title: An enzymatic technique to facilitate air separation of the stroma-Descemet's membrane junction.
abstract: OBJECTIVE
  To describe an enzymatic technique that facilitates air separation of Descemet's membrane from the corneal stroma.
  
  
  METHODS
  Fresh human corneoscleral tissue was mounted on an artificial anterior chamber. In a control group, air was injected into the stroma. A second group received a stromal injection of 2.5 mg/mL collagenase type 2 in balanced salt solution that was left in the stroma for 1 hour and 15 minutes. A third group received an injection of 2.5 mg/mL collagenase type 2 in balanced salt solution followed 1 hour and 15 minutes later by an injection of air into the stroma. All injections were performed with a 27-gauge needle into the deep stroma without penetrating Descemet's membrane. Anterior segment optical coherence tomography (AS-OCT), histologic examination, and electron microscopy of the junction between the stroma and Descemet's membrane were performed. The trypan blue exclusion and TUNEL assays were used to study endothelial cell viability after collagenase incubation.
  
  
  RESULTS
  Injection of air or collagenase into the deep corneal stroma did not result in a reproducible separation of the stroma-Descemet's junction. In contrast, the stroma was easily and reproducibly separated from Descemet's membrane with a combination of intrastromal collagenase and air injection. The separation was confirmed by using light and electron microscopy. The cleavage plane seemed to be located between the junction of the posterior stroma and the anterior banded layer of Descemet's membrane. Trypan blue staining demonstrated the viability of endothelial cells after collagenase incubation. TUNEL assay confirmed excellent viability after collagenase+air separation.
  
  
  CONCLUSIONS
  This technique facilitates the separation of Descemet's membrane from the stroma without affecting endothelial cell viability.
authors.affiliation: Department of Ophthalmology, University of Mississippi Medical Center, Jackson, Mississippi, USA. edgarespanamd@gmail.com
authors.forename: Edgar M
authors.initials: EM
authors.lastname: Espana
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
authors.forename: Jonathan
authors.initials: J
authors.lastname: Fratkin
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Henegar
mesh_terms: D002470:Cell Survival; D017364:Collagenases / Q000008:administration & dosage*; D003319:Corneal Stroma / Q000473:pathology / Q000601:surgery*; D016039:Corneal Transplantation / Q000379:methods*; D003886:Descemet Membrane / Q000187:drug effects* / Q000601:surgery / Q000648:ultrastructure; D004728:Endothelium, Corneal / Q000648:ultrastructure; D006801:Humans; D007267:Injections; D008854:Microscopy, Electron; D015203:Reproducibility of Results; D016707:Tissue Fixation / Q000379:methods*; D041623:Tomography, Optical Coherence
chemical_list: D017364:Collagenases