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.

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.

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.

Localization of nocistatin-binding sites in mice brain and spinal cord using a biotinylated nocistatin probe.

Nocistatin and nociceptin/orphanin FQ are two neuropeptides processed from the same precursor prepronociceptin. They have opposing roles in nociception and several other biological functions. Whereas the location and structure of the nociceptin/orphanin FQ receptors has been defined, the location of the nocistatin receptors remains unknown. In the course of this study, we synthesized a novel probe for histochemistry by linking biotin to the N terminus of nocistatin, and purified this with high-pressure liquid chromatography and confirmed the structure by mass spectrometer. Using this probe, we found nocistatin-binding sites in the cerebral cortex and the dorsal horn nucleus of the spinal cord. We also found that the nocistatin-binding sites were in the cell body, whereas the nociceptin/orphanin FQ binding sites were on the fibrous processes.

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.

Supraspinal nocistatin and its amide derivative antagonize the hyperalgesic effects of nociceptin in mice.

Nocistatin (NST) and nociceptin (NCP)/orphanin FQ are new neuropeptides derived from the same precursor molecule, and which are involved in pain transmission. Nocistatin has been shown to antagonize several effects of nociceptin by acting on a different receptor. We examined the effects of supraspinal nocistatin and nocistatin amide, and their interaction with nociceptin on nociceptive behavior in mice, using hotplate response times. We found that both nocistatin and nocistatin amide did not change the response time compared to control mice, whereas increasing doses of nociceptin caused progressive shortening of response times. Nocistatin and nocistatin amide were both able to antagonize the hyperalgesic effect of nociceptin. The effect of nocistatin amide was longer lasting and more potent, suggesting that the C-terminal free carboxyl group of nocistatin is not necessary for its biological activity, and that the amide derivative may be more biologically stable.

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.

Expression and purification of a neuropeptide nocistatin using two related plant viral vectors.

Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors.