Cerebrospinal fluid of chronic osteoarthritic patients induced interleukin-6 release in human glial cell-line T98G.

BACKGROUND: Chronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored. METHODS: Fifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1ß and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression. RESULTS: Significant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients' CSF in the presence of LPS. The IL-6 levels showed positive association with pain score (adjusted B estimate = 10.1 (95% Confidence Interval 4.3-15.9); p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase. CONCLUSION: Higher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of "unknown molecule" in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed.

Malignant Hyperthermia and Ryanodine Receptor Type 1 Gene (RyR1) Mutation in a Family in Singapore.

INTRODUCTION: Sporadic clinical episodes of malignant hyperthermia (MH) that develop during general anaesthesia (GA) have been reported in Singapore. However, there is no published local report of a confirmed case of MH susceptibility (MHS) by skeletal muscle contracture tests and/or molecular tests. MATERIALS AND METHODS: We report 2 patients from an extended family who developed signs of clinical MH while under GA. The MH episodes were successfully treated with intravenous dantrolene sodium. Sequence analysis of the entire Ryanodine Receptor Type 1 (RyR1) coding gene was carried out in an index patient. RESULTS: The index patient was found to carry a c.7373G>A (p.Arg2458His) mutation in exon 46. This particular mutation satisfies the criteria for a MHS causative mutation. Hence, the index patient was considered to be MHS and did not need to undergo further muscle contracture testing. The same mutation was also found in 3 other members of his extended family. CONCLUSION: This is the first report of a Singaporean family with at least 4 members carrying a MH-causative mutation in RyR1 gene. This report serves to highlight the existence of the putative gene for MH in Singapore, and the need for clinical vigilance during anaesthesia involving the use of triggering agents.

Cerebrospinal fluid of postherpetic neuralgia patients induced interleukin-6 release in human glial cell-line T98G.

Chronic intractable pain caused by postherpetic neuralgia (PHN) can be alleviated by intrathecal (i.t.) steroid therapy. We investigated the possibility that interleukin-6 (IL-6) release in an in vitro system could be a potential marker for evaluating the effectiveness of i.t. steroid therapy in PHN patients. We studied 32 patients who received a course of i.t. injection of water-soluble dexamethasone. Their therapeutic index was calculated as such: ((Pain score before treatment - Pain score after treatment)÷Pain score before treatment)×100%, and they were divided into two groups, therapy effective (index>50%) and ineffective (index<50%). Cerebrospinal fluid (CSF) from the patients was used to stimulate cultures of T98G glioblastoma cells, and the subsequent IL-6 release was measured by enzyme-linked immunosorbent assay (ELISA). Our results showed that the CSF triggered IL-6 release from T98G cells in a volume-dependent manner. IL-6 release was significantly lower when using CSF from the therapy effective patient group (p<0.001) compared to the therapy ineffective group. In particular, therapy effective patients had less IL-6 release even before treatment as compared to therapy ineffective patients. In the therapy effective group, in vitro steroid treatment suppressed the CSF's IL-6 releasing effect almost completely, whereas in the therapy ineffective group, the IL-6 release was significantly reduced but remained detectable. These in vitro tests may provide an objective evaluation on the efficacy of i.t. steroid therapy administered to PHN patients.

[Impacts on the skin temperature by the different distances of moxibustion: discussion on the safe distance of moxibusiton].

OBJECTIVE: To observe the impacts of the different distances of moxibustion on local skin temperature and provide a safy distance of moxibustion. METHODS: Three healthy adult volunteers were included. The pure moxa stick (without other herbs mixed together) was used. The moxa-stick moxibustion and the mild moxibustion (with moxa box) were applied to Zusanli (ST 36) on the right side and Guanyuan (CV 4) respectively. The distance from moxibustion to the local skin was 2 cm, 3 cm and 4 cm separately. The moxibustion time was limited by 3 cm stick burned out. The infrared thermography was adopted to record and store thermal images and made the systematic analysis. The same trial was repeated on the second day. The means of the skin temperature measured at each acupoint each time was taken as the results for the analysis. RESULTS: (1) Moxibustion with moxa box at Guanyuan (CV 4): at the distance of 4 cm, the local skin temperature at over 44 degrees C [(44.1 +/- 1.3)-(46.7 +/- 1.5) degrees C)] lasted 7 min; at the distance of 3 cm, the local skin temperature at over 44 degrees C [(44.1 +/- 1.3)-(49.3 +/- 2.0) degrees C] lasted about 10 min and that at over 49 degrees C [(49.0 +/- 2.1)-(49.3 +/- 2.0) degrees C)] lasted 2 min; at the distance of 2 cm, the observation could not be followed due to local burning pain. (2) Moxa-stick moxibustion at Zusanli (ST 36): at the distance of 4 cm, the skin temperature was ranged from (40.0 +/- 2.0) degrees C to (44.9 +/- 2.3) degrees C; at the distance of 3 cm, in 1 min of moxibustion, the skin temperature increased over 44 degrees C, sustaining in the range from (45.9 +/- 3.0) degrees C to (47.8 +/- 2.0) degrees C; at the distance of 2 cm, the observation could not be followed due to local burning pain. CONCLUSION: In moxibustion, the closer the moxa stick to the skin is, the higher the local skin temperature is. No matter with stick moxibustion or box moxibustion, the distance of moxa stick to the skin should be in the range from 3 to 4 cm.

Elevated prepronociceptin, nociceptin/orphanin FQ and nocistatin concentrations in rat chronic constriction nerve injury and diabetic neuropathic pain models.

Nociceptin/orphanin FQ (N/OFQ) and nocistatin are derived from the same precursor peptide, prepronociceptin. N/OFQ and nocistatin have been postulated to participate in pain modulation. In this study, we investigated whether the prepronociceptin, N/OFQ and nocistatin concentrations in the brain and spinal cord would be altered in chronic constriction injury and diabetic rat neuropathic pain models. Total brain and spinal cord lysates as well as serum from rats that had undergone chronic constriction injury and streptozocin-induced diabetic neuropathy were used to determine the concentrations of three peptides using competitive radioimmunoassay. We found that N/OFQ and prepronociceptin concentrations were significantly raised in both rat neuropathic pain models. Nocistatin was raised in the brains of post traumatic neuropathy pain rats. Overall, our data have demonstrated for the first time that prepronociceptin, N/OFQ and nocistatin concentrations are significantly altered at different tissues of two rat neuropathy pain models.

Singaporean nurses' provision of guidance to parents on non-pharmacological postoperative pain-relief methods: An educational intervention study.

This study examined the impact of an educational intervention (booklet distribution and lectures) on Singaporean nurses' provision of guidance to parents on the use of non-pharmacological methods of pain relief for their child's postoperative pain. Using a quasi-experimental one-group pre- and post-test study design, 134 and 112 registered nurses completed the questionnaires pre- and post-test, respectively. More than 75% of the nurses "always" guided parents to use breathing techniques, relaxation, positioning, comforting/reassurance, helping with activities of daily living, and creating a comfortable environment in the pretest and touch, presence, and distraction in addition to the aforementioned methods in the post-test. The nurses' provision of guidance to parents on all non-pharmacological methods increased, but statistically significant increases only were found in relation to massage and positive reinforcement. The results suggested that the educational intervention had some impact on nurses' provision of guidance to parents on the use of non-pharmacological methods of pain relief for children's postoperative pain. Continuing education in pain management should be provided to nurses in order to equip them with the knowledge to improve their practice.

The use of nonpharmacological methods for children's postoperative pain relief: Singapore nurses' perspectives.

PURPOSE: The purpose of this study was to examine nurses' use of nonpharmacological methods for school-age children's postoperative pain relief. DESIGN AND METHODS: A survey was conducted in 2008 with a convenience sample of 134 registered nurses from 7 pediatric wards in Singapore. RESULTS: Nurses who were younger, had less education, lower designation, less working experience, and no children of their own used nonpharmacological methods less frequently. PRACTICE IMPLICATIONS: Nurses need training and education on nonpharmacological pain relief methods, particularly on methods that have been shown to be effective in prior research but that were less often used by nurses in this study: massage, thermal regulation, imagery, and positive reinforcement.

A standardized transcutaneous electric acupoint stimulation for relieving tobacco urges in dependent smokers.

The efficacy of acupuncture in smoking cessation, and its effect on the urge to smoke are unclear. We evaluated the effect of a standardized protocol of transcutaneous electric acupoint stimulations (TEAS) on alleviating the urge to smoke. Ninety-eight smokers were recruited in two double-blind studies. Participants abstained from smoking for 26 h, and were randomized to receive TEAS alternating between 2 and 100 Hz at four acupoints (LI4 and PC8, PC6 and TE5) at four different intensities (10, 5, Intermittent 5 or 0 mA). The urge to smoke was assessed by the Questionnaire of Smoking Urges (QSU-Brief). In Experiment 1, the 10 mA group (n = 20) was compared with the 5 mA group (n = 20); the increase in smoking urges did not differ significantly. Considering the possibility that 5 mA may be an active intervention, in Experiment 2, a true placebo (0 mA), and a proxy of placebo [Intermittent 5 mA (i5 mA)] were compared with 10 mA TEAS. In this experiment, 10 mA (n = 20) TEAS showed a tendency to alleviate smoking urges compared with 0 mA (n = 16), and i5 mA (n = 19) TEAS. Only when the data of smokers with Fagerstöm Test for Nicotine Dependence score ≥5 were analyzed that the difference between the 10 mA group and the control group (0 and i5 mA) became significant. Based on these preliminary findings, we conclude that TEAS applied on the skin may antagonize the increase in urge to smoke in abstinent-dependent smokers. It seems warranted to assess the efficacy of TEAS in smoking cessation clinical trials involving a larger population of dependent smokers.

Nurses' use of non-pharmacological methods in children's postoperative pain management: educational intervention study.

AIM: This paper is a report of study of the impact of an educational intervention in pain management on nurses' self-reported use of non-pharmacological methods for children's postoperative pain relief and their perceptions of barriers that limited their use of these methods. BACKGROUND: Non-pharmacological methods have been shown to be effective in relieving pain; however, many barriers, including lack of knowledge, limit nurses' use of these methods. Pain education is a promising strategy for changing nursing practice, but only a few authors have examined the effectiveness of educational interventions for nurses to help relieve children's postoperative pain. METHODS: A quasi-experimental one-group pre- and post-test design was used. Questionnaire surveys were conducted with a convenience sample of 108 Registered Nurses in two public hospitals in Singapore in 2008. RESULTS: Statistically significant increases were found in nurses' self-reported use of imagery, positive reinforcement, thermal regulation, massage and positioning in the postintervention survey. Before the intervention, these methods were less frequently used compared to other methods. Heavy workload/lack of time and the child's inability to cooperate were the most commonly reported barriers at pre- and post-test. CONCLUSION: The educational intervention had a positive effect on nurses' use of several non-pharmacological methods. Regular dissemination of updated information to nurses on these pain management methods is recommended to maintain the positive changes. Nevertheless, education alone was not sufficient to optimize nurses' use of these methods, as various barriers limited their practice.

Simultaneous analysis of D- and L-serine in cerebrospinal fluid by use of HPLC.

BACKGROUND: D-Serine is a coagonist for the glycine-binding site of the N-methyl-D-aspartate receptors and has been implicated in various neuropsychiatric functions such as learning, memory, and nociception, as well as schizophrenia and Alzheimer disease. We developed an HPLC method for D- and L-serine in cerebrospinal fluid (CSF). METHODS: The dabsylated racemic serine peak, automatically collected using a previously reported HPLC separation process for CSF amino acids, was desalted and subjected to a chiral resolution HPLC step with a Sumichiral column using an ultraviolet-visible detector. RESULTS: The limits of quantification (signal-to-noise ratio = 10) for D- and L-serine were 0.8 and 1.3 micromol/L, respectively. The mean imprecision values (CVs) for within-day measurements of D- and L-serine were 2.1% and 1.8%, respectively, and for between-day were 6.2% and 6.6%. Mean recovery of CSF serine (sum of D-serine + L-serine) applied to the Sumichiral column was 87%. The mean (SD) d-serine concentrations in 45 CSF samples obtained from 16 patients with chronic pain due to degenerative osteoarthritis of the knees, 16 with postherpetic neuralgia, and 13 with no pain were, respectively, 3.97 (0.44), 1.85 (0.21), and 2.72 (0.32) micromol/L. CONCLUSION: D- and L-serine can be quantified with ultraviolet-visible detection of dabsyl derivatives. The dabsyl derivatives are stable and allow duplicate analysis of CSF samples in multisample runs.

Levels of neuropeptides nocistatin, nociceptin/orphanin FQ and their precursor protein in a rat neuropathic pain model.

Neuropeptides nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are related to pain modulation. The amounts of these peptides and their precursor protein, prepronociceptin (ppN/OFQ) in the brain, spinal cord and serum samples of rats with partial sciatic nerve ligation (PSNL) were compared with those in naïve rats using radioimmunoassay (RIA). There was a significant rise in the levels of ppN/OFQ, N/OFQ and NST in the brains of PSNL rats. Their spinal cords showed significantly increased ppN/OFQ and NST levels but no change in N/OFQ levels. The PSNL rats also had increased serum NST (statistically significant) and N/OFQ (statistically insignificant) with decreased ppN/OFQ suggesting important roles of these peptides in neuropathic pain mechanism.

Nocistatin and its derivatives antagonize the impairment of short-term acquisition induced by nociceptin.

We studied the effects of human nocistatin, a mature form of human nocistatin of 17 amino acid length (nocistatin 17), and the amide derivative of nocistain 17 (nocistatin amide), and nociceptin/orphanin FQ on short-term acquisition in mice using a multi trial passive avoidance protocol. Nociceptin 1 nmol administered by i.c.v. injection 15 min beforehand increased the number of trials required to achieve the learning objective and decreased the step through latency times in the first, second and third test trials. Nocistatin and nocistatin 17 on their own did not affect acquisition, but were able at doses of 4 nmol to antagonize the impairment caused by nociceptin 1 nmol. Nocistatin amide on its own also did not impair acquisition and at a lower dose of 1 nmol was able to completely antagonize nociceptin. [N-Phe(1)]-nociceptin (1-13) amide, a selective opioid receptor-like 1 (ORL1) receptor antagonist, could also antagonize the effect of nociceptin, confirming that nociceptin's effect is induced via the ORL1 receptor. The results support suggestions that both nocistatin and nociceptin have roles in learning and memory, with nocistatin working as a functional antagonist of nociceptin. The shorter mature human nocistatin peptide had similar activity to the larger peptide, and its amide derivative may be more potent.

Changes in amino acids and nitric oxide concentration in cerebrospinal fluid during labor pain.

This study analyzes the relationship between amino acids and pain perception during active labor. Cerebrospinal fluid (CSF) levels of the excitatory amino acids (EAAs)-glutamate, aspartate and their amide forms, inhibitory amino acids (IAAs)-glycine, gamma-amino butyric acid (GABA) and taurine and nitric oxide (NO) related compounds-arginine and citrulline (by-product of NO synthesis) were compared between pregnant women at term pregnancy with labor pain (n = 38) and without labor pain (Caesarian section; n = 30). The levels of aspartate, glycine, GABA and citrulline were significantly higher; whilst taurine was significantly lower in the labor pain group. These findings suggest that aspartate and NO are associated with labor pain. An inhibitory role for the IAA taurine and a pronociceptive role for glycine in labor pain are proposed.

Cyclooxygenase-1 inhibition shortens the duration of diazepam-induced loss of righting reflex in mice.

Cyclooxygenase-1 (COX-1) inhibition by a selective inhibitor valeryl salicylate, or nonselective inhibitors at 10 mg/kg, including aspirin, ibuprofen, indomethacin, and picroxicam, attenuated by 29%-46% the duration of loss of righting reflex induced by diazepam (20 mg/kg) in mice. On the other hand, arachidonic acid (20 mg/kg) increased the duration of diazepam-induced loss of righting reflex by 48%. This effect of arachidonic acid was abolished by aspirin. However, aspirin at 10 mg/kg also did not alter the effects of diazepam (5 mg/kg) on spontaneous activity and rotarod performance. These findings strongly suggest that one or more COX products, most likely prostaglandins, play a significant role in modulating the hypnotic effect of diazepam. Elucidating the mechanism involved may further our understanding of the pharmacology of benzodiazepines.

Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC.

Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5+/-2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.

Perceptions of complementary and alternative medicine amongst medical students in Singapore--a survey.

BACKGROUND: In view of the current upsurge of interest in, practice of, and research into, complementary and alternative medicine (CAM) worldwide and locally, a survey was conducted to gauge the understanding, interest and knowledge of CAM amongst medical students in a local university. METHODS: A total of 555 first to fifth year medical students completed a questionnaire (54% response rate) designed to assess their knowledge, beliefs and attitudes to CAM in general and 16 common CAM therapies. RESULTS: Acupuncture was the best known therapy, with 57% claiming to know at least something about it. No students claimed they knew a lot about chiropractic, osteopathy, Ayuverdic medicine, homeopathy and naturopathy, and many had not ever heard of these therapies. Knowledge of commonly held beliefs about the 16 CAM modalities was generally poor, even for modalities which students claimed to know most about. A significant number of students had knowledge about CAM that was erroneous. Lack of scientific support was considered to be the main barrier to implementation of CAM. Attitudes to CAM were positive, with 92% believing that CAM includes ideas and methods from which conventional medicine can benefit, 86% wishing to know more about CAM and 91% stating that CAM would play an important role in their future medical practice. CONCLUSION: As the public's use of various healing practices outside conventional medicine accelerates, ignorance about these practices by the country's future medical practitioners risks broadening the communication gap between the public and the profession that serves them. The majority of medical students recognise this risk and are keen to bridge this gap.