Evaluation of bone toxicity in various bones of aged rats

Abstract

The aim of the present study was to provide a method for evaluating bone toxicity induced by drugs in various bones in aged rats. Male Crl:CD (SD) rats at 46 weeks of age were administered 15 mg/m(2) body surface area of doxorubicin, which effects the growth plate in weanling rats, weekly for 9 weeks by intravenous injection, and the femur, sternum, humerus and tibia were examined histopathologically. In the doxorubicin-treated group, thinning of the growth plate was remarkably observed in the proximal tibia and humerus; however, these changes were not observed in other regions. In addition, the osteoclast number per bone perimeter in the proximal tibia was significantly higher than others in control aged rat. Thus, recognizing the various histological reactions related to the time of epiphyseal closure is important for evaluating bone toxicity in aged rats.

Keywords: bone toxicity; doxorubicin; epiphyseal closure; growth plate; rat.

Fig. 1.

Histopathological changes of the growth plate in aged rats. The growth plates (GP) of the (a) humerus, (b) femur and (c) sternum are shown for the control group. The GP of the (d) humerus, (e) femur and (f) sternum are shown for the DOX-treated group. (a–c) The GP in the humerus is thicker than those of the femur and sternum in the control. (d) In comparison of the humerus between the control and DOX-treated animals, the GP in the DOX-treated group was obviously thinner than that in the control. In addition, the zone of hypertrophy and calcification and the zone of cartilage degeneration decreased or disappeared. (e) Remarkable thinning of the GP was not observed in the femur of the DOX-treated group, and the zone of hypertrophy and calcification and the zone of cartilage degeneration almost disappeared in both groups, though the zone of cartilage degeneration partially remained in the femur. (f) The thinning of the GP was not remarkably observed in the sternum of the DOX-treated group, and the zone of hypertrophy and calcification and the zone of cartilage degeneration practically disappeared in both groups. Moreover, the osteogenic zone was thick in both groups. HE staining. Scale bars indicate 100 µm.

Fig. 2.

Growth plate heights of the distal femur, sternum, proximal tibia and humerus after treatment with DOX in aged rats. The growth plates of the tibia and humerus in the DOX-treated group became significantly thinner than those in the control group. Values are means ± SEM (n=3–4 animals/group). * p<0.05 for comparisons of the humerus between the DOX-treated group and control group. ** p<0.01 for the comparisons of the tibia between the DOX treated group and control group.

Fig. 3.

Ob.N/B.Pm of the distal femur, sternum, proximal tibia and humerus after treatment with DOX in aged rats. The Ob.N/B. Pm of the femur and sternum in the DOX-treated group tend­ed to be higher than those in the control group. Values are means ± SEM (n=3–4 animals/group).

Fig. 4.

Oc.N/B.Pm of the distal femur, sternum, proximal tibia and humerus after treatment with DOX in aged rats. The Oc.N/B. Pm of the tibia in the control group was significantly higher than that of the femur in the control group. Values are means ± SEM (n=3–4 animals/group). # p<0.05 for comparisons be­tween the femur and tibia in the control group.

Fig. 5.

Changes of histopathological and immunohistochemical staining in chondrocytes of the proliferative zone in the growth plate of the tibia. (a) The growth plates (GP) of the young control animals were positive for PCNA expression in the nuclei of the chondrocytes. (b) The GP of the aged control animals showed no obvious staining for PCNA. (c) The GP of the aged DOX-treated animals showed no obvious staining for PCNA. (d) The growth plates of the young animals were relatively thick (HE staining). (e) The GP of the aged control animals were thinner than those of the young animals, particularly in the zone of hypertrophy and calcification and the zone of cartilage degeneration (HE staining). (f) The GP of the DOX-treated animals were much thinner than those of the aged control animals, and in approximately half of the animals, the zone of hypertrophy and calcification and the zone of cartilage degeneration had decreased or disappeared (HE staining). Scale bars indicate 100 µm.