Changsheng Huang

Intrathecal 5-azacytidine inhibits global DNA methylation and methyl- CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury.

The pathogenesis of neuropathic pain remains largely unknown. Epigenetic mechanisms may play a major role in regulating expression of pro- or antinociceptive genes. DNA methylation is a major epigenetic mechanism in vertebrates, and methyl- CpG-binding protein 2 (MeCP2) is directly involved in methylation-mediated gene silencing. To determine how changes in global DNA methylation and MeCP2 expression occur following chronic constriction injury (CCI) and how repression of DNA methylation affects these changes and attenuates neuropathic pain, we used intrathecal 5-azacytidine, a DNA methyltransferase inhibitor, in CCI rats. Rats received 0.9% saline or 5-azacytidine (10μmol·d(-1)) via spinal injection once daily from day 3 to day 14 after CCI surgery. Global DNA methylation and MeCP2 expression increased in the spinal cord in CCI rats on day 14 after CCI surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were attenuated by intrathecal 5-azacytidine from day 5 to day 14 after CCI surgery. The increases in global DNA methylation and MeCP2 expression in the spinal cord in CCI rats were also significantly inhibited by intrathecal 5-azacytidine. These results demonstrate that increased global DNA methylation and MeCP2 expression in the spinal cord after nerve damage may play an important role in neuropathic pain. 5-azacytidine shows potential for treating neuropathic pain.

Curcumin Alleviates Neuropathic Pain by Inhibiting p300/CBP Histone Acetyltransferase Activity-Regulated Expression of BDNF and Cox-2 in a Rat Model

The management of neuropathic pain is still a major challenge because of its unresponsiveness to most common treatments. Curcumin has been reported to play an active role in the treatment of various neurological disorders, such as neuropathic pain. Curcumin has long been recognized as a p300/CREB-binding protein (CBP) inhibitor of histone acetyltransferase (HAT) activity. However, this mechanism has never been investigated for the treatment of neuropathic pain with curcumin. The aim of the present study was to investigate the anti-nociceptive role of curcumin in the chronic constriction injury (CCI) rat model of neuropathic pain. Furthermore, with this model we investigated the effect of curcumin on P300/CBP HAT activity-regulated release of the pro-nociceptive molecules, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (Cox-2). Treatment with 40 and 60 mg/kg body weight curcumin for 7 consecutive days significantly attenuated CCI-induced thermal hyperalgesia and mechanical allodynia, whereas 20 mg/kg curcumin showed no significant analgesic effect. Chromatin immunoprecipitation analysis revealed that curcumin dose-dependently reduced the recruitment of p300/CBP and acetyl-Histone H3/acetyl-Histone H4 to the promoter of BDNF and Cox-2 genes. A similar dose-dependent decrease of BDNF and Cox-2 in the spinal cord was also observed after curcumin treatment. These results indicated that curcumin exerted a therapeutic role in neuropathic pain by down-regulating p300/CBP HAT activity-mediated gene expression of BDNF and Cox-2.

p300 exerts an epigenetic role in chronic neuropathic pain through its acetyltransferase activity in rats following chronic constriction injury (CCI)

BackgroundNeuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300), as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI). To test this hypothesis, two different approaches were used in this study: (I) down-regulating p300 with specific small hairpin RNA (shRNA) and (II) chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646.ResultsUsing the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2), a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord.ConclusionsThe results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.

Cyclooxygenase inhibitors suppress the expression of P2X3 receptors in the DRG and attenuate hyperalgesia following chronic constriction injury in rats

Recent evidence suggests that P2X(3) receptors express abundantly in nociceptive sensory neurons and play an important role in neuropathic pain. Upregulation of prostaglandin E2 (PGE2) after nerve injure is involved in the pathogenesis of neuropathic pain. An increase of P2X(3) receptors after chronic constriction injury (CCI) to the sciatic nerve has also been reported, the mechanisms are not known clearly. In this study, we examined the effects of systemic administration of cyclooxygenase (COX) inhibitors on analgesia and the expression of P2X(3) receptors in the dorsal root ganglia (DRG) in CCI rats. Rats received 0.9% saline, the nonselective COX inhibitor ibuprofen (40mgkg(-1)day(-1)) or the selective COX-2 inhibitor celecoxib (30mgkg(-1)day(-1)) by gavage twice daily from 3 to 14 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI were markedly attenuated by celecoxib from 5 to 14 days after surgery, and relieved by ibuprofen treatment from 7 to 10 days after surgery. The increase of P2X(3) receptors in the DRG in CCI rats on day 14 after surgery was also significantly inhibited; the effect of ibuprofen was stronger than that of celecoxib. These results demonstrate that up-regulated COX/PGE2 after nerve damage may play an important role in neuropathic pain. They are highly involved in the expression of P2X(3) receptors in the DRG in CCI rats.

Resveratrol attenuates morphine antinociceptive tolerance via SIRT1 regulation in the rat spinal cord.

In recent years, researchers have begun to pay more attention to the role of Sirtuin 1 (SIRT1, a class III histone deacetylase) in pain. However, little research has been conducted examining the involvement of SIRT1 in chronic morphine tolerance. The aim of this study was to investigate the role of spinal SIRT1 and acetyl-histone H3(Ac-H3) in chronic morphine tolerance in rats. Chronic morphine tolerance was induced by twice-daily intrathecal (i.t.) injections of morphine (10μg) for 6 days. Control rats received normal saline (NS). Resveratrol (Res, a SIRT1 stimulant, 30μg i.t.) or dimethyl sulfoxide (DMSO, 10μl i.t.) was then injected on days 7-13. The thermal paw withdrawal threshold was assessed to determine the analgesic effects of morphine (10μg). qRT-PCR, western blotting and immunohistochemistry were used to detect the expression of SIRT1 and global Ac-H3. Administration of morphine for 6 days induced a stabilized antinociceptive tolerance, down-regulated SIRT1 expression and up-regulated Ac-H3 expression in the spinal dorsal horn. Resveratrol treatment from day 7 to 13 increased SIRT1 expression, suppressed global Ac-H3 expression compared to the morphine tolerance (MT) group, and significantly reversed morphine antinociceptive tolerance. These results suggest that resveratrol reversed morphine tolerance by upregulating the expression of SIRT1 in the spinal dorsal horn. SIRT1 and global Ac-H3 in the spinal cord may play an important role in the mechanisms of chronic morphine tolerance.

Microglial polarization dynamics in dorsal spinal cord in the early stages following chronic sciatic nerve damage

Peripheral nerve injury can lead to activation of spinal microglia, which can mediate neuroinflammation and contribute to neuropathic pain following nerve injury. Activated microglia may manifest with either pro-inflammatory M1 phenotype or anti-inflammatory M2 phenotype, which may lead to detrimental or beneficial roles in the nervous system. In this study, microglia numbers, morphology and gene profiles were examined in the dorsal spinal cord of rats over 14 days following sciatic nerve chronic constriction injury (CCI). The morphology of some microglia changed from a surveying to an activated state within 1 day of CCI. Neuropathic pain developed within seven to 14 days following injury and microglia numbers were increased, with almost all in the dorsal spinal cord morphologically defined as activated. At day one after CCI, both M1 and M2 microglia-related genes were increased but only M1 microglia-related genes remained elevated at day seven and 14 thereafter. These results indicate that both M1 and M2 microglia were activated in the dorsal spinal cord one day after CCI but the microglia skewed towards M1 phenotype during the following seven and 14 days.

Normalizing GDNF Expression in the Spinal Cord Alleviates Cutaneous Hyperalgesia but not Ongoing Pain in a Rat Model of Bone Cancer Pain.

Bone cancer pain (BCP) is the most common complication in patients with bone cancer. Glial cell line‐derived neurotrophic factor (GDNF) is believed to be involved in chronic pain conditions. In this article, the expression and roles of GDNF were studied in a rat model of BCP induced by tibia injection of Walker 256 rat mammary gland carcinoma cells. Significant mechanical and thermal hyperalgesia and ongoing pain were observed beginning as early as day 5 post injection. The expression level of GDNF protein examined on day 16 after tibia injection was decreased in the L3 dorsal root ganglion (DRG) and lumbar spinal cord, but not in other spinal levels or the anterior cingulate cortex. Phosphorylation of Ret, the receptor for GDNF family ligands, was also decreased. Furthermore, normalizing GDNF expression with lentiviral vector constructs in the spinal cord significantly reduced mechanical and thermal hyperalgesia, spinal glial activation, and pERK induction induced by tibia injection, but did not affect ongoing pain. Together these findings provide new evidence for the use of GDNF as a therapeutic treatment for bone cancer pain states.

Temporal Distribution of p300/CBP Immunoreactivity in the Adult Rat Spinal Dorsal Horn Following Chronic Constriction Injury (CCI)

Abstractp300 and its homolog cyclic AMP response element binding protein (CBP) are coactivators that were identified to participate in many biological processes including neural development and cognition. Their roles within the rodent spinal cord have not been reported systematically; in this study, their spatiotemporal distribution in the spinal cord of adult rat following chronic constriction injury (CCI) was studied. p300 and CBP expressed predominantly in nuclei in the gray matter of rat spinal cord. Rats undergoing CCI surgery showed increased p300/CBP immunoreactivity (IR) compared with normal control and sham-operated rats. The number of IR cells reached the peak at day 14 following CCI compared with those on day 3, 7, and 21, accompanied with significant behavioral changes of neuropathic pain. Cell-type determination by immunofluorescence at day 14 following CCI revealed that p300 and CBP expressed in neurons, but not in astrocytes or microglial cells. These results suggest that p300 and CBP are probably involved in the maintenance of neuropathic pain on spinal cord level. Furthermore, p300 and CBP may serve as a sensor only in neurons but not in astrocytes or microglia cells in the adult rat spinal cord.

Different symptoms of neuropathic pain can be induced by different degrees of compressive force on the C7 dorsal root of rats.

BACKGROUND CONTEXT Neuropathic pain after nerve injuries is characterized by positive and negative sensory symptoms and signs. The extent of sensory fiber loss after nerve injuries has been demonstrated to correlate with symptoms of neuropathic pain by quantitative sensory testing and confirmed by biopsies of small nerve fibers. However, the relationship between the pathologic changes of large nerves on injuries and resulting pain symptoms remains unclear. PURPOSE To investigate the relationship between the extent of dorsal root injury and resulting symptoms of neuropathic pain. STUDY DESIGN Nerve injury and assessment of the following pain-related behaviors and neuropathologic changes. METHODS A total of 24 adult male Sprague-Dawley rats weighing 250 to 300 g were randomly divided into three groups (n=8 each): sham group operated on but without nerve compression, 70 gf group, and 180 gf group; a compression force of 70 or 180 g was applied to the right C7 dorsal root, separately. Threshold thermal and mechanical pains were measured before surgery (baseline) and on the first, third, fifth, and seventh day after surgery. On the seventh day after surgery, all rats were killed, and the structural alterations of nerve fibers within the compressed areas were examined. RESULTS A compression force of 70 g resulted in hyperalgesia, whereas a compression force of 180 g induced hypoalgesia in the ipsilateral forepaw in response to both mechanical and thermal stimulations within 7 days after injury. Light microscopy and electron microscopy revealed a mild to moderate sensory fiber loss after 70-gf compression and a more severe sensory fiber loss after 180-gf compression. CONCLUSIONS Transient injuries on sensory fibers can produce either positive or negative symptoms of neuropathic pain, and the different extent of sensory fiber loss after different degrees of injuries might account for the varied resulting symptoms of neuropathic pain.

Swimming exercise ameliorates neurocognitive impairment induced by neonatal exposure to isoflurane and enhances hippocampal histone acetylation in mice.

Isoflurane-induced neurocognitive impairment in the developing rodent brain is well documented, and regular physical exercise has been demonstrated to be a viable intervention for some types of neurocognitive impairment. This study was designed to investigate the potential protective effect of swimming exercise on both neurocognitive impairment caused by repeated neonatal exposure to isoflurane and the underlying molecular mechanism. Mice received 0.75% isoflurane exposures for 4h on postnatal days 7, 8, and 9. From the third month after anesthesia, the mice were subjected to regular swimming exercise for 4weeks, followed by a contextual fear condition (CFC) trial. We found that repeated neonatal exposure to isoflurane reduced freezing behavior during CFC testing and deregulated hippocampal histone H4K12 acetylation. Conversely, mice subjected to regular swimming exercise showed enhanced hippocampal H3K9, H4K5, and H4K12 acetylation levels, increased numbers of c-Fos-positive cells 1h after CFC training, and less isoflurane-induced memory impairment. We also observed increases in histone acetylation and of cAMP-response element-binding protein (CREB)-binding protein (CBP) during the swimming exercise program. The results suggest that neonatal isoflurane exposure-induced memory impairment was associated with dysregulation of H4K12 acetylation, which may lead to less hippocampal activation following learning tasks. Swimming exercise was associated with enhanced hippocampal histone acetylation and CBP expression. Exercise most likely ameliorated isoflurane-induced memory impairment by enhancing hippocampal histone acetylation and activating more neuron cells during memory formation.