A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.
The level of wakefulness is one of the major factors affecting nociception and pain. Stress-induced analgesia supports an animal’s survival via prompt defensive responses against predators or competitors. Previous studies have shown the pharmacological effects of orexin peptides on analgesia. However, orexin neurons contain not only orexin but also other co-transmitters such as dynorphin, neurotensin and glutamate. Thus, the physiological importance of orexin neuronal activity in nociception is unknown. Here we show that adult-stage selective ablation of orexin neurons enhances pain-related behaviors, while pharmacogenetic activation of orexin neurons induces analgesia. Additionally, we found correlative activation of orexin neurons during nociception using fiber photometry recordings of orexin neurons in conscious animals. These findings suggest an integrative role for orexin neurons in nociceptive perception and pain regulation.
BackgroundSeveral etiological reports have shown that chronic pain significantly interferes with sleep. Inadequate sleep due to chronic pain may contribute to the stressful negative consequences of living with pain. However, the neurophysiological mechanism by which chronic pain affects sleep-arousal patterns is as yet unknown. Although serotonin (5-HT) was proposed to be responsible for sleep regulation, whether the activity of 5-HTergic neurons in the dorsal raphe nucleus (DRN) is affected by chronic pain has been studied only infrequently. On the other hand, the recent development of optogenetic tools has provided a valuable opportunity to regulate the activity in genetically targeted neural populations with high spatial and temporal precision. In the present study, we investigated whether chronic pain could induce sleep dysregulation while changing the activity of DRN-5-HTergic neurons. Furthermore, we sought to physiologically activate the DRN with channelrhodopsin-2 (ChR2) to identify a causal role for the DRN-5-HT system in promoting and maintaining wakefulness using optogenetics.ResultsWe produced a sciatic nerve ligation model by tying a tight ligature around approximately one-third to one-half the diameter of the sciatic nerve. In mice with nerve ligation, we confirmed an increase in wakefulness and a decrease in non-rapid eye movement (NREM) sleep as monitored by electroencephalogram (EEG). Microinjection of the retrograde tracer fluoro-gold (FG) into the prefrontal cortex (PFC) revealed several retrogradely labeled-cells in the DRN. The key finding of the present study was that the levels of 5-HT released in the PFC by the electrical stimulation of DRN neurons were significantly increased in mice with sciatic nerve ligation. Using optogenetic tools in mice, we found a causal relationship among DRN neuron firing, cortical activity and sleep-to-wake transitions. In particular, the activation of DRN-5-HTergic neurons produced a significant increase in wakefulness and a significant decrease in NREM sleep. The duration of NREM sleep episodes was significantly decreased during photostimulation in these mice.ConclusionsThese results suggest that neuropathic pain accelerates the activity of DRN-5-HTergic neurons. Although further loss-of-function experiments are required, we hypothesize that this activation in DRN neurons may, at least in part, correlate with sleep dysregulation under a neuropathic pain-like state.
Muscle cramps are one of the most common complications of hemodialysis (HD), and often are a source of great pain in spite of various clinical measures. The traditional herbal medicine, shao-yao-gan-cao-tang (Japanese name: Shakuyaku-kanzo-to), consists of equal amounts of paeony and licorice roots, and has been used in Japan and China for muscle pain or skeletal muscle tremors. To determine whether this medicine is able to prevent frequent and unendurable muscle cramps in patients undergoing HD, Shakuyaku-kanzo-to at 6 g per day was prospectively administered for 4 weeks to five patients on HD who were suffering from frequent muscle cramps. The frequency and severity of cramping before and after the treatment treatment were carefully observed and compared. Skeletal muscle cramps completely disappeared in two of the treated patients after the start of oral administration of Shakuyaku-kanzo-to. Moreover, the frequency of cramping was significantly decreased in two of the remaining three patients after persistent administration. The severity of muscle cramps was also decreased by this treatment in the responsive patients. No serious side effects were detected during the treatment period. The inhibitory effect of Shakuyaku-kanzo-to on muscle contraction was also experimentally examined by using phrenic nerve-diaphragm preparations from male Wistar rats. Differences between the twitch responses were determined when the diaphragms and the nerves were stimulated in the presence and absence of the extract of Shakuyaku-kanzo-to. The results demonstrated that extracts of paeony and licorice roots inhibit contraction of skeletal muscles in rats. Taken together, we suggest that administration of Shakuyaku-kanzo-to is a safe, effective treatment for preventing muscle cramps in patients undergoing HD.
Opioids are effective analgesics for the management of moderate to severe cancer pain. Here we show that κ opioid receptor (KOR) agonists act as anti-angiogenic factors in tumors. Treatment with KOR agonists, U50,488H and TRK820, significantly inhibited human umbilical vein endothelial cell (HUVEC) migration and tube formation by suppressing VEGFR2 expression. In contrast, treatment with a μ opioid receptor agonist, DAMGO, or a δ opioid receptor agonist, SNC80, did not prevent angiogenesis in HUVECs. Lewis lung carcinoma (LLC) or B16 melanoma grafted in KOR knockout mice showed increased proliferation and remarkably enhanced tumor angiogenesis compared with those in wild type mice. On the other hand, repeated intraperitoneal injection of TRK820 (0.1–10 μg/kg, b.i.d.) significantly inhibited tumor growth by suppressing tumor angiogenesis. These findings indicate that KOR agonists play an important role in tumor angiogenesis and this knowledge could lead to a novel strategy for cancer therapy.
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