title: Quantitative analyses of retinal vascular area and density after different methods to reduce VEGF in a rat model of retinopathy of prematurity.
abstract: OBJECTIVE
  Targeted inhibition of Müller cell (MC)-produced VEGF or broad inhibition of VEGF with an intravitreal anti-VEGF antibody reduces intravitreal neovascularization in a rat model of retinopathy of prematurity (ROP). In this study, we compared the effects of these two approaches on retinal vascular development and capillary density in the inner and deep plexi in the rat ROP model.
  
  
  METHODS
  In the rat model of ROP, pups received 1 μL of (1) subretinal lentivector-driven short hairpin RNA (shRNA) to knockdown MC-VEGFA (VEGFA.shRNA) or control luciferase shRNA, or (2) intravitreal anti-VEGF antibody (anti-VEGF) or control isotype goat immunoglobulin G (IgG). Analyses of lectin-stained flat mounts at postnatal day 18 (p18) included: vascular/total retinal areas (retinal vascular coverage) and pixels of fluorescence/total retinal area (capillary density) of the inner and deep plexi determined with the Syncroscan microscope, and angles between cleavage planes of mitotic vascular figures labeled with anti-phosphohistone H3 and vessel length.
  
  
  RESULTS
  Retinal vascular coverage and density increased in both plexi between p8 and p18 in room air (RA) pups. Compared with RA, p18 ROP pups had reduced vascular coverage and density of both plexi. Compared with respective controls, VEGFA.shRNA treatment significantly increased vascular density in the deep plexus, whereas anti-VEGF reduced vascular density in the inner and deep plexi. Vascular endothelial growth factor-A.shRNA caused more cleavage angles predicting vessel elongation and fewer mitotic figures, whereas anti-VEGF treatment led to patterns of pathologic angiogenesis.
  
  
  CONCLUSIONS
  Targeted treatment with lentivector-driven VEGFA.shRNA permitted physiologic vascularization of the vascular plexi and restored normal orientation of dividing vascular cells, suggesting that regulation of VEGF signaling by targeted treatment may be beneficial.
authors.affiliation: Department of Ophthalmology, The John Moran Eye Center, University of Utah, Salt Lake City, Utah.
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Yang
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Jiang
authors.forename: John
authors.initials: J
authors.lastname: Flannery
authors.forename: Scott
authors.initials: S
authors.lastname: Hammond
authors.forename: Tal
authors.initials: T
authors.lastname: Kafri
authors.forename: Sai Karthik
authors.initials: SK
authors.lastname: Vemuri
authors.forename: Bryan
authors.initials: B
authors.lastname: Jones
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Hartnett
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism; D063928:Ependymoglial Cells / Q000378:metabolism; D055785:Gene Knockdown Techniques; D058449:Intravitreal Injections; D008940:Mitotic Index; D018919:Neovascularization, Physiologic / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015861:Retinal Neovascularization / Q000503:physiopathology; D012171:Retinal Vessels / Q000502:physiology*; D012178:Retinopathy of Prematurity / Q000503:physiopathology*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat
keywords: VEGF; intrvitreal neovscularization; rat model of retinopathy of prematurity; vascular density; vascular extent