Clinically and neurologically normal male beagle dogs were used at 2-3 years (young adult dogs, n=12) and 10-12 years (aged dogs, n=12) of age, with normal values in blood count, chemistry, gas analysis and serum electrolytes. The animal protocol used in this study was reviewed and approved based on ethical procedures and scientific care by the Kangwon National University-Institutional Animal Care and Use Committee (KIACUC-12-0010).

For histochemical analysis, young adult and aged dogs (n=7 in each group) were anesthetized with zoletil50 (8 mg/kg) and xylazine (2 mg/kg) mixture and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The cervical (C₆-C₈), thoracic (T₉-T₁₀) and lumbar (L₅-L₆) spinal cord were removed and posfixed in the same fixative for 12 h. The spinal cord tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter the frozen tissues were serially sectioned on a cryostat (Leica, Wetzlar, Germany) into 30 µm in thickness, and the sections were then collected into six-well plates containing PBS.

To obtain the accurate data for immunohistochemistry, the free-floating sections were carefully processed under the same conditions. Immunohistochemistry for α-syn, glial fibrillary acidic protein (GFAP, a marker for astrocytes) and ionized calcium-binding adapter molecule-1 (Iba-1, a marker for microglia) was performed under the same conditions in dogs of different ages in order to examine whether the degree of immunohistochemical staining was accurate. The sections were sequentially treated with 0.3% H₂O₂ and 10% normal goat serum. They were then incubated with diluted sheep anti-α-syn (diluted 1:1000, Abcam, Cambridge, MA), mouse anti-GFAP (diluted 1:800, Chemicon, Temecula, CA) and rabbit anti-Iba-1 (diluted 1:800, Wako, Richmond, VA), respectively, and subsequently exposed to biotinylated rabbit anti-sheep, goat anti-rabbit or horse anti-mouse IgG and streptavidin peroxidase complex (1:200, Vector, Burlingame, CA). They were then visualized by staining with 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in 0.1 M Tris-HCl buffer (pH 7.2) and mounted on gelatin-coated slides. The sections were mounted in Canada balsam (Kanto) following dehydration. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. The negative control test resulted in the absence of immunoreactivity in all structures.

To confirm change in α-syn, GFAP and Iba-1 levels in the cervical, thoracic and lumbar spinal cord between the adult and aged dog, the animals (n=5 in each group) were sacrificed and used for the western blot analysis. In brief, the tissues were homogenized in 50 mM PBS (pH 7.4) containing EGTA (pH 8.0), 0.2% NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM PMSF, and 1 mM DTT. After centrifugation, the protein level in the supernatants was determined using a Micro BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). Aliquots containing 20 µg of total protein were boiled in a loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% SDS, 0.3% bromophenol blue, and 30% glycerol. The aliquots were then loaded onto a polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Crop, East Hills, NY, USA). To reduce background staining, the membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20, followed by incubation with sheep anti-α-syn (diluted 1:1000, Abcam, Cambridge, MA), rabbit anti-Iba-1 (diluted 1:1000, Wako, Richmond, VA), and mouse anti-GFAP (diluted 1:1000, Chemicon, Temecula, CA), peroxidase-conjugated donkey anti-sheep, rabbit or mouse IgG (Sigma, St. Louis, MO, USA) and ECL kit (Pierce Chemical). The result of western blot analysis was scanned, and densitometric analysis for the quantification of the bands was done using Scion Image software (Scion Corp., Frederick, MD), which was used to count relative optical density (ROD): A ratio of the ROD was calibrated as %, with the adult-group designated as 100%.

Every statistical analysis was carried out using GraphPad Prism 4.0 (GraphPad Software, USA). We used Kruskal-Wallis one way analysis of variance (ANOVA) on ranking test statistical significance in the number of cell, ROD and dimensions of gray matter and white matter. Data are expressed as the mean±SEM. The data were evaluated by student's t-test. Statistical significance was considered at P<0.05.

In the adult dog, α-syn immunoreactivity was easily detected in neuropil of all the laminae of the cervical, thoracic and lumbar spinal cord, and immunoreactivity was somewhat different according to the laminae; α-syn-immunoreactive profiles were mainly found in laminae I-III, and medial part of laminae X and laminae IX of motor nuclei (Figure 1A, 2A and 3A). In addition, in the thoracic level, α-syn immunoreactivity was also detected in intermediolateral nucleus in lamina VII (Figure 2A and 2b). α-Syn immunoreactivity was not detected in cell bodies and dendrites of neurons in the cervical and lumbar spinal cord (Figure 2 and 3).

In the aged group, the distribution pattern of α-syn-immunoreactive structures in the cervical and lumbar spinal cord was similar to that in the adult dog (Figure 1B and 3B), however, in the thoracic level, α-syn immunoreactivity was easily detected in the cytoplasm of many neurons, especially in laminae I-III, intermediolateral nucleus (autonomic nucleus) and motor nuclei (Figure 2B and 2f). Although immunoreactivity of α-syn was newly expressed in many neurons in the thoracic spinal cord (Figure 2B), the general pattern of α-syn immunoreactivity was not significantly changed in the cervical and lumbar spinal cord in the aged dogs compared to that in the adult group (Figure 1B and 3B).

α-Syn protein levels in the aged spinal cord were not found to be significantly changed in the cervical, thoracic and lumbar spinal cord compared to those in the adult group (Figure 4), although α-syn immunoreactivity was newly expressed in motor neurons in the aged thoracic spinal cord.

GFAP-immunoreactive astrocytes were observed mainly in the gray matter in the cervical, thoracic and lumbar spinal cord, and GFAP immunoreactivity was not apparently changed in the aged group compared to that in the adult group (data not shown).

Iba-1-immunoreactive microglia were observed throughout the gray matter in the cervical, thoracic and lumbar spinal cord of both the groups, and Iba-1 immunoreactivity was somewhat increased in the aged group compared to that in the adult group (data not shown).