C57BL/6 female mouse of 8-week-old obtained from Charles River Laboratories (Bundang, Korea). All the animals were housed in standard cages under a 12-hour light-dark cycle, with food and drinking water available ad libitum. All animals and experiment procedures were kept in accordance with the Association of Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The study protocol was reviewed and approved by the institutional animal care and use committee of the Soonchunhyang University Bucheon Hospital (No. SCHBC-animal-2013-12).

The MNU (Sigma-Aldrich, St. Louis, MO, USA) was kept at −20°C in the dark. Prior to its use, MNU powder was dissolved in physiologic saline, and a single intraperitoneal injection of MNU (60 mg/kg of bodyweight) was given to induce retinal degeneration in each of 15 mice to be used for immunohistochemical testing (n = 3 in each of the five stages [at days 1, 3, 5, 7, and 21]) and in each of the 15 mice to be used for western blot analysis (n = 3 in each of the five stages). The animals were sacrificed at 1, 3, 5, 7, and 21 days after the injection. Six additional age-matched, untreated control animals (3 mice for the immunohistochemical testing and 3 mice for the western blot analysis) were kept under the same conditions and were examined using the same methods and procedures that were used for the MNU-treated animals.

For cryosectioning, the eyes were enucleated and the anterior segments including the cornea, iris, and lens were removed. The posterior eye cups were fixed in 4% paraformaldehyde (PFA) for 1 hour at 4°C and then immersed in 30% sucrose overnight at 4°C, and embedded and frozen in frozen section compound (Leica Biosystems, Richmond, IL, USA). Completely frozen molds were stored at −70°C until used. Sagittally oriented, 8 µm sections were prepared and only the central sections, which included the optic nerve head were used for measurement purposes. All slides were air-dried for 1 day.

To identify the cellular responses in the retina after MNU-induced retinal degeneration, we obtained primary antibodies against nestin (mouse monoclonal, 1:500; Millipore, Billerica, MA, USA), Iba-1 (a marker for microglia; rabbit polyclonal, 1:1,000; Wako Pure Chemicals, Osaka, Japan) (15), CD11b (a marker for monocyte/macrophage; rat monoclonal, 1:1,000; AbD Serotec, Oxford, UK) (16), F4/80 (a marker for monocyte/macrophage; rat monoclonal, 1:2,000; AbD Serotec) (17), and GFAP (a marker for Müller cells; rabbit polyclonal, 1:500; Millipore) (18). Secondary antibodies included Alexa Fluor-488 conjugated goat anti-mouse for nestin; Alexa Fluor-568 anti-rat for CD11b and F4/80; and Alexa Fluor-568 anti-rabbit for GFAP and Iba-1(1:2,000; Molecular Probes, Eugene, OR, USA). For immunofluorescence staining, the cryosections were permeabilized with 0.1% Triton-X100 (Sigma-Aldrich) in 5% goat serum diluted in phosphate buffered saline (PBS; pH 7.4) for 1 hour, followed by overnight incubation with primary antibodies in 5% goat serum at 4°C. The slides were then washed three times for 5 minutes in PBS, and were further incubated with the appropriate secondary antibodies diluted to 1:2,000 in 5% goat serum. Finally, nuclei were counterstained with 4-6-diamino-2-phenylindole (DAPI) and the sections were mounted using fluorescent mounting medium (Dako, Glostrup, Denmark). Additionally, the cellular markers Iba-1, CD11b, and F4/80 have a cross reaction of staining monocytes, microglia, and macrophages, these cells likely represent overlapping spectra of cells of the same lineage. Therefore, we defined microglia as ramified or amoeboid cells in the neural retina and along the retinal vessels, monocytes as round cells in the vessels or still attached to the vessel walls, and macrophages as ramified or amoeboid cells in the nonneural tissue (17).

After the careful dissection of the retinas from the eye cups, the retinal tissue was flash frozen in −80°C liquid nitrogen until samples at all time points were collected. A 100 µL ice-cold extraction buffer containing 10 mM of Tris-HCl (pH = 7.5), 5 mM of ethylenediaminetetraacetic acid (EDTA), 200 mM of NaCl, 1 mM of phenylmethylsulfonyl fluoride, 1 µg/mL of leupeptin, and 28 µg/mL of aprotinin was added to both retinal tissues from one mouse. The retinal samples were sonicated and centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was collected, and protein concentrations were measured according to the manufacturer's instructions (BCA Protein Assay Kit; Thermo Scientific, Rockford, IL, USA). Proteins (30 µg of each sample) were fractionated on 8% polyacrylamide precast gels (Bio-Rad, Cambridge, MA, USA), and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked for 2 hours at room temperature in Tris-buffered saline (TBS) containing 5% skim milk and were then incubated with nestin (mouse monoclonal, 1:5,000; Millipore) or GFAP antibody (rabbit polyclonal, 1:1,000; Sigma-Aldrich) overnight at 4°C. The membranes were finally incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Sigma-Aldrich) for 2 hours at room temperature. Enhanced chemoluminescence (ECL) detection of nestin or GFAP was performed according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). The expression of those markers was used to quantify and compare them with each other using ImageJ software, version 1.47c (Image Processing and Analysis in Java; National Institutes of Health, Hamilton, MO, USA).

Cells positive for Iba-1, CD11b, F4/80, and nestin were counted in the MNU-treated retinal sections. Cell counts were performed for different time periods. The total number of immunofluorescent-labeled cells in whole retinal sections that contained the optic nerve head was counted manually, and two experienced counters were given the unlabeled sample slides to prevent bias.

The Mann-Whitney U test was used to assess differences in cell counts at the five designated time points after MNU administration. Statistical analysis was conducted using SPSS Statistics Version 21 (IBM, Somers, NY, USA). All tests were two-tailed, and differences having P values of less than 0.05 were considered statistically significant.

Intraperitoneal MNU injection was used to induce retinal degenerative changes. At 3 days after MNU injection, retinal thickness had obviously decreased owing to the selective loss of photoreceptor cells, and the arrangement of retinal neurons in the outer nuclear layer (ONL) was distorted (Fig. 1). Progressive loss of photoreceptor cells led to a nearly complete loss of ONL by day 7 PI (Fig. 1).

In the normal adult mouse retina, nestin was rarely expressed except for some vascular profiles in the outer plexiform layer (OPL) (Fig. 1).

Nestin expressing Iba-1+ microglia with amoeboid shape were identified mainly in outer plexiform layer at 1 day after retinal degeneration induced by MNU injection (Fig. 1). Nestin expressing microglia, previously located mainly in the OPL, had migrated into ONL. The number of microglia showing nestin expression was markedly increased at day 3 PI (Fig. 1). At day 5 PI, cell dimensions of nestin+ microglia were more extended (Fig. 1). At day 7 PI, nestin expressing microglia were significantly decreased compared with day 5 PI, and the immunoreactivity of nestin in the whole retina was also significantly diminished (Fig. 1). By 21 days after MNU, most of the retinal neurons nuclei were degraded and nestin expression had decreased to the baseline levels.

Expression of CD11b and F4/80 were rarely identified in the physiologic state prior to the MNU injection except for their expression in the endothelium (Figs. 2 and 3). At day 1 after MNU injection, nestin+/CD11b+ cells were evident in the OPL. Nestin+/CD11b+ cells migrated into the ONL at day 3PI (Fig. 2), peaked at day 5 (Fig. 2), and decreased thereafter (Fig. 2). F4/80+ cells observed in the subretinal space revealed oval or spindle shaped cytoplasm without ramified processes at day 1 PI (Fig. 3). The number of amoeboid nestin+/F4/80+ cells increased markedly from day 3 to day 5 PI and these cells were heavily confined within the subretinal space and OPL (Fig. 3). Although the number of F4/80+ cells decreased thereafter, it was relatively preserved at 7 days after MNU injection (Fig. 3).

GFAP labeled Müller cells were distinctly increased at 1 day after MNU (Fig. 4). Nestin immunoreactivity of GFAP+ astrocytes in the ganglion cell layer (GCL) and Müller cells was progressively increased at day 3 PI (Fig. 4), and revealed peak activity at day 5 (Fig. 4). After that, the number of nestin+/GFAP+ Müller cells was markedly reduced. However, GFAP expression remained in an activated state in the retinal tissue at day 7 (Fig. 4) and day 21 after MNU despite the diminution in retinal thickness (Fig. 4).

We used western blot analysis to quantify the expression of nestin and GFAP. Nestin showed increased immunoreactivation at day 1 PI. The expression of nestin peaked at day 3 PI and declined at day 7 PI. At day 21 PI, the level declined to baseline when compared with the control retinas (Fig. 5). GFAP expression initially showed a gradual increase for the first 3 days after the MNU injection, and this level was maintained from day 3 to day 7 PI. GFAP expression decreased at day 21 PI; however, unlike nestin expression, GFAP expression was higher than that in the control retina (Fig. 5).

The number of nestin-expressing cells labeled with CD11b, Iba-1, and F4/80 was calculated in the retinal sections. After the MNU injection, the number of microglia/monocyte/macrophage marker-positive cells was increased at day 1 after MNU, peaked at day 5 (P < 0.001), and then decreased significantly at day 7 PI (P < 0.001) (Fig. 6). The number of nestin co-expressing microglia/ monocytes/macrophages was also increased at day 1 PI. The number of nestin+/Iba-1+ and nestin+/F4/80+ cells peaked at day 3 PI (P < 0.001). The number of nestin+/Iba-1+ cells decreased significantly at day 5 (P < 0.001), and the number of nestin+/F4/80+ cells was significantly reduced at day 7 (P < 0.001). The number of nestin+/CD11b+ cells peaked at day 5 PI (P < 0.001) and then decreased at day 7 (P < 0.001) (Fig. 6B).