August 2015
Volume 56, Issue 9
Free
Glaucoma  |   August 2015
Evaluation of Chitosan/Aptamer Targeting TGF-β Receptor II Thermo-Sensitive Gel for Scarring in Rat Glaucoma Filtration Surgery
Author Affiliations & Notes
  • Xiaoyan Zhu
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Duo Xu
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Xiaomin Zhu
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Lei Li
    Department of Molecular Biology Center, State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Haijun Li
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Feng Guo
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Xia Chen
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Yan Tan
    Department of Molecular Biology Center, State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Lin Xie
    Department of Ophthalmology Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
  • Correspondence: Lin Xie, Department of Ophthalmology, DaPing Hospital, Third Military Medical University, No. 10, Changjiang Branch Road, Chongqing, PR China 400042; xielin_66@163.com
Investigative Ophthalmology & Visual Science August 2015, Vol.56, 5465-5476. doi:10.1167/iovs.15-16683
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      Xiaoyan Zhu, Duo Xu, Xiaomin Zhu, Lei Li, Haijun Li, Feng Guo, Xia Chen, Yan Tan, Lin Xie; Evaluation of Chitosan/Aptamer Targeting TGF-β Receptor II Thermo-Sensitive Gel for Scarring in Rat Glaucoma Filtration Surgery. Invest. Ophthalmol. Vis. Sci. 2015;56(9):5465-5476. doi: 10.1167/iovs.15-16683.

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

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Abstract

Purpose: This study was designed to develop a chitosan (CS) thermo-sensitive gel combined with aptamer S58 targeting transforming growth factor-beta receptor II (TGF-β RII) and to investigate the antifibrotic effects of CS/S58 gel in a rat glaucoma filtration surgery (GFS) model.

Methods: In vitro aptamer S58 release rate from the CS/S58 gel were detected, and the effect of mitomycin-C (MMC), TGF-β2, CS, or CS/S58 gel on wound healing were investigated in a rat GFS model by detecting scar-related factors and the involved inflammatory response. The levels of collagen I and α-smooth muscle actin (α-SMA) were detected by immunohistochemistry and Western blotting.

Results: The control and TGF-β2 eyes exhibited densely packed collagen fibers with no evidence of filtration after day 7. The pronounced increase in filtration efficiency was associated with thinner fibers, and a loosely organized subconjunctival matrix was observed in CS/S58 gel–treated eyes. The levels of collagen I and α-SMA were downregulated in CS/S58 gel–treated eyes. Conjunctival fibroblast proliferation and the inflammation response were also suppressed in the CS/S58 gel–treated group.

Conclusions: This study presents evidence that the antifibrotic effect of chitosan in combination with aptamer S58 is superior to chitosan alone in a rat GFS model. Chitosan/S58 gel may be considered to be a promising antifibrotic agent for a local drug therapy.

Glaucoma filtration surgery (GFS) is a major treatment for glaucoma that may decrease the IOP by draining the aqueous humor to the subconjunctival space, forming a bleb; subconjunctival scarring of the filtering bleb is the most important cause of the surgery failure. Although applications of mitomycin-C (MMC) or 5-flurouracil (5-FU) have been demonstrated to inhibit scarring and improve GFS outcomes in large prospective randomized trials, sight-threatening complications greatly hinder their clinical use.1 Thus, new antifibrotic agents capable of inhibiting postoperative scarring are urgently needed. 
Ample evidence supports that expression of TGF-β activates the proliferation, migration, synthesis of extracellular matrix components by human Tenon's fibroblasts (HTF), and excessive production of granulation tissue constituents, such as collagen, leading to scar formation.24 Studies have indicated that TGF-β may be an effective strategy to reduce scarring by impairing TGF-β activity through neutralization with antibodies,5,6 inactivation by proteoglycans-like decorin,7,8 and blockage of function by exogenous receptors.7,8 Despite the discovery of a number of active compounds that could serve as therapeutics, notably few candidates have had clinical success. The compound's poor activity in vivo is most often attributed to their low bioavailability, the extent and rate at which a drug reaches at target tissue. In addition, the bioavailability of traditional ocular drug delivery systems for treatment, such as eye drops, is poor due to the rapid loss and low corneal penetration of drugs. Thus, readministration is often required, which can lead to decreased patient compliance. 
In the past decades, chitosan-based drug delivery systems in various physical gel forms have been developed,9,10 As a natural polymer, chitosan thermosensitive hydrogels have gained especially great interest in pharmaceutics due to their nontoxicity, biodegradability, biocompatibility, and antimicrobial activity. Chitosan-based hydrogels are also promising biomaterials that can provide sustained, local delivery of agents, such as proteins, peptides, oligonucleotides, and genes, for therapeutic applications.11 Specific examples of chitosan-based hydrogels can be observed in cancer therapeutics,12 subcutaneous release, and oral delivery.13,14 The uses of chitosan-based hydrogels in wound dressing applications have also been widely studied.15,16 Studies have demonstrated that chitosan can protect encapsulated DNA from nuclease degradation17 and inhibit the proliferation of keloid fibroblasts.18 Therefore, chitosan-based hydrogels should be considered as an ocular drug delivery system for GFS. Our previous study revealed that nucleic acid aptamers targeting TGF-β receptor II (TβRII) were isolated and developed in vitro by systematic evolution of ligands by exponential enrichment (SELEX). We demonstrated in vitro that aptamer S58 could inhibit TGF-β2–induced myofibroblast transdifferentiation in HTFs,19 and a novel chitosan nanoparticle-S58 complexes was synthesized to preserve and prolong the efficacy of S58 for aptamer delivery in vitro.20 
In this study, we suggest aptamer S58-conjugated chitosan-based hydrogel (CS/S58) as a type of injectable agent to obtain a controlled delivery system in vivo. The present study investigated whether application of chitosan gel with or without aptamer S58 improved bleb survival in a rat GFS model and compared its effectiveness with the currently used postoperative antiscarring agent MMC. Histologic assessments were conducted following GFS to investigate wound healing and the inflammatory response to the CS or CS/S58 hydrogel. 
Materials and Methods
Materials
Aptamer S58 targeting human TGF-β RII (sense strand: 5′-ACATTGCTGCGTGATCGCCTCACATGGGTTTGTCTGGTCGATTTGGAGGTGGTGGGTGGC-3′) was synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China). Chitosan (deacetylation degree > 90%) was purchased from Shanghai Sangon Biotechnology Co. Ltd. Disodium β-GP (glycerol 2-phosphate disodium salt hydrate; cell culture grade) was obtained from Sigma Aldrich (Poole, UK). Antibodies against the following proteins were used: and α-smooth muscle actin (α-SMA; Abcam, Hong Kong, China), collagen I (Abcam), Ki67 (Abcam), CD45 (Abcam), β-actin (Sigma-Aldrich), horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology Co. Ltd., Beijing, China), and secondary antibodies conjugated to either AlexaFlour-488 or AlexaFluor-594 (Invitrogen, Eugene, OR, USA). The TUNEL cell analysis kit was provided by the Beyotime Institute of Biotechnology (Haimen, China). 
Preparation of the Chitosan/S58 (CS/S58) Hydrogel and S58 Release Analysis
The chitosan/β-glycerophosphate solution (CS/GP) was prepared following the method described previously.12 A 2% (wt/vol) chitosan solution was prepared stirring powdered chitosan in 0.5% (vol/vol) aqueous acetic acid at room temperature overnight. The insoluble particles were removed by filtration. A 50% (wt/vol) β-GP solution was prepared in distilled water and sterilized using PES syringe filters (0.22 μm) and stored at 4°C. The final pH value of the chitosan/β-GP solutions ranged from 6.5 to 7.2. The solutions were subsequently placed in 10-mL tubes and stored at 4°C. The aptamer S58 solution with a concentration of 100 μM was prepared following instructions from Sangon Biotechnology. The S58 solution was added to the CS/GP solution at a volume ratio of 1:10, and the mixture was vortexed for 1 minute and further incubated for 2 to 3 minute at 37°C to ensure CS/S58 hydrogel formation. 
For the aptamer S58 release study, the CS/S58 hydrogels (1 mL) were immersed in 100 μL distilled water at 37°C for different times, and then, the mixture was centrifuged at 11,180g for 10 minutes. The DNA concentration of the supernatant was analyzed, which accounted for the released S58, using a UV/Vis scanning spectrophotometer (JP; Shimadzu, Kyoto, Japan). To determine the integrity of the released S58, the samples were analyzed by 4% agarose gel electrophoresis in Tris-boric acid-EDTA (TBE) buffer.20 Electrophoresis was formed at a constant voltage of 110 V for 80 minutes, and then the gels were stained by SYBR Gold nucleic acid stain and visualized using an imager (Gel Doc XR; Bio-Rad, Hercules, CA, USA). 
Rat Model of Glaucoma Filtration Surgery
Adult male Sprague-Dawley rats (8–10 weeks old, 200–300 g) were used. All experiments with animals were approved by the Institutional Animal Care of Third Military Medical University and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were anaesthetized by intraperitoneal (i.p.) injection of 10 mg/mL chloral hydrate (Sangon Biotechnology Co., Ltd.) before the operation was conducted. Additional topical anaesthesia was provided in the form of 0.5% oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co., Ltd. Osaka, Japan). Glaucoma filtration surgery was performed on the left eyes of rats, as previously described by Douglas et al.21 
A limbus-based conjunctival flap was created 3 to 5 mm behind the limbus by making a conjunctival incision and elevating the underlying Tenon's capsule by blunt dissection. A full-thickness scleral tunnel was then created with a 25-G needle that was inserted into the anterior chamber (AC), taking great care to avoid the iridal blood vessels. Rats exhibiting any hyphema were not included. Viscoelastic solution (Curamed Ophthalmics, Inc., Vianen, The Netherlands) was injected through the needle to maintain the AC. A beveled 30-G micro cannula (Fine Science Tools, Foster City, CA, USA) was then inserted through the scleral tunnel. The micro cannula was fixed at the limbus, and then, the conjunctiva and Tenon's capsule were closed using a 10-0 (0.1 metric) Ethicon monofilament nylon suture (Ethicon, Inc., Guangzhou, China). The eyes exhibiting subsequent slippage or dislocation of the cannula were excluded. 
The rats operated eyes were treated with TGF-β2 (PeproTech, Rocky Hill, NJ, USA), MMC (Kyowa Hakko Kirin Co., Ltd., Shizuoko, Japan), CS or CS/S58 gel separately. Transforming growth factor–β2 was applied at 2 ng/mL, and MMC was applied at 0.4 mg/mL with a small piece of cellulose sponge for 1 minute. Then irrigation of the treated area was performed with 2 mL 0.9% sodium chloride using a syringe. While CS or CS/S58 gel (50 μL) was placed under the dissected conjunctiva, secured and closed by a 10-0 suture. Control conditions were performed on the opposing eyes. Each group treatment was performed on at least 12 eyes. 
Clinical Examination and Analysis of Blebs
The rats were anaesthetized prior to IOP measurement. Intraocular pressures were measured in each rat with a handheld commercial rebound tonometer (TonoLab; Icare, Espoo, Finland) according to the manufacturer's instructions. All IOP measurements were taken 5 to 10 times in each eye, and the mean IOP value of the operated eyes was expressed as a percentage over that of the unoperated eyes. 
Slit-lamp examination and anterior segment optical coherence tomography (AS-OCT) were performed on subconjunctival blebs. Bleb survival was based on the clinical appearance and masked grading of the bleb. A bleb was judged to be failed if the surgical site appeared flat and vascularized by slit-lamp analysis. 
Histology and Immunofluorescent Analysis
Rats were euthanized on day 7 after surgery. The eyes were enucleated for immediate fixation. The eyes were fixed in 4% paraformaldehyde and paraffin embedded. The samples were sectioned (4 μm) with the Microm HM550 (Carl Zeiss Ltd., Oberkochen, Germany). The sections were washed with xylene twice for 15 minutes each, and then, the slides were immersed in a series of ethanol solutions for 5 minutes each (twice in 100% (vol/vol), once in 95%, once in 80%, once in 70%), followed by 2-minute exchanges of water. The sections were stained with hematoxylin and eosin (H&E) for histologic analysis. To assess collagen matrix, picrosirius red staining was performed according to the introduction and visualized by microscopy (Olympus IX71; Olympus America, Inc., Center Valley, PA, USA). Following rehydration, the slides were stained by picrosirius red solution (0.1% Sirius red and green in saturated picric acid) for 1 hour, washed with water, 70% ethanol solution for 20 seconds, 100% ethanol solution for 20 seconds, and xylene for 30 seconds, dehydrated, and mounted. 
Antigen epitope retrieval was undertaken by immersing the slides after rehydration in 0.01 M sodium citrate buffer solution (pH 6), followed by heat treatment in a microwave oven at 100°C for 30 minutes. The slides were then rinsed with PBS (pH 7.2) for 15 minutes, followed by blocking with 3% H2O2 for 15 minutes, washed with PBS for three times for 5 minutes each, and then blocked with 5% (wt/vol) bovine serum albumin (BSA) in PBS for 30 minutes at 37°C. The slides for CD45 or Ki67 were treated with 0.3% Triton X-100 for 20 minutes at room temperature (RT) before being blocked with BSA. The slides were subsequently incubated with primary antibodies diluted in PBS at 4°C overnight. 
For immunofluorescent analysis for collagen I and α-SMA, the slides were washed three times with PBS before incubation with secondary antibodies conjugated to either AlexaFluor-488 or AlexaFluor-594 (Invitrogen). The slides for Ki67 and CD45 were washed with PBS and then incubated for 30 minutes with biotinylated secondary antibodies, rinsed three times with PBS, and incubated with streptavidin-HRP for 30 minutes. The slides were rinsed with PBS before color development with 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution (Zhongshan Goldenbridge Biotechnology Co., Ltd.), counterstained with hematoxylin for 3 to 5 minutes and rinsed with deionized water. The sections were visualized using an optical microscope. 
Western Blot Analysis
Rats were euthanized on day 28 after surgery, and the conjunctiva tissues at the surgical sites were dissected and cut off, then stored at −70°C for Western blot analysis. The conjunctival tissues were homogenized and lysed in Laemmli buffer (Bio-Rad), followed by boiling and centrifugation to obtain lysates. Protein concentrations were determined using a BCA protein assay kit. Proteins were separated on 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blotting was performed as previously described,19 using antibodies against α-SMA and collagen I. Quantification was performed by measuring intensity of the signals using Quantity One, version 4.6.2 software (Bio-Rad). 
TUNEL Analysis
End-labeling of exposed 3′-OH ends of DNA fragments in cryosections and cultured mouse conjunctival fibroblasts was performed with the DeadEnd Fluorometric TUNEL System according to manufacturer's instructions (Promega, Madison, WI, USA). Staining of the cell nucleus was achieved by mounting the TUNEL-stained cryosections in hematoxylin for 3 to 5 minutes and rinsing with deionized water. The sections were visualized by microscopy (Olympus IX71). 
Statistical Analysis
All experiments were conducted at least in triplicate; data are expressed as the mean ± SD. Comparisons between groups were conducted using one-way ANOVA, followed by Student's t-test. Statistically significant differences were considered at P less than 0.05. All statistical computations were performed using SPSS, version 13.0 (SPSS, Inc., Chicago, IL, USA). 
Results
Incorporation and Release of Aptamer S58 With CS/S58 Gel
The incorporated aptamer S58 complexes could be released in a sustained manner for at least 2 weeks, with gradual decrease in release rate with time. No apparent burst release was observed. Approximately 53% of the loaded S58 was released in the first 5 days, and more than 80% was released at 14 days (Fig. 1B). The structural integrity of the released S58 was examined using gel electrophoresis after 1, 3, 5, 7, 14, and 28 days (Fig. 1A). 
Figure 1
 
(A) Electrophoretic mobility of aptamer S58 from the CS/S58 gels. (B) Cumulative release of aptamer S58 from the CS/S58 gels (mean ± SD, n = 3). M, marker.
Figure 1
 
(A) Electrophoretic mobility of aptamer S58 from the CS/S58 gels. (B) Cumulative release of aptamer S58 from the CS/S58 gels (mean ± SD, n = 3). M, marker.
Bleb Survival and Vascularity
Blebs were elevated and avascular during the initial 3 days after surgery and became flat, more vascularized and smaller in size in appearance by day 28 (Fig. 2A). The area of the blebs was calculated, and the IOPs were recorded. The mean bleb size on day 28 is expressed as a percentage of the size at 3 days post surgery. The bleb size of CS/S58 gel-treated group was 36 ± 9% on day 28 compared with 17 ± 7% in the control group. The MMC-treated group achieved 30 ± 7%, the CS-treated group achieved 28 ± 8 %, and the TGF-treated group achieved 15 ± 5% (Fig. 2C). As shown by the morphologic features of the blebs in Figure 2, a characteristically thin bleb without hyperemia was observed in the CS/S58-treated eyes. We expressed the IOP of the operated eye as a percentage of the IOP of the unoperated eye of each animal at each time point. As shown in Figure 2D, the mean percentage IOP of the control, TGF-β2–, MMC-, CS-, and CS/S58-treated groups were 95 ± 14%, 103 ± 12%, 84 ± 21%, 78 ± 18% and 72 ± 20%, respectively, on day 28. No significant differences between the MMC- and CS-treated groups were observed. Treatment with CS or MMC led to a significant improvement in bleb survival to 50% (4 of 8), and combined treatment with S58 resulted in 88% (7 of 8) bleb survival on day 28. In contrast, none of the TGF-β2–treated (n = 8) and only one of eight of the control blebs survived to day 28. No complications, such as development of corneal epithelial toxicity, endophthalmitis, or cystic avascular bleb, were observed in the rats with CS alone or in combination with S58 throughout the study. 
Figure 2
 
Morphology and functional analysis of the blebs. (A) Slit-lamp examination of the surgical sites revealed the presence of blebs in the conjunctiva of the treated eyes (the area of blebs is outlined by dotted lines). (B) The mean bleb size is expressed as a percentage of the size at 3 days post surgery (n = 8). (C) Anterior-segment OCT imaging of the rat conjunctiva confirmed bleb survival. The control, TGF-β2–, MMC-, CS- and CS/S58 gel–treated rat eyes were examined on days 3 and 28 after glaucoma filtration surgery (GFS). The location of the bleb is indicated by a white box. (D) The IOPs of the operated and unoperated eyes in each group (n = 8) were measured at least six times. Each value represents the mean IOP percentage of the operated eye over the mean IOP of the unoperated eyes of the same animal at each time point. The numbers indicate the mean IOP percentage (n = 8).
Figure 2
 
Morphology and functional analysis of the blebs. (A) Slit-lamp examination of the surgical sites revealed the presence of blebs in the conjunctiva of the treated eyes (the area of blebs is outlined by dotted lines). (B) The mean bleb size is expressed as a percentage of the size at 3 days post surgery (n = 8). (C) Anterior-segment OCT imaging of the rat conjunctiva confirmed bleb survival. The control, TGF-β2–, MMC-, CS- and CS/S58 gel–treated rat eyes were examined on days 3 and 28 after glaucoma filtration surgery (GFS). The location of the bleb is indicated by a white box. (D) The IOPs of the operated and unoperated eyes in each group (n = 8) were measured at least six times. Each value represents the mean IOP percentage of the operated eye over the mean IOP of the unoperated eyes of the same animal at each time point. The numbers indicate the mean IOP percentage (n = 8).
Further analysis by AS-OCT was also performed to evaluate bleb survival. Optical coherence tomography measurements on days 3 and 28 after surgery confirmed the slit-lamp observations. Blebs were elevated and avascular during the initial 3 days after surgery and progressively reduced in size, becoming flat, and more vascularized in appearance by day 28. Limited elevated blebs were observed in MMC- and CS/S58-treated eyes at day 28 (Fig. 2B). 
Histologic Characteristics
Representative H&E staining and picrosirius red staining images of the specimens are shown in Figures 3A through 3F. The inflammatory response to surgery with different pretreatment processes was evaluated after 7 days. A typical infiltration of granulocytes and macrophages could be observed at this stage of wound healing. Varying inflammatory responses to surgery were observed with the different pretreatment processes. The varying thickness in conjunctival tissue at the surgery site provided an initial indication of the inflammatory response level. A typical inflammatory response to TGF-β2 treatment was observed (Fig. 3C), whereas the width of the granulation tissue in the subconjunctival space at the surgery site was thicker than that observed for other groups. Fibroblast infiltration and newly formed blood vessels could also be visualized. In contrast, fibroblast infiltration was mostly adjunct to the conjunctival epithelium in the MMC-treated group (Fig. 3D). In CS- and CS/S58-treated eyes (Figs. 3E, 3F), the outermost connective tissue surrounding the blebs was more loosely arranged, and scattered fibroblasts and inflammatory cells, such as lymphocytes, macrophages, and occasional eosinophils, were observed surrounding the CS particles. 
Figure 3
 
Hematoxylin and eosin staining and picrosirius red-stained sections of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Picrosirius red staining revealed the differences in the collagen matrices of the surgical sites in each rat eye. Scale bars: 100 μm. CS, chitosan.
Figure 3
 
Hematoxylin and eosin staining and picrosirius red-stained sections of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Picrosirius red staining revealed the differences in the collagen matrices of the surgical sites in each rat eye. Scale bars: 100 μm. CS, chitosan.
Picrosirius red staining indicated collagen deposition in the surgical sites. The subconjunctival space of the surgical sites in all of the treated eyes was thicker than that in normal eyes without any treatment, as shown in Figure 3A. Thick strands of fibrous material, which predominantly consisted of collagen fibers, were observed in the subconjunctival space overlying the sclera in the control and TGF-β2–treated eyes (Figs. 3B, 3C). The surgical sites were densely compacted with thick, well-aligned collagen fibers, resembling a scar. In contrast, the blebs in the CS- and CS/S58-treated eyes contained few and thin, loosely assembled collagen fibers, noticeably yellow-green birefringent, suggesting the preponderance of immature fibers in an expanded noncollagenous subconjunctival space (Figs. 3E, 3F). The MMC-treated eyes exhibited loosely arranged episclera with outer dense collagen fibers (Fig. 3D). 
Histologic Markers of Fibrosis in Conjunctival Blebs
Excess production and deposition of the extracellular matrix (ECM) proteins induced by TGF-β2 play an important role in the development of conjunctival fibrosis. To further determine differences in the ECM of the conjunctiva, immunofluorescence analysis for collagen I and α-SMA expression and location was performed. 
In the normal eyes, thin and fine strands of collagen I with few α-SMA active fibroblasts overlying the sclera were observed (Figs. 4A, 5A). Transforming growth factor–β2 induced α-SMA expression in conjunctival fibroblasts as previously reported.19 The TGF-β2–treated eyes also exhibited more enhanced staining for collagen I and α-SMA subconjunctiva at day 7 after surgery (Figs. 4C, 5C) compared with the control group (Figs. 4B, 5B). The MMC-, CS- and CS/S58-treated groups had remarkably reduced collagen I staining intensity (Figs. 4D, 4F, 5D, 5F). In addition, a significant decrease in the number of α-SMA positive myofibroblasts was observed in the CS/S58 gel–treated eyes (Fig. 5F). 
Figure 4
 
Expression and immunolocalization of collagen I of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of collagen I in conjunctival tissues at the surgical sites at day 28. Nor, normal; Con, control; CS, chitosan; CS/S58, chitosan/aptamer, S58; S, subconjunctival tissue.
Figure 4
 
Expression and immunolocalization of collagen I of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of collagen I in conjunctival tissues at the surgical sites at day 28. Nor, normal; Con, control; CS, chitosan; CS/S58, chitosan/aptamer, S58; S, subconjunctival tissue.
Figure 5
 
Expression and immunolocalization of α-SMA of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of α-SMA in conjunctival tissues at the surgical sites at day 28. White arrowheads indicate the chitosan gel particles.
Figure 5
 
Expression and immunolocalization of α-SMA of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of α-SMA in conjunctival tissues at the surgical sites at day 28. White arrowheads indicate the chitosan gel particles.
Western Blotting Analysis
Western blotting analysis of the tissue samples at day 28 was conducted to detect col I and α-SMA content. Col I and α-SMA levels in the TGF group increased within 28 days after surgery and remained higher than in the control group. In contrast, col I and α-SMA expression was significantly lower in the MMC, CS, and CS/S58 group compared with controls. No significant differences were observed between the MMC- and CS gel–treated eyes. In contrast, the effect of CS/S58 depletion on the synthesis of collagen I and α-SMA was striking; α-SMA levels remained constant at a low level until day 28 (Figs. 4G, 5G). 
These observations suggest that collagen deposition might be altered in CS/S58-treated eyes. The survival of the bleb is partially due to reduced deposition of collagen fibers and α-SMA–positive myofibroblasts, which lead to deficient maturation of the scar at the wound site. 
Recruitment of Inflammatory Cells to the Conjunctival Bleb
The inflammatory response to the surgical site was also evaluated by investigating the CD45 expression. The sections were stained for CD45, which is commonly used as a marker of inflammation and presents on the surface of nucleated cells, including neutrophils, B cells, T cells, and macrophages. Few conjunctival cells of normal eyes were positive for CD45 (Fig. 6A). CD45 expression was primarily detected in the subconjunctival space at the wound site of the control and TGF-β2–treated eyes, especially in the tissue surrounding the implanted cannula (Figs. 6B, 6C). In the MMC group, CD45-positive cells were observed around the MMC-treated episclera (Fig. 6D). In the CS-treated group, CD45-positive cells were predominantly detected around the chitosan particles; in contrast, few CD45-positive cells were detected in the CS/S58-treated eyes (Figs. 6E, 6F). 
Figure 6
 
Recruitment of inflammatory cells to the operated sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Immunostaining for CD45 leukocytes revealed the presence of inflammatory cells in the subconjunctival sites. Scale bars: I, 100 μm; II, 20 μm.
Figure 6
 
Recruitment of inflammatory cells to the operated sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Immunostaining for CD45 leukocytes revealed the presence of inflammatory cells in the subconjunctival sites. Scale bars: I, 100 μm; II, 20 μm.
The Proliferation and Apoptosis of Conjunctiva Cells at the Wound Site
The presence of proliferative cells at surgical sites was investigated by immunostaining for Ki67 expression to verify the mechanism of CS/S58 in wound healing and to determine any toxic or side effects. At day 7 after surgery, Ki67-positive cells were observed near the implanted cannula in the control group (Fig. 7B) and near the subconjunctival space of the TGF-β2–treated eyes (Fig. 7C), suggesting that there were wound-activated proliferating cells at the surgical sites. Much fewer Ki67-positive staining was observed in the conjunctival cells treated with MMC or CS/S58 (Figs. 7D, 7F). In contrast, Ki67-positive cells were predominately observed around the chitosan particles in the CS group (Fig. 7E). 
Figure 7
 
Immunostaining of Ki67 revealed the presence of proliferative cells at the surgical sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: I, 100 μm; II, 20 μm.
Figure 7
 
Immunostaining of Ki67 revealed the presence of proliferative cells at the surgical sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: I, 100 μm; II, 20 μm.
TUNEL analysis showed that only a small number of conjunctival cells were apoptotic in MMC-treated eyes or around the chitosan particles in CS-treated eyes, whereas no apoptotic conjunctival cells were observed in the control, TGF-β2– or CS/S58-treated eyes (Fig. 8). 
Figure 8
 
Apoptosis of conjunctiva cells at the surgical sites. Few apoptotic (black arrows: TUNEL-positive) cells, which would appear as brown cells, were observed in control and TGF-β2–treated eyes. A number of apoptotic cells were observed in MMC-treated conjunctival sites and around the chitosan particles in CS-treated eyes. Few apoptotic cells were observed in CS/S58-treated eyes. Scale bars: 20 μm.
Figure 8
 
Apoptosis of conjunctiva cells at the surgical sites. Few apoptotic (black arrows: TUNEL-positive) cells, which would appear as brown cells, were observed in control and TGF-β2–treated eyes. A number of apoptotic cells were observed in MMC-treated conjunctival sites and around the chitosan particles in CS-treated eyes. Few apoptotic cells were observed in CS/S58-treated eyes. Scale bars: 20 μm.
Discussion
Excessive postoperative scarring at the conjunctiva and sclerostomy sites is associated with poor postoperative pressure control. This study demonstrated that novel therapeutic application of chitosan/S58 gel inhibited conjunctival scarring and concomitantly preserved bleb survival in a rat GFS model, with inhibited inflammation response. The pronounced increase in filtration efficiency was associated with thinner fibers, and loosely organized subconjunctival matrix was observed in CS/S58 gel–treated eyes. The antifibrotic effect of the chitosan gel in combination with aptamer S58 was superior to chitosan gel alone for antifibrotic treatment in rats GFS model. 
The TGF-β protein family has been shown to be one of the most potent stimulators of conjunctival scarring after glaucoma filtration surgery, and these proteins can be secreted by multiple cell types, such as platelets, fibroblasts, and macrophages.22 They act as a chemokine for fibroblasts, induce the differentiation of myofibroblasts, regulate collagen synthesis, and modulate matrix turnover. A previous study demonstrated that TGF-β activates the production of granulation tissue, which consists of fibroblasts and myofibroblasts, and eventually leads to ECM over-accumulation and scar deposition.23 Our previous study also showed that TGF-β2 could induce α-SMA expression and the transdifferentiation of conjunctival fibroblasts to myofibroblasts with high contractility.19 In agreement with previous studies, we observed the accumulation of collagen fibres and proliferation of α-SMA–positive fibroblasts in the subconjunctival space in TGF-β–treated rat eyes with elevated IOP and limited bleb sizes. The results confirmed that bleb failure in glaucoma filtration surgery was primarily due to the excessive accumulation of collagen and ECM organization in the subconjunctival space, and high levels of TGF-β activity were associated with scarring. 
The present study showed a reduction in collagen fibers and the assembly of a loosely organized stromal network in the subconjunctival space in eyes treated with MMC. Mitomycin-C is a bioreductive alkylating agent that undergoes metabolic reductive activation and has various oxygen tension–dependent cytotoxic effects on cells, including the cross-linking of DNA.24 The intraoperative regimen of MMC has gained favor due to the convenience of a single treatment and the delivery of lower intraocular pressure in certain eyes.25 We demonstrated that intraoperative application of MMC extended the bleb survival period to 28 days and resulted in reduced collagen deposition compared with control eyes. However, even short exposure to MMC results in irreversible local tissue destruction. The profound cytotoxic effect of MMC in the proliferation phases of Tenon's fibroblasts has also been demonstrated in mouse,26 rabbit,27 and monkey GFS models.28 In the rat GFS model, the densities of fibroblasts and areas of collagen fiber in conjunctival lesions were significantly decreased in MMC-treated eyes compared with control eyes (Fig. 3). The lack of proliferation was verified by the lack of Ki67-positive staining in the cells treated with MMC compared with control eyes (Fig. 7). Karyopyknosis and a number of apoptotic cells (TUNEL-positive cells) were observed in the subconjunctival tissue of MMC-treated eyes (Fig. 8). 
Chitosan-based gels have been widely studied in the biomedical field for local therapeutic agent delivery, gene therapy,9 and wound healing.29 Making use of its in situ gelling properties, chitosan can be applied and distributed on the ocular surface in almost liquid form, thereafter transitioning to gel status. Due to its nontoxicity, permeation enhancing properties and physicochemical characteristics, chitosan-based hydrogel is a suitable material for the design of ophthalmic drug delivery systems.30 However, the limited colloidal stability of chitosan-based drug delivery systems is known to induce immunogenicity. Previous studies have demonstrated that chitosan can enhance the functions of inflammatory cells, such as polymorphonuclear leukocytes (PMN), macrophages, and fibroblasts,10 and stimulate macrophages to produce growth factors, such as TGF-β and platelet-derived growth factor (PDGF).31 L929 mouse fibroblasts were cultured with chitosan, and no significant differences in the amount of ECM were observed between the control and chitosan groups, indicating that chitosan does not directly accelerate ECM production by fibroblasts; ECM production may be increased by growth factors, such as TGF-β and PDGF.31 In present study, CS/GP gel was applied in the conjunctiva during surgery; no significant differences were observed in the amount of collagen I and α-SMA proteins between the CS gel and MMC-treated eyes in the rat GFS model (Figs. 4, 5). In addition the CS/GP gel did not accelerate ECM production by fibroblasts in the conjunctiva in rats compared with the control. Therefore, these effects of chitosan should be considered prior to drug delivery. 
In this study, aptamer S58-conjugated chitosan-based (CS/S58) hydrogel was developed and directly used for conjunctiva wound healing in the rat GFS model; a prolonged ocular residence time of agents and improved therapeutic efficacy could be achieved. Aptamer S58 targeting TβRII was previously reported to inhibit TGF-β–induced human Tenon fibroblasts (HTFs) transdifferentiation in vitro.19 In rat GFS models, the filtration blebs were elevated and avascular for the initial 3 days following surgery and reduced progressively in size, becoming flat and more vascularized in appearance by day 7. However, limited elevated blebs were observed in CS/S58 gel–treated eyes at day 28 (Figs. 2A, 2B). To assess the effects of the CS/S58 gel on conjunctival wound healing, we examined the expression and location of collagen I and α-SMA in terms of wound response compared with CS gel. Alpha-SMA and collagen I expression was significantly reduced in the conjunctiva in CS/S58 gel–treated eyes with prolonged bleb survival compared with the CS group. Alpha-SMA is a marker of myofibroblasts that is a contractile and secretory cell type differentiated from fibroblasts induced by TGF-β.32 A significant decrease in the number of α-SMA–positive myofibroblasts was also observed in CS/S58 gel–treated eyes (Fig. 5F). The morphologic appearance of the filtering bleb and IOP also showed modulation of the wound healing effects of CS/S58 gel (Fig. 2). These results indicated that the CS/S58 gel may have a relatively inhibitory effect on TGF-β and that the antiscarring effect may have resulted from the sustained release of aptamer S58 from the hydrogel. 
When the operation is made and micro cannula is implanted, an acute inflammatory response starts, and infiltration of granulocytes and macrophages can be observed at this stage of wound healing, especially in TGF-β–treated eyes. Upon implantation of a foreign material, injury to the vasculature occurs, resulting in hemorrhage, infiltration of neutrophils, activation of the complement cascade, and almost induce inflammation and fibrosis.33 In addition, chitosan is a macrophage-activating agent and accelerates cytokine production from macrophages. Nishimura et al.34 reported that chitosan stimulates the production of IL-1 by macrophages. Immunohistochemical staining for CD45 expression, a marker of inflammation, was performed at the surgical sites. The limited colloidal stability of chitosan-based drug delivery systems is known to induce immunogenicity.35 As shown in Figure 6, CD45+ cells were observed around the implanted micro cannula and the chitosan particles in the subconjunctival space; fibroblast infiltration and newly formed blood vessels could also be observed. 
Highly deacetylated (DD) chitosan (>85%) is soluble only up to a pH of 6.5. Chitosan is soluble in aqueous dilute acid when the degree of DD of chitin reaches approximately 50%. Low levels of β-GP solution were required to increase the pH of an acidic chitosan solution up to 7, at which the chitosan solution underwent thermal gelation at 37°C. The chitosan/β-GP gel is biocompatible and allows cells to remain viable.12 However, high concentrations of β-GP significantly affect cell viability.36 It is necessary to assess the tolerance of the CS or CS/S58 gels in ocular tissue. Physical hydrogels of chitosan only composed of chitosan and water were processed and applied to treat full-thickness burn injuries, and chitosan materials were well tolerated and promoted good tissue regeneration.37 In the rat GFS model, TUNEL analysis showed that only a number of conjunctival cells were apoptotic in MMC-treated eyes or around the chitosan particles in CS gel–treated eye, whereas few apoptotic conjunctival cells were observed in the CS/S58-treated eyes (Fig. 8). CS/S58 hydrogel showed excellent ocular tolerance. Moreover, the hydrogels were decreased with prolonged postsurgery time, verifying that the hydrogels were biodegraded in the rats' eyes. 
This study provides evidence that combined chitosan and aptamer S58 offers superior antifibrotic effect over monotherapy in a rat model of glaucoma filtration surgery. The study also provides an opportunity for further developments in antifibrotic drug therapy. 
Acknowledgments
The authors thank their coworkers at Molecular Biology Centre, Institute of Surgery Research, for sharing equipment and reagents. 
Supported by National Natural Science Foundation of China (Nos. 81170852, 81470629; Chongqing, China) and Natural Science Foundation of Chongqing (CSTC2013jjB10030; Chongqing, China). 
Disclosure: X. Zhu, None; D. Xu, None; X. Zhu, None; L. Li, None; H. Li, None; F. Guo, None; X. Chen, None; Y. Tan, None; L. Xie, None 
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Figure 1
 
(A) Electrophoretic mobility of aptamer S58 from the CS/S58 gels. (B) Cumulative release of aptamer S58 from the CS/S58 gels (mean ± SD, n = 3). M, marker.
Figure 1
 
(A) Electrophoretic mobility of aptamer S58 from the CS/S58 gels. (B) Cumulative release of aptamer S58 from the CS/S58 gels (mean ± SD, n = 3). M, marker.
Figure 2
 
Morphology and functional analysis of the blebs. (A) Slit-lamp examination of the surgical sites revealed the presence of blebs in the conjunctiva of the treated eyes (the area of blebs is outlined by dotted lines). (B) The mean bleb size is expressed as a percentage of the size at 3 days post surgery (n = 8). (C) Anterior-segment OCT imaging of the rat conjunctiva confirmed bleb survival. The control, TGF-β2–, MMC-, CS- and CS/S58 gel–treated rat eyes were examined on days 3 and 28 after glaucoma filtration surgery (GFS). The location of the bleb is indicated by a white box. (D) The IOPs of the operated and unoperated eyes in each group (n = 8) were measured at least six times. Each value represents the mean IOP percentage of the operated eye over the mean IOP of the unoperated eyes of the same animal at each time point. The numbers indicate the mean IOP percentage (n = 8).
Figure 2
 
Morphology and functional analysis of the blebs. (A) Slit-lamp examination of the surgical sites revealed the presence of blebs in the conjunctiva of the treated eyes (the area of blebs is outlined by dotted lines). (B) The mean bleb size is expressed as a percentage of the size at 3 days post surgery (n = 8). (C) Anterior-segment OCT imaging of the rat conjunctiva confirmed bleb survival. The control, TGF-β2–, MMC-, CS- and CS/S58 gel–treated rat eyes were examined on days 3 and 28 after glaucoma filtration surgery (GFS). The location of the bleb is indicated by a white box. (D) The IOPs of the operated and unoperated eyes in each group (n = 8) were measured at least six times. Each value represents the mean IOP percentage of the operated eye over the mean IOP of the unoperated eyes of the same animal at each time point. The numbers indicate the mean IOP percentage (n = 8).
Figure 3
 
Hematoxylin and eosin staining and picrosirius red-stained sections of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Picrosirius red staining revealed the differences in the collagen matrices of the surgical sites in each rat eye. Scale bars: 100 μm. CS, chitosan.
Figure 3
 
Hematoxylin and eosin staining and picrosirius red-stained sections of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Picrosirius red staining revealed the differences in the collagen matrices of the surgical sites in each rat eye. Scale bars: 100 μm. CS, chitosan.
Figure 4
 
Expression and immunolocalization of collagen I of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of collagen I in conjunctival tissues at the surgical sites at day 28. Nor, normal; Con, control; CS, chitosan; CS/S58, chitosan/aptamer, S58; S, subconjunctival tissue.
Figure 4
 
Expression and immunolocalization of collagen I of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of collagen I in conjunctival tissues at the surgical sites at day 28. Nor, normal; Con, control; CS, chitosan; CS/S58, chitosan/aptamer, S58; S, subconjunctival tissue.
Figure 5
 
Expression and immunolocalization of α-SMA of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of α-SMA in conjunctival tissues at the surgical sites at day 28. White arrowheads indicate the chitosan gel particles.
Figure 5
 
Expression and immunolocalization of α-SMA of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: 100 μm. (G) Western blot analysis of α-SMA in conjunctival tissues at the surgical sites at day 28. White arrowheads indicate the chitosan gel particles.
Figure 6
 
Recruitment of inflammatory cells to the operated sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Immunostaining for CD45 leukocytes revealed the presence of inflammatory cells in the subconjunctival sites. Scale bars: I, 100 μm; II, 20 μm.
Figure 6
 
Recruitment of inflammatory cells to the operated sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Immunostaining for CD45 leukocytes revealed the presence of inflammatory cells in the subconjunctival sites. Scale bars: I, 100 μm; II, 20 μm.
Figure 7
 
Immunostaining of Ki67 revealed the presence of proliferative cells at the surgical sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: I, 100 μm; II, 20 μm.
Figure 7
 
Immunostaining of Ki67 revealed the presence of proliferative cells at the surgical sites of normal (A), control (B), TGF-β2– (C), MMC- (D), CS- (E), and CS/S58- (F) treated eyes at 7 days post surgery. Scale bars: I, 100 μm; II, 20 μm.
Figure 8
 
Apoptosis of conjunctiva cells at the surgical sites. Few apoptotic (black arrows: TUNEL-positive) cells, which would appear as brown cells, were observed in control and TGF-β2–treated eyes. A number of apoptotic cells were observed in MMC-treated conjunctival sites and around the chitosan particles in CS-treated eyes. Few apoptotic cells were observed in CS/S58-treated eyes. Scale bars: 20 μm.
Figure 8
 
Apoptosis of conjunctiva cells at the surgical sites. Few apoptotic (black arrows: TUNEL-positive) cells, which would appear as brown cells, were observed in control and TGF-β2–treated eyes. A number of apoptotic cells were observed in MMC-treated conjunctival sites and around the chitosan particles in CS-treated eyes. Few apoptotic cells were observed in CS/S58-treated eyes. Scale bars: 20 μm.
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