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Application of an organotypic ocular perfusion model to assess intravitreal drug distribution in human and animal eyes 
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Anterior bead distribution as a function of size is conserved between live and perfused pig eyes. ( a ) Representative image of a yellow pellet (asterisk) and needle trace (arrow) after 4 h of perfusion indicates where two sizes of fluorescent <t>microbeads,</t> 20 nm (green) and 2000 nm (red), were deposited into the inferior vitreous in the dissected posterior eye cup ( n = 3). ( b ) Representative higher magnification stereomicroscope images suggested the 20 nm beads (green) dispersed more than the 2000 nm size (red), visible as a green corona around the pellet (arrows). ( c ) Outflow tissues in sections through the anterior segment were assessed in confocal projections following ITV microbead injections. Only the smaller 20 nm beads (green) were visible in the trabecular meshwork and angular aqueous plexus (asterisks), which was co-stained for CD31 (white, merge), while the larger 2000 nm beads (red) did not appear in this compartment ( n = 3, arrow indicates the location of the AC for orientation). Note that the orientation and location of the probed tissues are also indicated in the electronic supplementary material, figure S6. A corresponding z-stack side projection was also assessed to ensure beads remained within the sectioned tissue plane. ( d ) Anterior microbead distribution patterns were also observed 4 h following injections in live pig eyes showing similar 20 nm bead depositions in outflow tissues (green), but no 2000 nm beads (red) (n = 3). ( e ) Corresponding fluorescent bead numbers were measured from serial aqueous fluid samples from the same perfused eyes collected at 2 h and 4 h following ITV bead injection and normalized as a percentage of injected beads. A progressive time-dependent increase in 20 nm microbeads was observed, but 2000 nm microbeads remained near baseline ( n = 3, linear regression shown, note; error bars smaller than the data point do not plot). ( f ) Raw microbead numbers measured from aqueous fluid samples at 4 h from the live pig study were compared with 4 h aqueous fluid samples from perfused ex vivo pig eyes, indicating comparable ratios between the 20 nm and 2000 nm sizes (n = 4). There were dramatically higher concentrations of 20 nm beads than 2000 nm in both cases. However, there was a small but significant difference between live and perfused 2000 nm samples. In comparison, controls with no active perfusion (control) showed virtually no aqueous signal detected ( n = 4). (Scale bars indicate 50 µm, data are mean ± s.e.m., *** p < 0.005, * p < 0.05, n.s.; not significant).
Application of an organotypic ocular perfusion model to assess intravitreal drug distribution in human and animal eyes 
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Anterior microbead distribution is conserved in perfused human eyes. ( a ) Representative confocal projections of sectioned human outflow tissues following 4 h perfusion ex vivo showed deposition of 20 nm beads (green), but no 2000 nm beads (red) in trabecular meshwork and SC as visualized by CD31 labelling (white, Merge), similar to the pig studies ( n = 3, arrow indicates the location of the AC for orientation). A side projection indicates beads remained in the tissue plane (Side, n = 3). ( b ) Similar to porcine samples, corresponding serial aqueous samples from perfused ex vivo human eyes exhibited a time-dependent increase in the percentage of 20 nm <t>microbeads,</t> but 2000 nm microbeads remained near baseline. For comparison, pig perfusion data are also plotted on the graph in dashed lines ( n = 3, linear regression shown, note; error bars smaller than the data point do not plot). ( c ) Comparisons at 4 h between analysed human and porcine aqueous samples show a similar ratio of raw numbers of microbead sizes, with dramatically higher concentrations of 20 nm beads than 2000 nm. The human perfusions resulted in small, but significantly lower concentrations of 20 nm beads than in the pig ( n = 3). (Scale bars indicate 50 µm, data are mean ± s.e.m., *** p < 0.001).
Application of an organotypic ocular perfusion model to assess intravitreal drug distribution in human and animal eyes 
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Retinal microbead distribution are preserved in perfused human and porcine eyes. Confocal projections of retinal sections were assessed from regions underlying the microbead pellet from perfused and live pig eyes, and ex vivo human eyes after 4 h perfusion. ( a ) Perfused pig retina images demonstrated the distribution of 20 nm beads (green) and fewer 2000 nm beads (red), with <t>microbeads</t> of both sizes generally excluded from penetrating into the retinal layers (indicated by text along the right-hand side of the merged image). A side projection was also assessed to ensure beads remained in the tissue plane ( n = 3). Comparable retinal microbead distribution patterns were observed at 4 h following injections in ( b ) live pig eyes ( n = 3) and ( c ) perfused human eyes ( n = 3), with very few of either size penetrating into the retinal layers. (Scale bars indicate 50 µm). ( d ) Representative confocal images of central retina sections from ex vivo pig eyes following 4 h perfusion, stained with antibodies to the markers R/G opsin, GS, Chx-10 and RBPMS ( n = 3, Scale bars indicate 50 μm, note the sections are oriented the same way, with the retinal layers indicated along with the right-hand panel). ( e ) Representative confocal images of central retina sections from ex vivo human eyes following 4 h perfusion, stained for the same markers as in ( d ) ( n = 3, Scale bars indicate 50 µm, retinal layers are indicated along with the right-hand panel). GCL; ganglion cell layer, INL; inner nuclear layer, ONL; outer nuclear layer, Vit; vitreous.
Application of an organotypic ocular perfusion model to assess intravitreal drug distribution in human and animal eyes Journal of the Royal Society Interface, 2022 Jan 26
"Anterior bead distribution as a function of size is conserved between live and perfused pig eyes. ( a ) Representative image of a yellow pellet (asterisk) and needle trace (arrow) after 4 h of perfusion indicates where two sizes of fluorescent <t>microbeads,</t> 20 nm (green) and 2000 nm (red), were deposited into the inferior vitreous in the dissected posterior eye cup ( n = 3). ( b ) Representative higher magnification stereomicroscope images suggested the 20 nm beads (green) dispersed more than the 2000 nm size (red), visible as a green corona around the pellet (arrows). ( c ) Outflow tissues in sections through the anterior segment were assessed in confocal projections following ITV microbead injections. Only the smaller 20 nm beads (green) were visible in the trabecular meshwork and angular aqueous plexus (asterisks), which was co-stained for CD31 (white, merge), while the larger 2000 nm beads (red) did not appear in this compartment ( n = 3, arrow indicates the location of the AC for orientation). Note that the orientation and location of the probed tissues are also indicated in the electronic supplementary material, figure S6. A corresponding z-stack side projection was also assessed to ensure beads remained within the sectioned tissue plane. ( d ) Anterior microbead distribution patterns were also observed 4 h following injections in live pig eyes showing similar 20 nm bead depositions in outflow tissues (green), but no 2000 nm beads (red) (n = 3). ( e ) Corresponding fluorescent bead numbers were measured from serial aqueous fluid samples from the same perfused eyes collected at 2 h and 4 h following ITV bead injection and normalized as a percentage of injected beads. A progressive time-dependent increase in 20 nm microbeads was observed, but 2000 nm microbeads remained near baseline ( n = 3, linear regression shown, note; error bars smaller than the data point do not plot). ( f ) Raw microbead numbers measured from aqueous fluid samples at 4 h from the live pig study were compared with 4 h aqueous fluid samples from perfused ex vivo pig eyes, indicating comparable ratios between the 20 nm and 2000 nm sizes (n = 4). There were dramatically higher concentrations of 20 nm beads than 2000 nm in both cases. However, there was a small but significant difference between live and perfused 2000 nm samples. In comparison, controls with no active perfusion (control) showed virtually no aqueous signal detected ( n = 4). (Scale bars indicate 50 µm, data are mean ± s.e.m., *** p < 0.005, * p < 0.05, n.s.; not significant). "
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