Following right femora fracture, x-ray images were immediately obtained by using X-ray machine (Proteus XR/a, GE, UK) to confirm both the intramedullary placement of the K-wire and the fracture (figure 1). Each rat was then separately housed

in a clean cage. To prevent infection antibiotic Baytril 5% at a dose of 10 mg/kg (Bayer, Thailand) was administered intramuscularly daily for seven days as well as daily dressing of the incision wound with Povidone Inhibitors,research,lifescience,medical iodine solution. On following day after femora fracture, the sham (SO) group (n=6) was started on supplement with normal saline as vehicle, while the ovariectomizedgroup (n=18) was further randomly assigned into three groups: (i) ovariectomized-control (OVXC) group treated with normal saline as vehicle; (ii) ovariectomized+estrogen replacement therapy Inhibitors,research,lifescience,medical (ERT) group, treated orally with conjugated equine estrogen (Premarin-Wyeth, Canada) at dose of 100 µg/kg/day,21 (iii) ovariectomized+ P.s (P.s) group, treated orally with P.s water extract at dose of 125 mg/kg.18 Following fracture, all the rats received the above treatment by oral gavage for another six weeks. After treatment, the rats were sacrificed with over dose of diethyl ether. The right femora were dissected from the hind limbs and

stained with Inhibitors,research,lifescience,medical Hemaetoxylin and Eosin (H&E) for histological assessment. Histological Analysis Using H&E Stain Tissue Preparation After the sacrifice, the right femora were dissected from the hind limbs of the rats, cleaned from soft tissues and the K-wires were removed. Neutral Inhibitors,research,lifescience,medical buffered formalin 10% was prepared and specimens of right femora were taken and fixed in that solution for at least 24 hours. Ethylenediaminetetracetic acid (EDTA) is a chelating agent, and has been the preferred decalcifying agents due to the facts that EDTA is gentle and slow acting, and preserves tissue components as compared to formic acid. All samples were decalcified by using EDTA 10% solution for 12 Inhibitors,research,lifescience,medical weeks as per previous protocol.22 In addition, the EDTA solution was changed every five days. The samples were placed in a warm

place and agitated daily to accelerate decalcification process. Decalcified bone samples were assessed by pricking with a sharp needle. As per previous protocols, following decalcification all bone samples were dehydrated to remove the water content using increasing 70% of Ethanol solution.23 Calpain Samples were immersed in equal parts of Alcohol: Toluene and samples were then cleared by Toluene. Finally bone samples were embedded in suitable containers with melted paraffin wax and stored at -4°C. Paraffin blocks were sectioned see more longitudinally by microtome (Leica RM 2235) at 5 µm thickness. The sections were stained with H&E stain and assessed by image analyzer. Figure 1 Radiograph image of right femur after fracture (A), and right femur sample harvested after sacrifice (B).