Time Course of Lens Epithelial Cell Behavior in Rabbit Eyes following Lens Extraction and Implantation of Intraocular Lens

Background After cataract surgery, some lens epithelial cells (LECs) transdifferentiate into myofibroblast-like cells, which causes fibric posterior capsule opacification (PCO). Residual LECs differentiate into lens fiber cells, forming Elschnig pearls with PCO. This study was carried out to identify the time course of both types of LEC behavior in rabbit eyes following lens extraction and implantation of an intraocular lens (IOL). Methods Phacoemulsification and implantation of posterior chamber IOLs were performed in rabbit eyes. Following enucleation, immunohistochemical methods were used to detect α-smooth muscle actin (α-SMA), a marker for myofibroblast-like cells, in the pseudophakic rabbit eyes. A mouse monoclonal antibody against α-SMA was used. Results Soon after the operation, the LECs migrated and covered the lens capsule. Thereafter, the LECs around the anterior capsular margin were always positive for α-SMA. However, the distributions of these cells were not consistent. In some specimens, α-SMA-positive LECs were present around the IOL optic early after surgery, but most of them had disappeared several weeks after the surgery. The residual cells induced fibrotic PCO. In the other specimens, most LECs around the IOL optic except the anterior capsular margin were negative for α-SMA. In the peripheral region covered by the peripheral anterior and posterior capsules, LECs on the posterior capsule always differentiated into lens fiber cells and formed a Soemmering ring. Thereafter, migration of lens fiber cells from the Soemmering ring and differentiation of LECs in situ on the central posterior capsule consisted of Elschnig pearls type of PCO. Conclusions Although postoperative LEC behavior is not consistent, residual α-SMA-positive LECs induced fibrotic PCO. The lens fiber cells that migrated from the peripheral capsular bag or that were differentiated in situ covered the central posterior capsule, forming Elschnig pearls with PCO.

To determine the precise time course of the LEC behavior after cataract surgery, it is important to examine how the two types of cells (α-SMA positive or negative cells) appear and cause PCO immediately after surgery until the development of PCO. However, it is difficult to obtain human donor capsular bags with implanted IOLs, especially early after cataract surgery. Although there is a possibility that an immunohistochemical study using a pseudophakic animal model can reveal the necessary information, studies have only been performed on specimens obtained 2 weeks after surgery [18]. To determine the precise time course of the LEC behavior after cataract surgery with IOL implantation, we conducted an immunohistochemical study in which we monitored the presence of α-SMA in rabbit eyes implanted with IOLs immediately after surgery until the development of PCO.

Methods
A total of 34 young female Japanese albino rabbits (1.0-1.5 kg) were studied. Each of the following agents was applied topically three times on the day of surgery to one eye of each rabbit: diclofenac sodium, tropicamide, phenylephrine hydrochloride, and nofloxacin. e animals were anesthetized with an intravenous administration of sodium pentobarbital (40 mg/kg) and atropine sulfate (0.02 mg/kg). A superior corneal incision was made with a 3 mm keratome. Continuous curvilinear capsulorhexis of the anterior capsule was then carried out. e lens nucleus was emulsified, and the residual cortex was removed with a phacoemulsifier. e corneal incision was enlarged, and an ophthalmic viscosurgical device (sodium hyaluronate, Johnson and Johnson Vision Care, Inc., New Brunswick, NJ) was injected into the anterior chamber and the lens capsular bag. An IOL (6 mm IOL with 3-piece haptics, Bausch and Lomb, Rochester, NY) was implanted in the bag. After removal of the viscoelastic material, the corneal incision was closed with a continuous 10-0 nylon suture. ese procedures adhered to the guidelines of the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research. e rabbits were humanely killed with an overdose of sodium pentobarbital at 1 d, 3 d, 5 d, 7 d, and 10 d and at 2 wks and 1 mo after the operation. At least two rabbits were studied at each time specified. e eyes were enucleated and immersed in 10% neutral buffered formalin. After fixation, the globe was sectioned at the equator. Specimens were dehydrated through a graded series of alcohols and embedded in paraffin. Tissue sections were then cut from these specimens.
We used a Histostain-SP kit (Invitrogen, Camarillo, CA, USA). e labeled streptavidin-biotin method was used for the immunohistochemical detection of α-SMA as described previously [24]. e primary antibody was a mouse monoclonal antibody directed against α-SMA (IgG2a, clone 1A4, code no. M851, Dakopatts, Denmark). Peroxidase activity of the secondary antibody was visualized by the addition of a solution containing 3-3'-diaminobenzidine hydrochloride (0.3 mg/ml), 0.005% hydrogen peroxide, and 50 mM TRIS-HCl buffer. e sections were then counterstained with hematoxylin. Mouse monoclonal IgG2a antibody (clone Dak-G05, code no. X943, Dakopatts) was used as a negative control. No immunoreaction was detected in the negative control. e sphincter and dilator muscles of the iris were used as internal positive controls [27].

Results
On postoperative day 1, the capsulotomy margin of the anterior capsule was attached to the IOL optic. LECs on the preequatorial anterior capsule became flattened and migrated toward the posterior capsule via the lens equator.
ese LECs showed negative staining for α-SMA (data not shown).
By postoperative day 3, a monolayer of LECs covered the inner surface of the central posterior capsule (Figure 1). ese LECs were also negative for α-SMA. On postoperative day 5, the flattened and multilayered LECs were positive for α-SMA. LECs around the capsulotomy margin of the anterior capsule were always α-SMApositive (Figures 2(a), 2(c), 2(d), 2(f )). On the other hand, in the peripheral space without IOL optics, the inner surfaces of the peripheral anterior and posterior capsules were always covered by a monolayer of LECs. ese peripheral capsules were attached to each other like a zipper, with the associated LECs staining negative for α-SMA (Figures 2(a) and 2(c)).
However, the specimens could be divided into two groups according to the distribution pattern of the α-SMApositive LECs around the IOL optics, which is consistent with a previous study by Matsushima et al. [18]. In the first group, the IOL optic was surrounded by α-SMA-positive LECs ("surrounding type"; Figures 2(a) and 2(b)). In the On postoperative day 10, the LECs around the capsulotomy margin of the anterior capsule were always α-SMApositive as were the specimens on postoperative day 5. In the peripheral space without IOL optics, α-SMA-negative LECs on the inner surfaces of the posterior capsules began to differentiate into lens fiber cells (Figures 3(a), 3(c), and 3(d)) to form Soemmering ring (Figure 3(a)). e distribution of α-SMA-positive LECs around the anterior capsular margin did not change in the restricted type but did in the surrounding type: these LECs were absent on some of the anterior and posterior capsules surrounding the IOL optics (Figures 3(a) and 3(b)).
On postoperative day 14, in the surrounding type of distribution ( Figure 4(a)), the separation between the IOL optic and Soemmering ring was lost. e size of the Soemmering ring increased, and some lens fiber cells within the Soemmering ring began to attach to the IOL optic. e cell-free area on the anterior and posterior capsules spread. e area on the posterior capsule attached to the α-SMApositive LECs decreased. Some posterior capsules were wrinkled without LECs.
In the restricted type of distribution pattern (Figures 4(b) and 4(c)), the differentiation of LECs into lens fiber cells under the IOL optic progressed to form the Elschnig pearls type of opacification.
One month after surgery, it became difficult to divide the specimens according to the distribution pattern of α-SMA-positive LECs because these LECs existed only in limited areas, such as the anterior capsular margin and some parts of the posterior capsule. Some areas of the anterior and posterior capsules surrounding the IOL optic without LECs, which had been observed on postoperative day 14, decreased. e LECs that covered the inner surface of the anterior capsule exhibited a normal cuboidal appearance. e peripheral capsular bag was filled with lens fiber cells and had formed a Soemmering ring. Lens fiber cells were present at the posterior capsule under the IOL optic and had formed Elschnig pearls type of opacification. Lens fiber cells were occasionally also present between the anterior surface of the IOL optic and the anterior capsule. Some of these lens fiber cells migrated beyond the anterior capsular margin and formed posterior synechia to the iris (Figures 5(a) and 5(b)). ese LECs and lens fiber cells were negative for α-SMA. When the central posterior capsule was completely covered by the lens fiber cells, i.e., the Elschnig pearls type of opacification, we could not ascertain whether these lens fiber cells resulted from the differentiation of the LECs on the posterior capsule in situ or whether they had migrated from the peripheral portion of the lens capsular bag, namely Soemmering ring. However, in some specimens, the lens fiber cells were over residual α-SMA-positive LECs, which formed fibrotic PCO accompanied by wrinkling of the posterior capsule ( Figure 5(c)). is observation implies that the LECs differentiated into lens fiber cells at the peripheral capsular bag and that these fiber cells migrated to the central posterior capsule and formed the Elschnig pearls type of opacification. e time course of the behavior of α-SMA-positive and negative LECs is summarized in Table 1.

Discussion
e present study demonstrated that undifferentiated LECs migrated and covered the lens capsule soon after IOL implantation. Some LECs subsequently differentiated into α-SMA-positive LECs by the fifth postoperative day. LECs at the anterior capsular margin were always positive for α-SMA, but those around the IOL optics were occasionally positive and they decreased within 2 weeks of the operation. On the other hand, in the peripheral space without IOL optics, the inner surface of the peripheral anterior and posterior capsules was always covered by a monolayer of LECs. ose on the peripheral posterior capsule differentiated into lens fiber cells and formed a Soemmering ring. On the other hand, the Elschnig pearls type of opacification on the central posterior capsule consisted of lens fiber cells migrating from the Soemmering ring and/or the differentiation of LECs in situ.
In this study, α-SMA-positive LECs appeared 5 days after surgery. e specimens from 5 days to 2 weeks after surgery were divided into two groups according to the LEC distribution. However, since the number of these LECs then decreased, it became difficult to group the specimens one month after surgery. Matsushima et al. [18] also reported that specimens of rabbit pseudophakic eyes 2 weeks after cataract surgery could be divided into two groups depending on whether the α-SMA-positive LECs separated the IOL optic edge from the peripheral lens fiber cells.
ese findings were consistent with our results. Moreover, they reported that the two groups were almost evenly distributed. Uusitalo and Kivelä [17] reported the distributions of the α-SMA-positive LECs were not unique to human pseudophakic eyes obtained at autopsy. Ness et al. [9] also found two types of histopathologic sections of human cadaver eyes. One had lens fiber cells within the Soemmering ring that were separated from the IOL optics by flattened LECs, the same as our "surrounding type," and the other showed lens fiber cells attached directly to the IOL optics, which resembles our "restricted type." However, the period from surgery to enucleation was unknown. We found it became difficult to divide the specimens according to the distribution pattern of α-SMA-positive LECs one month after surgery because these LECs existed only in limited areas, such as the anterior capsular margin in both types. erefore, there is a possibility that the latter showed changes obtained late after surgery.
It is unclear how the distribution of α-SMA-positive LECs becomes divided into two groups. Matsushima et al. [18] reported that IOL materials did not affect it. In aphakia eyes after cataract surgery, α-SMA-positive LECs were also present around the anterior capsular margin [24]. Tan et al. [28] reported that the presence of α-SMA-positive LECs was dependent on the size of the anterior capsulorhexis: α-SMA-positive LECs were not observed with a 2.0 mm diameter anterior capsulorhexis but were observed with a 4.0 or 6.0 mm diameter. ere is a possibility that the size of the anterior capsulorhexis might affect the distribution of α-SMA-positive LECs.
On the other hand, the LECs around the anterior capsular margin were always positive for α-SMA after the fifth postoperative day. In human cadaver eyes, the LECs around the anterior capsular margin were always positive for α-SMA [11,12]. Transforming growth factor-β (TGF-β), which is present in the aqueous and vitreous humor [29][30][31][32], induces the myodifferentiation of LECs in vitro and in vivo [2][3][4][33][34][35]. An anterior capsular margin is a place that comes into direct contact with the aqueous humor. TGF-β in the aqueous humor may influence the myodifferentiation of LECs around the anterior capsular margin.
In this study, most α-SMA-positive LECs around IOL optics except for the anterior capsular margin were diminished, which formed a cell-free area on the capsule around the IOL. In an immunohistochemical examination using a whole mount of a human donor capsular bag implanted with an IOL, the central posterior capsules were largely free of LECs, and in the other area, there were α-SMA-positive LECs that detached from the wrinkled areas around the optic edge [12] and showed cell degeneration [11]. In anterior capsular opacification around the IOL optic of monkey eyes, flattened LECs were initially abundantly observed, but degeneration and cellular debris were observed 2 months after the operation [21]. e migration of LECs in humans starts within days after cataract surgery, with the disappearance of LECs occurring between 30 and 90 days postoperatively in 87% of patients with implantation of polyacrylic IOLs, 8% with silicone IOLs, and 15% with PMMA IOLs [36]. ese findings suggest that the cell-free area appeared due to cell regression after the LECs had covered it. We previously reported that the disappearance of myofibroblast-like LECs occurred at the margin of the anterior capsule in aphakic rabbit eyes due in part to apoptosis [37]. In human eyes, LECs undergo apoptosis in the early phase after cataract surgery [22].
Although further studies are required, there is a possibility that the formation of a cell-free area is related to cell death.
In this study, in the peripheral space without IOL optics, the inner surfaces of the peripheral anterior and posterior capsules were covered by a monolayer of LECs 5 days after cataract surgery. en, LECs on the posterior capsule differentiated into lens fiber cells and formed a Soemmering ring as previously reported [6,12]. After that, we observed  differentiated lens fibers not only between the IOL optic and the anterior capsule but also over the residual α-SMApositive LECs, which means that differentiated lens fibers can migrate onto the central posterior capsule from the peripheral capsular bag. ey induced the formation of the Elschnig pearls type of PCO. Even if the Soemmering ring had been separated from the central posterior capsule by α-SMA-positive LECs, this separation was broken by the increase in the size of the Soemmering ring. ese findings are consistent with previous studies with human pseudophakic eyes. Volk et al. [38] showed reopening of the anterior and posterior capsular fusion at the IOL optic edge when lens fiber cells from the Soemmering ring pushed out, and they migrated on the IOL optic under the anterior capsule but also on the posterior capsule behind the IOL optic, as shown by optical coherence tomography. On the other hand, it was previously reported that the Elschnig pearls type of PCO is produced by the differentiation of LECs into lens fibers on the posterior capsule in situ [23,39]. Such differentiation occurred in the present study in the case of the restricted type. erefore, the formation of the Elschnig pearls type of PCO, which indicates the presence of lens fibers between the posterior capsule and the IOL optic, may come from not only the differentiation of LECs in situ but also migration from the peripheral capsular bag, namely the Soemmering ring.

Conclusions
e present study showed that the behaviors of the α-SMApositive LECs were not consistent, and some of them disappeared, forming cell-free areas on the posterior capsule. On the other hand, α-SMA-negative LECs formed the Elschnig pearls type of PCO by both differentiation on the central posterior capsule under the IOL optic and also by migration from the Soemmering ring. Although further investigations are required to clarify the mechanism of these phenomena, these findings may lead to the prevention of PCO.

Data Availability
All relevant data are included within the article and are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.