Sprague-Dawley rats were purchased from Daehan Biolink (Korea) and bred in our animal facility. Five- to six-week-old male rats weighing 121.1 ± 19.2 g were used throughout the experiments. The animals were housed in cages in a standard barrier facility and maintained on a 12-h light:12-h dark cycle at 23℃. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care, as found in the NIH guidelines (USA).

Irradiation was carried out using a ⁶⁰Co γ-ray source (370 TBq; Co-60 Irradiation Facility, Applied Radiological Science Research Institute, Cheju National University, Korea). Animals were anesthetized with chloral hydrate (375 mg/kg body weight, peritoneal injection), and the whole bodies of the rats were irradiated, each with a single dose of 8 Gray (Gy). Groups were subsequently killed on days 1, 4, and 9 PI (n = 5 per each time point), respectively. Control animals were not irradiated.

In order to study the expression of caveolin-1 in the spinal cords, tissues were sampled on days 0 (control), 1, 4, and 9 PI (n = 5 per each time point). The spinal cords were removed and frozen at -70℃ for protein analysis. Some pieces of spinal cords were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4. Specimens were processed using standard paraffin embedding methods, and were cut to a thickness of 5 µm for routine hematoxylin-eosin staining and immunohistochemical studies.

The specificity of rabbit polyclonal anti-caveolin-1 (N-20) (Santa Cruz Biotechnologies, USA) has been well characterized by the manufacturer. The N-20 was an affinity-purified rabbit antibody raised against a peptide mapping at the N-terminus of caveolin-1 of human origin according to the data sheet supplied by the manufacturer. Mouse monoclonal anti-GFAP (Sigma, USA) was used for the detection of astrocytes. Mouse monoclonal anti-beta actin was also obtained from Sigma (USA).

Spinal cords were homogenized in lysis buffer (40 mM Tris, 120 mM NaCl, 0.1% Nonidet P-40, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) with 20 strokes in a homogenizer. The homogenates were transferred into microtubes and centrifuged at 12,000 rpm for 20 min; the supernatant was then harvested.

For the immunoblot assay, supernatant containing 20 µg of protein was loaded into each lane of a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Schleicher & Schuell BioScience, USA). The residual binding sites on the membrane 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 h and then incubated with each primary antibody, including anti-caveolin-1 (1 : 1000) and anti-GFAP (1 : 20,000), for 2 h. The blots were washed three times in TBS containing 0.1% Tween-20, and were then probed with appropriate secondary antibodies including horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Vector, USA) for 1 h. The blots were developed using enhanced chemiluminescence (ECL) reagents according to the instructions of the manufacturer. After visualization with ECL, the antibodies were stripped from the membranes, which were reprobed with monoclonal anti-beta actin antibody (Sigma, USA). The density of each band obtained by Western blot analysis was measured with a scanning laser densitometer (GS-700; Bio-Rad, USA) and analyzed using Molecular Analyst software (Bio-Rad, USA). The ratios of caveolin-1/beta-actin were compared. The results were analyzed statistically by one-way ANOVA followed by the Newman-Keuls test. In all cases, p < 0.05 was considered significant.

After deparaffinization and hydration, the sections were treated with 0.3% hydrogen peroxide in deionized water for 20 min to block endogenous peroxidase. Some paraffin sections for anti-caveolin-1 immunostaining were boiled in 10 mmol/L sodium citrate (pH 6.0) for 10 min at 95℃. After three washes with PBS, the sections were exposed to 10% normal goat serum and then incubated with each primary antiserum including rabbit polyclonal anti-caveolin-1 (1 : 200) for 1 h at room temperature. After three washes, the appropriate biotinylated secondary antibody and the avidin-biotin-peroxidase complex (ABC) from the Elite kit (Vector, USA) were added sequentially. Peroxidase was developed with a diaminobenzidine tetrahydrochloride (DAB) substrate kit (Vector, USA). The sections were counterstained with hematoxylin prior to mounting.

For double-staining of two antigens in the same section, double-immunofluorescence was applied using fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (1 : 50 dilution; Sigma, USA) and tetramethyl rhodamine isothiocyanate (TRITC)-labeled goat anti-mouse IgG (1 : 50 dilution; Sigma, USA) secondary antibodies in order to co-localize caveolin-1 and GFAP (1 : 800) in the same cell.

Western blot analysis of caveolin-1 showed that the expression of caveolin-1 significantly increased (fold increase [mean ± SE]: 4.1 ± 1.9, p < 0.05) at day 1 PI compared to those of normal spinal cords, and returned to the level of normal controls at days 4 and 9 PI (Fig. 1). This finding suggests that caveolin-1 is constitutively expressed in the normal spinal cord of the rat, and that it transiently increases in the spinal cord following irradiation.

Western blot analysis of GFAP expression in the spinal cords of normal and irradiated rats is shown in Fig. 2. GFAP immunoreactivity was significantly increased at day 1 PI (1.51 ± 0.06, p < 0.05) and day 4 PI (1.99 ± 0.2, p < 0.01) compared to those of normal spinal cords, and declined thereafter to the normal level at 9 PI (Fig. 2). This finding suggests that irradiation stimulates the expression of GFAP in the spinal cords of rats.

Immunohistochemical analysis was applied to visualize the cell phenotype expressing caveolin-1 in the spinal cords of normal control and irradiated rats. In the normal control rats, caveolin-1 was weakly immunostained in some vascular endothelial cells, neurons, and some glial cells (Fig. 3A&C). The immunoreactivity of caveolin-1 was intense in some vascular endothelial cells, neurons, and glial cells from the spinal cords of irradiated rats at day 1 PI (Fig. 3B&D). These findings indicate that various CNS cells, including glial cells, are responsive to irradiation with a concomitant increase of caveolin-1.

A double-labeling experiment was performed to identify the cell phenotype expressing caveolin-1, and to determine whether hypertrophied astrocytes contain enhanced levels of caveolin-1. This is based on the knowledge that astrogliosis is a prominent feature of radiation injury. Using double-immunofluorescence, both caveolin-1- (Fig. 4A) and GFAP- (Fig. 4B) positive astrocytes were rarely observed in the normal spinal cords (Fig. 4C), while the number of caveolin-1- (Fig. 4D) and GFAP- (Fig. 4E) positive cells was more abundant at day 1 PI (Fig. 4F). These findings suggest that the expression of caveolin-1 is increased in hypertrophied astrocytes in the spinal cord after irradiation.