Effect of fixation pressure on juxtacanalicular tissue and Schlemm's canal

Purpose. A quantitative study was performed to compare the effect of two commonly used fixation pressures, 0 mm Hg (immersion) and 10 mm Hg (perfusion), on the porosity of the juxtacanalicular tissue and the size of Schlemm's canal. Methods. Twelve pairs of human eyes were studied by fixing one eye with perfusion fixation and the fellow eye with immersion fixation, Morphometric analysis of the juxtacanalicular tissue and Schlemm's canal was performed. Outflow resistance was calculated from these measurements and compared with the measured outflow resistance obtained in six eyes. Results. Schlemm's canal was narrowed in perfusion-fixed eyes, with a 47% smaller cross-sectional area than in immersion-fixed eyes (P = 0.04). Juxtacanalicular tissue of perfusion-fixed eyes had a 13.4% increase in the relative amount of empty space when compared with immersion-fixed fellow eyes (P = 0.04), Solid tissue components were almost equally divided among amorphous basement membrane, tendon and sheath material, and cytoplasm. No obvious washout of extracellular material was noted in perfused tissue. Measured outflow resistance was 100 times larger than outflow resistance of the juxtacanalicular tissue calculated from histologic measurements, Conclusions. Perfusion fixation at physiologic intraocular pressure caused a 47% decrease in the area of Schlemm's canal and a mean increase of 13.4% in the relative amount of empty space in the juxtacanalicular tissue compared with immersion-fixed fellow eves. Perfusion of fixative did not appear to cause washout of extracellular material. Perfusion-fixed tissue appears preferable for studies of Schlemm's canal and for ultrastructural studies of the aqueous outflow pathways within the juxtacanalicular tissue.