Roles of estrogen on sensory nerve innervation of bone in osteoprosis-induced female rats

Abstract

entation. Sixty female Wistar rats aged 6 months were randomly assigned into 5 groups. Group 1-4, which were OVX-, E2 1μg, E2 5 μg or E2 15 μg, underwent bilateral ovariectomy and received daily subcutaneous injection of 10% DMSO in propelyne glycol the drug vehicle, 1, 5 or 15 μg/kg BW of 17-β-estradiol, respectively. Group 5 was sham operated received the same treatment as ovariectomy group but ovaries were not removed. All of them were treated for ninety consecutive days. During the treatment, daily feed intake and body weight were measured. At the end of the treatment, all rats were anesthetized and sacrificed by cardiac puncture. Blood sample was collected to analyze for calcium and phosphorus concentration in serum. Some part of blood sample was allowed to clot, and serum was collect and kept at -20 oC for bone-specific alkaline phosphatase (AP) measurement. Uterine weight was determined immediately after removal. The left femur was kept at -20 oC for measurement of bone mass density (BMD) and calcium content using dual-energy x-ray absorptiometry (DEXA) and atomic absorption spectrophotometry, respectively. The right femur was prepared for immunohistochemistry study (IHC). We showed that body weight gain with no alteration of feed intake in OVX group was increased compared with sham group (p<0.05). The changes were prevent by daily estrogen injection at dose of 1, 5 or 15 μg/kg BW (p<0.05). Ovariectomy for 90 days without estrogen supplementation in the present study of pubertal rats significantly reduced the total bone mass density but not bone calcium content. The bone turnover marker, both total and bone specific AP were highest in OVX group. The evidence suggested that bone loss was evident after 3 months ovariectomy. The part of femoral bone of the OVX group that predominantly showed lowest BMD was the distal part. The IHC was also performed by using the neuronal and sensory neurotransmitter marker antibodies, anti-PGP 9.5 and anti-calcitonin gene related peptides (anti-CGRP) and anti-substance P antibody, respectively to stain on the semi-thin section of femoral bone, followed with an appropriate secondary antibody conjugated with fluorochrome, FITC or Texas red. The IHC results revealed a dramatic decrease of PGP-9.5, CGRP and SP-immunoreactivity (ir) in the distal part of femur of OVX group. The up-regulation of CGRP- ir but not SP-ir was indicated in the proximal part of femur which did not show evident of bone loss in the OVX group. All of these changes prevented by daily estrogen injection (1 μg/kg BW). The results of this study indicated the significance of nerve containing CGRP in the regulation of bone metabolism to protect against bone loss.