Ultrastructural Findings in the Primate Eye After Intravitreal Injection of Bevacizumab SWAANTJE PETERS, PETER HEIDUSCHKA, SYLVIE JULIEN, FOCKE ZIEMSSEN, HEIKE FIETZ, KARL ULRICH BARTZ-SCHMIDT, THE TÜBINGEN BEVACIZUMAB STUDY GROUP, AND ULRICH SCHRAERMEYER ● PURPOSE: To examine the ultrastructural effect of intravitreal bevacizumab on primate eyes with particular focus set on the choriocapillaris and to examine the influence of vascular endothelial growth factor (VEGF) inhibition on endothelial cell fenestration. ● DESIGN: Animal study. ● METHODS: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated and prepared for light and electron microscopy on days one, four, seven, and 14. Control eyes remained untreated. Choriocapillaris endothelial cell fenestrations were quantified. ● RESULTS: Choriocapillaris endothelial cell fenestra- tions were significantly reduced after intravitreal injec- tion of bevacizumab. Fenestration was lowest on day four (15.9  6.7 per 25 m) and increased again from days seven to 14, but was still significantly lower than in the control (66.2  9.5 per 25 m). Densely packed thrombocytes and leukocytes regionally occluded the choriocapillaris lumen of treated eyes. On day one an increased number of leukocytes filled in the choriocapil- laris lumen. Photoreceptors were damaged in two of 40 light microscopic sections. On days one to seven, choroi- dal melanocytes contained giant melanosomes. None of these described features was found in controls. ● CONCLUSIONS: Intravitreal bevacizumab causes ultra- structural changes in the choriocapillaris of primate eyes. A significant reduction of choriocapillaris endothelial cell fenestrations is seen as early as 24 hours after injection and their number increases again after two weeks. These findings may play a role in the early clinical effect of intravitreal bevacizumab for macular edema. Because an increased risk of circulation disturbances in the chorio- capillaris cannot be excluded, patients should be carefully monitored. (Am J Ophthalmol 2007;143:995–1002. © 2007 by Elsevier Inc. All rights reserved.) B EVACIZUMAB, A HUMANIZED MONOCLONAL ANTI- vascular endothelial growth factor (VEGF) antibody, is US Food and Drug Administration approved for the adjuvant antiangiogenic treatment of metastatic colorectal cancer, but gets increasingly used as an off-label therapy in ophthalmology. Recent clinical data of the intravitreal use show excellent results in the treatment of choroidal neovas- cularization (CNV) 1 and macular edema. 2 Therefore the treatment with intravitreal bevacizumab has quickly spread worldwide. Useful databases have been built in the internet to register clinical reports of side effects. 3 But because it is still an off-label therapy, basic research is still necessary to support the clinical use or reveal potential secondary effects. We chose monkey eyes for our study because they are closer related to the human pathophysiology than rat or mouse. 4 Light and electron microscopy of the retina and choroid was performed after intravitreal injection of bevacizumab and in control eyes. Particular focus was set on the choriocapillaris, the source of CNV and aiming point of an anti-VEGF therapy in age-related macular degeneration (AMD). METHODS ● ANIMALS: Four Cynomolgus monkeys (ages 8 to 9 years, supplied by Nafovanny, Vietnam) for the experi- ments, plus one Cynomolgus monkey as control were raised in standard conditions with veterinarian attendance in the Covance Laboratories (Muenster, Germany). ● INTRAVITREAL INJECTION OF BEVACIZUMAB: Bev- acizumab (Avastin ® , Roche, 25 mg/ml) for intravenous use was aliquoted into tuberculin syringes under sterile condi- tions by our pharmacy. The aliquots of 50 l contained 1.25 mg bevacizumab and were stored continuously at 4 C to 8 C until use. The animals were anesthetized with ketamine (10 mg/kg) plus xylazine (2 mg/kg). Before injection the eyes were examined for any signs of inflam- mation. Pupils were dilated (tropicamide, phenylephrine) and anesthetized (oxybuprocainhydrochloride). The con- junctival and corneal surface was disinfected (povidone iodine 10%). After sterile coating and insertion of a lid speculum, 1.25 mg bevacizumab were injected into the vitreous cavity using a 27-gauge canula. When removing the cannula, the injection site was compressed with forceps Accepted for publication Mar 7, 2007. From the University Eye Hospital Tübingen (S.P., P.H., S.J., F.Z., H.F., K.U.B.-S., Tübingen Bevacizumab Study Group, U.S.); and Steinbeis Transfer Centre for Pathology and Toxicology of the Eye (U.S.), Tübingen, Germany. Inquiries to Swaantje Peters, University Eye Hospital Tübingen, Schleichstr. 12, D-72076, Tübingen, Germany; e-mail: Swaantje.Peters@ googlemail.com © 2007 BY ELSEVIER INC.ALL RIGHTS RESERVED. 0002-9394/07/$32.00 995 doi:10.1016/j.ajo.2007.03.007