Weight-bearing asymmetry and vertical activity differences in a rat model of post- traumatic knee osteoarthritis  C.B. Hamilton, M.A. Pest, V. Pitelka, A. Ratneswaran, F. Beier, B.M. Chesworth  Osteoarthritis and Cartilage  Volume 23, Issue 7, Pages 1178-1185 (July 2015) DOI: 10.1016/j.joca.2015.03.001 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Schematic of experimental protocol. Three months old male Sprague–Dawley rats were acclimatized for 1 week. They then underwent ACLT + pMMx (n = 6), sham surgery (n = 5), or were naïve (n = 5). At the same time, a drug delivery pump with distilled water as vehicle was installed under the skin in the midsaggital region of the back. Pump revision surgery was done at 4 weeks after induction surgery to install a newly filled pump to replace the exhausted pump. The rats were followed for 8 weeks after ACLT + pMMx or sham surgery, and static weight-bearing and exploratory motor behaviour was assessed at five and three post-surgery time points, respectively. Two alternate groups were followed for 4 weeks post-surgery, to access early knee structural changes. Osteoarthritis and Cartilage 2015 23, 1178-1185DOI: (10.1016/j.joca.2015.03.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Evidence of OA in ACLT + pMMx operated rats as shown by histology. (A) Representative toluidine blue stained sections of knee joints demonstrates histological changes congruent with early to mid OA in ACLT + pMMx (n = 6) but not sham operated animals (n = 5). Cartilage degeneration (red arrowheads) and osteophyte development (yellow asterisks) are indicated. (B) At 4 and (C) 8 weeks post-surgery, ACLT + pMMx rats showed significantly higher OARSI cartilage degeneration scores in the MTP and MFC of operated knee joints. **P < 0.005, Mann–Whitney statistical test. The values are presented as mean with 95% CI for each study group. T = medial tibia, F = medial femur. Osteoarthritis and Cartilage 2015 23, 1178-1185DOI: (10.1016/j.joca.2015.03.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Percent ipsilateral weight-bearing between rat groups. Percent ipsilateral weight-bearing assessed static weight-bearing as a likely indicator of pain. Percent ipsilateral weight-bearing was consistently statistically significantly lower for the ACLT/pMMx rats when compared to the sham operated rats. *At each time point, the groups had statistically significant difference at adjusted P < 0.001. The values are presented as means with 95% CI for each study group at each assessed post-surgery time point. Osteoarthritis and Cartilage 2015 23, 1178-1185DOI: (10.1016/j.joca.2015.03.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Vertical episode count differed between rat groups. Vertical episode count was one of eighteen variables used to assess dynamic pain-related behaviour. It is one of the three vertical activity variable that consistently showed statistically significantly lower values for the ACLT + pMMx rats when compared to the sham operated rats from 4 through 8 weeks post-surgery. *At each time point, the groups had statistically significant difference at adjusted P < 0.05. The values are presented as means with 95% CI for each study group at each assessed post-surgery time point. Osteoarthritis and Cartilage 2015 23, 1178-1185DOI: (10.1016/j.joca.2015.03.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Supplementary Fig. 1 No evidence of OA found in the lateral knee joint compartments of ACLT + pMMx operated rats. (A) Representative toluidine blue stained sections of knee joints demonstrates healthy lateral compartment cartilage in anterior cruciate ligament transection and partial medial meniscectomized (ACLT, n = 6) and sham operated animals (n = 5). (B) At 4 and (C) 8 weeks post-surgery, ACLT + pMMx rats showed very minor but statistically significantly higher OARSI cartilage degeneration scores in the lateral tibial plateau (LTP) and lateral femoral condyle (LFC) of operated knee joints. (D) Representative toluidine blue stained sections of the medial knee joint synovium shows minor fibrosis in ACLT + pMMX rats. (E) Representative toluidine blue stained sections of the lateral knee joint synovium shows comparable histology between ACLT + pMMX and sham operated rats. *P < 0.05 **P < 0.005, Mann–Whitney statistical test. The values are presented as mean with 95% CI for each study group. T = lateral tibia, F = lateral femur. Osteoarthritis and Cartilage 2015 23, 1178-1185DOI: (10.1016/j.joca.2015.03.001) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions