Rabbit Model Of Trabeculectomy Research Articles

The Inhibitory Effect of Connective Tissue Growth Factor Antibody on Postoperative Fibrosis in a Rabbit Model of Trabeculectomy.

To compare the efficacy of subconjunctival injection of an anti-connective tissue growth factor antibody (anti-CTGF) versus mitomycin-C (MMC) and placebo in reducing scar formation in a rabbit model of trabeculectomy. A total of 14 rabbits were included. Nine rabbits underwent trabeculectomy with subconjunctival injections of either anti-CTGF antibody, MMC, or balanced salt solution (BSS), each administered in three eyes, before peritomy. The anti-CTGF group received a repeated dose of the antibody five days after surgery. All nine rabbits were euthanized on day 14; the globes were stained with hematoxylin & eosin, Masson's Trichrome, and immunohistochemistry for detecting alpha-smooth muscle (α-SMA) actin. RNA extraction was performed on five eyes of the remaining rabbits which included one eye without any surgery, one eye 5 hr after trabeculectomy without any injection, one eye five days after trabeculectomy without any injection, and two eyes five days after trabeculectomy with administration of MMC and BSS, respectively. The mean bleb area in the anti-CTGF, MMC, and control groups was 3.8 1.45, 5.9 1.4, and 3.5 1.9 mm2, respectively. Collagenous tissue was found to occupy the bleb area by 13.7%, 13.5%, and 18.5%, respectively. This ratio was significantly higher in the BSS group (P = 0.04). The expression of CTGF mRNA after 5 hr and five days in eyes undergoing trabeculectomy were significantly more pronounced as compared to the unoperated eye. The mean H-SCORE of α-SMA-immune reactive cells calculated as the grade of staining multiplied by the percentage of immune stained cells was 14.6, 10.22, and 140.58 in the anti-CTGF, MMC, and control groups, respectively. While the control eyes had a significantly higher score (Ps 0.001), the anti-CTGF and MMC groups were comparable (P = 0.87). Based on the results of this animal study, the anti-CTGF antibody injection resulted in a significant reduction in collagenous tissue and myofibroblast cells after trabeculectomy.

Effects of mammalian target of rapamycin inhibitors on fibrosis after trabeculectomy

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matrix (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impediments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-β1) in cultured human conjunctival fibroblast (HCF) cells. The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-β1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-β1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-β1-induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-β1 induction, TGF-β1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.

Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model

To investigate the effect of silibinin in myofibroblast transdifferentiation and in animal trabeculectomy models. The effect of silibinin on the expression of α-smooth muscle actin (α-SMA) and vimentin in response to transforming growth factor-β1 (TGF-β1) was determined in human tenon fibroblasts (HTFs). Cell migration and collagen contraction arrays were used to demonstrate the functionality of silibinin-modulated HTFs. ELISA analysis was used to determine the effect of silibinin on the release of type 1 collagen and connective tissue growth factor (CTGF). The effect of silibinin on the activation of the TGF-β receptor-related pathway was evaluated by Western blotting. A rabbit model of trabeculectomy was established to assess the effect of silibinin in vivo. TGF-β1 elevated the expression of α-SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF-β1 increased cell migration and collagen contraction of HTFs, which were also suppressed by silibinin. The production of both CTGF and type 1 collagen in TGF-β1-treated HTFs was inhibited by silibinin. The effects of silibinin on TGF-β1-stimulated HTFs were mediated via the down-regulation of TGF-β receptor-related SMAD signalling pathways. In the rabbit model of trabeculectomy, silibinin increased the period of decreasing intraocular pressure after trabeculectomy and reduced the production of collagen and α-SMA at the site of blebs in vivo. Silibinin inhibited the TGF-β receptor-related signalling pathway in TGF-β-treated HTFs and several of the downstream events associated with myofibroblast transdifferentiation. Silibinin also improved the outcome of trabeculectomies by reducing the fibrotic response in the bleb tissue in vivo.

The role of different VEGF isoforms in scar formation after glaucoma filtration surgery

Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. Our previous studies showed a differential role of VEGF isoforms in retinal physiological angiogenesis. We also demonstrated that non-selective inhibition of VEGF by bevacizumab had a beneficial effect on surgical outcome after glaucoma filtration surgery by reducing angiogenesis as well as fibrosis. However, the function of the VEGF isoforms in pathological angiogenesis and wound healing in the eye still remains unidentified. This study was designed to elucidate the differential roles of VEGF isoforms in scar formation after trabeculectomy. Furthermore, we also investigated whether pegaptanib (Macugen™, Pfizer), an aptamer which specifically blocks VEGF165, could improve surgical outcome by reducing postoperative scarring. VEGF-R2 and neuropilin-1 (NRP-1) expression was analyzed in vitro by RT-PCR, and were found to be expressed at higher levels in human umbilical vein endothelial cells (HUVEC) as compared to Tenon fibroblasts (TF). The effect of the different VEGF isoforms (VEGF121, VEGF165 and VEGF189) and pegaptanib on cell proliferation was determined via WST-1 assay. Endothelial cell proliferation was stimulated after addition of VEGF121 and VEGF165, whereas VEGF121 and VEGF189 increased fibroblast growth. These effects on proliferation were associated with an activation of the ERK pathway, as revealed using the TransAM c-Myc assay. Inhibition of the ERK pathway, by PD98059 administration, significantly reduced VEGF isoform induced cell growth. A dose-dependent reduction of endothelial cell proliferation was observed after pegaptanib administration, while only the highest dose was able to inhibit fibroblast growth. Next, the in vivo effect of pegaptanib was investigated in a rabbit model of trabeculectomy. The surgical outcome was evaluated by performing clinical investigations (IOP, bleb area, height and survival), as well as histomorphometric analyses of angiogenesis (CD31), inflammation (CD45) and fibrosis (Sirius Red). A single postoperative application of pegaptanib had a beneficial impact on surgical outcome, mainly by reducing angiogenesis, but not inflammation or collagen deposition. Repeated injections slightly improved surgical outcome, but again solely by reducing angiogenesis. In summary, our results revealed that the VEGF isoforms play a differential role in ocular wound healing: VEGF165 and VEGF121 predominantly affect blood vessel growth, whereas VEGF189 is rather involved in fibrosis, an important process in wound healing.

Anti-scar effect of bevacizumab by multiple-time and high-dose subconjunctival administration following trabeculectomy in rabbits

Background Bevacizumab is primarily aimed at pathologic angiogenesis for off-label uses such as the treatment of ocular neovascular disorders.However,as a new anti-fibrotic and anti-angiogenic agent following trabeculectomy,the safety and efficacy of bevacizumab by multiple-time and high-dose subconjunctival injection are still under study.Objective This study was to assess the safety and efficacy of bevacizumab after multiple-time and high-dose subconjunctival injections.Methods Regular trabeculectomy filtration surgery was performed on both eyes of 18 clean New Zealand White rabbits 0.1ml of bevacizumab(25g/L) was subconjunctivally injected intraoperatively and 3,5,7 days postoperatively in the left eyes of rabbits,and no any intervene in the right eyes were as normal controls.Bleb morphology was examined every 2 days and graded based on Moorefield's criteria and compared between the bevacizumab-treated eyes and normal saline(NS) eyes.The animals were sacrificed at 10,20 and 30 days after surgery respectively.The histopathological changes of the blebs were detected by hematoxylin and eosin stain to evaluate the cellular element around the bleb,and Masson stain was used to assess the degree of fibroblast proliferation.The degree of vascularity of bleb was identified by anti-vWf stain.Approval of this protocol was obtained and permitted from People's Hospital Institutional Animal Care and Use Committee of Peking University.The use of experimental animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Compared to the NS-treated eyes,bevacizumab-treated eyes showed the larger and more diffusely elevated blebs with the significant difference(2.48±0.22cm2 versus 1.73±0.27cm2,t=5.194,P＜0.05).The survival time of the filtration bleb in bevacizumab panel was longer in bevacizumab-treated eyes compared to control eyes,showing a significantly difference between them(21.0±1.56 days versus 12.5±1.97 days,t=3.830,P=0.005).Histological and immunohistochemical analysis confirmed that bleb and adjacent conjunctiva vascularity(A value) was significantly less in bevacizumab-treated eyes than that in control eyes at 20 days after surgery with the difference value 14320.7±4134.9(t=12.275,P＜0.05),and fibroblast deposition value was evidently diminished after bevacizumab treatment at 30 days following surgery in comparison with control eyes with the mean difference 0.27±0.03(t=15.980,P＜0.05＝.Conclusion Repeated subconjunctival injection of bevacizumab can effectively prolong the survival time of bleb in a rabbit model of trabeculectomy and limit the degree and area of vascularization in 30 days following surgery.Bevacizumab inhibit fibroblast-meditated tissue formation significantly in the later phase of vascularization after trabeculectomy. Key words: Trabeculectomy; Bleb; Bevacizumab; Fibrosis; Vascularization; Subconjunctival injection

The role of LOX and LOXL2 in wound healing after glaucoma filtration surgery

Abstract Purpose Lysyl oxidase (LOX) and lysyl oxidase‐like protein 2 (LOXL2) are involved in the crosslinking of collagen and elastin. We have shown that LOX and LOXL2 were upregulated after glaucoma surgery. The aim of this study was to investigate the efficacy of anti‐LOX (M64) and of anti‐LOXL2 (M20) antibodies (Arresto Biosciences) in a rabbit model of trabeculectomy. Methods Treatment with the antibodies or control (PBS) was initiated on day 0 after surgery by giving an intracameral injection (0.6 mg) and a subconjunctival (SC) injection (0.3 mg). Thereafter the antibodies were injected twice a week SC (0.3 mg) until 30 days after the surgery. The outcome of the treatment was studied by clinical investigation (IOP, bleb area and bleb survival) and by analysis of angiogenesis (CD31), inflammation (CD45) and collagen deposition (Sirius Red). Results Both antibodies were able to significantly improve surgical outcome by increasing bleb area and bleb survival compared to control (p<0.001). Analyses of the different stainings showed a significant reduction in angiogenesis (47%; p=0.01), inflammation (34%; p=0.0003) and fibrosis (16%; p=0.01), respectively, in the anti‐LOXL2 treated group compared to control. Anti‐LOX antibody resulted in a significant reduction only in collagen deposition (22%; p=0.0009 compared to control). Conclusion These results showed that LOX and LOXL2 are important in ocular wound healing. We also provide evidence on effectiveness of repeated anti‐LOXL2 injections after trabeculectomy. The anti‐LOXL2 antibody was able to improve surgical outcome by reducing angiogenesis, as well as inflammation and collagen deposition. These new insights may have important therapeutic implications for glaucoma surgery. Commercial interest

Is inhibition of VEGF165 sufficient to inhibit scar formation after trabeculectomy?

Abstract Purpose We have previously shown that VEGF plays a role in scar formation after glaucoma surgery and that inhibition of all VEGF‐isoforms by bevacizumab is able to reduce scar formation. This study was designed to elucidate the exact role of VEGF165 in scar formation after trabeculectomy. The effect of pegaptanib (Pfizer), which specifically blocks VEGF165, was investigated in vitro and in vivo. Methods The effect of pegaptanib on Tenon fibroblasts and HUVEC in vitro was determined using a WST‐1 proliferation assay. The effect of the aptamer was also investigated in vivo in a rabbit model for glaucoma surgery by studying angiogenesis, inflammation and collagen deposition. Results A dose‐dependent reduction of HUVEC proliferation was seen after pegaptanib administration in vitro (P<0.05 with a dose of at least 0,3 mg/ml). A concentration of 2 mg/ml pegaptanib was necessary to inhibit the proliferation of Tenon fibroblasts. The aptamer also significantly reduced blood vessel density 3 days after surgery in a rabbit model of trabeculectomy (P=0.001). There were no significant differences in inflammation and collagen deposition in the treated eyes compared to control. Conclusion Whereas HUVEC cells were inhibited by pegaptanib in a dose‐dependent way, Tenon fibroblasts were only inhibited at the highest dose. A single administration of pegaptanib at the time of trabeculectomy reduced postoperative angiogenesis, but not inflammation or collagen deposition. Further studies using repeated pegaptanib injections are necessary to investigate whether the lack of effect of pegaptanib on scarring was due to a shorter working time of pegaptanib compared to bevacizumab, or due to the difference in their effect on the various VEGF isoforms.

Microplasmin improves surgical outcome in a rabbit model for trabeculectomy

Abstract Purpose This study was designed to study the efficacy and safety of Microplasmin as an anti‐scarring agent after trabeculectomy in a rabbit model. Methods The effect of Microplasmin was investigated in vivo in a rabbit model for glaucoma surgery. Clinical outcome measures were intra‐ocular pressure, bleb area and survival, side effects on slit lamp examination. Moreover, (immuno‐) histochemical analysis of the eyes was performed, with quantification of inflammation (CD 45) and collagen deposition (Trichrome and Sirius Red). In the first experiment (n=10), Microplasmin anterior chamber injection was compared to placebo injection. In the second experiment (n=3), topical Microplasmin drops were compared to placebo drops. In the third experiment (n=5) the combination of Microplasmin anterior chamber injection and topical drops was compared to placebo injection and drops. All experiments were conducted in a masked observator way. Results Microplasmin significantly augmented the bleb area and survival in a rabbit model of trabeculectomy after a single anterior chamber injection or combination therapy (injection combined with drops) compared to control. Collagen deposition was borderline reduced after Microplasmin administration compared to control. No significant changes in inflammation were noticed in the anterior chamber or in the conjunctiva. Conclusion Microplasmin single injection or combination with postoperative drops improved the outcome after trabeculectomy. In a rabbit model, larger blebs were produced for a longer period compared to control, and collagen deposition tended to decrease in this small series.

Inhibition of Vascular Endothelial Growth Factor Reduces Scar Formation after Glaucoma Filtration Surgery

Filtration failure due to excessive postoperative scarring remains a major problem after glaucoma surgery. The authors have investigated whether glaucoma and filtration surgery are associated with increased levels of vascular endothelial growth factor (VEGF), and whether a humanized monoclonal antibody against VEGF, bevacizumab, can reduce postoperative scar formation and improve surgical outcome. The levels of VEGF in samples of aqueous humor were measured by ELISA. The expression of the VEGF receptors Flt-1 and KDR was analyzed in cultured Tenon fibroblasts by real-time RT-PCR and Western blotting. The effect of VEGF and bevacizumab on Tenon fibroblasts in vitro was determined using a proliferation assay. The in vivo effect of the antibody was investigated in a rabbit model of trabeculectomy by measuring the intraocular pressure (IOP) and bleb area, and by immunohistological analysis of angiogenesis, inflammation, and fibrosis. VEGF levels were increased significantly in the aqueous humor of glaucoma patients and rabbits that had undergone surgery. Both VEGF receptors were expressed on Tenon fibroblasts. Fibroblast proliferation in vitro was stimulated by delivery of VEGF, and was inhibited by administration of bevacizumab. The antibody also reduced angiogenesis and collagen deposition significantly, and improved the outcome of glaucoma surgery in rabbits. VEGF was upregulated in the aqueous humor of glaucoma patients and in the rabbit model, and it stimulated fibroblast proliferation in vitro. This suggests that it is involved in the scarring process after filtration surgery. Bevacizumab reduced the proliferation of fibroblasts in vitro and improved surgical outcome.

Inhibition of vascular endothelial growth factor reduces scar formation after glaucoma filtration surgery

Abstract Purpose In 30‐50%, glaucoma filtration surgery fails due to excessive postoperative scarring. This study was designed to elucidate the role of vascular endothelial growth factor (VEGF) in fibrosis after glaucoma surgery. In addition, the effects of the monoclonal humanized VEGF‐antibody bevacizumab (AvastinTM, Genentech) on fibroblast proliferation and outcome after trabeculectomy were studied. Methods The effect of VEGF and bevacizumab on Tenon fibroblasts in vitro was determined using a Tenon fibroblast mediated proliferation assay. The effect of the antibody was also investigated in vivo in a rabbit model for glaucoma surgery by measuring intra‐ocular pressure (IOP) and bleb area, and by (immuno‐)histological analysis of inflammation and fibrosis. VEGF‐concentration after bevacizumab‐administration was measured in samples of aqueous humor by ELISA. Results The proliferation of human and rabbit Tenon fibroblasts in vitro was stimulated by VEGF‐delivery and inhibited by bevacizumab‐administration. The antibody also significantly improved glaucoma surgery outcome, more specific the bleb area, in a rabbit model of trabeculectomy. Inflammation and collagen deposition were significantly reduced after bevacizumab treatment as compared to sham injections. VEGF was significantly reduced in aqueous humor after bevacizumab‐administration. Conclusion VEGF stimulates in vitro fibroblast proliferation suggesting that it plays a role in scarring after filtering surgery. Furthermore, the monoclonal humanized VEGF‐antibody reduces in vitro fibroblast proliferation and improves surgical outcome in vivo in a rabbit model of trabeculectomy.

Effects of Interleukin-1 Blockers on Ophthalmic Wound Healing in a Rabbit Model of Trabeculectomy

A trabeculectomy model was used to test the efficacy of some Interleukin-1 blockers on the modulation of ophthalmic wound healing in the pigmented rabbit. The corticosteroid, methyl prednisolone, was used as a positive control and was effective in prolonging the time to failure of the trabeculectomy by 21%. The experimental compounds CK-17, CK-101A, CK-102 and CK-103A were more efficacious than vehicle control at prolonging the number of days before failure of the trabeculectomy by 55%, 55%, 30% and 79%, respectively. The CK- compounds increased the amount of time before failure of the fistula, with no side effects at the doses tested to date.