Table 1.
Primary and secondary antibodies.
Table 2.
Primer sequences and amplification efficiencies for qPCR.
Fig 1.
Longitudinal cryosections of immunolabeled aquaporin-4 and phalloidin on human (A-E), and porcine (B) optic nerve head tissue.
Minimal label for AQP4 (green) is visible in the lamina cribrosa (between dotted white lines in A, B; seen at higher power in C and E. AQP4 label is prominent in the retinal nerve fiber layer, prelaminar area and myelinated optic nerve in each species. Phalloidin labeling of F-actin (red) is prominent in the prelamina, lamina and myelinated ON (D, E). AQP4 labeling is visible within axon bundles and pronounced along the edge of axons bundles and at the pia surface. DAPI (blue) identifies cell nuclei. Scale Bar: 200 μm (A, D), 50 μm (B, C, E).
Fig 2.
Longitudinal sections of porcine optic nerve head labeled for aquaporin 1 (AQP1, red) and aquaporin-9 (AQP9, green) and DAPI (blue).
Labeling of AQP1 and AQP9 was substantial at the internal limiting membrane (A), but minimal throughout the lamina and myelinated optic nerve, except in the walls of larger blood vessel walls as seen in higher power images (B, C). Scale Bar: 100 μm (A), 50 μm (B, C).
Fig 3.
Longitudinal (A-C) and cross-sections (D-I) of porcine optic nerve head labeled for aquaporin 4 (AQP4, green), myelin basic protein (MBP, red) and DAPI (blue).
In longitudinal section, A, the lamina cribrosa is indicated by the zone between dotted white lines. Label for AQP4 is present in the retina and prelaminar region, absent in the lamina, and begins again coincident with the initial zone of MBP labeling. The lamina cribrosa in cross-section (D,E) is devoid of both AQP4 and MBP. In F and G, the section has lamina in the inferior area and the initial myelinated optic nerve present in the upper portion, showing that MBP staining begins just anterior to that of AQP4. The myelinated optic nerve (H, I) labels for both AQP4 and MBP. Scale Bar: 200 μm (A, D, E, F, G, H, I), 50 μm (B), 25 μm (C).
Fig 4.
Longitudinal (A, B) and cross-sections (C-F) of porcine tissue labeled for aquaporin 4 (AQP4, green), glial fibrillary acidic protein (GFAP, red) and DAPI (blue).
Labeling for GFAP is visible throughout (A-C), in retina through the lamina and into the myelinated nerve, while AQP4 is only visible in the retina and myelinated nerve. Cross-sections of the lamina region (D, E) show label for GFAP but not for AQP4. Higher power cross-section of the transition zone from lamina to myelin shows co-incident labeling for GFAP and AQP4 in central lamina (F). In myelinated nerve, both labels are present, but are somewhat more distinct in position from each other (G). High power image (40x) shows the GFAP and AQP4 co-localization (yellow/orange) in astrocytic cells within and along the periphery of nerve bundles. Scale Bar: 150 μm (A, B), 50 μm (C, D, E), 25 μm (F, G) 10 μm (H).
Fig 5.
Labeling for α-dystroglycan (αDG, green) in human (A, B) and porcine (C, D) optic nerve head region in longitudinal sections (A, B, C) and cross-section (D).
αDG is present throughout the retina, pre-lamina, lamina, and myelinated nerve. It is more evident in the periphery of axon bundles, basement membrane and bordering blood vessels. DAPI (blue). Scale Bar: 200 μm (A), 50 μm (B), 25 μm (C, D).
Fig 6.
Semi-quantification of aquaporin-4 labeling in 6 regions of porcine ONH region.
Values in choroid, sclera, and lamina are minimal, not significantly different from each other, while each was significantly lower than retina, myelin transition zone (MTZ), and myelinated optic nerve (MON). Mean ± standard errors. Dotted line identifies the AQP4 background level in choroid. †p < 0.05, ††p < 0.01 for difference from control choroid. *p < 0.05, **p < 0.01 for difference from lamina.
Fig 7.
Relative mRNA expression of AQP4 (A), GFAP (B), and CD68 (C) in regions of the porcine retina and optic nerve head.
All samples were normalized to the geometric mean of the corresponding housekeeping gene values. R, Retina; L, Lamina; MTZ, myelin transition zone; MON, myelinated optic nerve. N = 4 replicates per region. Standard deviation error bars. Data was considered statistically significant if p < 0.05; * (0.033), ** (0.0021), *** (0.0002), **** (0.0001).
Fig 8.
Western blot of aquaporin-4 (AQP4) on lamina and myelinate nerve (MON) of porcine tissue.
Three biological replicates with three samples of lamina (lane 1,2,3) and three biological replicates with three samples of MON (lane 4,5,6) were processed. AQP4 was only detected in the MON samples, while none detected in the lamina tissues. All 6 lanes were positive for β-actin.