Chung-Ren Lin

Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats

BACKGROUND Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal. METHODS Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h⁻¹) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg⁻¹) was performed to assess morphine dependence. RESULTS In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose-response curve, as measured by per cent maximal possible effect (MPE), such that the AD₅₀ was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal. CONCLUSIONS We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.

Intrathecal miR-183 delivery suppresses mechanical allodynia in mononeuropathic rats

Members of the miR‐183 family are unique in that they are highly abundant in sensory organs. In a recent study, significant downregulation was observed for miR‐96 and miR‐183 in the L5 dorsal root ganglion (DRG) 2 weeks after spinal nerve ligation (SNL). In this study, we focused on miR‐183, which is the most regulated member of the miR‐183 family, to look at the specific role on neuropathic pain. Persistent mechanical allodynia was induced with the L5 SNL model in 8‐week‐old male Sprague‐Dawley rats. Paw withdrawal thresholds in response to mechanical stimuli were assessed with Von Frey filaments. Expression of miR‐183 in the L5 DRG was assessed with quantitative real‐time polymerase chain reaction (qPCR) analysis. Lentivirions expressing miR‐183 were injected intrathecally into SNL rats. Changes in mechanical allodynia were assessed with Von Frey filaments. In addition, changes in the predicted target genes of miR‐183 were assessed with qPCR. L5 SNL produced marked mechanical allodynia in the ipsilateral hindpaws of adult rats, beginning at postoperative day 1 and continuing to day 14. L5 SNL caused significant downregulation of miR‐183 in adult DRG cells. Intrathecal administration of lentivirions expressing miR‐183 downregulated SNL‐induced increases in the expression of Nav1.3 and brain‐derived neurotrophic factor (BDNF), which correlated with the significant attenuation of SNL‐induced mechanical allodynia. Our results show that SNL‐induced mechanical allodynia is significantly correlated with the decreased expression of miR‐183 in DRG cells. Replacement of miR‐183 downregulates SNL‐induced increases in Nav1.3 and BDNF expression, and attenuates SNL‐induced mechanical allodynia.

GADD45A protects against cell death in dorsal root ganglion neurons following peripheral nerve injury.

A significant loss of neurons in the dorsal root ganglia (DRG) has been reported in animal models of peripheral nerve injury. Neonatal sensory neurons are more susceptible than adult neurons to axotomy‐ or nerve growth factor (NGF) withdrawal‐induced cell death. To develop therapies for preventing irreversible sensory cell loss, it is essential to understand the molecular mechanisms responsible for DRG cell death and survival. Here we describe how the expression of the growth arrest‐ and DNA damage‐inducible gene 45α (GADD45A) is correlated with neuronal survival after axotomy in vivo and after NGF withdrawal in vitro. GADD45A expression is low at birth and does not change significantly after spinal nerve ligation (SNL). In contrast, GADD45A is robustly up‐regulated in the adult rat DRG 24 hr after SNL, and this up‐regulation persists as long as the injured fibers are prevented from regenerating. In vitro delivery of GADD45A protects neonatal rat DRG neurons from NGF withdrawal‐induced cytochrome c release and cell death. In addition, in vivo knockdown of GADD45A expression in adult injured DRG by small hairpin RNA increased cell death. Our results indicate that GADD45A protects neuronal cells from SNL‐induced cell death.

Epigenetic histone methylation regulates transforming growth factor β-1 expression following bile duct ligation in rats

BackgroundMultiple mechanisms contribute to the liver fibrosis following cholestasis. Recent research has focused on the role of transforming growth factor β-1 (TGF-β1) in the progression of fibrosis. The aim of our study is to examine the role of epigenetic chromatin marks, such as histone H3 lysine methylation (H3Kme), in bile duct ligation (BDL)-induced TGF-β1 gene expression in rat liver.MethodsTime course of methylated-histone H3 and SET7/9 recruitment were determined by chromatin immunoprecipitation in livers from BDL rats on days 1, 4, 9 and 14. Levels of TGF-β1 and SET7/9 were determined by western blots. The effect of SET7/9 knockdown on BDL-induced expression of TGF-β1, serum enzymes and liver collagen content was studied in vivo.ResultsResults showed that BDL increased the expression of the TGF β-1. Increased levels of active chromatin marks (H3K4me1, H3K4me2, and H3K4me3) and decreased levels of repressive marks (H3K9me2 and H3K9me3) in TGF-β1 promoter accompanied the changes in expression of the TGF β-1. BDL also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to the promoter. SET7/9 gene knockdown with siRNAs significantly attenuated BDL-induced TGF-β1 gene expression, serum enzymes and liver collagen content.ConclusionsTaken together, these results show the functional role of epigenetic chromatin histone H3Kme in BDL-induced TGF-β1 expression. Pharmacologic and other therapies that reverse these modifications could have potential hepatoprotective effects for BDL-induced cirrhosis.

Sonoporation-mediated gene transfer into adult rat dorsal root ganglion cells

BackgroundGene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment. However, effective transfection of postmitotic neurons remains a challenge. The aim of this study was to develop a method for gene transfer into rat primary dorsal root ganglion neurons using sonoporation.MethodsDissociated cells from adult rat dorsal root ganglion (DRG) cells were sonicated for 1-8 s at 2.5-10 W to determine the optimal ultrasound duration and power for gene transfection and cell survival. Transfection efficiency was compared between sonoporation, liposome and lentiviral vector gene transfer techniques.ResultsThe optimum ultrasound intensity was 5 W for 2 s and yielded an efficiency of gene transfection of 31% and a survival rate of 35%.ConclusionsSonoporation can be optimized to minimize cell death and yield a high percentage of transfected neurons and that this technique can be easily applied to primary cultures of rat dorsal root ganglion neurons.

Pulsed radiofrequency treatment attenuates increases in spinal excitatory amino acid release in rats with adjuvant-induced mechanical allodynia.

Excitatory amino acids (EAAs) play a critical role in the development of peripheral tactile and thermal hypersensitivity after the induction of paw inflammation in rats. We used a spinal microdialysis model to examine the effect of complete Freund’s adjuvant (CFA)-induced inflammation on the spinal release of EAAs and assessed the antinociceptive effect of pulsed radiofrequency (PRF). CFA was injected into the plantar surface of the left hind paw to induce inflammation. Either the sciatic nerve of adult CFA rats in the mid-thigh, or the L4 anterior primary ramus just distal to the intervertebral foramen was treated with PRF (20 ms, 500 kHz pulses) at a rate of 2 Hz and a maximum temperature of 42°C. Concentrations of amino acids in the dialysate from the spinal microdialysis catheter and mechanical paw withdrawal threshold were determined to evaluate the analgesic effect of PRF. An intraplantar injection of CFA induced a significant release of glutamate, aspartate, and citrulline for 7 days. The behavior tests showed that PRF administered to the anterior ramus, just distal to the intervertebral foramen, significantly reduced mechanical allodynia, and microanalysis showed a significant suppression of EAAs and citrulline release. The antiallodynic effect of PRF was observed the day following CFA injection and maintained for 7 days. We showed that PRF administered adjacent to the dorsal root ganglion suppresses the release of EAAs, which may account for the PRF antiallodynic properties observed in adjuvant-induced inflammation.

Altered neuronatin expression in the rat dorsal root ganglion after sciatic nerve transection.

BackgroundSeveral molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among them, neuronatin (Nnat) was the one which expression was significantly up-regulated. Nnat was identified as a gene selectively expressed in neonatal brains and markedly reduced in adult brains. The present study investigated whether the expression of Nnat correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve.MethodsWestern blotting, immunohistochemistry, and the Randall and Selitto test were used to study the protein content, and subcellular localization of Nnat in correlation with pain-related animal behavior.ResultsIt was found that after nerve injury, the expression of Nnat was increased in total protein extracts. Unmyelinated C-fiber and thinly myelinated A-δ fiber in adult dorsal root ganglions (DRGs) were the principal sub-population of primary afferent neurons with distributed Nnat. The increased expression of Nnat and its subcellular localization were related to mechanical hyperalgesia.ConclusionsThe results indicated that there was significant correlation between mechanical hyperalgesia in axotomy of sciatic nerve and the increased expression of Nnat in C-fiber and A-δ fiber of adult DRG neurons.

Pulsed radiofrequency attenuates diabetic neuropathic pain and suppresses formalin-evoked spinal glutamate release in rats

BACKGROUND: Pulsed radiofrequency (PRF) has been used to treat chronic pain for years, but its effectiveness and mechanism in treating diabetic neuropathic pain are still unexplored. The aim of this study was to elucidate the modulation of diabetic neuropathic pain induced by streptozotocin and the release of spinal excitatory amino acids by PRF. METHODS: Diabetes was induced by intraperitoneal administration of streptozotocin. Pulsed radiofrequency was applied to L5 and L6 dorsal roots at 42 °C for 2 min. The responses of all of the groups to thermal, mechanical and cold stimuli were measured for a period of 6 d after this process. Seven days after PRF treatment, intrathecal microdialysis was used to examine the effect of pulsed radiofrequency on the formalin-evoked spinal release of excitatory amino acids and concurrent behaviour responses from diabetic rats. RESULTS: Three weeks after intraperitoneal streptozotocin treatment and before PRF application, mechanical, thermal and cold hypersensitivity occurred. Application of PRF significantly alleviated hyperglycaemia-induced mechanical, thermal and cold hypersensitivity and also attenuated the increase in formalin-evoked CSF glutamate concentration, compared with sham treated diabetic rats. CONCLUSION: It may be concluded that PRF has an analgesic effect on neuropathic pain by suppressing the nociception-induced release of excitatory neurotransmitters. PRF may provide a novel promising therapeutic approach for managing diabetic neuropathic pain.

Anesthetic breathing circuit obstruction mimicking severe bronchospasm: An unusual manufacturing defect

We report an unexpected ventilation difficulty with an anesthetic breathing circuit in a pediatric patient receiving left herniorrhaphy. A manufacturing defect in a limb of the anesthetic breathing circuit caused this problem. This defect induced a high-pitched, wheezing-like sound, which was difficult to differentiate from a hyper-reactive airway, commonly seen in pediatric patients with recent upper respiratory tract infection. We recommend that the patency of the anesthetic breathing circuit should routinely be examined before connecting it to the anesthesia machine.

750. Coelectrotransfer to Spinal Cord of Three Plasmids Coding for Pain Killer Gene and Regulatory Factors of the Tetracycline-Inducible System To Achieve Tightly Regulated Expression, Inhibition of Thermal Hyperalgesia, and Mechanical Allodynia

We describe an approach employing intrathecal plasmid electrotransfer to deliver secretable forms beta-endorphin, which can produce profound and long lasting analgesia in neuropathic rats. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. The regulation of beta-endorphin expression was first assessed in spinal neuronal culture, then we electrotranfected this plasmid into the spinal cord of mononeuropathic rats and evaluated the analgesic potential of this therapy in vivo by thermal and mechanical withdrawal latency. Intraperitoneal injections of doxycycline were made to elucidate the possible exogenous upregulation of transfected beta-endorphin gene expression in vivo. The levels of beta-endorphin were analysed by intrathecal microdialysis and RIA. We found that he expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) both in vitro and vivo. The beta-endorphin protein was secreted into CSF at a level of 45 pmol/L in dialysate, which was sufficient to inhibit neuropathic pain. In conclusion, we have shown that controllable expression of beta-endorphin can be obtained following intrathecal electrotransfer of a tetracycline-inducible, three-plasmid-based system. Beta-endorphin protein expression following intrathecal electrotransfer allweviates siatic nerve ligation-induced limb pain. Although physiological minute-to-minute beta-endorphin regulation may not be attained, this approach suffice as a sole or complementary treatment for chronic pain.