In order to describe intragraft chimerism in detail, we apply a new method with laser capture microdissection of accurately selected areas of new bone formation in bone allotransplants. We aim to describe the lineage of cells in allotransplants as compared to isotransplants and study its progress over time. National Institutes of Health guidelines were followed and approval was obtained from our Institutional Animal Care and Use Committee. A VBAT model previously designed in our laboratory was used (Fig. 1A). Eleven female Dark Agouti
rats (DA, RT1a) served as donors in the allotransplant groups. Ten female Piebald Viral Glaxo rats (PVG; RT1c) served as donors in the isotransplant groups. Male Piebald Virol Glaxo rats (PVG; RT1c) served as recipient U0126 price rats, providing a major histocompatibility mismatch for the DA donor rats. In the allotransplant group, 22 PVG rats CH5424802 datasheet were included with survival at two different time points: 4 weeks (group A, n = 11) and 18 weeks (group B, n = 11). Twenty PVG rats were allocated to the isotransplant groups with two survival periods: 4 weeks (group C, n = 10) and 18 weeks (group D, n = 10). Rats were allocated randomly to each
group. The female donor rat was anesthetized with ketamine (90 mg/kg IM) and xylazine (10 mg/kg IM) and the right femur was dissected with its nutrient vascular pedicle including the proximal common iliac artery and vein for later anastomosis. Next, the proximal and distal parts of the femur were resected, leaving a 20 mm femoral diaphyseal segment with its pedicle.
The intramedullary canal was reamed and the pedicle rinsed with heparinized saline. Next, a male PVG rat was anesthetized and the right femoral artery and vein were ligated. End to end anastomosis was performed. The contralateral saphenous arteriovenous bundle was dissected and implanted filipin into the full length of the donor bone intramedullary canal. The allotransplant was wrapped in a silicone sheath and placed in an abdominal subcutaneous pocket. Rats in all groups received daily intramuscular injections of FK-506 (1 mg/kg/day IM; Tacrolimus, Fujisawa Pharmaceutical Co., Osaka, Japan) during the first 2 weeks postoperatively. Animals were given calcein green and tetracycleine orange fluorescent labels 14 and 2 days, respectively, prior to sacrifice. These labels are absorbed in active bone remodeling areas, which allow clear microscopic identification of these areas (Fig. 1B). Rats were anesthetized with ketamine (90 mg/kg IM) and xylazine (10 mg/kg IM). To ensure that cortical bone was completely cleared from blood cells that could interfere with accurate cell heritage quantification, the vena cava and aorta were cannulated and the lower extremity irrigated with heparinized saline.