Lin-Cheng Yang

Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat

N-methyl-D-aspartate (NMDA) receptor activation, at the level of the spinal cord, has been shown to play an important role in the facilitation of nociception in several animal models. However, the use of NMDA antagonists as analgesics is limited by serious side effects due to nonselective effects among the NMDA receptor subtypes. Recent discoveries revealed that the transfection of small interfering RNAs (siRNAs) into animal cells resulted in the potent, long-lasting, post-transcriptional silencing of specific genes. Thus, we investigated the effect of intrathecal (i.t.) injection of siRNAs targeting NMDA-R2B receptor subunit protein (NR2B) receptors, a subunit of NMDA receptor, for the modulation of pain. The results indicate that the use of siRNA targeting the NR2B subunit not only decreased the expression of NR2B mRNA and its associated protein, as demonstrated by real-time PCR and Western blotting, but also abolished formalin-induced pain behaviors in rat model. The peak effect occurred on day 3 for mRNA and day 7 for its protein, following i.t. injection of 5 μg of siRNA-NR2B. These data prove the feasibility of i.t. siRNAs in the investigation of functional gene expression in the context of whole animal behavior for the management of chronic pain.

Intrathecal bupivacaine with morphine or neostigmine for postoperative analgesia after total knee replacement surgery

PurposeTo compare the postoperative analgesic efficacy and safety of intrathecal (IT) néostigmine and IT morphine in patients undergoing total knee replacement under spinal anesthesia. Methods: Sixty patients scheduled for elective total knee replacement under spinal anesthesia were randomly divided into three equal groups which received IT 0.5% hyperbaric bupivacaine 15 mg with either normal saline 0.5 ml_, néostigmine 50 μg, or morphine 300μg. The maximal level of sensory block, duration of analgesia, time to use of rescue analgesics, the overall 24-hr and four-hour interval visual analogue scale (VAS) pain score, and the incidence of adverse effects were recorded for 24 hr after administration.ResultsThere was no significant difference in maximal level of sensory block among the three groups. The morphine group had a later onset of postsurgical pain and longer time to first rescue analgesics than the néostigmine group (P < 0.05). Overall 24-hr VAS pain scores were significantly higher in the saline groupvs the morphine and néostigmine groups (P < 0.05). Motor block lasted significantly longer in the néostigmine group than in the morphine and saline groups (P < 0.05). The incidence of adverse effects was similar in the néostigmine and morphine groups except for pruritus (70%) occurring more frequently in the morphine group than in the néostigmine and saline groups (0%; P < 0.05). Overall satisfaction rates were better in the néostigmine group than in the morphine and saline groups (P < 0.05).ConclusionsIT néostigmine 50 μg produced postoperative analgesia lasting about seven hours with fewer side effects and better satisfaction ratings than IT morphine 300 μg.RésuméObjectifComparer l’efficacité et l’innocuité analgésique postopératoire de l’administration intrathécale (IT) de néostigmine et de morphine chez des patients devant subir une arthroplastie totale du genou sous rachianesthésie.MéthodeSoixante patients devant recevoir une prothèse totale de genou sous rachianesthésie ont été répartis au hasard en trois groupes égaux. Ils ont reçu 15 mg de bupivacaine hyperbare IT à 0,5 % et, soit 0,5 mL de solution salée, soit 50 μg de néostigmine, soit 300 μg de morphine. Le niveau maximal du bloc sensitif, la durée de l’analgésie, l’heure des premières demandes d’analgésiques de secours, les scores de douleur des 24 h d’observation et de chaque intervalle de quatre heures selon l’échelle visuelle analogique (EVA) et l’incidence d’effets indésirables ont été enregistrés pendant 24 h après l’administration médicamenteuse.RésultatsLe niveau maximal de blocage sensitif n’a pas présenté de différence intergroupe significative. Chez les patients avec morphine, la douleur post-chirurgicale s’est installée plus tard et leur première demande d’analgésie de secours a donc eu lieu plus tard que chez les patients avec néostigmine (P < 0,05). Les scores de douleur à l’EVA ont été, sur 24 h, signifcativement plus élevés avec la solution salée vs la morphine ou la néostigmine (P < 0,05). La durée du blocage moteur a été signifcativement plus longue avec la néostigmine qu’avec la morphine ou la solution salée (P < 0,05). L’incidence d’effets indésirables a été similaire avec la néostigmine et la morphine, sauf pour le prurit (70%) qui a été plus fréquent avec la morphine qu’avec la néostigmine ou la solution salée (0 %; P < 0,05). Le taux de satisfaction générale a été meilleur avec la néostigmine qu’avec la morphine ou la solution salée (P < 0,05).

Electroporation for direct spinal gene transfer in rats

We investigated the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of pE-GFP C1 vector, followed by five electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting. At day 1, 3 or 7 following electroporation a clear GFP expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and Western blotting. The expression of GFP protein was peaked between 3 and 7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery and have a potential to be used clinically, especially, acute or chronic pain.

Gene-gun particle with pro-opiomelanocortin cDNA produces analgesia against formalin-induced pain in rats

Endogenous opioid peptides play an essential role in the intrinsic modulation and control of inflammatory pain, and could be therapeutically useful. These opioid peptides are synthesized as parts of larger precursor molecules. One such precursor molecule is pro-opiomelanocortin (POMC). In this study, we developed a gene-gun method for the transfer of POMC cDNA in vivo, and investigated its therapeutic effect on inflammatory pain in a rat model of formalin-induced pain. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered to the skin of rats by gene gun. Three days after gene-gun injection, 1% formalin was injected. Endorphin levels were measured in the serum and skin after the formalin test, and skin histology was used to detect endorphin after green fluorescent protein (GFP; control) or POMC cDNA transfer. There was no significant difference in the results of acute nociceptive tests between the experimental and control groups. There was also no difference in response between the groups to phase 1 of the formalin test. However, rats which received POMC cDNA via gene-gun injection showed a significantly reduced response in phase 2 of the formalin test. Endorphin immunoreactivity in the skin increased approximately three- to four-fold in experimental animals compared with GFP-treated controls at day 3 after injection. The phase 2 response in animals treated with formalin and naloxone did not differ significantly from the control, implying that the analgesic effects of POMC cDNA particle injection in phase 2 of the formalin test are reversed by naloxone. There are two major findings from this study. First, in vivo DNA delivery by gene gun to the skin is feasible. Second, the production of β-endorphin is insufficient to block phasic pain, but is effective against sensitization of the afferent neurons during phase 2 of the formalin test.

Effect of pretreatment with ketorolac on propofol injection pain.

Background:  Pain on injection is still a major problem with propofol. We performed this study to compare different doses of intravenous (i.v.) ketorolac with and without venous occlusion and its effect on the incidence and the severity of the pain after propofol injection.

Intrathecal spinal progenitor cell transplantation for the treatment of neuropathic pain

Injury to, or dysfunction of, the nervous system can lead to spontaneous pain, hyperalgesia, and/or allodynia. It is believed that the number and activity of GABAergic neurons gradually decreases over the dorsal horn. Glutamic acid decarboxylase (GAD) immunocompetence has been demonstrated on spinal progenitor cells (SPCs) cultivated in vitro. The intrathecal implantation of these cultivated progenitor cells may provide a means of alleviating neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the hind paw of rats. SPCs (1 × 106) were implanted intrathecally on the third day after the CCI surgery. The behavioral response to thermal hyperalgesia was observed and recorded during the 14 days postsurgery. Various techniques were utilized to trace the progenitor cells, confirm the differentiation, and identify the neurotransmitters involved. GAD immunoactivity was revealed for 65% of the cultivated spinal progenitor cells in our study. We also determined that transplanted cells could survive more than 3 weeks postintrathecal implantation. Significant reductions were demonstrated for responses to thermal stimuli for the CCI rats that had received intrathecal SPC transplantation. A novel intrathecal delivery with SPCs reduced CCI-induced neuropathic pain.

Altered synaptophysin expression in the rat spinal cord after chronic constriction injury of sciatic nerve

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Abeta-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. Synaptophysin is a presynaptic vesicle protein, useful in the identification of synaptogenesis. Here we investigated whether synaptogenesis as measured by the expression of synaptophysin protein correlates with symptoms of neuropathic pain in rats with a chronic constriction injury (CCI) of the sciatic nerve. We used immunohistochemistry, Western immunoblotting and densitometry to study the distribution of synaptophysin and to quantify relative protein. Synaptophysin was increased in the ipsilateral dorsal horn with a peak level on day 14 and returned to baseline on day 21 post-CCI. Synaptophysin levels temporally correlated with thermal hyperalgesia but not with tactile allodynia. Our results indicate that thermal hyperalgesia in CCI significantly correlates with synaptogenesis within the superficial layers of the dorsal horn.

Intramuscular electroporation with the pro-opiomelanocortin gene in rat adjuvant arthritis

Endogenous opioid peptides have an essential role in the intrinsic modulation and control of inflammatory pain, which could be therapeutically useful. In this study, we established a muscular electroporation method for the gene transfer of pro-opiomelanocortin (POMC) in vivo and investigated its effect on inflammatory pain in a rat model of rheumatoid arthritis. The gene encoding human POMC was inserted into a modified pCMV plasmid, and 0–200 μg of the plasmid-POMC DNA construct was transferred into the tibialis anterior muscle of rats treated with complete Freunds adjuvant (CFA) with or without POMC gene transfer by the electroporation method. The safety and efficiency of the gene transfer was assessed with the following parameters: thermal hyperalgesia, serum adrenocorticotropic hormone (ACTH) and endorphin levels, paw swelling and muscle endorphin levels at 1, 2 and 3 weeks after electroporation. Serum ACTH and endorphin levels of the group into which the gene encoding POMC had been transferred were increased to about 13–14-fold those of the normal control. These levels peaked 1 week after electroporation and significantly decreased 2 weeks after electroporation. Rats that had received the gene encoding POMC had less thermal hypersensitivity and paw swelling than the non-gene-transferred group at days 3, 5 and 7 after injection with CFA. Our promising results showed that transfer of the gene encoding POMC by electroporation is a new and effective method for its expression in vivo, and the analgesic effects of POMC cDNA with electroporation in a rat model of rheumatoid arthritis are reversed by naloxone.

Anesthetic effect of epidural anesthesia with cephalad or caudad catheterization for ankle surgery or hemorrhoidectomy

Background:  The larger size of the first sacral nerve root has been reported to be an unfavorable factor leading to sacral sparing in epidural anesthesia. Previous studies have shown that an adequate analgesic effect of the epidural block was achieved with the catheter placement in the caudal direction. In this study, the anesthetic effect of epidural anesthesia with catheter placement of a cephalic or caudad direction was compared in ankle and hemorrhoid surgery.

Changes in the levels of nitric oxide synthase and protein kinase C gamma following kainic acid receptor activation in the rat spinal cord.

In this study, we evaluated the levels of nitric oxide synthase, both neuronal and induced (nNOS and iNOS, respectively), cyclooxygenase-1 and 2 (COX-1 and COX-2) and protein kinase C gamma (PKCgamma) and correlated these with algogenic behavior following spinal kainic acid (KA) receptor activation in rats. Thirty adult male Sprague-Dawley rats were randomly assigned into six groups (n=5). Groups A, B, and C received 0.5 g kainic acid intrathecally and were analyzed at 3, 6, 24 h after injection, respectively. Groups D, E, and F received saline and were analyzed at 3, 6, 24 h after injection, respectively. We observed for behavioral changes in the rats following intrathecal KA injection and analyzed the protein levels of NOS, COX and PKCgamma by Western blotting techniques. Importantly, we clarified the potential roles of PKCgamma in the regulation of nNOS and COX-2 following intrathecal injection with KA in the rat spinal cord. COX-2 protein was detected but not significantly changed in the lumbosacral spinal cord at 3, 6, and 24 h following intrathecal KA injection (P>0.05). In contrast, nNOS protein was detected at higher levels in comparison with normal spinal cord at 6 and 24 h after intrathecal administration of KA (P<0.05). PKCgamma also increased significantly at 3, 6, and 24 h after intrathecal KA injection when compared with the baseline level (P<0.05). On the other hand, COX-1 and iNOS were not detected in either normal or KA treated spinal cords. These results provide strong in vivo evidence to support the idea that nNOS but not COX-2, plays an important role in spinal KA receptor activation. Furthermore, up-regulation of PKCgamma is involved in KA induced algogenic behavior in rats.