Serum keratan sulfate transiently increases in the early stage of osteoarthritis during strenuous running of rats: protective effect of intraarticular hyaluronan injection

Wistar rats 16 to 18 weeks of age (Sankyo Labo Service, Tokyo, Japan) were used for the experiments. All experiments were conducted in accordance with the institutional guidelines for the care and use of experimental animals. Rats were divided into three groups: no running group (0 km, n = 5); only strenuous running group (30 km, n = 8); and strenuous running and hyaluronan injection group (15 km, n = 3; 30 km, n = 5).

For strenuous running exercise, a rodent treadmill machine (MK-680R5; ME Service., Tokyo, Japan) was used with a 5% incline (Figure 1a). The MK-680R5 has been designed to compulsively make animals exercise by electrical shock delivered to the animals without failure by the adoption of a shock generator scrambler. The rats were acclimated to the treadmill by gradually increasing the running speed and time as follows: day 1, 10 minutes at 10 m/min; day 2, 15 minutes at 12 m/min; day 3, 20 minutes at 15 m/min; day 4, 30 minutes at 18 m/min; and day 5, 35 minutes at 20 m/min. On day 8 and thereafter, the rats were forced to run for 55 minutes a day at 20 m/min, with the first 10 minutes consisting of a 12 m/min warm-up. Rats ran 30 km in 6 weeks, as shown in Figure 1b[18]. For the hyaluronan injection group (n = 5), 100 μl hyaluronan (average molecular weight = 8 × 10⁵ Da; Seikagaku Corp., Tokyo, Japan) containing 1 mg in formulated concentrate was injected into the right knee. The injections were performed initially at 5 days and every 1 week thereafter under anesthesia of 10 mg sodium pentobarbital (Dainippon Sumitomo Pharma, Osaka, Japan) by intraperitoneal injections. As a control for hyaluronan treatment, saline or PBS was not injected into the left knee to avoid the possibility that they might enhance osteoarthritis [19, 20].

Femoral and tibial condyles were carefully dissected separately without damaging the cartilage surface, and were then stained with India ink to identify the location, size and severity of cartilage degeneration. Macroscopic pictures were taken using a specification MPS-7 (Sugiura Laboratory Inc., Tokyo, Japan), a dedicated medical photography platform. Digital images were taken using a Nikon Coolpix 4500 digital camera (Nikon, Tokyo, Japan).

The rats were sacrificed with an overdose of sodium pentobarbital. Both femurs and tibias were fixed in 4% paraformaldehyde at pH 7.4 for 3 days, were decalcified in 20% ethylenediamine tetraacetic acid solution for 21 days, and were then embedded in paraffin wax. Femurs and tibias were sectioned sagittally at 5 μm and stained with safranin-O. Histological sections were visualized using an Olympus IX71 microscope (Olympus, Tokyo, Japan). Each section was evaluated with the Mankin's histological grading system (Mankin's score: 0 to 14) for articular cartilage degeneration [21].

Sections were deparaffinized, washed in PBS, and pretreated with 0.4 mg/ml proteinase K (DAKO, Carpinteria, CA, USA) in Tris–HCl buffer for 15 minutes at room temperature for optimal antigen retrieval. Endogenous peroxidases were quenched using 0.3% hydrogen peroxide in methanol for 20 minutes at room temperature. The sections were rinsed once in PBS for 5 minutes and were briefly blocked with 10% normal horse serum (Vector Laboratories, Burlingame, CA, USA) to avoid nonspecific binding of the antibody. The sections were then incubated in monoclonal anti-keratan sulfate antibody (5-D-4, 1:100 dilution with PBS containing 1% BSA; Seikagaku Corp.) or anti-mouse monoclonal antibody against human type II collagen (1:200 dilution with PBS containing 1% BSA; Daiichi Fine Chemical, Toyama, Japan) at room temperature for 60 minutes. After rinsing in PBS, the tissues were incubated with biotinylated horse anti-mouse IgG secondary antibody (Vector Laboratories) for 30 minutes at room temperature. The slides were again immersed in PBS and were incubated for another 30 minutes with Vectastain ABC reagent (Vector Laboratories). Finally, the sections were shortly counterstained with hematoxylin, dehydrated and mounted in a xylol-soluble mount (Vitro-Clud; R. Langenbrinck, Emmendingen, Germany) [12, 22, 23].

To avoid circadian variation of keratan sulfate in serum, blood was collected at between 3:00 and 4:00 pm, 1 hour after finishing strenuous running. Approximately 500 μl blood was aspirated with a 27-gauge needle from the tail vein of the rats. The blood was kept at 4°C for 2 hours and was centrifuged at 2,000 rpm for 15 minutes at 4°C. The serum was separated, allocated into 100 μl, and kept frozen at -70°C. To avoid an influence of freeze–thaw, the serum was used for the analyses without refreezing. Every rat started strenuous running on Monday and had its blood aspirated every Friday. Each 200 μl serum was diluted with 800 μl distilled water, digested with 100 μl of 2.0% Actinase E (Kaken Pharmaceutical., Tokyo, Japan) at 55°C for 24 hours, and was heated at 100°C for 10 minutes. The whole solution was applied to Q Sepharose 0.15 M sodium chloride (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK), and was extracted with 50 mM Tris–HCl buffer (pH 8.6) containing 2 M sodium chloride. The extracted material was desalinated with PD-10 (GE Healthcare), dried, and dissolved again by 0.2 ml distilled water. Then 1 mU Keratanase II (Seikagaku Corp.) was added, followed by addition of 40 μl of 100 mM sodium acetate buffer (pH 6.0), and the mixture was incubated at 37°C for 3 hours.

The sample was ultrafiltered using an Ultrafree C3GC system whose molecular weight cutoff was 10,000 daltons (Japan Millipore, Tokyo, Japan). The filtrate, which contained monosulfate disaccharide and disulfate disaccharide derived from keratan sulfate, was analyzed by HPLC with the column packed with polyamine-bound silica (YMC gel PA-120; YMC Ltd, Kyoto, Japan). The monosulfate disaccharide and disulfate disaccharide were eluted with a gradient of 0 to 100 mM sodium sulfate for 45 minutes at a flow rate of 0.5 ml/min. To elute from the column, 100 mM sodium tetraborate buffer (pH 9.0) containing 1% 2-cyanoacetamide was added at a flow rate of 0.5 ml/min. The mixture was passed through poly(ether ether ketone) (PEEK; Victrex, Lancashire, UK) tubing with a 0.5 mm diameter and a 10 m length in a dry fluoromonitor (excitation, 331 nm; emission, 383 nm). The area of each peak corresponding to monosulfate disaccharide and to disulfate disaccharide was calculated by Borwin-HSS2000 software (Jasco, IJsselstein, Netherlands) and was converted to the amount of the corresponding disaccharides against the area of standard monosulfate disaccharide and disulfate disaccharide (Seikagaku Corp.) [12].

Strenuous running exercise affected the articular cartilage. The cartilage surfaces of both the lateral femoral condyle and the lateral tibial plateau were irregular after 30 km of strenuous running (Figure 2). A weekly intraarticular hyaluronan injection helped maintain the smoothness of the surface of the articular cartilage (Figure 2). Histological analyses demonstrated that 30 km of strenuous running induced depletion of the articular cartilage matrix (Figure 3a) and worsened the Mankin's score (Figure 3b). Hyaluronan treatment suppressed the degeneration of the articular cartilage (Figure 3c).

Sequential serum concentrations of keratan sulfate were examined. In the control group (the only strenuous running group), the concentration transiently peaked at 3 to 4 weeks (Figure 4, left). Hyaluronan treatment appeared to suppress the keratan sulfate concentration (Figure 4, center). The average of the maximum keratan sulfate concentration during the 5-week period in each rat in the hyaluronan group was significantly lower than that in the control group (Figure 4, right).

Keratan sulfate and type II collagen were expressed in cartilage of rats before strenuous running and were still present at 3 weeks after 15 km of strenuous running. Keratan sulfate expression decreased and was hardly observed at 6 weeks after 30 km of strenuous running in the control group. The type II collagen-positive area decreased along with the cartilage area, but the expression could be still observed in the remaining cartilage in the control group after 30 km of strenuous running. Hyaluronan treatment suppressed the loss of keratan sulfate and type II collagen after 30 km of strenuous running (Figure 5).