D several enzymes (marked by a black arrow in Fig. 3B1 and listed in Table 1) in positions that did not vary substantially between the experimental groups. One spot (framed by a black circle) was strikingly only present in hypertensive samples (Fig. 3B1), and was absent in all of the other experimental groups in normotensive animals (Fig. 3B2) a, animals with iridectomy (Fig. 3B3), and those treated with Ti (Fig. 3B3, B4), Ti/Tr (Fig. 3B5), Ti/D (Fig. 3B6), and Ti/B (Fig. 3B7). Subsequent MALDI-MS confirmed that the spot corresponded to crybb2. Crybb3 (framed by a circle) was present equally in all groups (Fig. 3B1 7), demonstrating the reproducibility of the applied method.Confirmation with Western blotting and immunohistochemistryAdditional WB was performed for crybb2, crybb3, crybL, crybH, cryc, crym, HSP-25, and HSP-70 on retinal samples to better characterize the changes in crystallin and HSP expression as seen through 2DE and MALDI-MS. Crybb2, with a molecular mass of 23 kDa, was markedly up-regulated in the hypertensive retina (7.461.1-fold; p,0.001), being only marginally present in the normotensive retina (1.3060.12-fold), and nearly totally absent in retinal samples treated with Ti (35.0060.07-fold), Ti/ Tr (20.060.1-fold), Ti/D (60.060.4-fold), and Ti/B (61.060.1fold; Fig. 4A, B). Crybb3 expression did not differ significantly among the groups (Fig. 4C, D). Consistent with crybb2, crybL was strongly 842-07-9 cost expressed in hypertensive samples (7.261.8-fold; p,0.001), while it was only slightly expressed in all other groups (Fig. 4E, F). There were no marked changes in the expressions of either crybH (Fig. 4G, H) or HSP-70 (Fig. 5A, B). Crym (Fig. 5C, D) and HSP-25 (Fig. 5E, F) were the most strongly expressed in the normotensive samples (p,0.001). Cryc expression resembled the expressions of crybb2 and crybL, showing markedly higher expression in hypertensive samples (10.8060.35-fold; p,0.001) than in the other groups (Fig. 5G, H). To confirm and 1676428 visualize crybb up-regulation within the retinal tissue, immunohistochemical staining was performed on normotensive and hypertensive retinal slices. IHC revealed up-regulation of crybb after IOP elevation relative 24272870 to normotensive samples (Fig. 6A, B). The signal increased with the duration of exposure to elevated IOP (Fig. 6C). Crybb signaling was higher at 28 days than at 7 days after IOP elevation, and crybb expression was localized predominantly in the RGC layer of retinal slices (Fig. 6B, C), indicating that RGCs are mainly adversely affected by elevated IOP in glaucoma.Figure 3. Two-dimensional gelelectrophoresis of retinal proteins. A Peptide mapping of a retina obtained from a Sprague-Dawley rat with untreated, sustained elevated IOP 8 weeks after IOP induction. Hypertensive retinal samples showed a marked increase in bb2crystallin (crybb2) expression (area marked by a black box in A, shown at higher magnification in B1 compared to a normotensive shamtreated retina, B2). The 4 weeks of IOP-lowering treatment with Lixisenatide site topical treatment with Ti (B3,B4), Ti/T (B5), Ti/D (B6), and Ti/B (B7) decreased crybb2 expression to baseline levels. The proteins identified, which are marked by an arrow in A and B1, are listed in Table 1 according to the number given. doi:10.1371/journal.pone.0049730.gThese recorded readings remained constant over the 4 weeks of antihypertensive treatment. IOP recordings are illustrated in detail in Fig. 1.Quantification of retinal ganglion cellsThe density of RGCs in retin.D several enzymes (marked by a black arrow in Fig. 3B1 and listed in Table 1) in positions that did not vary substantially between the experimental groups. One spot (framed by a black circle) was strikingly only present in hypertensive samples (Fig. 3B1), and was absent in all of the other experimental groups in normotensive animals (Fig. 3B2) a, animals with iridectomy (Fig. 3B3), and those treated with Ti (Fig. 3B3, B4), Ti/Tr (Fig. 3B5), Ti/D (Fig. 3B6), and Ti/B (Fig. 3B7). Subsequent MALDI-MS confirmed that the spot corresponded to crybb2. Crybb3 (framed by a circle) was present equally in all groups (Fig. 3B1 7), demonstrating the reproducibility of the applied method.Confirmation with Western blotting and immunohistochemistryAdditional WB was performed for crybb2, crybb3, crybL, crybH, cryc, crym, HSP-25, and HSP-70 on retinal samples to better characterize the changes in crystallin and HSP expression as seen through 2DE and MALDI-MS. Crybb2, with a molecular mass of 23 kDa, was markedly up-regulated in the hypertensive retina (7.461.1-fold; p,0.001), being only marginally present in the normotensive retina (1.3060.12-fold), and nearly totally absent in retinal samples treated with Ti (35.0060.07-fold), Ti/ Tr (20.060.1-fold), Ti/D (60.060.4-fold), and Ti/B (61.060.1fold; Fig. 4A, B). Crybb3 expression did not differ significantly among the groups (Fig. 4C, D). Consistent with crybb2, crybL was strongly expressed in hypertensive samples (7.261.8-fold; p,0.001), while it was only slightly expressed in all other groups (Fig. 4E, F). There were no marked changes in the expressions of either crybH (Fig. 4G, H) or HSP-70 (Fig. 5A, B). Crym (Fig. 5C, D) and HSP-25 (Fig. 5E, F) were the most strongly expressed in the normotensive samples (p,0.001). Cryc expression resembled the expressions of crybb2 and crybL, showing markedly higher expression in hypertensive samples (10.8060.35-fold; p,0.001) than in the other groups (Fig. 5G, H). To confirm and 1676428 visualize crybb up-regulation within the retinal tissue, immunohistochemical staining was performed on normotensive and hypertensive retinal slices. IHC revealed up-regulation of crybb after IOP elevation relative 24272870 to normotensive samples (Fig. 6A, B). The signal increased with the duration of exposure to elevated IOP (Fig. 6C). Crybb signaling was higher at 28 days than at 7 days after IOP elevation, and crybb expression was localized predominantly in the RGC layer of retinal slices (Fig. 6B, C), indicating that RGCs are mainly adversely affected by elevated IOP in glaucoma.Figure 3. Two-dimensional gelelectrophoresis of retinal proteins. A Peptide mapping of a retina obtained from a Sprague-Dawley rat with untreated, sustained elevated IOP 8 weeks after IOP induction. Hypertensive retinal samples showed a marked increase in bb2crystallin (crybb2) expression (area marked by a black box in A, shown at higher magnification in B1 compared to a normotensive shamtreated retina, B2). The 4 weeks of IOP-lowering treatment with topical treatment with Ti (B3,B4), Ti/T (B5), Ti/D (B6), and Ti/B (B7) decreased crybb2 expression to baseline levels. The proteins identified, which are marked by an arrow in A and B1, are listed in Table 1 according to the number given. doi:10.1371/journal.pone.0049730.gThese recorded readings remained constant over the 4 weeks of antihypertensive treatment. IOP recordings are illustrated in detail in Fig. 1.Quantification of retinal ganglion cellsThe density of RGCs in retin.
