Glaucoma Filtration Surgery In Rabbits Research Articles

VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model.

To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model. Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31). Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P<0.001, P < 0.001) and vs. control (P<0.001, P<0.001). After 30days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P<0.001), and control (P<0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P<0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978). Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

MiR-29b Downregulation by p53/Sp1 Complex Plays a Critical Role in Bleb Scar Formation After Glaucoma Filtration Surgery.

To investigate the function and mechanism of tumor protein p53 in pathological scarring after glaucoma filtration surgery (GFS) using human Tenon's fibroblasts (HTFs) and a rabbit GFS model. The expression of p53 in bleb scarring after GFS and transforming growth factor-β (TGF-β)-induced HTFs (myofibroblasts [MFs]) was examined by western blot and immunochemical analysis. The interaction between p53 and specificity protein 1 (Sp1) was investigated by immunoprecipitation. The role of p53 and Sp1 in the accumulation of collagen type I alpha 1 chain (COL1A1) and the migration of MFs was evaluated by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), wound healing, and Transwell assay. The regulatory mechanisms among p53/Sp1 and miR-29b were detected via qRT-PCR, western blot, luciferase reporter assay, and chromatin immunoprecipitation assay. The therapeutic effect of mithramycin A, a specific inhibitor of Sp1, on scarring formation was evaluated in a rabbit GFS model. p53 was upregulated in bleb scar tissue and MFs. p53 and Sp1 form a transcription factor complex that induces the accumulation of COL1A1 and promotes the migration of MFs through downregulation of miR-29b, a known suppressor of COL1A1. The p53/Sp1 axis inhibits miR-29b expression by the direct binding promoter of the miR-29b gene. Mithramycin A treatment attenuated bleb scar formation in vivo. The p53/Sp1/miR-29b signaling pathway plays a critical role in bleb scar formation after GFS. This pathway could be targeted for therapeutic intervention of pathological scarring after GFS. Our research indicates that inhibition of p53/Sp1/miR-29b is a promising therapeutic strategy for preventing post-GFS pathological scarring.

Artesunate protects against ocular fibrosis by suppressing fibroblast activation and inducing mitochondria-dependent ferroptosis.

Artesunate, a derivative from extracts of Artemisia annua, has recently been reported to alleviate fibrosis recently. Here, in this study, we sought to determine the anti-fibrosis effect of artesunate in rabbit glaucoma filtration surgery (GFS) model and illuminate underlying mechanisms. Our results showed that artesunate subconjunctival injection alleviated bleb fibrosis by inhibiting fibroblast activation and inducing ferroptosis. Further mechanistic investigation in primary human ocular fibroblasts (OFs) showed that artesunate abrogated fibroblast activation by inhibiting TGF-β1/SMAD2/3 and PI3K/Akt pathways and scavenged OFs by inducing mitochondria-dependent ferroptosis. Mitochondrial dysfunction, mitochondrial fission, and iron-dependent mitochondrial lipid peroxidation were observed in artesunate-treated OFs. Besides, mitochondria-localized antioxidants inhibited artesunate-induced cell death, suggesting a critical role of mitochondria in artesunate-induced ferroptosis. Our study also found that expression of mitochondrial GPX4 but no other forms of GPX4 was decreased after artesunate treatment and that mitochondrial GPX4 overexpression rescued artesunate-induced lipid peroxidation and ferroptosis. Other cellular ferroptosis defense mechanisms, including cellular FSP1 and Nrf2, were also inhibited by artesunate. In conclusion, our study demonstrated that artesunate protects against fibrosis through abrogation of fibroblast activation and induction of mitochondria-dependent ferroptosis in OFs, which may offer a potential treatment for ocular fibrosis.

The anti-scar effect of tyrosine-kinase inhibitor nintedanib in experimental glaucoma filtration surgery in rabbits

PurposeTo investigate the efficacy of nintedanib on preventing postoperative scar in formation following glaucoma filtering surgery (GFC) in rabbits in comparison with Mitomycin-C (MMC). DesignExperimental Animal Study. Methods24 New Zealand rabbits were divided randomly into 3 groups as Sham, Nindetanib and MMC(n = 8). Limbal-based trabeculectomy was performed on the right eyes of the rabbits. Left eyes that did'nt undergo surgery were included in the control group (n = 8). Following surgery, Intraocular pressures (IOP), postoperative complications and morphological changes in the bleb were evaluated. On the 28th day, eight eyes from each group were enucleated and histologically and immunohistochemically analyzed. Matrix metalloproteinase-2 (MMP-2), Transforming Growth Factor-1 (TGF-B1) and alpha-smooth muscle actin (a-SMA) were evaluated. ResultsIt was observed that nintedanib has no side effects and reduces subconjunctival fibrosis. Postoperative IOP values in the Nindetanib group were lower than the other groups (p < 0.05). The longest bleb survival was observed in the Nintedanib group and the shortest in the Sham group (p < 0.001). Conjunctival vascularity and inflammation was reduced in the Nintedanib group compared to the Sham group (p < 0.05). The highest subconjunctival fibrosis was observed in the Sham group and the least in the Nintedanib group (p < 0.05). Although the fibrosis score was found lower in the Nintedanib group compared to the MMC(p > 0.05). α-SMA TGF-β1, MMP-2 expressions were similar in Nintedanib and MMC groups (p > 0.05), however, it was observed that significantly decreased in both groups compared to Sham group (p < 0.05). ConclusionIt has been observed that Nindetanib suppress fibroblast proliferation Thus, It may be a drug that can prevent subconjunctival fibrosis in GFC.

Bio-modulation of scaring Glaucoma Filtration Surgery using a novel application of coated magnesium

Filtration surgery still plays a mainstream role of treatment for glaucoma. Postoperative scarring is the main cause of surgical failure. This study evaluated the biocompatibility and anti-proliferative properties of pure magnesium with three different coatings, which are hydroxyapatite (HA), dicalcium phosphate dihydrate (DCPD) and DCPD + stearic acid (SA), in a primary culture of human tenon's capsule fibroblasts (HTCFs) and in rabbit Glaucoma Filtration Surgery. Titanium and glass were used as controls in vitro , and trabeculectomy was used as control in vivo . The results show the number and shape of HTCFs seeded on different coatings showed less quantity and poor cell morphology. Each type of coated magnesium demonstrated significantly decreased metabolic activity of HTCFs. DCPD + SA showed higher cytotoxicity than the other coatings. Significant inhibition of proliferation was observed with the DCPD + SA coating. The expression of α-SMA was decreased in the cells when seeded on all of the coated magnesium disks. In vivo , no obvious adverse effects were observed after operation. No significant difference existed for any of the different samples regarding different ion concentrations in the aqueous humor. The inflammatory response in the titanium, DCPD and DCPD + SA treated eyes was more intense than in the trabeculectomy alone and HA groups. Western Blot analysis showed that collagen-1 and α-SMA expression was significantly lower in the titanium, HA, DCPD and DCPD+SA groups compared with the control. Different coatings on magnesium were able to affect the corrosive properties, which in turn, influenced the morphology and function of HTCFs. HA coated magnesium may be considered a very promising biodegradable material for the next generation of glaucoma drainage devices.

AR12286 Alleviates TGF-β-Related Myofibroblast Transdifferentiation and Reduces Fibrosis after Glaucoma Filtration Surgery.

Scar formation can cause the failure of glaucoma filtration surgery. We investigated the effect of AR12286, a selective Rho-associated kinase inhibitor, on myofibroblast transdifferentiation and intraocular pressure assessment in rabbit glaucoma filtration surgery models. Cell migration and collagen contraction were used to demonstrate the functionality of AR12286-modulated human conjunctival fibroblasts (HConFs). Polymerase chain reaction quantitative analysis was used to determine the effect of AR12286 on the production of collagen Type 1A1 and fibronectin 1. Cell migration and collagen contraction in HConFs were activated by TGF-β1. However, compared with the control group, rabbit models treated with AR12286 exhibited higher reduction in intraocular pressure after filtration surgery, and decreased collagen levels at the wound site in vivo. Therefore, increased α-SMA expression in HConFs induced by TGF-β1 could be inhibited by AR12286, and the production of Type 1A1 collagen and fibronectin 1 in TGF-β1-treated HConFs was inhibited by AR12286. Overall, the stimulation of HConFs by TGF-β1 was alleviated by AR12286, and this effect was mediated by the downregulation of TGF-β receptor-related SMAD signaling pathways. In vivo results indicated that AR12286 thus improves the outcome of filtration surgery as a result of its antifibrotic action in the bleb tissue because AR12286 inhibited the TGF-β receptor-related signaling pathway, suppressing several downstream reactions in myofibroblast transdifferentiation.

Effect of connective tissue growth factor gene editing using adeno-associated virus-mediated CRISPR-Cas9 on rabbit glaucoma filtering surgery outcomes.

Suppressing excessive wound healing responses is critical to ensure surgical success in glaucoma filtration surgery (GFS). Currently used adjunctive materials can lead to side effects due to the nonselectivity in cell inhibition and may require repeated applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system may become a compelling opportunity in glaucoma surgery due to its high selectivity and permanent effect. Connective tissue growth factor (CTGF) is one of the most potent stimulators of tissue fibrosis in the eye. Therefore, we tested the effect of CTGF suppression using the CRISPR-Cas9 system on GFS fibrosis. We used an adeno-associated virus (AAV)-CRISPR-Cas9 system and confirmed successful CTGF suppression was achieved in fibroblasts in vitro through western blot analysis and deep sequencing. In the in vivo intereye-comparison rabbit GFS model, CRISPR-CTGF-treated eyes showed significantly better survival of the surgery site, less subconjunctival fibrosis, limited collagen deposition, and reduced cellularity than untreated eyes. Our results suggest a new possibility of CRISPR-Cas9-mediated CTGF suppression to improve human GFS outcomes.

Microspheres scar formation glaucoma filtration surgery area therapeutic effect—Based on rapamycin-chitosan-calcium alginate sustained-release

To study the efficacy of rapamycin (RAPA)-chitosan (CS)-calcium alginate (CA) sustained-release microspheres on scar formation in a rabbit model of glaucoma filtration surgery. Eighty New Zealand white rabbits were randomly divided into four groups and a glaucoma filtration model was established by scleral bite through the eyes. RAPA-CS-CA sustained-release microspheres were implanted in the right eye of 40 rabbits (Group A) and CS blank sustained-release microspheres were implanted in the left eye (Group B). Another 40 rabbits were treated with a 0.2 g·L-1 RAPA cotton sheet in the right eye (Group C) and the left eye underwent a simple sclerotomy (Group D). The intraocular pressure, filter bleb, anterior chamber inflammation, complications, and corneal endothelial cell density were observed after the operation. Rabbits were euthanized for pathological examination 7 days, 14 days, and 21 days after the operation. The drug loading rate of RAPA-CS-CA sustainedrelease microspheres was (34.58±1.47)% and the encapsulation rate was (56.23±1.55)%. The microsphere release in vitro was relatively stable. The release rate of the microspheres during the burst was only 16.54%. After 49 days, the cumulative release rate of the microspheres reached 94.07% and the sustained release effect was significant within 45 days. Group A maintained low-level intraocular pressure for the longest period of time, followed by Group C, and then Group B and D. The survival time of filter vesicles in Group A was longer than that in other groups. There were no postoperative complications in each group. The conjunctival epithelium of Group A had better integrity and fewer subconjunctival fibroblasts than other groups. There was no obvious inflammation or infiltration around the filtering mouth and there were fewer new collagen fibers. RAPA-CS-CA slow-release microspheres safely and effectively inhibited the proliferation of fibroblasts and neonatal collagen fibers in rabbit glaucoma filtration surgery and significantly improved the success rate of glaucoma filtration surgery.

Effects of rosiglitazone/PHBV drug delivery system on postoperative fibrosis in rabbit glaucoma filtration surgery model

The aim of this study is to investigate the effects and toxicities of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV)-loading rosiglitazone on preventing scar formation after glaucoma filtration surgery (GFS) in the rabbit model. Rosiglitazone/PHBV drug delivery system was prepared via electrospinning. Release behavior of RSG/PHBV membrane was evaluated by high-performance liquid chromatography. The different concentration membranes were implanted under the conjunctiva of the rabbit’s eyes (RSG/PHBV groups). Also, MMC-soaked sponges were placed under the conjunctiva of the eyes (positive group) for 3 min. Intraocular pressures and bleb features were then assessed for 4 weeks postoperative. Bleb sections were stained with HE, Masson’s trichrome and α smooth muscle action (αSMA) immunohistochemistry. The protein expression of collagen I, αSMA, and connective tissue growth factor in the bleb area were then quantified. The following results were observed: (1) the concentration of rosiglitazone would not affect the morphology of RSG/PHBV membrane. (2) RSG/PHBV membrane would effective and safety prevent the formation of fibrosis after GFS in the rabbit model. Implantation of RSG/PHBV membrane prevents scar formation after GFS. What’s more, it ameliorated toxicity to conjunctiva and cornea compared with the placement of MMC. The RSG/PHBV membrane would be a more effectivity and safer strategy than MMC.

Anti-scarring effect of rapamycin in rabbits following glaucoma filtering surgery

To study the anti- scarring effect of rapamycin in rabbits receiving glaucoma filtering surgery. Ninety-six Chinchilla rabbits were randomized equally into 3 rapamycin treatment groups and one control group. All the rabbits underwent trabeculectomy, after which the rabbits in the 3 rapamycin groups were treated with eye drops containing 1%, 3%, or 5% rapamycin in the operated eyes, and those in the control groups were given castor oil 4 times a day. The intraocular pressure (IOP) and inflammatory reaction in the treated eyes were observed, and the PCNA-positive cells in the filtering bleb were detected using immunohistochemistry. RTFs isolated from the Tenon's capsule of the rabbits were cultured in vitro, and the expressions of caspase-3, caspase-8, and caspase-9 in the fibroblasts were detected after treatment with different concentrations of rapamycin. The IOP was significantly lower in rapamycin-treated group than in the control group after the surgery (P &lt; 0.05). The counts of the PCNA-positive cells were significantly lower in rapamycin-treated rabbits than in the control group (P &lt; 0.05). Rapamycin treatment dose-dependently increased the expressions of caspase-3 and caspase- 9 at both the mRNA (P &lt; 0.001) and protein (P &lt; 0.001) levels without causing significant changes in the expressions of caspase-8. Rapamycin can inhibit excessive proliferation of the fibroblasts in the filtering bleb to reduce scar formation after glaucoma filtration surgery in rabbits. Rapamycin also increases the expressions of caspase-3 and caspase-9 to induce apoptosis of the RTFs.

The Effect of Dry Eye Disease on Scar Formation in Rabbit Glaucoma Filtration Surgery

The success rate of glaucoma filtration surgery is closely related to conjunctival inflammation, and the main mechanism of dry eye disease (DED) is inflammation. The aim of this study was to evaluate the effect of DED on bleb scar formation after rabbit glaucoma filtration surgery. Sixteen New Zealand white rabbits were randomly divided into control and DED groups. A DED model was induced by twice-daily topical administration of 0.1% benzalkonium chloride (BAC) drops for three weeks. Ocular examinations were performed to verify the DED model. Surgical effects were assessed, and histologic assessments were performed on the 28th postoperative day. Higher fluorescein staining scores, lower basal tear secretion levels and goblet cell counts, and increased interleukin 1β (IL-1β) levels were observed in the DED group. The DED eyes displayed significantly higher intraocular pressure (IOP)% on the 14th postoperative day; a smaller bleb area on days 14, 21 and 28; and a shorter bleb survival time. Moreover, proliferating cell nuclear antigen (PCNA) and alpha-smooth muscle actin (α-SMA) levels were significantly increased in the DED group. These results demonstrate that DED promotes filtering bleb scar formation and shortens bleb survival time; these effects may be mediated via IL-1β.

α5β1‐Integrin inhibitor (CLT‐28643) effective in rabbit trabeculectomy model

Glaucoma filtration surgery (GFS) fails due to fibrosis. The α5β1-integrin plays a pivotal role in fibrosis, angiogenesis and inflammation. This is the first experiment evaluating the prevention of fibrosis after GFS by a specific small molecule α5β1-integrin inhibitor (CLT-28643). Twenty-four rabbits received trabeculectomy on their right eyes. The rabbits were randomized into three groups of eight eyes each. CLT-28643 was given as a single subconjunctival injection intraoperatively to two of the right eye groups followed by postoperative vehicle eye drops (CLT+ group) or CLT-28643 eye drops 4times daily (CLT++ group). A third group received mitomycin-C (MMC) intraoperatively (sponge application, 0.04%, 2min) followed by vehicle eye drops postoperatively. The control-surgery group consisted of 12 left eyes having trabeculectomy with no adjunctive therapy. The remaining 12 left eyes formed the untreated group. Clinical assessment included intraocular pressure (IOP) measurement, slit-lamp examination (including bleb survival and morphology) and bleb photography. The rabbits were killed after four weeks for histology. Both CLT-28643-treated groups showed significantly prolonged bleb survival, and better bleb score compared to the control-surgery group. At end of the study, most functioning blebs were found in the MMC group (MMC group 75%; CLT+ group 12.5%, CLT++ group 25%; CLT+ group 12.5%, control-surgery group 0%). CLT-28643 was non-toxic and well tolerated. This rabbit GFS study indicates that inhibition of α5β1-integrin by the novel α5β1-integrin antagonist CLT-28643 significantly improved the outcome. The effect of a single intro-operative application of CLT-28643 seems to be inferior to 0.04% MMC.

Evaluation of an injectable thermosensitive hydrogel as drug delivery implant for ocular glaucoma surgery.

In this study, a biodegradable thermo-sensitive hydrogel from poly(trimethylene carbonate)15-F127-poly(trimethylene carbonate)15 (PTMC15-F127-PTMC15) was designed and evaluated as an injectable implant during ocular glaucoma filtration surgery in vivo and in vitro. Mitomycin C (MMC) was loaded into this hydrogel for controlled released to prolong the efficacy and to reduce the long-term toxicity. The properties of the hydrogel were confirmed using 1H NMR and gel permeation chromatography (GPC). Compared to the Pluronic F127 hydrogel, the PTMC15-F127-PTMC15 hydrogel showed a good solution-gel transition temperature at 37°C, a lower work concentration of 5% w/v and a longer mass loss time of more than 2 weeks. The in vitro study showed that the drug could be released from PTMC15-F127-PTMC15 (5% w/v) hydrogel for up to 16 days with only 57% of drug released in the first day. Moreover, the cell toxicity, which was tested via LDH and ANNEXIN V/PI, decreased within 72 h in human tenon's fibroblast cells (HTFs). The in vivo behavior in a rabbit glaucoma filtration surgery model indicated that this hydrogel loaded with 0.1 mg/ml MMC led to a better functional bleb with a prolonged mean bleb survival time (25.5±2.9 days). The scar tissue formation, new collagen deposition and myofibroblast generation appeared to be reduced upon histological and immunohistochemistry examinations, with no obvious side effects and inflammatory reactions. The in vitro and in vivo results demonstrated that this novel hydrogel is a safe and effective drug delivery candidate in ocular glaucoma surgery.

Mechanism of anti-scarring effect of retinoic acid drug delivery system in glaucoma filtration surgery in rabbits

Objective To investigate the mechanism of postoperative anti-scarring effect of retinoic acid (RA) drug delivery system (RADDS) on a rabbit model of glaucoma filtration surgery.Methods Glaucoma filtration surgery was performed on both eyes of 25 New Zealand white rabbits.The left eyes received RADDS (RA + PLGA),15 right eyes only randomly received drug delivery system (PLGA),and remaining 10 right eyes served as null vehicle (NV).The rabbits were killed on the day 7,14,30,60and 90 after the surgery.Both eyes were enucleated and tissue sections were immunohistochemically stained for proliferating cell nuclear antigen (PCNA) and transforming growth factor-52 (TGF-β2) of fibroblasts in surgery sites of rabbits' eyes.Semi-quantification of TGF-β2 mRNA was performed using reverse transcription-polymerase chain reaction (RT-PCR).Results The RA drug delivery system reduced fibroblast activity,with a significant reduction in the number and expression of PCNA in fibroblasts (P ＜ 0.01).RADDS treatment could significantly reduce the expression of TGF-β2 protein and mRNA (P ＜0.01).Conclusion The effect of postoperative anti-scarring of RA drug delivery system was mediated by inhibiting proliferation of fibroblasts and altering the expression of TGF-β2. Key words: Retinoic acid ; Glaucoma filtration surgery; Scarring; Proliferating cell nuclear antigen; Transforming growth factor-β

Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery

The purpose of this study is to investigate a thermoreversible gel using Pluronic® F-127 to deliver an activin receptor-like kinase 5 (ALK-5) inhibitor SB-505124 in glaucoma filtration surgery (GFS). The gel was characterized for in vitro drug release and viscosity studies. Cytotoxicity of Pluronic® F-127 was examined by MTT assay using cultured rabbit subconjunctival fibroblasts. In addition, Pluronic® F-127 gel (18% w/v) containing 5 mg of SB-505124 was applied at the surgical site in an in vivo rabbit GFS model. In the in vitro viscosity study, the gel showed a change in viscosity (from 1000 cps to 45,000 cps) from low temperature (10°C) to body temperature (37°C). The in vitro drug release study demonstrated 100% drug release within 12 h. The gel did not show cytotoxicity to the cultured rabbit subconjunctival cells by MTT assay. In the in vivo rabbit GFS model, the drug was successfully delivered by injection and no severe post-surgical complications were observed. A thermoreversible gel system with SB-505124 was successfully prepared and delivered for the rabbit GFS model, and it may provide a novel delivery system in GFS.

Filtering bleb area and intraocular pressure following subconjunctival injection of CTGF antibody after glaucoma filtration surgery in rabbits.

To study the role of connective tissue growth factor (CTGF) antibody in inhibiting bleb scarring after glaucoma filtration surgery (GFS) in rabbit model. GFS was performed on both eyes in five rabbits. One eye of each rabbit was chosen randomly as antibody group and received subconjunctival injection of 0.1mL CTGF antibody (50mg/L) immediately after GFS applied and on the 5 th day after GFS. The other eye of each rabbit as control group was received subconjunctival injection of 0.1mL PBS at the same time as antibody group. On postoperative days 1, 3, 5, 7, 10, and 14, the appearance of filtrating blebs was observed under slit lamp, the area and the intraocular pressure (IOP) were measured with micrometer and applanation tonometer, respectively. On postoperative days 1, 3, 5, 7, 10, and 14, areas of filtrating blebs in antibody group were all larger comparing with the control group (P<0.05) and IOPs of antibody group were lower than the control group (P<0.05). Subconjunctival injection of CTGF antibody can maintain larger bleb area and lower IOP after GFS in rabbit.

Evaluation of Sustained Release of PLC-Loaded Prednisolone Acetate Microfilm on Postoperative Inflammation in an Experimental Model of Glaucoma Filtration Surgery

Purpose: To evaluate the effect of a biodegradable microfilm with sustained release of prednisolone acetate (PA) on postoperative wound healing after experimental glaucoma filtration surgery (GFS).Methods: Biodegradable microfilms composed of poly (d-, l-lactide-co-caprolactone) (PLC) were fabricated and then pre-loaded PA-20% total weight. Fourteen New Zealand White rabbits were randomly divided into 3 treatment groups: GFS alone (n = 4), GFS with PLC microfilms (n = 4) and GFS with PA-loaded microfilm (n = 6). Microfilms were inserted subconjunctivally, adjacent to the filtering surgical site. We monitored all eyes with slit-lamp examination, bleb photography and anterior segment optical coherence tomography (AS-OCT). Histology with immunohistochemistry was performed to determine the presence of any inflammation.Results: Prednisolone acetate 20%-loaded microfilms exhibited steady, sustained release in vitro. Eyes implanted with PA-loaded microfilms showed a significantly better bleb survival (100% vs. 37.5%, p < 0.001) and reduced bleb vascularity (58%; 95% CI 54–62% vs. 30%; 95% CI 23–37%, p = 0.001) compared to the control at 30 days postoperatively. Histology and immunohistochemistry demonstrated less T-cell infiltration in the eye implanted with PA-loaded microfilms.Conclusion: Subconjunctival insertion of a PA-loaded biodegradeable microfilm exhibit sustained release of PA to reduce postoperative inflammation and prolong bleb survival in rabbit GFS.

A Thin Honeycomb-patterned Film as an Adhesion Barrier in an Animal Model of Glaucoma Filtration Surgery

To evaluate the effectiveness and safety of a thin honeycomb-patterned biodegradable film for glaucoma filtration surgery in rabbits. A 7 microm-thick film made from poly(L-lactide-co-epsilon-caprolactone) was placed in the subconjunctival space in one eye of rabbits, with or without full thickness filtration surgery. The film had a honeycomb-patterned surface that faced the subconjunctival Tenon tissue and the other side was smooth. Filtration surgery was also performed in the fellow eye, which received either no adjunctive treatment or 0.4 mg/mL mitomycin C (MMC; n=6 each). Intraocular pressure (IOP) measurements and bleb evaluations using ultrasound biomicroscopy were performed periodically for 28 days after surgery followed by histologic observation. Postoperative IOPs of the film-treated eyes were significantly lower than that of control eyes from day 10 to day 28 (P<0.05), but were not significantly different from those of MMC-treated eyes. The subconjunctival filtration space, detected by ultrasound biomicroscopy, disappeared in 5 control eyes, 1 MMC-treated eye, but none of the film-treated eyes. A bleb leak occurred postoperatively in 2 MMC-treated eyes. Histologically, in eyes without filtration surgery, fibrotic tissue with the film partly attached to it was noted on the honeycomb side, but was minimal on the sclera that faced the smooth side of the film. In eyes with filtration surgery, the honeycomb-patterned film lined the inner bleb wall with minimal inflammatory reaction. The thin honeycomb-patterned film that attached to the inner bleb wall worked as an adhesion barrier in glaucoma filtration surgery in rabbits, which is worthy of further investigation.

Expression of Matrix Metalloproteinases in Wound Healing after Glaucoma Filtration Surgery in Rabbits

Purpose: To investigate the protein and mRNA expressions of matrix metalloproteinases (MMPs), gelatinolytic activity and localization of MMP activity in wounds after glaucoma filtration surgery in rabbits. Methods: Sixty eyes of 30 rabbits were removed 1, 3, 7, 14 and 120 days after the surgery and used for this experiment. Protein and mRNA expressions were analyzed by immunohistochemistry and laser capture microdissection/real-time RT-PCR, respectively. The gelatinolytic activity was analyzed by gelatin zymography and the localization was studied using in situ zymography. Results: By immunohistochemistry, expression of MMP-1, MMP-2, MMP-3, MMP-9 and MT1-MMP was detected in the wounds, most markedly 3 days after the surgery. MMP-positive cells were predominantly macrophages. Expression of MMP-9 and MT1-MMP mRNAs was verified by RT-PCR. Gelatinolytic activities corresponding to proMMP-2 and the active form of MMP-2 were detected in the wounds 3 and 7 days after surgery. In situ zymography localized gelatinolytic activities at the wound site. These activities were almost completely abolished by an MMP inhibitor, indicating that the gelatinolytic activity belongs to metalloproteinases. Conclusions: MMPs, particularly MMP-2/MT1-MMP, play important roles in the degradation of the extracellular matrix in the wound healing process after glaucoma filtration surgery and may represent an important target for therapeutic intervention after glaucoma filtration surgery.

Transcript Expression of Matrix Metalloproteinases in the Conjunctiva following Glaucoma Filtration Surgery in Rabbits

Purpose: To determine the changes in the expression of transcripts of matrix metalloproteinases (MMPs) and their tissue inhibitor (TIMP) in the conjunctiva following glaucoma filtration surgery (GFS). Methods: The reverse transcription-polymerase chain reaction method was performed on tissue specimens obtained from adult rabbits undergoing GFS at various postoperative time points. Results: MMP 1 decreased more than 7-fold from its baseline level during the first 2 weeks following surgery. MMP 2 decreased on the 1st postoperative day, but increased on day 2, and remained elevated for 2 weeks. The postoperative level of MMP 14 did not change much from baseline except that noted on day 9. The TIMP 1 expression showed an early biphasic pattern. MMP 3 and MMP 9 were not detected in all the specimens. Conclusions: The transcript expression of MMPs and TIMP 1 in the conjunctiva is altered following GFS, with each gene product demonstrating a unique temporal pattern.