Original Paper
Ophthalmologica
Received: December 2, 2014 Accepted after revision: December 29, 2014 Published online: February 17, 2015
Ophthalmologica DOI: 10.1159/000371901
The Effects of Ozurdex® (Dexamethasone Intravitreal Implant) on Experimental Proliferative Vitreoretinopathy Hsi-Kung Kuo a, b Yi-Hao Chen a, b Pei-Chang Wu a, b Yu-Hsia Kuo a a Department of Ophthalmology, Kaohsiung Chang Gung Memorial Hospital, and b Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC
Abstract Purpose: To investigate a new sustained-release formulation of dexamethasone (Ozurdex®) for inhibiting proliferative vitreoretinopathy (PVR) and its effect on the expression of retinal glial reaction and inflammation in experimental PVR eyes. Methods: We used 30 pigmented rabbits for this study. One week after gas compression, the eyes were injected with 5 × 104 retinal pigment epithelial cells into the vitreous cavity to induce PVR. Concurrently, one eye also received an intravitreal injection of Ozurdex; the other eye was used as a control. PVR was graded by indirect ophthalmoscopy on days 1, 3, 7, 14, 21, and 28. The expression of the retinal glial reaction and inflammation in experimental PVR eyes were evaluated by Western blot analysis. Results: PVR severity increased gradually and peaked after 14 days, and no differences in PVR severity between the study and control groups were observed at any time point. The expression of glial fibrillary acid protein (GFAP) increased on days 7 and 14 in both the PVR control and study groups. While the use of Ozurdex in the study group showed less GFAP expression, this difference was not significant. The expression of tumor
© 2015 S. Karger AG, Basel 0030–3755/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/oph
necrosis factor (TNF)-α and interleukin (IL)-6 significantly increased on days 7 and 14 in PVR control eyes. There was a significant difference in TNF-α between PVR control eyes and Ozurdex-treated eyes on days 7 (p < 0.001) and 14 (p = 0.019). Ozurdex in the study group showed lower IL-6 expression; however, this difference was not significant on days 7 (p = 0.063) and 14 (p = 0.052). Conclusions: The intravitreal injection of Ozurdex suppressed the expression of inflammatory markers; however, it did not mitigate the severity of experimental PVR in this animal model. © 2015 S. Karger AG, Basel
Introduction
Rhegmatogenous retinal detachment (RD) is a severe sight-threatening disease. The primary surgical reattachment rate is currently 75–90%, and final retinal reattachment failure is mostly due to the development of proliferative vitreoretinopathy (PVR) [1–3]. This process is characterized by uncontrolled cellular proliferation on both surfaces of the detached retina, on the posterior vitreous face, and within the vitreous base resulting in the
H.-K.K. and Y.-H.C. contributed equally to this work.
Hsi-Kung Kuo, MD Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital 123 Ta-Pei Road, Niao-Sung District, Kaohsiung 833, Taiwan (ROC) E-Mail hsikung @ cgmh.org.tw
Downloaded by: NYU Medical Center Library 128.122.253.228 - 5/11/2015 9:17:12 AM
Key Words Proliferative vitreoretinopathy · Steroid · Ozurdex® · Inflammation
Materials and Methods Animals Pigmented rabbits weighing between 2 and 2.5 kg were used. All animals were sedated using intramuscular injections of zoletil (15 mg/kg) and xylazine (10 mg/kg). The rabbit eyes were dilated with one drop each of 0.5% tropicamide and 2.5% phenylephrine HCl. The animals were killed by intravenous injection of an overdose of lidocaine HCl. Both eyes instead of one eye were used for decreasing the number of animals and reducing individual variation. One eye was used for the study group and the other for the control group. Totally, there were 20 animals for efficacy study and 10 (8 for PVR and 2 as normal controls) for Western blot studies. This experiment was approved by the animal ethical committee of Kaohsiung Chang Gung Memorial Hospital. Efficacy Study PVR Induction. Rabbit RPE cells were prepared from primary culture as described before [14, 15]. PVR was induced by injection of rabbit RPE cells after gas compression of the vitreous [14, 15]. Each eye of every experimental rabbit was injected intravitreally through the pars plana with 0.1 ml 50% C3F8 (gas compression)
2
Ophthalmologica DOI: 10.1159/000371901
after anterior chamber paracentesis. Seven days later, each eye was treated again: 5 × 104 RPE cells in 0.1 ml Hanks’ buffered salt solution (HBSS) was injected into the vitreous cavity after anterior chamber paracentesis. One eye was used for the study group and the other eye for the control group. The eyes without manipulation were used as normal controls for Western blot study. Drug Injection and Observation. The study eyes (right eye of each rabbit) received one intravitreal injection of Ozurdex at the time of RPE cell injection. All animals received follow-up examinations by slit lamp biomicroscopy and indirect ophthalmoscopy to evaluate the development and progression of PVR on days 1, 3, 7, 14, 21, and 28 after drug injection. The animals were sacrificed after 28 days. The severity of the PVR was graded on a scale of 0–5. Briefly, the stages were defined as follows: stage 0, normal; stage 1, intravitreous membranes; stage 2, focal traction, localized vascular engorgement and elevation; stage 3, localized medullary ray detachment; stage 4, total medullary ray detachment and peripheral RD, and stage 5, total RD and retinal folds. The same procedure was used to prepare the samples for studying glial and inflammatory marker expression except that the animals were sacrificed earlier on days 7 and 14 postinjection [8, 15]. Western Blot Analysis of Retinal Glial and Inflammatory Cell Expression Total protein was extracted from dissected retina samples using tissue protein extraction reagent (Pierce Co., Rockford, Ill., USA). The extracted samples were resolved by SDS-PAGE on 15% gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked overnight at 4 ° C with 5% dry milk in phosphate-buffered saline (PBS) with 0.1% Tween and then incubated for 1 h at room temperature with primary antibodies, including anti-actin (monoclonal, clone C4; Millipore, Billerica, Mass., USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (monoclonal, clone 6C5; Abcam, Cambridge, UK), anti-glial fibrillary acid protein (GFAP) (monoclonal, clone GA5; Millipore), anti-interleukin (IL)-6 (polyclonal, Abcam), and antitumor necrosis factor (TNF)-α (polyclonal; Santa Crus, Dallas, Tex., USA). The membranes were then washed and incubated for 1 h at room temperature with 1: 10,000 horseradish peroxidaseconjugated anti-mouse or anti-rabbit IgG as the secondary antibody. The bound antibodies were detected using a Western blot analysis detection system (ECL Plus; Amersham Biosciences Inc., Piscataway, N.J., USA).
Statistical Analyses The nonparametric Mann-Whitney test was used to compare the degree of PVR and the differences in expression levels as determined by Western blotting. Statistical significance was defined as a p value
Ophthalmologica
Received: December 2, 2014 Accepted after revision: December 29, 2014 Published online: February 17, 2015
Ophthalmologica DOI: 10.1159/000371901
The Effects of Ozurdex® (Dexamethasone Intravitreal Implant) on Experimental Proliferative Vitreoretinopathy Hsi-Kung Kuo a, b Yi-Hao Chen a, b Pei-Chang Wu a, b Yu-Hsia Kuo a a Department of Ophthalmology, Kaohsiung Chang Gung Memorial Hospital, and b Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC
Abstract Purpose: To investigate a new sustained-release formulation of dexamethasone (Ozurdex®) for inhibiting proliferative vitreoretinopathy (PVR) and its effect on the expression of retinal glial reaction and inflammation in experimental PVR eyes. Methods: We used 30 pigmented rabbits for this study. One week after gas compression, the eyes were injected with 5 × 104 retinal pigment epithelial cells into the vitreous cavity to induce PVR. Concurrently, one eye also received an intravitreal injection of Ozurdex; the other eye was used as a control. PVR was graded by indirect ophthalmoscopy on days 1, 3, 7, 14, 21, and 28. The expression of the retinal glial reaction and inflammation in experimental PVR eyes were evaluated by Western blot analysis. Results: PVR severity increased gradually and peaked after 14 days, and no differences in PVR severity between the study and control groups were observed at any time point. The expression of glial fibrillary acid protein (GFAP) increased on days 7 and 14 in both the PVR control and study groups. While the use of Ozurdex in the study group showed less GFAP expression, this difference was not significant. The expression of tumor
© 2015 S. Karger AG, Basel 0030–3755/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/oph
necrosis factor (TNF)-α and interleukin (IL)-6 significantly increased on days 7 and 14 in PVR control eyes. There was a significant difference in TNF-α between PVR control eyes and Ozurdex-treated eyes on days 7 (p < 0.001) and 14 (p = 0.019). Ozurdex in the study group showed lower IL-6 expression; however, this difference was not significant on days 7 (p = 0.063) and 14 (p = 0.052). Conclusions: The intravitreal injection of Ozurdex suppressed the expression of inflammatory markers; however, it did not mitigate the severity of experimental PVR in this animal model. © 2015 S. Karger AG, Basel
Introduction
Rhegmatogenous retinal detachment (RD) is a severe sight-threatening disease. The primary surgical reattachment rate is currently 75–90%, and final retinal reattachment failure is mostly due to the development of proliferative vitreoretinopathy (PVR) [1–3]. This process is characterized by uncontrolled cellular proliferation on both surfaces of the detached retina, on the posterior vitreous face, and within the vitreous base resulting in the
H.-K.K. and Y.-H.C. contributed equally to this work.
Hsi-Kung Kuo, MD Department of Ophthalmology Kaohsiung Chang Gung Memorial Hospital 123 Ta-Pei Road, Niao-Sung District, Kaohsiung 833, Taiwan (ROC) E-Mail hsikung @ cgmh.org.tw
Downloaded by: NYU Medical Center Library 128.122.253.228 - 5/11/2015 9:17:12 AM
Key Words Proliferative vitreoretinopathy · Steroid · Ozurdex® · Inflammation
Materials and Methods Animals Pigmented rabbits weighing between 2 and 2.5 kg were used. All animals were sedated using intramuscular injections of zoletil (15 mg/kg) and xylazine (10 mg/kg). The rabbit eyes were dilated with one drop each of 0.5% tropicamide and 2.5% phenylephrine HCl. The animals were killed by intravenous injection of an overdose of lidocaine HCl. Both eyes instead of one eye were used for decreasing the number of animals and reducing individual variation. One eye was used for the study group and the other for the control group. Totally, there were 20 animals for efficacy study and 10 (8 for PVR and 2 as normal controls) for Western blot studies. This experiment was approved by the animal ethical committee of Kaohsiung Chang Gung Memorial Hospital. Efficacy Study PVR Induction. Rabbit RPE cells were prepared from primary culture as described before [14, 15]. PVR was induced by injection of rabbit RPE cells after gas compression of the vitreous [14, 15]. Each eye of every experimental rabbit was injected intravitreally through the pars plana with 0.1 ml 50% C3F8 (gas compression)
2
Ophthalmologica DOI: 10.1159/000371901
after anterior chamber paracentesis. Seven days later, each eye was treated again: 5 × 104 RPE cells in 0.1 ml Hanks’ buffered salt solution (HBSS) was injected into the vitreous cavity after anterior chamber paracentesis. One eye was used for the study group and the other eye for the control group. The eyes without manipulation were used as normal controls for Western blot study. Drug Injection and Observation. The study eyes (right eye of each rabbit) received one intravitreal injection of Ozurdex at the time of RPE cell injection. All animals received follow-up examinations by slit lamp biomicroscopy and indirect ophthalmoscopy to evaluate the development and progression of PVR on days 1, 3, 7, 14, 21, and 28 after drug injection. The animals were sacrificed after 28 days. The severity of the PVR was graded on a scale of 0–5. Briefly, the stages were defined as follows: stage 0, normal; stage 1, intravitreous membranes; stage 2, focal traction, localized vascular engorgement and elevation; stage 3, localized medullary ray detachment; stage 4, total medullary ray detachment and peripheral RD, and stage 5, total RD and retinal folds. The same procedure was used to prepare the samples for studying glial and inflammatory marker expression except that the animals were sacrificed earlier on days 7 and 14 postinjection [8, 15]. Western Blot Analysis of Retinal Glial and Inflammatory Cell Expression Total protein was extracted from dissected retina samples using tissue protein extraction reagent (Pierce Co., Rockford, Ill., USA). The extracted samples were resolved by SDS-PAGE on 15% gels and then transferred to polyvinylidene difluoride membranes. The membranes were blocked overnight at 4 ° C with 5% dry milk in phosphate-buffered saline (PBS) with 0.1% Tween and then incubated for 1 h at room temperature with primary antibodies, including anti-actin (monoclonal, clone C4; Millipore, Billerica, Mass., USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (monoclonal, clone 6C5; Abcam, Cambridge, UK), anti-glial fibrillary acid protein (GFAP) (monoclonal, clone GA5; Millipore), anti-interleukin (IL)-6 (polyclonal, Abcam), and antitumor necrosis factor (TNF)-α (polyclonal; Santa Crus, Dallas, Tex., USA). The membranes were then washed and incubated for 1 h at room temperature with 1: 10,000 horseradish peroxidaseconjugated anti-mouse or anti-rabbit IgG as the secondary antibody. The bound antibodies were detected using a Western blot analysis detection system (ECL Plus; Amersham Biosciences Inc., Piscataway, N.J., USA).
Statistical Analyses The nonparametric Mann-Whitney test was used to compare the degree of PVR and the differences in expression levels as determined by Western blotting. Statistical significance was defined as a p value
