PS11-24. Multi-dose Clodronate Disodium Use on Osteoclast Recruitment and Apoptosis in Juvenile Quarter Horses.

Abstract Clodronate disodium (CD) is a bone modulating bisphosphonate approved for navicular disease in horses > 4 years of age. Anecdotal reports of use in juvenile horses undergoing exercise leads to concerns regarding impacts of CD on skeletal development and bone healing. Therefore, the objectives were to determine the effects of CD administration on osteoclast recruitment, number, and apoptosis, hypothesizing that immunohistochemical markers of osteoclast recruitment would remain the same but osteoclast apoptosis would be higher following multiple doses of CD. To test this, 16 juvenile Quarter Horses were stratified by age (499 ± 12 d), BW (335 ± 31 kg), sex (n = 9 geldings; n = 7 fillies), and initial bone optical density and randomly allocated to treatment groups. Treatments included four-doses CD (4X; n = 8; d 0, 42, 84, and 126; 1.8 mg/kg BW CD Osphos®) and control (CON; n = 8; isovolumetric saline). Horses were housed individually in stalls and exercised 5 d/wk using a free stall exerciser in a phase-based progressive workload. Phase I (d 0-84) simulated sales preparation. Phase II (d 85-168) mimicked early training. Tuber coxae bone biopsies were performed on d 84 (left) and on d 168 (ipsilateral and contralateral). Samples were paraffin embedded and stained for receptor activator of NF-kB ligand (RANKL) and apoptosis. Resulting images captured at 40x magnification were analyzed for percent area positively stained for RANKL (recruitment), number of osteoclasts, and percent of apoptotic osteoclasts. Data were analyzed using PROC MIXED in SAS for main effects of treatment, sample site, and treatment x sample site interaction. There were no treatment or sample site effects for RANKL (P > 0.19). There was a treatment x sample site interaction for percent area positively stained for RANKL (P < 0.01), where RANKL tended to be higher in 4X in the d 168 contralateral samples compared to CON, but was lower in 4X compared to CON in the ipsilateral samples where metabolic activity is highest at the healing site of initial biopsy. There were no treatment differences or interactions for number of osteoclasts or percent apoptotic osteoclasts (P > 0.11). There were differences between sample sites (P < 0.01) where there were more osteoclasts and a higher percentage of apoptotic osteoclasts in ipsilateral samples. The results indicate that osteoclast recruitment critical for bone resorption was reduced at a healing site in horses administered 4 doses of clodronate. Additionally, more osteoclasts and higher percent of apoptotic osteoclasts in bone that had previously undergone biopsy highlight the importance of osteoclasts’ role to repair damaged bone. Based on these results, the current study does not support the use of bisphosphonates such as CD in juvenile horses. Funding: AQHF#406, AFRI#2021-67015-34079