Development of a novel analgesic for neuropathic pain targeting brain-derived neurotrophic factor.

Effective treatment of neuropathic pain is challenging as its underlying mechanism remains largely unknown. Recently, the participation of brain-derived neurotrophic factor (BDNF) in neuropathic pain has been attracting increased attention. BDNF binds to a member of the tyrosine kinase receptor family, the TrkB receptor, that is specific for BDNF and is the transmembrane receptor on the posterior horn of spinal cord. In the present study, we purified two proteins that included the BDNF-binding domain of TrkB (eTrkB) and eTrkB coupled with a liposomal outer surface (liposomal eTrkB) in order to inhibit the BDNF-TrkB pathway in neuropathic pain. Results of the pull-down assay showed that eTrkB was bound to BDNF. We investigated the neuropathic pain suppression effect of this purified protein by its intrathecal administration in a rat neuropathic pain model. Mechanical and thermal hyperalgesia induced by L5 lumbar nerve ligation was markedly suppressed by treatment with eTrkB protein. Furthermore, we showed a prolonged algetic inhibition by liposomal eTrkB protein treatment. In conclusion, this study suggests that eTrkB, which sequesters endogenous BDNF and inhibits the BDNF-TrkB pathway, may prove to be a novel analgesic to treat neuropathic pain.

Five-aza-2'-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells.

DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.

Up-regulation of brain-derived neurotrophic factor in the dorsal root ganglion of the rat bone cancer pain model.

Metastatic bone cancer causes severe pain, but current treatments often provide insufficient pain relief. One of the reasons is that mechanisms underlying bone cancer pain are not solved completely. Our previous studies have shown that brain-derived neurotrophic factor (BDNF), known as a member of the neurotrophic family, is an important molecule in the pathological pain state in some pain models. We hypothesized that expression changes of BDNF may be one of the factors related to bone cancer pain; in this study, we investigated changes of BDNF expression in dorsal root ganglia in a rat bone cancer pain model. As we expected, BDNF mRNA (messenger ribonucleic acid) and protein were significantly increased in L3 dorsal root ganglia after intra-tibial inoculation of MRMT-1 rat breast cancer cells. Among the eleven splice-variants of BDNF mRNA, exon 1-9 variant increased predominantly. Interestingly, the up-regulation of BDNF is localized in small neurons (mostly nociceptive neurons) but not in medium or large neurons (non-nociceptive neurons). Further, expression of nerve growth factor (NGF), which is known as a specific promoter of BDNF exon 1-9 variant, was significantly increased in tibial bone marrow. Our findings suggest that BDNF is a key molecule in bone cancer pain, and NGF-BDNF cascade possibly develops bone cancer pain.

Antinociceptive effects of intrathecal landiolol injection in a rat formalin pain model.

Perioperative beta-blocker administration has recently been recommended for patients undergoing cardiac or other surgery due to the beneficial cardiovascular effects of these agents. In addition, some studies have reported that perioperatively administered beta-blockers also have analgesic effects. In this study, to investigate the antinociceptive effects and the analgesic profile of landiolol, we examined the effects of intrathecal landiolol administration on nociceptive pain behavior and c-fos mRNA expression (a neural marker of pain) in the spinal cord using a rat formalin model. We found that pain-related behavior was inhibited by intrathecal landiolol administration. Moreover, the increase in c-fos mRNA expression on the formalin-injected side was less pronounced in rats administered landiolol than in saline administered controls. Thus, intrathecal administration of landiolol exhibited antinociceptive effects. Further investigation of the antinociceptive mechanism of landiolol is required.

Expression changes of the neuregulin 1 isoforms in neuropathic pain model rats.

The neuregulin1 (Nrg1) gene that is expressed in the dorsal root ganglion (DRG) contains an EGF-like domain, which is known to be a direct ligand for ErbB3 and ErbB4. Multiple splice variants of the Nrg1 gene are broadly classified into 3 groups by structural features (type I, type II and type III) and their functions differ in various tissues. The Nrg1 gene has emerged as a key mediator of axon-Schwann cell interactions and as a regulator of Schwann cell development. The Nrg1 gene is indicated as a promising growth factor for neuronal development. However, the function of the Nrg1 in pain has not been clarified. We therefore, examined the expression profiles of each type of the Nrg1 transcript in the bilateral L4/L5 DRGs using L5 spinal nerve ligation (SNL) model rats and complete Freund's adjuvant (CFA) model rats. Behavior tests have shown typical mechanical hyperalgesia in both the L5SNL model and the CFA model. In the L5SNL model, expression of the Nrg1 type I and type II were significantly increased in the L5 DRG. On the other hand, the expression of the Nrg1 type III was decreased in the L5 DRG. We demonstrated that the expression changes of the Nrg1 isoforms in the ipsilateral DRGs were preferentially related to the response to nerve injury. Our findings suggest that the aberrant expression may play an important role in nerve injury, regeneration and subsequent neuropathic pain on the L5SNL.

Decoy strategy targeting the brain-derived neurotrophic factor exon I to attenuate tactile allodynia in the neuropathic pain model of rats.

The mechanism underlying neuropathic pain is still largely unclear. Recently, much attention has been focused on the role of brain-derived neurotrophic factor (BDNF) as a neuromodulator in the spinal cord. We previously reported that the expression of Bdnf exon I mRNA was remarkably up-regulated in the dorsal root ganglion (DRG) neurons with the rat L5 spinal nerve ligation (SNL) model. In the present study, we investigated whether neuropathic pain response would be reduced by the inhibition of the Bdnf exon I in the rat SNL model. We identified the promoter region of exon I and synthesized the decoy ODNs targeting the region. Reverse transcription-polymerase chain reaction analysis confirmed that the decoy ODN treatment reduced SNL-induced Bdnf exon I mRNA up-regulation in ipsilateral L4 and L5 DRGs. Furthermore, post-treatment with the decoy ODNs significantly attenuated SNL-induced tactile allodynia. This study suggested that decoy ODNs targeting the Bdnf exon I might provide a novel analgesic strategy for the treatment of neuropathic pain.

Effects of selective spinal nerve ligation on acetic acid-induced nociceptive responses and ASIC3 immunoreactivity in the rat dorsal root ganglion.

We investigated changes in pain behavior after injection of acetic acid in the hindpaws of rats with L5 spinal nerve ligation (SNL)-induced neuropathy. We also examined immunoreactivity for acid-sensing ion channel 3 (ASIC3) in the dorsal root ganglion (DRG) of rats with L5 SNL. Two weeks after SNL, the withdrawal threshold to a mechanical stimulus was significantly lower in the SNL group than in the sham-operated group (n=9 per group, P<0.01). After acetic acid injection, spontaneous pain responses in the SNL group were significantly increased compared to those in the sham-operated group (n=5, P<0.05). L5 SNL significantly increased the proportion of total ASIC3-immunoreactive (ir) neurons in the ipsilateral L4 DRG compared to that in sham-operated rats (n=4, P<0.01). Analysis of cell size showed that the proportion of large (>1200 mm(2)) ASIC3-ir neurons in the ipsilateral L4 DRG significantly increased after L5 SNL (P<0.05). In the ipsilateral L5 DRG, the proportion of ASIC3-ir neurons was not significantly affected by treatment. However, L5 SNL significantly increased (P<0.01) the proportion of small (<1200 mm(2)) ASIC3-ir neurons and significantly decreased (P<0.01) the proportion of large ASIC3-ir neurons compared to proportions in sham-operated animals. These findings suggest that ASIC3 is associated with hyperalgesia in response to a chemical stimulus in the L5 SNL rat model.

Expression changes of multiple brain-derived neurotrophic factor transcripts in selective spinal nerve ligation model and complete Freund's adjuvant model.

Brain-derived neurotrophic factor (BDNF) expression changes in the dorsal root ganglion (DRG) and spinal cord in some pain models. Recently, rat BDNF transcripts containing novel 5' untranslated exons were identified and characterized, and a new numbering system for rat BDNF exons was introduced. We examined the expression profiles of these novel BDNF transcripts in bilateral L4/5 DRGs in an L5-selective spinal nerve ligation (SSNL) model and bilateral L5 DRGs in a complete Freund's adjuvant (CFA) model of rats. L5SSNL increased significantly (P<0.05) the expression of total BDNF mRNA and exon I, IIA, IIB, IIC, III, IV, VI, and IXA transcripts in ipsilateral L4 DRG. Although expression of total BDNF mRNA remained unchanged in ipsilateral L5 DRG in the L5SSNL model, expression of exon I transcript increased significantly (P<0.05) and that of exon IV transcript decreased significantly (P<0.05). The expression profiles of the variant exons in ipsilateral L4 DRG of the L5SSNL model were quite similar to those in ipsilateral DRG of the CFA model, and exon I transcript was the most common BDNF mRNA in these DRGs. Although L5SSNL increased significantly (P<0.05) the expression of total BDNF mRNA and exon IIC and IXA transcripts in the contralateral L4/5 DRGs, CFA treatment did not alter the expression of total BDNF mRNA or specific transcripts in the contralateral DRGs. These findings suggest that exon I plays an important role in the increase in BDNF expression in ipsilateral DRGs regardless of condition.

Expression profiles of BDNF splice variants in cultured DRG neurons stimulated with NGF.

Expression of brain-derived neurotrophic factor (BDNF) mRNA is increased in the dorsal root ganglion (DRG) in response to peripheral inflammation. Nerve growth factor (NGF) from inflammatory tissue is thought to induce expression of BDNF. Recently, it was reported that the BDNF gene has eight non-coding exons that are transcribed independently into several splice variants. Expression of these splice variants in DRG neurons stimulated with NGF has not been studied. We examined changes in expression of BDNF splice variants in a rat model of peripheral inflammation and in cultured DRG neurons exposed to NGF. Total BDNF mRNA was increased by inflammation in vivo and by NGF in vitro. Among all splice variants, exon 1-9 showed the greatest increase in expression in both experiments. Our results indicate that exon 1-9 contributes to changes in total BDNF levels and may play an important role in the acute response of DRG to NGF.

Altered response to formalin by L5 spinal nerve ligation in rats: a behavioral and molecular study.

BACKGROUND: The status of neuropathic pain alters the responsiveness to formalin injection in rats. However, the mechanism by which this alteration occurs is unknown. METHODS: We used immunocytochemistry to examine the expression of brain-derived neurotrophic factor (BDNF) and calcitonin gene-related peptide (CGRP) in the spinal cord of rats with L5 spinal nerve ligation (SNL)-induced neuropathy, and investigated the expression of c-Fos in the spinal cord after injection of formalin in the hindpaw of rats with SNL. RESULTS: Four weeks after SNL, the withdrawal threshold was significantly lower in the SNL group than in the sham-operated (sham) group (n = 12 per group, P < 0.05). In the SNL group, expression of BDNF in the L4 (P < 0.05) and L5 (P < 0.01) superficial dorsal horn was significantly decreased compared to that in the sham group. CGRP protein in the L5 but not in the L4, dorsal horn was significantly decreased compared to that in the sham group (P < 0.01). After formalin injection, spontaneous pain responses in the SNL group were significantly decreased compared to those in the sham group (P < 0.05). Immunolabeling for c-Fos was significantly decreased in the L4 and L5 dorsal horn in the SNL group (P < 0.01). CONCLUSION: Our examination of c-Fos distribution indicates that decreased neuronal activity in the spinal cord in response to inflammatory pain may be important for altering the perception of acute pain. Decreased BDNF expression in response to SNL-induced neuropathy may be involved in this alteration.

The effects of continuous epidural anesthesia and analgesia on stress response and immune function in patients undergoing radical esophagectomy.

We investigated whether perioperative extensive epidural block (C3-L) affects postoperative immune response in patients undergoing radical esophagectomy. Patients undergoing radical esophagectomy were randomly assigned to either general anesthesia with continuous epidural infusion via 2 epidural catheters that was continued for postoperative analgesia (group E, n = 15) or intraoperative general anesthesia and postoperative IV morphine analgesia (group G, n = 15). Plasma levels of stress hormones, cytokines, C-reactive protein (CRP), leukocyte counts, and distribution of lymphocyte subsets were assessed before and after surgery and on postoperative days (PODs) 1 and 3. In comparison with group E, significant increases in plasma epinephrine level at the end of surgery (P < 0.05) and norepinephrine level at the end of surgery (P < 0.01) and on POD1 (P < 0.01) and POD3 (P < 0.01) and significant decrease in cluster of differentiation (CD4/CD8 ratio) at the end of surgery (P < 0.05) were observed in group G. However, there were no significant differences in other variables between groups. In both groups, plasma cortisol, adrenocorticotropic hormone, interleukin (IL)-1beta, IL-6, IL-10, and CRP levels were increased after surgery (each group P < 0.01) and IL-1beta, IL-6, IL-10, and CRP were still increased on POD1 and POD3 (each change, each group P < 0.01). Leukocyte counts were increased on POD1 (each group P < 0.05) and POD3 (each group P < 0.01). The proportion of lymphocytes decreased from the end of surgery to POD3 (each group P < 0.01). The proportion of B cells was increased on POD1 (each group P < 0.01); that of natural killer cells was decreased at POD1 and POD3 (each group P < 0.01). We conclude that tissue damage and inflammation apparently overcome the effects of extensive epidural block on stress response and immune function in radical esophagectomy.

Total spinal anesthesia provides transient relief of intractable pain.

PURPOSE: Intentional total spinal anesthesia (TSA) has been used for intractable pain treatment. However, the long-term effect of pain-relief is controversial. We investigate the short- and long-term effects of pain-relief by TSA. METHODS: Twelve patients with intractable pain participated in a crossover study. All participants received two different treatments in random order at a 30-day interval: i.v. infusion with 300 mg of lidocaine (i.v.-Lido), and TSA with 20 mL of 1.5% lidocaine (TSA-Lido). Pain level at rest was scored with the visual analogue scale (VAS: 0-100), and blood pressure and heart rate were measured before and at two hours, 24 hr, seven days, and 30 days after treatment. Plasma lidocaine concentrations were measured at 0.5, one, and two hours. RESULTS: Heart rate and mean arterial pressure during or after TSA-Lido were similar to those before TSA-Lido. Plasma lidocaine concentrations were similar between the two treatments. No significant difference in any value occurred in the i.v.-Lido treatment. VAS were similar before both treatments (87 +/- 6 for TSA-Lido; 86 +/- 7 for i.v.-Lido). After TSA-Lido, VAS decreased significantly until day seven (two hours, 17 +/- 22, P < 0.01; 24 hr, 43 +/- 20, P < 0.01; seven days, 66 +/- 16, P < 0.01). However, VAS returned to the pre-block values 30 days after TSA-Lido. CONCLUSION: Intractable pain was decreased significantly for several days after TSA, but pain-relief was not sustained.