September 2010
Volume 51, Issue 9
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Effects of Nuclear Factor-κB Small Interfering RNA on Posterior Capsule Opacification
Author Affiliations & Notes
  • Hae-Young Lopilly Park
    From the Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea;
  • In-Tae Kim
    From the Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea;
  • Kyung-Min Lee
    Chan's Eye Hospital, Seoul, Korea; and
  • Jun-Sub Choi
    From the Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea;
  • Myung-Ok Park
    BiopolyMed, Inc., Seoul, Korea.
  • Choun-Ki Joo
    From the Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea;
  • Corresponding author: Choun-Ki Joo, Department of Ophthalmology, Seoul St. Mary's Hospital, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea; ckjoo@catholic.ac.kr
Investigative Ophthalmology & Visual Science September 2010, Vol.51, 4707-4715. doi:10.1167/iovs.09-4984
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      Hae-Young Lopilly Park, In-Tae Kim, Kyung-Min Lee, Jun-Sub Choi, Myung-Ok Park, Choun-Ki Joo; Effects of Nuclear Factor-κB Small Interfering RNA on Posterior Capsule Opacification. Invest. Ophthalmol. Vis. Sci. 2010;51(9):4707-4715. doi: 10.1167/iovs.09-4984.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The effects of small interfering (si)RNA of nuclear factor kappa B (NF-κB) on the development of posterior capsule opacification (PCO) were investigated both in vitro and in vivo in rabbits.

Methods.: After application of p105 NF-κB siRNA to lens epithelial cells (LECs), Western blot analyses were performed to detect p105 and p50 NF-κB and a scratch assay was used to determine cell migration. In the capsular bag model, immunocytochemistry was performed to determine expression of p50 NF-κB and Western blot analyses for the presence of epithelial-to-mesenchymal transition (EMT) markers. Two sequences of p105 NF-κB siRNA were used in cataract surgery in 15 New Zealand White rabbits. PCO grading was conducted by slit lamp biomicroscopy and a computer-based PCO grading program. One month after surgery, the eyes of the rabbits were enucleated, and sections were prepared for examination of the posterior capsule and other ocular tissues by light microscopy.

Results.: Application of p105 NF-κB siRNA to LECs decreased p105 NF-κB and p50 NF-κB expression, and migration of LECs was shown to be inhibited on the scratch assay. In the capsular bag model, the LEC count was significantly decreased, and immunocytochemistry showed reduced p50 NF-κB expression on the posterior capsule. EMT markers were significantly decreased after application of p105 NF-κB siRNA in the capsular bag model. In the in vivo study in rabbits, p105 NF-κB siRNA effectively decreased PCO, as determined by both slit lamp examination and the PCO grading program.

Conclusions.: NF-κB seems to be related to migration and proliferation of LECs. NF-κB siRNA was effective in inhibiting the migration and proliferation of LECs in vitro and decreased PCO formation after cataract surgery in an in vivo rabbit model.

The most common long-term complication of modern cataract surgery is posterior capsule opacification (PCO). 15 Since the first intraocular lens (IOL) implantation, technology has undergone a wide variety of improvements to reduce the incidence of PCO. However, the incidence is still approximately 50% in adults and 100% in children. 4,68 Decreased visual acuity induced by PCO is reported to occur in 20% to 40% of patients 2 to 5 years after surgery. 9 PCO usually causes a decrease in visual acuity by directly blocking the visual axis, and indirect complications, such as striae or folds in the posterior capsule or traction-induced IOL malposition can occur. Visual symptoms may vary widely in proportion to the degree of PCO. 
PCO is usually caused by the proliferation, migration, epithelial-to-mesenchymal transition (EMT), collagen deposition, and lens fiber regeneration of residual lens epithelial cells (LECs). 1013 It appears that cataract surgery induces a wound-healing response in the lens, and leftover LECs proliferate and migrate across the posterior capsule and undergo lens fiber regeneration and EMT. Usually, proliferation of the remaining LECs starts within a few hours after cataract surgery. 14 Proliferation and migration of LECs may precede EMT, and the two events are thought to be independently regulated. 15,16 Therefore, postsurgical medical inhibition of LEC proliferation and migration and EMT would be an option for preventing PCO. 
The transcriptional factor nuclear factor-kappa B (NF-κB) plays an important role in the control of cellular growth, and its translocation causes stimulation or inhibition of cell death in various cell types. 1724 The NF-κB family includes RELA (p65), NF-κB1 (p50; p105), NF-κB2 (p52; p100), c-REL, and RELB1 and -2. These proteins have a structurally conserved amino terminal region of 300 amino acids, which contains the dimerization, nuclear localization, and DNA-binding domains. 25,26 NF-κB exists in the cytoplasm as heterodimers or homodimers of Rel-related proteins. The predominant form of NF-κB is composed of NF-κB1 (p50) and RelA (p65) and is associated with the inhibitor κB as an inactive form. NF-κB is activated by various stimuli, including inflammatory cytokines, phorbol esters, bacterial toxins, viruses, UV, and various types of stress or injury. 2733 When NF-κB is activated, it undergoes phosphorylation and subsequent proteolytic degradation, resulting in the release of NF-κB, its translocation to the nucleus, and transcriptional activation of its target genes. 34 NF-κB is thought to be related to cancer pathogenesis, 35 immune system function, 36 and chronic inflammation in various cells. 34 In addition, various drugs used to treat human inflammatory diseases have effects on NF-κB activity. Furthermore, the feasibility and efficacy of specific inhibition of NF-κB activity have been demonstrated in several animal models of inflammatory disease. 37  
We focus on NF-κB because of its relation in cellular proliferation, differentiation, and apoptosis. There were no previous reports about the role of NF-κB in LEC migration and proliferation leading to PCO. Our previous studies indicated that NF-κB has a stimulating effect on human LEC proliferation and that LEC proliferation is reduced by NF-κB inhibitors in vitro. Also these inhibitors blocked LEC migration from the equatorial to the posterior capsule. 21,22  
In the present study, we used p105 NF-κB siRNA as an inhibitor of NF-κB and investigated its effects on the development of PCO during cataract surgery in a rabbit model. siRNA is widely accepted as an essential molecular tool for the validation of genes that potentially regulate cellular metabolism. Previous reports have indicated that siRNA can be used to suppress targets in gene signaling and that siRNA incorporated into a protein complex recognizes and cleaves target mRNA. 38,39 This method allows the selection of several sequences assumed to be the most effective in the study. We also chose two p105 NF-κB siRNA sequences shown to be most effective in inhibiting LEC migration and proliferation in cell culture and the capsular bag model. 
Specifically, the p50 NF-κB subtype is inhibited by p105 NF-κB siRNA, which inhibits the precursor of p50 NF-κB. The p50 protein is generated by proteolytic processing of the precursor p105 protein. 40 Each member of the NF-κB family, with the exception of RELB, can form homodimers as well as heterodimers with one another. The main activated form of NF-κB is a heterodimer of the p65 subunit associated with either a p50 or p52 subunit. 25,26 Subtypes p50 and p65 are expressed widely in various cell types. However, p65 is mainly related to cell survival signaling, and p50 is related to immunity and inflammation. 25,26,34 Therefore, we inhibited the p50 subtype in our PCO prevention study, especially the precursor form, p105. 
Materials and Methods
Observing the Migration of HLE B-3 Cells after Application of p105 NF-κB siRNA
Culture of HLE B-3 Cells.
In this study, we used the human lens epithelial (HLE) B-3 cell line, which was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in culture medium consisting of minimal essential medium (MEM 1:1; Ham's F12) supplemented with 20% fetal bovine serum (FBS), 5 μg/mL insulin, 0.1 μg/mL cholera toxin, and 0.5% dimethyl sulfoxide (DMSO) in a humidified 5% CO2 incubator at 37°C. Before p105 NF-κB siRNA inoculation, the HLE B-3 cells (5 × 104 cells/well) were cultured in six-well plates. After 48 hours, the cultured cells received scratch injury. 
Inoculation of p105 NF-κB siRNA.
Cultures of HLE B-3 cells in the treatment groups were treated with nonsilencing siRNA and eight types of p105 NF-κB siRNA sequence; control cultures were left untreated. The p105 NF-κB siRNA was designed from the published sequences (NM003998; Table 1). The sequence of nonsilencing siRNA was 5′-CCUACGCCACCAAUUUCGU-3′, and the cell cultures treated with it were designated the nonsilencing siRNA group. 
Table 1.
 
Eight Sequences of p105 NF-κB siRNA
Table 1.
 
Eight Sequences of p105 NF-κB siRNA
Number p105 NF-κB siRNA Sequence
1 5′-GCCAAAGAAGGACATGATAAA-3′
2 5′-TGGCAGGTATTTGACATATTA-3′
3 5′-TCTGATGATTTACTAGCACAA-3′
4 5′-ATGTGAAGCTGCAGCTGTATA-3′
5 5′-AGAAATTCCTGATCCAGACAA-3′
6 5′-GATCCTTCTTTGACTCATACA-3′
7 5′-GCCTGAATCATTCTCGATTTA-3′
8 5′-CTACGTTCCTATTGTCATTAA-3′
siRNA was complexed with PEG-CS (chitosan)-PLR (poly-l-arginine) at a volume ratio of 1:1 (10 μL of PEG-CS-PLR:10 μL of siRNA:20 ng/mL) for cell treatment. In addition, aliquots of 20 μL of siRNA polymer complexes were treated in each cell culture well. The PEG-CS-PLR complex is a pegylated chitosan-PLR complex by polyethylene glycol (molecular weight, 2000; Sigma-Aldrich, St. Louis, MO). CS-PLR was prepared by mixing 1 mL of CS solution (2 mg/mL, 12.4 mM pyranose) and 0.7 mg of PLR. 
Scratch Assay for Cell Migration.
The LEC migration assay was performed by the scratch technique. In cultures that were at approximately 95% confluence, a standard 200-μL yellow tip was drawn across the center of each well to produce the wound. After the wells were carefully washed to remove detached cells, the wounded LECs were cultured for 24 hours in culture medium supplemented with 5% serum. The medium was changed to reduced-serum medium (Opti-Mem; Invitrogen, Carlsbad, CA) before siRNA treatment. After 30 minutes, the wounded cells were treated with eight p105 NF-κB siRNA sequences, PEG-CS-PLR polymer as a control, and nonsilencing siRNA. To compare the motility of each cell line, we replaced the medium of the cultured cells with medium containing 5% FBS after 2 hours. After a 24-hour incubation, the cells were washed twice with phosphate-buffered saline (PBS), fixed with 10% formalin solution, and stained with 0.1% crystal violet. Three microscopic fields were evaluated for each group. 
Observing p105 and p50 NF-κB Expression in LECs after Application of p105 NF-κB siRNA in HLE B-3 Cell Culture
After various treatments, peripheral monolayers were rinsed with cold PBS and lysed with lysis buffer (20 mM Tris [pH 7.4]), 150 mM NaCl2, 1 mM ethylene glycol tetraacetic acid [EGTA], 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, and 10 μg/mL aprotinin). Identical amounts of protein lysates were resolved by 4% to 12% SDS-PAGE, followed by electroblotting onto nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% skimmed milk in PBS and then probed with p105 NF-κB antibody (1:5000). Donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP; 1:10,000) was used, and the immunoblots were detected with an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). 
In Vitro Capsular Bag Model
Making the Capsular Bag Model.
Twenty eyes of 10 pigs were obtained from an abattoir. After the cornea was removed, capsular bags were prepared by hydrodissection followed by capsulorhexis. The original shape of the capsule was maintained by inserting a tension ring (CTR98-A; Lucid Korea, Seoul, Korea) into the capsular bag. After treatment with p105 NF-κB siRNA or no treatment, the capsular bags were incubated in M199 medium for 3 weeks in an atmosphere of 5% CO2 at 38°C. The medium was changed every 72 hours. LEC proliferation was observed with a phase-contrast microscope (Axiovert S100; Carl Zeiss Meditec, Oberkochen, Germany). The model was established, and the observation replicated three to four times in each treatment. 
Immunocytochemistry of the Capsular Bag.
After 3 weeks in culture, the capsular bags were fixed for 2 hours in 10% formalin, washed for 10 minutes with PBS, and incubated with blocking solution containing 2% bovine serum albumin. Subsequently, the specimens were washed and incubated with anti-p50 primary antibody (1:200) for 1 hour at room temperature. After they were washed in PBS, the specimens were allowed to react with anti-rabbit IgG rhodamine-conjugated secondary antibody (1:500) for 1 hour at room temperature and counterstained with Hoechst. The stained specimens were examined with a fluorescence microscope (Axiovert S100; Carl Zeiss Meditec) and digital images were saved. 
Western Blot Analysis of the Capsular Bag for EMT and Proliferation Markers.
Capsular bags were rinsed with cold PBS and lysed with lysis buffer (20 mM Tris [pH 7.4]), 150 mM NaCl2, 1 mM ethylene glycol tetraacetic acid [EGTA], 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, and 10 μg/mL aprotinin). Identical amounts of protein lysates were resolved by 4% to 12% SDS-PAGE, followed by electroblotting onto nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% skimmed milk in PBS and then probed with fibronectin, α-SMA, and PCNA antibody (1:5000). Donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP; 1:10,000) was used, and the immunoblots were detected with an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). 
Cataract Surgery in Rabbits, Performed with p105 NF-κB siRNA
Fifteen New Zealand White rabbits weighing between 2.3 and 3.0 kg were used. All rabbits were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animal protocols were approved and monitored by the Animal Care Committee of the Research Laboratory of Experimental Animals in the College of Medicine, Catholic University of Korea. Specimens were divided randomly into four groups, each of which consisted of six eyes. Phacoemulsification and surgery were performed by the same surgeon in all cases (ITK) in a blind manner with regard to the treatment groups. 
Surgical Procedures.
The animals were anesthetized with 50 mg/kg ketamine hydrochloride and 10 mg/kg xylazine intramuscularly. Proparacaine hydrochloride ophthalmic solution was administered for topical anesthesia. Pupil dilation was obtained by repeated instillation of 1% cyclopentolate hydrochloride and 2.5% phenylephrine hydrochloride. The ocular area was disinfected with povidone iodine, and the lids were retracted with a wire lid speculum. A clear corneal incision was made with a 3.0-mm blade. Sodium hyaluronate was injected into the anterior chamber, and continuous curvilinear capsulorhexis was performed with capsular forceps, in a diameter of 5.0 to 6.0 mm. Phacoemulsification was performed, and the nucleus was removed with an ultrasound power of 20%, aspiration flow rate of 20 mL/min, and vacuum of 100 to 150 mm Hg. All cortical materials were removed carefully by irrigation/aspiration. Sodium hyaluronate was used to fill the capsulorhexis opening and anterior part of the capsular bag to prevent leakage of the drugs. Chondroitin sulfate was injected beneath the endothelium to protect the corneal endothelium. In the control group, the capsular bag was irrigated with 0.1 mL of PEG-CS-PLR polymer. In the nonsilencing siRNA group, the capsular bag was irrigated with nonsilencing siRNA. In two treatment groups, p105 NF-κB siRNA sequence 1 (concentration 200 picomoles) or p105 NF-κB siRNA sequence 8 (concentration 200 picomoles) was used to irrigate the capsular bag. These groups were designated the siRNA sequence 1 group and siRNA sequence 8 group, respectively. The NF-κB siRNA sequences 1 and 8 were chosen based on the results of cell culture and capsular bag model study (see the Results and Discussion sections for explanations). The drugs were administered with an anterior chamber cannula beneath the injected sodium hyaluronate. After 1 minute, the capsular bag was irrigated via irrigation and aspiration. Sodium hyaluronate was injected into the bag and an intraocular lens (SA60AT; Alcon Laboratories, Fort Worth, TX) was inserted into the bag. Viscoelastics were removed, and the corneal incision was sutured with 10-0 nylon sutures. After surgery, 0.3% ofloxacin and 1% prednisolone eyedrops were instilled four times a day. 
Assessment of Posterior Capsular Opacification.
The amount of PCO was determined by slit lamp biomicroscopy and POCOman software (St. Thomas Hospital and King's College, London, UK) 41 at 1, 2, 3, and 4 weeks after surgery. By slit lamp biomicroscopy, PCO was clinically graded on a scale of 0 to 3 as follows: 0, clear, no visible proliferative tissue on the peripheral or central posterior capsule; 1, mild, proliferative tissue in the peripheral area only; 2, moderate, sparse proliferative tissue on the peripheral and central capsules; and 3, dense, diffuse, and thick opacification on the entire capsule. 42  
Assessment of Central Corneal Thickness and Anterior Chamber Inflammation.
Central corneal thickness and anterior chamber inflammation were assessed weekly after surgery. Central corneal thickness was determined as the mean of three repeated measurements of each eye by ultrasonic pachymetry. Inflammation in the anterior chamber was graded on a scale of 0 to 4 by counting cells in a 3- × 1-mm area by slit lamp biomicroscopy: 0, rare cells; 1, 5 to 10 cells; 2, 11 to 20 cells; 3, 21 to 50 cells; and 4, >50 cells. 42 PCO grading and determination of anterior chamber inflammation by slit lamp biomicroscopy were performed independently by two observers. The grade was rechecked in cases of disagreement between the two observers. 
Histologic Examinations of the Posterior Capsule and Other Ocular Tissues.
One month after surgery, the rabbits were killed with an overdose of pentobarbital and their globes were enucleated. The eyes were fixed in 4% paraformaldehyde for 24 hours. The eyes were then sectioned in the coronal plane just anterior to the equator. The anterior segments were dehydrated in ethanol and embedded in paraffin. Sagittal sections were stained with hematoxylin and eosin. PCO and ocular tissues, including the cornea, iris, ciliary bodies, and choroid, were observed by light microscopy. 
Statistical Analysis
Statistical analysis was performed using Scheffé's post hoc test for comparison of PCO and anterior chamber inflammation between groups. Analysis of variance (ANOVA) was used to compare central corneal thickness between the groups (SPSS, ver. 11.0; SPSS Inc., Chicago, IL). The data of each group were compared and are expressed as the mean ± SD, with P < 0.05 taken to indicate significance. 
Results
Inhibition of p105 and p50 NF-κB Expression in LECs
Western blot analysis was performed to evaluate the effects of eight sequences of p105 NF-κB siRNA on inhibition of p105 and p50 NF-κB expression. As shown in Figure 1, all p105 NF-κB siRNA sequences reduced the levels of p105 and p50 NF-κB expression compared with control and nonsilencing siRNA groups. In particular, sequences 1, 2, 3, 7, and 8 resulted in lower levels of p105 and p50 NF-κB expression than the other sequences. 
Figure 1.
 
Western blot analysis to determine the expression of NF-κB p105 and p50 in LECs. All p105 NF-κB siRNA sequences reduced the expression of p105 NF-κB compared with control and nonsilencing siRNA groups. In particular, sequences 1, 2, 3, 7, and 8 showed lower levels of p105 NF-κB (arrows). α-Tubulin is also shown as a protein volume indicator. CON, control group; NS, nonsilencing siRNA group.
Figure 1.
 
Western blot analysis to determine the expression of NF-κB p105 and p50 in LECs. All p105 NF-κB siRNA sequences reduced the expression of p105 NF-κB compared with control and nonsilencing siRNA groups. In particular, sequences 1, 2, 3, 7, and 8 showed lower levels of p105 NF-κB (arrows). α-Tubulin is also shown as a protein volume indicator. CON, control group; NS, nonsilencing siRNA group.
Inhibition of the Migration of LECs in the Wound-Healing Assay
To confirm the relations between p105/p50 NF-κB inhibition and LEC migration, we examined the effects of NF-κB inhibitors on cultured HLE B-3 cells after a scratch injury using five sequences (p105 siRNA sequences 1, 2, 3, 7, and 8). As shown in Figure 2, the wounds in the control and nonsilencing siRNA groups were healed almost completely after 24 hours. However, the migration of HLE B-3 cells was decreased in all p105 siRNA sequence groups compared with that in the control and nonsilencing siRNA groups. In addition, the effect was greater in the NF-κB siRNA sequence 1 and 8 groups, indicating higher degrees of suppression of cell migration than the other siRNA sequences. The dose-dependent effect of the sequence 1 and 8 of p105 NF-κB siRNA were also compared by scratch assay. There was no significant inhibitory effect at a concentration of 50 pmol. However, the inhibitory effect of p105 NF-κB siRNA became apparent from 100 pmol, and the effect was significant over the concentration of 200 pmol in both sequences (Fig. 3). 
Figure 2.
 
Microscopy of wounded human LECs (HLE B-3). The HLE B-3 cells were wounded with a small yellow tip in culture dishes. Wounds in the control and nonsilencing siRNA groups were healed almost completely after 24 hours. Migration of HLE B-3 cells was significantly inhibited in the presence of p105 NF-κB siRNA sequences 1 and 8 relative to the other sequences (2, 3, and 7; bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 2.
 
Microscopy of wounded human LECs (HLE B-3). The HLE B-3 cells were wounded with a small yellow tip in culture dishes. Wounds in the control and nonsilencing siRNA groups were healed almost completely after 24 hours. Migration of HLE B-3 cells was significantly inhibited in the presence of p105 NF-κB siRNA sequences 1 and 8 relative to the other sequences (2, 3, and 7; bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 3.
 
Dose-dependent effects of the two sequences of p105 NF-κB siRNA were determined. The most effective p105 NF-κB siRNA sequences (sequence 1 and 8) were chosen by the results of Western blot analysis and scratch assay. The inhibitory effect of p105 NF-κB siRNA was apparent at concentrations over 100 pmol in both sequences. NS, nonsilencing siRNA group.
Figure 3.
 
Dose-dependent effects of the two sequences of p105 NF-κB siRNA were determined. The most effective p105 NF-κB siRNA sequences (sequence 1 and 8) were chosen by the results of Western blot analysis and scratch assay. The inhibitory effect of p105 NF-κB siRNA was apparent at concentrations over 100 pmol in both sequences. NS, nonsilencing siRNA group.
Inhibition of LEC Proliferation by p105 NF-κB siRNA in a Capsular Bag Model
Our findings were applied to a capsular bag model. We cultured capsular bags with or without p105/p50 NF-κB inhibitor. On microscopic examination, p105 NF-κB siRNA was shown to inhibit the growth of LECs after 3 weeks. However, LECs showed vigorous proliferation and migration over the capsulorhexis line in the control and nonsilencing groups at 3 weeks (Fig. 4). Counting the LECs in the posterior capsule indicated that p105 NF-κB siRNA sequences 1 and 8 significantly inhibited the growth of LECs relative to the control and nonsilencing groups (P < 0.05). In the groups treated with p105 NF-κB siRNA sequences 1 and 8, the mean number of cells in the posterior capsule were 227 ± 23 (43.6% of the control group count; n = 5) and 216 ± 19 (41.4% of control group count; n = 5), respectively. However, in the control and nonspecific siRNA groups, the number of cells in the posterior capsule were 521 ± 27 (n = 5) and 504 ± 29 (n = 5), respectively. 
Figure 4.
 
Microscopy of LECs in a capsular bag. Each photograph was taken 21 days after capsular bag culture, with or without p105 NF-κB inhibitor, sequences 1 and 8 of p105 NF-κB siRNA. On microscopic examination at 14 days without the inhibitor, control and nonsilencing siRNA groups showed vigorous proliferation beyond the capsulorhexis line, whereas sequences 1 and 8 p105 NF-κB siRNA inhibited LEC proliferation and migration. These p105 NF-κB inhibitors significantly decreased the number of cells in the posterior capsule (bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 4.
 
Microscopy of LECs in a capsular bag. Each photograph was taken 21 days after capsular bag culture, with or without p105 NF-κB inhibitor, sequences 1 and 8 of p105 NF-κB siRNA. On microscopic examination at 14 days without the inhibitor, control and nonsilencing siRNA groups showed vigorous proliferation beyond the capsulorhexis line, whereas sequences 1 and 8 p105 NF-κB siRNA inhibited LEC proliferation and migration. These p105 NF-κB inhibitors significantly decreased the number of cells in the posterior capsule (bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Immunocytochemistry of the Capsular Bag
Immunostaining was performed to verify the expression of p50 NF-κB protein and suppression of p50 NF-κB by p105 NF-κB siRNA in proliferating LECs at 3 weeks in the capsular bag. In the p105 NF-κB siRNA sequence 1 and 8 groups, p50 NF-κB was not present in the proliferating LECs, indicating inactivity of the p50 precursor (p105). In contrast, cells in control and nonspecific groups showed p50 NF-κB expression, indicating p50 precursor activity in proliferating cells (Fig. 5). 
Figure 5.
 
Immunocytochemistry of NF-κB. Light photomicrographs of Hoechst nuclear staining (top) and p50 NF-κB staining (bottom) after 3 weeks in culture. p50 NF-κB was observed in LECs of control and nonsilencing groups. However, p50 NF-κB was not observed in p105 NF-κB siRNA-treated groups (sequences 1 and 8, both). CON, control; NS, nonsilencing siRNA group.
Figure 5.
 
Immunocytochemistry of NF-κB. Light photomicrographs of Hoechst nuclear staining (top) and p50 NF-κB staining (bottom) after 3 weeks in culture. p50 NF-κB was observed in LECs of control and nonsilencing groups. However, p50 NF-κB was not observed in p105 NF-κB siRNA-treated groups (sequences 1 and 8, both). CON, control; NS, nonsilencing siRNA group.
Western Blot Analysis of the Capsular Bag for EMT and Proliferation Markers
Besides the effect on migration and proliferation of the LECs, Western blot analysis for the EMT markers (fibronectin and α-SMA) and the proliferation marker (PCNA) were done to find out the effect of p105 NF-κB siRNA on EMT and proliferation. As shown in Figure 6, p105 NF-κB siRNA sequences reduced the levels of fibronectin and α-SMA expression compared with that of the control and nonsilencing siRNA groups. The proliferation marker PCNA was also reduced with p105 NF-κB siRNA sequences 1 and 8. 
Figure 6.
 
Western blot analysis of the markers of EMT. The EMT markers (fibronectin and α-SMA) and the proliferation marker (PCNA) were analyzed in a capsular bag model and in normal pig lens epithelial cells (LEP). Both the EMT and proliferation markers were decreased after application of p105 NF-κB siRNA sequences 1 and 8 in a capsular bag. Actin is shown as a protein volume indicator. CON, control; NS, nonsilencing siRNA group.
Figure 6.
 
Western blot analysis of the markers of EMT. The EMT markers (fibronectin and α-SMA) and the proliferation marker (PCNA) were analyzed in a capsular bag model and in normal pig lens epithelial cells (LEP). Both the EMT and proliferation markers were decreased after application of p105 NF-κB siRNA sequences 1 and 8 in a capsular bag. Actin is shown as a protein volume indicator. CON, control; NS, nonsilencing siRNA group.
Cataract Surgery in Rabbits, with p105 NF-κB siRNA
Assessment of PCO by Slit Lamp Biomicroscopy.
The development of PCO was assessed after cataract surgery by using p105 NF-κB siRNA. At 1 month after surgery, PCO grading was performed by slit lamp biomicroscopy. In the control group, three eyes showed moderate PCO and three eyes demonstrated severe PCO. The mean PCO score was 2.50 ± 0.76. In the nonsilencing group, one eye showed mild PCO, two eyes showed moderate PCO, and three eyes showed severe PCO. The mean PCO score in the nonsilencing group was 2.43 ± 0.44. In the siRNA sequence 1 group, four eyes were clear, one eye showed mild PCO, and one eye showed moderate PCO. The mean PCO score in this group was significantly decreased to 0.51 ± 0.33. In the siRNA sequence 8 group, two eyes were clear, two eyes showed mild PCO, and two eyes showed moderate PCO. The mean PCO score was 0.99 ± 0.46, also indicating a significant decrease of PCO in that group (Table 2). 
Table 2.
 
Comparison of PCO Scores by Slit Lamp Biomicroscopy at 1 Month after Surgery
Table 2.
 
Comparison of PCO Scores by Slit Lamp Biomicroscopy at 1 Month after Surgery
Score CON NS siRNA Seq. 1 siRNA Seq. 8
0: Clear 4 2
1: Mild 1 1 2
2: Moderate 3 2 1 2
3: Severe 3 3
Mean 2.50 ± 0.76 2.43 ± 0.44 0.51 ± 0.33 0.99 ± 0.46
Central Corneal Thickness and Anterior Chamber Reaction.
No significant differences were observed in central corneal thickness or anterior chamber inflammation after surgery in the four groups (Tables 3, 4). 
Table 3.
 
Comparison of Central Corneal Thickness in Each Group
Table 3.
 
Comparison of Central Corneal Thickness in Each Group
Time after Surgery CON NS siRNA Seq. 1 siRNA Seq. 8 P
1 week 451.50 ± 70.28 461.25 ± 50.41 450.23 ± 65.34 442.20 ± 70.28 0.380
2 weeks 418.75 ± 23.21 423.23 ± 17.53 424.45 ± 37.05 420.55 ± 44.25 0.525
3 weeks 420.75 ± 93.91 422.55 ± 64.33 424.33 ± 17.76 414.52 ± 63.21 0.577
4 weeks 414.25 ± 40.62 416.75 ± 32.42 417.47 ± 27.60 411.30 ± 37.23 0.420
Table 4.
 
Comparison of Anterior Chamber Inflammation in Each Group
Table 4.
 
Comparison of Anterior Chamber Inflammation in Each Group
Time after Surgery CON NS siRNA Seq. 1 siRNA Seq. 8 P
1 week 2.00 ± 0.93 1.98 ± 0.66 1.75 ± 0.88 2.00 ± 0.70 0.483
2 weeks 0.75 ± 0.75 0.93 ± 0.84 0.75 ± 0.50 1.00 ± 0.55 0.352
3 weeks 0.50 ± 0.43 0.60 ± 0.41 0.50 ± 0.52 0.50 ± 0.44 0.411
4 weeks 0.25 ± 0.27 0.23 ± 0.37 0.25 ± 0.41 0.25 ± 0.37 0.780
Assessment of PCO.
The PCO grade was compared among the four groups with POCOman software 41 (Fig. 7). Although no significant differences were observed among the groups at 1 or 2 weeks after surgery, significant differences in PCO score were observed at 3 and 4 weeks after surgery. PCO scores in the control group and nonsilencing siRNA group were 1.15 ± 0.71 and 1.19 ± 0.32, respectively, at 3 weeks after surgery. The PCO score was significantly decreased in the p105 NF-κB siRNA treatment groups, with PCO scores of 0.25 ± 0.17 and 0.42 ± 0.40 in the p105 NF-κB siRNA sequence 1 and 8 groups, respectively. At 4 weeks after surgery, PCO scores were 1.51 ± 0.53 and 1.44 ± 0.20 in the control group and nonsilencing siRNA group, respectively. However, the PCO scores were significantly decreased in the p105 NF-κB siRNA sequence 1 and 8 groups, with values of 0.28 ± 0.31 and 0.62 ± 0.45, respectively (Fig. 8). 
Figure 7.
 
PCO grade compared using POCOman software. Figures show the rabbit posterior capsule at 1 week (A) and 1 month after surgery (B) in each group. PCO decreased in the p105 NF-κB siRNA group. CON, control; NS, nonsilencing siRNA group.
Figure 7.
 
PCO grade compared using POCOman software. Figures show the rabbit posterior capsule at 1 week (A) and 1 month after surgery (B) in each group. PCO decreased in the p105 NF-κB siRNA group. CON, control; NS, nonsilencing siRNA group.
Figure 8.
 
Comparison of PCO scores by POCOman software at each observation period. At 3 and 4 weeks after surgery, significant differences were observed in PCO scores in the p105 NF-κB applied groups (sequences 1 and 8) compared with the control and nonsilencing siRNA groups. CON, control; NS, nonsilencing siRNA group.
Figure 8.
 
Comparison of PCO scores by POCOman software at each observation period. At 3 and 4 weeks after surgery, significant differences were observed in PCO scores in the p105 NF-κB applied groups (sequences 1 and 8) compared with the control and nonsilencing siRNA groups. CON, control; NS, nonsilencing siRNA group.
Histologic Examination of the Posterior Capsule and Other Ocular Tissues
The posterior capsule and the corneal endothelium were observed under a light microscope. In the control and nonsilencing siRNA groups, proliferative LECs and a thickened posterior capsule were observed. However, the p105 NF-κB siRNA treatment groups showed a significantly reduced number of LECs in the posterior capsule. No abnormalities were observed in the corneal endothelium in the group treated with p105 NF-κB siRNA (Fig. 9). 
Figure 9.
 
Light microscopy of the posterior capsule and cornea endothelium after cataract surgery. Proliferating LECs and a thickened posterior capsule were observed in the control and nonsilencing siRNA groups. However, the p105 NF-κB siRNA-treated group showed a significantly reduced number of LECs in the posterior capsule. No abnormalities were observed in the corneal endothelium of the p105 NF-κB siRNA-treated group. CON, control; NS, nonsilencing siRNA group.
Figure 9.
 
Light microscopy of the posterior capsule and cornea endothelium after cataract surgery. Proliferating LECs and a thickened posterior capsule were observed in the control and nonsilencing siRNA groups. However, the p105 NF-κB siRNA-treated group showed a significantly reduced number of LECs in the posterior capsule. No abnormalities were observed in the corneal endothelium of the p105 NF-κB siRNA-treated group. CON, control; NS, nonsilencing siRNA group.
Discussion
PCO can be reduced by thorough cortical cleanup, atraumatic surgery with a continuous curvilinear capsulorhexis (CCC), IOL implantation in the bag, reducing postoperative inflammation, and improving IOL materials and designs. 4345 These improvements have served mainly to delay the onset of PCO. However, PCO is still a problem, and the only effective treatment of PCO is Nd:YAG laser capsulotomy. Although this procedure is easy and quick, there are several complications and a considerable cost burden. Therefore, understanding of the pathogenic mechanism of PCO and development of an alternative medical treatment of PCO is of critical importance. Some in vitro and in vivo studies have indicated the possible efficacy of antimitotics and antimetabolites, such as mitomycin-C, daunomycin, 5-fluorouracil, and colchicines. EDTA, retinoic acid, cyclosporin A, diclofenac, and corticosteroids have also been shown to control PCO. 4553 However, appropriate concentrations for use in vivo have not been determined. The risk of toxic effects on surrounding intraocular tissues has restricted their clinical use. 
Our previous study in a capsular bag model indicated that NF-κB activation and proliferation of LECs are related. 21 The LECs in the proliferating region showed nuclear localization of NF-κB, indicating that activation of NF-κB occurred in proliferating cells. When NF-κB inhibitors, such as NF-κB SN50 peptide or 200 μM sulfasalazine, were used, the NF-κB level decreased and LEC proliferation in the capsular bag model was inhibited. For further in vivo study with rabbits, 200 μM of sulfasalazine was used; however, undesirable toxic effects on the cornea and marked anterior chamber inflammation occurred. We diluted the concentration to 100 μM and then to 50 μM. Toxic effects were also observed with 100 μM, and no toxic effects were observed with 50 μM sulfasalazine. However, the inhibiting effect of 50 μM sulfasalazine was weak, and the PCO grade was not significantly decreased compared with that of the control group (results not shown). This experience led us to seek a safer and more effective method of decreasing NF-κB in LECs. 
RNA interference has become an almost standard method for in vitro knockdown of any target gene of interest. The major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. Numerous studies targeting liver, kidney, lung, ear, heart, tumors, blood, and the central nervous system have been conducted. Different modes of administration and various siRNA formulations have already shown promising results. Advantages of applying siRNAs include the relatively easy chemical synthesis of small RNA molecules, safety, and lower probability of nonspecific side effects. Using siRNA for NF-κB, we attempted to reduce the expression of NF-κB and therefore NF-κB-dependent signaling in LECs. In addition, we explored the role of NF-κB siRNA in cell culture and a capsular bag model. The most effective NF-κB siRNA sequences and a concentration of 200 pmol were chosen and applied during cataract surgery in a rabbit model. Specifically, the p50 NF-κB subtype was inhibited by p105 NF-κB siRNA, which inhibits the precursor of p50 NF-κB. 
Western blot analysis of the LECs indicated that p105 and p50 NF-κB protein levels decreased after the application of eight types of p105 NF-κB siRNA. With the application of p105 NF-κB siRNA, both p105 and p50 NF-κB protein levels were reduced by transcriptional inhibition, as p105 NF-κB is a precursor of p50 NF-κB. The migration of LECs was also inhibited by the application of p105 NF-κB siRNA to HLE B-3 cells. These results indicated that p105 NF-κB siRNA effectively inhibits translation of p105 and p50 NF-κB and decreases NF-κB protein levels in LECs, leading to a decrease in the migration of LECs in cell culture. In the capsular bag model study, p105 NF-κB siRNA sequences 1 and 8 reduced LEC proliferation. In addition, immunocytochemical analysis of the capsule showed decreased p50 staining. In the in vivo study performed in New Zealand White rabbits, the use of NF-κB siRNA during cataract surgery effectively decreased PCO, as determined by both slit lamp examination and the POCOman software 41 assessment. 
NF-κB siRNA did not significantly change central corneal thickness and anterior chamber inflammation from those in the controls. In addition, the histologic appearance of the corneal endothelium was similar to that of the other groups. These results suggest that using p105 NF-κB siRNA in vivo may be safe for other ocular tissues. With NF-κB siRNA, we found no complications in other ocular tissues. NF-κB siRNA targets the gene in the cell nucleus and dividing cells such as LECs, which may be more susceptible to the inhibitory effect of NF-κB siRNA than quiescent confluent cells, such as those in the corneal endothelium. A careful surgical technique with administration of the drugs into the capsular bag using a cannula beneath the injected sodium hyaluronate may minimize the toxicity to other ocular tissues. 
The inhibitory effect on migration was greater with p105 NF-κB siRNA sequence 1 (5′-GCCAAAGAAGGACATGATAAA-3′) than with sequence 8 (5′-CTACGTTCCTATTGTCATTAA-3′). The cell count in the posterior capsule of the capsular bag model showed that siRNA sequence 8 had a slightly stronger inhibitory effect on cell proliferation than did siRNA sequence 1. The in vivo results indicated that PCO was significantly decreased by siRNA sequence 1 in comparison to siRNA sequence 8. It seemed that siRNA sequence 1 was more effective in inhibiting LEC migration and siRNA sequence 8 was more effective in inhibiting LEC proliferation. Suppressing LEC proliferation and migration may be an effective strategy for preventing PCO; it may disturb the early phase of PCO formation and stop further mechanisms that promote EMT. These two sequences of p105 NF-κB siRNA also reduced the levels of fibronectin and α-SMA expression, showing the potential to inhibit EMT during PCO formation. However, further studies investigating the underlying mechanism of NF-κB in LEC proliferation and migration are needed, with the confirmation of the changes induced by NF-κB siRNA. Fibroblast growth factor-2, 54 hepatocyte growth factor, 54,55 and transforming growth factor-β 5658 have been shown to initiate LEC proliferation. Fibroblast growth factor-2, epithelial growth factor, and matrix metalloproteinases 59,60 have been related to LEC migration. Confirming these changes in relation to NF-κB siRNA may be of value. Also studying changes of key signaling pathways for EMT may provide more information to understand the pathogenesis of PCO. 
In summary, NF-κB is related to the migration and proliferation of LECs. Suppression of the NF-κB pathway using NF-κB siRNA was effective in inhibiting the migration and proliferation of LECs in cell cultures and the capsular bag model. There were results showing NF-κB siRNA as a potential inhibitor of EMT in PCO formation. In addition, PCO formation after cataract surgery in rabbits was effectively reduced by this treatment. No toxicity occurred in other ocular tissues, and the procedure was simple and easy to perform during cataract surgery, making it applicable to routine cataract surgery for the prevention of PCO. 
Footnotes
 Supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A092258).
Footnotes
 Disclosure: H.-Y.L. Park, None; I.-T. Kim, None; K.M. Lee, None; J.S. Choi, None; M.-O. Park, None; C.-K. Joo, None
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Figure 1.
 
Western blot analysis to determine the expression of NF-κB p105 and p50 in LECs. All p105 NF-κB siRNA sequences reduced the expression of p105 NF-κB compared with control and nonsilencing siRNA groups. In particular, sequences 1, 2, 3, 7, and 8 showed lower levels of p105 NF-κB (arrows). α-Tubulin is also shown as a protein volume indicator. CON, control group; NS, nonsilencing siRNA group.
Figure 1.
 
Western blot analysis to determine the expression of NF-κB p105 and p50 in LECs. All p105 NF-κB siRNA sequences reduced the expression of p105 NF-κB compared with control and nonsilencing siRNA groups. In particular, sequences 1, 2, 3, 7, and 8 showed lower levels of p105 NF-κB (arrows). α-Tubulin is also shown as a protein volume indicator. CON, control group; NS, nonsilencing siRNA group.
Figure 2.
 
Microscopy of wounded human LECs (HLE B-3). The HLE B-3 cells were wounded with a small yellow tip in culture dishes. Wounds in the control and nonsilencing siRNA groups were healed almost completely after 24 hours. Migration of HLE B-3 cells was significantly inhibited in the presence of p105 NF-κB siRNA sequences 1 and 8 relative to the other sequences (2, 3, and 7; bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 2.
 
Microscopy of wounded human LECs (HLE B-3). The HLE B-3 cells were wounded with a small yellow tip in culture dishes. Wounds in the control and nonsilencing siRNA groups were healed almost completely after 24 hours. Migration of HLE B-3 cells was significantly inhibited in the presence of p105 NF-κB siRNA sequences 1 and 8 relative to the other sequences (2, 3, and 7; bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 3.
 
Dose-dependent effects of the two sequences of p105 NF-κB siRNA were determined. The most effective p105 NF-κB siRNA sequences (sequence 1 and 8) were chosen by the results of Western blot analysis and scratch assay. The inhibitory effect of p105 NF-κB siRNA was apparent at concentrations over 100 pmol in both sequences. NS, nonsilencing siRNA group.
Figure 3.
 
Dose-dependent effects of the two sequences of p105 NF-κB siRNA were determined. The most effective p105 NF-κB siRNA sequences (sequence 1 and 8) were chosen by the results of Western blot analysis and scratch assay. The inhibitory effect of p105 NF-κB siRNA was apparent at concentrations over 100 pmol in both sequences. NS, nonsilencing siRNA group.
Figure 4.
 
Microscopy of LECs in a capsular bag. Each photograph was taken 21 days after capsular bag culture, with or without p105 NF-κB inhibitor, sequences 1 and 8 of p105 NF-κB siRNA. On microscopic examination at 14 days without the inhibitor, control and nonsilencing siRNA groups showed vigorous proliferation beyond the capsulorhexis line, whereas sequences 1 and 8 p105 NF-κB siRNA inhibited LEC proliferation and migration. These p105 NF-κB inhibitors significantly decreased the number of cells in the posterior capsule (bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 4.
 
Microscopy of LECs in a capsular bag. Each photograph was taken 21 days after capsular bag culture, with or without p105 NF-κB inhibitor, sequences 1 and 8 of p105 NF-κB siRNA. On microscopic examination at 14 days without the inhibitor, control and nonsilencing siRNA groups showed vigorous proliferation beyond the capsulorhexis line, whereas sequences 1 and 8 p105 NF-κB siRNA inhibited LEC proliferation and migration. These p105 NF-κB inhibitors significantly decreased the number of cells in the posterior capsule (bottom; P < 0.05). CON, control; NS, nonsilencing siRNA group.
Figure 5.
 
Immunocytochemistry of NF-κB. Light photomicrographs of Hoechst nuclear staining (top) and p50 NF-κB staining (bottom) after 3 weeks in culture. p50 NF-κB was observed in LECs of control and nonsilencing groups. However, p50 NF-κB was not observed in p105 NF-κB siRNA-treated groups (sequences 1 and 8, both). CON, control; NS, nonsilencing siRNA group.
Figure 5.
 
Immunocytochemistry of NF-κB. Light photomicrographs of Hoechst nuclear staining (top) and p50 NF-κB staining (bottom) after 3 weeks in culture. p50 NF-κB was observed in LECs of control and nonsilencing groups. However, p50 NF-κB was not observed in p105 NF-κB siRNA-treated groups (sequences 1 and 8, both). CON, control; NS, nonsilencing siRNA group.
Figure 6.
 
Western blot analysis of the markers of EMT. The EMT markers (fibronectin and α-SMA) and the proliferation marker (PCNA) were analyzed in a capsular bag model and in normal pig lens epithelial cells (LEP). Both the EMT and proliferation markers were decreased after application of p105 NF-κB siRNA sequences 1 and 8 in a capsular bag. Actin is shown as a protein volume indicator. CON, control; NS, nonsilencing siRNA group.
Figure 6.
 
Western blot analysis of the markers of EMT. The EMT markers (fibronectin and α-SMA) and the proliferation marker (PCNA) were analyzed in a capsular bag model and in normal pig lens epithelial cells (LEP). Both the EMT and proliferation markers were decreased after application of p105 NF-κB siRNA sequences 1 and 8 in a capsular bag. Actin is shown as a protein volume indicator. CON, control; NS, nonsilencing siRNA group.
Figure 7.
 
PCO grade compared using POCOman software. Figures show the rabbit posterior capsule at 1 week (A) and 1 month after surgery (B) in each group. PCO decreased in the p105 NF-κB siRNA group. CON, control; NS, nonsilencing siRNA group.
Figure 7.
 
PCO grade compared using POCOman software. Figures show the rabbit posterior capsule at 1 week (A) and 1 month after surgery (B) in each group. PCO decreased in the p105 NF-κB siRNA group. CON, control; NS, nonsilencing siRNA group.
Figure 8.
 
Comparison of PCO scores by POCOman software at each observation period. At 3 and 4 weeks after surgery, significant differences were observed in PCO scores in the p105 NF-κB applied groups (sequences 1 and 8) compared with the control and nonsilencing siRNA groups. CON, control; NS, nonsilencing siRNA group.
Figure 8.
 
Comparison of PCO scores by POCOman software at each observation period. At 3 and 4 weeks after surgery, significant differences were observed in PCO scores in the p105 NF-κB applied groups (sequences 1 and 8) compared with the control and nonsilencing siRNA groups. CON, control; NS, nonsilencing siRNA group.
Figure 9.
 
Light microscopy of the posterior capsule and cornea endothelium after cataract surgery. Proliferating LECs and a thickened posterior capsule were observed in the control and nonsilencing siRNA groups. However, the p105 NF-κB siRNA-treated group showed a significantly reduced number of LECs in the posterior capsule. No abnormalities were observed in the corneal endothelium of the p105 NF-κB siRNA-treated group. CON, control; NS, nonsilencing siRNA group.
Figure 9.
 
Light microscopy of the posterior capsule and cornea endothelium after cataract surgery. Proliferating LECs and a thickened posterior capsule were observed in the control and nonsilencing siRNA groups. However, the p105 NF-κB siRNA-treated group showed a significantly reduced number of LECs in the posterior capsule. No abnormalities were observed in the corneal endothelium of the p105 NF-κB siRNA-treated group. CON, control; NS, nonsilencing siRNA group.
Table 1.
 
Eight Sequences of p105 NF-κB siRNA
Table 1.
 
Eight Sequences of p105 NF-κB siRNA
Number p105 NF-κB siRNA Sequence
1 5′-GCCAAAGAAGGACATGATAAA-3′
2 5′-TGGCAGGTATTTGACATATTA-3′
3 5′-TCTGATGATTTACTAGCACAA-3′
4 5′-ATGTGAAGCTGCAGCTGTATA-3′
5 5′-AGAAATTCCTGATCCAGACAA-3′
6 5′-GATCCTTCTTTGACTCATACA-3′
7 5′-GCCTGAATCATTCTCGATTTA-3′
8 5′-CTACGTTCCTATTGTCATTAA-3′
Table 2.
 
Comparison of PCO Scores by Slit Lamp Biomicroscopy at 1 Month after Surgery
Table 2.
 
Comparison of PCO Scores by Slit Lamp Biomicroscopy at 1 Month after Surgery
Score CON NS siRNA Seq. 1 siRNA Seq. 8
0: Clear 4 2
1: Mild 1 1 2
2: Moderate 3 2 1 2
3: Severe 3 3
Mean 2.50 ± 0.76 2.43 ± 0.44 0.51 ± 0.33 0.99 ± 0.46
Table 3.
 
Comparison of Central Corneal Thickness in Each Group
Table 3.
 
Comparison of Central Corneal Thickness in Each Group
Time after Surgery CON NS siRNA Seq. 1 siRNA Seq. 8 P
1 week 451.50 ± 70.28 461.25 ± 50.41 450.23 ± 65.34 442.20 ± 70.28 0.380
2 weeks 418.75 ± 23.21 423.23 ± 17.53 424.45 ± 37.05 420.55 ± 44.25 0.525
3 weeks 420.75 ± 93.91 422.55 ± 64.33 424.33 ± 17.76 414.52 ± 63.21 0.577
4 weeks 414.25 ± 40.62 416.75 ± 32.42 417.47 ± 27.60 411.30 ± 37.23 0.420
Table 4.
 
Comparison of Anterior Chamber Inflammation in Each Group
Table 4.
 
Comparison of Anterior Chamber Inflammation in Each Group
Time after Surgery CON NS siRNA Seq. 1 siRNA Seq. 8 P
1 week 2.00 ± 0.93 1.98 ± 0.66 1.75 ± 0.88 2.00 ± 0.70 0.483
2 weeks 0.75 ± 0.75 0.93 ± 0.84 0.75 ± 0.50 1.00 ± 0.55 0.352
3 weeks 0.50 ± 0.43 0.60 ± 0.41 0.50 ± 0.52 0.50 ± 0.44 0.411
4 weeks 0.25 ± 0.27 0.23 ± 0.37 0.25 ± 0.41 0.25 ± 0.37 0.780
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