Effects Of Estrogen Replacement On Osteoporosis In Ovariectomized Rats Accompanied With Unilateral Disuse

Part Ⅰ. Establishment and evaluation of osteoporosis model by sciatic neurectomyBackground:Mechanical loading plays a crucial role in the maintenance of normal bone remodeling and structure. The reductions in mechanical loading such as immobilization result in a dramatic loss of bone mass and deterioration of bone microarchitecture, which is known as disuse osteoporosis. There are various methods to establish osteoporosis model by disuse. Sciatic neurectomy (SN) is one of the most common methods.Purpose:The purpose of this part was to evaluate the effect of SN on the rat activity, bone mass, and bone microarchitecture in male and female rats.Methods:Thirty-two male and thirty-two female Sprague-Dawley rats were separately divided into two groups randomly (n= 16/group):one group were subjected to SN and subdivided into short-term disuse group (disused for 2 weeks; 2w-DIS, n= 8) and long-term disuse group (disused for 8 weeks; 8w-DIS, n= 8), the other group were subdivided into two control groups (2w- and 8w-CTRL, n= 8/group), matching the disuse groups, respectively. At 1 day,2 weeks and 8 weeks post-surgery, the activity of the hindlimb in the animals was evaluated. The bone mass and microarchitecture of the proximal tibia were investigated by micro-Computed Tomography (micro-CT) and histology at 2 weeks and 8 weeks after surgery.Results:(1)The mobility of hip flexion, hip stretch, and knee stretch in both male and female disuse rats was influenced in the early phase after surgery, and the evaluation scores were decreased. However, at 2 weeks post-surgery, the mobility recovered to the normal level, and the result was the same at 8 weeks after surgery. The mobility of ankle flexion, ankle stretch, and knee flexion was completely lost throughout the entire study with evaluation scores of 0. (2) Micro-CT analysis showed that the bone volume and trabecular thickness (Tb.Th) in both male and female disuse rats were significantly decreased compared with those in the corresponding control rats. The male and female rats showed similar results by micro-CT. (3) Histological analysis confirmed bone volume in the disuse rats (2w-and 8w-DIS) in either male or female rats was significantly decreased compared with those in the time-matched control rats, which was consistent with micro-CT analysis.Conclusion:(1) Sciatic neurectomy causes a considerable loss of bone mass and deterioration of bone microarchitecture in the animal model. Sciatic neurectomy is an effective method to establish disuse-osteoporosis model. (2) The establishment of disuse-osteoporosis by sciatic neurectomy shows no significant gender bias.Part Ⅱ. Site-specific bone response to mechanical unloading in disuse-osteoporosisBackground:Sclerostin, coded by SOST gene, is an osteocyte-specific cysteine knot-secreted glycoprotein and a potent inhibitor of bone formation. Mechanical unloading induces different changes of sclerostin expression in the previous studies. The effect of mechanical unloading on the SOST expression is controversial.Purpose:Most reported studies regarding the changes caused by mechanical unloading were only based on a single site. Considering that the longitudinal bone growth leads to cells of different age with different sensitivity to unloading, we hypothesized that bone turnover in response to unloading is site-specific.Methods:Thirty-two male Sprague-Dawley rats were randomly divided into four groups (n=8/group):2w-CTRL,2w-DIS,8w-CTRL,8w-DIS, which were consistent with Part Ⅰ. We evaluated the bone mass and microarchitecture at various sites of tibia by micro-Computed Tomography (micro-CT) and histology. At different sites or system, we investigated sclerostin/SOST expression by immunohistochemistry, quantitative reverse transcription polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA); evaluated tartrate resistant acid phosphatase 5b (TRAP 5b) by ELISA and TRAP staining. A reliable marker of the resorbing activity of osteoclasts, CTx, was also evaluated by ELISA.Results:(1) Micro-CT and histological analysis confirmed the bone volume at different sites of tibia in the disuse rats was significantly decreased compared with control rats. (2) Compared with the control groups, SOST mRNA expression in the diaphysis was down-regulated at both week 2 and 8 post-surgery. On the contrary, the percentage of sclerostin-positive osteocytes showed an up-regulated response in the 5-6 mm region from the growth plate in 2w- and 8w-DIS tibia, while the percentage had no significant difference in the 2.5-3.5 mm region. Nevertheless, in 0.5-1.5 mm region, the percentage of sclerostin-positive osteocytes had no significant difference between 2w-DIS and 2w-CTRL group, decreased at 8 weeks after disuse compared with 8w-CTRL group, and elevated in 8w-CTRL group compared with 2w-CTRL group.(3)There were different responses of osteoclasts (OC) to mechanical unloading at different sites. In primary spongiosa, Oc.S/N.Oc (osteoclast surface/osteoclast number) and Oc.S/BS (osteoclast surface/bone surface) in 2w- and 8w-DIS groups increased significantly and N.Oc/B.Pm (osteoclast number/bone perimeter) has no significant difference, compared with corresponding CTRL groups. In secondary spongiosa, Oc.S/N.Oc, Oc.S/BS, and N.Oc/B.Pm in 2w-DIS group were significantly increased compared with the corresponding CTRL groups, while there were no significant difference between 8w-DIS and 8w-CTRL groups. The marker of osteoclast number, serum TRAP 5b, showed no significant difference between 2w-DIS and 2w-CTRL, but was significantly lower in 8w-DIS than in 8w-CTRL. The marker of osteoclast activity, CTx, showed a significant elevation in 2w-DIS group compared with 2w-CTRL group, while there was no significant difference between 8w-DIS and 8w-CTRL groups.Conclusion:(1) The bone mass and bone microarchitecture at different sites of tibia have similar tendencies of bone loss. (2) There are site specific and time specific sclerostin/SOST expression by osteocytes in the mechanical unloading tibia. (3) Osteoclasts at different sites of tibia show different responses to mechanical unloading. Our data indicate that unloading-induced changes in bone turnover are probably site specific.Part Ⅲ. Impacts on bone tissue by the combination of estrogen-deficiency and disuseBackground:Estrogen and mechanical loading play integral roles in the maintenance of bone. Osteoporosis affects a large number of women aged 50 years and older. It is estimated that 90% of women aged 75 years and older suffer osteoporosis. Frequent occurrences of cardia-cerebrovascular disease and traffic accidents result in large numbers of old women with hemiplegic, paraplegic and bedridden. However, few studies explored the changes of osteoporosis by the combination of estrogen-deficiency and immobility.Purpose:The objective of the current study was to evaluate the ongoing development of osteoporosis induced by the ovariectomy (OVX) combined with unilateral sciatic neurectomy (SN) in a rat model, and investigate the ongoing changes of bone markers in this process.Method:Ninety-six female Sprague-Dawley rats were divided randomly into three groups (n= 32/group):OVX combined with unilateral SN (OVX+SN), sham-OVX combined with unilateral SN (sham-OVX+SN) and sham operation as control (CTRL). Eight rats from each group were separately euthanized at days 3,7,14, and 28 post-surgery. The ongoing changes on the parameters of bone mass and trabecular microarchitecture (BV/TV, BS/TV, BS/BV, Tb.Th, Tb.N and Tb.Sp) were analyzed using micro-Computed Tomography (micro-CT) and histology. Sclerostin/SOST ongoing expression was investigated by immunohistochemistry, quantitative reverse transcription polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). The ongoing changes of osteoclasts (Oc.S/N.Oc, Oc.S/BS and N.Oc/B.Pm) were evaluated by TRAP 5b (tartrate resistant acid phosphatase 5b) staining and ELISA. In addition, serum markers of bone resorption, including C-terminal telopeptides of type Ⅰ collagen (CTx), receptor activator for nuclear factor κB ligand (RANKL), adiponectin, and vascular endothelial growth factor (VEGF), were quantified by ELISA.Result:(1) Micro-CT analysis showed that BV/TV, Tb.Th, and Tb.N in sham-OVX+SN and OVX+SN rats declined gradually, and Tb.Sp elevated gradually after surgery. The parameters analyzed by micro-CT in OVX+SN group were more significant than those in sham-OVX group. BV/TV by histological analysis indicated similar results with micro-CT analysis. (2) Sclerostin expression by immunohistochemistry analysis showed in sham-OVX+SN rats, there was an ongoing-increased trend and significant elevation until 28 days post-surgery compared with CTRL; in OVX+SN rats, significant increase had appeared at 7 days after surgery and remain until to the end of the study. SOST mRNA in diaphysis of tibia indicated that in OVX+SN group, an increase appeared at 7 days post-surgery and a reduction at 28 days; in the sham-OVX+SN group, there was significant elevation at 14 days. Serum sclerostin showed no significant difference in both sham-OVX+SN and OVX+SN at 3,7,14,28 days after operation. (3) Compared with corresponding CTRL group, Oc.S/N.Oc, Oc.S/BS related to osteoclasts in OVX+SN group showed gradual increase and significant difference from 3 days to 14 days post-surgery, and reduced at 28 days; N.Oc/B.Pm became significantly high at 7 days post-surgery, and no significant difference at 3,14,28 days. In sham-OVX+SN rats, Oc.S/N.Oc, Oc.S/BS elevated gradually after surgery, and Oc.S/N.Oc became significantly different at 28 days, and Oc.S/BS showed no significant difference; while there was no significant difference in N.Oc/B.Pm between 3 days and 28 days post-surgery. (4) In OVX+SN group, serum CTx became elevated gradually, reached to peak at 14 days and reduced to normal level at 28 days. In sham-OVX+SN group, there was no significant difference in serum CTx. Compared with CTRL, serum RANKL showed no significant difference in sham-OVX+SN rats, showed significant elevation in OVX+SN at 28 days post-surgery.Conclusion:(1) The combination of estrogen-deficiency and mechanical unloading results in dramatic and significant bone loss and microarchitecture deterioration. (2) The surface area of single osteoclasts and the activity of osteoclasts are dramatically increased by estrogen-deficiency and mechanical unloading. (3) The sclerostin expression on osteocytes is elevated and accelerated by estrogen-deficiency and mechanical unloading.Part Ⅳ:Effects of estrogen replacement on osteoporosis in ovariectomized rats accompanied with unilateral disuseBackgroud:It is widely known that estrogen replacement prevents bone loss resulting from estrogen deficiency in postmenopausal women. There are large numbers of postmenopausal women companied with immobility due to the frequent occurrences of cardia-cerebrovascular disease and traffic accidents. It is clinically important to determine whether estrogen replacement has a positive effect on postmenopausal women combined with mobility difficulties, due to the potential risks of estradiol.Purpose:The objective of the current study was to investigate the effect of estradiol replacement on osteoporosis induced by the ovariectomy (OVX) combined with unilateral sciatic neurectomy (SN) in a rat model.Method:One hundred and twenty-eight female Sprague-Dawley rats were randomly divided into four groups (n= 32/group):OVX combined with unilateral SN treated with 17β-estradiol (E2) (OVX+SN+E2); the same three groups in the Part Ⅲ, including OVX+SN, sham-OVX+SN and CTRL. Eight rats from each group were separately euthanized at days 3,7,14, and 28 post-surgery. The same analytical methods were used in Part Ⅲ.Results:(1) Micro-CT analysis showed that BV/TV, Tb.Th and Tb.N in sham-OVX+SN, OVX+SN and OVX+SN+E2 rats decreased gradually, and Tb.Sp elevated gradually after surgery. E2 treatment of OVX+SN rats improved the preservation of the bone volume fraction (BV/TV) in the tibias at day 14 post-surgery, which was 46%(micro-CT) and 58%(histology) higher in OVX+SN+E2 rats than that in OVX+SN rats, Tb.Th 14% higher, Tb.N 47% higher, and Tb.Sp 24% lower. However, BV/TV, Tb.Th, Tb.N, and Tb.Sp were at similar level at day 28 post-surgery between OVX+SN+E2 and OVX+SN. (2) Expression of sclerostin in tibias of OVX+SN rats analyzed by immunohistochemistry was significantly elevated at day 7 post-surgery compared with the CTRL, while OVX+SN with E2 replacement appeared significant increase until day 14 post-surgery. SOST mRNA in diaphysis in OVX+SN and OVX+SN+E2 groups showed similar tendency. (3) The relative parameters of osteoclasts, Oc.S/N.Oc, Oc.S/BS, and N.Oc/B.Pm in OVX+SN+E2, showed similar tendency to those in sham-OVX+SN group, but appeared to have completely different trend compared with OVX+SN group. Oc.S/N.Oc in sham-OVX+SN and OVX+SN+E2 increased gradually after surgery, while there was significant increase in OVX+SN+E2 between 7 day and 28 days. The ongoing increased trend of Oc.S/BS in OVX+SN+E2 was also similar to sham-OVX+SN, while the former became significant elevated at day 28 post-surgery and the latter showed no significant difference. N.Oc/B.Pm in both OVX+SN+E2 and sham-OVX+SN groups showed no significant difference at day 3,7,14,28 post-surgery. (4) Serum CTx in OVX+SN rats was elevated gradually after surgery, and became significantly increased at 14 days post-surgery, while decreased at 28 days. However, there was no significant difference in serum CTx level in both OVX+SN+E2 and sham-OVX+SN groups compared with CTRL group. Compared with CTRL, serum RANKL showed significant increase in OVX+SN+E2 and OVX+SN rats at day 28 post-surgery, while there were no significant difference in sham-OVX+SN rats.Conclusion:(1) Estrogen replacement could transiently protect against bone loss in OVX rats combined with mechanical unloading. However, the longer-term administration of estrogen replacement should be cautious. Bone markers, CTx and RANKL, might imply the extent of bone destruction. (2) The up-regulation of sclerostin expression appears to be transiently delayed by E2 treatment in our models. (3) In OVX+SN model, the surface area of single osteoclasts and the activity of osteoclasts were suppressed with estrogen replacement.