Chien-Hui Yang

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 Complete Freund’s Adjuvant–Induced Epigenetic Suppression of Potassium Chloride Cotransporter 2 Expression

Background Pulsed radiofrequency (PRF) treatment offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freunds adjuvant (CFA)-induced inflammatory pain. Epigenetic modification of potassium-chloride cotransporter 2 (KCC2) gene expression was examined to elucidate the potential contributing mechanism. Methods Male Sprague-Dawley rats were injected with CFA into the plantar surface of the left hind paw to induce inflammation. PRF (20 minutes of 500-kHz RF pulses, delivered at a rate of 2 Hz, maximum temperature 42ºC) was delivered to the L5 and L6 anterior primary ramus just distal to the intervertebral foramen of adult CFA or saline rats. The hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before and after CFA. Acetyl-histone H3 and H4 was determined by chromatin immunoprecipitation in spinal dorsal horn. KCC2 expression was determined by Western blot. Inhibitory synaptic function was evaluated by patch clamp in lamina II neurons. Results KCC2 gene expression was suppressed through histone hypoacetylation, resulting in decreased efficacy of GABAergic signaling in CFA rats. PRF increased histone acetylation and KCC2 expression, partially restored the GABA synaptic function, and relieved sensitized pain behavior. Conclusion These findings suggest that PRF might be an alternative therapy for inflammatory pain. One of the underlying mechanisms is through modification of KCC2, which is an important determinant for the efficacy of inhibitory neurotransmission in the spinal cord, and its expression levels are regulated by histone acetylation epigenetically following inflammation.

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.