Inhibitory Effectiveness in Delayed-Rectifier Potassium Current Caused by Vortioxetine, Known to Be a Novel Antidepressant.

Vortioxetine (VOR) is recognized to exert antidepressant actions. However, whether this drug modifies ionic currents in excitable cells remains unclear. The aim of this study was to explore the electrophysiological effects of VOR and other related compounds in pituitary GH3 cells and in Neuro-2a cells. VOR suppressed the delayed-rectifier K+ current (IK(DR)) in a concentration-, time-, and state-dependent manner. Effective IC50 values needed to inhibit peak and sustained IK(DR) were computed to be 31.2 and 8.5 µM, respectively, while the KD value estimated from minimal binding scheme was 7.9 µM. Cell exposure to serotonin (10 µM) alone failed to alter IK(DR), while fluoxetine (10 µM), a compound structurally similar to VOR, mildly suppressed current amplitude. In continued presence of VOR, neither further addition of propranolol nor risperidone reversed VOR-mediated inhibition of IK(DR). Increasing VOR concentration not only depressed IK(DR) conductance but also shifted toward the hyperpolarized potential. As the VOR concentration was raised, the recovery of IK(DR) block became slowed. The IK(DR) activated by a downsloping ramp was suppressed by its presence. The inhibition of IK(DR) by a train pulse was enhanced during exposure to VOR. In Neuro-2a cells, this drug decreased IK(DR). Overall, inhibitory effects of VOR on ionic currents might constitute another underlying mechanism of its actions.

TGF-ß1 in plasma and cerebrospinal fluid can be used as a biological indicator of chronic pain in patients with osteoarthritis.

INTRODUCTION: Previous studies have demonstrated that cytokines, transforming growth factor (TGF-ß1), and brain-derived neurotrophic factor (BDNF) can impact the intensity of pain in rodents. However, the roles of cytokines, TGF-ß1 and BDNF in humans with chronic pain in osteoarthritis remains unclear, and no comparison between plasma and central cerebral spinal fluid (CSF) has been conducted. METHODS: Patients with osteoarthritis who were scheduled to receive spinal anesthesia were enrolled. The intensity of pain was evaluated with a visual analogue scale (VAS). In addition, patients with genitourinary system (GU) diseases and without obvious pain (VAS 0-1) were included as a comparison (control) group. The levels of TGF-ß1, BDNF, tumor necrosis factor-α (TNF-α), and interleukin (IL)-8 within the CSF and plasma were collected and evaluated before surgery. RESULTS: The plasma and CSF TGF-ß1 levels were significantly lower in the osteoarthritis patients with pain (VAS ≥ 3) than in the GU control patients. Downregulation of plasma BDNF was also found in osteoarthritis patients with pain. The Spearman correlation analysis showed that the VAS pain scores were significantly negatively correlated with the levels of TGF-ß1 in the CSF of patients with osteoarthritis. However, there was no significant correlations between the pain scores and the levels of BDNF, TNF-α, and IL-8 in either the CSF or plasma. CONCLUSIONS: TGF-ß1 but not BDNF, TNF-α, or IL-8 may be an important biological indicator in the CSF of osteoarthritis patients with chronic pain.

Effective Perturbations of the Amplitude, Gating, and Hysteresis of IK(DR) Caused by PT-2385, an HIF-2α Inhibitor.

PT-2385 is currently regarded as a potent and selective inhibitor of hypoxia-inducible factor-2α (HIF-2α), with potential antineoplastic activity. However, the membrane ion channels changed by this compound are obscure, although it is reasonable to assume that the compound might act on surface membrane before entering the cell´s interior. In this study, we intended to explore whether it and related compounds make any adjustments to the plasmalemmal ionic currents of pituitary tumor (GH3) cells and human 13-06-MG glioma cells. Cell exposure to PT-2385 suppressed the peak or late amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner, with IC50 values of 8.1 or 2.2 µM, respectively, while the KD value in PT-2385-induced shortening in the slow component of IK(DR) inactivation was estimated to be 2.9 µM. The PT-2385-mediated block of IK(DR) in GH3 cells was little-affected by the further application of diazoxide, cilostazol, or sorafenib. Increasing PT-2385 concentrations shifted the steady-state inactivation curve of IK(DR) towards a more hyperpolarized potential, with no change in the gating charge of the current, and also prolonged the time-dependent recovery of the IK(DR) block. The hysteretic strength of IK(DR) elicited by upright or inverted isosceles-triangular ramp voltage was decreased during exposure to PT-2385; meanwhile, the activation energy involved in the gating of IK(DR) elicitation was noticeably raised in its presence. Alternatively, the presence of PT-2385 in human 13-06-MG glioma cells effectively decreased the amplitude of IK(DR). Considering all of the experimental results together, the effects of PT-2385 on ionic currents demonstrated herein could be non-canonical and tend to be upstream of the inhibition of HIF-2α. This action therefore probably contributes to down-streaming mechanisms through the changes that it or other structurally resemblant compounds lead to in the perturbations of the functional activities of pituitary cells or neoplastic astrocytes, in the case that in vivo observations occur.

Morphine produces better thermal analgesia in young Huntington mice and are associated with less neuroinflammation in spinal cord.

BACKGROUND: Huntington's disease (HD) is an inherited disease characterized by both mental and motor dysfunctions. Our previous studies showed that HD mice demonstrate a diminished pain response. However, few studies have focused on the relationship between HD and morphine analgesia. The purpose of this study is to investigate and compare the analgesic effects of morphine in HD and wild-type (WT) mice. METHODS: We used clinically similar transgenic HD mice (7-10 weeks of age with motor dysfunction at 8-9 mo of age) carrying a mutant Huntington CAG trinucleotide repeats to evaluate morphine analgesia. The morphine (10 mg/kg subcutaneously) analgesia was evaluated with a tail-flick in hot water (52°C). Mice spinal cords were harvested at the end of the analgesia studies. An immunofluorescence assay and western blotting were used to identify changes in the cells and cytokines. RESULTS: Our data demonstrate that preonset young HD mice exhibited a better analgesic response to morphine than the WT mice. Western blotting and an immunohistological examination of the lumbar spinal cord tissue indicated less activation of glial cells and astrocytes in the HD mice compared with the WT mice. The production levels of tumor necrosis factor α and interleukine-1ß were also lower in the young HD mice. CONCLUSION: Our data demonstrate better morphine analgesic and less pain-related cytokine responses at the spinal cord level for HD mice. Further studies are needed to determine the morphine analgesia mechanism in HD.

Mortality Risks for Dialysis Patients: A Nationwide Population-Based Study.

OBJECTIVE: Several studies have demonstrated increased postoperative mortality rates in patients on chronic hemodialysis compared with non-dialyzed patients. However, limited studies have examined factors that may contribute to postoperative mortality. METHODS: In this retrospective cohort study, data were collected from 9,140 dialysis and 45,725 non-dialysis patients undergoing surgery between 2007 to 2009 from Taiwan's National Health Insurance Registry Database. Patient demographics, comorbidities, and anesthesia duration were used to compare 30-day postoperative mortality differences in dialysis patients. RESULTS: Dialysis patients undergoing first-time surgery were significantly older, more likely male, and possessed more comorbidities. Overall, dialysis patients had significantly higher all-cause postoperative mortality (odds ratio, 15.005; 95% confidence interval, 11.917-18.893). Gender (hazard ratio [HR], 0.762), age (HR, 1.012), longer duration of inhalation general anesthesia (HR, 1.113), and comorbidities of hypertension (HR, 0.759), diabetes (HR, 1.339), congestive heart failure (HR, 1.232), coronary artery disease (HR, 1.326), cerebral vascular accident (HR, 1.312), intracranial hemorrhage (HR, 6.765), gastrointestinal bleeding (HR, 1.396), and liver cirrhosis (HR, 2.027), independently increased postoperative mortality risk in dialysis patients. Of the comorbidities, intracranial hemorrhage posed the greatest risk. CONCLUSION: Patient demographics, anesthesia factors, and comorbidities help dialysis patients understand their postoperative mortality. These potential risk factors also inform anesthesiologists and surgeons weight perioperative conditions in dialysis patients before surgery.

The analgesic effect of propofol associated with the inhibition of hypoxia inducible factor and inflammasome in complex regional pain syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) is related to microcirculation impairment caused by tissue hypoxia and peripheral cytokine overproduction in the affected human limb and chronic post-ischemic pain (CPIP) is considered as an animal model for this intractable disease. Previous studies suggest that the pathogenesis of CPIP involves the hypoxia inducible factor-1α (HIF-1α) and an exaggerated regional inflammatory and free radical response. The inhibition of HIF-1α is known to relieve CPIP. So, propofol, as a free radical scavenger, is very likely to be beneficial in terms of relieving CPIP. METHODS: We set up a CPIP model using the hindpaw of mice. We administered propofol (10 mg/kg) just after the reperfusion period (early stage) and also on the second day (late stage), as treatment. The analysis evaluated the expression of HIF-1α, free radicals, and inflammasome. RESULTS: Propofol administration produced obvious analgesia in both mechanical and thermal evaluation in the early stage of CPIP (2 h after reperfusion). Only a mild analgesic effect was found in the late stage (48 h later after reperfusion). In the early stage, the expression of HIF-1α and the inflammasome marker (NALP1) along with caspase-1 were suppressed by propofol. The free radical level also decreased in the propofol group. But those molecular changes were not founded in the late stage of CPIP. CONCLUSION: Our data demonstrated that propofol produces mice analgesia in the early stage of CPIP and this effect is associated with inhibition of free radical, hypoxia inducible factor and inflammasome.

Effectiveness of nalbuphine, a κ-opioid receptor agonist and µ-opioid receptor antagonist, in the inhibition of INa , IK(M) , and IK(erg) unlinked to interaction with opioid receptors.

Nalbuphine (NAL) is recognized as a mixer with the κ-opioid receptor agonist and the µ-opioid receptor antagonist. However, whether this drug causes any modifications in neuronal ionic currents is unclear. The effects of NAL on ionic currents in mHippoE-14 hippocampal neurons were investigated. In the whole-cell current recordings, NAL suppressed the peak amplitude of voltage-gated Na+ current (INa ) with an IC50 value of 1.9 µM. It shifted the steady-state inactivation curve of peak INa to the hyperpolarized potential, suggesting that there is the voltage dependence of NAL-mediated inhibition of peak INa . In continued presence of NAL, subsequent application of either dynorphin A1-13 (1 µM) or naloxone (30 µM) failed to modify its suppression of peak INa . Tefluthrin (Tef; 10 µM), a pyrethroid known to activate INa , increased peak INa with slowed current inactivation; however, further application of NAL suppressed Tef-mediated suppression of peak INa followed by an additional slowing of current inactivation. In addition, NAL suppressed the amplitude of M-type K+ current [IK(M) ] with an IC50 value of 5.7 µM, while it slightly suppressed erg-mediated and delayed-rectifier K+ currents. In the inside-out current recordings, NAL failed to modify the activity of large-conductance Ca2+ -activated K+ channels. In differentiated NG108-15 neuronal cells, NAL also suppressed the peak INa , and subsequent addition of Tef reversed NAL-induced suppression of INa . Our study highlights the evidence that in addition to modulate opioid receptors, NAL has the propensity to interfere with ionic currents including INa and IK(M) , thereby influencing the functional activities of central neurons.

Rib soft fixation produces better analgesic effects and is associated with cytokine changes within the spinal cord in a rat rib fracture model.

Traumatic rib fracture can cause severe pain and is usually associated with the depression of respiratory drive followed by severe respiratory complications. It is critical for patients with rib fracture to receive adequate analgesia. However, strong opioids and other analgesics often produces side effects and may even cause respiratory suppression. Meanwhile, rib fixation now has become a popular method for treating rib fracture patients. However, the actual molecular mechanism leading to its effectiveness as an analgesia has not been fully investigated, and the best analgesic method for its use in rib fracture patients has not yet been determined. We developed a new animal model for rib fracture and evaluated changes in pain severity after rib fixation. Our data indicated significantly better analgesic behavior if a soft string rib fixation is performed, which is associated with cytokine (interleukine-6 and interleukine-10) decreases in the spinal cord and co-localization with glia cells. Our results provided a treatment suggestion for rib fracture patients and the possible molecular mechanism for the analgesic effects. Further molecular mechanisms and the best therapeutic methods are still needed for this severe painful condition.

Differential suppression of delayed-rectifier and inwardly rectifier K+ currents by a group of ent-kaurane-type diterpenoids from Croton tonkinensis, in microglial cells.

Croton is an extensive flowering plant genus in the spurge family, Euphorbiaceae. Three croton compounds with the common ent-kaurane skeleton were purified from Croton tonkinensis. By using patch-clamp recording technique, we thoroughly examined the effect of a group of croton compounds, croton-01 (ent-18-acetoxy-7α-hydroxykaur-16-en-15-one), croton-02 (ent-7α,14ß-dihydroxykaur-16-en-15-one), and croton-03 (ent-1ß-acetoxy-7α,14ß-dihydroxykaur-16-en-15-one), on the membrane current in SM826 and BV2 microglial cells. Although neither voltage-gated Na+ nor Ca2+ currents were present in these cells, both delayed-rectifier K+ outward (IK(DR)) and inwardly rectifying K+ currents (IK(IR)) were readily detected. Croton-03 differentially caused inhibition of IK(DR) or IK(IR) in a concentration-dependent manner. According to a minimal scheme, the shortening of the time constant in either the IK(DR)-related block or IK(IR) caused by different concentrations of croton-03 was quantitatively estimated with a dissociation constant of 6.45 and 29.5 µM, respectively. In SM826 cells differentiated with ß-amyloid, inhibitory action on these K+ currents remained unaltered. In ultraviolet C-irradiated cells, the magnitude of IK(IR) was still decreased by addition of croton-03. Therefore, our study suggests that these ent-kaurane diterpenoids ought to somehow act on the cellular mechanisms by which they influence the functional activities of microglial cells.

Concerted suppression of Ih and activation of IK(M) by ivabradine, an HCN-channel inhibitor, in pituitary cells and hippocampal neurons.

Ivabradine (IVA), a heart-rate reducing agent, is recognized as an inhibitor of hyperpolarization-activated cation current (Ih) and also reported to ameliorate inflammatory or neuropathic pain. However, to what extent this agent can perturb another types of membrane ion currents in neurons or endocrine cells remains to be largely unknown. Therefore, the Ih or other types of ionic currents in pituitary tumor (GH3) cells and in hippocampal mHippoE-14 neurons was studied with or without the presence of IVA or other related compounds. The IVA addition caused a time- and concentration-dependent reduction in the amplitude of Ih with an IC50 value of 0.64 µM and a KD value of 0.68 µM. IVA (0.3 µM) shifted the Ih activation curve to a more negative potential by approximately 8 mV, despite no concomitant change in the gating charge. Additionally, IVA was found to increase M-type K+ current (IK(M)) together with a rightward shift in the activation curve. In cell-attached current recordings, IVA (3 µM) applied to the bath increased the open probability of M-type K+ channels; however, it did not modify single-channel conductance of the channel. In current-clamp voltage recordings, IVA suppressed the firing of spontaneous action potentials in GH3 cells; and, further addition of linopirdine attenuated its suppression of firing. In hippocampal mHippoE-14 neurons, IVA also effectively increased IK(M) amplitude. In summary, both inhibition of Ih and activation of IK(M) caused by IVA can synergistically combine to influence electrical behaviors in different types of electrically excitable cells occurring in vivo.

Parecoxib, a selective blocker of cyclooxygenase-2, directly inhibits neuronal delayed-rectifier K+ current, M-type K+ current and Na+ current.

Parecoxib, a prodrug of valdecoxib, is a selective inhibitor of cyclooxygenase-2 and widely used for traumatic and postoperative patients to avoid opioid-induced side effects. It is a potent analgesic and has a role in multimodal analgesic and enhanced recovery after surgery. Whether parecoxib exerts any actions on these types of ionic currents remains unclear. In this study, we investigated whether it exerts any effects on ion currents in differentiated NG108-15 neuronal cells. Cell exposure to parecoxib (1-30 µM) caused a reversible reduction in the amplitude of IK(DR) with an IC50 value of 9.7 µM. The time course for the IK(DR) inactivation in response to a long-lasting pulse was changed to the biexponential process during cell exposure to 3 µM parecoxib. Other agents known to inhibit the cyclooxygenase activity have minimal effects on IK(DR). Parecoxib enhanced the degree of excessive accumulative inhibition of IK(DR) inactivation evoked by a train of brief repetitive stimuli. This compound suppressed the amplitude of M-type K+ current. It depressed the peak amplitude of voltage-gated Na+ current with no change in the current-voltage relationship of this current. However, it did not have any effect on hyperpolarization-activated cation current. No change in the expression level of KV3.1 mRNA was detected in the presence of parecoxib. The effects of parecoxib on ion currents are direct and unrelated to its inhibition of the enzymatic activity of cyclooxygenase-2. The inhibition of these ion channels by parecoxib may partly contribute to the underlying mechanisms by which it affects neuronal function in vivo.

Magnetic field distribution modulation of intrathecal delivered ketorolac iron-oxide nanoparticle conjugates produce excellent analgesia for chronic inflammatory pain.

BACKGROUND: Nanoparticles have become one of the most promising among the potential materials used for biomedical applications. However, few researchers have focused on their effects on analgesia. Despite the fact that various nanoparticles have been evaluated for drug delivery and MRI imaging contrast enhancement in clinical settings, no reports have investigated the in vivo synergy of ketorolac iron-oxide nanoparticle conjugates to improve the analgesic effect. METHODS: Ketorolac conjugated magnetic iron oxide nanoparticles (Keto-SPIO) were synthesized via two-stage additions of protective agents and chemical co-precipitation. ICR mice were used to develop inflammatory pain models induced by Complete Freund's adjuvant (CFA) injection in the hind paw. Different magnet field strengths and polarities were applied to the spinal cord after injecting Keto-SPIO into the theca space. Analgesia behavior was evaluated with the up-down method via von Frey microfilament measurement. Spinal cord tissues were harvested at the end analgesia time point upon induction of the inflammatory pain. The presence of the two cyclooxygenases (COX) in the spinal cord was examined via Western blotting to quantify the changes after intra-thecal Keto-SPIO administration. RESULTS: Intrathecal Keto-SPIO administration demonstrated a magnetic field-dependent analgesia effect in CFA pain model with a significant reduction in COX expression. CONCLUSIONS: Our results indicated that intrathecal administration of the Keto-SPIO combined magnet field modulated delivery significantly promoted an analgesia effect with suppression of COX in the mice inflammatory pain model.

Huntington Mice Demonstrate Diminished Pain Response in Inflammatory Pain Model.

BACKGROUND: Huntington disease (HD) affects the nervous system and leads to mental and motor dysfunction. Previous studies have shown that HD is caused by the exon 1 region of the huntingtin (HTT) gene having expanded CAG trinucleotide repeats. However, few studies have focused on the relationship between HD and pain. The purpose of this study is to investigate the relationship between HD and pain response. METHODS: We used clinical similar transgenic HD mice carrying a mutant HTT exon 1 containing 84 CAG trinucleotide repeats to evaluate the relationship between HD and pain. Inflammatory pain models were induced by either formalin or complete Freund adjuvant injection over the hind paw. Spinal cord, dorsal root ganglion, and paw skin tissues were harvested at the end of the behavioral inflammatory pain studies. Immunofluorescence assay, Western blotting, and enzyme-linked immunosorbent assay were used to identify changes in cells and cytokines. RESULTS: Our data demonstrate that preonset HD mice exhibited less pain behavior than wild-type (WT) mice in both young (n = 11 [WT], 13 [HD]) and aged (n = 8 [WT], 9 [HD]) mice. Western blotting and immunohistological examination of lumbar spinal cord tissue and dorsal root ganglion indicate less activation of glial cells and astrocytes in young HD mice (n = 6-7) compared to that in WT mice (n = 6-7). The production levels of tumor necrosis factor-α, interleukin-1ß, and substance P were also lower in young HD mice (n = 6-7). CONCLUSIONS: Our data demonstrate less pain behavior and pain-related cytokine response at the spinal cord level for HD mice compared to those for WT mice. Further studies are needed for determining the mechanism as to how mutant HTT leads to altered pain behavior and pain-related cytokine response.

The analgesia efficiency of ultrasmall magnetic iron oxide nanoparticles in mice chronic inflammatory pain model.

Few studies have investigated the effects of iron oxide nanoparticles (NPs) on analgesia. We developed inflammatory pain models via complete Freund's adjuvant injection over the hind paw in CD1 mice. Various doses of magnetite (Fe3O4) NPs were injected into the paw. Analgesia behavior was checked with von Frey microfilament and thermal irradiation measurements. Paw skin tissues were harvested at the maximal analgesia time point. The presence of activated white cells (CD68, myeloperoxidase) and free radical (reactive oxygen species, ROS) production was also checked. Western blotting was used to identify the changes of ROS production enzymes. Fe3O4 NPs demonstrated a dose-related analgesia effect with significant reduction in inflammatory cells, pro-inflammatory markers, and ROS production in the lesion paw. ROS production enzyme expression also declined. The results indicate that local Fe3O4 NP administration induced significant analgesia via attenuation of inflammatory cell infiltration and pro-inflammatory signaling as well as scavenging of microenvironment free radicals in a mouse inflammatory pain model.

Transcutaneous electrical nerve stimulator of 5000 Hz frequency provides better analgesia than that of 100 Hz frequency in mice muscle pain model.

Transcutaneous electrical nerve stimulators (TENSs) have been proved to be effective in muscle pain management for several decades. However, there is no consensus for the optimal TENS program. Previous research demonstrated that a 100 Hz TENS (L-TENS) provided better analgesia than a conventional TENS (< 5 Hz). However, no research compared a higher-frequency (> 100 Hz) TENS with a 100 Hz TENS. We used a 5000 Hz (5 kHz) frequency TENS (M-TENS) and an L-TENS to compare analgesic effect on a mice skin/muscle incision retraction model. Three groups of mice were used (sham, L-TENS, and M-TENS) and applied with different TENS programs on Day 4 after the mice skin/muscle incision retraction model; TENS therapy was continued as 20 min/d for 3 days. Mice analgesic effects were measured via Von Frey microfilaments with the up-down method. After therapy, mice spinal cord dorsal horn and dorsal root ganglion (DRG) were harvested for cytokine evaluation (tumor necrosis factor-α and interleukin-1ß) with the Western blotting method. Our data demonstrated that the M-TENS produced better analgesia than the L-TENS. Cytokine in the spinal cord or DRG all expressed lower than that of the sham group. However, there is no difference in both cytokine levels between TENSs of different frequencies in the spinal cord and DRG. We concluded that the M-TENS produced faster and better mechanical analgesia than the L-TENS in the mice skin/muscle incision retraction model. Those behavior differences were not in accordance with cytokine changes in the spinal cord or DRG.

The effect of propofol and sevoflurane on antioxidants and proinflammatory cytokines in a porcine ischemia-reperfusion model.

OBJECTIVES: Ischemia-reperfusion (IR) features massive oxidative stress of tissues and cytokine response. Propofol and sevoflurane, both of which are commonly used anesthetics, are thought to have different antioxidant activities. The aim of this study is to delineate the influence of these two drugs on the production of free radicals and proinflammatory cytokines in IR conditions via in vitro and in vivo models. METHODS: An in vitro IR model was performed by incubating porcine cells (including mononuclear cells, and coronary and aortic smooth muscle cells) with either propofol 25 µM or sevoflurane 2% in the hypoxia chamber (1% O2, 37°C) for 1 hour, followed by room temperature air for 2 hours. Reactive oxygen species (ROS) and tumor necrosis factor-α (TNF-α) were also measured via flow cytometry and enzyme-linked immunosorbent assay methods, respectively. Ten pigs were used for the in vivo study. After anesthesia with either propofol (10-15 mg/kg/h) or sevoflurane (2%), internal carotid and femoral arterial catheters were inserted for direct blood pressure monitoring and blood sampling. The IR models were produced via descending thoracic aorta clamping for 1 hour and declamping for 2 hours during the procedure for left ventricular assist device implantation. Blood serum was sampled from upper and lower body vessels for ROS and TNF-α evaluation via thiobarbituric acid reacting substances method and enzyme-linked immunosorbent assay, respectively. RESULTS: The results showed significant reduction of both ROS and TNF-α levels in the propofol group in vitro IR model. However, there was no difference in lipid peroxidation and TNF-α level between propofol and sevoflurane for the in vivo IR model. CONCLUSION: We concluded that propofol, compared with sevoflurane, can significantly inhibit ROS formation on a cell level. In addition, propofol can significantly inhibit TNF-α formation of monocytes and coronary smooth muscle cells but not aortic smooth muscle cells.

Hypoxia inducible factor-1α inhibition produced anti-allodynia effect and suppressed inflammatory cytokine production in early stage of mouse complex regional pain syndrome model.

Complex regional pain syndrome (CRPS) is related to microcirculation impairment associated with tissue hypoxia and peripheral cytokine overproduction in the affected limb. Previous studies suggest that the pathogenesis involves hypoxia inducible factor-1α (HIF-1α) and exaggerated regional inflammatory response. 1-methylpropyl 2-imidazolyl disulfide (PX-12) acts as the thioredoxin-1 (Trx-1) inhibitor and decreases the level of HIF-1α, and can rapidly be metabolized for Trx-1 redox inactivation. This study hypothesized that PX-12 can decrease the cytokine production for nociceptive sensitization in the hypoxia-induced pain model. CD1 mice weighing around 30 g were used. The animal CRPS model was developed via the chronic post-ischaemic pain (CPIP) model. The model was induced by using O-rings on the ankles of the mice hind limbs to produce 3-h ischaemia-reperfusion injury on the paw. PX-12 (25 mg/kg, 5 mg/kg) was given through tail vein injection immediately after ischaemia. Animal behaviour was tested using the von Frey method for 7 days. Local paw skin tissue was harvest from three groups (control, 5 mg/kg, 25 mg/kg) 2 h after injection of PX-12. The protein expression of interleukin-1ß (IL-1ß) and HIF-1α was analysed with the Western blotting method. Mice significantly present an anti-allodynia effect in a dose-related manner after the PX-12 administration. Furthermore, PX-12 not only decreased the expression of HIF-1α but also decreased the expression of IL-1ß over the injured palm. This study, therefore, shows the first evidence of the anti-allodynia effect of PX-12 in a CPIP animal model for pain behaviour. The study concluded that inhibition of HIF-1α may produce an analgesic effect and the associated suppression of inflammatory cytokine IL-1ß in a CPIP model.

Visualizing the antivascular effect of bortezomib on the hypoxic tumor microenvironment.

Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib. It was shown in vitro by Western blot, flow cytometry, and ELISA that bortezomib accumulated HIF-1α in non-functional forms and blocks its hypoxia response in human colorectal cancer cell lines. Ex vivo experiments were performed with fluorescent immunohistochemical staining techniques using multiple endogenous and exogenous markers to identify hypoxia (pimonidazole, HRE-TKeGFP), blood flow/permeability (Hoechst 33342), micro-vessels (CD31 and SMA), apoptosis (cleaved caspase 3) and hypoxia response (CA9). After bortezomib administration, overall apoptosis index was significantly increased and blood perfusion was dramatically decreased in tumor xenografts. More importantly, apoptosis signals were found preferentially located in moderate and severe pretreatment hypoxic regions in both tumor and endothelial cells. Meanwhile, DCE MRI examinations showed that the tumor blood flow and permeability decreased significantly after bortezomib administration. The present study revealed that bortezomib reduces tumor hypoxia response and blood perfusion, thus, presenting antivascular properties. It will be important to determine the hypoxic/perfusion status pre- and during treatment at further translational studies.

Hypoxia-targeted triple suicide gene therapy radiosensitizes human colorectal cancer cells.

The hypoxic microenvironment, an important feature of human solid tumors but absent in normal tissue, may provide an opportunity for cancer-specific gene therapy. The purpose of the present study was to investigate whether hypoxia-driven triple suicide gene TK/CD/UPRT expression enhances cytotoxicity to ganciclovir (GCV) and 5-fluorocytosine (5-FC), and sensitizes human colorectal cancer to radiation in vitro and in vivo. Stable transfectant of human colorectal HCT8 cells was established which expressed hypoxia-inducible vectors (HRE-TK/eGFP and HRE-CD/UPRT/mDsRed). Hypoxia-induced expression/function of TK, CD and UPRT was verified by western blot analysis, flow cytometry, fluorescent microscopy and cytotoxicity assay of GCV and 5-FC. Significant radiosensitization effects were detected after 5-FC and GCV treatments under hypoxic conditions. In the tumor xenografts, the distribution of TK/eGFP and CD/UPRT/mDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC and GCV in mice in combination with local irradiation resulted in tumor regression, as compared with prodrug or radiation treatments alone. Our data suggest that the hypoxia-inducible TK/GCV+CDUPRT/5-FC triple suicide gene therapy may have the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo.

PURPOSE: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. MATERIALS AND METHODS: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. RESULTS: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. CONCLUSIONS: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.