## 4 Discussion
Ovariectomy resulting in estrogen deficiency caused the significant reduction of BMD in both lumbar spine and femurs. Lack of estrogen changes the course of bone metabolism/mineralization, inducing excessive osteoclast activity and reducing calcium deposition in bone, resulting in weakening of the trabecular bone that forms the porous bone network beneath calcified cartilage to support load-bearing by the joints. Changing the microscopic structure in this area can cause the development of bone cysts, osteophyte formation, flattening and deforming of the subchondral articular contour and all these features are recognized as osteoarthritis [58]. Despite the fact that the rat model does not completely mimic human OA because of differences in growth plate activity during adulthood, patterns of cartilage loading, and spontaneous intrinsic healing of cartilage lesions [59], rats in addition to mice and rabbits [60] are the main animal models used in OA research. Their main advantages are cost efficiency and manageability, and the rat model provides crucial evidence for further study in other larger animals. This species is not considered as a spontaneous OA model like guinea pigs, STR/1N mice, or C57/BL mice, but it is commonly used in a chemical induction model using intraarticular injection of adjuvant, collagen, iodoacetate, or papain [60; 61]. Just in the last decade, high-energy-diet-induced OA models have been developed, first in mice [62], then rabbits [63], and most recently rats [15]. As a new animal model, evidence is still inconsistent. Our study revealed interesting and novel information about this model. First, young female rats (26 weeks of age) did not spontaneously evolve explicit pathological lesions in knee joints; however, the older rats (48 weeks of age) revealed some pathological changes by the Mankin score. Even though the lesions in the cartilage layers appeared to be only a modest feature of OA, the changes were significant after time; a similar finding has been reported [64]. This means that age also contributes to OA development at subclinical levels in the rat models. At 48 weeks of age, all rats showed at least mild features of cartilage attrition. As low grade inflammation due to HFHS diet and bone loss from estrogen deficiency progressed, cartilage destruction became more pronounced and distinct only in one particular group. This cumulative evidence can explain the multiple phenotypes of osteoarthritis observed in this rat model. Interestingly, plasma concentrations of CTX-II, the cartilage degradation marker, did not correlate with the pathological changes in knee joints. In a matching study, we did detect a significant increase of CTX-II in younger rats correlating with obesity and the initiation of Met-OA [31]. A similar study showed that serum CTX-II dramatically increased in OVX rats within 3 weeks of surgery, then declined to the baseline level six weeks later OVX [65]. Likewise, [PERSON] et al. [66] monitored serum CTX-II in 5-month-old ovariectomized rats and found that CTX-II transiently increased by approximately 50% within 2 weeks after surgery and then declined afterward to 50% below baseline at week 8. CTX-II in the sham rats also declined as the same level of the OVX rats. This indicates that the sensitivity of serum CTX-II as a cartilage marker is of less value in rats older than 6 months. This ambiguity is likely due to the limitation of the model, as mature rats retain growth plate function. To overcome this drawback, CTX-II levels should be monitored immediately after the second week of surgery. Another limitation of this study is due to the lack of revelation in the precise pathological mechanisms driving the development of the disease and the anti-osteoarthritis effect of GSM. This may be an interesting gap for other studies in the future. However, no adverse effects of GSM were noted, suggesting that the findings in the current pre-clinical study warrant clinical trials with whole GSM powder.