53.2.4 Histological Examination and Immunohistochemistry

Hematoxylin-eosin (H-E) staining, BrdU incorporation and immunohistochemistry for nestin was performed.

53.3 Results

53.3.1 Fundus Examination and Histology of the Retina

Fundus examination showed an enlargement of the size of the optic nerve head and the size of the retinal vessels around the optic disc (Fig. 53.1a and b). Histological examination showed thicker retina in p27(–/–) (Fig. 53.1c) mice when compared to those of p27(–/+) (not shown) or wild type (Fig. 53.1d) mice. The thicker layers were observed in almost all layers in the retina.

Fig. 53.1 Fundus photograph and histological examination. Fundus photograph ((a) and (b)) and histology ((c) and (d)) show enlargement of optic nerve head and thicker retina in p27(–/–) mice. Bars indicated 100 μm

53.3.2 ERG

When we performed ERG, statistically significant decreases of a and b waves amplitudes were observed in p27(–/–) mice when compared to those of wild mice (Table 53.1). These findings were clearer in 12 month-old mice than in 3 month-old mice.

53.3.3 BrdU Incorporation

MNU injection showed retinal degeneration similar to that reported by others (Fig. 53.2a and b) (Nambu et al. 1997). We found that the degree of retinal degeneration seemed almost the same between p27(–/–) and wild type mice. When we examined BrdU incorporation, BrdU positive cells were observed at the ONL (Fig. 53.2c and d). Although we did not perform quantitative analysis, there seemed to be no difference between wild and p27(–/–) mice.

Fig. 53.2 BrdU incorporation and immunohistochemistry of nestin 3 M after MNU injection BrdU positive cells were observed at the ONL ((a): wild mice, (b) p27(–/–)). Nestin immunoreactivity was observed at inner retinal layer and outer plexiform layer. ((c) wild mice, (d) p27(–/–) mice). (e) and (f) show H-E finding, respectively

53.3.4 Immunohistology of Nestin

Although we did not perform quantification, nestin-like immunoreactivity seemed to show no differences between p27(–/–) and wild type mice (Fig. 53.2e and f).

53.4 Discussion

Acute damage stimulates Müller glia to re-enter the cell cycle, and induce expression of CASH-1, Pax6 and Chx10, transcription factors expressed by embryonic retinal progenitors. Fischer and coworker reported that, in response to damage, Müller glia was a potential source of neural regeneration (Fischer et al. 2001). Cdk inhibitors have been reported to stop cell-cycle activity during development and in maintaining cells in a terminally differentiated state, demonstrating a cell-selective expression pattern in various organs (Nakayama et al. 1996). When we performed histological examination, we found thick retina and optic nerves in p27kip1 (–/–) mice. These results may demonstrate the failure of the retina to differentiate. Interestingly, mice with thick retina did not show a normal response by ERG examination, even though each cell in the retina seemed to be normal. The results may relate to other reports that mice homozygote for p27 mutation had severe hearing loss and their organ of Corti exhibited an increase in the number of inner and outer hair cells (Kanzaki et al. 2006). These results may show that adequate cell differentiation and normal cell number is important for normal retinal function.

When we generated retinal degeneration by MNU injection, as was reported previously (Nambu et al. 1997), although we found retinal degeneration, we could not find any differences between p27(–/–) and wild mice. When we examined the BrdU incorporation and nestin expression in the retina, we also could not find any differences between p27(–/–) and control wild mice. These results may show that under our experimental condition, the suppression of cell cycle molecules p27kip1 on its own may not induce retinal regeneration.

In conclusion, the CDKs inhibitor, p27(–/–) itself may induce retinal abnormalities, both histologically and electrophysiologically. However, p27(–/–) itself did not induce retinal regeneration in our experimental condition.

References

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