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The precise mechanism whereby mitomycin C enhances IOP reduction in glaucoma filtering surgery still eludes us. Ten rabbits received full-thickness Nd:YAG laser sclerostomy ab interno and adjunctive intraoperative treatment with mitomycin C (MMC) applied topically over the intact conjunctiva (0.5 mg ml-1 for 5 min). A systematic ultrastructural analysis of the fistulas and surrounding tissue was then conducted in conjunction with clinical observations, over the ensuing 10 weeks. In order to investigate also the extent to which MMC impedes fistula occlusion in the absence of percolating aqueous humour, we created non-perforating ('half-thickness') sclerostomies ab interno in three additional rabbits, one with and two without MMC therapy. Transconjunctival MMC application resulted in no serious complications. Eight of the ten full-thickness fistulas remained patent throughout the study, maintaining significant IOP reduction; the other two sclerostomies were compromised by iris incarceration. The MMC-treated, half-thickness canal remained as a tissue-free cul de sac; the two non-treated ones became completely occluded within one week without having recourse to extraocular cell populations. MMC suppressed the migration and proliferation of fibroblasts, macrophages and clump cells from the episclera, sclera, ciliary body and iris root. Repolymerization of heat-damaged collagen was abortive; neosynthesis was not observed. Myofibroblasts were encountered in the vicinity of the sclerostomy canals, and, after the fifth week, these cells were also found to be deployed as a canal-lining layer, delimiting the lumen from the surrounding stroma along most of the fistula length. Towards the external ostium, this layer of myofibroblasts was incomplete or absent. Near the internal ostium, lining cells were derived from the corneal endothelium. The transconjunctival mode of applying MMC appears to be efficient. This antifibrotic drug exerts its inhibitory influence by suppressing not only cell migration and proliferation, but also phagocytic and synthetic activities. However, exposed tissues are not acellular, and amongst the populations present, myofibroblasts are found to dominate the scene. The canal-delimiting cellular lining may play a role in maintaining fistula patency in MMC-treated eyes.