Inhibition of Connexin 43 and Phosphorylated NR2B in Spinal Astrocytes Attenuates Bone Cancer Pain in Mice

Abstract

Bone cancer pain (BCP) is common in patients with advanced cancers when the tumors are metastasized to bone. The limited understanding of the complex pathogenesis of BCP leads to the poor effectiveness of clinical treatment. Previous studies have shown that astrocyte-specific connexin (Cx) 43, a forming protein of gap junction (GJ) and hemichannel, and N-methyl-D-aspartate receptors (NMDARs), especially the phosphorylated NMDAR 2B subunit (NR2B) phosphorylated NR2B (p-NR2B) subunit are involved in BCP. However, the relationship between Cx43 and p-NR2B in BCP remains unclear. In the present study, we investigated the expressions of Cx43, glial fibrillary acidic protein (GFAP, a marker of astrocytes), and p-NR2B in the spinal dorsal horn (SDH) in a mouse model of BCP established by intra-femural inoculation of Lewis lung carcinoma (LLC) cells via intrathecal (ith) injection of the GJ/hemichannel blocker carbenoxolone (CARB) and the NMDAR antagonist MK801, respectively. We found that the characters of BCP were mimicked by intra-femural inoculation of LLC cells in mice, and the expressions of Cx43, GFAP and p-NR2B in BCP mice were remarkably increased in a time-dependent manner from day 7 to day 21 after cell inoculation with a gradual aggravate in spontaneous pain and mechanical allodynia. Furthermore, Cx43 was predominantly expressed in the spinal astrocytes. Both CARB and MK801 inhibited the expressions of Cx43, GFAP and p-NR2B with attenuated pain hypersensitivity in BCP mice. In addition, Cx43 was co-localized with p-NR2B in the SDH, which further evidenced the presence of functional NR2B in the spinal astrocytes in BCP mice. Our findings demonstrate that inhibition of Cx43 and p-NR2B in spinal astrocytes could attenuate BCP in mice and Cx43 and p-NR2B in the astrocytes of the SDH may play an important role via their combination action in the development and maintenance of BCP in mice. These results may provide a potential therapeutic target in the prevention and/or treatment of BCP.

LSO:Ce Inorganic Scintillators are Biocompatible with Neuronal and Circuit Function

“…Slices were incubated with and without 0.25 to 0.5 mg/mL of LSO:Ce radioluminescent particles for 0, 1, 3, and 6 hours. Slices were fixed overnight at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS) and immunohistochemistry was performed based on a modified protocol (Dissing-Olesen and MacVicar, 2015; Miller et al, 2017; Yang et al, 2018). Briefly, the free-floating fixed slices were washed 5 times for 10 minutes in PBS.…”

Role of Connexins in Chronic Pain and Their Potential as Therapeutic Targets for Next-Generation Analgesics

“…Cx43 expression appears to be upregulated within the spinal cord grey matter following a spinal cord injury, oxaliplatin and bortezomib-induced peripheral neuropathy. [37][38][39][40][41][42][43]…”

“…Norimitsu Nociceptive hypersensitivity observed in these models is ameliorated by intrathecal treatment with the gap junction blocker carbenoxolone (CBX). [39][40][41][42][43] Intrathecal treatment of CBX does not affect pain perception in healthy animals; thus, it appears that channel functioning in the chronic pain state appears to be different from that in the healthy state. In addition to the ipsilateral spinal dorsal horn, a unilateral CCI increases Cx43 protein expression within the sciatic nerve and in the dorsal root ganglion.…”

“…79) Surprisingly, the expression of Cx36 is not changed in spinal dorsal horn in several models of chronic pain, such as spinal cord injury, spinal nerve ligation, oxaliplatin-induced neuropathic pain and hind paw carrageenan treatment. 39,40,42,51) As there are few studies that have examined the role of Cx36 in chronic pain, the mechanism that regulates Cx36 expression has yet to be elucidated. As Cx36 is mainly expressed in neurons, a change in Cx36 expression would involve not only local neuronal functioning but also neuronal circuits and networks.…”

“…In fact, previous studies have shown that protein expression of Cx32 is not changed in spinal dorsal horn following spinal cord injury, oxaliplatin and carrageenan treatment. 39,40,42) In other models, there appears to be a role of neural Cx32. Qin et al showed increased numbers of heterotypic gap junction channels between astrocytic Cx43 and neuronal Cx32, and this response paralleled the induction of heat hyperalgesia following subcutaneous injection of formalin into the rat hindpaw.…”