The skeletal system is a common site for cancer metastasis, among which the most common site is the vertebrae, followed by the pelvis and long bones [1]. When tumors metastasize to bone, stimulation of tumor cells leads to local inflammation, which increases the activity of osteoclasts, osteoclast activity, leading to bone destruction. At the same time, nerve endings are damaged by tumor invasion. Therefore, both inflammatory and neuropathic injuries exist in bone cancer lesions, so bone cancer pain is a special chronic pain with characteristics of both inflammatory and neuropathic pain[1–2]. In this study, according to the method of Liu et al. [21], SHZ-88 breast cancer cells were directly injected into the tibial medulla cavity of female SD rats. The injection hole was sealed with sterile bone wax to restrict tumor cells within the bone marrow and prevent tumor from invading surrounding soft tissues. As a result, tibial bone destruction and hyperalgesia were observed in rats inoculated with SHZ-88 cells, suggesting that the bone cancer pain model was successfully established.
HCN channels are widely distributed in sensory neurons of the peripheral and central nervous system. Previous studies have shown that HCN channels of dorsal root ganglion and spinal cord are involved in the occurrence of pathological pain [9–10, 29–31]. It is well known that the midline structure of brain constitutes the endogenous analgesic system. Among them, the midbrain periaqueductal gray (PAG), especially ventrolateral region (vlPAG), is an important neural circuit for regulating pain in the endogenous analgesic system [32–33]. Ding et al. and Zhang et al. found that microinjection of HCN channel blocker ZD7288 into amygdala and/or thalamus significantly reduced mechanical and thermal hyperalgesia in CCI rats [17, 34]. Recent studies have shown that HCN1 and HCN2 channels are expressed in vlPAG [35–37]. Whether HCN channels in vlPAG are involved in the occurrence of bone cancer pain remains unclear. In this study, the expression of HCN1 and HCN2 channels and the amplitude of Ih current all increased in vlPAG of rats with bone cancer pain, indicating that bone cancer can lead to the overactivation of HCN channels. In addition, intravlPAG injection of HCN channels antagonist ZD7288 can significantly reduce mechanical and thermal hyperalgesia in bone cancer rats, further suggesting that the hyperactivity of HCN channel promotes bone cancer pain.
Abnormal ectopic firing of sensory neurons is involved in the pathogenesis of pain states. Studies have shown that the hyperactivity of HCN channels drive abnormal discharge, resulting in hyperexcitability of sensory neurons, which contributes to the development of pathological pain [24, 38]. Moreover, studies have shown that blockage of HCN channels significantly reduce the ectopic discharges and pain behaviors caused by inflammation and peripheral nerve injury [9, 15, 19, 31, 39–43]. In this study, the amplitude of Ih in vlPAG neurons of bone cancer rats was significantly upregulated, leading to hyperexcitability of vlPAG neurons, which may impair the function of descending endogenous analgesia system and promote bone cancer pain.
It is reported that overactivation of cAMP-PKA signaling promotes the occurrence of pathological pain [44]. The cAMP-PKA signaling is also an important modulator of HCN channel activation. As a positive HCN channel modulator, cAMP promotes the opening of HCN channels by affecting the structure of the C-linker and cyclic nucleotide-binding domain [28]. In this study, the content of cAMP in vlPAG of bone cancer rats was significantly increased, which means that the increased cAMP level may promote the opening of HCN channels, resulting in hyperexcitability of vlPAG neurons, which contributes to bone cancer pain. On the other hand, intravlPAG injection of HCN channel antagonist ZD7288 attenuated the increase of cAMP in vlPAG of rats with bone cancer, which also indirectly indicates that the increase of cAMP in vlPAG plays a role through HCN channel. Recent studies have shown that HCN channel is not only regulated by cAMP, but also adversely affects intracellular cAMP level. For example, Ma et al. [24] found that intrathecal administration of HCN channel blockers ZD7288 attenuated the increase of cAMP levels in the spinal dorsal cord of diabetic neuropathic pain rats. Our previous study also showed that intrathecal injection of ZD7288 reduced the elevation of cAMP in DRG in rats with sciatic nerve injury [45]. How does HCN channel affect intracellular cAMP levels? Huang et al. [46] report that the opening of HCN channels can increase [Ca2+]i and further Ca2+/calmodulin-dependent activation of adenylatecyclase, leading to the elevation of cAMP. The elevation of [Ca2+]i induced by HCN channel activation may be that Na+ influx due to HCN channel opening leads to membrane depolarization, which leads to the opening of voltage-dependent calcium channel and the increase of [Ca2+]i [47]. For example, Felix et al. [48] found that HCN channel antagonist ZD7288 could block the T-type Ca2+ currents in mouse spermatogenic cells and HEK-293 cells. In this study, whether HCN channels affect cAMP concentration by altering [Ca2+]i and further Ca2+/calmodulin-dependent activation of adenylatecyclase in vlPAG neurons remains to be further confirmed.
In conclusion, in this study, HCN1 and HCN2 channels expression and Ih in vlPAG neurons were significantly increased when mechanical and thermal hyperalgesia occurred in tibial carcinoma rats inoculated with SHZ-88 breast cancer cells. Furthermore, blockage of HCN channel in vlPAG by intravlPAG injection of ZD7288 (HCN channels antagonist) significantly alleviated mechanical and thermal hyperalgesia in tibial cancer rats, suggesting that the hyperactivity of HCN channel may be involved in the development of bone cancer pain. In addition, in this study, the level of cAMP in vlPAG of tibial cancer rats was significantly increased, and intravlPAG injection of ZD7288 reduced the elevation of cAMP. Since cAMP is an important factor promoting the activation of HCN channel, it is suggested that the elevation of cAMP promote the opening of HCN channel, lead to hyperexcitability of vlPAG neurons in bone cancer rats, which promotes the occurrence of bone cancer pain.