Changes in Intraocular pressure in Glaucoma treated vs treated eyes
Intraocular pressure showed an increase in the postoperative period with a spike at 1 hour postoperative (60 mmHg) that reduced in the first day. Posteriorly, a stabilization was found between the groups and the values were similar between the glaucoma model groups in the first week; average values ranked in the glaucoma group 21.8±0.6 mmHg, glaucoma-genipin group 25.5±0.5 mmHg and glaucoma-BAPN group 22.5±0.7 mmHg. Then, there was a reduction of IOP in the glaucoma-BPNA group and an increase in the glaucoma-genipin group. In the second week, there was an increase in the glaucoma-genipin group vs. glaucoma and there was a reduction of IOP in the glaucoma-BAPN group. However, those values were not significantly different in the ANOVA analysis. This difference between groups (reduction in glaucoma-BAPN and increase in glaucoma-genipin) remained significantly elevated in weeks 2, 3 and 4; the IOP values at this point were 19.8±1.1 mmHg in group 1 (glaucoma), 22.8±0.58 mmHg in group 2 (glaucoma+genipin), 16.1±0.4 mmHg in group 3 (glaucoma+BAPN), 8.8±0.7 mmHg in group 4 (genipin), 8.3±0.2 mmHg in group 5 (BAPN) and 8.5±0.43 mmHg in untreated eyes. A significant difference was found between glaucoma groups (ANOVA on Ranks p value 0.05) (Figure 1, Table 2).
Changes in the optic nerve
The longitudinal optic nerve sections showed a disorganized structure of the optic nerve in the glaucoma (Figure 2A) and glaucoma-genipin treated eyes (Figure 2B) vs glaucoma-BAPN (Figure 2C); in the glaucoma stiffened eyes, the destruction of the structures was evident, as was the loss of bundles in the optic nerve in comparison with the control (Figure 2D). These bundles were well preserved in the control (Figure 2D) and severely altered in the glaucoma (Figure 2A) and glaucoma-genipin groups (Figure 2B) with clearance of the cytoplasm, degenerative changes and microglial cells (asterisks). A Schnabel cavernous optic atrophy like, is observed and characterized with the loss of myelin and axons and preservation of septa with a spongiform appearance and was evident in the glaucoma and glaucoma-genipin groups. Glaucoma-BAPN showed organization of optic nerve bundles with minimal microglial cells (Figure 2C).
Changes in retina and nerve fiber evaluation
At week 4, retinal cell layers were evaluated using the retina cross sections stained with H&E and a comparison between all groups. The normal retina was observed in the control group (Figure 3d), and thinning of the retina was observed in glaucoma (Figure 3a) along with alterations in ONL, OPL, IPL and a reduction in RGC with a loss of inner nuclear cells layer and gliosis-like shape; a loss of the retinal structure was observed in the glaucoma-genipin group (Figure 3b) while a complete evaluation of all layers is observed in the glaucoma-BAPN (Figure 3c), genipin (Figure 3e) and BAPN (Figure 3f).
Bielschowsky's silver staining shows preservation of the inter-neuronal unions and axonal staining in the normal retina (Figure 4D), genipin alone (Figure 4E) and BAPN alone (Figure 4F), as well as in the glaucoma-BAPN group (Figure 4C); in the glaucoma group (Figure 4A) and glaucoma-genipin group (Figure 4B), severe fiber distortion and a loss of the axonal union was observed in the central area of the retina. Staining with GFAP antibody in the glaucoma-genipin (Figure 5B) and glaucoma (Figure 5A) groups was evident in the nerve fiber layer (NFL), ganglion cell layer (GCL), and projections to inner plexiform layer (IPL), while there was minimal staining in the NFL in the control group (Figure 5D) and glaucoma-BAPN group (Figure 5C).
Changes in Gene Expression and effects of BAPN and genipin in glaucoma eyes
Gene expression of C3 complement was up-regulated in glaucoma +6.1±0.4 in comparison with the control group; a significative down-regulation observed in the BAPN/glaucoma group and in the genipin/glaucoma group. No differences were noted in the BAPN and genipin group (Figure 6A).
The GFAP gene was up-regulated in glaucoma by +1.4±0.02 in comparison to the control group, as well as in BAPN (+1.3±0.9) and was down-regulated in the glaucoma-genipin group (-19±0.9), BAPN/glaucoma group (-33±2.0) and in the genipin group (-16±1.4) (Figure 6B).
Cpg15 gene was down regulated in glaucoma by -1.8±0.7 in comparison with the control group, as well as in the BAPN-glaucoma group by -20±2.0 and in the genipin-glaucoma group by -14±0.9; in the genipin group, this was reduced by -2.7±1.4 and was increased in the BAPN group by +3.7±1.0 (Figure 6C).
Ywhab gene was up-regulated in the glaucoma group by 1.8±0.27 and down-regulated in the glaucoma-genipin and glaucoma-BAPN group, as well as severely up-regulated in the genipin group (77±1.42) (Figure 7A).
The Ywhaz gene was down-regulated in glaucoma by -5.3±0.4 in comparison to the control group, and was also down-regulated in the BAPN/glaucoma group -21±2.1; in BAPN alone, a significant reduction was also observed by -30±1.2, while an increase in expression was observed in the genipin/glaucoma group by +3.7±0.95 and in the genipin group by +10.3±1.4 (Figure 7B).