Protective effects on the retina after ranibizumab treatment in an ischemia model

PLoS One. 2017 Aug 11;12(8):e0182407. doi: 10.1371/journal.pone.0182407. eCollection 2017.

Abstract

Retinal ischemia is common in eye disorders, like diabetic retinopathy or retinal vascular occlusion. The goal of this study was to evaluate the potential protective effects of an intravitreally injected vascular endothelial growth factor (VEGF) inhibitor (ranibizumab) on retinal cells in an ischemia animal model via immunohistochemistry (IF) and quantitative real-time PCR (PCR). A positive binding of ranibizumab to rat VEGF-A was confirmed via dot blot. One eye underwent ischemia and a subgroup received ranibizumab. A significant VEGF increase was detected in aqueous humor of ischemic eyes (p = 0.032), whereas VEGF levels were low in ranibizumab eyes (p = 0.99). Ischemic retinas showed a significantly lower retinal ganglion cell number (RGC; IF Brn-3a: p<0.001, IF RBPMS: p<0.001; PCR: p = 0.002). The ranibizumab group displayed fewer RGCs (IF Brn-3a: 0.3, IF RBPMS: p<0.001; PCR: p = 0.007), but more than the ischemia group (IF Brn-3a: p = 0.04, IF RBPMS: p = 0.03). Photoreceptor area was decreased after ischemia (IF: p = 0.049; PCR: p = 0.511), while the ranibizumab group (IF: p = 0.947; PCR: p = 0.122) was comparable to controls. In the ischemia (p<0.001) and ranibizumab group (p<0.001) a decrease of ChAT+ amacrine cells was found, which was less prominent in the ranibizumab group. VEGF-receptor 2 (VEGF-R2; IF: p<0.001; PCR: p = 0.021) and macroglia (GFAP; IF: p<0.001; PCR: p<0.001) activation was present in ischemic retinas. The activation was weaker in ranibizumab retinas (VEGF-R2: IF: p = 0.1; PCR: p = 0.03; GFAP: IF: p = 0.1; PCR: p = 0.015). An increase in the number of total (IF: p = 0.003; PCR: p = 0.023) and activated microglia (IF: p<0.001; PCR: p = 0.009) was detected after ischemia. These levels were higher in the ranibizumab group (Iba1: IF: p<0.001; PCR: p = 0.018; CD68: IF: p<0.001; PCR: p = 0.004). Our findings demonstrate that photoreceptors and RGCs are protected by ranibizumab treatment. Only amacrine cells cannot be rescued. They seem to be particularly sensitive to ischemic damage and need maybe an earlier intervention.

MeSH terms

  • Amacrine Cells / drug effects
  • Amacrine Cells / metabolism
  • Animals
  • Aqueous Humor / metabolism
  • Calcium-Binding Proteins / metabolism
  • Cell Count
  • Cholinergic Neurons / drug effects
  • Cholinergic Neurons / metabolism
  • Disease Models, Animal
  • Glial Fibrillary Acidic Protein / metabolism
  • Humans
  • Ischemia / drug therapy*
  • Ischemia / pathology
  • Mice
  • Microfilament Proteins / metabolism
  • Microglia / drug effects
  • Microglia / metabolism
  • Photoreceptor Cells, Vertebrate / drug effects
  • Photoreceptor Cells, Vertebrate / metabolism
  • Protective Agents / pharmacology
  • Protective Agents / therapeutic use*
  • Protein Binding / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Ranibizumab / pharmacology
  • Ranibizumab / therapeutic use*
  • Rats
  • Reperfusion Injury / pathology
  • Retina
  • Retinal Ganglion Cells / drug effects
  • Retinal Ganglion Cells / metabolism
  • Rhodopsin / metabolism
  • Synapses / drug effects
  • Synapses / metabolism
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Aif1 protein, rat
  • Calcium-Binding Proteins
  • Glial Fibrillary Acidic Protein
  • Microfilament Proteins
  • Protective Agents
  • RNA, Messenger
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A, rat
  • Rhodopsin
  • Ranibizumab

Grant support

The study was in part supported by Novartis Pharma GmbH and the spotting unit was funded by PURE, a project of North Rhine-Westphalia, a federal German state, Germany. No additional external funding was received for this study. The funders had no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication.