Table 1.
Fluorescent microspheres of varying size and modification used in this study.
Fig 1.
Segmental flow in perfused human eyes.
En face image of a whole human eye labeled with Qtracker-655 (red) during perfusion. The perfused tissue was cut into approximately 8 wedges and all were imaged en face by confocal microscopy. En face images were positionally labeled and then photo-merged digitally to create a collage using Adobe Photoshop software. Regions of high and low labeling demonstrate the segmental nature of outflow with labeled brackets indicating examples of each region. Gaps reflect razor blade cut sites. Blue is autofluorescence of the TM tissue.
Fig 2.
Mapping the flow regions in high and low flow areas of the TM.
Human TM sections from high flow (A-C), and medium—low (D-F) flow regions labeled with 200 nm fluorescent amine-modified microspheres (green) are shown en face (A and D) or as frontal sections (B, C, E, F). Confocal images of frontal sections show labeling at increasing levels of magnification. High flow regions are shown to contain micro sub-regions of high and low flow (arrows) when viewed at higher magnification (C). Blue color is due to autofluorescence of the TM. Scale bars = 100 μm. OW = Outer wall, SC = Schlemm’s canal, IW = Inner wall.
Fig 3.
Micro-scale distribution of fluorescent microspheres in a high flow region.
High flow regions as shown in Fig 2 were imaged at higher magnification. Relative fluorescent intensity (RFU) was measured across 500 μm and plotted using Image J software. A periodic pattern of micro-flow regions alternating high and low regions (arrows) at approximately every 50–100 μm is shown and originated from a macro high flow TM region. Scale bar = 100 μm.
Fig 4.
Amine- and carboxy-modified fluorescent microspheres (200 nm) colocalize in high and low flow regions of the TM.
Anterior segments were sequentially perfused with 200 nm amine-modified (red) and carboxy-modified (green) fluorescent microspheres prior to fixation and confocal imaging. Confocal images show overlapping red and green in both high (A, C, D) and low (B) flow regions. Immunostaining for versican (gray) localizes to the inner wall and outer wall of Schlemm’s canal and to the JCT region of the TM (A-D). Boxed area in A was zoomed and red and green channels were separated to visualize overlap with versican (gray) (C, D). Pearson’s correlation coefficients (Pcc) were measured using Imaris software in order to determine the amount of colocalization between two signals €. The amine- and carboxy-modified fluospheres colocalize in both high and low flow regions of the TM; n = 3 for each pairing. Scale bars = 20 μm. OW = Outer wall, SC = Schlemm’s canal, IW = Inner wall, JCT = Juxtacanalicular TM.
Fig 5.
Collector channels are present in high and low flow areas of the TM.
Representative images are shown of high (A) and low (B) flow regions of the TM after perfusion with 200 nm amine-modified fluorescent microspheres (red). Fluorescent microspheres appear to accumulate in areas near collector channels (arrows) in both regions. Scale Bar = 50 μm. OW = Outer wall, SC = Schlemm’s canal, IW = Inner wall. Blue is PECAM immunostaining to aid in the visualization of Schlemm’s canal and collector channels, particularly in low flow regions.
Fig 6.
Quantitative PCR array of TM from high flow regions in comparison with low flow regions.
Human TM’s were dissected from perfused anterior segments, RNA was isolated, reverse transcribed, and measured using the human extracellular matrix and adhesion molecule quantitative PCR array. Fold change gene expression is shown as either enriched in high flow regions (values greater than 1.0) or enriched in low flow regions (values less than 1.0). All fold changes greater than 1.5 fold and less than 0.5 were considered to be biologically significant. SAM (significance analysis of microarrays), version 4.01, with 4 biological replicates, was used to determine statistically significant fold gene expression changes for HF regions in comparison with LF regions. The raw data using the delta delta Ct values for HF and LF regions was normalized, then subjected to SAM analysis. All fold change genes shown here were determined to be statistically significant by SAM, and biologically significant, by our 1.5 and 0.5 fold criteria. Error bars are the s.e.m.; n = 4 high flow and low flow regions from 4 individual donor eyes.
Fig 7.
Immunostaining for select ECM proteins in high and low flow regions of the TM.
Tissues were perfused with 200 nm amine-modified fluorescent microspheres (red) to separate high (A, C, E) and low (B, D, F) flow regions. Frontal sections were immunostained (green) with antibodies to Type VI collagen (A, B), SPARC (C, D) or SPP1 (E, F) and imaged by confocal microscopy. Scale bar = 50 μm. OW = Outer wall, SC = Schlemm’s canal, IW = Inner wall.
Fig 8.
MMP3 immunostaining and MMP activity in high and low flow regions of human TM.
Frontal sections of high (A) and low (B) flow regions of human TM from perfused anterior segment organ culture were immunostained with an antibody to MMP3 (green). DAPI is the nuclear stain (blue). Scale bar = 30 μm. OW = Outer wall, SC = Schlemm’s canal, IW = Inner wall, JCT = Juxtacanalicular TM. MMP activity in high and low flow regions of human eyes (C). Results are shown as relative fluorescent units (RFUs) normalized to total protein (μg/ml) in each sample. “N” for each region is listed in the graphs.