leading to a reduction in contrast sensitivity, halo effect and lack of binocular vision (3). Despite improvements in the techniques used in cataract surgery and in lens design, statistically, it was reported to occur after surgery in approximately 11.8% of cases within the first year, in almost 20.7% by 3 years, and in 28.4% by 5 years (4). The overall occurrence is as high as 50% in adults and 100% in children from 2 months to 5 years after surgery (5). In addition to diminishing the patient's quality of life, PCO makes it difficult to examine the fundus and to perform specialist ophthalmic examinations, such as optical coherence tomography.
Postoperative capsular opacification is mainly caused by migration and proliferation of residual lens epithelial cells (LECs) after cataract surgery. It occurs more frequently and is more severe in younger patients as they possess increased numbers of LECs and greater mitotic activity. Therefore, along with development of improved operating techniques, research is underway to develop an effective and safe method for preventing PCO.
The aim of this research was to systematize the current knowledge concerning the prevention of PCO and to present scientific hypotheses that may contribute to reducing

Treatment
The most common procedure performed to improve visual acuity that provides an insight into the fundus of an eye with PCO, is laser capsulotomy (15). This procedure entails making an opening in the posterior capsule on the visual axis. Although widely used, laser capsulotomy is associated with complications, which range from a mild shortterm increase in intraocular pressure to severe visual impairment due to retinal detachment, endophthalmitis, cystoid macular edema, or macular holes (16,17). Trinavarat et al. noticed that laser capsulotomy may lead to IOL damage, subluxation, dislocation, and secondary glaucoma (18). Moreover, laser capsulotomy is not compatible with the new generation of accommodative lenses, which require the presence of a lens capsule to function (19). Laser treatment also places a significant financial burden on health care systems and is not widely available in developing countries. Therefore, the second course of action is to search for methods to prevent secondary cataracts. Until now, numerous in vitro and experimental animal studies have been conducted, but many have reported undesirable effects on the corneal endothelium (20,21). Various trials of antiproliferative therapy have been performed in cells cultured in vitro and lens capsule models or in vivo (in rabbits, monkeys, and humans) (22)(23)(24)(25). Active agents are delivered to LECs via various carriers (including ophthalmic viscoelastic devices, drug delivery systems [DDSs], or slow-release implants) or can be delivered directly to the cell culture medium during in vitro experiments. Various techniques have also been developed for intraoperative application of pharmacological agents to lens cells, including adding them to infusion fluids, using devices that confine them in the capsular space, or specially coating IOLs with the test substance (26).

Etiopathogenesis
Breakdown of the blood-aqueous humor barrier during cataract surgery causes an immune response to a foreign body. Epithelial-mesenchymal transition (EMT) then occurs in cells in the lens capsule. The main cause of PCO after cataract surgery is migration and proliferation of LECs. The average number of LECs is around 4,000-5,000/mm², depending on patient age, with a significant decrease in density in those aged 80 years or older. LECs consist of a single layer of cuboidal-cylindrical cells attached to the posterior surface of the anterior lens capsule. A foreign body, such as an IOL, induces an inflammatory reaction, which includes multinuclear leukocytes, giant cells, and fibroblasts, in the anterior chamber in the immediate postoperative period (27,28). These cells produce cytokines, including transforming growth factor-beta (TGF-β), interleukin-1, interleukin-6, basic fibroblast growth factor (FGF), and tumor necrosis factor-alpha (TNF-α), which activate transformation of LECs, proliferation, metaplasia around the equator of the anterior capsule, and migration towards the posterior capsule, leading to thickening and hypertrophy. This process is characterized by fibrosis and contraction reactions due to the activity of actin filaments (29). Moreover, immunohistochemical studies have identified extracellular matrix (ECM), fibronectin, and collagen molecules on the surface of the IOL (30,31). Collagen deposition on the IOL and capsule can cause clouding and edema in the posterior lens capsule (31).
The extent to which the IOL becomes coated by LECs depends on the material used in the IOL, its surface (31), the shape and diameter of the optic, the mechanical properties of the haptics (30), and the implantation methods used (32). Two types of PCO are caused by two different types of epithelial cells. The first type of PCO involves anterior epithelial cells located in the central zone of the anterior capsule which consist of LECs that are relatively inactive mitotically. When stimulated, these cells are converted to myofibroblasts, causing fibrous metaplasia. Cells may also migrate towards the posterior capsule, where they proliferate and undergo hypertrophy and hyperplasia on the capsule surface, causing opacification (33). The second type of PCO occurs when pluripotent cells gather around the equator of the capsule. When activated by interleukins, these cells migrate posteriorly. They do not undergo fibrosis but instead form large, balloon-shaped, or clustered Wedl or bladder cells. They undergo constant mitosis, proliferation, hypertrophy, and hyperplasia, creating clouding (34). They can also participate in the formation of the fibrous form of PCO through the process of fibrous metaplasia, appearing as fibrous membranes. Metaplasia and inflammatory cells of the iris and ciliary body as well as fibrinous exudate also stimulate development of PCO (35). Furthermore, the residual cortical fibers may regenerate, creating pathological clouding known as Soemmering's ring and Elschnig's pearls (31).

In vitro research
In vitro research uses cells derived directly from human or animal lenses. These cells perfectly reflect the processes occurring during formation of PCO because of the original phenotype of the epithelium and their greater sensitivity to external factors. The proliferation and differentiation capacity of cultured LECs can be assessed using microscopy (26). Alternatively, cells can be obtained by culture of a cell line; these cells are more stable and can provide information about cellular processes, but do not completely reflect all processes occurring under real biological conditions.

Inhibition of TGF-β
Given that TGF-β plays a key role in formation of PCO, research efforts have focused on inhibiting its action. Sun et al. (36) performed a study in which they grew human lens epithelial (HLEB3) cells in vitro and stimulated them with TGF-β2. The cells then transformed into fibroblast-like cells and the bonds between them loosened, causing them to migrate (i.e., undergo EMT). Application of a SNAIL siRNA polyclonal antibody released from a coated IOL inhibited the EMT process, mainly via inhibition of migration and adhesion of LECs. Collison et al. (37) investigated the effect of an anti-TGF-β monoclonal antibody (CAT-152) on progression of PCO. In their experiment, ex vivo human lens capsules were incubated with various concentrations of TGF-β with or without CAT-152. Incubation of the lens capsule with TGF-β resulted in an increase in migration and differentiation of LECs, which in turn resulted in shrinkage of the capsule and formation of PCO. Addition of CAT-152 inhibited this effect.
Yang et al. (38) evaluated the ability of pirfenidone at concentrations of 0.25, 0.5, and 1.0 mg/mL to inhibit migration and differentiation of human LECs in vitro using the SRA01/04 cell line. Cell viability was assessed by trypan blue staining, and the level of lactate dehydrogenase released from lysed cells was measured. After 24 h, dose-dependent inhibition of proliferation, migration, and EMT was compared between the different doses of pirfenidone. The authors concluded that pirfenidone inhibits TGF-β2-induced cell proliferation and migration, most likely by downregulating the TGF-β receptor (and Smad2, Smad3, and Smad4) in human LECs.
Lovastatin also has the potential to inhibit TGF-β (39). The mechanism of action of lovastatin involves blocking RhoGTPase, which stimulates the TGF-β-dependent conversion of LECs. In a study performed using a porcine model, LECs were pre-treated with lovastatin and then incubated with TGF-β for 24 h. Unlike the control group, the lovastatin group showed no increase in mRNA expression, protein production, or increased collagen contractility. These findings suggest that lovastatin could be considered for prevention of PCO (39).
Kubo et al. described the roles of TGF and fibroblast growth factor (FGF)-2 and their relationship to tropomyosin in regulation of EMT in lens capsule cells and the development of PCO (40). These authors identified two types of TGF-β-dependent differentiation of LECs, with or without FGF-2, based on research performed in mice and rats. The first type led to differentiation of cells in the lens capsule towards tropomyosin-positive myofibroblasts showing an epithelial-to-myofibroblast transition (EMyoT) morphology without the presence of FGF-2. The second type differentiated into fibroblastic tropomyosinnegative cells induced by FGF-2 with simultaneous administration of TGF-β2. Increased expression of tropomyosin may be associated with progression of EMyoT in mice, PCO in rats, and healing of LECs in mice. Tropomyosin is an important marker of PCO and constitutes a therapeutic target in the wound healing process and in neoplastic invasion. Further investigation of the mechanism by which tropomyosin is regulated will provide valuable insights into its inhibitors and introduce new methods of treatment and prevention of EMT-dependent diseases.
Hypoxia-inducible factor (HIF)-1α is degraded after cataract surgery due to an increase in pO2. TGF-β2, in turn, increases the activity of HIF-1α in cells after cataract surgery. Nahomi and Nagaraj evaluated the role of HIF-1α in EMT mediated by TGF-β2 in cultured FHL124 human LECs (41). TGF-β2 was shown to increase the concentration of HIF-1α during EMT in FHL124 cells, while an attempt to increase HIF-1α expression using a prolyl hydroxylase inhibitor did not induce EMT in these cells. Moreover, KC7F2, an HIF-1α protein translation inhibitor, inhibited TGF-β2-mediated EMT in FHL124 cells. These findings indicate that HIF-1α has a significant role in TGF-β2-mediated EMT of LECs, which may be important in prevention of PCO.
Taiyab et al. evaluated the interaction between β-catenin (involved in non-canonical signal transduction) and Smad3 (required in canonical signaling) in TGF-β-induced EMT regulation using an ex vivo rat lens model (42). Lens cells were tested to show the interaction between the three TGF-β-dependent EMT pathways using the alpha-smooth muscle actin (α-SMA) marker. In their study, inhibition of the Smad3 pathway protected against translocation of β-catenin and E-cadherin and blocked TGF-β-dependent EMT in lens cells. Inhibition of β-catenin and Smad3 reduced the amount of TGF-β receptor mRNA present. The results of that study indicate a close relationship between interaction in the β-catenin/Smad3 line and regulation of the EMT process in the lens and could lead to development of an agent for prevention and treatment of PCO.

Proteasome inhibition
Efforts have also been made to influence other transmitters responsible for development of PCO, including the proteasome inhibitor MG-132 (43). Proteasomes are intracellular proteins, the main function of which is to remove damaged or defective proteins. Proteasomes regulate the cell cycle, signal transduction and transcription, apoptosis, oncogenesis, migration, antigen presentation, and selective degradation of damaged proteins via the ubiquitin pathway. Their specific functions depend on the cell type, proliferative activity, growth factors, and type of compound used. Proteasome inhibition downregulates the activity of matrix metalloproteinase (MMP)-2 and MMP-9, which are responsible for the release of FGF-2 (44). Proteasome inhibitors have been found to reduce proliferation of LECs in the presence or absence of growth factors that are normally secreted during and after surgery. Therefore, proteasome inhibitors could be used to prevent PCO (43).

Disruption of cell adhesion
Mibefradil is a T-type antagonist of the calcium channels present in cell membranes. As a non-selective calcium channel blocker, it causes depolarization of cell membranes. Moreover, it reduces the expression of integrins, resulting in inhibition of proliferation and induction of apoptosis. Its use in the prevention of PCO is based on the hypothesis that it inhibits the pathways mediating cell adhesion. The effect of mibefradil dihydrochloride was tested in primary human LECs obtained after cataract surgery and in a human lens cell line (HLE-B3) (45). During the surgical procedure, fragments of the lens present on the anterior capsule were collected and subjected to cell culture for 1-2 weeks. They were then separated from the capsule and transferred to growth medium. Next, the cells were treated with the drug for 24 h. Apoptosis of cells and proliferation and migration of integrins were analyzed by flow cytometry and verified by confocal microscopy. The earliest signs of mibefradil-induced apoptosis of human LECs were observed after 4 h of incubation. These signs were accompanied by a significant reduction in cell size and initiation of apoptosis by phosphatidylserine switch in the plasma membrane. DNA fragmentation in the cell nucleus and fragmentation of cytoskeletal actin were observed; this phenomenon is known as anoikis (Greek for "homelessness"). Apoptosis is correlated with inhibition of integrin expression, decreased proliferation, and depolarization of cell membranes. This research suggests that depolarization of the human LEC membrane and inhibition of integrin expression leads to a decrease in cell adhesion and apoptosis, which may inhibit development of PCO. Inhibition of integrins prevents LECs from adhering to the lens capsule and leading to PCO.
Sureshkumar et al. (19) tested the ability of drugs that rapidly inhibit the actin cytoskeleton network to prevent PCO in the human lens capsule. Two agents were used, namely, H-7, a broad-spectrum serine-threonine kinase inhibitor, and latrunculin (LAT)-B, a macrolide isolated from the sea sponge Latrunculia (Negombata) magnifica, both of which are less toxic than ethylenediaminetetraacetic acid (EDTA) and antimetabolites that act in a similar fashion (46). These substances differ slightly in their mechanism of action in that H-7 reduces focal cell adhesion, damages cell membranes, and causes relaxation of fibers whereas LAT-B damages direct intercellular connections. In their study, Sureshkumar et al. (19) tested 48 human eye lens capsules that were collected 24 h postmortem. After extracapsular cataract extraction was performed, the pouches were incubated in three types of solutions: H-7 (50, 100, or 300 μM, n=13), 1% balanced saline solution (BSS; the medium for H-7, n=12), LAT-B (2, 5, or 10 μM, n=11), and 0.25% dimethyl sulfoxide (the substrate for LAT-B, n=12). The cells were then observed with photographic documentation for 28-30 days. In the control group, opacities formed on the lens capsule, leading to shrinkage of the capsule on days 3-5, which became more marked at the end of the observation period. No free or dead cells were observed in the surrounding fluid. In the group incubated with H-7, no PCO was detected during the observation period, whereas in the liquid medium, drifting free cells were visible (probably LECs that could not adhere to the capsule). Inhibition of growth of LECs on the capsule occurred at each drug concentration used. In the LAT-B group, the results varied according to concentration used. LECs were visible at a dose of 2 µM, some were visible, and some were not at a dose of 5 µM, and none were seen at a dose of 10 µM. There were no visible free cells floating in the medium in any subgroup. The differences in efficacy between the substances tested suggest that focal cell adhesion has a greater role than intercellular junctions in formation of PCO. However, H-7 also inhibited wound healing in a rat cornea model (47), which would be an undesirable effect during postoperative recovery.

EGFR as a potential target for prevention of PCO
The epidermal growth factor receptor (EGFR) is of interest to researchers because it activates cascades that play a key role in the formation of PCO (48,49). Stimulation of this receptor activates the tyrosine kinase cascade, which alters gene expression and increases cell activity and growth. Erlotinib and gefitinib are EGFR inhibitors that were registered and approved by the US Food and Drug Administration in 2003 and 2004, respectively, for the treatment of locally advanced or metastatic non-small cell lung cancer in combination with cisplatin and other cytostatic agents, as well as advanced pancreatic cancer in 2007 (50).
The potential role of EGFR inhibitors in the prevention of PCO has been tested in vitro in human LECs (an HLE-B3 cell line) and spontaneously immortalized fetal cells (an FHL-124 cell line) (47,48). Their influence on the ability of these cells to proliferate, migrate, spread, and contract was investigated. At the same time, ex vivo observations were performed on human lens capsules. Cataract surgery was performed on the eyes of cadaveric donors 24 h postmortem. Pairs of collected anterior lens capsules were tested: one was incubated with a gefitinib/erlotinib solution and the other served as a control. Migration capacity was assessed as the ability of crystal violet-stained HLE-B3 cells to pass through a membrane with 8 µm pores and spread. PCO fundamentally involves proliferation of multiplied cells from the equator of the anterior lens capsule to the visual axis, which causes a decrease in visual acuity. Both EGFR inhibitors decreased chemotactic migration, proliferation, and contractility of lens cells in vitro. In both cell culture and donor tissues, the migration of gefitinib-treated cells was prolonged from an average of 5.8 days to 10.6 days. Although no cytotoxicity was observed, these agents may cause trichomegaly, trichiasis, dysfunctional dry eye syndrome, and corneal ulceration with longterm oral administration.

In vitro research involving photodynamic therapy
In vitro research also involves observation of tissues rather than individual cell types. Using this method, all or a portion of the lens is collected from a human or animal donor. Human tissue culture is the model closest to in vivo conditions and can provide information on migration, proliferation, and differentiation of LECs (26). However, a relatively long shelf-life and the need for preservatives can alter cell viability.
Photodynamic therapy with bacteriochlorin has been studied as a method for prevention of PCO. Bacteriochlorin is obtained from the photosynthetic bacterium Rhodospirillum rubrum and acts as a photosensitizing agent (51). Both photodynamic therapy and bacteriochlorin are safe when used separately but have a damaging effect on cells in combination and in the presence of oxygen. This treatment strategy has been successfully used in the treatment of skin cancer, esophageal and bladder cancer, and non-cancerous diseases, such as arteriosclerosis and rheumatoid arthritis (52).
In a study by Fisher et al. (52), 106 human eyes were collected ex vivo and subjected to extracapsular cataract extraction. In the experimental group, bacteriochlorin was administered at concentrations of 50, 25, 12.5, 10, 6.25, 3.12, and 1.6 µg/mL for 10, 8, 6, 4, or 2 min with irradiation using laser diode light at a wavelength of 760 nm for periods of 12, 10, 5, or 2 min. Control groups were treated with bacteriochlorin only or light only. After 7 days of tissue culture, LECs underwent histological evaluation. In the study group that was incubated with 10 µg/mL bacteriochlorin for 10 min followed by irradiation for 15 min, proliferation activity and cell growth on the lens capsule was completely inhibited. After 7 days, no formation or growth of LECs was observed. The cell nuclei did not show any staining or chromatin condensation. However, there were visible signs of apoptosis, and the cytoplasm showed signs of severe disorganization. These features were also observed, but to a lesser extent, in cells exposed to lower concentrations of bacteriochlorin and irradiation for a shorter period. In the control groups, LECs developed as a compact layer and contained heterochromatin-packed and euchromatin-packed nuclei and a welldifferentiated cytoplasm with many cell organelles. The toxicity of bacteriochlorin to corneal endothelial cells and the ciliary body, as well as the duration of therapy, have yet to be established. Furthermore, the treatment period was extended by 25 min because this is the optimal application of both treatments. Bacteriochlorin could be administered as an intraoperative injection with application of light occurring after surgery.

Ex vivo tests with heat shock protein 90
Heat shock protein (HSP)90 is known to be involved in regulation of cell proteostasis in the presence of pathological factors. However, its role in PCO is not known. Li et al. (53) investigated the potential therapeutic use of HSP90 in PCO using an LEC line and an ex vivo rat lens capsule. Protein expression in response to application of tanespimycin (17-AAG), an HSP90 inhibitor, was assessed by immunoblotting and real-time polymerase chain reaction (RT-PCR) and apoptosis by the TdT-mediated dUTP nick-end labeling (TUNEL) method. Tanespimycin was found to suppress proliferation of LECs and to inhibit the viability of the LECs remaining in the capsule. Moreover, tanespimycin induced apoptosis, which was characterized by an increase in reactive oxygen species levels, apoptotic DNA damage, and activation of caspase-3 and caspase-9. HSP90 has been reported to play a role in regulation of the EGFR and TGF-β receptor (TGFR) signaling pathways. Inhibition of HSP90 by tanespimycin led to destabilization of EGFR and inhibition of TGFβ-mediated phosphorylation processes (47,48). These data suggest that stimulation of HSP90 protects the cells remaining in the epithelium of the lens capsule from oxidative stress and is also involved in regulation of cell proliferation, EMT, and migration of rat LECs through the EGFR and TGFR signaling pathways. Therefore, treatment with tanespimycin suppresses PCO and may be a candidate agent for prevention of PCO (47,48).
While in vitro studies are valuable, not all pathways of proliferation and migration, or contraction of the lens capsule, are reproducible under laboratory conditions. Moreover, PCO in humans and laboratory animals may be stimulated to different degrees postoperatively depending on the operative course (55). Therefore, in vivo tests are required.

Research in laboratory animals
In vivo animal models play an essential role in clinical practice by enabling the mapping of the processes occurring in living tissues. The most used animal model for PCO is the rabbit eye. This model has the advantages of the lens being similar in size to the human lens coupled with the ability to implant an artificial lens. The disadvantage is that the metabolic pathways in the rabbit eye are slightly different from those in the human eye (56). Rabbit eyes are suitable for assessment of the effectiveness of treatments that are potentially able to prevent PCO because opacities develop rapidly, usually within weeks after cataract surgery. Furthermore, the postoperative inflammatory reaction is rapid, and elevated levels of inflammatory markers persist for up to 9 weeks after surgery (57).
Antimetabolite agents have been widely studied for their ability to prevent PCO. They inhibit differentiation and proliferation of many cell types, including mitosis in LECs. Inan et al. (21) investigated the effect of retinoic acid and mitomycin C when administered intraoperatively to rabbit eyes during phacoemulsification. Of all retinoids, retinoic acid has the strongest inhibitory properties with respect to cell proliferation and collagen production by reducing production of TGF-β and it is widely used to treat skin conditions and cancer. In ophthalmology, it has applications in xerophthalmia and experimental modulation of corneal wound healing. It has also demonstrated potential to inhibit cells in the retinal pigment epithelium during in vitro studies of PCO in laboratory animals (21,22). Mitomycin C is an antineoplastic antibiotic derived from Streptomyces caespitosus. It is an alkylating agent rather than an antimetabolite and selectively inhibits DNA replication, mitosis, and protein synthesis. In the case of PCO, its action is mainly based on inhibition of fibroblast proliferation. In one study (22), rabbits were divided into three groups. In group I, retinoic acid was administered at a dose of 25 µg during hydrodissection and 225 µg into the lens capsule after aspiration of the cortical masses. In group II, mitomycin C was administered at a dose of 0.04 mg; although this dose is lower than that considered to be toxic (3.0 mg/mL), two subjects developed severe corneal edema, which led to their exclusion from further investigation. Group III served as the control group (22). The PCO levels in groups I and II were significantly lower than those in the control group, with significantly fewer complications.
The same authors extended their research to include three other substances (21). That is, they investigated the effect of EDTA, arginine-glycine-aspartic acid (RGD) peptide, diclofenac, and dexamethasone in a rabbit eye model. EDTA is used mainly for diagnostic purposes. In the case of PCO, it works by chelating calcium ions, thereby inhibiting migration of LECs. When included as part of in vitro studies, it was used to separate epithelial cells from the basal membrane (58). RGD, by contrast, inhibits integrins by competitively binding to the b-chain present in the ECM. Therefore, the LECs did not adhere to the lens capsule. After 3 months, the eyes underwent histological examination. All three substances had a significant inhibitory effect on formation of PCO in comparison with the control group. Mitomycin C was the most effective, followed by RGD, EDTA, and diclofenac. There was no statistically significant difference in the PCO scores between the test groups. The least effective was dexamethasone, although the PCO score in this group was significantly lower than that in the control group (20). The most common postoperative complication was mild to moderate corneal edema, except for two eyes in the mitomycin C group, one eye in the EDTA group, and one eye in the diclofenac group, edema persisted for more than a week after surgery. Inflammatory cells were also observed in the aqueous humor of two eyes in the control group and one in the EDTA group. 5-Fluorouracil (5-FU) is a widely researched agent, and much hope has been raised in the context of PCO inhibition. It is an antimetabolite that inhibits DNA synthesis and is active in the second phase of the cell cycle. It inhibits fibroblast proliferation, but adherence and migration remain unchanged.
Pandey et al. (59) designed a ring that slowly released 5-FU and implanted it into the lens capsule after the phacoemulsification procedure. This ring is composed of a copolymer of hydroxypropyl methacrylate and methyl methacrylate (75:25) with a water content of 19%, a cross-sectional diameter of 9.7 mm when dry and 10.2 mm when hydrated, and a thickness of 0.53 mm. It contained 300 g of 5-FU coated with poly(lactide-co-glycolide) (PLGA) to slow the release of the drug. Seventeen New Zealand rabbits were divided into three groups (group 1, no ring implanted; group 2, the ring was implanted but without 5-FU; group 3, the ring containing the slow drug-release system was implanted). After 8 weeks, statistically significant differences in the incidence of PCO were found between group 1 and the other two groups but not between groups 2 and 3. No damage was found in fragments of the retina or cornea that were collected to assess the cytotoxic effects of 5-FU on the surrounding tissues. However, the rabbit cornea has much greater regenerative ability than the human cornea (60). (61). A combination of methotrexate and actinomycin D inhibited formation of PCO in both models. However, these findings were not confirmed by an in vivo study in monkeys (62).

The effect of actinomycin on PCO was investigated by Nibourg et al. in a rabbit lens model (26) and by Sternberg et al. in an ex vivo human lens
Rapamycin (sirolimus) is a macrocyclic triene antibiotic with immunosuppressive, anti-inflammatory, and antiproliferative properties and is produced by Streptomyces hyslopius isolated from soil samples. Liu et al. (5) conducted a prospective randomized study of rapamycin in 60 New Zealand white rabbits. The animals were divided into three groups depending on the type of lens implanted: group A, a conventional lens; group B, a lens coated with a poly(lactic-co-glycolic acid) carrier only; and group C, a rapamycinloaded PLGA-coated IOL. Histological observations showed that group C had the lowest accumulation of lenticular material on the posterior capsule, less inflammation in the anterior chamber after surgery and less frequent and delayed PCO when compared with groups A and B. Rapamycin was detected in the anterior chamber through day 7 after surgery but not in peripheral blood.
Nishi et al. conducted a similar study (63), which showed that implantation of an indomethacin-soaked IOL significantly reduced formation of PCO in phacoemulsified rabbit eyes during 12 months of follow-up. All indomethacin was released from the IOLs in BSS within 24 h. In another study, the same authors implanted a disc that gradually released indomethacin for 3 weeks after phacoemulsification surgery. Although inflammation of the anterior chamber was significantly reduced, indomethacin had no inhibitory effect on formation of PCO (64).
Heparin is another agent that has been used in animal studies. It is known to inhibit proliferation of LECs and fibroblasts, inhibit deposition and adhesion of platelets, macrophages, and fibroblasts on the surface of the posterior capsule, and reduce the activation of granulocytes, leading to reduced inflammation and activation of clot dissolution (2,9). In one study, Xie et al. investigated the value of PLGA, a macromolecular carrier characterized by delayed release and good biodegradability, in 50 New Zealand white rabbits. The rabbits were divided into five groups: in group A, saline drops were administered; in group B, a DDS with the carrier was implanted into the posterior chamber of the eye; in group C, heparin eye drops were placed in the eye; in group D, the DDS containing heparin was implanted in the subconjunctiva; and in group E, the DDS containing heparin was implanted into the posterior chamber of the eye. In all groups receiving heparin in any form, fewer and later signs of PCO were observed, while in group E, PCO did not occur in most cases (n=6/10) during the entire 12-week observation period (66).
According to some researchers, only 5% of the volume of a drug administered via the conjunctiva can penetrate the eye due to the blood-aqueous humor barrier (4). This limitation led to further research with the purpose of developing a DDS containing low-molecular-weight heparin for implantation in the posterior chamber as a safe and effective method of preventing PCO (67).
A postmortem study of 1% lidocaine without preservative in rabbit eyes by Vargas et al. (68) showed that hydrodissection can reduce the number of viable LECs by reducing the adherence of cells to the basement membrane, damaging their desmosomal junctions, or by direct toxic effects. Sixteen eyes were phacoemulsified and divided into two groups of 8: in group I, the anterior lens capsule was irrigated with 1% preservative-free lidocaine solution and in group II the anterior lens capsule was irrigated with BSS for 1, 2, or 5 min. The capsules were stained with trypan blue and alizarin red. Photographic documentation was obtained and assessed for LEC damage. Fragments of the capsules from group I showed cytotoxic activity, which was manifested by blue-stained nuclei. The number of dead LECs increased with increasing duration of contact with lidocaine. This phenomenon was not observed in group II, regardless of irrigation time. Subsequently, in group II, four eyes were hydrodissected with 1% lidocaine and the other four with BSS followed by further standard phacoemulsification steps. The degree of LEC adhesion to the anterior capsule was assessed histologically and in the group that underwent hydrodissection with lidocaine, the anterior capsules were almost completely LEC-free. In the group treated with BSS, a normal layer of LECs was observed on the surface of the capsule. In the subjective opinion of the surgeon, the process of cortical mass removal during irrigation aspiration was easier in the group in which hydrodissection was performed with lidocaine. This effect can be explained by the weakening of cellular adhesion in the area of desmosomes, and thus decreased cell adherence. Furthermore, in vitro, lidocaine reduces production of leukotriene B4 and interleukin-1 and inhibits proliferation of fibroblasts, which may reduce formation of PCO. However, these promising results were not confirmed in human eyes, where it was shown that lidocaine did not reduce the incidence of PCO nor the inflammatory reaction in the anterior chamber in comparison with the control group (68).

Drug delivery via silicone capsule
Maloof et al. created a sealed capsule irrigation device, the Perfect Capsule™ (Milvella Pty. Ltd., Epping, NSW, Australia). It is a foldable silicone disc 0.7 mm thick and 7 mm in diameter with contoured irrigation and a system that allows the lens capsule to be closed during the operation by creating negative pressure, adhering it tightly to the anterior capsule (69). Th device enables safe delivery and use of pharmacological agents (70) during cataract surgery while isolating the inner space of the capsule from the surrounding tissues. The system allows for an experienced surgeon to safely rinse the intracapsular space after a sufficiently long application of a selected chemical preparation..
Distilled water is a hypotonic solution that, through osmosis, can cause degeneration of hydrops, detachment of LECs from the basement membrane, nuclear disintegration, and cell lysis (71). Crowston et al. suggested that use of distilled water leads to rapid lysis of LECs in vitro without damaging the lens capsule, and can therefore be used in airtight irrigation systems during cataract surgery (71). Using this technique in a rabbit model, they removed the lens by phacoemulsification and then irrigated the inner space with Triton X-100 and distilled water in group II without sealed-capsule irrigation (SCI) and in group III with SCI. Group I was the control group without irrigation (72). After histological examination of the corneal endothelium, Descemet's membrane, iris, and retina, they found that in groups I and III, there were no statistically significant deviations from the normal condition within the groups or between groups. However, in group II, they found extensive loss of corneal endothelial cells with remnants of scattered nuclei on Descemet's membrane and lesions within the iris pigment layer. The lens capsule was wrinkled and contained both damaged and undamaged LECs with extensive cell lysis in the retina.
To exclude the influence of the IOL on formation of PCO, the eyes were left aphakic and the anterior capsule was closed with a miniature capsulorhexis valve manufactured specifically for their study. The observation period was 5 weeks (or less if advanced PCO was observed). All study groups developed PCO, but at different rates. PCO occurred first in the SHA group after 1 day. PCO also occurred in the eyes treated with EDTA, mitomycin C, acetic acid 0.3% or 3%, distilled water (in younger animals), BSS, acetic acid 0.003%, 5-FU 10 mM, EDTA, and distilled water (in older animals) after 47 days. Other postoperative complications included corneal edema at the port site in the acetic acid group at both concentrations and appearance of inflammatory cells in the aqueous humor in the EDTA group. Eyes exposed to distilled water showed the most rapid regeneration and had the least severe inflammatory reaction after surgery. The results of this study did not confirm previous reports of the ability of EDTA to inhibit formation of PCO (14). In the EDTA group, appearance of PCO was somewhat delayed; however, by day 28, it was the same as that in the control group. 5-FU was not as effective as in other research that showed its effectiveness to be 5 times greater than that in a control group (74). These reports and the results of other studies of the effectiveness of distilled water in inhibiting the formation of PCO led to new studies in a human model.

Controlled cell apoptosis
The problem of cataract and secondary clouding of the posterior capsule post-removal also occurs in dogs, reaching a frequency of 100% (75). The pathomechanism is the same as that in humans. In a study by Pot et al. (75), selenium was applied to dog's lens capsules after euthanasia. Selenium is a catalyst for formation of oxygen free radicals, which cause apoptosis. After reduction, the selenium anion enters a thiol oxidation reaction, the products of which are superoxide (O2 -) radicals and hydrogen peroxide. Cells that die by apoptosis are removed by macrophages. The danger of widespread apoptosis is eliminated by the short decay time of the particles (<50 ms) and their small range of spread (<40 µm) (76). The lens cells were cultured in vitro and divided into three groups. Group I cells were placed on a selenocystamine-coated IOL, group II cells were placed on an IOL without a coating, and group III cells were not placed on either type of IOL. The cells were observed and photographed for 10 days. In group I, the first symptoms of PCO appeared only on day 6 (on day 1 in the other groups) and were less severe. A cytotoxic effect was observed only at points where there was contact between the cells and the IOL.

Steroidal and non-steroidal anti-inflammatory drugs
A study assessed the effect of steroidal and non-steroidal anti-inflammatory drugs on formation of PCO in lenses explanted from rats (77). The potential of these agents to prevent PCO derives from their ability to affect production of TGF-β and bFGF. Research has shown that these proteins play a key role in formation of PCO (28,78). TGF-β and bFGF inhibit production of prostaglandins through different metabolic pathways. Nonsteroidal anti-inflammatory drugs directly inhibit release of the cyclooxygenase (COX) 1 and COX 2 isoforms. The action of steroids is more complex and involves inhibition of phospholipase A2, a key enzyme in the synthesis of arachidonic acid, which is a precursor to prostaglandins and leukotrienes. Moreover, they participate in regulation of COX expression. Both groups of drugs are commonly used to prevent inflammation following cataract surgery.
In a study by Symonds et al., lenses from Wistar rats were cultured with addition of TGF-β and bFGF to recreate the conditions under which PCO is formed (77). The explanted lenses were then treated with dexamethasone and diclofenac. Both agents inhibited formation of PCO by preventing the spread of abnormal cells along the lens capsule and accumulation of type I collagen in the ECM, with a progressive decrease in the number of abnormal cells. However, the smaller size of the rodent eye requires use of a different cataract removal technique. The inability to perform accurate continuous capsulorhexis or implant an artificial lens are serious limitations. There are also differences in the metabolic pathways involved (77,78).
Zukin et al. studied aldose reductase activity inhibition to attenuate induction of EMT markers in an in vivo model of cataract surgery (79). Modified extracapsular lens extraction was performed in C57BL/6 mice and in mice with increased or decreased expression of aldose reductase. Immunofluorescence staining was used to assess the presence of markers of PCO postoperatively. Quantitative changes in expression of vimentin, fibronectin, E-cadherin, and α-SMA after surgery were evaluated by RT-PCR. Sorbinil, an aldose reductase inhibitor, was administered postoperatively. Changes in the number of EMT markers were assessed by RT-PCR. Inhibition of aldose reductase reduced the postoperative changes characteristic of EMT and protected against induction of lens fibrin cell markers.

Clinical trials
Koopmans et al. demonstrated that the differences in metabolic pathways between species was the reason that pharmacological agents that have a positive effect in preventing PCO in animals are effective in humans. Therefore, the effectiveness of an individual substance must be evaluated in clinical trials (62). Rękas et al. (80) assessed the ability of distilled water to inhibit formation of PCO. Using a procedure previously described by Maloof et al. (69), they examined 59 patients who underwent phacoemulsification, thorough polishing of the posterior capsule, and implantation of an IOL. Anticipating that it would not be possible to completely remove the LECs mechanically, additional irrigation of the inner surface of the capsule with distilled water suing the tight Perfect Capsule system was performed for 3 min in a second group of 29 eyes. A period of 3 min was used based on a preliminary finding that application of distilled water for 3 minutes causes cytolysis of cells with a probability of 70.8%, (i.e., 3.51 times greater than would be achieved in 1 min). The purpose of using distilled water was to remove and/or significantly damage as many LECs as possible on the inner surface of the lens capsule to limit their proliferation and migration, thereby preventing secondary cataract formation. Irrigation allowed clean, transparent pouches to be obtained from the lens without the residues of lens masses and LECs visible under the operating microscope.
In this study, use of a liquid formulation made it possible to safely reach every part of the capsule. Application of distilled water (a hypo-osmotic fluid) damaged the LECs by osmosis, leading to complete cytolysis or significant cell damage (72). The few residual LECs were damaged and had low mitotic, proliferative, and migratory capacity, which reduced the risk of developing PCO. Twenty-four months after the procedure, PCO was less advanced in the eyes treated with distilled water and occurred in a smaller area of the capsule than in the eyes in which only mechanical cleaning of the capsule was performed. The safety profile in both groups was the same. There was no statistically significant between-group differences in the rate of complications such as corneal edema, Descemet's membrane folds, corneal epithelial erosion, or postoperative astigmatism. Endothelial cell loss was slightly higher in the distilled water group; however, this finding could not be attributed to the use of distilled water alone and was more likely due to mechanical manipulation during insertion and removal of the Perfect Capsule system. Unlike in the study by Crowston et al. (71), use of distilled water in vitro did not produce signs of damage to the lens capsule on histological examination. Therefore, it seems that this substance can be used in SCI systems during cataract surgery. The disadvantage of its use in SCI is the extended operating time, additional manipulations in the anterior chamber during implantation and explantation of the Perfect Capsule system, as well as the cost of purchasing the system (2). D'Antin et al. compared the effect of hydrogen peroxide and distilled water on the progression of PCO (81). Human lens capsules were divided into three groups-one treated with hydrogen peroxide, another with distilled water, and a control group. The lens capsules in the active treatment groups were rinsed for 5 min in 30 mM hydrogen peroxide solution or distilled water. Samples were then grown for a month, during which time microscopic images were obtained. Specimens were subjected to overall histological evaluation with hematoxylin-eosin staining and immunohistochemical evaluation of α-SMA, Ki-67, and vimentin. The authors observed growth retardation in the groups treated with hydrogen peroxide and distilled water in comparison with the control group, with no significant differences in growth between the two substances.
Joshi and Hussain studied the effect of irrigation of the lens capsule with trypan blue (0.06%) on PCO in 100 phacoemulsified eyes from 50 patients with senile cataracts (54). One eye in each patient was randomized to one of two groups. The study group received a 0.2-mL injection of trypan blue into the lens capsule after cleaning of the cortical layer. The control group (the second eye of the same patient) received a 0.2-mL injection of BSS administered in a similar manner to the trypan blue. PCO was assessed in the central zone of the implanted lens using customized computer software at 6, 12, 24, and 36 months postoperatively. At 6 months, there was a reduction in the degree of PCO in the trypan blue group. However, no treatment effect was observed at 36 months.
Rajesh et al. assessed the effect of rotation of the implanted lens/IOL by 360° during cataract phacoemulsification (82). After placing/inserting the lens/IOL in the capsule, the lens was rotated 90° using a Sinskey hook inserted through the corneal incision. The anterior chamber was filled with viscoelastic. Another Sinskey hook was then inserted through the side port, and the lens was rotated 180°. A third hook was introduced through the port on the opposite side, and the IOL was rotated 90°. The effect was analyzed after 6, 12, 24 and 36 months in the group with IOL rotation and compared with a control group in which no rotation was performed. Postoperative follow-up was performed by slit-lamp examination in mydriasis. Images were assessed with the aid of a computer program to examine the density of PCO. The median PCO scores at 6, 12, and 24 months were significantly lower in the rotation group than in the control group. There was no significant difference between the two groups during observation months 24-36. The authors concluded that rotation of the IOL in the capsule is a simple and safe method for preventing PCO and requires no additional tools or operator skills.

Local administration of steroid and non-steroidal anti-inflammatory drugs after surgery
Use of anti-inflammatory drugs for prevention of PCO is also under investigation. These agents inhibit production of prostaglandin E2, which plays a role in formation of PCO.
In vitro studies have confirmed that diclofenac suppresses proliferation and metaplasia of LEC fibers (83) and inhibits synthesis of prostaglandin E2 and collagen (84). However, the data from in vivo studies are inconclusive.
Seki et al. showed that administration of diclofenac three times daily for a year inhibited formation of PCO (85). In contrast, a prospective randomized, double-blind study by Neumayer et al. that investigated the effect of a topical combination of diclofenac and prednisolone on formation of Elschnig's pearls found no significant difference between the active and placebo groups (86). Moreover, other long-term studies of diclofenac and dexamethasone did not confirm a benefit during 2-3 years of observation (87)(88)(89). Similar results were obtained in a study comparing diclofenac with betamethasone (90); however, the duration of administration was shorter (a maximum of 3 months) in that study. Another retrospective study in 120 patients compared the occurrence of PCO 3 years after treatment with diclofenac 0.1% or ketorolac tromethamine 0.5% and found no significant between-group difference in frequency of PCO (12% in both groups). However, when compared to occurrence rate in the general population, this is a favorable result (91).
Tranilast (N-[3',4'-dimethoxycinnamoyl]-anthranilic acid) inhibits the collagen synthesis that occurs because of proliferation of fibroblasts from hypertrophic scars or keloids without affecting the healing processes in normal, healthy skin. This oral antiallergic agent reduces vascular permeability and inhibits the release of chemokines, such as TNF-β, from mast cells and other inflammatory cells. Tranilast drops are widely used in Japan for allergic conjunctivitis and have few known side effects (92).
Miyazawa et al. hypothesized that the inflammatory process would favor penetration of tranilast into damaged tissues and found high concentrations of the drug in the aqueous humor up to 30 min after intraocular administration in rabbits that had undergone phacoemulsification 4 weeks earlier (93).
A prospective, randomized, double-blind study by Tobari et al. (92) evaluated the ability of tranilast drops to prevent PCO in humans when administered into the conjunctival sac. Forty-four eyes that had undergone cataract phacoemulsification were randomized postoperatively to administration of tranilast 0.5% or placebo (vehicle only) four times a day for 3 months. Standard postoperative ofloxacin and dexamethasone therapy was also provided for 1 week after surgery. Patients were examined and photographic documentation was obtained 1 week, 1 month, and 3 months after the procedure. The intensity of the PCO was determined by the degree of light scattering at 10 points in the posterior capsule, and the result was calculated by the algorithm in digital form. After 3 months of observation, the PCO level was significantly higher in the placebo group.
However, the trial design had no effect on corneal wound healing. Most above-mentioned studies are summarized in Table 1.

Discussion
Efforts to prevent PCO are ongoing. However, the pharmacological agents used intraoperatively should be effective and safe. The outstanding issue is the toxicity of substances used to treat LECs and their affect on other structures and tissues of the eye, in particular the corneal endothelial cells. Several pharmacological agents have been found to damage LECs in both animals and humans. The type and extent of damage varies according to type of pharmacological preparation and duration of exposure. The more cytotoxic the agent, the more rapid and greater the damage to LECs. Therefore, despite the promising results of in vitro studies, the use of chemicals (namely 5-FU and mitomycin C) that damage LECs by inhibiting their proliferation is limited. Non-steroidal anti-inflammatory drugs are safer because they have the potential to reduce PCO, especially when used in sustained-release systems, at lower concentrations and have a longer duration of action. Their main mechanism of action is toxic damage to LECs. Furthermore, in vitro experiments using substances that inhibit TGF-β have yielded promising results. However, at this stage, disruption of metabolic pathways is certain to affect other tissues as well, and the in vivo consequences are difficult to predict. Compounds affecting LECs by means of osmotic pressure, such as 23.4% sodium chloride and distilled water, offer greater possibilities for use in clinical practice. The use of a technique that enables easy, rapid, and safe delivery to the inside of the lens capsule and removal of toxic substances, allowing for selective damage to LECs, may be the best way to overcome the present difficulties in preventing PCO.

Conclusion
Despite many efforts, an effective, economical, and optimal pharmacological method for prevention of PCO that is accessible and could be used widely in patients is yet to be developed. The application of hydrogels, which have attractive physical properties (i.e., porosity), may be an option for use in DDSs for sustained release of the loaded drug while reducing its toxic and side effects on endothelial cells. Further research is needed to