J Korean Med Sci.  2017 Feb;32(2):343-351. 10.3346/jkms.2017.32.2.343.

Nestin Expression in the Adult Mouse Retina with Pharmaceutically Induced Retinal Degeneration

• Moon CH
• Cho H
• Kim YK
• Park TK

• KMID: 2364181
• DOI: http://doi.org/10.3346/jkms.2017.32.2.343

Abstract

The present study investigated the temporal pattern and cellular localization of nestin in the adult mouse retina with pharmaceutically induced retinal degeneration using N-methyl-N-nitrosourea (MNU). After a single intraperitoneal injection of MNU in 8-week-old C57BL/6 mice, the animals were sacrificed at 1, 3, 5, 7, and 21 days (n = 6, in each stage). The eyes were examined by means of immunohistochemical tests using nestin, ionized calcium-binding adaptor molecule (Iba-1), CD11b, F4/80, and glial fibrillary acidic protein (GFAP). Western blot analysis and manual cell counting were performed for quantification. Nestin expression was increased after MNU administration. Nestin+/Iba-1+ cells were migrated into outer nuclear layer (ONL) and peaked at day 3 post injection (PI). Nestin+/CD11b+ cells were also mainly identified in ONL at day 3 PI and peaked at day 5. Nestin+/F4/80+ cells were shown in the subretinal space and peaked at day 3 PI. Nestin+/GFAP+ cells were distinctly increased at day 1 PI and peaked at day 5 PI. The up-regulation of nestin expression after MNU administration in adult mouse retinal microglia, and monocyte/macrophage suggests that when retinal degeneration progresses, these cells may revert to a more developmentally immature state. Müller cells also showed reactive gliosis and differentiational changes.

Keyword

Nestin; N-methyl-N-nitrosourea (MNU); Retinal Degeneration; Mouse

MeSH Terms

Adult*
Animals
Blotting, Western
Cell Count
Glial Fibrillary Acidic Protein
Gliosis
Humans
Injections, Intraperitoneal
Methylnitrosourea
Mice*
Microglia
Nestin*
Retina*
Retinal Degeneration*
Retinaldehyde*
Up-Regulation
Glial Fibrillary Acidic Protein
Methylnitrosourea
Retinaldehyde

Figure

• 

  Fig. 1 Nestin and Iba-1 expression in control and degenerated adult mouse retina. Immunofluorescent labeling with nestin (green), Iba-1(red), and DAPI (blue) is shown. DAPI was used for nuclei staining to visualize the retinal layers. The decrease in thickness of the ONL due to selective loss of photoreceptor cells was becoming obvious by day 3 after MNU injection. Progressive loss of photoreceptor cells ultimately led to a complete loss of the ONL at day 7 PI. Immunoreactivity of nestin was increased at day 1 PI, peaked at day 3, and declined gradually until day 21. MNU treatment resulted in migrating nestin+/Iba-1+ cells into the ONL. The number of nestin+/Iba-1+ cells peaked at day 3 PI. After that, the number of nestin+/Iba-1+ cells diminished progressively, particularly at day 7, and the cells were sparsely distributed by day 21. Arrows indicate nestin/Iba-1 co-immunolabeling. Iba-1 = ionized calcium-binding adaptor molecule, DAPI = 4-6-diamino-2-phenylindole, ONL = outer nuclear layer, MNU = N-methyl-N-nitrosourea, PI = post injection, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer.

• 

  Fig. 2 Nestin and CD11bexpression in control and degenerated adult mouse retina. Immunofluorescent labeling with nestin (green), CD11b (red), and DAPI (blue) is shown. Arrows indicate nestin/CD11b co-immunolabeling. After a single injection of MNU, nestin+/CD11b+ cells migrated into the ONL at day 3 and showed a peak at day 5. The number of nestin+/CD11b+ cells diminished progressively, particularly after 7 days, with sparse distribution by day 21. DAPI = 4-6-diamino-2-phenylindole, ONL = outer nuclear layer, MNU = N-methyl-N-nitrosourea, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer.

• 

  Fig. 3 Nestin and F4/80 expression in control and degenerated adult mouse retina. Immunofluorescent labeling with nestin (green), F4/80 (red), and DAPI (blue) is shown. Arrows indicate nestin and F4/80 co-immunolabeling. The number of amoeboid nestin+/F4/80+ cells increased markedly from day 3 to day 5 post injection and decreased thereafter. DAPI = 4-6-diamino-2-phenylindole, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer.

• 

  Fig. 4 Nestin and GFAP expression in control and degenerated adult mouse retina. Immunofluorescent labeling with nestin (green), GFAP (red), and DAPI (blue) is shown. Arrows indicate nestin/GFAP co-immunolabeling. The number of nestin+/GFAP+ Müller cells increased progressively at day 3 after MNU treatment, peaked at day 5, and decreased thereafter. GFAP-positive cells remained in an activated state at day 7 and day 21 post injection, despite the diminution in retinal thickness. GFAP = glial fibrillary acidic protein, DAPI = 4-6-diamino-2-phenylindole, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer.

• 

  Fig. 5 Western blot analyses of nestin and GFAP expression. (A) Significantly increased immune-reactivity of nestin at day 1 and significantly decreased to mostly the level of control group at 21 days after MNU injection. (B) Immune-reactivity of GFAP showed gradually increasing for 3 days after MNU injection, and declined at 21 days. Nevertheless, GFAP remained in activated state compared to control group even after 21 days after MNU injection. GFAP = glial fibrillary acidic protein, MNU = N-methyl-N-nitrosourea.

• 

  Fig. 6 Cell counts of Iba-1+, CD11b+, F4/80+, and nestin+ cells. (A) Iba-1+, CD11b+, F4/80+ cells were peaked at 5 days after injection. (B) Nestin/Iba-1 co-immunolabelling cells and Nestin/F4/80 co-immunolabelling cells were peaked at 3 days after injection. Nestin/CD11b co-immunolabelling cells were peaked at 5 days after injection. Iba-1 = ionized calcium-binding adaptor molecule, MNU = N-methyl-N-nitrosourea.

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