Sheng-Xi Wu

Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3.

The role of apoptosis in the pathogenesis of intervertebral disc degeneration (IDD) remains enigmatic. Accumulating evidence has shown that the apoptotic machinery is regulated by miRNAs. We hypothesized that miRNAs might contribute to apoptosis in IDD. We have found that 29 miRNAs were differentially expressed and miR‐155 was down‐regulated in degenerative nucleus pulposus (NP). The deregulation of miR‐155 was further verified using real‐time PCR (0.56 fold, p < 0.05). Bioinformatics target prediction identified FADD and caspase‐3 as putative targets of miR‐155. Furthermore, miR‐155 inhibited FADD and caspase‐3 expression by directly targeting their 3′‐UTRs, which was abolished by mutation of the miR‐155 binding sites. In vitro up‐regulation of miR‐155 in human NP cells by transfection with lentiviral pre‐miR‐155 resulted in repression of FADD and caspase‐3; whereas knockdown of miR‐155 with lentiviral antigomiR‐155 led to over‐expression of FADD and caspase‐3. Also, Fas‐mediated apoptosis was increased when antagonizing miR‐155 and decreased when using pre‐miR‐155 in human NP cells. In addition, we presented direct evidence of NP cells undergoing apoptosis in IDD tissues using transmission electron microscopy analysis. Moreover, a combination of in situ hybridization (ISH) and immunohistochemistry (IHC) revealed that miR‐155 expressed in the cytoplasm of human NP cells with reverse correlation with FADD and caspase‐3. In summary, this is the first study addressing the underlying mechanisms of IDD in terms of apoptosis and miRNAs. Furthermore, caspase‐3 is identified as a novel target of miR‐155. Our results suggest that deregulated miR‐155 promotes Fas‐mediated apoptosis in human IDD by targeting FADD and caspase‐3, implicating an aetiological and therapeutic role of miR‐155 in IDD. Copyright

Crosstalk between Spinal Astrocytes and Neurons in Nerve Injury-Induced Neuropathic Pain

Emerging research implicates the participation of spinal dorsal horn (SDH) neurons and astrocytes in nerve injury-induced neuropathic pain. However, the crosstalk between spinal astrocytes and neurons in neuropathic pain is not clear. Using a lumbar 5 (L5) spinal nerve ligation (SNL) pain model, we testified our hypothesis that SDH neurons and astrocytes reciprocally regulate each other to maintain the persistent neuropathic pain states. Glial fibrillary acidic protein (GFAP) was used as the astrocytic specific marker and Fos, protein of the protooncogene c-fos, was used as a marker for activated neurons. SNL induced a significant mechanical allodynia as well as activated SDH neurons indicated by the Fos expression at the early phase and activated astrocytes with the increased expression of GFAP during the late phase of pain, respectively. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or astroglial toxin L-α-aminoadipate (L-AA) reversed the mechanical allodynia, respectively. Immunofluorescent histochemistry revealed that intrathecal administration of c-fos ASO significantly suppressed activation of not only neurons but also astrocytes induced by SNL. Meanwhile, L-AA shortened the duration of neuronal activation by SNL. Our data offers evidence that neuronal and astrocytic activations are closely related with the maintenance of neuropathic pain through a reciprocal “crosstalk”. The current study suggests that neuronal and non-neuronal elements should be taken integrally into consideration for nociceptive transmission, and that the intervention of such interaction may offer some novel pain therapeutic strategies.

Changes of the expression of 5-HT receptor subtype mRNAs in rat dorsal root ganglion by complete Freund's adjuvant-induced inflammation.

By using the reverse transcriptase polymerase chain reaction technique, the expression of 5-hydroxytryptamine (5-HT) receptor subtype mRNAs in the rat lumbar dorsal root ganglion (DRG) was investigated following unilateral injection of complete Freunds adjuvant (CFA) into the rat hind paw. The results showed that 5-HT(1A), 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(2A), 5-HT(3), 5-HT(4), 5-HT(5A) and 5-HT(7) receptor subtypes were present in the rat lumbar DRG. CFA injection resulted in a significant increase in mRNA level of 5-HT(1A), 5-HT(1B), 5-HT(1F), 5-HT(2A), 5-HT(3), 5-HT(4) and 5-HT(7) receptor subtypes and a marked induction of 5-HT(2C) subtype mRNA in the DRG. The present results suggest the important roles for these 5-HT receptor subtypes in generating peripheral nociceptive signaling and provide evidence to elucidate the mechanism of 5-HT in nociception.

Vesicular glutamate transporter immunoreactivity in the central and peripheral endings of muscle-spindle afferents

Expression of vesicular glutamate transporters (VGLUTs: VGLUT1, VGLUT2 and VGLUT3) in muscle spindle afferents was examined in rats. VGLUT1 immunoreactivity was detected in the sensory endings on the equatorial and juxta-equatarial regions of intrafusal fibers as well as in many axon terminals within lamina IX of the spinal cord. VGLUT1 might be expressed not only in the central axon terminals but also in the peripheral sensory endings of muscle-spindle afferents.

Changes of 5-HT receptor subtype mRNAs in rat dorsal root ganglion by bee venom-induced inflammatory pain

The reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to examine the changes of the expression of 5-hydroxytryptamine (5-HT) receptors in the rat lumbar dorsal root ganglion (DRG) following subcutaneous bee venom (BV) injection into the plantar surface of the unilateral hindpaw. In the DRG ipsilateral to the BV injection, significant increase of mRNA levels of 5-HT(1A), 5-HT(1B), 5-HT(2A) and 5-HT(3) receptor subtypes were observed at 1 and 4h after the BV injection, while increase of 5-HT(2C), 5-HT(4), 5-HT(6) and 5-HT(7) receptor subtype mRNAs was detected at 4h only. No such changes were observed in the expressions of 5-HT(1D), 5-HT(1F) and 5-HT(5A) receptor subtype mRNAs. Upregulation of 5-HT(1A), 5-HT(1B) and 5-HT(2A) receptor subtype mRNAs was also observed in the contralateral DRG at 4 h. The presence of 5-HT(1E), 5-HT(2B) and 5-HT(5B) receptor subtype mRNAs was not detected in the rat DRG. The present results suggest that different sets of 5-HT receptor subtypes work at different stages of the inflammatory pain induced by subcutaneous BV injection.

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain.

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

A NMDA Receptor Antagonist, MK-801 Impairs Consolidating Extinction of Auditory Conditioned Fear Responses in a Pavlovian Model

Background In auditory fear conditioning, repeated presentation of the tone in the absence of shock leads to extinction of the acquired fear responses. The glutamate N-methyl-D-aspartate receptor (NMDAR) is thought to be involved in the extinction of the conditioned fear responses, but its detailed role in initiating and consolidating or maintaining the fear extinction memory is unclear. Here we investigated this issue by using a NMDAR antagonist, MK-801. Methods/Main Findings The effects of immediate (beginning at 10 min after the conditioning) and delayed (beginning at 24 h after conditioning) extinctions were first compared with the finding that delayed extinction caused a better and long-lasting (still significant on the 20th day after extinction) depression on the conditioned fear responses. In a second experiment, MK-801 was intraperitoneally (i.p.) injected at 40 min before, 4 h or 12 h after the delayed extinction, corresponding to critical time points for initiating, consolidating or maintaining the fear extinction memory. i.p. injection of MK-801 at either 40 min before or 4 h after delayed extinction resulted in an impairment of initiating and consolidating fear extinction memory, which caused a long lasting increased freezing score that was still significant on the 7th day after extinction, compared with extinction group. However, MK-801 administered at 12 h after the delayed extinction, when robust consolidation has been occurred and stabilized, did not affect the established extinction memory. Furthermore, the changed freezing behaviors was not due to an alteration in general anxiety levels, since MK-801 treatment had no effect on the percentage of open-arm time or open-arm entries in an Elevated Plus Maze (EPM) task. Conclusions/Significance Our data suggested that the activation of NMDARs plays important role in initiation and consolidation but not maintenance of fear extinction memory. Together with the fact that NMDA receptor is very important for memory, our data added experimental evidence to the concept that the extinction of conditioned fear responses is a procedure of initiating and consolidating new memory other than simply “erasing” the fear memory.

Spinal high-mobility group box 1 contributes to mechanical allodynia in a rat model of bone cancer pain.

Mechanisms underlying bone cancer-induced pain are largely unknown. Previous studies indicate that neuroinflammation in the spinal dorsal horn is especially involved. Being first reported as a nonhistone chromosomal protein, high-mobility group box 1 (HMGB1) is now implicated as a mediator of inflammation. We hypothesized that HMGB1 could trigger the release of cytokines in the spinal dorsal horn and contribute to bone cancer pain. To test this hypothesis, we first built a bone cancer pain model induced by intratibal injection of Walker 256 mammary gland carcinoma cells. The structural damage to the tibia was monitored by radiological analysis. The mechanical allodynia was measured and the expression of spinal HMGB1 and IL-1beta was evaluated. We observed that inoculation of cancer cells, but not heat-killed cells, induced progressive bone destruction from 9 d to 21 d post inoculation. Behavioral tests demonstrated that the significant nociceptive response in the cancer cells-injected rats emerged on day 9 and this kind of mechanical allodynia lasted at least 21 d following inoculation. Tumor cells inoculation significantly increased HMGB1 expression in the spinal dorsal horn, while intrathecal injecting a neutralizing antibody against HMGB1 showed an effective and reliable anti-allodynia effect with a dose-dependent manner. IL-1beta was significantly increased in cancer pain rats while intrathecally administration of anti-HMGB1 could decrease IL-1beta. Together with previous reports, we predict that bone cancer induces HMGB1 production, enhancing spinal IL-1beta expression and thus modulating spinal excitatory synaptic transmission and pain response.

Temporal changes of astrocyte activation and glutamate transporter-1 expression in the spinal cord after spinal nerve ligation-induced neuropathic pain.

Astrocyte activation is involved in the neuropathic pain. As a glutamate scavenger, the glutamate transporter‐1 (GLT‐1) is exclusively expressed on the astrocytes and probably correlates with astrocyte activation. In the present study, we attempted to clarify the temporal changing courses of astrocyte activation and GLT‐1 expression, as well as their correlations induced by a neuropathic pain model, namely, spinal nerve ligation (SNL) in which rapidly appearing (<3 days) and persistent (>21 days) mechanical allodynia and thermal hyperalgesia were presented. Immunofluorescent staining showed that GLT‐1 was expressed exclusively in most (not all) of the astrocytes, even when the GLT‐1 expression reached its peak. The expression of GLT‐1 displayed an interesting biphasic change, with an initial up‐regulation followed by a down‐regulation after SNL. Our results also demonstrated that SNL induced a marked and long‐term (>21 days) activation of astrocytes in the ipsilateral spinal dorsal horn. These results suggest that astrocyte activation, the change of GLT‐1 expression and the potential relationship between them might play key roles in the induction and/or maintenance of neuropathic pain. The present results provide novel clues in understanding the mechanisms underlying the involvement of astrocytes and GLT‐1 in the neuropathic pain. Anat Rec, 291:513–518, 2008.

Independent inputs by VGLUT2- and VGLUT3 - positive glutamatergic terminals onto rat sympathetic preganglionic neurons

To characterize glutamatergic axon terminals onto sympathetic preganglionic neurons (SPNs), we visualized immunohistochemically three vesicular glutamate transporters (VGLUTs) in the intermediolateral cell column (IML) of rat thoracic spinal cord. VGLUT2 and VGLUT3 immunoreactivities but not VGLUT1 immunoreactivity were distributed in the IML and found in terminals making asymmetric synapses and apposed to dendrites immunopositive for choline acetyltransferase, an SPN marker. VGLUT2 and VGLUT3 immunoreactivities were not co-localized with each other. A population of VGLUT2-immunoreactive but not VGLUT3-immunoreactive terminals were adrenergic or noradrenergic. Some of VGLUT3-immunoreactive but not VGLUT2-immunoreactive terminals contained serotonin. These results indicate at least two independent glutamatergic terminal populations, which include a distinct monoaminergic subpopulation, making excitatory inputs onto SPNs.