**4.1 Methods**

#### **4.1.1 Induction of antigen-induced arthritis**

The AIA rabbit model was developed as described by Consden and colleagues (Consden et al., 1971). Briefly, rabbits were anesthetized by an intravenous injection of pentobarbital sodium and were immunized by 1.2 ml intradermal injections of 6 mg/ml ovalbumin (Sigma-Aldrich, St Louis, MO, USA) in saline emulsified with an equal volume of TiterMax Gold (TiterMax, Norcross, GA, USA). The rabbits were re-immunized in the same manner 30 days later. Seven days after the second immunization, the rabbits underwent skin testing following a 0.1 ml intradermal injection of a solution of 200 μg/ml ovalbumin in saline. Animals exhibiting a welt of 13 mm or greater after 24 hours were confirmed as 'immunized'. Twelve days after the second immunization, the 'immunized' rabbits were anesthetized and arthritis was induced by 0.5 ml bilateral knee intra-articular injections of a solution of 20 mg/ml ovalbumin in saline.

#### **4.1.2 Treatment protocol**

Twenty-four hours after arthritis induction, the rabbits were anesthetized and different doses of ADM (1 ng to 3 μg; Peptide Institute Inc., Osaka, Japan) dissolved in 0.3 ml saline were injected into the knee joint spaces or 0.3 ml saline was injected into the contralateral knee joint spaces as controls. For time-course experiments, ADM and saline were injected into the knee joint spaces daily for 7 days and 20 days. The rabbits were sacrificed on day 8 (*n* = 5 in each group) and day 21 (*n* = 3 in each group).

#### **4.1.3 Joint swelling**

To evaluate the grade of arthritis/inflammation, joint swelling was assessed by measuring the maximum diameter of the swollen joint using calipers. The swelling was compared with that at the same level on the contralateral knee, treated with saline.

#### **4.1.4 Histological evaluation**

For histological evaluation, rabbits were given an overdose of pentobarbital 8 days and 21 days after arthritis induction. The infrapatellar fat pads were harvested from dissected knees and were cut longitudinally, perpendicular to the patella ligament in the middle of the infrapatellar fat pad. The tissues were fixed in 10% buffered formaldehyde and embedded in paraffin wax, and sections 3 μm thick were obtained. The specimens were stained with H & E and Mallory–Azan stains. The area of the infrapatellar fat pad was measured using AxioVision software (release 4.3; ZEISS, Oberkochen, Germany).

These observations lead us to conclude that ADM inhibited the secretion of IL-6 and

Besides the findings in our study, a similar report stated that ADM reduced constitutive production of IL6 from RA synoviocytes in a dose-dependent manner. A high concentration of ADM (>10-8 mmol/l) significantly reduced constitutive production of IL6 compared with a low concentration of ADM (<10-9 mmol/l) (p=0.0029) (Nanke et al., 2003). Then, we tried

**4. Adrenomedullin injection to the knee in antigen-induced arthritis (AIA) in** 

The AIA rabbit model was developed as described by Consden and colleagues (Consden et al., 1971). Briefly, rabbits were anesthetized by an intravenous injection of pentobarbital sodium and were immunized by 1.2 ml intradermal injections of 6 mg/ml ovalbumin (Sigma-Aldrich, St Louis, MO, USA) in saline emulsified with an equal volume of TiterMax Gold (TiterMax, Norcross, GA, USA). The rabbits were re-immunized in the same manner 30 days later. Seven days after the second immunization, the rabbits underwent skin testing following a 0.1 ml intradermal injection of a solution of 200 μg/ml ovalbumin in saline. Animals exhibiting a welt of 13 mm or greater after 24 hours were confirmed as 'immunized'. Twelve days after the second immunization, the 'immunized' rabbits were anesthetized and arthritis was induced by 0.5 ml bilateral knee intra-articular injections of a

Twenty-four hours after arthritis induction, the rabbits were anesthetized and different doses of ADM (1 ng to 3 μg; Peptide Institute Inc., Osaka, Japan) dissolved in 0.3 ml saline were injected into the knee joint spaces or 0.3 ml saline was injected into the contralateral knee joint spaces as controls. For time-course experiments, ADM and saline were injected into the knee joint spaces daily for 7 days and 20 days. The rabbits were sacrificed on day 8

To evaluate the grade of arthritis/inflammation, joint swelling was assessed by measuring the maximum diameter of the swollen joint using calipers. The swelling was compared with

For histological evaluation, rabbits were given an overdose of pentobarbital 8 days and 21 days after arthritis induction. The infrapatellar fat pads were harvested from dissected knees and were cut longitudinally, perpendicular to the patella ligament in the middle of the infrapatellar fat pad. The tissues were fixed in 10% buffered formaldehyde and embedded in paraffin wax, and sections 3 μm thick were obtained. The specimens were stained with H & E and Mallory–Azan stains. The area of the infrapatellar fat pad was measured using AxioVision software (release 4.3; ZEISS, Oberkochen, Germany).

regulated the IL-6 production in the cultured RA synoviocytes.

to inject ADM into the knee in antigen-induced arthritis in rabbits.

**4.1.1 Induction of antigen-induced arthritis** 

solution of 20 mg/ml ovalbumin in saline.

(*n* = 5 in each group) and day 21 (*n* = 3 in each group).

that at the same level on the contralateral knee, treated with saline.

**4.1.2 Treatment protocol** 

**4.1.3 Joint swelling** 

**4.1.4 Histological evaluation** 

**rabbits** 

**4.1 Methods** 

Inflammatory cells, including lymphocytes and plasma cells, were counted in the superficial and deep portions of the infrapatellar fat pads (three fields under ×200 magnification in each portion) in H & E-stained specimens. The inflammatory cell count was performed by two independent observers.

To measure the collagen volume, the images of sections with Mallory–Azan stain were projected onto a color imaging analysis system (Mac SCOPE version 2.3.2; Mitani, Fukui, Japan). In each section, 10 separate sites were analyzed at ×40 magnification. The collagen volume fraction was obtained by calculating the mean ratio of connective tissue to the total tissue area.
