title: Trypan blue staining for capsulorhexis: ultrastructural effect on lens epithelial cells and capsules.
abstract: OBJECTIVE
  To evaluate the ultrastructural effect of trypan blue 0.1% staining for capsulorhexis on lens epithelial cells (LECs) and capsules.
  
  
  METHODS
  Division of Ophthalmology, University of São Paulo, São Paulo, Brazil.
  
  
  METHODS
  Before capsulorhexis, patients were randomly assigned to 1 of 2 groups. Trypan blue 0.1% staining was performed in the treatment group. No trypan blue was used in the control group. Samples of capsules with LECs were fixed and analyzed with routine optical microscopy techniques, immunohistochemistry for beclin-1 expression (a marker of autophagy), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling to detect apoptosis, and transmission electron microscopy (TEM). Morphometric analyses were performed, and the 2 sets of data were compared.
  
  
  RESULTS
  Each group comprised 15 patients. Cell death by autophagy and apoptosis was observed in the treatment group but not in the control group. The TEM images of subcapsular epithelium cells showed mitochondrial rupture, dilation of the cisterns of the endoplasmic reticulum, increased cytoplasmic and nuclear electron density, and abnormalities in the nuclear profile of trypan blue-stained cells. Morphometric analysis showed statistically significant differences between the 2 groups in the longest nuclear axes and the ratio between the total nuclear perimeter and the cell area (P = .03). The difference in capsule thickness between groups was not significant.
  
  
  CONCLUSIONS
  Trypan blue caused LEC death, which supports the hypothesis that staining with trypan blue 0.1% can help reduce the incidence of posterior capsule opacification after cataract surgery.
  
  
  BACKGROUND
  No author has a financial or proprietary interest in any material or method mentioned.
authors.affiliation: From the Division of Ophthalmology (Portes, Monteiro), Hospital das Clínicas, University of São Paulo Medical School, São Paulo, and the Biological Sciences Institute (Almeida, Allodi, Miguel), Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: André Luís F
authors.initials: AL
authors.lastname: Portes
authors.forename: Andréa C
authors.initials: AC
authors.lastname: Almeida
authors.forename: Silvana
authors.initials: S
authors.lastname: Allodi
authors.forename: Mário L R
authors.initials: ML
authors.lastname: Monteiro
authors.forename: Nádia C O
authors.initials: NC
authors.lastname: Miguel
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D000071186:Beclin-1; D019923:Capsulorhexis; D004396:Coloring Agents / Q000494:pharmacology*; D053938:DNA Fragmentation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020287:In Situ Nick-End Labeling; D007903:Lens Capsule, Crystalline / Q000187:drug effects / Q000648:ultrastructure*; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D018918:Phacoemulsification; D011446:Prospective Studies; D013194:Staining and Labeling; D014343:Trypan Blue / Q000494:pharmacology*
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D004396:Coloring Agents; D008565:Membrane Proteins; D014343:Trypan Blue