Molecular evidence of senescence in corneal endothelial cells of senescence-accelerated mice

Abstract

Purpose: To investigate senescent evidence in corneal endothelial cells (CECs) of the senescence-accelerated mouse (SAM), which is considered a suitable animal model for the further study of the senescent mechanism in CECs.

Methods: Thirty-six male mice from a senescence resistant mouse strain (SAM R1) and a senescence-prone strain (SAM P8) at various ages (1, 6, and 12 months) were analyzed in this study. The endothelial cell density (ECD) and cell viability were detected using trypan blue and alizarin red dyes while the senescent cells were observed by senescence-associated beta-galactosidase (SA-beta-Gal; pH 6.0) staining. In addition, ultrastructure was observed using an electron microscope. The senescence-related genes (p16(INK4a), p19(ARF), p21(WAF1/CIP1), and p53) in the CECs were visualized via immunohistochemistry and were quantitatively detected using real-time polymerase chain reaction (PCR). Signal proteins of phospho-extracellular signal-regulated kinase 1/2 (p-ERK 1/2) were detected by western blot analysis.

Results: Our results indicated that the ECD values decreased with increasing age in both the SAM-R1 and SAM P8 series where the values in the older SAM p8 series decreased even lower than in the older SAM R1 series. The mean decreased rate was 2.276% per month in the SAM R1 and 2.755% per month in the SAM P8 series. In addition, changes in the senescence-like ultrastructure were observed in the CECs of both strains, and the increase in the positive staining of SA-beta-Gal was observed in both strains as well. It is worth noting that such changes were more significant in the SAM P8 strain. Immunohistochemical detection assays indicated the expression of p-ERK 1/2, p16(INK4a), p19(ARF), p21(WAF1/CIP1), and p53 (nuclear localization for each) in each age group analyzed. Furthermore, the results of real-time PCR studies showed an increase in the expression of p16(INK4a) mRNA as a function of age in the SAM R1 strain and in the early senescence stage of the SAM P8 strain in addition to an increase in the expression of p21(WAF1/CIP1) and p53 mRNA as a function of age in the SAM P8 strain (no significant increase was observed in the SAM R1 strain). Additional results from western blot analysis demonstrated an age-related increase in the quantity of the p-ERK 1/2 proteins in both strains.

Conclusions: The SAM R1 and SAM P8 strains represent suitable models for the study of CEC senescence in vivo. In addition, the progression of cellular senescence in CECs occurs more quickly in the SAM P8 strain as opposed to the SAM R1 strain. Our results also indicate that the p16(INK4a) signaling pathway may play a key role in the early stages of senescence in CECs while the p53/p21(WAF1/CIP1) signaling pathway may exert its principle effect in the late stages of senescence in CECs. Further study is still required about the role of the mitogen-activated protein kinase (MAPK) signaling cascade in the process of senescence in CECs.

Figure 1

Corneal endothelium of the SAM strains stained with alizarin red and trypan blue for detection of the cell morphology, density and viability. A: The endothelium of a one-month-old specimen from the SAM R1 strain is shown. Most of the cells are regular hexagon. Magnification: 400X. B: The endothelium of a six-month-old specimen from the SAM P8 strain shows a significant number of polygonal cells. Magnification: 400X. C: The endothelium of a 12-month-old specimen from the SAM P8 strain is displayed. The arrows indicate the hyperplasia of the tunica vasculosa on the endothelial surface layer. The covering of the tunica vasculosa causes most of the cells displayed to look unclear. The blue lines (black arrow) indicate the hyperplastic blood vessel. Magnification: 100X.

Figure 2

Changes in the endothelial cell density values at various ages for each experimental group. The results indicate a significant decrease in the ECD values of the SAM R1 strain at six months (p=0.010 versus one-month-old specimen) and at 12 months (p=0.000 versus both one- and six–month-old specimens). There is also a significant decrease in the ECD values of the SAM P8 specimens at six months (p=0.000 versus one-month-old specimen) and at 12 months (p=0.000 versus both one- and six-month-old specimens). In addition, there is no significant difference in the ECD values at one month between the SAM R1 and the SAM P8 strains (p=0.293), but there is a significant decrease in the ECD values of both six- and 12–month-old specimens between the SAM R1 and the SAM P8 strains (p=0.010 and p=0.042, respectively). The results indicate the decrease of ECD in SAM P8 is faster than in SAM R1. An asterisks mark a statistically significant difference (p<0.05).

Figure 3

Analysis of transmission electron microscope imaging. The cornea from one-month-old mice of the SAM R1 and the SAM P8 experimental groups are shown (A and B, respectively). The thickness of the endothelium was greater than or equal to the thickness of the Descemet’s membrane. The microvilli were abundant (locations indicated by arrows). C: Analysis of six-month-old specimens from the SAM R1 experimental group shows that the thickness of endothelium was also less than or equal to the thickness of the descemet’s membrane. D: Analysis of six-month-old specimens from the SAM P8 experimental group shows that the thickness of the endothelium decreased significantly and that many of the interstices were located in the interlamination. E: Analysis of 12-month-old specimens from the SAM R1 experimental group showed that the thickness of the endothelium was uneven and that many of the interstices could be observed. F: Analysis of the 12-month-old specimens from the SAM P8 experimental group showed that the thickness of endothelium was very thin. Scale bars: 1 μm. En: endothelium; De: Descemet’s membrane; St: stroma.

Figure 4

Analysis of scanning electron microscope imaging. Analysis of one-month-old specimens from the SAM R1 and the SAM P8 experimental groups (A and B, respectively) displayed the morphology of endothelial cells, which was hexagonal and uniform, with numerous microvilli located on the surface. Analysis of six-month-old specimens from the SAM R1 and the SAM P8 experimental groups (C and D, respectively) showed that the endothelial cells became larger and pantomorphic. Analysis of 12-month-old specimens from the SAM R1 and the SAM P8 experimental groups (E and F, respectively) showed more polygonal cells, and these cells also became larger. Scale bars: 10 μm.

Figure 5

Staining of senescence-associated β-galactosidase activity in corneal endothelial cells of the SAM R1 and the SAM P8 strains at various ages. The positive stainings are shown in blue. The staining of the SAM R1 strain at one month, six months, and 12 months are shown (A, C, and E, respectively). The staining of the SAM P8 strain at one month, six months, and 12 months are also displayed (B, D,and F, respectively). There were few to no positive stainings for SA-β-Gal activity in panels A and B while there were more multifocal and intense positive stainings in panels C, D, E, and F. Magnification: 400X.

Figure 6

Average percentage of SA-β-Gal-positive cells at each grade in various experimental groups. The classification standard of SA-β-Gal activity is according to Tatsuya et al. [49]. This graph shows that few to no positive SA-β-Gal staining was observed in the endothelium in corneas from younger mice. In corneas from older mice, the percentage of CECs staining positive for SA-β-Gal was higher in the P8 strain than in the R1 strain.Statistical analysis is shown in Table 6.

Figure 7

Immunohistochemical staining of p-ERK 1/2, p16^INK4a, p19^ARF, p21^WAF1/CIP1, and p53 in corneal endothelial cells. The arrows indicate the positive staining in the endothelial nuclei at various ages of each experimental group A: p-ERK 1/2, SAM R1 strain at one month; B: p16^INK4a, SAM P8 strain at one month; C: p19^ARF, SAM P8 strain at 12 months; D: p21^WAF1/CIP1, SAM P8 strain at six months; E: p53, SAM R1 strain at 12 months. F: The negative control incubated with PBS instead of a primary antibody, SAM P8 strain at six months. Magnification: 400X.

Figure 8

Quantitative real-time polymerase chain reaction analyses of mRNA expression of senescence-related genes after normalization to GAPDH in the corneal endothelial cells of SAM R1 and P8 strains at various ages. A: Quantitative comparison of the fold difference in the expression of p16^INK4a mRNA is shown at various ages in each experimental group. The results indicate that the expression of p16^INK4a was significantly increased from six months old to 12 months old in the SAM R1 strain experimental group (p=0.027 and p=0.000, respectively). In the SAM P8 strain experimental group, a significant increase was observed at six months (p=0.000 when compared to the one-month-old control group) and a significant decrease was observed at 12 months (p=0.000 when compared to the value at six months), although comparison between the 12-month- and 1-month-old values still resulted in a statistically significant increase (p=0.008). B: Quantitative comparison of the fold difference in the expression of p19^ARF mRNA is shown at various ages. The results indicate that the expression of p19^ARF mRNA decreased significantly at six months in the SAM R1 strain experimental group (p=0.000). C: Quantitative comparison of the fold difference in the expression of p21^WAF1/CIP1 mRNA is shown. The results indicated that the expression of p21^WAF1/CIP1 was significantly higher at 12 months in the SAM P8 strain experimental group (p=0.007). D: Quantitative comparison of the fold difference in the expression of p53 mRNA is shown. The results indicate that the expression of p53 decreased significantly at six months of age in the SAM R1 strain experimental group, although no significant difference was observed at 12 months of age (when compared to the value at one month; p=0.000, p=0.927, respectively). However, the expression of p53 increased significantly at 12 months of age in the SAM P8 strain experimental group (p=0.000). There were three individual corneas used in each experimental group, and each cDNA sample had been detected three times. An asterisk marks a statistically significant difference (p<0.05).

Figure 9

Western blot analyses of the expression levels of the p-ERK 1/2 protein in the corneal endothelial cells at various ages of the SAM R1 and SAM P8 strains. A: The expressions of p-Erk1/2 and Erk1/2 proteins in 1-month-, 6-month-, and 12-month-old specimens of the SAM-R1 and the SAM P8 strains are displayed. B: Quantitative comparison of the fold changes in the expression of p-Erk1/2 after normalization to the Erk1/2 protein is shown at various ages in each experimental group. The results show that p-ERK1/2 significantly increased at 12 months of age in both the R1 and P8 strains, which indicate that the activation level of Erk1/2 protein is much higher in the late senescent CECs. There were three individual corneas used in each experimental group, and each protein sample had been detected three times. An asterisk marks a statistically significant difference (p<0.05).