This is a pilot, prospective, interventional, case-control study investigating aqueous levels of vascular endothelial growth factor (VEGF) in eyes with AMD-related neovascularization treated with intravitreal bevacizumab at the Medical Retina Department, University of Molise, Campobasso.

This was a pilot, prospective, interventional, case-control study investigating aqueous levels of VEGF in eyes with AMD-related NV treated with intravitreal bevacizumab.

Classification of neovascularization was based on its anatomic localization as ascertained by multimodal imaging employing FA (fluorescein angiography), ICGA (indocyanine green angiography) and OCT (optical coherence tomography) as recently proposed by Freund et al.

Diagnostic procedures and follow-up At baseline all the patients underwent BCVA measurement using early treatment diabetic retinopathy study (ETDRS) chart at 4 meters, fundus biomicroscopy, FA, ICGA and sd-OCT. All examinations were repeated in occasion of the follow-up appointments with exception of angiographic tests.

Angiographic tests and OCTs scans were recorded using Spectralis sd-OCT (Spectralis SD-OCT, Heidelberg Engineering, Heidelberg, Germany).

CMT was calculated after acquiring a sequence of 145 horizontal sections recorded in the high-resolution mode (1024 A-scans/30 degrees) and covering an area of 20 degrees (horizontal) × 15 degrees (vertical) with a distance of ~30 µm between individual sections. On follow-up examinations, the imaging processing software allowed reevaluation at exactly the same location.

The images were then processed by the "Thickness Map" analysis program. Field 1 of the map analysis protocol (central 1 mm) was used for central retinal thickness calculations.

Aqueous Sampling and Bevacizumab Injections All patients with AMD-related neovascularization received three intravitreal injections of 1.25 mg/0.05 ml of bevacizumab (Avastin; Genentech Inc, South San Francisco, California, USA) at baseline, month 1 and month 2.

Before injection, topical anesthesia was induced by tetracaine (1%) eye drops. Povidone-iodine was applied to the eyelid margins, the lashes, the conjunctiva bulbi and the fornices. After application of a sterile drape, a lid speculum was inserted.

Immediately before each intravitreal injection, aqueous sampling was performed by aspirating 0.05-0.1 ml of aqueous using a 30-gauge needle connected to a tuberculin syringe at the temporal limbus. All the samples were collected in the operating theatre under operating microscope control.

Intravitreal bevacizumab (IVB) injection was then performed using a 30-gauge needle in the inferotemporal quadrant at 3.5 mm to 4 mm posterior to the limbus.

The undiluted aqueous samples were transferred into sterile containers and immediately stored in a -80°C freezer until analysis.

Control Group Reference samples were obtained from 14 age-matched patients undergoing cataract surgery.

Aqueous humor samples were obtained in the same fashion described above for eyes with wet-AMD. The undiluted aqueous samples were transferred into sterile containers and immediately stored in a -80°C freezer until analysis.

Vascular Endothelial Growth Factor Assay Collected samples were gradually equilibrated to room temperature before beginning the assay and diluted 1:1 with the sample diluent provided by the manufacturer.

The VEGF content was determined on 50 µl of diluted sample with a human VEGF ELISA kit (EHVEGF, Pierce Biotechnology, Rockford, Illinois, USA) according to the manufacturer's instruction and using an extended standard curve including a 16, 8 and 4 pg/ml standard point. All assays were performed in duplicate. The minimum detectable concentration of VEGF was 3.5 pg/ml. Values inferior to 3.5pg/ml were considered equal to 1 for statistical analysis.

Inclusion Criteria:

• wet age-related macular degeneration type 1, 2 or 3

Exclusion Criteria:

• BCVA at baseline less than 1.0 logMAR
• any previous treatment of the neovascular lesion
• previous vitrectomy
• laser coagulation within the last 3 months
• previous participation in any studies using investigational drugs within 3months preceding day 0
• intraocular surgery (including cataract surgery) in the study eye within 3 months preceding day 0
• glaucoma in the study eye; diabetes mellitus
• use of immunosuppressive drugs
• malignant tumors of any location