OVX mice had lower pain threshold on the paw-flick test and von Frey test, 4, 6, 8, and 10 weeks after surgery, compared with the sham mice (Fig. 2.2a, b), and the rotarod test showed a lower threshold 6 and 8 weeks after surgery (Fig. 2.2c). OVX mice were treated with BP (OVX + BP) for 2 weeks. The pain thresholds of the OVX mice improved significantly in all tests after administrating BP, and these effects remained 4 weeks after discontinuing BP treatment (Fig. 2.2a-c). The decreased pain threshold value due to OVX was improved significantly 2 weeks after administering BP (OVX + BP). These effects maintained until 10 weeks after discontinuing BP treatment [22].

The serum TRAP5b was significantly related to the pain threshold value, with correlation on the paw-flick test, on the von Frey test, and on the rotarod test. Furthermore, BP’s reduction in pain-like behavior was associated with decreased serum TPAP5b levels.

The number of c-Fos immunoreactive neurons, a marker of nociception, in laminae I–II of the dorsal horn of the spinal cord was significantly greater in the OVX mice than in the sham mice (Fig. 2.3). Those neurons were significantly lower in the OVX + BP mice than in the OVX mice (Fig. 2.3). These results, therefore, suggest that OVX mice with osteoporosis have greater skeletal pain than normal mice. In addition, the higher serum TRAP5b levels associated with OVX decreased significantly following BP treatment and were significantly correlated with pain threshold values [22], further suggesting that the skeletal pain accompanying osteoporosis is possibly associated with osteoclastic activity.

Several studies show that estrogen decrease associated with OVX increases skeletal pain by augmenting nociceptive pathway excitability in the peripheral and central neuronal systems [23–25]. BP may also directly suppress the release of neurotransmitters or inflammatory mediators in the nervous system [26–29]. Estrogen decrease due to OVX may have caused skeletal pain by directly influencing the nervous system in our study. However, more than 80 % of intravenously administered BP is taken up by bone tissue within 24 h and remains in the tissue for a long duration [30]. Furthermore, our results suggest that the BP improvement of pain-like behavior was maintained even after discontinuing treatment for more than 4 weeks (Fig. 2.2). Therefore, the pain-like behavior associated with OVX and the inhibitory effects of BP are likely reflected by bone metabolic changes.

Histochemical findings in bone tissue demonstrated that the number of TRAP-positive osteoclasts in OVX mice was increased in comparison with that in sham mice (Fig. 2.4). In contrast, the number of TRAP-positive osteoclasts in OVX + BP was lower than that in OVX mice (Fig. 2.4). The V-ATPase expression was greater in OVX mice than that in sham or OVX + BP mice, respectively (Fig. 2.5). These findings, which osteoclast activity and V-ATPase expression were increased in OVX mice, indicated that the skeletal pain accompanying osteoporosis is possibly associated with the acidic microenvironment resulting from osteoclast activation [22].

With regard to the induction of pain through acid-sensing nociceptors, two main classes of these nociceptors, transient receptor potential channel vanilloid subfamily member 1 (TRPV1) and acid-sensing ion channels (ASICs), are expressed in the sensory neurons innervating the bone [34, 35] and elicit pain signals when activated by acid stimuli [33, 34]. TRPV1 is a member of a family of polymodal and nonselective cation channels that are predominantly expressed by sensory nerve fibers and sensitized by protons [21] and improved the threshold [22]. ASICs are a major group of acid-sensing nociceptors and are expressed in sensory neurons innervating the bone and known to be related to the acid-induced pain [34, 35] in a similar manner as another important nociceptor TRPV1 [21, 22]. According to our study, the antagonists of TRPV1 or ASIC significantly improved the threshold value for pain-like behavior in OVX mice (Fig. 2.6) [22 and unpublished data]. In general, the secretion of protons by osteoclasts through V-ATPase is known as an acidification pathway during bone resorption [32]. We also demonstrated that a V-ATPase inhibitor significantly improved pain-like behavior in OVX mice (unpublished data). These results support our hypothesis that the acidic microenvironment produced by osteoclasts activates acid-sensing nociceptors and contributes to bone pain.

Extracellular adenosine triphosphate (ATP) and P2X receptors , which is a P2X receptor ligand, are implicated in nociceptive signaling under both normal and pathologic pain states. P2X receptors, which include seven subunits (P2X1–P2X7) belonging to the ligand-gated ion channel family, are activated by extracellular ATP [38, 39]. In the periphery, ATP can be released as a result of tissue injury, visceral distension, or sympathetic activation and can excite nociceptive primary afferents by acting as homomeric P2X3 or heteromeric P2X2/3 receptors [38]. Recent studies have demonstrated that the level of ATP is increased by osteoclast activation under a high bone turnover state in osteoporosis and that several P2X receptor subunits exist in bone tissue [40–42]. Particularly, many studies have demonstrated that P2X7 receptor expressed in osteoclasts or osteoblasts and had significant roles in bone homeostasis or in association with pathogenesis of osteoporosis [43–46]. However, there are relatively few studies concerning expression and function of P2X2 [45] or P2X3 receptors in the bone. A recent study has indicated that a P2X3 receptor could contribute to bone cancer pain through the upregulation of increased local ATP levels in bone tissue [42]. P2X3 receptor, a specific ATP-sensitive ligand-gated ion channel, is selectively localized on peripheral and central processes of sensory afferent neurons and participates in the role of nociceptive signaling [47, 48]. The P2X3 receptor is natively expressed as a functional homomer and as a heteromultimeric combination with the P2X2 receptor [47–49]. Based on the results of those studies, we also hypothesize that increased local ATP underlying a high bone turnover state in osteoporosis activates the P2X receptor in bone tissue, and those activated P2X2/3 might has a role in the induction of skeletal pain.