Max Warga

Effects of bevacizumab on retinal function in isolated vertebrate retina.

Background: Bevacizumab (Avastin) is a recombinant protein that targets vascular endothelial growth factor (VEGF). In vitro, bevacizumab inhibits VEGF induced cell proliferation and tissue factor production. Abnormal angiogenesis involving VEGF is a central event during the development of choroidal neovascularisation (CNV). The present study was designed to evaluate the short term toxic effects of bevacizumab on retinal function for a therapeutic intraocular application. Methods: Isolated bovine retinas were perfused with an oxygen pre-incubated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using silver/silver chloride electrodes. Bevacizumab was added in different concentrations to the nutrient solution for 45 minutes. Thereafter the retina was reperfused for 60 minutes with normal nutrient solution. The percentage of a-wave and b-wave reduction during the application of bevacizumab was calculated and compared to control recordings. Results: During the application of three different concentrations of bevacizumab (0.08 mg/ml, 0.25 mg/ml, 0.8 mg/ml) no significant reduction of the a-wave and b-wave amplitude was observed. During the washout, the ERG amplitudes were unchanged. Conclusion: The present study suggests that an intraocular application of 0.25 mg/ml bevacizumab for the treatment of CNV is reasonable. No significant short term effects of bevacizumab on retinal function were detected, but long term effects cannot be excluded.

The retinal tolerance to bevacizumab in co‐application with a recombinant tissue plasminogen activator

Aim: To investigate the retinal toxicity of bevacizumab in co-application with a commercially available recombinant tissue plasminogen activator (rt-PA), and to facilitate a new therapeutic concept in the treatment of massive subretinal haemorrhage caused by neovascular age-related macular degeneration (AMD). Methods: Isolated bovine retinas were perfused with an oxygen-preincubated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes. Bevacizumab (0.25 mg/ml) and rt-PA (20 μg/ml) were added to the nutrient solution for 45 min. Thereafter, the retina was reperfused for 60 min with normal nutrient solution. Similarly, the effects of rt-PA (20 μg/ml, 60 μg/ml and 200 μg/ml) on the a- and b-wave amplitudes were investigated. The percentages of a- and b-wave reduction during application and at washout were calculated. Results: During application of bevacizumab (0.25 mg/ml) in co-application with 20 μg/ml (rt-PA), the ERG amplitudes remained stable. The concentrations of rt-PA alone (20 μg/ml and 60 μg/ml) did not induce significant reduction of the b-wave amplitude. In addition, 20 μg/ml rt-PA did not alter the a-wave amplitude. However, 60 μg/ml rt-PA caused a slight but significant reduction of the a-wave amplitude. A full recovery was detected for both concentrations during the washout. At the highest tested concentration of 200 μg/ml rt-PA, a significant reduction of the a- and b-wave amplitudes was provoked during the exposure. The reduction of ERG amplitudes remained irreversible during the washout. Conclusion: The present study suggests that a subretinal injection of 20 µg/ml rt-PA in co-application with bevacizumab (0.25 mg/ml) for the treatment of massive subretinal haemorrhage seems possible. This is a safety study. Therefore, we did not test the clinical effectiveness of this combined treatment.

Sutureless amniotic membrane fixation using fibrin glue for ocular surface reconstruction in a rabbit model.

Purpose: Amniotic membrane transplantation has become an important treatment option for corneal surface reconstruction. However, suture fixation of the transplant has various disadvantages like corneal irritation, scarring, graft loss due to membrane shrinkage, and the need for subsequent suture removal. Replacement of sutures by bioadhesives might be an advantageous alternative. This controlled study was designed to evaluate a new sutureless technique for amniotic membrane fixation onto the corneal surface by using fibrin glue. Methods: Standardized disks of cryopreserved amniotic membranes were transplanted onto the deepithelialized cornea of 12 rabbits using either conventional suture fixation or a new fibrin glue technique. The rabbits were followed-up with slit-lamp examination and fluorescein staining until epithelialization was completed. Consecutively, the rabbits were killed and the eyes processed for histology and immunohistochemistry for cytokeratin-3. Results: All membranes of both groups stayed in place throughout the follow-up time and showed a progressive graft epithelialization that was completed after 12 days. Whereas suture-fixated membranes showed progressive tissue shrinkage, fibrin-glued sheets remained unaltered. In the bioadhesive group, histology revealed a smooth fibrin layer in the graft-host interface and a continuous, stratified layer of cytokeratin-3 expressing corneal epithelial cells on the membrane surface. In contrast, suture-fixated membranes showed contracted and prominent membrane edges with epithelial ingrowth into the submembrane interface. Conclusion: Our results demonstrate the general feasibility of reproducible and reliable sutureless amniotic membrane fixation onto the corneal surface in rabbits. Stable adherence is maintained until epithelialization is completed. The sutureless technique gives sufficient manipulation time for the sheet before the final cross-linking process is completed. Furthermore, several advantageous characteristics could be demonstrated as increased biocompatibility, better epithelialization pattern and the lack of membrane shrinkage.

The effects of triamcinolone crystals on retinal function in a model of isolated perfused vertebrate retina

A good clinical experience of intravitreal triamcinolone acetonide (TA) has been reported in several studies, but there are growing indications that epiretinal crystals of TA exhibit retinal toxicity. To investigate the effects of TA on retinal function we used a model of an electrophysiological in vitro technique for testing retinal toxicity. Isolated bovine retinas were perfused with an oxygen saturated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes. After reaching stable ERG-amplitudes TA at the maximum solubility equilibrium (36 microg/ml) was either applied to the nutrient solution for 45 min or TA was administered epiretinally at concentrations (1 mg/ml, 4 mg/ml, 8 mg/ml, 20 mg/ml and 40 mg/ml) above the maximum solubility equilibrium to assure direct contact of the TA crystals with the isolated perfused retinas. After that the retinas were reperfused for 75 min with the standard nutrient solution. The percentage of a- and b-wave reduction directly after the application and at the washout was calculated. To assess the effects of TA at the level of the ganglion cell layer a Viability/Cytotoxicity Kit for mammalian cells was used. No changes of the ERG-amplitudes were detected during the exposure to 36 microg/ml TA for 45 min (b-wave: 9.6 microV+/-2.1 vs. 8 microV+/-2.1 (p=0.135); a-wave: -11 microV+/-2.7 vs. -10.6 microV+/-2.3 (p=0.889)) and at the washout (b-wave: 8 microV+/-2.1 vs. 8.3 microV+/-2.4 (p=0.18); a-wave: -10.6 microV+/-2.3 vs. -12 microV+/-2.6 (p=0.225)). At concentrations higher than 1mg/ml TA induced a decrease of the a- and b-wave in a concentration dependent manner. These changes were reversible for concentrations of TA up to 20mg/ml (b-wave: 9 microV+/-2.4 vs. 6.6 microV+/-2.5 (p=0.08); a-wave: -11.4 microV+/-2.0 vs. -11.2 microV+/-2.2 (p=0.37)), but irreversible at 40 mg/ml even at the end of the washout (b-wave: 9.8 microV+/-1.9 vs. 3 microV+/-1.7 (p=0.009); a-wave: -9.8 microV+/-2.1 vs. -2.6 microV+/-2.1 (p=0.001)). Histological examination of the preparations revealed a dramatic ganglion cell death, in which an application of 20mg/ml and 40 mg/ml TA led to a 60.53% (p=0.013) and 82.35% (p=0.002) ganglion cell death, respectively. The epiretinal application of 4 mg/ml TA and higher resulted in distinct effects on the ERG of the isolated perfused retinas. Ganglion cell death was induced at a concentration of 20mg/ml and higher. TA shows an asymmetric and partly high concentrated distribution after intravitreal application. Therefore, we consider concentrations of 4 mg/ml and higher might be toxic and should be avoided in clinical use.

Primary silicone oil tamponade in the management of severe intraocular foreign body injuries: an 8-year follow-up.

Purpose: To evaluate the long-term outcome of pars plana vitrectomy and primary silicone oil tamponade in patients with severe intraocular foreign body (IOFB) injuries and high risk of proliferative vitreoretinopathy (PVR). Methods: This retrospective consecutive study included 23 patients with severe IOFB injuries who had extensive lacerations including sclera, choroid, and retina, and were complicated by predictive factors for elevated proliferative activity and an unfavorable outcome. All patients underwent pars plana vitrectomy, removal of the IOFB, and primary silicone oil tamponade and were followed up for a mean 8.9 years. Main functional outcome was assessed as final best-corrected visual acuity. Anatomic success was defined as permanent retinal attachment. Results: PVR occurred in 70% of all eyes and required 16 revisions. Silicone oil was removed in 78% of the eyes after a mean tamponade duration of 9.1 months. Complete retinal attachment was achieved in 83% of the eyes. Three eyes developed a persisting hypotony that was stabilized under permanent silicone oil. Functional stabilization was observed in the third year resulting in a final visual acuity of 20/630. Useful vision of better than 20/400 could be preserved in 55% of the patients. Only one eye underwent a late enucleation after 6.8 years. Conclusions: Primary silicone oil stabilizes the retina during the critical period of active PVR and may limit the visual loss in selected high-risk eyes in the long term.

Does intravitreal injection of bevacizumab have an effect on the blood‐aqueus barrier function?

Bevacizumab (Avastin), a humanised monoclonal anti-VEGF antibody originally developed for intravenous therapy of metastatic cancer, is now being used as off-label therapy in age related macular degeneration.1 The cost effective drug promises not only anti-angiogenic capabilities in neovascular eye disease, but has also anti-exudative effects by lowering transendothelial permeability of blood vessels.2 Blood-aqueous barrier (BAB) breakdown has been reported after intraocular application of lyophilised rhuFab formulations.3 Anterior chamber inflammation is one of the most frequently reported adverse events (19%) after pegaptanib injections.4 We wanted to post these safety issues for bevacizumab by objective detection of aqueous protein concentration. The anterior chamber reaction was analysed in 60 patients receiving 1.25 mg of bevacizumab for age …

Neuroprotective effects of a taurine-containing irrigation solution for vitrectomy.

Background: During pars plana vitrectomy, the retina is exposed to several iatrogenic risk factors, including excitotoxicity. A taurine-containing irrigation solution for pars plana vitrectomy (PURI PROTECT) has been developed and is claimed to have neuroprotective properties. Methods: Retinal ganglion cells (RGC-5) and retinal whole mounts were incubated in standard irrigation solution (SIS) and SIS supplemented with 3 mM taurine (SIS-taurine). Excitotoxicity was induced by the addition of 8, 10, and 12 mM or 250 &mgr;M glutamate. Cell viability and cell survival were assessed by the MTT test and Annexin-V/propidium iodide flow cytometry. Whole mounts were stained with the Live/Dead staining assay. Pars plana vitrectomy with SIS or SIS-taurine was performed in rabbits. Animals were followed-up by electroretinography. Results: RGC-5 incubated in SIS-taurine showed a 4.3-fold (P < 0.0005) better overall cell viability and an up to 8.5-fold (P < 0.05) increased cell survival under excitotoxic conditions compared with that incubated in SIS. Whole mounts incubated in SIS-taurine showed a 1.7-fold (P < 0.0005) and 1.6-fold (P < 0.0005) better cell survival under excitotoxic and nonexcitotoxic conditions, respectively. In the immediate postoperative period, b-wave amplitudes were significantly better in animals operated with SIS-taurine compared with control (P < 0.01). Conclusion: A taurine-containing irrigation solution may protect retinal ganglion cells against excitotoxicity.

Experimental Implantation and Long-term Testing of an Intraocular Vision Aid in Rabbits

OBJECTIVE To develop an intraocular vision aid to provide artificial vision in severely traumatized eyes, where neuroretinal function could be preserved but irreversible anterior segment opacification resulted in blindness. METHODS The basis of an intraocular vision aid is in principle a telemetric circuit to bridge the opaque cornea and to allow for artificial light stimulation of the retina. The visual prosthesis comprises an external high-dynamic range complementary metal oxide semiconductor camera and digital signal processing unit and an intraocular miniaturized light-emitting diode array to project the image onto the retina. For in vivo testing of long-term function and biocompatibility, silicone-encapsulated active photodiodes were implanted in 13 pigmented rabbits and were followed up for up to 21 months. RESULTS Lens extraction and stable fixation of the device in the ciliary sulcus were successful in all cases. For up to 21 months inductive energy transmission and wireless stimulation of the implants could be maintained. Electrophysiologic data and histology demonstrated a good tissue biocompatibility in the long-term follow-up. CONCLUSION The results demonstrate the general feasibility and biocompatibility to implant and fixate an intraocular light-emitting diode prosthesis. Inductive energy transmission to the intraocular device and wireless light stimulation are assured in the long term but depend on meticulous water-impermeable encapsulation of the delicate microelectronic components. Clinical Relevance An intraocular vision aid compound system with a high-resolution light-emitting diode matrix might be a future treatment option to restore vision in blind eyes with severe anterior segment disorders.

Safety Profile Of A Taurine Containing Irrigation Solution (acriprotect) In Pars Plana Vitrectomy

Purpose: To evaluate the clinical safety of a taurine containing irrigation solution (AcriProTect) before its routine application in pars plana vitrectomy (PPV). Methods: Twenty-five patients who underwent PPV were included in this prospective interventional clinical study. Standard irrigation solution containing the addendum 3 mmol/L taurine was used during PPV. Postoperative follow-up visits included a standard eye examination, corneal thickness measurements, endothelial cell counts, determination of the foveal thickness by optical coherence tomography (OCT), and an electrophysiologic examination. For statistical analysis Wilcoxon test was used. Results: Significant improvement of visual acuity (VA) was observed at the 3- and 6-month controls (P = 0.024; P = 0.002 for 3 and 6 months, respectively). Endothelial cell counts and corneal thickness at 3 and 6 months were not significantly different from preoperative values (P = 0.204; P = 0.126 for endothelial cell count and P = 0.475; P = 0.095 for corneal thickness at 3 and 6 months, respectively). Both scotopic and photopic Ganzfeld electroretinography and electro-oculography did not show significant changes during the follow-up. No increase in complication rate was detected. Conclusions: The investigation demonstrates a good biocompatibility of taurine-containing irrigation solution developed for vitrectomy in humans concomitant with habitually observed good functional outcome.