Effect of aflibercept on proliferative vitreoretinopathy: Proteomic analysis in an experimental animal model.

PURPOSE: The aim of this study was to monitor inflammatory, proliferative and progressive effects of proliferative vitreoretinopathy (PVR) and aflibercept treatment in dispase induced PVR rat model by proteomic analysis. MATERIAL AND METHODS: A total of 35 male Long Evans pigmented rats were divided into three groups, namely, PVR (dispase+saline), PVR+aflibercept (dispase+aflibercept) and control. The PVR group received 2 µl of 0.03 IU/µl dispase and 2 µl saline, the PVR+aflibercept group received 2 µl of 0.03 IU/µl and 2 µl of 40 mg/ml aflibercept at the first day of the experiment. At the end of the 6th week all retina and vitreous specimens were collected by evisceration and transferred to the proteomics laboratory for analysis. Proteomic analysis by 2D gel electrophoresis coupled with MALDI-TOF/TOF was performed. RESULTS: In the PVR and PVR+aflibercept group 16 different proteins that were identified to be differentially regulated in comparison to the control group. In the PVR+aflibercept group, ENO1, ENO2, LDH-B, PEBP-1 and GS levels were higher than the PVR group. In addition, the association of proteins such as UCHL, PEBP1, PDHB and ENO1 with PVR has been demonstrated for the first time. CONCLUSION: STRING analysis elucidated the functional protein-protein interaction among the differentially regulated proteins and highlighted that those proteins mainly played roles in carbon and nucleotide metabolisms. Functional analysis of the differentially regulated proteins indicated the presence of inflammation, gliosis and retinal damage in the PVR group. Aflibercept treatment had pronounced effect on prevention of inflammation and retinal damage while causing a slight increase in gliosis. However, aflibercept treatment was not effective enough to normalize the levels of differentially regulated proteins of the PVR group. Therefore, we predict that the treatment dose of aflibercept used in this study was below of its ideal concentration and should be increased in the future studies. The differential regulation of these structural proteins in this study should shed some light to the mechanism of glial wound formation in the retina and guide future treatment modalities.

A Mouse Model of Proliferative Vitreoretinopathy Induced by Intravitreal Injection of Gas and RPE Cells.

Purpose: Develop a reproducible proliferative vitreoretinopathy (PVR) mouse model that mimics human PVR pathology. Methods: Mice received intravitreal injections of SF6 gas, followed by retinal pigment epithelial cells 1 week later. PVR progression was monitored using fundus photography and optical coherence tomography imaging, and histologic analysis of the retina as an endpoint. We developed a PVR grading scheme tailored for this model. Results: We report that mice that received gas before retinal pigment epithelial injection developed more severe PVR. In the 1 × 104 retinal pigment epithelial cell group, after 1 week, 0 of 11 mice in the no gas group developed grade 4 or greater PVR compared with 5 of 13 mice in the SF6 gas group (P = 0.02); after 4 weeks, 3 of 11 mice in the no gas group developed grade 5 or greater PVR compared with 11 of 14 mice in the SF6 gas group (P = 0.01). We were able to visualize contractile membranes both on the retinal surface as well as within the vitreous of PVR eyes, and demonstrated through immunohistochemical staining that these membranes expressed fibrotic markers alpha smooth muscle actin, vimentin, and fibronectin, as well as other markers known to be found in human PVR membranes. Conclusions: This mouse PVR model is reproducible and mimics aspects of PVR pathology reported in the rabbit PVR model and human PVR. The major advantage is the ability to study PVR development in different genetic backgrounds to further elucidate aspects of PVR pathogenesis. Additionally, large-scale experiments for testing pharmacologic agents to treat and prevent PVR progression is more feasible compared with other animal models. Translational Relevance: This model will provide a platform for screening potential drug therapies to treat and prevent PVR, as well as elucidate different molecular pathways involved in PVR pathogenesis.

The effect of infliximab and octreotide on cytokine levels experimental proliferative vitreoretinopathy.

Purpose: To investigate the efficiency of intravitreal octreotide, which has previously been shown to have benefits in the treatment of proliferative vitreoretinopathy (PVR), and intravitreal infliximab as a novel option in an experimental dispase-induced PVR model.Methods: A total of 28 pigmented guinea pigs were divided into four groups, and each group consisted of seven subjects. Group 1 (control) was treated with a 0.2 mL saline solution intravitreally from 1.5 mm behind the limbus. Group 2 (sham) was treated with 0.07 IU/0.1 mL dispase 0.1 mL saline solution using the same method. Group 3(infliximab) received 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL infliximab, and group 4(octreotide) was treated with 0.07 IU/0.1 mL dispase and 1 mg/0.1 mL octreotide. An intravitreal injection of infliximab and octreotide was administered to groups 3 and 4 two times during the experiment. The subjects were held for a 10-week period to await for the formation of PVR. At the end of ten weeks, the eyes were enucleated, and tumour necrosis factor-alpha (TNF-α), interleukin 1(IL-1), interleukin 6 (IL-6), transforming growth factor (TGF-ß), and platelet-derived growth factor (PDGF) and levels in homogenised retina tissue were measured using the enzyme linked-immuno-sorbent assay (ELISA) method.Results: Retinal TNF-α, IL-1, IL-6, and PDGF levels had significantly decreased in treatment groups compared to the sham group (p < 0.05). The decrease in the level of TGF-ß was not statistically significant between the treatment and the sham groups (p > 0.05).Conclusions: Intravitreal infliximab can inhibit the development of PVR and reduce levels of cytokine, which plays an essential role in the pathogenesis of PVR. The results of our study suggest that it may be possible to identify the ideal adjuvant pharmacological drugs that are effective in preventing PVR.

Experimental proliferative vitreoretinopathy in rabbits by delivery of bioactive proteins with gelatin microspheres.

Proliferative vitreoretinopathy (PVR) is a challenging pathological condition, often causing failure of retinal detachment surgery. The purpose of this study was to evaluate the feasibility of a delivery system of bioactive proteins using anionic and cationic gelatin microspheres and to establish a new PVR model in rabbits by intraocular sustained delivery of basic fibroblast growth factor (bFGF) and interferon-beta (IFNß). Anionic and cationic gelatin microspheres were prepared and immersed in bFGF and IFNß solution, respectively, to yield a polyion complex between gelatin matrix and a bioactive protein. The bFGF-impregnated microspheres were injected into the subretinal space in rabbit eyes. At week 2, the IFNß-impregnated microspheres also were injected into the same space. Control eyes received gelatin microspheres without bFGF or IFNß, or both. The eyes then were observed for 8 weeks by ophthalmoscopy, fundus photography, and fluorescein angiography. The eyes also were evaluated histologically. In the group with both bFGF and IFNß, the number of eyes with more severe PVR increased over time. Histologic examination showed retinal folds. In contrast, no proliferative changes were seen in any control groups. Subretinal implantation of bFGF and IFNß-impregnated gelatin microspheres induced reproducible PVR in rabbit eyes. This study guaranteed delivery of bioactive proteins with gelatin microspheres.

An immunohistochemical analysis of a rat model of proliferative vitreoretinopathy and a comparison of the expression of TGF-ß and PDGF among the induction methods.

Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment surgery or ocular trauma. Our previous study indicated that intravitreal co-injection of retinal pigmented epithelial (RPE)-J cells and platelet-rich plasma (PRP) (not RPE-J cells or PRP alone) in Wistar rat eyes can successfully induce a model of PVR. But which cells are involved in this process and why different induction methods, intravitreal injection of RPE-J cells or/and PRP, induced a different situation remain to be unknown. In this study, immunohistochemistry was performed to identify the main cell types involved in this process. The expression levels of transforming growth factor (TGF)-ß2, platelet-derived growth factor (PDGF)-AA and PDGF-BB were tested using enzyme-linked immunosorbent assay (ELISA). The results showed that RPE cells, glial cells, fibroblasts and macrophages took part in the pathogenesis of this model. The expression levels and durations of TGF-ß2 and PDGF-BB partially explained the different results induced by the different induction methods. This provides an experimental proof for attenuation of the experimental PVR by targeting at a specific cells or growth factor.

The relation between bevacizumab injection and the formation of subretinal fibrosis in diabetic patients with panretinal photocoagulation.

BACKGROUND AND OBJECTIVE: To report the development of subretinal fibrosis after the injection of intravitreal bevacizumab in eyes with proliferative diabetic retinopathy (PDR) refractory to panretinal laser photocoagulation (PRP). PATIENTS AND METHODS: Twenty-one eyes of 15 patients treated with PRP and intravitreal injection of bevacizumab were included in this study. The clinical outcomes of 21 eyes having subretinal fibrosis after intravitreal bevacizumab injection were reviewed. RESULTS: There were 9 men and 6 women with a mean age of 51.3 +/- 8.9 years. All eyes had PDR refractory to panretinal photocoagulation and were treated with at least one intravitreal injection of 1.25 mg of bevacizumab (mean number of injections: 1.8). Before injection, there was subretinal fibrosis in 5 eyes and vitreoretinal traction in 19 eyes. After a mean follow-up period of 7 months, the development or progression of subretinal fibrosis was detected in all eyes. CONCLUSION: Intravitreal injection of bevacizumab may cause formation or progression of subretinal fibrosis in patients with PDR refractory to PRP.

Estudo experimental da inibição da proliferação vitreorretiniana pelo uso da hiperecina / Experimental study of vitreoretinal proliferation inhibition by the use of hypericin

OBJETIVOS: Induzir a produção de membranas vitreorretinianas em modelo de trauma ocular animal. Avaliar a inibição do desenvolvimento da proliferação vitreorretiniana (PVR) com o uso de hiperecina. MÉTODOS: Estudo Experimental. Foram utilizados 19 coelhos machos pigmentados adultos com peso entre 2.000 e 3.000 gramas. Todos submetidos a modelo de trauma com dispase associada à diatermia da retina para indução de membranas de PVR. Separados randomicamente para receberem hiperecina (10 µM em 0,1 ml) ou solução salina (0,1 ml) como placebo. Avaliados clinicamente no sétimo, décimo quarto, vigésimo primeiro e vigésimo oitavo dias de pós-operatório com oftalmoscopia indireta e retinografia colorida digitalizada. O grau de PVR foi classificado em estágios (de 0 a 7) segundo Hida e colaboradores. RESULTADOS: A formação de membranas esteve presente em 79 por cento dos olhos, sendo 100 por cento nos olhos do grupo placebo e 60 por cento nos olhos do grupo tratamento (hiperecina). A comparação entre as médias dos estágios de PVR entre os grupos mostrou diferença estatisticamente significativa, com valor p=0,0321 pelo teste Wilcoxon. CONCLUSÕES: O modelo de trauma com uso de dispase e diatermia da retina produz membranas vitreorretinianas. A hiperecina mostrou-se eficaz na diminuição do aparecimento e progressão do PVR.

PURPOSE: To produce proliferative vitreoretinopathy (PVR) in an animal ocular trauma model. To evaluate the inhibition of (PVR) emergence and progression by hypericin. METHODS: Experimental Study. Nineteen pigmented male adult rabbits weighing between 2,000 and 3,000 grams were used in this study. All of them were submitted to trauma model with dispase and retinal diathermy to induce PVR membranes formation. They were randomly assigned to receive hypericin (10 µM in 0.1 ml) or saline solution (0.1 ml) as placebo. They were evaluated clinically in the seventh, fourteenth, twenty-first and twenty-eighth postoperative days with indirect ophthalmoscopy and digital color retinography. The PVR degree was classified according to Hida (0 to 7). RESULTS: Membranes formation was present in 79 percent of the eyes; being 100 percent in the eyes of placebo group and 60 percent in the eyes of treatment group (hypericin). The comparison between PVR phases averages within the groups showed a statistically significant difference between the two groups, with a p value of 0.0321 for Wilcoxon test. CONCLUSIONS: The trauma model with dispase and retinal diathermy produces vitreoretinal membranes. Hypericin was considered effective in PVR emergence and progression decrease.

Acute contraction of the proliferative membrane after an intravitreal injection of bevacizumab for advanced retinopathy of prematurity.

BACKGROUND: Despite the recent reports describing the benefits of the intravitreal injection of bevacizumab (IVB) to treat ocular neovascular disorders, including retinopathy of prematurity (ROP), the possible adverse effects of this therapy must also be described. We report here a case of advanced ROP which showed an acute contraction of the proliferative membrane after an intravitreal injection of bevacizumab. METHODS: A female infant born at 23 weeks of gestation with a birth weight of 598 g was referred to the ophthalmologist at 4 weeks of age. With funduscopic examinations, broad avascular retinas were found in both eyes. Since the ROP had progressed to stage 3, zone 1 with plus disease in both eyes, retinal photocoagulation was performed at 10 weeks of age. Despite the adequate photocoagulation therapy, the proliferation progressed further, and partial tractional retinal detachment (TRD) occurred in the right eye, classified as stage 4A with plus disease. After extensive discussion with the parents about the risks and benefits of IVB as an alternative therapy, they consented to the treatment. Under general anesthesia, an intravitreal injection of 0.4 mg bevacizumab was performed at 14 weeks of age. RESULTS: The following day, the vascular component of the fibrovascular membrane (FVM) regressed, and acute fibrosis occurred. However, the ring-shaped FVM contracted centripetally, which caused a deterioration of the TRD. The contraction of the FVM progressed until 7 days after IVB, and resulted in a funnel-like retinal detachment at the posterior retina. The other eye also showed TRD at 19 weeks of age classified as stage 4B, which necessitated a vitrectomy. No systemic complications were noted before and after the treatment. CONCLUSIONS: IVB is a useful therapy to maintain aggressive ROP. However, IVB might cause TRD progression in some specific cases.

Further evidence for proinflammatory nature of perfluorohexyloctane in the eye.

BACKGROUND: The authors present a clinicopathological report of their initial experience with perfluorohexyloctane (F6H8), a novel semifluorinated liquid fluorocarbon developed as a long-term vitreous substitute. METHODS: A retrospective observational review was performed of five patients in whom F6H8 had been used for management of rhegmatogenous retinal detachment. Surgical specimens taken from two patients at the time of F6H8 removal were also submitted for histopathological, immunohistochemical and electron microscopic analysis. RESULTS: Clinical and histological analysis of the present small case series confirmed the propensity of F6H8 to emulsify, and suggested a probable biological reaction to F6H8. Surrounding and engulfing the F6H8 were numerous cells morphologically in keeping with macrophages. Immuno-histochemistry confirmed macrophage phenotype but electron microscopic evaluation showed epithelial ultra-structural features. It is suggested that the finding of macrophagic phenotype in cells with epithelial ultra-structure provides further evidence for a continuum of phenotypic differentiation of the pigment epithelial cells as part of the repair and regeneration that is the proliferative vitreo-retinopathy (PVR) response. CONCLUSIONS: The data do not indicate any benefit of F6H8 over other perfluorocarbons for use in short-term post-operative intraocular tamponade. Although early experience suggests that F6H8 use in primary vitrectomy with minimal PVR is acceptable and produces temporary inflammatory effects only, these cases can often be successfully managed by conventional scleral buckling techniques, or vitrectomy with standard tamponading agents, without the need for F6H8 and subsequent extra surgical procedures. Furthermore in eyes already predisposed to inflammation through prior surgery and/or presence of PVR, the inflammatory effects were not insignificant. The use of F6H8 is not recommended in the clinical setting, except as part of a controlled trial subject to the approval of an ethics committee and informed consent.

Clinical findings on the use of long-term heavy tamponades (semifluorinated alkanes and their oligomers) in complicated retinal detachment surgery.

BACKGROUND: Heavy tamponades for pathologies in the lower part of the retina are a new development, and different tamponades have recently come into clinical use: semifluorinated alkanes (F(6)H(6), F(6)H(8)) and their oligomers (OL62HV). METHOD: Nine patients had been operated on using F(6)H(8) (n=5) and by OL62HV (n=4). In all cases the reasons for using the tamponades were complicated retinal detachments in the lower part. In three cases the use was primary and in six cases tamponades were used after reoperations. In all cases the endotamponade was removed within 6 weeks. Fluorescein angiography (FLA) was performed in the F(6)H(8) group. RESULTS: In the F(6)H(8)group dispersion developed in two of the three aphacic patients. In two out of five cases soft epiretinal membranes and cellular material could be found between the substance and the lower periphery. In two membranes examined by light microscopy, cystic cells and amorphous material could be found. In one case (PDRP, aphacic) cyclophotocoagulation had to be performed because of persistent elevated IOP. FLA was unremarkable. In the OL62HV group, severe recurrent PVR reaction occurred in the lower periphery (2/4) and unusual precipitates were observed (4/4). In one case, after a normal postoperative period (VA 0.05 after 5 days) an extensive cellular reaction on the complete surface of the tamponade occurred. After 5 weeks VA was no light perception. During removal of the oligomer unusual adherent cellular components were found on the surface of the retina. The retina appeared necrotic, showed constricted retinal vessels and there was optic atrophy. Histologically, fluffy epiretinal material and a lens capsule obtained from one eye filled with OL62HV resembled the appearance with F(6)H(8). CONCLUSION: Heavy endotamponades on the basis of semifluorinated alkanes can lead to an unusual biological reaction and need further investigation before clinical use.

Ribozyme to proliferating cell nuclear antigen to treat proliferative vitreoretinopathy.

PURPOSE: A DNA-RNA chimeric ribozyme was developed that targets the mRNA of a cell cycle regulatory protein, proliferating cell nuclear antigen (PCNA). The hypothesis was that inhibition of PCNA, essential in DNA replication, would decrease the proliferation of cells that are involved in formation of granuloma after surgical procedures in the eye. The ability of intravitreous injection of this ribozyme to prevent or inhibit development of proliferative vitreoretinopathy (PVR) was tested in a dispase-induced rabbit PVR model. METHODS: Rabbit genomic DNA encoding PCNA was cloned and sequenced. The cleavage of rabbit PCNA by the chimeric ribozyme was tested in vitro. Delivery of the ribozyme to rabbit retinal pigment epithelial (RPE) or fibroblast cells and its effects on proliferation of fibroblasts were examined. The stability of the ribozyme in vitreous fluid and serum was studied as well. In the dispase-induced rabbit model of PVR, the ability of the PCNA ribozyme to prevent or inhibit development of PVR and retinal detachment (RD) was tested. Experimental groups receiving intravitreous PCNA ribozyme, with or without a lipid vehicle, were compared with sham-treated control groups. Progression of PVR in rabbit eyes was followed by indirect ophthalmic examination and observations documented by fundoscopic photography, gross pathology, and histopathology. RESULTS: The chimeric ribozyme targeted a specific sequence in the rabbit PCNA that was identical with that in the human. In vitro cleavage assays confirmed the ability of the ribozyme to cleave the mRNA of PCNA. The catalytic efficiency in vitro, calculated as k(2)/K(m)(app), was 0.26 microM(-1) x min(-1). In vitro studies with fluoresceinated ribozyme indicated that lipid vehicles facilitated delivery of the ribozyme into cells causative of PVR (RPE and fibroblasts); however, the PCNA ribozyme decreased the proliferation of fibroblasts, with or without lipid vehicle. The ribozyme displayed good stability in vitreous fluid, whereas, it degraded quite rapidly in serum. In animal experiments, rabbits in sham-treated groups usually exhibited development of severe PVR characterized by focal traction or RD. Animals in the PCNA ribozyme-treated groups usually did not exhibit an RD. If they did have RD, it was small and localized, or focal tractions developed that did not progress to the degree that the sham-treated animal eyes did over the follow-up period. The in vivo use of a lipid delivery vehicle resulted in a precipitate; however, an effective naked ribozyme dose was identified that did not cause this side effect. CONCLUSIONS: In addition to validating the newly developed dispase PVR rabbit model, the results indicate that ribozyme targeted against the cell cycle agent PCNA is efficacious in the treatment or prevention of PVR in the rabbit eye. These experiments suggest that chimeric ribozyme targeted against PCNA may have a therapeutic or preventative role in humans.

Upregulation of extracellular ATP-induced Müller cell responses in a dispase model of proliferative vitreoretinopathy.

PURPOSE: To test whether in an animal model of proliferative vitreoretinopathy (PVR) the Müller glial cells displayed an upregulation of purinergic P2 receptor-mediated responses. METHODS: PVR was induced by intravitreal injection of the proteolytic enzyme, dispase, in the eyes of adult rabbits. The developing PVR was examined ophthalmoscopically. After 3 weeks, small retinal pieces were wholemounted and used for calcium imaging, freshly dissociated Müller cells were subjected to calcium imaging, and patch-clamp recordings were made. The presence of P2 receptor-mediated Ca(2+) responses was determined both directly--that is, fluorometrically--and indirectly, by electrophysiological recording of Ca(2+)-activated K(+) currents. RESULTS: According to earlier observations in another model of retinal detachment and PVR, the reactive Müller cells displayed hypertrophy, downregulation of inwardly rectifying K(+) currents, and depolarization of the resting membrane potential, all dependent on the severity of the PVR. Further, significant PVR-induced increase was observed in the number of Müller cells responding to adenosine 5'-triphosphate (ATP), with a transient elevation of their [Ca(2+)](i). If isolated Müller cells were exposed to ATP, 13% of the control cells, but 29% (moderate PVR) or 53% (massive PVR) of the reactive cells, showed fluorometric Ca(2+) increases. An increase of Ca(2+)-activated K(+) currents was measured in 11% of the control cells, but in 83% (moderate PVR) and 90% (massive PVR) of the reactive cells. Confocal images of retinal wholemounts revealed similar results. Because similar responses were elicited by uridine triphosphate (UTP), the dominant involvement of metabotropic (P2Y type) purinergic receptors is suggested. CONCLUSIONS: An upregulation of purinergic receptors is part of the reactive changes of Müller cells during PVR. It is suggested that ATP-evoked Ca(2+) responses may support the proliferation of Müller cells during PVR.

A new model of proliferative vitreoretinopathy.

PURPOSE: To design a new model of proliferative vitreoretinopathy (PVR) that would not rely on the addition of exogenous cells. The release of endogenous cells from surrounding attachments seems to be an early event in the pathogenesis of PVR. Because the proteolytic enzyme dispase dissociates tissues, the hypothesis was that an intraocular injection of dispase could trigger events that would cause PVR. The requirement for a surgical retinal break at the time of dispase injection was also examined. METHODS: One eye of Dutch Belted rabbits was injected with 0.003 U to 1.0 U dispase in the subretinal space or vitreous cavity. Control rabbits received a saline injection. An intentional retinal tear was created in animals in some groups. Observations were made for at least 10 weeks after surgery. RESULTS: Proliferative vitreoretinopathy developed in response to subretinal or intravitreal dispase, with or without creation of a controlled retinal break. Increased severity of PVR correlated with increasing doses of dispase. Evidence of PVR included preretinal membranes, distortion of myelin wings and retinal vessels, fixed retinal folds, and traction retinal detachment. Proliferative vitreoretinopathy did not develop in saline-treated control animals. CONCLUSIONS: Dispase initiated the development of PVR without the addition of exogenous cells, growth factors, or cytokines typically found in PVR membranes. A cascade of events was probably triggered by dispase, causing native cells and factors to produce PVR. The dispase model of PVR was technically easy to perform, permitted a clear view of the retina, and had a high success rate in development of PVR.