Eyes from 2-day-old, 5-week-old, and 6-month-old female Landrace pigs (n=4-5 per group) were obtained from a local farm. The eyes were enucleated and the anterior segment of the globe was dissected. The retinas were detached carefully from the eyes in phosphate buffered saline (PBS; pH 7.4), fixed in 10% neutral-buffered formalin in 0.1 M PBS for histological analysis, and deep-frozen at -80℃ until used for Western blot analysis. Retinas recovered from female Sprague Dawley rats (7 to 8-week-old, 160-200 g) were used as a reference to compare the expression of CBP. All retinas had normal histological features. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals at Jeju National University.

Western blot analysis of the pig retinas was performed as reported previously [11], with minor modifications. Briefly, retinal tissues were thawed in lysis buffer consisting of 20 mM HEPES, pH 7.2 (Sigma-Aldrich, St. Louis, MO, USA), 1% Triton X-100 (AMRESCO, Solon, OH, USA), 1% deoxycholate (Sigma-Aldrich), 0.1% sodium dodecyl sulfate (SDS; Bio-Rad, Hercules, CA, USA), 150 mM NaCl (Junsei, Tokyo, Japan), 10 µg/ml leupeptin (Sigma-Aldrich), 10 µg/ml aprotinin (Sigma-Aldrich), and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Tissues were given 10 strokes in a Dounce homogenizer. After incubation for 60 minutes in an ice-bath, the homogenates were each centrifuged at 14,000 rpm for 20 minutes, and the supernatant was harvested. For the immunoblot assay, supernatant containing 40 µg of protein was loaded into individual lanes and subjected to 6% SDS-polyacrylamide gel electrophoresis. The resolved proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA). The residual binding sites on the membranes were blocked by incubation with 5% nonfat milk in Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 1 hour at room temperature (RT). Subsequently, the membranes were incubated with rabbit anti-CBP (1 : 1,000, C-20 [sc-583], Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours at RT. The membranes were washed three times in TBS containing 0.1% Tween-20 before being incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1 : 2,000, Santa Cruz Biotechnology) for 1 hour at RT. The membranes were developed using an enhanced chemiluminescence reagent kit (Amersham Pharmacia Biotech, Uppsala, Sweden), employed according to the manufacturer's instructions, and exposed to medical X-ray film (Agfa Gevaert, Mortsel, Belgium). After imaging, the membranes were stripped and reprobed using monoclonal anti-β-tubulin antibody as the primary antibody (Sigma-Aldrich) using a similar protocol to that described above. The optical density (OD/mm²) of each band was measured with a GS-700 scanning laser densitometer (Bio-Rad) and the values are presented as the mean±standard error of the mean. The ratios of the density of the CBP band to that of the β-actin band were compared using Molecular Analyst software (Bio-Rad).

Immunohistochemistry of the pig retinas was performed as reported previously [12], with minor modifications. Five micrometer-thick paraffin wax embedded tissue sections were deparaffinized and rehydrated by routine protocols before being exposed to citrate buffer (0.01 M, pH 6.0) and heated in an autoclave for 10 minutes. They were then incubated in 0.3% hydrogen peroxide in methyl alcohol for 20 minutes to block the endogenous peroxidase activity. After three washes with PBS, the sections were incubated with 10% normal goat serum (Vector ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) diluted in PBS, which was followed by incubation with rabbit polyclonal anti-CBP (1 : 400). After three washes in PBS, the sections were incubated with biotinylated anti-rabbit IgG (ABC Elite Kit, Vector Laboratories). After three washes in PBS the sections were incubated with avidin-biotin peroxidase complexes were formed using an Elite kit (Vector Laboratories). The peroxidase reaction was developed with a diaminobenzidine substrate kit (Vector Laboratories), and sections were counterstained with hematoxylin before mounting. As a negative control, the primary antiserum was omitted for a few test sections in each experiment; no specific labeling of cell bodies or fibers was detected in these sections (Fig. 2D). To examine the phenotype of CBP immunoreactive cells, double immunofluorescence was applied using cell-type-specific markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), protein kinase C, alpha (PKCα), and parvalbumin for glial cells, Müller cells, bipolar cells, and amacrine cells, respectively. First, the sections prepared as described previously were incubated with rabbit anti-CBP overnight at 4℃, followed by biotinylated anti-rabbit IgG, and tetramethylrhodamine isothiocyanate (TRITC)-labeled streptavidin (Zymed, South San Francisco, CA, USA). The slides were then incubated with mouse monoclonal anti-GFAP (1 : 400, Sigma-Aldrich), anti-GS (1 : 5,000, Chemicon International, Temecula, CA, USA), anti-PKCα (Santa Cruz Biotechnology), or anti-parvalbumin (Sigma-Aldrich) overnight at 4℃, followed by fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Sigma-Aldrich), for 1 hour at RT. Double immunofluorescencestained specimens were examined using a FV500 laser confocal microscope (Olympus, Tokyo, Japan).

The data was analyzed using one-way ANOVA followed by a Student-Newman-Keuls post hoc test for multiple comparisons. In all cases, P<0.05 was considered statistically significant.

CBP was strongly detected in the retinas of 2-day-old piglets (density value 6.8±1.63 OD/mm²) and was decreased significantly in the retinas of 5-week-old (2.23±0.44; P<0.05 vs. 2-day-old piglets) and 6-month-old pigs (2.34±0.57; P<0.05 vs. 2-day-old piglets) (Fig. 1).

The retinas of 2-day-old, 5-week-old, and 6-month-old pigs consisted of multiple layers, including the optic nerve fiber layer (ONF), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear cell layer (INL), outer plexiform layer (OPL), outer nuclear cell layer (ONL), and rod and cone layer (RCL). In the retinas of 2-day-old piglets, diffuse CBP immunoreactivity was detected in the IPL, OPL, and RCL. CBP was immunostained intensely in the glial process in the ONF, GCL, and IPL (Fig. 2A, E). Moderate CBP immunoreactivity was detected in the ganglion cells in the GCL (Fig. 2A, E). Additionally, intense CBP immunostaining was seen in some cells in the INL (Fig. 2A, H). Conversely, weak CBP immunostaining was identified in the ONL (Fig. 2A). In the retinas of 5-week-old (Fig. 2B, F, I) and 6-month-old pigs (Fig. 2C, G, J), CBP was immunostained in the ONF, GCL, IPL, INL, OPL, and ONL. CBP localization was similar in the retinas of 2-day-old piglets, but the CBP immunoreactivity in the ganglion cells was decreased (Fig. 2B, C). CBP immunoreactivity was also detected in the RCL. In the retinas of adult rats, CBP immunoreactivity was detected in ganglion cells in the GCL and some cells in the INL (Fig. 2K). Interestingly, there was no CBP immunostaining in the glial processes in the GCL or IPL, or in any neuronal or glial cells in the OPL, ONL, or RCL (Fig. 2K).

We performed double immunofluorescence staining with both GFAP (Fig. 3), and GS (Fig. 4) to ascertain the precise localization of CBP in the glial cells in the retina.

GFAP immunofluorescence was very rarely detected in the retinas of 2-day-old piglets, and it increased in the retinas of 5-week-old and 6-month-old pigs (Fig. 3). However, CBP immunoreactivity in the RCL was occasionally co-localized in a few GFAP-positive glial cells in the retinas of 5-week-old and 6-month-old pigs (Fig. 3). Most GS immunofluorescence was detected in the GCL and INL, but was limited in the IPL in the retinas of 2-day-old piglets (Fig. 4). Interestingly, most of the area in the GCL consisted of GS-positive Müller cells in the retinas of 2-day-old piglets (Fig. 4), indicating that the phenotype of glial cells in the piglets was GS-positive Müller cells, not GFAP-positive glial cells (Fig. 3). In the retinas of 5-week-old and 6-month-old pigs, GS immunoreactivity was seen in the GCL, IPL, and INL (Fig. 4). Most GS immunoreactivity in the GCL was co-localized with CBP in the GCL in all-age retinas, but occasionally co-localized with CBP in the IPL and INL (Fig. 4).

Next, we performed double immunofluorescence staining with both PKCα and parvalbumin to ascertain the precise localization of CBP in the bipolar cells, and amacrine cells in the INL, respectively. In the retina of all age pigs, CBP immunofluorescence in the INL co-localized with PKCα-positive bipolar cells (Fig. 5). Interestingly, some CBP immunoreactivity was detected in the processes of PKCα-positive bipolar cells in the retinas of 5-week-old pigs (Fig. 5), but immunoreactivity was not detected in the parvalbuminpositive amacrine cells in the retinas of all age pigs (data not shown).