title: Noninvasive Quantification of Retinal Microglia Using Widefield Autofluorescence Imaging.
abstract: To validate widefield autofluorescence (AF) in vivo imaging of the retina in mice expressing green fluorescent protein (gfp) in microglia, and to monitor retinal microglia reconstitution in vivo after lethal irradiation and bone marrow transplantation.
  
  
  
  Transgenic Cx3cr1gfp/gfp and wildtype Balb/c mice were used in this study. A confocal scanning laser ophthalmoscope was used for AF imaging with a 55° and a widefield 102° lens. Intrasession reproducibility was assessed for each lens. To investigate reconstitution in vivo, bone marrow from Cx3cr1gfp/gfp mice was used to rescue lethally irradiated wildtype mice. Data were compared to confocal microscopy of retinal flat mounts.
  
  
  
  Both the 55° and the 102° lens produced high resolution images of retinal microglia with similar microglia density. However, compared to the 55° lens, the widefield 102° lens captured approximately 3.6 times more microglia cells (1515 ± 123 cells versus 445 ± 76 cells [mean ± SD], for 102° and 55°, respectively, P < 0.001). No statistical difference in the number of gfp positive cells within corresponding areas was observed within the same imaging session. Imaging of microglia reconstitution showed a similar time course compared to flat mount preparations with an excellent correlation between microglia cell numbers in AF and gfp-stained flat mounts (R = 0.92, P < 0.0001).
  
  
  
  Widefield AF imaging of mice with gfp expressing microglia can be used to quantify retinal microglia. In vivo microglia counts corresponded very well with ex vivo counts on retinal flat mounts. As such, AF imaging can largely replace ex vivo quantification.
authors.affiliation: Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Despina
authors.initials: D
authors.lastname: Kokona
authors.affiliation: Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Nadia
authors.initials: N
authors.lastname: Schneider
authors.affiliation: Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Helena
authors.initials: H
authors.lastname: Giannakaki-Zimmermann
authors.affiliation: Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Joel
authors.initials: J
authors.lastname: Jovanovic
authors.affiliation: Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebneter
authors.affiliation: Department of Ophthalmology and Department of Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Zinkernagel
mesh_terms: D000818:Animals; D002452:Cell Count; D005260:Female; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D017628:Microglia / Q000378:metabolism / Q000473:pathology* / Q000528:radiation effects; D018613:Microscopy, Confocal; D061848:Optical Imaging / Q000379:methods*; D011833:Radiation Injuries, Experimental; D012160:Retina / Q000378:metabolism / Q000473:pathology* / Q000528:radiation effects; D012164:Retinal Diseases / Q000209:etiology / Q000378:metabolism / Q000473:pathology*
chemical_list: D049452:Green Fluorescent Proteins