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 for the experiments. The study was carried out in accordance with the internationally accepted principles for laboratory animal use and care as per NIH guidelines (USA).

The rats were divided into two equal groups and anesthetized with chloral hydrate (375 mg/kg body weight, peritoneal injection). The rats from one group were subjected to whole-body irradiation with 15 Gray in a single fraction (n = 7). Irradiation was carried out using a ⁶⁰Co γ-ray source (10,000 Ci; Co-60 Irradiation Facility, Applied Radiological Science Research Institute, Cheju National University, Korea). The rats from the other group were not irradiated and used as controls (n = 7).

The rats were sacrificed at 24 h post-irradiation (PI) under ether anesthesia. The spinal cords were removed immediately after death and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4 to allow for paraffin embedding, or were quick-frozen and stored for later use in immunoblotting.

Rabbit polyclonal anti-p-caveolin-1 (Tyr 14; Santa Cruz, USA), rabbit polyclonal anti-fibronectin (Santa Cruz, USA), and mouse monoclonal anti-beta-actin (Sigma, USA) antibodies were used in this study. The astrocytes and macrophages were identified by applying the mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (Sigma, USA) and mouse monoclonal anti-rat macrophage (ED 1; Serotec, UK), respectively. ED1 was also used to estimate the level of microglia and macrophage activation in a Western blot analysis since ED1 recognizes the rat macrophage lysosomal membrane antigen [2]. Biotinylated isolectin B4 (IB4) derived from Griffonia simplicifolia (Sigma, USA) was used to detect the vascular endothelial cells and activated microglia [6].

A Western blot analysis was performed as previously described [6]. Briefly, the tissue was homogenized in a lysis buffer containing protease and a phosphatase inhibitor. The proteins were resolved by SDS-PAGE (12% acrylamide) and transferred to a nitrocellulose membrane (Schleicher & Schuell, USA). The blots were blocked with 5% skim milk in TTBS (TBS with 0.1% Tween 20) for 1 h, washed, and then incubated with primary antibodies overnight. The blots were washed three times with TTBS and incubated for 1 h with HRP-conjugated anti-rabbit or mouse IgG antibodies (Vector, USA). The immunoreactive bands were developed using a chemiluminescent substrate (WEST-one Kit; iNtRON Biotech, Korea).

To assess the immunohistochemistry, paraffin-embedded spinal cord sections (5 µm) were deparaffinized, treated with a citrate buffer (0.01 M, pH 6.0) in a microwave for 10 min, and then treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min to block the endogenous peroxidase activity. After three washes with PBS, the sections were incubated with 10% normal goat serum and then with polyclonal anti-p-caveolin-1 for 1 h at room temperature (RT). The immunoreactivity was visualized using an avidin-biotin peroxidase complex (Vector Elite; Vector, USA) and the peroxidase reaction was developed using a diaminobenzidine substrate kit (Vector, USA).

The cell phenotype of p-caveolin-1 expression was examined by applying double immunofluorescence using anti-GFAP. The paraffin sections were reacted sequentially with primary rabbit anti-p-caveolin-1 followed by fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (1 : 50 dilution; Sigma, USA). The sections were then incubated with anti-GFAP followed by tetramethyl rhodamine isothicyanate (TRITC)-labeled goat anti-mouse IgG (1 : 50 dilution; Sigma, USA). To observe the co-localization of p-caveolin-1 and IB4 in the spinal cords, the sections were reacted with biotinylated IB4 (Sigma, USA), followed by TRITC-labeled streptavidin (Zymed, USA). Next, the sections were then reacted with the rabbit anti-p-caveolin-1, followed by a reaction with FITC-labeled goat anti-rabbit IgG (Sigma, USA). For the reduction of lipofuscin autofluorescence, the sections were washed in PBS (three times for 1 h) at RT, dipped briefly in distilled H₂O, treated with 10 mM CuSO₄ in an ammonium acetate buffer (50 mM CH₃COONH₄, pH 5.0) for 20 min, dipped again briefly in distilled H₂O, and then returned to PBS. Following this, the double immunofluorescence-stained specimens were examined by laser confocal microscopy (FV500; Olympus, Japan).

The results are expressed as the mean ± SE of the number of determinations indicated. The statistical significance of differences was determined using analysis of variance. Significance was accepted at p <0.05.

A Western blot analysis showed that the level of p-caveolin-1 expression was significantly higher in the spinal cord at 24 h PI (0.267 ± 0.036; n = 7 rats; p < 0.05 vs. normal controls) than in the normal controls (density value, 0.066 ± 0.015 OD/mm²; n = 7 rats) (Fig. 1A).

A semiquantitative analysis of the macrophage marker using ED1 was performed to show the activation of microglial cells. The immunoblot of the rat macrophage lysosomal membrane antigen detected by ED1 showed significantly higher levels in the spinal cord at 24 h PI (0.012 ± 0.001; n = 7 rats; p < 0.05 vs. normal controls) than in the normal controls (0.004 ± 0.001 OD/mm²; n = 7 rats) (Fig. 1B).

A Western blot analysis was used to measure the level of fibronectin in order to determine if there was any leakage of blood fibronectin in the spinal cords with irradiation. The level of fibronectin immunoreactivity was significantly higher in the irradiated spinal cords at day 1 PI (0.927 ± 0.068; n = 7 rats; p < 0.05 vs. normal controls) than in the normal control spinal cords (0.436 ± 0.027 OD/mm²; n = 7 rats) (Fig. 1C).

Weak p-caveolin-1 immunoreactivity was constitutively detected in some vascular endothelial, glial, and ependymal cells in the spinal cords of the normal rats (Figs. 2A & B). In the irradiated spinal cords, p-caveolin-1 was found to be intensely immunostained in the ramified glial cells as well as in some vascular endothelial and ependymal cells (Figs. 2C & D).

A double-labeling experiment was performed in the spinal cords to determine the cell phenotype of p-caveolin-1 expression. The immunoreactivity of p-caveolin-1 in the parenchyma was co-localized in some IB4-positive microglia and vascular endothelial cells (Figs. 3A-C) but was scarce in the astrocytes (Figs. 3D-F). This suggests that most p-caveolin-1 positive cells are ramified microglial cells.