Rabbit Tenon's Fibroblasts Research Articles

Pirfenidone inhibits proliferation of rabbit tenon fibroblasts by down-regulating TGF-β3 in the TGF-β/Smad pathway

To investigate the molecular mechanism by which pirfenidone inhibits scar formation through the TGF-β/Smad pathway. Cultured rabbit tenon fibroblasts (RTFs) were treated with different concentrations of pirfenidone to determine its initial active concentration and optimum concentration of pirfenidone for inhibiting RTF proliferation using CCK-8 assay. In RTFs treated with pirfenidone at the initial and optimal concentrations, expressions of TGF-β3, collagen I and collagen III were examined with both immunofluorescence assay and Western blotting, and their mRNA expression levels were detected using RT-PCR. The initial and optimal concentrations of pirfenidone for inhibiting RTF proliferation were 0.1 mg/mL and 0.27 mg/mL, respectively. In RTFs treated with pirfenidone at the two concentrations for 24 h, both immunofluorescence assay and Western blotting showed significantly lowered protein expressions of TGF-β3, collagen I and collagen III as compared with those in the control group (P < 0.05). The mRNA expressions of TGF-β3, collagen I and collagen III in the RTFs were also significantly lowered after treatment with pirfenidone at the initial and optimal concentrations (P < 0.05). Pirfenidone concentration-dependently inhibits the proliferation of RTFs possibly by down-regulating the expression of TGF-β3 in the TGF-β/Smad pathway.

Forkhead domain inhibitory-6 attenuates subconjunctival fibrosis in rabbit model with trabeculectomy

Antiproliferative therapies are crucially important for improving the success rate of the glaucoma filtration surgeries. In this study, we investigated the potential efficacy of Forkhead Domain Inhibitory-6 (FDI-6) in inhibiting post-trabeculectomy subconjunctival fibrosis. In vitro, the effect of FDI-6 (10 μM) on fibrotic response and its underlying mechanism were investigated in rabbit tenon's fibroblasts (RTFs) treated with or without transforming growth factor-β1 (TGF-β1, 20 ng/mL). In vivo, FDI-6 (40 μM) was injected subconjunctivally to a rabbit trabeculectomy model. Intraocular pressure (IOP) changes were monitored within the 14-day period post-surgery. Bleb morphology and subepithelial fibrosis at the operating area were evaluated with slit lamp and confocal microscopic examinations and with histologic examinations. The results showed that, in cell culture studies, FDI-6 suppressed the proliferation, migration, collagen gel contraction and the expression levels of fibronectin (FN) and α-smooth muscle actin (α-SMA) in RTFs with TGF-β treatment by down-regulating the TGF-β1/Smad2/3 signaling pathway. In animal studies, the IOPs of the FDI-6-treated group were significantly lower than those of the saline-treated group after trabeculectomy. The FDI-6-treated eyes showed a better bleb appearance with fewer blood vessels compared to the saline-treated eyes. The analysis of confocal microscopy in vivo and histopathology revealed that subconjunctival fibrosis after trabeculectomy was significantly attenuated in the FDI-6-treated group compared to the controls. In conclusion, our studies indicate that FDI-6 exerts an inhibitory effect on subconjunctival fibrosis caused by trabeculectomy, holding potentials as a new antiproliferative agent used in anti-glaucoma filtration surgeries in the future.

Development of a novel CsA-PLGA drug delivery system based on a glaucoma drainage device for the prevention of postoperative fibrosis

The formation of a scar after glaucoma surgery often leads to unsuccessful control of intraocular pressure, and should be prevented by using a variety of methods. We designed and developed a novel drug delivery system (DDS) comprising cyclosporine A (CsA) and poly(lactic-co-glycolic acid) (PLGA) based on a glaucoma drainage device (GDD) that can continuously release CsA to prevent postoperative fibrosis following glaucoma surgery. The CsA@PLGA@GDD DDS was observed by field emission scanning electron microscopy and revealed an asymmetric pore structure. Thermogravimetric analysis was performed to measure the weight loss and evaluate the thermal stability of the CsA@PLGA@GDD DDS. The in vitro drug release profile of the DDS was studied using high performance liquid chromatography, which confirmed that the DDS released CsA at a stable rate and maintained adequate CsA concentrations for a relatively long time. The biocompatibility of the DDS and the inhibitory effects on the postoperative fibrosis were investigated in vitro using rabbit Tenon's fibroblasts. The in vivo safety and efficacy of the DDS were examined by implanting the DDS into Tenon's capsules in New Zealand rabbits. Bleb morphology, intraocular pressure, anterior chamber reactions, and anterior chamber angiography were studied at a series of set times. The DDS kept the filtration pathway unblocked for a longer time compared with the control GDD. The results indicate that the CsA@PLGA@GDD DDS represents a safe and effective strategy for preventing scar formation after glaucoma surgery.

Development of a novel injectable drug delivery system for subconjunctival glaucoma treatment

In this study we present the development of an injectable polymeric drug delivery system for subconjunctival treatment of primary open angle glaucoma. The system consists of hyaluronic acid sodium salt (HA), which is commonly used in ophthalmology in anterior segment surgery, and an isocyanate-functionalized 1,2-ethylene glycol bis(dilactic acid) (ELA-NCO). The polymer mixtures with different ratios of HA to ELA-NCO (1/1, 1/4, and 1/10 (v/v)) were investigated for biocompatibility, degradation behavior and applicability as a sustained release system. For the latter, the lipophilic latanoprost ester pro-drug (LA) was incorporated into the HA/ELA-NCO system.In vitro, a sustained LA release over a period of about 60days was achieved. In cell culture experiments, the HA/ELA-NCO (1/1, (v/v)) system was proven to be biocompatible for human and rabbit Tenon's fibroblasts. Examination of in vitro degradation behavior revealed a total mass loss of more than 60% during the observation period of 26weeks.In vivo, LA was continuously released for 152days into rabbit aqueous humor and serum. Histological investigations revealed a marked leuko-lymphocytic infiltration soon after subconjunctival injection. Thereafter, the initial tissue reaction declined concomitantly with a continuous degradation of the polymer, which was completed after 10months.Our study demonstrates the suitability of the polymer resulting from the reaction of HA with ELA-NCO as an injectable local drug delivery system for glaucoma therapy, combining biocompatibility and biodegradability with prolonged drug release.

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.

Controlled Delivery of 5-Chlorouracil Using Poly(Ortho Esters) in Filtering Surgery for Glaucoma

To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

Inhibition of Tenon's fibroblast proliferation and enhancement of filtration surgery in rabbits with cytosine arabinoside.

Our purpose was to study the antiproliferative effect of cytosine arabinoside (Ara-C) on rabbit Tenon's fibroblasts and the efficacy of Ara-C as an adjunctive antifibrosis treatment for glaucoma filtration surgery in the rabbit eye. Rabbit Tenon's fibroblasts were exposed to 1 microg/ml of Ara-C for various time intervals, then cell number and viability was assessed at different time points. Following posterior lip sclerectomy, rabbit eyes were treated with 10 mg subconjunctival Ara-C daily for 7 days, then every other day for 7 days. Rabbit fibroblasts exposed to 1 microg/ml Ara-C for 1 hour showed no significant decrease in cell number compared with control. Continuous or 24-hour incubation of fibroblasts with Ara-C was lethal to the cells. Exposure of cells to Ara-C for 3-, 6-, and 9-hour intervals caused significant reduction of cell proliferation. Pulsed treatment of cells with 6 hour exposure to 1 microg/ml Ara-C every 3 days caused prolonged suppression of cell proliferation. Following posterior lip sclerectomy in rabbits, topical instillation of Ara-C drops at varying concentrations and dosing intervals did not cause any significant lowering of intraocular pressure compared with control eyes, although bleb survival was prolonged in eyes treated with Ara-C (P < 0.01). In rabbit eyes treated postoperatively with subconjunctival injections of 10 mg Ara-C every other day for two weeks, the mean intraocular pressure was significantly decreased and the bleb survival time was significantly prolonged (P < 0.0067) compared with control eyes. In conclusion, Ara-C inhibits rabbit Tenon's fibroblast proliferation in vitro. Postoperative subconjunctival injection of Ara-C results in improved bleb function after filtration surgery in the rabbit.

Normal rabbit aqueous humour, fibronectin, and fibroblast conditioned medium are chemoattractant to Tenon's capsule fibroblasts.

Some of the chemotactic and chemokinetic properties of rabbit Tenon's fibroblasts were examined in a 48-well micro-chemotaxis chamber. Normal rabbit aqueous humour, fibronectin, and fibroblast conditioned medium were used in the assay, and all were shown to be chemoattractant. In addition, aqueous humour was shown to be powerfully chemotactic. Since the failure of human trabeculectomies is associated with migration of fibroblasts to the operation site, the study of the chemoattractant influences acting on these cells may allow manipulation of their behaviour in order to influence favourably the outcome of surgery.