LABORATORY SCIENCE
Effect of total lens epithelial cell destruction on intraocular lens fixation in the human capsular bag David J. Spalton, FRCS, FRCP, Sarah L. Russell, PhD, Richard Evans-Gowing, Julie A. Eldred, PhD, I. Michael Wormstone, PhD, FRSM
PURPOSE: To evaluate the effect of complete destruction of lens epithelial cells (LECs) in the capsular bag on intraocular lens (IOL) stability. SETTING: School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. DESIGN: Comparative evaluation. METHODS: An in vitro organ culture model using the bag–zonule–ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and fiber extraction were performed, and an Acrysof IOL was implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining it in 6 mL Eagle minimum essential medium supplemented with 5% v/v fetal calf serum and 10 ng/mL transforming growth factor-b2 for 3 weeks or more. One bag of each pair was treated with 1 mM thapsigargin to destroy all LECs. Observations of LEC growth were captured by phase-contrast microscopy, IOL stability by video microscopy, and endpoint analysis through scanning electron microscopy and immunocytochemistry. RESULTS: The LECs in control capsular bags migrated centrally, closing the bag and fixating the IOL between the anterior and posterior capsules, as seen clinically. These events were not observed in the thapsigargin-treated group. After a period of controlled orbital movement, the IOL in the control group stabilized quicker than in the treated bags. There was no IOL rotation in the bag; however, the IOLs in the treated group rocked with axial movement. CONCLUSIONS: The LECs appeared to aid stabilization of current IOL designs in the capsular bag. The results have clinical implications for IOL design and for strategies to prevent posterior capsule opacification. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2014; 40:306–312 Q 2014 ASCRS and ESCRS
Despite improvements in intraocular lens (IOL) design and cataract surgery, posterior capsule opacification (PCO) remains a significant clinical problem.1–3 In 2010, the Medicare system in the United States reimbursed surgeons $2498 million for cataract surgery and $222 million for neodymium:YAG (Nd:YAG) laser capsulotomy. Over that year, the ratio of capsulotomy to cataract surgery was approximately 27% (including capsulotomies from surgery performed in previous years). Posterior capsule opacification is a particular problem in pediatric eyes, in eyes with coexisting pathology such as uveitis, and now in eyes with multifocal
306
Q 2014 ASCRS and ESCRS Published by Elsevier Inc.
IOLs. Capsular bag fibrosis after surgery restricts the elasticity of the capsule and this is probably the major barrier to development of a truly accommodating IOL. Hydrophobic IOL materials,3 high-quality posterior square-edged IOL profiles4 with a 360-degree barrier, and a capsulorhexis lying on the anterior IOL surface5 all lead to a clinically significant reduction in postoperative PCO1,2; however, the failure of present technology to eradicate PCO has led to further research of its prevention by destroying lens epithelial cells (LECs), typically through pharmacologic means,6,7 by their mechanical destruction and removal,8 or by mechanically preventing access to the posterior
0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2013.06.030
LABORATORY SCIENCE: EFFECT OF LEC DESTRUCTION ON IOL STABILITY
capsule.9,10 At present, pharmacologic solutions have not reached clinical application because of the risk for collateral damage elsewhere in the eye. Mechanical devices designed to remove LECs have never attained clinical practice because of increased surgical time, expense, and lack of clinical effect, and while IOL designs such as the bag-in-the-lens or posterior capsulorhexis with optic capture prevent LECs from reaching the visual axis, the eyes can develop a Soemmerring ring in the equatorial bag over a longterm follow-up.11 Although complete LEC destruction might seem desirable for the total prevention of PCO, a lack of LECs in the bag could be complicated by instability of the IOL in the capsular bag and possibly by longterm degenerative changes in the bag if the LECs are required to maintain the capsule membrane. This study, using an organ-culture system of human capsular bags prepared from donor eyes, was designed to examine the effect of total LEC destruction on IOL stability in the bag. MATERIALS AND METHODS Capsular Bag Preparation
307
made to polish the anterior capsule to remove its LECs. A single-piece Acrysof IQ Aspheric Natural IOL (Alcon Laboratories, Inc.) was implanted in each capsular bag, and the specimen was transferred to a tissue culture dish. Using this method, the lens capsule remained intact and the capsular bag was freely suspended from the zonule in a very physiologic way, simulating phacoemulsification surgery and avoiding artifactual areas of capsule contact or collapse. All capsular bag preparations were maintained in EMEM supplemented with 5% fetal calf serum, 10 ng/mL transforming growth factor-b2, and 50 mg/mL gentamicin (all from Sigma-Aldrich). One bag of each pair of eyes was treated with 1 mM thapsigargin (Sigma-Aldrich), a calcium ATPase inhibitor, to destroy all LECs.6,14 Ongoing observations were captured using phasecontrast microscopy. At the endpoint, IOL–capsular bag stability was assessed using video microscopy by placing the specimen over a grid placed on a Titramax 100 shaker (Heidolph Instruments) and illuminated from above using a fiber-optic light source. Images were captured during and after a 10-second period of controlled orbital movement (a 1.5 mm vibration orbit at 500 rpm). The video images were then reviewed using Quicktime Pro software (Apple Inc.). The point at which IOL–capsular bag stability was reached was determined when no change in IOL position was observed in consecutive frames.
Immunocytochemistry
The model previously described by Liu et al.12 and developed by Cleary et al.13 was used. Donor eyes were obtained within 48 hours postmortem with national research ethics committee approval and were used in accordance with the tenets of the Declaration of Helsinki. The procedure involved isolating the ciliary body–zonule–lens complex from pairs of donor eyes, washing the lens briefly with Eagle minimum essential medium (EMEM), and then securing the ciliary body to a silicone ring using entomological pins before capsulorhexis creation and lens fiber mass removal by hydroexpression. Residual lens material was then removed by irrigation/aspiration; no attempt was
Submitted: April 10, 2013. Final revision submitted: June 26, 2013. Accepted: June 28, 2013.
All reagents were from Sigma-Aldrich (unless otherwise stated). The IOLs were removed from capsular bag preparations, which were then fixed for 30 minutes in 4% formaldehyde in phosphate-buffered saline (PBS). Preparations were washed for 3 minutes 15 minutes in PBS containing 0.02% w/v bovine serum albumin and 0.05% v/v octylphenoxypolyethoxyethanol (Igepal). The F-actin cytoskeleton and chromatin were stained with Texas Red-X-phalloidin (Molecular Probes) and 40 ,6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature. The stained preparations were again washed extensively, floated onto microscope slides, and mounted in Hydromount mounting medium (National Diagnostics). Images were viewed with an epifluorescence microscope and Axiovision software (both Zeiss).
Scanning Electron Microscopy
From the King Edward VII Hospital (Spalton), London, and the IOL Research Group (Russell, Evans-Gowing, Eldred, Wormstone), School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. Supported by Fight for Sight, London, and the Humane Research Trust, Stockport, Cheshire, United Kingdom . The staff of the East Anglian and Bristol Eye Banks provided donor material for this study. Presented in part at the XXX Congress of the European Society of Cataract & Refractive Surgeons, Milan, Italy, September 2012. Corresponding author: I. Michael Wormstone, PhD, FRSM, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail: [email protected].
Specimens were fixed in 4% formaldehyde in PBS. Specimens were dehydrated in a graded ethanol series, critically point dried with carbon dioxide, and then coated with 7 to 10 nm of platinum–gold. They were inspected and photographed with a scanning electron microscope (Philips SEM 505, Philips Industries).
RESULTS Three donor pairs were used, 2 women with an age at death of 50 years and 84 years, respectively, and 1 man aged 74 years. Table 1 shows the observations in each capsular bag preparation. Soon after the surgeries, viable populations of cells were observed attached to the anterior lens capsule in all bags. In each case, the IOL was well positioned and the capsulorhexis edge
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Table 1. Research outcomes for the 6 human capsular bag preparations used in this study. Capsular Bag 1A 1B 2A 2B 3A 3B
Thapsigargin Treated
360 Capsulorhexis Contact on IOL
Viable Cells After Surgery
Viable Cells at Endpoint
Evidence of AC/PC Adherence
No Yes No Yes No Yes
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes
Yes No Yes No Yes No
Yes No Yes No Yes No
AC Z anterior capsule; IOL Z intraocular lens; PC Z posterior capsule
could be seen in association with 360 degrees of the IOL optic. Within the first week of culture, LECs maintained in standard culture conditions showed recolonization of denuded regions of the anterior capsule and growth on the peripheral posterior capsule. However, in the presence of thapsigargin, the remaining cell populations on the anterior capsule began to look distressed and there was no evidence of growth on the posterior capsule. With continued culture, cells in the control preparations grew across the lens capsule and the anterior surface of the IOL (Figure 1, A to C). It was also evident that adhesion between the anterior capsule and posterior capsule had taken place (Figures 1, B, and 2, A and B) and that the anterior capsule had sealed to the anterior IOL surface (Figures 1, C, and 2, A and B), which effectively shrink-wrapped the IOL in all control eyes. In capsular bags treated with thapsigargin, all cells ultimately died (Figure 1, D, E, and F). As a consequence, no adhesion between the anterior capsule and posterior capsule or between the anterior capsule and IOL was formed (Figures 1, E and F, and 2, C). Electron microscopy showed adherence of the anterior capsule at the capsulorhexis margin to the IOL in control preparations (Figure 3, A), which is in contrast to an acellular capsulorhexis margin in the thapsigargin-treated eyes (Figure 3, B). Immunohistochemistry showed dynamic actin organization of LECs growing on the lens capsule and anterior surface of the IOL (Figure 4, A and B), whereas these areas was acellular in thapsigargintreated eyes (Figure 4, C). To determine whether the inability to form capsule– capsule and capsule–IOL adhesion could affect the stability of the IOL within the bag, the capsular bags were subjected to a defined period of orbital shaking and the period to stability was determined using video microscopy. The IOLs in the control bags stabilized more quickly (P%.05, Student t test) (Figure 5). The IOLs showed no tendency to rotate in the x–y plane in the control or thapsigargin-treated capsular bags.
However, the IOL in the thapsigargin-treated bags could be seen to seesaw in the z-plane against a pivotal axis, which was created by physical interactions of opposing haptics with the capsular bag (Figure 6). DISCUSSION Current strategies have significantly reduced the need for Nd:YAG laser capsulotomy but have not
Figure 1. Comparison of control and thapsigargin-treated capsular bags after a 24-day culture period. A and D: Phase micrographs showing viable cells on the anterior capsule adjacent to the haptics in control capsular bags and cell death in the thapsigargin-treated group. B: Adhesion between the anterior and posterior capsules in control preparations. C: Creation of a cell-mediated seal of the anterior capsule and IOL surface in control preparations. E: Failure to form capsule–capsule adhesion in the thapsigargin-treated group. F: Failure to achieve adhesion following thapsigargin treatment. Arrows indicate the capsulorhexis margin.
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Figure 3. Scanning electron micrographs showing the interaction of the anterior capsule, LECs, and the anterior IOL surface in control preparation (A) and thapsigargin-treated preparation (B). The control cells have grown from the capsulorhexis onto the IOL surface, forming a seal; however, in the thapsigargin-treated group, no seal can be observed. Note scale bar representative of both images.
Figure 2. Low-power modified dark-field images showing gross changes in control and thapsigargin-treated capsular bags. A and B: Distinct regions of adhesion (arrows) between the anterior capsule and posterior capsule can be observed in control preparations. C: No adhesion between opposing capsule surfaces can be seen in thapsigargin-treated capsular bags.
eliminated the problem. Reducing the impact of PCO on patients is of great practical importance, and new approaches must be developed. One proposed
approach is complete destruction of the LECs in the capsular bag; however, if a safe and reliable method can be developed to achieve this goal, it is still important to consider the effect of total cell loss on IOL stability and its interaction with the capsular bag. In a closed-bag system, lens cells grow on the capsule, changing its properties. The IOL will be fixated by LEC growth around the IOL haptics and optic with adhesion between the anterior and posterior capsule. Contact with the IOL will be enhanced, and subsequent fibrotic changes and the transformation of LECs into myofibroblasts will shrink-wrap the capsule around the IOL, further stabilizing its position in the bag. This study found that in the absence of LECs, the anterior and posterior capsules failed to adhere to the IOL, leaving the bag open. Rotatory movement itself did not affect IOL positioning in
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Figure 5. Assessment of the ability to stabilize the IOL in control and thapsigargin-treated capsular bags after a defined period of orbital shaking. The data are expressed as mean G SEM (n Z 3). The asterisk indicates a significant difference between the groups (P%.05, Student t test) (Tg Z thapsigargin).
z-axis. Such movement would produce intolerable visual symptoms and could conceivably lead to abrasion of the bag over the long term. In this study, we used single-piece Acrysof IOLs with a C-loop haptic design and a diameter of 13.0 mm. With bag fibrosis after surgery, these haptics provide a broad area of contact (approximately 45 degrees) with the equatorial bag; however, in the absence of LECs, these haptics essentially provide 2 opposing points of contact with the equatorial region of the capsular bag, giving rise to a pivotal axis. A different haptic design, such as a 4-point design, might be more stable. It is also worth considering the effect total LEC loss may have on the capsule itself. The capsule is a basement membrane consisting largely of type IV collagen secreted by LECs15 and increases in thickness throughout life.16 A potential concern is that a lack of cells will lead to degeneration of the capsule, possibly by enhanced mechanical abrasion from IOL movement; this in turn could lead to increasing fragility with IOL subluxation or dislocation. The major group of enzymes associated with degradation of extracellular matrix components are matrix metalloproteinases (MMPs).17–19 Disruption to the eye, such as cataract surgery, will elevate MMP levels, at least in
Figure 4. Epifluorescence micrographs showing actin distribution on the anterior lens capsule (AC) and anterior intraocular lens (IOL) in control preparation (A and B) and thapsigargin-treated preparation (C). The arrows indicate the capsulorhexis margin.
the x–y axis, and the IOL maintained a stable location in the bag in the absence of cells. However, in the cell-free bags, the IOL wobbled in the anteroposterior
Figure 6. Intraocular lens movement along a pivotal axis, which is established by points of stability between opposing haptics and the capsular bag.
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the short term.20,21 However, the lens capsule is one of the thickest basement membranes in the body,15 and it is unlikely that significant thinning of this structure would occur. This study suggests that LECs have an important role in stabilizing an IOL after surgery and an alternative strategy that modulates LEC function rather than destroying them would be appealing; there is some clinical and experimental evidence to suggest that this is a possibility. Clinical experience has shown that the accommodating dual-optic Synchrony IOL (Abbott Medical Optics, Inc.) has a very low PCO rate.22,23 A unique feature of this IOL is that the capsular bag is held open after surgery by the optic components so there is free communication of the bag with aqueous. Such open-bag designs have been shown experimentally in animal eyes to reduce PCO significantly.24,25 Also, recent results in a study using a capsular ring device26 that keeps the bag open suggests that this very significantly reduces PCO in human eyes. In the case of open-bag devices, the interactions with the cell-lined anterior capsule allied to continuous ring designs could be sufficient to provide IOL stability. An absence of cells could render such designs more likely to undergo rotational movement. Specific IOL designs based on an open-bag principle are expected to be evaluated in imminent clinical trials.
2. 3.
4.
5.
6.
7.
8.
9.
10.
WHAT WAS KNOWN Complete LEC destruction is a possible strategy for the total prevention of PCO.
11.
12.
WHAT THIS PAPER ADDS The LECs appeared to aid stabilization of current IOL designs in the capsular bag. In an acellular capsular bag, there was minimal rotation of the IOL in the x–y plane. However, there was greater movement in the z-plane against a pivotal axis, which is created by physical interactions of opposing haptics with the capsular bag. This study indicates IOL/haptic designs have to be developed to improve stability in an acellular capsular bag, which will improve the therapeutic value of future strategies to prevent PCO.
13.
14.
15. 16.
17.
18.
REFERENCES 1. Awasthi N, Guo S, Wagner BJ. Posterior capsular opacification: a problem reduced but not yet eradicated. Arch Ophthalmol 2009; 127:555–562. Available at: http://archopht.jamanetwork.com/
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data/Journals/OPHTH/10060/emo80002_555_562.pdf. Accessed September 6, 2013 Wormstone IM, Wang L, Liu CS. Posterior capsule opacification. Exp Eye Res 2009; 88:257–269 Findl O, Buehl W, Bauer P, Sycha T. Interventions for preventing posterior capsule opacification. Cochrane Database System Rev 2010; Issue 2, Art. No. CD003738. Summary available at: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003738. pub3/pdf/abstract. Accessed September 6, 2013 Nanavaty MA, Spalton DJ, Boyce J, Brain A, Marshall J. Edge profile of commercially available square-edged intraocular lenses. J Cataract Refract Surg 2008; 34:677–686 Hollick EJ, Spalton DJ, Ursell PG, Pande MV, Barman SA, Boyce JF, Tilling K. The effect of polymethylmethacrylate, silicone, and polyacrylic intraocular lenses on posterior capsular opacification 3 years after cataract surgery. Ophthalmology 1999; 106:49–54; discussion by RC Drews, 54–55 Duncan G, Wormstone IM, Liu CS, Marcantonio JM, Davies PD. Thapsigargin-coated intraocular lenses inhibit human lens cell growth. Nat Med 1997; 3:1026–1028 € u € _ € U, € Ozt lu L, Ozer €rk F, Kaynak S, Kurt E, Emirog Inan U E, _ €ler C. Prevention of posterior capsule opacification Ilker SS, Gu by intraoperative single-dose pharmacologic agents. J Cataract Refract Surg 2001; 27:1079–1087 Mamalis N, Grossniklaus HE, Waring GO III, Werner L, Brubaker J, Davis D, Espander L, Walker R, Thyzel R. Ablation of lens epithelial cells with a laser photolysis system: histopathology, ultrastructure, and immunochemistry. J Cataract Refract Surg 2010; 36:1003–1010 De Groot V, Leysen I, Neuhann T, Gobin L, Tassignon M-J. One-year follow-up of bag-in-the-lens intraocular lens implantation in 60 eyes. J Cataract Refract Surg 2006; 32:1632–1637 Menapace R. Routine posterior optic buttonholing for eradication of posterior capsule opacification in adults; report of 500 consecutive cases. J Cataract Refract Surg 2006; 32: 929–943; erratum, 1410 Werner L, Tassignon M-J, Zaugg BE, de Groot V, Rozema J. Clinical and histopathologic evaluation of six human eyes implanted with the bag-in-the-lens. Ophthalmology 2010; 117:55–62 Liu CS, Wormstone IM, Duncan G, Marcantonio JM, Webb SF, Davies PD. A study of human lens cell growth in vitro; a model for posterior capsule opacification. Invest Ophthalmol Vis Sci 1996; 37:906–914. Available at: http://www.iovs.org/content/37/5/ 906.full.pdf. Accessed September 6, 2013 Cleary G, Spalton DJ, Zhang J-J, Marshall J. In vitro lens capsule model for investigation of posterior capsule opacification. J Cataract Refract Surg 2010; 36:1249–1252 Wang L, Wormstone IM, Reddan JR, Duncan G. Growth factor receptor signaling in human lens cells: role of the calcium store. Exp Eye Res 2005; 80:885–895 Danysh BP, Duncan MK. The lens capsule. Exp Eye Res 2009; 88:151–164 Barraquer RI, Michael R, Abreu R, Lamarca J, Tresserra F. Human lens capsule thickness as a function of age and location along the sagittal lens perimeter. Invest Ophthalmol Vis Sci 2006; 47:2053–2060. Available at: http://www.iovs.org/content/47/5/2053.full.pdf. Accessed September 6, 2013 Mandal M, Mandal A, Das S, Chakraborti T, Sajal C. Clinical implications of matrix metalloproteinases. Mol Cell Biochem 2003; 252:305–329 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases; structure, function, and biochemistry. Circ Res 2003; 92:827–839. Available at: http:// circres.ahajournals.org/content/92/8/827.full.pdf. Accessed September 6, 2013
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19. Wride MA, Geatrell J, Guggenheim JA. Proteases in eye development and disease. Birth Defects Res C Embryo Today 2006; 78:90–105 20. Di Girolamo N, Verma MJ, McCluskey PJ, Lloyd A, Wakefield D. Increased matrix metalloproteinases in the aqueous humor of patients and experimental animals with uveitis. Curr Eye Res 1996; 15:1060–1068 21. Wormstone IM, Tamiya S, Anderson I, Duncan G. TGF-b2induced matrix modification and cell transdifferentiation in the human lens capsular bag. Invest Ophthalmol Vis Sci 2002; 43:2301–2308. Available at: http://www.iovs.org/cgi/reprint/43/ 7/2301. Accessed September 6, 2013 22. Ossma IL, Galvis A, Vargas LG, Trager MJ, Vagefi MR, McLeod SD. Synchrony dual-optic accommodating intraocular lens. Part 2: pilot clinical evaluation. J Cataract Refract Surg 2007; 33:47–52 23. Werner L, Pandey SK, Izak AM, Vargas LG, Trivedi RH, Apple DJ, Mamalis N. Capsular bag opacification after experimental implantation of a new accommodating intraocular lens in rabbit eyes. J Cataract Refract Surg 2004; 30:1114–1123 24. Leishman L, Werner L, Bodnar Z, Ollerton A, Michelson J, Schmutz M, Mamalis N. Prevention of capsular bag opacifica-
tion with a modified hydrophilic acrylic disk-shaped intraocular lens. J Cataract Refract Surg 2012; 38:1664–1670 25. Hara T, Hara T, Sakanishi K, Yamada Y. Efficacy of equator rings in an experimental rabbit study. Arch Ophthalmol 1995; 113:1060–1065 26. Hara T, Hara T, Narita M, Hashimoto T, Motoyama Y, Hara T. Long-term study of posterior capsular opacification prevention with endocapsular equator rings in humans. Arch Ophthalmol 2011; 129:855–863. Available at: http://archopht.jamanetwork. com/data/Journals/OPHTH/22539/ecs15001_855_863.pdf?resultClickZ3. Accessed September 6, 2013
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First author: David J. Spalton, FRCS, FRCP King Edward VII Hospital, London, United Kingdom
Effect of total lens epithelial cell destruction on intraocular lens fixation in the human capsular bag David J. Spalton, FRCS, FRCP, Sarah L. Russell, PhD, Richard Evans-Gowing, Julie A. Eldred, PhD, I. Michael Wormstone, PhD, FRSM
PURPOSE: To evaluate the effect of complete destruction of lens epithelial cells (LECs) in the capsular bag on intraocular lens (IOL) stability. SETTING: School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. DESIGN: Comparative evaluation. METHODS: An in vitro organ culture model using the bag–zonule–ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and fiber extraction were performed, and an Acrysof IOL was implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining it in 6 mL Eagle minimum essential medium supplemented with 5% v/v fetal calf serum and 10 ng/mL transforming growth factor-b2 for 3 weeks or more. One bag of each pair was treated with 1 mM thapsigargin to destroy all LECs. Observations of LEC growth were captured by phase-contrast microscopy, IOL stability by video microscopy, and endpoint analysis through scanning electron microscopy and immunocytochemistry. RESULTS: The LECs in control capsular bags migrated centrally, closing the bag and fixating the IOL between the anterior and posterior capsules, as seen clinically. These events were not observed in the thapsigargin-treated group. After a period of controlled orbital movement, the IOL in the control group stabilized quicker than in the treated bags. There was no IOL rotation in the bag; however, the IOLs in the treated group rocked with axial movement. CONCLUSIONS: The LECs appeared to aid stabilization of current IOL designs in the capsular bag. The results have clinical implications for IOL design and for strategies to prevent posterior capsule opacification. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2014; 40:306–312 Q 2014 ASCRS and ESCRS
Despite improvements in intraocular lens (IOL) design and cataract surgery, posterior capsule opacification (PCO) remains a significant clinical problem.1–3 In 2010, the Medicare system in the United States reimbursed surgeons $2498 million for cataract surgery and $222 million for neodymium:YAG (Nd:YAG) laser capsulotomy. Over that year, the ratio of capsulotomy to cataract surgery was approximately 27% (including capsulotomies from surgery performed in previous years). Posterior capsule opacification is a particular problem in pediatric eyes, in eyes with coexisting pathology such as uveitis, and now in eyes with multifocal
306
Q 2014 ASCRS and ESCRS Published by Elsevier Inc.
IOLs. Capsular bag fibrosis after surgery restricts the elasticity of the capsule and this is probably the major barrier to development of a truly accommodating IOL. Hydrophobic IOL materials,3 high-quality posterior square-edged IOL profiles4 with a 360-degree barrier, and a capsulorhexis lying on the anterior IOL surface5 all lead to a clinically significant reduction in postoperative PCO1,2; however, the failure of present technology to eradicate PCO has led to further research of its prevention by destroying lens epithelial cells (LECs), typically through pharmacologic means,6,7 by their mechanical destruction and removal,8 or by mechanically preventing access to the posterior
0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2013.06.030
LABORATORY SCIENCE: EFFECT OF LEC DESTRUCTION ON IOL STABILITY
capsule.9,10 At present, pharmacologic solutions have not reached clinical application because of the risk for collateral damage elsewhere in the eye. Mechanical devices designed to remove LECs have never attained clinical practice because of increased surgical time, expense, and lack of clinical effect, and while IOL designs such as the bag-in-the-lens or posterior capsulorhexis with optic capture prevent LECs from reaching the visual axis, the eyes can develop a Soemmerring ring in the equatorial bag over a longterm follow-up.11 Although complete LEC destruction might seem desirable for the total prevention of PCO, a lack of LECs in the bag could be complicated by instability of the IOL in the capsular bag and possibly by longterm degenerative changes in the bag if the LECs are required to maintain the capsule membrane. This study, using an organ-culture system of human capsular bags prepared from donor eyes, was designed to examine the effect of total LEC destruction on IOL stability in the bag. MATERIALS AND METHODS Capsular Bag Preparation
307
made to polish the anterior capsule to remove its LECs. A single-piece Acrysof IQ Aspheric Natural IOL (Alcon Laboratories, Inc.) was implanted in each capsular bag, and the specimen was transferred to a tissue culture dish. Using this method, the lens capsule remained intact and the capsular bag was freely suspended from the zonule in a very physiologic way, simulating phacoemulsification surgery and avoiding artifactual areas of capsule contact or collapse. All capsular bag preparations were maintained in EMEM supplemented with 5% fetal calf serum, 10 ng/mL transforming growth factor-b2, and 50 mg/mL gentamicin (all from Sigma-Aldrich). One bag of each pair of eyes was treated with 1 mM thapsigargin (Sigma-Aldrich), a calcium ATPase inhibitor, to destroy all LECs.6,14 Ongoing observations were captured using phasecontrast microscopy. At the endpoint, IOL–capsular bag stability was assessed using video microscopy by placing the specimen over a grid placed on a Titramax 100 shaker (Heidolph Instruments) and illuminated from above using a fiber-optic light source. Images were captured during and after a 10-second period of controlled orbital movement (a 1.5 mm vibration orbit at 500 rpm). The video images were then reviewed using Quicktime Pro software (Apple Inc.). The point at which IOL–capsular bag stability was reached was determined when no change in IOL position was observed in consecutive frames.
Immunocytochemistry
The model previously described by Liu et al.12 and developed by Cleary et al.13 was used. Donor eyes were obtained within 48 hours postmortem with national research ethics committee approval and were used in accordance with the tenets of the Declaration of Helsinki. The procedure involved isolating the ciliary body–zonule–lens complex from pairs of donor eyes, washing the lens briefly with Eagle minimum essential medium (EMEM), and then securing the ciliary body to a silicone ring using entomological pins before capsulorhexis creation and lens fiber mass removal by hydroexpression. Residual lens material was then removed by irrigation/aspiration; no attempt was
Submitted: April 10, 2013. Final revision submitted: June 26, 2013. Accepted: June 28, 2013.
All reagents were from Sigma-Aldrich (unless otherwise stated). The IOLs were removed from capsular bag preparations, which were then fixed for 30 minutes in 4% formaldehyde in phosphate-buffered saline (PBS). Preparations were washed for 3 minutes 15 minutes in PBS containing 0.02% w/v bovine serum albumin and 0.05% v/v octylphenoxypolyethoxyethanol (Igepal). The F-actin cytoskeleton and chromatin were stained with Texas Red-X-phalloidin (Molecular Probes) and 40 ,6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature. The stained preparations were again washed extensively, floated onto microscope slides, and mounted in Hydromount mounting medium (National Diagnostics). Images were viewed with an epifluorescence microscope and Axiovision software (both Zeiss).
Scanning Electron Microscopy
From the King Edward VII Hospital (Spalton), London, and the IOL Research Group (Russell, Evans-Gowing, Eldred, Wormstone), School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. Supported by Fight for Sight, London, and the Humane Research Trust, Stockport, Cheshire, United Kingdom . The staff of the East Anglian and Bristol Eye Banks provided donor material for this study. Presented in part at the XXX Congress of the European Society of Cataract & Refractive Surgeons, Milan, Italy, September 2012. Corresponding author: I. Michael Wormstone, PhD, FRSM, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail: [email protected].
Specimens were fixed in 4% formaldehyde in PBS. Specimens were dehydrated in a graded ethanol series, critically point dried with carbon dioxide, and then coated with 7 to 10 nm of platinum–gold. They were inspected and photographed with a scanning electron microscope (Philips SEM 505, Philips Industries).
RESULTS Three donor pairs were used, 2 women with an age at death of 50 years and 84 years, respectively, and 1 man aged 74 years. Table 1 shows the observations in each capsular bag preparation. Soon after the surgeries, viable populations of cells were observed attached to the anterior lens capsule in all bags. In each case, the IOL was well positioned and the capsulorhexis edge
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Table 1. Research outcomes for the 6 human capsular bag preparations used in this study. Capsular Bag 1A 1B 2A 2B 3A 3B
Thapsigargin Treated
360 Capsulorhexis Contact on IOL
Viable Cells After Surgery
Viable Cells at Endpoint
Evidence of AC/PC Adherence
No Yes No Yes No Yes
Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes
Yes No Yes No Yes No
Yes No Yes No Yes No
AC Z anterior capsule; IOL Z intraocular lens; PC Z posterior capsule
could be seen in association with 360 degrees of the IOL optic. Within the first week of culture, LECs maintained in standard culture conditions showed recolonization of denuded regions of the anterior capsule and growth on the peripheral posterior capsule. However, in the presence of thapsigargin, the remaining cell populations on the anterior capsule began to look distressed and there was no evidence of growth on the posterior capsule. With continued culture, cells in the control preparations grew across the lens capsule and the anterior surface of the IOL (Figure 1, A to C). It was also evident that adhesion between the anterior capsule and posterior capsule had taken place (Figures 1, B, and 2, A and B) and that the anterior capsule had sealed to the anterior IOL surface (Figures 1, C, and 2, A and B), which effectively shrink-wrapped the IOL in all control eyes. In capsular bags treated with thapsigargin, all cells ultimately died (Figure 1, D, E, and F). As a consequence, no adhesion between the anterior capsule and posterior capsule or between the anterior capsule and IOL was formed (Figures 1, E and F, and 2, C). Electron microscopy showed adherence of the anterior capsule at the capsulorhexis margin to the IOL in control preparations (Figure 3, A), which is in contrast to an acellular capsulorhexis margin in the thapsigargin-treated eyes (Figure 3, B). Immunohistochemistry showed dynamic actin organization of LECs growing on the lens capsule and anterior surface of the IOL (Figure 4, A and B), whereas these areas was acellular in thapsigargintreated eyes (Figure 4, C). To determine whether the inability to form capsule– capsule and capsule–IOL adhesion could affect the stability of the IOL within the bag, the capsular bags were subjected to a defined period of orbital shaking and the period to stability was determined using video microscopy. The IOLs in the control bags stabilized more quickly (P%.05, Student t test) (Figure 5). The IOLs showed no tendency to rotate in the x–y plane in the control or thapsigargin-treated capsular bags.
However, the IOL in the thapsigargin-treated bags could be seen to seesaw in the z-plane against a pivotal axis, which was created by physical interactions of opposing haptics with the capsular bag (Figure 6). DISCUSSION Current strategies have significantly reduced the need for Nd:YAG laser capsulotomy but have not
Figure 1. Comparison of control and thapsigargin-treated capsular bags after a 24-day culture period. A and D: Phase micrographs showing viable cells on the anterior capsule adjacent to the haptics in control capsular bags and cell death in the thapsigargin-treated group. B: Adhesion between the anterior and posterior capsules in control preparations. C: Creation of a cell-mediated seal of the anterior capsule and IOL surface in control preparations. E: Failure to form capsule–capsule adhesion in the thapsigargin-treated group. F: Failure to achieve adhesion following thapsigargin treatment. Arrows indicate the capsulorhexis margin.
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Figure 3. Scanning electron micrographs showing the interaction of the anterior capsule, LECs, and the anterior IOL surface in control preparation (A) and thapsigargin-treated preparation (B). The control cells have grown from the capsulorhexis onto the IOL surface, forming a seal; however, in the thapsigargin-treated group, no seal can be observed. Note scale bar representative of both images.
Figure 2. Low-power modified dark-field images showing gross changes in control and thapsigargin-treated capsular bags. A and B: Distinct regions of adhesion (arrows) between the anterior capsule and posterior capsule can be observed in control preparations. C: No adhesion between opposing capsule surfaces can be seen in thapsigargin-treated capsular bags.
eliminated the problem. Reducing the impact of PCO on patients is of great practical importance, and new approaches must be developed. One proposed
approach is complete destruction of the LECs in the capsular bag; however, if a safe and reliable method can be developed to achieve this goal, it is still important to consider the effect of total cell loss on IOL stability and its interaction with the capsular bag. In a closed-bag system, lens cells grow on the capsule, changing its properties. The IOL will be fixated by LEC growth around the IOL haptics and optic with adhesion between the anterior and posterior capsule. Contact with the IOL will be enhanced, and subsequent fibrotic changes and the transformation of LECs into myofibroblasts will shrink-wrap the capsule around the IOL, further stabilizing its position in the bag. This study found that in the absence of LECs, the anterior and posterior capsules failed to adhere to the IOL, leaving the bag open. Rotatory movement itself did not affect IOL positioning in
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Figure 5. Assessment of the ability to stabilize the IOL in control and thapsigargin-treated capsular bags after a defined period of orbital shaking. The data are expressed as mean G SEM (n Z 3). The asterisk indicates a significant difference between the groups (P%.05, Student t test) (Tg Z thapsigargin).
z-axis. Such movement would produce intolerable visual symptoms and could conceivably lead to abrasion of the bag over the long term. In this study, we used single-piece Acrysof IOLs with a C-loop haptic design and a diameter of 13.0 mm. With bag fibrosis after surgery, these haptics provide a broad area of contact (approximately 45 degrees) with the equatorial bag; however, in the absence of LECs, these haptics essentially provide 2 opposing points of contact with the equatorial region of the capsular bag, giving rise to a pivotal axis. A different haptic design, such as a 4-point design, might be more stable. It is also worth considering the effect total LEC loss may have on the capsule itself. The capsule is a basement membrane consisting largely of type IV collagen secreted by LECs15 and increases in thickness throughout life.16 A potential concern is that a lack of cells will lead to degeneration of the capsule, possibly by enhanced mechanical abrasion from IOL movement; this in turn could lead to increasing fragility with IOL subluxation or dislocation. The major group of enzymes associated with degradation of extracellular matrix components are matrix metalloproteinases (MMPs).17–19 Disruption to the eye, such as cataract surgery, will elevate MMP levels, at least in
Figure 4. Epifluorescence micrographs showing actin distribution on the anterior lens capsule (AC) and anterior intraocular lens (IOL) in control preparation (A and B) and thapsigargin-treated preparation (C). The arrows indicate the capsulorhexis margin.
the x–y axis, and the IOL maintained a stable location in the bag in the absence of cells. However, in the cell-free bags, the IOL wobbled in the anteroposterior
Figure 6. Intraocular lens movement along a pivotal axis, which is established by points of stability between opposing haptics and the capsular bag.
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the short term.20,21 However, the lens capsule is one of the thickest basement membranes in the body,15 and it is unlikely that significant thinning of this structure would occur. This study suggests that LECs have an important role in stabilizing an IOL after surgery and an alternative strategy that modulates LEC function rather than destroying them would be appealing; there is some clinical and experimental evidence to suggest that this is a possibility. Clinical experience has shown that the accommodating dual-optic Synchrony IOL (Abbott Medical Optics, Inc.) has a very low PCO rate.22,23 A unique feature of this IOL is that the capsular bag is held open after surgery by the optic components so there is free communication of the bag with aqueous. Such open-bag designs have been shown experimentally in animal eyes to reduce PCO significantly.24,25 Also, recent results in a study using a capsular ring device26 that keeps the bag open suggests that this very significantly reduces PCO in human eyes. In the case of open-bag devices, the interactions with the cell-lined anterior capsule allied to continuous ring designs could be sufficient to provide IOL stability. An absence of cells could render such designs more likely to undergo rotational movement. Specific IOL designs based on an open-bag principle are expected to be evaluated in imminent clinical trials.
2. 3.
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WHAT WAS KNOWN Complete LEC destruction is a possible strategy for the total prevention of PCO.
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WHAT THIS PAPER ADDS The LECs appeared to aid stabilization of current IOL designs in the capsular bag. In an acellular capsular bag, there was minimal rotation of the IOL in the x–y plane. However, there was greater movement in the z-plane against a pivotal axis, which is created by physical interactions of opposing haptics with the capsular bag. This study indicates IOL/haptic designs have to be developed to improve stability in an acellular capsular bag, which will improve the therapeutic value of future strategies to prevent PCO.
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15. 16.
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REFERENCES 1. Awasthi N, Guo S, Wagner BJ. Posterior capsular opacification: a problem reduced but not yet eradicated. Arch Ophthalmol 2009; 127:555–562. Available at: http://archopht.jamanetwork.com/
311
data/Journals/OPHTH/10060/emo80002_555_562.pdf. Accessed September 6, 2013 Wormstone IM, Wang L, Liu CS. Posterior capsule opacification. Exp Eye Res 2009; 88:257–269 Findl O, Buehl W, Bauer P, Sycha T. Interventions for preventing posterior capsule opacification. Cochrane Database System Rev 2010; Issue 2, Art. No. CD003738. Summary available at: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003738. pub3/pdf/abstract. Accessed September 6, 2013 Nanavaty MA, Spalton DJ, Boyce J, Brain A, Marshall J. Edge profile of commercially available square-edged intraocular lenses. J Cataract Refract Surg 2008; 34:677–686 Hollick EJ, Spalton DJ, Ursell PG, Pande MV, Barman SA, Boyce JF, Tilling K. The effect of polymethylmethacrylate, silicone, and polyacrylic intraocular lenses on posterior capsular opacification 3 years after cataract surgery. Ophthalmology 1999; 106:49–54; discussion by RC Drews, 54–55 Duncan G, Wormstone IM, Liu CS, Marcantonio JM, Davies PD. Thapsigargin-coated intraocular lenses inhibit human lens cell growth. Nat Med 1997; 3:1026–1028 € u € _ € U, € Ozt lu L, Ozer €rk F, Kaynak S, Kurt E, Emirog Inan U E, _ €ler C. Prevention of posterior capsule opacification Ilker SS, Gu by intraoperative single-dose pharmacologic agents. J Cataract Refract Surg 2001; 27:1079–1087 Mamalis N, Grossniklaus HE, Waring GO III, Werner L, Brubaker J, Davis D, Espander L, Walker R, Thyzel R. Ablation of lens epithelial cells with a laser photolysis system: histopathology, ultrastructure, and immunochemistry. J Cataract Refract Surg 2010; 36:1003–1010 De Groot V, Leysen I, Neuhann T, Gobin L, Tassignon M-J. One-year follow-up of bag-in-the-lens intraocular lens implantation in 60 eyes. J Cataract Refract Surg 2006; 32:1632–1637 Menapace R. Routine posterior optic buttonholing for eradication of posterior capsule opacification in adults; report of 500 consecutive cases. J Cataract Refract Surg 2006; 32: 929–943; erratum, 1410 Werner L, Tassignon M-J, Zaugg BE, de Groot V, Rozema J. Clinical and histopathologic evaluation of six human eyes implanted with the bag-in-the-lens. Ophthalmology 2010; 117:55–62 Liu CS, Wormstone IM, Duncan G, Marcantonio JM, Webb SF, Davies PD. A study of human lens cell growth in vitro; a model for posterior capsule opacification. Invest Ophthalmol Vis Sci 1996; 37:906–914. Available at: http://www.iovs.org/content/37/5/ 906.full.pdf. Accessed September 6, 2013 Cleary G, Spalton DJ, Zhang J-J, Marshall J. In vitro lens capsule model for investigation of posterior capsule opacification. J Cataract Refract Surg 2010; 36:1249–1252 Wang L, Wormstone IM, Reddan JR, Duncan G. Growth factor receptor signaling in human lens cells: role of the calcium store. Exp Eye Res 2005; 80:885–895 Danysh BP, Duncan MK. The lens capsule. Exp Eye Res 2009; 88:151–164 Barraquer RI, Michael R, Abreu R, Lamarca J, Tresserra F. Human lens capsule thickness as a function of age and location along the sagittal lens perimeter. Invest Ophthalmol Vis Sci 2006; 47:2053–2060. Available at: http://www.iovs.org/content/47/5/2053.full.pdf. Accessed September 6, 2013 Mandal M, Mandal A, Das S, Chakraborti T, Sajal C. Clinical implications of matrix metalloproteinases. Mol Cell Biochem 2003; 252:305–329 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases; structure, function, and biochemistry. Circ Res 2003; 92:827–839. Available at: http:// circres.ahajournals.org/content/92/8/827.full.pdf. Accessed September 6, 2013
J CATARACT REFRACT SURG - VOL 40, FEBRUARY 2014
312
LABORATORY SCIENCE: EFFECT OF LEC DESTRUCTION ON IOL STABILITY
19. Wride MA, Geatrell J, Guggenheim JA. Proteases in eye development and disease. Birth Defects Res C Embryo Today 2006; 78:90–105 20. Di Girolamo N, Verma MJ, McCluskey PJ, Lloyd A, Wakefield D. Increased matrix metalloproteinases in the aqueous humor of patients and experimental animals with uveitis. Curr Eye Res 1996; 15:1060–1068 21. Wormstone IM, Tamiya S, Anderson I, Duncan G. TGF-b2induced matrix modification and cell transdifferentiation in the human lens capsular bag. Invest Ophthalmol Vis Sci 2002; 43:2301–2308. Available at: http://www.iovs.org/cgi/reprint/43/ 7/2301. Accessed September 6, 2013 22. Ossma IL, Galvis A, Vargas LG, Trager MJ, Vagefi MR, McLeod SD. Synchrony dual-optic accommodating intraocular lens. Part 2: pilot clinical evaluation. J Cataract Refract Surg 2007; 33:47–52 23. Werner L, Pandey SK, Izak AM, Vargas LG, Trivedi RH, Apple DJ, Mamalis N. Capsular bag opacification after experimental implantation of a new accommodating intraocular lens in rabbit eyes. J Cataract Refract Surg 2004; 30:1114–1123 24. Leishman L, Werner L, Bodnar Z, Ollerton A, Michelson J, Schmutz M, Mamalis N. Prevention of capsular bag opacifica-
tion with a modified hydrophilic acrylic disk-shaped intraocular lens. J Cataract Refract Surg 2012; 38:1664–1670 25. Hara T, Hara T, Sakanishi K, Yamada Y. Efficacy of equator rings in an experimental rabbit study. Arch Ophthalmol 1995; 113:1060–1065 26. Hara T, Hara T, Narita M, Hashimoto T, Motoyama Y, Hara T. Long-term study of posterior capsular opacification prevention with endocapsular equator rings in humans. Arch Ophthalmol 2011; 129:855–863. Available at: http://archopht.jamanetwork. com/data/Journals/OPHTH/22539/ecs15001_855_863.pdf?resultClickZ3. Accessed September 6, 2013
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First author: David J. Spalton, FRCS, FRCP King Edward VII Hospital, London, United Kingdom
