Aldose reductase inhibition enhances lens regeneration in mice

Abstract

After cataract surgery, epithelial cells lining the anterior lens capsule can transition to one of two divergent pathways, including fibrosis which leads to posterior capsular opacification (PCO), or lens fiber cell differentiation which leads to regeneration of lens material. We previously showed that the PCO response can be suppressed with aldose reductase (AR) inhibitors. In this present study we show that AR inhibition, both genetic and pharmacologic with Sorbinil, can augment the process of lens regeneration. Extracapsular lens extraction (ECLE) was carried out in C57BL/6 (WT), AR overexpression (AR-Tg), and AR knockout (ARKO) mice, and in some cases in mice treated with the AR inhibitor sorbinil. Whole eyes were harvested approximately 8 weeks after ECLE and evaluated by histological analysis and immunostaining for the fiber cell marker γ-crystallin. All eyes examined for lens regeneration were paraffin embedded for serial sectioning to produce three-dimensional reconstructed models of lens morphology and size. We observed that AR-null mice respond to ECLE by regenerating a lens-like structure with a circular shape and array of cell nuclei reminiscent of the lens bow region typical of the native mammalian lens. Although WT and AR-Tg eyes also produced some regenerated lens material after ECLE, their structures were consistently smaller than ARKO regenerated lenses. WT mice treated with sorbinil showed higher levels of lens regeneration after ECLE compared to WT mice, as assessed by size and three-dimensional morphology. Altogether, this study adds evidence for a critical role for AR in the response of lens epithelial cells to cataract extraction and lens regeneration.

1. Introduction

The leading cause of visual impairment worldwide is cataract, the age-related clouding of the ocular lens, accounting for 10.8 million cases of blindness and an additional 35.1 millions cases of visual impairment.¹ Correspondingly, cataract removal is among the most common surgeries in the world. During cataract surgery, the opaque lens is removed from its surrounding collagenous capsule and replaced with a clear, artificial intraocular lens. The most common postoperative complication of this procedure is posterior capsular opacification (PCO), occurring in up to 20–30% of cases.² This process is thought to occur due to surgical trauma causing a proliferation of residual anterior lens epithelial cells (LECs) that are inevitably left within the lens capsule after surgery. PCO has two distinct histologic phenotypes: fibrotic and pearl-type PCO.³ Fibrotic PCO is caused by an epithelial-to-mesenchymal transition (EMT) of LECs which obstruct the visual axis by migrating along the posterior capsule, taking on a myofibroblastic phenotype, and causing wrinkling and fibrosis of the otherwise smooth capsule.⁴ The central mediator of the EMT process is transforming growth factor-β (TGF-β),⁴ which in turn modulates SMAD protein signaling,^5–7 eventually leading to formation of spindle-like myofibroblast cells⁸ and expression of EMT markers like α-smooth muscle actin (α-SMA), fibronectin, and vimentin.^9–13 Pearl-type PCO, on the other hand, is characterized by LEC differentiation into fiber-like cells containing high levels of crystallin proteins, which normally account for about 90% of the contents of the native human lens.¹⁴ Interestingly, when the anterior LECs are preserved following lens extraction, lens fiber mass regeneration has been demonstrated in numerous species, including humans.¹⁵ Of note, both PCO subtypes can coexist in the same capsule and can both be triggered simultaneously in response to cataract surgery.¹⁶

A growing body of evidence suggests that the enzyme aldose reductase (AR) plays an important role in PCO. AR is well known as a central regulator of the polyol pathway, through which it can increase the production of reactive oxygen species (ROS)¹⁷ and in turn cause LEC growth and a fibrotic PCO-like phenotype.¹⁸ Various experimental models have demonstrated that this PCO phenotype can be prevented with antioxidants^19–21 and that AR inhibition can decrease ROS.^22–24 We have previously linked AR and PCO by showing that AR inhibition suppresses TGF-β mediated expression of fibrotic PCO biomarkers in cell culture.²³ Recently, we demonstrated similar inhibition of EMT biomarkers and the histologic manifestations of fibrotic PCO in an in vivo surgical model.²⁵ Taken together, these studies support the notion that AR inhibition can suppress the fibrotic PCO response after lens extraction.

Lens fiber cell differentiation leading to pearl-type PCO is the second possible fate of LECs following lens extraction.¹⁶ Similarly, lens regeneration is another possible long-term outcome after lens extraction.¹⁵ While a definitive causal link between early lens fiber differentiation and later lens regeneration has not been shown in mammals, these studies raise the question of what factors influence the LEC fate toward EMT and fibrotic PCO versus lens fiber differentiation or lens regeneration. As noted above, while AR inhibition suppresses EMT markers after lens extraction, we recently found that AR inhibition does not suppress the early postoperative expression of the lens fiber markers αA-crystallin and aquaporin‒0.²⁵ As an extension of this observation, in this present study we demonstrate that AR inhibition can augment lens regeneration in a surgical model in mice.

2. Methods and Materials

2.1. Transgenic Mice

All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Colorado Institutional Animal Care and Use Committee. C57BL/6 wild-type (WT) mice were acquired from The Jackson Laboratory (Bar Harbor, ME, USA). Both the AR knockout strain (ARKO) and the AR overexpression strain (Par40 strain of AR transgenic mice, AR-Tg) were produced as previously described.^26–28

2.2. Lens Extraction Surgical Model

Lens extraction was performed in mice using a modified extracapsular lens extraction technique (ECLE) based on previously described methods.25,29–31 The anterior LEC have been shown to be critical to lens regeneration, and their preservation surgically is important in optimizing regeneration.¹⁵ To this end, corneal and capsular incisions were made peripherally in a semicircular manner as described below. Adult mice were anesthetized with 80 mg/kg ketamine and 5 mg/kg xylazine. One eye of each mouse was dilated using several drops of topical phenylephrine and tropicamide. A peripheral, semicircular corneal incision was made using a disposable ophthalmic knife. The incision extended approximately 120–150 degrees with a radius of approximately 0.5 mm from the center of the cornea. Following reinflation of the anterior chamber with an ophthalmic viscoelastic agent, an incision of similar size and shape was made in the anterior capsule. A viscoelastic cannula was used to instill physiological saline solution into the capsular space to hydro-dissect the lens fiber mass away from the capsule. To further preserve anterior LEC integrity, additional saline was instilled into the capsule, posterior to the lens, facilitating gentle extrusion of an intact lens mass from the capsule. This was followed by careful irrigation of the capsule to remove any residual lens material, particularly lens cortex, until a clear view to the posterior segment of the eye was achieved. A viscoelastic agent was then injected into the capsule and anterior chamber to re-inflate the eye and maintain its structural integrity postoperatively. The corneal incision was closed using 11–0 nylon sutures. At about 2 months postoperatively, animals were euthanized and whole eyes were removed, with the un-operated contralateral eye serving as an experimental control. The eyes were then processed for histology as described below. Surgery, euthanasia, dissection, and processing were performed in parallel for each experiment to ensure consistency and comparability. Furthermore, the surgeon was masked to the genetic strain of each mouse during operations. For experiments involving sorbinil ([4S]-6-Fluoro-2,3-dihydro-spiro[4H-1-benzopyran-4,40-imidazolidine]-20,50-dione), WT mice were treated from birth via their drinking water, at a concentration of 25mg/mL sorbinil was provided by Pfizer Central Research (Groton, CT, USA).

2.3. Immunohistochemistry

After dissection whole eyes were embedded in paraffin. Each tissue block was then sectioned at a thickness of 6 μm. Every section was examined with a light microscope until it contained either capsule or regenerated lens, which was then mounted on a glass slide as the “first” section. Then every fifth section was slide-mounted until tissue sections no longer contained capsule or regenerated lens (the “last” section). All slides were then processed for 3-dimensional analysis (described below). For confirmation of fiber cell characteristics, sections from the approximate middle of each eye (ARKO, ARTg, WT, WT with sorbinil) were stained for γ-crystallin. Sections were first processed by antigen retrieval and blocking of endogenous peroxidase activity. Sections were then incubated with a 1:200 dilution γ-crystallin in OP Quanto Diluent (Thermo Fisher Scientific, Waltham, MA, USA) in phosphate buffered saline (PBS) for 1 hour. After three washes with PBS, the sections were incubated with biotinylated secondary antibody (Vector, Burlingame, CA, USA) for 1 hour, then HRP conjugated streptavidin (Vector, Burlingame, CA, USA) for 30 minutes and DAB (Vector, Burlingame, CA, USA) for 10 minutes. Images were acquired immediately after processing.

2.4. 3-Dimensional Reconstruction from Serial Sections

General workflow for our 3-dimensional reconstruction data is outlined in Figure 1. After serial sectioning and placement of sections onto glass slides, photographs were captured via a dissecting microscope of each section. Consistent magnification and focusing settings were used throughout the imaging and were noted for later conversions between pixels and length units. After this digitization step, images were aligned via the SIFT algorithm plugin within FIJI image analysis software (http://fiji.sc [in the public domain]; an open source image processing application based on the National Institutes of Health software ImageJ, http://imagej.nih.gov/ij/ [in the public domain]). This alignment algorithm has been employed in three-dimensional reconstruction applications of serial histological sections in other tissues.32 The aligned images were then stacked within the imaging software, and a mask was drawn manually around the regenerated lens material within each image of each section. Then appropriate spacing parameters were entered (6 μm x 5 sections = 30 μm between each image) and the stack was converted to a three—dimensional image using the 3D Viewer plugin within FIJI. Area calculations were obtained from each section using standard pixel measuring tools within the application, and then converted to μm based on microscope parameters. The area of each masked section (representing only the regenerated lens material) was then multiplied by the distance between each section (30 μm). The sum of volumes was used as an estimate for the total regenerated lens volume. Because unoperated control lenses were of regular and symmetrical shape, they were not processed with the same serial sectioning method. Lenses were dissected from the whole eyes after paraffin processing and photographed in a similar fashion under dissecting microscope (see Figure 1). Lenses were assumed to have an ellipsoid shape, so all three lens radii were measured in FIJI as described above, converted to μm from pixels, and volumes were calculated using the ellipsoid volume formula $\frac{4}{3}\pi abc$. Because of likely differences in baseline eye size between each mouse, postoperative eye volumes were calculated as a fraction of the unoperated eye volume. These fractions were then normalized to the percentage regenerated in WT mice to obtain the final comparative data. This was done because paraffinization likely had a different effect on the regenerated lens material as compared to native lens material, meaning the absolute percentage regenerated in any of the strains is likely not as relevant as the comparison of growth between strains. All volumes are reported in μL.

Figure 1. Workflow of three-dimensional reconstruction analysis.

After surgery and paraffin embedding, experimental eyes (red background) were serially sectioned at a thickness of 6μm, and every fifth section was slide-mounted and digitized via light microscopy. These images were then stacked and aligned with the imaging software FIJI, and a mask was created manually around each regenerated lens. The calculated areas of the outlined lenses were multiplied by the distance between sections to produce an estimate of the volume of each section, which was then combined for a sum calculated volume estimate. Additionally, built-in FIJI functions were employed for three-dimensional visualization. For control eyes (blue background), whole lenses were dissected after paraffin processing, measured via light microscopy and then digitized. These native lenses were assumed to be ellipsoid in shape, so volume was computed via the appropriate formula.

3. Results

3.1. AR overexpression, AR knockout, and wild-type mice all regenerate a γ-crystallin positive, intracapsular lens-like structure two months after lens extraction

We performed a modified lens extraction procedure on WT, AR overexpression (AR-Tg), and AR knockout (ARKO) mice. The procedure was aimed at maximally preserving the anterior LECs, which is critical for optimal lens regeneration.15 After two months, eyes were harvested, sectioned, and stained for the lens fiber cell marker γ-crystallin. Representative transverse sections through the center of the eye are shown in Figure 2. The regenerated lens-like mass is apparent just behind the cornea, as each mouse strain retained recognizable anatomy postoperatively. All lens-like masses were γ-crystallin positive, confirming that cells within the regenerated material have characteristics of lens fiber cells. This regenerative capacity and the time-course of regeneration is consistent with prior studies demonstrating lens regeneration in rodents.25,33 While these experiments were carried out to confirm that cells in the regenerated tissue contain typical crystallin proteins, it did not escape our notice that the regenerated lenses were consistently much larger in post-surgical eyes from AR-null mice.

Figure 2. Two-month postoperative lenses demonstrate γ-crystallin positive (brown), regenerated intracapsular lens masses.

The crystallin positivity confirms that the intracapsular mass is, in fact, lens material. ARKO mice have a shape more reminiscent of native lenses compared to WT and AR-Tg. “L” labels the regenerated lens mass. Scale bar = 500 μm.

3.2. Genetic knockout of AR produces larger regenerated lenses than WT and genetic overexpression of AR produces smaller regenerated lenses than WT

Because lenses regenerate in irregular shapes postoperatively, individual histologic sections are inadequate to accurately assess volume and morphology. To obtain proper dimensions, we performed serial sectioning of regenerated lenses, followed by digitization of these sections, manual demarcation of regenerated lens in each digital image, and then computational estimation of lens volume (as described in the Methods). Table 1 shows the calculated volumes of the regenerated lenses in each mouse strain. The regeneration volume measurements are reported as a fraction of the unoperated (native) eye, as each individual mouse has a differently sized native lens. Furthermore, because paraffin processing may differentially alter the size of a native lens compared to a regenerated lens, the absolute values of the fractional regeneration among individual mice in a given strain is likely not as informative as the differences in the regeneration between the mouse strains. Thus, we normalized the fractional regeneration in AR mutant strains to that of WT mice, allowing more accurate comparisons. Overall, we saw that ARKO mice had the greatest degree of regeneration, at about 70% greater than WT. By contrast, AR-Tg had about 20% less regeneration compared to WT. Treatment of WT mice with the pharmacologic AR inhibitor sorbinil led to regenerated lenses about 50% larger than WT controls not treated with sorbinil. Statistical comparisons between groups revealed a significant difference between ARKO and AR-Tg (p=0.0299). However, other comparisons between groups did not reach statistical significance.

Table 1.

Size analysis of regenerated lenses two months post-lens extraction.

	Regenerated Volume (ul)	Fraction of Native Lens	Normalized Fractional Regeneration
ARKO (n=4)	0.47 ± 0.068	0.116	1.7 ± 0.26
WT (n=2)	0.26 ± 0.193	0.069	1.0
AR-Tg (n=3)	0.19 ± 0.051	0.057	0.8 ± 0.26
WT + sorbinil (n=2)	0.39 ± 0.046	0.104	1.5 ± 0.17

3.3. All mouse strains produce lenses of similar shape two months after lens extraction

We also processed the digitized images of the serial sections to create a 3-dimensional reconstruction of the regenerated lenses (Figure 3). Each strain shows an area of adhesion between the anterior and posterior capsules that does not contain regenerated lens material. This “donut-hole” corresponds to the site of the capsule incision, where postoperative scarring is maximal. However, at two months postoperatively this is an expected morphology, as has been previously demonstrated in rodent lens regeneration models.^25,33 While the three-dimensional images in Figure 3 are presented to demonstrate the overall gross morphology of regenerated lenses, size comparisons between strains cannot be drawn since the images are not presented at the same scale.

Figure 3. Three-dimensional computationally reconstructed regenerated lenses.

All regenerated lenses display a similar ellipsoid shape with a prominent “donut hole” at the location of the capsular incision, likely a result of postoperative fibrosis that is maximal at this spot. Of note, lenses are not to scale.

4. Discussion

Lens epithelial cells have the capacity to undergo two very different responses after lens extraction in cataract surgery: either a fibrotic response leading to PCO or a process of lens fiber differentiation leading to possible lens regeneration.¹⁶ Interestingly, not only can these phenotypes co-exist in the same lens capsule, but they are also triggered and mediated by the same molecular signaling pathways.³⁴ There is a well-established body of literature demonstrating the paradigm of incidental surgical trauma inducing TGFβ signaling, leading to EMT and fibrotic PCO. However, much less is known about the precise mechanism that govern lens fiber cell differentiation and lens regeneration after lens extraction, particularly in mammals. Furthermore, to our knowledge there is no study identifying a factor that can push the postoperative LEC response toward lens regeneration. This present study is perhaps the first to identify AR as such a factor. While our prior work demonstrated that AR inhibition can suppress the fibrotic response and preserve the early lens fiber cell response,²⁵ this present work expands on this notion by showing that AR inhibition also augments lens regeneration in the longer term. Although these results do not provide definitive evidence, this work adds to the paradigm that fiber cell differentiation can lead to lens regeneration in a manner that can be modulated and perhaps optimized. While much additional work is required, our study is the first to present a mechanism of augmenting or accelerating lens regeneration.

Many possible mechanisms may be responsible for the observation that AR inhibition augments lens regeneration. It is well established that AR inhibition suppresses the fibrotic EMT response. By extension, it is possible that blocking this pathway simply increases the biochemical flux through the alternative, fiber cell pathway. As noted previously, both fiber cell differentiation and EMT proceed via the same signaling cascades but involve differential phosphorylation of different downstream kinases.³⁴ It is also possible that AR inhibition contributes to differential phosphorylation of these kinases to promote a signaling program that favors lens regeneration and lens fiber cell differentiation. Indeed, our lab has previously shown that AR activity affects the phosphorylation state of downstream TGF-β signaling targets.²⁶ Also, some have suggested the anterior lens epithelium harbors endogenous stem cells or stem-like cells.^15,35 While there is perhaps no current evidence to suggest a direct connection between AR and stem cell like properties, AR has a very well-established role in oxidative stress in numerous tissues.^36–39 Thus, a more favorable oxidative environment may contribute to persistence of these stem-like cells, which are known to decrease with age.³⁵ Clearly significant additional experimentation will be required to elucidate these pathways.

While modulation of lens regeneration is certainly of scientific interest, it may have tangible clinical applications. The clearest application is presbyopia, whereby the aging lens losses the ability to change shape (accommodation), preventing patients from visualizing near objects clearly. This condition has a significant economic and quality-of-life burden globally,^40,41 and an accommodating intraocular lens has long been viewed as the holy grail in cataract surgery innovation.⁴² Indeed, a regenerated lens may have the potential to restore accommodation. Obviously much additional work is needed to understand the mechanism of lens regeneration and methods to optimize and accelerate it, and the role of AR is perhaps an initial step towards this end.

Highlights.

• Lens epithelial cells have the capacity to regenerate fiber cells following cataract surgery.

• Drug inhibition of aldose reductase facilitates lens regeneration.

• Mice with a genetic deficiency in aldose reductase show greater lens regeneration.