Aqueous Vascular Endothelial Growth Factor (VEGF) Levels in Type 3 Neovascularization
Condition: Age-related Macular Degeneration
MeSH terms: Macular Degeneration; Neovascularization, Pathologic
Study type: Interventional

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Aqueous sample; Aqueous sample; Aqueous sample; Aqueous sample
Intervention type(s): Procedure; Procedure; Procedure; Procedure
Intervention description(s): Aqueous sample (0.1ml of aqueous)+ intravitreal injection of bevacizumab (1.25mg/0.05ml); Aqueous sample (0.1ml)+intravitreal injection of bevacizumab (1.25mg/0.05ml); Aqueous sample (0.1ml)+ intravitreal injection of bevacizumab (1.25mg/0.05ml); Aqueous sample (0.1ml)
Other intervention name(s): Avastin; Avastin; Avastin; Avastin
Intervention MeSH terms: Bevacizumab
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)