title: Small interfering RNA-mediated suppression of Ccl2 in Müller cells attenuates microglial recruitment and photoreceptor death following retinal degeneration.
abstract: BACKGROUND
  The recruitment and activation of inflammatory cells is thought to exacerbate photoreceptor death in retinal degenerative conditions such as age-related macular degeneration (AMD). We investigated the role of Müller cell-derived chemokine (C-C motif) ligand (Ccl)2 expression on monocyte/microglia infiltration and photoreceptor death in light-mediated retinal degeneration, using targeted small interfering (si)RNA.
  
  
  METHODS
  Adult Sprague-Dawley rats were injected intravitreally with 1 μg of either Ccl2 siRNA or scrambled siRNA, and were then exposed to 1000 lux of light for a period of 24 hours. The mice were given an overdose of barbiturate, and the retinas harvested and evaluated for the effects of bright-light exposure. Ccl2 expression was assessed by quantitative PCR, immunohistochemistry, and in situ hybridization. Monocytes/microglia were counted on retinal cryostat sections immunolabeled with the markers ED1 and ionized calcium binding adaptor (IBA)1, and photoreceptor apoptosis was assessed using terminal dUTP nick end labeling.
  
  
  RESULTS
  Intravitreal injection of Ccl2 siRNA significantly reduced the expression of Ccl2 following light damage to 29% compared with controls. In retinas injected with Ccl2 siRNA, in situ hybridization and immunohistochemistry on retinal cryostat sections showed a substantial decrease in Ccl2 within Müller cells. Cell counts showed significantly fewer ED1-positive and IBA1-positive cells in the retinal vasculature and outer nuclear layer of Ccl2 siRNA-injected retinas, compared with controls. Moreover, there was significantly less photoreceptor apoptosis in Ccl2 siRNA-injected retinas compared with controls.
  
  
  CONCLUSIONS
  Our data indicate that Ccl2 expression by Müller cells promotes the infiltration of monocytes/microglia, thereby contributing to the neuroinflammatory response and photoreceptor death following retinal injury. Modulation of exaggerated chemokine responses using siRNA may have value in reducing inflammation-mediated cell death in retinal degenerative disease such as AMD.
authors.affiliation: The John Curtin School of Medical Research, College of Medicine, Biology and Environment, The Australian National University, Building 131, Garran Rd, Canberra, ACT 0200, Australia. matt.rutar@anu.edu.au
authors.forename: Matt
authors.initials: M
authors.lastname: Rutar
authors.forename: Riccardo
authors.initials: R
authors.lastname: Natoli
authors.forename: Jan M
authors.initials: JM
authors.lastname: Provis
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects* / Q000528:radiation effects; D018932:Chemokine CCL2 / Q000378:metabolism*; D004195:Disease Models, Animal; D053331:Ectodysplasins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D058449:Intravitreal Injections; D008027:Light / Q000009:adverse effects; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000473:pathology / Q000528:radiation effects; D009457:Neuroglia / Q000187:drug effects* / Q000528:radiation effects; D010786:Photoreceptor Cells / Q000187:drug effects / Q000473:pathology* / Q000528:radiation effects; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology* / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D012162:Retinal Degeneration / Q000188:drug therapy / Q000209:etiology / Q000473:pathology*
chemical_list: C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D018932:Chemokine CCL2; D053331:Ectodysplasins; C506019:Eda protein, mouse; D008840:Microfilament Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering