Inner Retinal Changes in Acute Experimental BRVO Treated With Bevacizumab or Triamcinolone Acetonide.

Purpose: Apoptosis is a key process in neural degeneration associated with retinal vascular diseases. Vascular endothelial growth factor (VEGF) antagonists, including bevacizumab, are used to treat macular edema in these diseases. As VEGF has a critical role in the preservation of retinal neuronal cells, this study investigates the effects of bevacizumab on neural damage in a pig model of branch retinal vein occlusion (BRVO) and compares it with triamcinolone acetonide (TA) which is reported to possess neuroprotective properties. Methods: Thirty-six pigs had a photothrombotic BRVO in both eyes. Six pigs were injected with bevacizumab in one eye and TA in the fellow eye, then they were sacrificed, the eyes enucleated, and retinas processed at 2, 6, 10, and 20 days, respectively, together with three pigs (six eyes) BRVO only and three normal pigs (six eyes). Neuronal degeneration (apoptosis) and associated inner retinal changes were determined by terminal deoxyynuclotidyl transferase dUTP nick-end labeling (TUNEL), histology, and immunohistochemistry for macrophages. Results: TUNEL labeling showed significantly higher apoptosis rates in the ganglion cell layer (GCL) and the inner nuclear layer (INL) in the bevacizumab-treated compared with the TA-treated retinas at 2, 10, and 20 day time points after occlusion (P < 0.05). Pyknotic cells were significantly higher in the GCL in bevacizumab-treated eyes at 6, 10, and 20 days and in the INL at 2 days compared to TA-treated retinas (P < 0.05). Macrophage infiltration was seen at all time points in both untreated and treated retinas with an absence of significance between bevacizumab- and TA-treated retinas (P > 0.05). Conclusions: Neurodegeneration in the BRVO acute phase is exacerbated by current standard treatments for BRVO. These results may have implications for the timing and treatment type. Translational Relevance: In the acute phase of BRVO, VEGF suppression with bevacizumab and to a lesser extent with triamcinolone exacerbates apoptosis in the inner retinal layers, which has implications for both the timing and choice of treatment.

Neuronal degeneration and associated alterations in cytokine and protein in an experimental branch retinal venous occlusion model.

The mechanisms of neuronal degeneration and associated acute alterations in intraretinal cytokine and protein levels remain poorly understood in variable ischaemic retinopathies such as in branch retinal vein occlusion (BRVO). Herein we investigate neuronal damage and alterations in retinal cytokines and proteins in a pig model of acute BRVO. Twelve pigs had a BRVO induced photothrombotically in both eyes. Three pigs (6 eyes) each at 2, 6, 10 and 20 days were sacrificed together with an additional 3 control (6 eyes), enucleated, retinas dissected and processed. Apoptosis in the inner retina was determined by terminal deoxyynuclotidyl transferase mediated dUTP nick end labelling (TUNEL) and histology. Expression of glial acidic fibrillary protein (GFAP), aquaporin-4 (AQP4), inward rectifier potassium channel 10 protein (Kir 4.1) encoded by KCNJ10 gene, vascular endothelial growth factor (VEGF), stromal derived growth factor-1α (SDF-1) encoded by CXCL12 gene and interleukin (IL) -6 and 8 were analysed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry. TUNEL labelling showed positive nuclei in the ganglion cell layer (GCL) and the inner nuclear layer (INL) which was significantly higher at 2 days after BRVO compared to other time points (p < 0.05). Analysis by RT-qPCR revealed that compared with controls, BRVO significantly increased mRNA expression of GFAP at 6, 10 and 20, AQP4 at 20, VEGF at 6, SDF-1 at 20 and IL-8 at 2 and 10 days respectively (p < 0.05): Kir 4.1 at 6, VEGF at 2 and 10, and IL-6 at 2 days were significantly decreased (p < 0.05). This study indicates that neural cell death occurs early in this acute model and the responses include inflammation and breakdown of osmohomeostasis as evidenced by the upregulation of GFAP and IL-8 and down regulation of Kir 4.1 associated with glyotic changes. Early short term VEGF upregulation seen may be related to involvement of Muller glial cells. These findings support the development of acute therapeutic strategies aimed at preservation of retinal neural cells as part of an overall management plan for BRVO.

Intravitreal tenecteplase (metalyse) for acute management of retinal vein occlusions.

PURPOSE: To determine the ability of an intravitreal injection of tenecteplase (TNK) to penetrate an intraretinal venous thrombus and its effectiveness in thrombolysis in a porcine model of branch retinal vein occlusion (BRVO). METHODS: Six pigs (group 1) were anesthetized, and a BRVO was induced photothrombotically in the left eye; immediately afterward, fluorescence-conjugated TNK (100 µg) was injected into both eyes, with enucleation at 24 hours. Retinal penetration was assessed on frozen sections by epifluorescence microscopy. A further six pigs (group 2) were anesthetized; BRVO was induced in both eyes, and TNK was injected into the vitreous in the left eye. Both eyes were harvested a week later. The area of the lasered site and an area away from the burn were dissected and processed in epoxy resin and stained for light or transmission electron microscopy. The percentage blockage, clot volume, cytostructure, and extent of thrombolysis by TNK were assessed. RESULTS: TNK penetrated the veins in both eyes of group 1 pigs, with more intense staining in the eyes with the occlusion. In group 2 eyes, thrombolysis was significant in the eyes injected with TNK (P = 0.03); blockage was seen in all six untreated eyes and one treated eye. Clot volume was significantly higher in untreated eyes (P = 0.028). Percentage blockage varied from 8.5% to 83.9%. Damage by TNK to the neural retina was not seen. There was no significant difference in cytostructure between treated and untreated eyes (P = 0.357). CONCLUSIONS: TNK was able to penetrate the retinal veins with and without an occlusion and effect lysis of BRVO, and did not cause damage to the retinal tissue. Intravitreal TNK may be useful as an acute treatment for RVOs of recent onset.

Evaluation of the ability of a photocoagulator to rupture the retinal vein and Bruch's membrane for potential vein bypass in retinal vein occlusion.

BACKGROUND AND OBJECTIVE: To evaluate the laser power required to rupture both the retinal vein and Bruch's membrane in a porcine model using a new laser system for treatment of retinal vein occlusion. MATERIALS AND METHODS: The retina was treated in areas with and without an overlying vein. Sections through the laser sites were stained for light and transmission electron microscopy. The percentage of the laser sites with rupture of the retinal vein and Bruch's membrane, the degree of tissue damage, and the diameter of the rupture in Bruch's membrane were determined. RESULTS: The rate of vein rupture was 75% at 3 W, and rupture of Bruch's membrane was achieved at all powers tested. The mean diameter of the rupture in Bruch's membrane increased with laser power. CONCLUSION: The Integre Duo laser system (Ellex Medical Lasers, Adelaide, Australia) is capable of rupturing the retinal vein and Bruch's membrane at lower powers than prior laser systems.

Intravitreal triamcinolone acetonide induced changes in the anterior segment in a pig model of branch retinal vein occlusion.

BACKGROUND: Triamcinolone acetonide (TA) has applications for the treatment of a large range of intraocular vascular diseases. The present study in pigs was performed to investigate histopathological and histochemical changes in the levels of myocilin deposition in the anterior segment in a model of branch retinal vein occlusion (BRVO) after vitreal administration of TA. METHODS: After ophthalmoscopic examination, intraocular pressure (IOP) measurement and fundus photography, a BRVO was created photothrombotically in each eye of six pigs, using argon green photocoagulation. The left eye was then injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined, animals sacrificed, and eyes enucleated and processed in paraffin and epoxy resin. Immunofluorescence cytochemistry on paraffin sections was performed to localise the distribution of myocilin in the anterior segment and histology by light and transmission electron microscopy on epoxy resin sections on TA-treated and untreated eyes. RESULTS: Histology revealed pathological changes in the TA-treated eye, including swollen mitochondria, layered long endoplasmic reticulum, pleomorphic nuclei, dense fibrillar extracelluar deposits and aggregates of unusual cell inclusions. Myocilin levels were significantly higher in the TA-treated eyes in the trabecular meshwork (p = 0.001), ciliary process (p = 0.011) and iris (p = 0.030) than in the untreated eyes. CONCLUSIONS: This study suggests that increased myocilin synthesis and related ultrastructural changes in the anterior segment after treatment with intravitreal TA in a porcine model of retinal oedema in BRVO may contribute to IOP elevation.

Effect of triamcinolone acetonide on vascular endothelial growth factor and occludin levels in branch retinal vein occlusion.

PURPOSE: To investigate the molecular mechanism by which triamcinolone acetonide (TA) may reduce edema in a porcine model of branch retinal vein occlusion (BRVO). DESIGN: Animal study. METHOD: After baseline ophthalmoscopic examination and fundus photography, a BRVO was created photothrombotically in each eye of 6 pigs, using argon green photocoagulation and intravenous Rose Bengal. Following this, the left eye was injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined. Fluorescein angiography, in addition to ophthalmoscopy and fundus photography, was performed. Following sacrifice of the animals, the eyes were enucleated and processed. The distribution of vascular endothelial growth factor (VEGF), occludin, and glial fibrillary acidic protein (GFAP) were localized by immunofluorescence cytochemistry on 10 microm frozen retinal sections of TA-treated and untreated eyes. RESULTS: Retinal VEGF levels were significantly lower in the TA-treated eyes as compared with the untreated eye (P = .002). Conversely occludin levels were significantly higher in the treated eye (P = .026). There was also a significant reduction in GFAP immunoreactivity in the Muller cells of the treated eyes (P = .015) with no statistical significance in the astrocytes (P = .065). CONCLUSION: Intravitreal TA down regulates VEGF, which may prevent a decrease in occludin and also inhibits an increase in GFAP expression in Muller cells. These events may contribute to a reduction in the blood retinal barrier breakdown that occurs in BRVO and promote resolution of the associated retinal edema.

Investigation of the safety of tenecteplase to the outer retina.

PURPOSE: To investigate the safety of the thrombolytic agent, tenecteplase to the outer retina in pig eyes. METHODS: Tenecteplase (50 microg) was injected into the subretinal space in one eye while the fellow eye received balanced salt solution. At 24 h, 1 week and 9 weeks the eyes were examined by indirect ophthalmoscopy and photographed. Animals were killed at 9 weeks, eyes enucleated and processed for light and transmission electron microscopy. Three locations within the area of the injection bleb were analysed. RESULTS: Retinal pigment epithelial defects, which appeared to be iatrogenic and confined to the injection site, were seen in most of the treated and control eyes. There was no significant difference in the degree of retinal damage between the two groups of eyes at the three sites examined (P > 0.05). CONCLUSION: Fifty micrograms of tenecteplase appears to be a safe dose with no evidence of toxicity to the outer retina and may have a potential role in the treatment of submacular haemorrhage.

A study of retinal penetration of intravitreal tenecteplase in pigs.

PURPOSE: To determine the degree of retinal penetration of intravitreal tenecteplase in a porcine model. METHODS: Fluorescence-tagged tenecteplase (50 microg in 0.1 mL physiologic saline) was injected into the vitreous of the right and left eyes at 24 and 6 hours before death, respectively, in six nonvitrectomized pigs. Retinal penetration was assessed on frozen sections by epifluorescence microscopy and statistical analysis was performed. Frozen sections of two eyes without injection (control) were also assessed. RESULTS: Labeling of fluorescence-tagged tenecteplase was seen in all the layers of the retina at both time points with more intense signal at 24 hours after injection (P<0.05). CONCLUSIONS: Fluorescence-tagged tenecteplase can penetrate all the layers of the retina of porcine eyes after intravitreal injection. Intravitreal tenecteplase may be useful in the in the management of subretinal hemorrhage.

Morphological and topographic effects on calcification tendency of pHEMA hydrogels.

Poly(2-hydroxyethyl methacrylate) hydrogels were prepared in the presence of varying concentrations of water, or a co-monomer ethoxyethyl methacrylate at different strengths of crosslinking agent ethylene glycol dimethacrylate. Calcification tendency and its correlation with monomer mixture composition, topography and porosity of these materials were investigated. Scanning (SEM) and transmission electron microscopy (TEM) was used to study topography and porosity respectively. Calcification and calcium diffusion ability in to the hydrogels were investigated by light microscopy, SEM and energy dispersive analysis of X-rays (EDAX) after incubation of the materials in a metastable calcifying solution for 48 days. Polymer and solvent volume fractions were also studied to determine if a correlation existed between porosity and calcification. Most of the series of hydrogels showed surface irregularities. Internal structure showed evidence of a porous structure in one of the series. Calcification studies indicated diffusion of calcium ions in some of the series. The diffusion of calcium is limited to 30-40 microm in most calcified specimens. For hydrogels that exhibited substantial surface irregularities and micro channels, the infiltration of calcium up to 200 microm was observed. Attempts to detect porosity by electron microscopy failed in some of the hydrogels due to difficulty in sample processing and sectioning. However, collaboration of the results with different techniques used, indicated that surface defects are the major contributors to calcium deposition. Decrease in porosity reduces the amount of calcium deposits and infiltration with decreasing solvent volume fraction which is associated with crosslinking concentration and initial water content of the polymer.

Histopathology of long-term Cardona keratoprosthesis: a case report.

PURPOSE: To report histologic findings of a Cardona keratoprosthesis (KPro) in an eye enucleated after 14 years. METHODS: Case report of a 56-year-old male whose KPro was implanted following bilateral chemical burns. Following enucleation, histopathology by light and transmission electron microscopy was performed. RESULTS: The prosthesis was retained in the eye until clinical complications including retinal detachment necessitated enucleation after 14 years. Histopathology confirmed a total retinal detachment, necrosis of the detached retina, inflammation, and foreign-body reaction in the Dacron mesh. Calcification was not seen. CONCLUSION: PMMA is a suitable material for an optic and provides for good postoperative acuity. The complex skirt area may be predisposed to long-term inflammation. Less complicated surgery and a simple inert device with a flange with good mechanical and biologic integration may help in reducing postoperative complications.

In vitro drug-induced spoliation of a keratoprosthetic hydrogel.

PURPOSE: To investigate in vitro the effects of selected drugs on the spoliation of poly(2-hydroxyethyl methacrylate) (PHEMA), a synthetic acrylic hydrogel currently used for the manufacture of a keratoprosthesis, AlphaCor. The experiments were carried out both in the presence of simulated aqueous humor (SAH) and in its absence. METHODS: Disks of PHEMA were incubated and shaken with 9 commonly prescribed drugs at 37 degrees C in sterile conditions for 1 week. Samples were incubated either in SAH only (controls), in each drug preparation, or in each drug for 1 week followed by 1 week in SAH. The drugs selected for this study were steroids (prednisolone, dexamethasone, fluorometholone, medroxyprogesterone), antiglaucoma drugs (timolol maleate and pilocarpine), and antibiotics (chloramphenicol, cephazolin, and ciprofloxacin), as commercially available formulations. Following incubation, the PHEMA specimens were examined visually and then histologically, after staining with alizarin red for the presence of calcium in the spoliating sediments/deposits. RESULTS: Although only 5 of the drug formulations (dexamethasone as Maxidex, fluorometholone as FML, pilocarpine as Isopto Carpine, chloramphenicol as Chlorsig, and medroxyprogesterone as Depo-Ralovera) induced spoliation of the hydrogel in the absence of SAH, all drugs induced spoliation after postincubation in SAH, and calcium was detected in the majority of samples. The deposits on the hydrogel specimens incubated first in cephazolin (as Cefazolin-BC), pilocarpine (as Isopto Carpine), and chloramphenicol (as Chlorsig) and then in SAH did not contain calcium, despite its presence in SAH. CONCLUSIONS: The study appears to confirm our earlier clinical observations that topical medication may play a role in the spoliation of the hydrogel ophthalmic devices. Presence of calcium in the deposits seems to be correlated to the nature of drug. Although the incidence of spoliation in real clinical situations is much lower than suggested by this extreme-case in vitro simulation, topical therapy after implantation of AlphaCor should be carefully considered, kept to the minimum required, and additive-free where possible.

The Chirila Keratoprosthesis: phase I human clinical trial.

OBJECTIVE: To undertake a preliminary safety and performance evaluation of an artificial cornea, the Chirila Keratoprosthesis, in human patients. DESIGN: A prospective, interventional case series. PARTICIPANTS: Fourteen consecutive patients with blindness of corneal origin not treatable by repeated standard penetrating keratoplasty. METHODS: Keratoprostheses were manufactured and implanted. The patients, all with preoperative visual acuity of light perception to count fingers (CF), were followed clinically in adherence to a protocol. MAIN OUTCOME MEASURES: Safety (keratoprosthesis retention, incidence of serious complications) and performance (visual acuity, comfort, appearance). RESULTS: Ninety-three percent of keratoprostheses were retained to the date of reporting, up to 2.5 years. One keratoprosthesis (7%) was removed in a manner that restored the patient's preoperative condition. All but one patient maintained their preoperative level of visual acuity or improved on it, with most achieving their estimated full potential visual acuity, (range, count fingers - 20/20). CONCLUSIONS: This keratoprosthesis is acceptably safe and has demonstrated an ability to restore vision in cases in which alternative management would have had a poor prognosis. More extensive trials are warranted.