Filtering Blebs and Aqueous Pathway: An Immunocytological and In Vivo Confocal Microscopy Study

Abstract

To characterize and understand, at the cellular level, the aqueous humor pathways after filtering surgery, using in vivo confocal microscopy and impression cytology (IC). Observational case series. Thirty-two blebs of 29 patients after trabeculectomy were retrospectively evaluated. In vivo confocal microscopy and immunofluorescence staining of IC samples taken on and around the bleb area were performed. Impression cytology samples were examined under confocal microscopy after goblet cell and inflammatory cell immunostaining with anti-MUC5AC and antivimentin antibodies, respectively. Eyes were classified into 3 groups: (1) functioning blebs (11 eyes), (2) nonfunctioning blebs (10 eyes), and (3) functioning blebs after mitomycin C application (12 eyes). Impression cytology specimens and in vivo confocal microscopy images were analyzed and compared in a masked manner. Conjunctival epithelium changes of each type of bleb were analyzed using both impression cytology specimens and in vivo confocal microscopy and correlated to clinical outcomes. In all IC specimens, numerous MUC5AC-positive cells were observed outside the edges of the blebs. Few MUC5AC-positive cells were observed at the surface of nonfunctioning blebs. Numerous goblet cells with immunostaining that was weak or limited to the membrane were clearly visible morphologically at the surface of functioning blebs (with and without adjunctive mitomycin C). Using in vivo confocal microscopy, all functioning blebs showed numerous intraepithelial optically empty microcysts, whereas nonfunctioning blebs had none or only a few. All blebs contained dendritiform inflammatory cells, especially after mitomycin C application. Impression cytology and in vivo confocal microscopy provide a new approach to filtering blebs. Microcysts observed at the surface of functioning blebs seemed to correspond to goblet cells, mostly containing aqueous humor instead of highly hydrophilic gel-forming mucins. Although this hypothesis requires further confirmation, the transcellular pathway of the aqueous humor could be hypothesized to occur at the level of goblet cells toward the ocular surface.