title: Factors influencing the retrograde labeling of retinal ganglion cells with fluorogold in an animal optic nerve crush model.
abstract: OBJECTIVE
  To investigate whether different crush durations or a different fluorogold (FG) injection timing can affect the efficiency of FG retrograde labeling of retinal ganglion cells (RGCs) in the optic nerve (ON) crush model.
  
  
  METHODS
  We performed the ON crush in rats with a clip at different durations or a jewel forceps to compare the effects of different crush methods with FG staining. RGC density was compared between the FG injection 1 week before the sacrifice of the animals (group A) and the injection before the crush experiment (group B). Double staining with CD11b and FG in the retinal sections was conducted to investigate the relationship between the overcounting of RGCs and microglia.
  
  
  RESULTS
  The FG-stained particles were significantly decreased at the distal part of the crush site compared to the proximal site of the ON with a crush duration of over 30 s or when crushed with the jewel forceps. Two weeks after ON crush, the RGC count was higher both in the central and mid-peripheral retinas in group B. The percentage of CD11b-stained cells among the FG-stained cells in the RGC layer of retinas in group B was higher than that of group A (34% in group B vs. 4% in group A, p = 0.0001). Overcounting of RGC density in group B was due to additional microglia with FG engulfing.
  
  
  CONCLUSIONS
  Our results suggest that each laboratory should test its setting conditions to avoid factors influencing the RGC density measurement before conducting ON crush experiments.
authors.affiliation: Institute of Eye Research, Buddhist Tzu Chi General Hospital, Tzu Chi University, Hualien, Taiwan, ROC.
authors.forename: Tzu-Lun
authors.initials: TL
authors.lastname: Huang
authors.forename: Sun-Ping
authors.initials: SP
authors.lastname: Huang
authors.forename: Chung-Hsing
authors.initials: CH
authors.lastname: Chang
authors.forename: Kung-Hung
authors.initials: KH
authors.lastname: Lin
authors.forename: Min-Muh
authors.initials: MM
authors.lastname: Sheu
authors.forename: Rong-Kung
authors.initials: RK
authors.lastname: Tsai
mesh_terms: D000818:Animals; D001370:Axonal Transport; D015415:Biomarkers / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D002452:Cell Count; D002470:Cell Survival; D004195:Disease Models, Animal; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D009409:Nerve Crush / Q000379:methods*; D020221:Optic Nerve Injuries / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000378:metabolism* / Q000473:pathology; D013194:Staining and Labeling; D013266:Stilbamidines / Q000378:metabolism*
chemical_list: C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D015415:Biomarkers; D039481:CD11b Antigen; D005456:Fluorescent Dyes; D013266:Stilbamidines