Xue-Bi Tian

Activation of CXCL10/CXCR3 Signaling Attenuates Morphine Analgesia: Involvement of Gi Protein

Morphine is a potent agonist of μ-opioid receptor and is widely used to relieve severe pain, including cancer pain. Some chemokines, for example, CX3CL1 and CCL2, participate in the regulation of opioid santinociception. In our previous study, we found overexpression of chemokine CXCL10/CXCR3 in spinal cord participated in the development of cancer-induced bone pain, so we supposed that CXCL10 may have influence in morphine analgesia in cancer pain relief. In this study, we found that a single dose of morphine could transiently increase the expression of CXCL10 in spinal cord. Blocking the function of CXCL10 enhanced morphine antinociception in cancer-induced bone pain rats. However, overexpression of CXCL10 induced acute algesia and decreased the analgesic effect of morphine in normal mice. The algesic effect of CXCL10 was blocked by inhibition of CXCR3 and Gi protein. These results suggested that CXCL10 in spinal cord serves as a novel negative regulator of morphine analgesia and provided evidence that activation of CXCL10/CXCR3 in spinal cord may attenuate antinociceptive potency of morphine in cancer pain relief.

Motor Cortex-Periaqueductal Gray-Spinal Cord Neuronal Circuitry May Involve in Modulation of Nociception: A Virally Mediated Transsynaptic Tracing Study in Spinally Transected Transgenic Mouse Model

Several studies have shown that motor cortex stimulation provided pain relief by motor cortex plasticity and activating descending inhibitory pain control systems. Recent evidence indicated that the melanocortin-4 receptor (MC4R) in the periaqueductal gray played an important role in neuropathic pain. This study was designed to assess whether MC4R signaling existed in motor cortex- periaqueductal gray- spinal cord neuronal circuitry modulated the activity of sympathetic pathway by a virally mediated transsynaptic tracing study. Pseudorabies virus (PRV)-614 was injected into the left gastrocnemius muscle in adult male MC4R-green fluorescent protein (GFP) transgenic mice (n = 15). After a survival time of 4–6 days, the mice (n = 5) were randomly assigned to humanely sacrifice, and spinal cords and brains were removed and sectioned, and processed for PRV-614 visualization. Neurons involved in the efferent control of the left gastrocnemius muscle were identified following visualization of PRV-614 retrograde tracing. The neurochemical phenotype of MC4R-GFP-positive neurons was identified using fluorescence immunocytochemical labeling. PRV-614/MC4R-GFP dual labeled neurons were detected in spinal IML, periaqueductal gray and motor cortex. Our findings support the hypothesis that MC4R signaling in motor cortex-periaqueductal gray-spinal cord neural pathway may participate in the modulation of the melanocortin-sympathetic signaling and contribute to the descending modulation of nociceptive transmission, suggesting that MC4R signaling in motor cortex- periaqueductal gray-spinal cord neural pathway may modulate the activity of sympathetic outflow sensitive to nociceptive signals.

An improved Tet-On System for gene expression in neurons delivered by a single lentiviral vector

In most cases, the successful application of gene therapy requires the development of vectors that can provide regulated control of therapeutic gene expression. We have reconstituted the Tet-On (tetracycline-regulated transgene expression) two-component system in a single lentiviral vector with insertion of a chicken chromatin insulator (cHS4) element between the two expression cassettes. Optimization of this vector included an improved reverse tetracycline-dependent trans-activator (rtTA) sequence developed through HIV viral evolution, and an rtTA-dependent, Tet-responsive element containing modifications of the tetO sequence (TRE-tight1) to improve leaky basal transcription. Transduction of HeLa cells with these lentiviral vectors resulted in a high level of rtTA expression in the presence of doxycycline. In neuronal cells, rtTA expression driven by a neuron-specific enolase (NSE) promoter was more efficient than gene expression from a murine cytomegalovirus promoter. Transgene expression from the NSE promoter also provided tightly regulated gene expression in neurons in vivo.

MHC-I promotes apoptosis of GABAergic interneurons in the spinal dorsal horn and contributes to cancer induced bone pain.

Cancer induced bone pain (CIBP) remains one of the most intractable clinical problems due to poor understanding of its underlying mechanisms. Recent studies demonstrate the decline of inhibitory interneurons, especially GABAergic interneurons in the spinal cord, can evoke generation of chronic pain. It has also been reported that neuronal MHC-I expression renders neurons vulnerable to cytotoxic CD8+ T cells and finally lead to neurons apoptosis in a variety neurological disorders. However, whether MHC-I could induce the apoptosis of GABAergic interneurons in spinal cord and contribute to the development of CIBP remains unknown. In this study, we investigated roles of MHC-I and underlying mechanisms in CIBP on a rat model. Our results showed that increased MHC-I expression on GABAergic interneurons could deplete GABAergic interneurons by inducing their apoptosis in the spinal dorsal horn of tumor-bearing rats. Pretreatment of MHC-I RNAi-lentivirus could prevent the apoptosis of GABAergic interneurons and therefore alleviated mechanical allodynia induced by tumor cells intratibial injection. Additionally, we also found that CD8+ T cells were colocalized with MHC-I and GABAergic neurons and presented a significant and persistent increase in the spinal cord of tumor-bearing rats. Taken together, these findings indicated that MHC-I could evoke CIBP by promoting apoptosis of GABAergic interneurons in the dorsal horn, and this apoptosis was closely related to local CD8+ T cells.

Slit2/Robo1 Mediation of Synaptic Plasticity Contributes to Bone Cancer Pain.

Synaptic plasticity is fundamental to spinal sensitivity of bone cancer pain. Here, we have shown that excitatory synaptogenesis contributes to bone cancer pain. New synapse formation requires neurite outgrowth and an interaction between axons and dendrites, accompanied by the appositional organization of presynaptic and postsynaptic specializations. We have shown that Slit2, Robo1, and RhoA act as such cues that promote neurite outgrowth and guide the axon for synapse formation. Sarcoma inoculation induces excitatory synaptogenesis and bone cancer pain which are reversed by Slit2 knockdown but aggravated by Robo1 knockdown. Synaptogenesis of cultured neurons are inhibited by Slit2 knockdown but enhanced by Robo1 knockdown. Sarcoma implantation induces an increase in Slit2 and decreases Robo1 and RhoA, while Slit2 knockdown results in an increase of Robo1 and RhoA. These results have demonstrated a molecular mechanism of synaptogenesis in bone cancer pain.

The optimal acupoint for acupuncture stimulation as a complementary therapy in pediatric epilepsy

Fig. 1. Diagrams of the locations of auricular acupuncture points (Shen Men). It is well known that the auricular branch of the vagus nerve innervates the auricular concha and so provides noninvasive or minimally invasive access to the vagus nerve [47]. Wang et al. [48] had demonstrated the use of ear acupuncture (Shen Men) for preoperative anxiety by placing a needle in the concha, an area innervated by the vagus nerve. Metaphysical theories of “auriculotherapy” claim that 168 body points on the ear are connected to various body locations. The optimal acupoint for acupuncture stimulation as a complementary therapy in pediatric epilepsy

Involvement of chemokine CXCL11 in the development of morphine tolerance in rats with cancer-induced bone pain.

Morphine is viewed as one of the classical treatments for intractable pain, but its role is limited by side effects, including analgesic tolerance. A few chemokines have been reported to be engaged in the mechanisms of morphine tolerance. However, the exact roles of CXC chemokine 11 (CXCL11) in chronic morphine tolerance remain unknown. In this study, Walker 256 mammary gland carcinoma cells were inoculated into the tibia of rats to provoke cancer‐induced bone pain. Then, morphine was intrathecally administered twice daily for seven consecutive days to induce drug tolerance. We found that the level of CXCL11 in lumbar spinal cord was increased during the development of morphine tolerance in cancer‐induced bone pain rats. Meanwhile, CXCL11 was co‐localized with markers of astrocytes and neurons in the spinal cord. Inhibition of CXCL11 by neutralizing antibodies could remarkably attenuate the degree of morphine tolerance and decrease the activation of astrocytes. Moreover, blocking astrocyte activation by d, l‐Fluorocitric acid could distinctly alleviate morphine tolerance and reduce the expression of CXCL11. Finally, morphine stimulation could induce the release of CXCL11 by cultured astrocytes and neurons in vitro. In summary, our results provide evidence that spinal CXCL11 plays a powerful modulatory role in the development of morphine tolerance through cross‐talking between astrocytes and neurons.

Enhancement of morphine analgesia and prevention of morphine tolerance by downregulation of β-arrestin 2 with antigene RNAs in mice

β-arrestin 2, a regulatory molecule of G protein-coupled receptor, has been proved to play an important role in regulating functions of mu opioid receptor. Changes of β-arrestin 2 expression might affect the function of mu opioid receptors and the effect of its agonists. In this study, antigene RNAs (agRNAs), which could selectively target gene transcription start sites and potently inhibit gene expression, were used to downregulate the expression of β-arrestin 2 to investigate its effects on morphine analgesia and tolerance in mice. After intracerebroventricular administration of recombinant lentivirus encoding β-arrestin 2 agRNAs to the mice, β-arrestin 2 expression was significantly decreased for more than 3 weeks. Mice treated with β-arrestin 2 agRNAs showed enhanced analgesic effects in response to morphine and failed to develop antinociceptive tolerance. These results suggest that inhibition of β-arrestin 2 in the brain with specific agRNAs can improve morphine efficacy, and consequently provide us a useful strategy for treatment of chronic intractable pain and morphine tolerance in vivo.

Specific patterns of spinal metabolites underlying α-Me-5-HT-evoked pruritus compared with histamine and capsaicin assessed by proton nuclear magnetic resonance spectroscopy

The mechanism behind itching is not well understood. Proton nuclear magnetic resonance (1H-NMR) spectroscopic analysis of spinal cord extracts provides a quick modality for evaluating the specific metabolic activity of α-Me-5-HT-evoked pruritus mice. In the current study, four groups of young adult male C57Bl/6 mice were investigated; one group treated with saline, while the other groups intradermally injected with α-Me-5-HT (histamine independent pruritogen), histamine (histamine dependent pruritogen) and capsaicin (algogenic substance), respectively. The intradermal microinjection of α-Me-5-HT and histamine resulted in a dramatic increase in the itch behavior. Furthermore, the results of NMR studies of the spinal cord extracts revealed that the metabolites show very different patterns for these different drugs, especially when comparing α-Me-5-HT and capsaicin. All the animals in the groups of α-Me-5-HT and capsaicin were completely separated using the metabolite parameters and principal component analysis. For α-Me-5-HT, the concentrations of glutamate, GABA, glycine and aspartate increased significantly, especially for GABA (increased 17.2%, p=0.008). Furthermore, the concentration of NAA increased, but there was no significant difference (increased 11.3%, p=0.191) compared to capsaicin (decreased 29.1%, p=0.002). Thus the application of magnetic resonance spectroscopy technique, coupled with statistical analysis, could further explain the mechanism behind itching evoked by α-Me-5-HT or other drugs. It can thus improve our understanding of itch pathophysiology and pharmacological therapies which may contribute to itch relief.

NRG1-ErbB signalling promotes microglia activation contributing to incision-induced mechanical allodynia

Spinal microglia activation is one of the pathologic mechanisms involved in post‐operative pain, which results from surgical injuries in skin, fascia, muscle and small nerves innervating these tissues. Recent research has shown that neuregulin‐1 (NRG1) and its receptor erythroblastosis oncogene B (ErbB) family mediate microglia proliferation and chemotaxis contributing to the development of neuropathic pain. However, it is unclear whether NRG1‐ErbB signalling contributes to incision‐induced mechanical allodynia.