Table 1.
List of primary antibodies and markers used in this study.
Figure 1.
Quantification of Connexins in Astrocytes Predominantly in the Parenchyma or Predominantly Associated with the Vasculature in Rat Retina and their Differences in Aging.
Changes in the number and plaque size of connexins in astrocytes predominantly in the parenchyma (A&C) and predominantly associated with the vasculature (B&D) in the retina as a function of age. At 3 months of age all connexins were detected in astrocytes predominantly in the parenchyma or predominantly associated with the vasculature. However, the plaque size of both Cx30 and Cx43 were significantly larger in the parenchyma compared to other Cx. Cx30 plaque size was larger than all other connexins at all ages associated with the vasculature. This suggest that Cx30 in young rats was the major connexin in the retina. With aging, Cx26 showed a transient increase at 9 months in the predominantly parenchymal astrocytes, but this was not sustained through 22 months. By 22 months, there was significantly more Cx30 both in the predominantly parenchymal astrocytes and astrocytes predominantly associated with the vasculature and the plaque size of Cx30 was significantly larger, compared to all other Cx. Each value represents the mean ± SEM of data from four retinas from different rats (n = 4) normalized to the total area for each retina. The mean number was also determined by counts in eight different fields of view in the central, midperipheral and peripheral regions of each retina at each age (A total of 24 fields of view per retina). One-way ANOVA with Bonferroni's post-hoc multiple comparison tests were applied for connexin plaque size and number. * denotes a statistical significance of P<0.05 for comparisons of each connexin to itself with age. E&F: the plaque number was multiplied by the size of plaque for each connexin at each time point. This confirmed our results in A–D that Cx30 was the predominant connexin associated with aging in retinal astrocytes.
Figure 2.
Connexin30 Expression in Astrocytes Predominantly in the Parenchyma or Predominantly Associated with the Vasculature in the Retina from Young and Old Rats.
Cx30 expression in astrocytes predominantly in the parenchyma (GFAP+) and predominantly associated with the vasculature (GFAP+/GS isolectin B4+) in the midperipheral retina of 3-month (young adult, A–C, G) and 22-month old (aged, D–F, H) rats. At all ages examined: Cx30 (red) labelled gap junctions in astrocytes predominantly in the parenchyma (large arrows) and predominantly associated with the vasculature (small arrows). GFAP labelled astrocytes (green, B & E, G & H) and GS isolectin B4 labelled blood vessels (purple, C & F, G & H). Double immunostaining of Cx30 with GFAP or GS isolectin B4 are shown in yellow and pink respectively (arrowheads, B & E, C & F, respectively). Colocalisation of all three markers is shown in white (large arrowheads, G & H). Scale bar, 50 µm (A–H).
Figure 3.
Quantification of Connexin Proteins in Young and Old Rat Retinas.
Western blots using antibodies to Cx26, Cx30, Cx43 and Cx45 in rat retinal homogenates. A. Immunoblot analysis with antibodies directed to Cx26, Cx30, GAPDH, Cx43, and Cx45 were specifically detected at 25–29, 33, 37, 43, 45–47 kDa respectively. B. Band integrated density value (IDV) of each astrocyte connexin expressed during aging (except 18 months which was omitted from analysis due to insufficient numbers in this group). Each value represents the mean ± SEM of data from three retinas from different rats (n = 3). Friedman's non-parametric and Dunn's multiple comparison tests were applied. * denotes a statistical significance of P<0.05 for comparisons of each connexin to itself with age.
Figure 4.
Six marker Immunohistochemistry of Connexins on Astrocytes Predominantly Associated with the Vasculature and Predominantly in the Parenchyma in the Retina of a Young Rat.
Cx26 (blue, A), Cx30 (red, B), Cx43 (green, C), Cx45 (pale brown, D) were colocalised (E) on GFAP positive-astrocytes (pale purple, F). Heterogeneity in gap junction plaques are indicated as follows: Cx26+/Cx43+ (large arrows), Cx26+/Cx45+ (curved arrows), Cx26+/Cx30+/Cx43+ (small arrows), Cx26+/Cx43+/Cx45+ (arrowheads). Scale bar, 50 µm (A–F).
Figure 5.
Six Marker Immunohistochemistry Connexins on Astrocytes Predominantly in the Parenchyma in the Retina of a Middle Aged Rat.
Cx26 (blue, A), Cx30 (red, B), Cx43 (green, C), Cx45 (pale brown, D) were colocalised (E) on GFAP positive-astrocytes (pale purple, F). Connexin heterogeneity in gap junction plaques are indicated as follows: Cx26+/Cx43+ (large arrows), Cx26+/Cx45+ (small arrows), Cx26+/Cx30+/Cx43+ (curved arrows), Cx26+/Cx43+/Cx45+ (arrowheads). Scale bar, 50 µm (A–F).
Figure 6.
Six Marker Immunohistochemistry of Connexins on Astrocytes Predominantly in the Parenchyma in the Retina of an Aged Rat.
Cx26 (blue, A), Cx30 (red, B), Cx43 (green, C), Cx45 (pale brown, D) were colocalised (E) on GFAP positive-astrocytes (pale purple, F). Connexin heterogeneity in gap junction plaques are indicated as follows: Cx26+/Cx43+ (large arrows), Cx26+/Cx45+ (small arrows), Cx26+/Cx30+/Cx43+ (curved arrows), Cx26+/Cx43+/Cx45+ (arrowheads). Scale bar, 50 µm (A–F).
Table 2.
All possible Cx protein (26, 30, 43 and 45) combinations in retinal astrocytes.
Figure 7.
Imaris Snapshot Images through the Entire Depth of the Retinal Nerve Fibre Layer.
Imaris snapshot images of confocal z-stack projections that visualise the entire depth of the retinal fibre layer where both astrocytes and blood vessels are situated. The overlapping immunohistochemistry represents connexin heterogeneity in gap junction plaques expressed by astrocytes and captured using high resolution three-dimensional projection, simulated fluorescence processing and orthogonal mode camera dialogue. The GFAP and GS isolectin B4 stain were omitted from these images to better illustrate the fluorescence of the connexins. A&C were taken in the central retinal region and B&D from a peripheral retinal region. A&C and B&D are a different angle of the same 3D analysis to show different connexin combinations as indicated by the arrows. Immunostaining of astrocyte connexins represents Cx26 (blue), Cx30 (red), Cx43 (green) and Cx45 (brown). Different colocalisation patterns of connexin proteins in diverse astrocyte connexin hemichannels were qualitatively analysed (1Cx protein, small arrows, 2Cx and 3Cx proteins, curved arrows, and 4Cx proteins, large arrowheads). All images are highly zoomed and rendered confocal z-slice projections. For qualitative purposes, displayed images were also constructed using automated scale bar, object frame (grid, tickmarks and box) and XYZ clipping plane (i.e. crops object below the plane). The set camera angle logarithmic functions were also applied to measure angle, elevation and azimuth parameters for each of the images represented. This was as follows: angle = −5.6521 (A), 4.4572 (B), −6.3247 (C), −0.0729 (D), elevation = 91.8453 (A), 88.6771 (B), 93.7761 (C), 90.0196 (D), azimuth = 119.9393 (A), −65.222 (B), 127.6513 (C), 93.3840 (D).
Figure 8.
Quantification of the Connexin Heterogeneity in Gap Junction Plaques During Aging in the Rat Retina.
Changes in the number and type of connexins in gap junction plaques in astrocytes predominantly in the parenchyma (A, C &E) and predominantly associated with the vasculature (B, D & F) in the retina as a function of age. In all graphs, each value represents the mean ± SEM of data from four retinas from different rats (n = 4). The mean number was also determined by counts in eight different fields of view in the central, midperipheral and peripheral regions of each retina at each age (a total of 24 fields of view per retina) and normalized to the total area for either the parenchyma or vasculature. In each graph, x-axis represents age in months, and y-axis represents number per retina (A–F). One-way ANOVA with Bonferroni's post-hoc multiple comparison tests were applied. * denotes a statistical significance of P<0.05 for comparisons of each connexin to itself with age. The horizontal line indicates all these Cx heterogeneous GJs at the particular age time point were significantly different compared to 3 months of age.