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Bone cancer pain (BCP) profoundly compromises the life quality of patients with bone metastases. Severe side effects of the drugs which were widely used and effective in the various stages of this condition results in a huge challenge for BCP treatment. Here, we investigated the antinociceptive effects of XPro1595, a soluble tumor necrosis factor (solTNF) inhibitor with considerable immunoregulatory efficacy, on BCP, as well as the underlying mechanisms within the spinal dorsal horn (SDH). Walker 256 mammary gland carcinoma cells were intratibially inoculated to induce BCP. Intrathecal administration of XPro1595 alleviated bone cancer-induced chronic pain in a dose-dependent manner, with an ED50 of 9.69 mg/kg. Bone cancer resulted in the activation of astrocytes and microglia in the SDH through the upregulation of mitogen-activated protein kinase (MAPK) pathways, which was accompanied by an over-expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. XPro1595suppressed bone cancer-evoked glial activation and the consequent neuroinflammation. These inhibitory effects of XPro1595 were, at least partially, mediated by a reduction in the phosphorylation of p38 MAPK in spinal glial cells. In conclusion, inhibition of spinal glia by XPro1595 may have utility in the treatment of bone cancer-induced neuroinflammation, and our results further implicate XPro1595 as a new promising therapeutic agent for BCP.

Combined the retrograde double tracing with immunofluorescence histochemical staining, we examined the neurons in the lateral parabrachial nucleus (LPB) sent collateral projections to the paraventricular thalamic nucleus (PVT) and central amygdaloid nucleus (CeA) and their roles in the nociceptive transmission in the rat. After the injection of Fluoro-gold (FG) into the PVT and tetramethylrhodamine-dextran (TMR) into the CeA, respectively, FG/TMR double-labeled neurons were observed in the LPB. The percentages of FG/TMR double-labeled neurons to the total number of FG- or TMR-labeled neurons were 6.18% and 9.09%, respectively. Almost all of the FG/TMR double-labeled neurons (95%) exhibited calcitonin gene-related peptide (CGRP) immunoreactivity. In the condition of neuropathic pain, 94% of these neurons showed FOS immunoreactivity. The present data indicates that some of CGRP-expressing neurons in the LPB may transmit nociceptive information toward the PVT and CeA by way of axon collaterals.

Aims The present study aims to test whether astrocytes contribute to glucocorticoid-mediated diabetic mechanical allodynia. Methods Streptozotocin (STZ)-induced diabetic rats were used in our study. The intrathecal operation was performed 21 days after the onset of diabetes. Diabetic mechanical allodynia was present 28 d after the onset of diabetes, and the mechanical threshold was tested using von Frey filaments. Immunohistochemistry, including immunofluorescent histochemical staining, was performed to observe the morphology of the spinal dorsal horn (SDH). Western blot analysis was employed as a semi-quantitative assay of the expression levels of GFAP and NDRG2 associated with diabetic mechanical allodynia. Results Diabetic rats displayed mechanical allodynia and activated astrocytes in the SDH 28 days after the onset of diabetes. This allodynia was attenuated by intrathecal administration of the astrocyte-specific inhibitor l -α-aminoadipate. In parallel, intrathecal injection of RU486, a glucocorticoid receptor antagonist, inhibited the activation of astrocytes in the SDH, alleviating the diabetes-induced mechanical allodynia. Furthermore, we found that dorsal horn astrocytes express abundant N-myc downstream-regulated gene 2 (NDRG2), which contributes to astrocyte reactivity. NDRG2 was over-expressed in activated astrocytes in diabetic rats with mechanical allodynia. Intrathecal injection of RU486 prevented the over-expression of NDRG2, which reversed the astrocyte reactivity and diabetic tactile allodynia. Conclusions These results suggest that glucocorticoid-mediated over-expression of NDRG2 may contribute to the activation of dorsal horn astrocytes, which play a crucial role in diabetic mechanical allodynia. Thus, inhibiting glucocorticoid receptors and/or astrocyte reactivity in the SDH may be a therapeutic strategy for treating diabetic tactile allodynia.

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) is characterized by a reduction in excitatory amino acid transporter 2 (EAAT2) expression and severe amino acid excitotoxicity. The aim of this study was to explore the neuroprotective effect of ceftriaxone (CEF), a potent compound that up-regulates EAAT2, against EBI and the potential mechanisms using in vitro experiments and a rat model of SAH. Intracisternal treatment with CEF significantly improved neurological outcomes and alleviated extracellular glutamate accumulation after SAH. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining and Western blot analysis of cleaved caspase 3 showed that CEF decreased hippocampal neuronal apoptosis following SAH. Immunofluorescent staining and Western blotting revealed that CEF significantly reversed the down-regulation of EAAT2 expression following SAH. In Morris water maze (MWM) tests, CEF remarkably ameliorated the SAH-induced cognitive dysfunction in spatial learning memory and reference memory. CEF promoted the nuclear translocation of p65 as well as the activation of Akt in hippocampal astrocytes in vitro and in vivo. These findings suggest that CEF may exert significant protective effects against EBI following SAH by modulating the PI3K/Akt/NF-κB signaling pathway.

The anterior cingulate cortex (ACC) has been implicated as a key structure in the affective component of pain (such as unpleasantness or aversion). Recent evidence suggests that activation of spinal astrocytes contributes to the development and maintenance of the sensory component of pain after peripheral inflammation. However, whether the astrocytes in the ACC contribute to the affective component of pain is unknown. In this study, we demonstrated that intraplantar administration of Complete Freund's Adjuvant (CFA) in rats induced mechanical allodynia and place escape/avoidance behavior, which reflects the aversion of mechanical nociceptive stimuli. A reverse transcriptase-polymerase chain reaction study showed a significant increase in the mRNA level of GFAP, an astrocytic marker in the bilateral ACC at 3 d and 14 d after CFA-induced peripheral inflammation. Similarly, Western blot also revealed enhanced expression of GFAP protein at 3 d and 14 d after CFA injection. Interestingly, intra-ACC injection of L-alpha-aminoadipate (L-α-AA), an astroglial toxin, inhibited the escape/avoidance behavior, but did not affect the paw withdrawal threshold at 3 d following CFA injection. All together, our results suggest that the astrocytes activation in the ACC may contribute to the affective component of pain.

Glutamate transmission from vestibular end organs to central vestibular nuclear complex (VNC) plays important role in transferring sensory information about head position and movements. Three isoforms of vesicular glutamate transporters (VGLUTs) have been considered so far the most specific markers for glutamatergic neurons/cells. In this study, VGLUT1 and VGLUT2 were immunohistochemically localized to axon terminals in VNC and somata of vestibular primary afferents in association with their central and peripheral axon endings, and VGLUT1 and VGLUT3 were co-localized to hair cells of otolith maculae and cristae ampullaris. VGLUT1 and VGLUT2 defined three subsets of Scarpa's neurons (vestibular ganglionic neurons): those co-expressing VGLUT1 and VGLUT2 or expressing only VGLUT2, and those expressing neither. In addition, many neurons located in all vestibular subnuclei were observed to contain hybridized signals for VGLUT2 mRNA and a few VNC neurons, mostly scattered in medial vestibular nucleus (MVe), displayed VGLUT1 mRNA labelling. Following unilateral ganglionectomy, asymmetries of VGLUT1-immunoreactivity (ir) and VGLUT2-ir occurred between two VNCs, indicating that the VNC terminals containing VGLUT1 and/or VGLUT2 are partly of peripheral origin. The present data indicate that the constituent cells/neurons along the vestibular pathway selectively apply VGLUT isoforms to transport glutamate into synaptic vesicles for glutamate transmission.