Activation of Apoptosis in a βB1-CTGF Transgenic Mouse Model

Abstract

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5-9/group) and quantitative real-time PCR analysis (n = 5-7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3⁺ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL⁺) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma.

Keywords: Bax/Bcl2; apoptosis; caspase 3; neurofilament H; primary open-angle glaucoma; synapse; βB1-CTGF.

Figure 1

Loss of neurofilament H. (A) Retinae were stained with antibodies against Brn-3a (green) and neurofilament H (red) at 5 and 10 weeks (n = 5–8/group). Cell nuclei were visualized with 4′,6 diamidino-2-phenylindole (DAPI) (blue). (B) The number of Brn3a⁺ cells remained unaltered in βB1-CTGF retinae in comparison to wildtype (WT) at 5 and 10 weeks of age in the total as well as in the central and peripheral retina. (C) RT-qPCR analyses were performed regarding the mRNA expression levels of the retinal ganglion cell (RGC) marker Pou4f1 at both ages (n = 5–7/group). In accordance with the immunohistological results, no alterations in Pou4f1 expression levels were noted in βB1-CTGF animals at the age of 5 and 10 weeks compared to in WT. (D) RT-qPCR analyses of Tubb3 were performed in 5- and 10-week-old βB1-CTGF and WT mice (n = 5/group). At 5 weeks, no changes were seen in Tubb3 mRNA expression levels. In contrast, a significant downregulation of Tubb3 mRNA was revealed in transgenic mice at 10 weeks of age (p = 0.09). (E) At 5 weeks, the staining area of neurofilament H remained unchanged in transgenic and WT animals. In contrast, a significant decrease in the neurofilament H⁺ area was detected in βB1-CTGF mice (p = 0.01). (F) The mRNA levels of Nefh (n = 5/group) were not altered in 5-week-old transgenic mice. However, a significant downregulation of Nefh mRNA levels was observed in βB1-CTGF mice at 10 weeks (p = 0.02). Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer. Values are mean ± SEM for immunohistology and median ± quartile + maximum/minimum for RT-qPCR. The dotted lines in C, D, and F represent the relative expression levels of the WT group. Scale bar: 20 μm. * p < 0.05, ** p < 0.01.

Figure 2

Increased apoptosis rate in retinal ganglion cells. (A) Retinae were stained with the apoptosis marker cleaved caspase 3 (red) in combination with Brn3a (green) at 5 and 10 weeks (n = 7–8/group). Furthermore, apoptotic cells were visualized using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining (n = 5/group). Cell nuclei were visualized with DAPI (blue). (B) Significantly more cleaved caspase 3⁺ RGCs were revealed in βB1-CTGF mice compared to in WT animals at 5 (p = 0.005) and 10 weeks (p = 0.02). (C) The mRNA expression levels of Casp3 were upregulated at 5 (p = 0.02) and 10 weeks (p = 0.03). (D) At 5 weeks, the mRNA expression levels of Bax/Bcl2 were not altered in βB1-CTGF mice. In contrast, a significant upregulation of Bax/Bcl2 levels was noted in 10-week-old transgenic animals (p = 0.04). (E) A higher number of TUNEL⁺ apoptotic cells in the ganglion cell layer was reveled in 5- (p = 0.03) and 10-week-old βB1-CTGF mice compared to in WT (p = 0.02). Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer. Values are mean ± SEM for immunohistology and median ± quartile + maximum/minimum for RT-qPCR. The dotted lines in C and D represent the relative expression levels of the WT group. Scale bar: 20 μm. * p < 0.05, ** p < 0.01.

Figure 3

Loss of synapses. (A) GABAergic synapses were labeled using gephyrin (green) and glutamatergic synapses were stained with Vglut1 (red), while cell nuclei were counterstained with DAPI (blue) at 5 and 10 weeks of age (n = 7–8/group). (B) At 5 weeks of age, a significantly decreased gephyrin⁺ intensity was revealed in βB1-CTGF mice (p = 0.02). At 10 weeks, the staining intensity reverted to the WT level. (C) The mRNA expression levels of Gphn (n = 5–7/group) showed no changes at 5 weeks, but a significant downregulation was shown in 10-week-old βB1-CTGF mice (p = 0.02). (D) Regarding Vglut1 staining, significantly decreased immunoreactivity was noted in transgenic mice at 5 (p = 0.007) and 10 weeks of age (p = 0.01). (E) RT-qPCR analyses detected no alterations in Slc17a7 (Vglut1) mRNA expression levels at both points in time (n = 5–7/group). (F) Exemplary, retinal cross sections were labeled with antibodies against ribeye (ribbon synapses; green) and PKCα (rod bipolar cells; red). DAPI counterstained cell nuclei (blue). At 5 and 10 weeks, βB1-CTGF and WT mice displayed distinct co-staining of both markers in the inner plexiform and outer plexiform layer. In the WT animals at both ages, the co-staining was more intense compared to transgenic mice. Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. Values are mean ± SEM for immunohistology and median ± quartile + maximum/minimum for RT-qPCR. The dotted lines in C and E represent the relative expression levels of the WT group. Scale bars: 20 μm. * p < 0.05, ** p < 0.01.

Figure 4

No alterations in the number of bipolar cells. (A) Cone bipolar cells were evaluated by using recoverin (red) and rod bipolar cells using PKCα staining (green; n = 7–9/group). Cell nuclei were labeled with DAPI (blue). (B) The analysis of the recoverin⁺ area revealed no significant difference in staining area in βB1-CTGF animals at 5 weeks. At the age of 10 weeks, there was a trend towards smaller cone, bipolar cell areas in βB1-CTGF mice compared to in WT mice (p = 0.052). (C) The Rcvrn mRNA expression levels were comparable between βB1-CTGF and WT animals at both ages (n = 5–7/group). (D) PKCα⁺ cell numbers were not altered in βB1-CTGF retinae compared to in WT at 5 and 10 weeks. (E) RT-qPCR analyses of Prkca mRNA expression levels showed no alterations at 5 and 10 weeks of age. (F) Additionally, the mRNA levels of Slc18a3 (encoding for the vesicular acetylcholine transporter) were not altered in 5- and 10-week-old βB1-CTGF animals. Abbreviations: INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. Values are mean ± SEM for immunohistology and median ± quartile + maximum/minimum for RT-qPCR. The dotted lines in C, E, and F represent the relative expression levels of the WT group. Scale bar: 20 µm.

Figure 5

Photoreceptors remain unaffected. (A) Rhodopsin (rods, green) and opsin (L-cones, red) staining of retinal sections were performed to evaluate photoreceptors (n = 7–9/group). DAPI was added to visualize cell nuclei (blue). (B) Comparable rhodopsin⁺ areas were detected in both groups at 5 and 10 weeks of age. (C) The RT-qPCR results showed no alteration in Rho mRNA expression levels in βB1-CTGF mice at the age of 5 and 10 weeks (n = 5–7/group). (D) The number of opsin⁺ cells remained unaltered in βB1-CTGF animals compared to in WT at both ages. Abbreviations: ONL = outer nuclear layer, OS = outer segment. Values are mean ± SEM for immunohistology and median ± quartile + maximum/minimum for RT-qPCR. The dotted line in C represents the relative expression level of the WT group. Scale bar: 20 µm.

Figure 6

No macroglia reaction. (A) Macroglia were analyzed using GFAP (red) and glutamine synthetase (green) at 5 and 10 weeks (n = 7–9/group). DAPI (blue) visualized cell nuclei. (B) The GFAP area analysis showed a similar GFAP signal area in βB1-CTGF retinae and WT retinae at 5 and 10 weeks. (C) Regarding glutamine synthetase, we observed no difference in the staining area in βB1-CTGF mice compared to in WT at both ages. Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer. Values are mean ± SEM. Scale bar: 20 µm.