EphrinB-EphB signaling regulates spinal pain processing via PKCγ.

Spinal ephrinB-EphB signaling is involved in the modulation of pain processing. The aim of the present study was to investigate whether protein kinase C-γ (PKCγ) acts as a downstream effector in regulating spinal pain processing associated with ephrinB-EphB signaling in mice. The intrathecal injection of ephrinB2-Fc, an EphB receptor activator, caused thermal hyperalgesia and mechanical allodynia, as well as increased activation of spinal PKCγ. Knockdown of spinal PKCγ prevented the pain behaviors induced by ephrinB2-Fc. Furthermore, the intrathecal injection of EphB2-Fc, an EphB receptor blocker, suppressed formalin-induced inflammatory, chronic constriction injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, in addition to reducing the activation of spinal PKCγ. Finally, the intrathecal injection of MK801, an N-methyl-D-aspartate (NMDA) receptor blocker, prevented the pain behaviors and spinal PKCγ activation induced by ephrinB2-Fc. Overall, the results confirm the important role of PKCγ in the regulation of spinal pain processing associated with ephrinB-EphB signaling.

COX-2 is required for the modulation of spinal nociceptive information related to ephrinB/EphB signalling.

BACKGROUND: EphB receptors and their ephrinB ligands are implicated in modulating spinal nociceptive information processing. Here, we investigated whether cyclooxygenase-2 (COX-2), acts as a downstream effector, participates in the modulation of spinal nociceptive information related to ephrinB/EphB signalling. METHODS: Thermal hyperalgesia and mechanical allodynia were measured by using radiant heat and von Frey filaments test, respectively. Real-time PCR (RT-PCR) was used to detect the expression of spinal COX-2 mRNA. Spinal COX-2 and extracellular signal-regulated kinase (ERK) protein were determined by Western blot analysis. RESULTS: Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal COX-2 mRNA and protein. Inhibition of spinal COX-2 prevented and reversed pain behaviours induced by the intrathecal injection of ephrinB2-Fc. Blockade of EphB receptors by intrathecal injection of EphB2-Fc reduced complete Freund's adjuvant (CFA)-induced inflammatory pain behaviours, which were accompanied by decreased expression of spinal COX-2 mRNA and protein. Furthermore, treatment with U0126, a mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor, suppressed spinal ERK activation and COX-2 mRNA and protein expression induced by intrathecal injection of ephrinB1-Fc. CONCLUSIONS: These results confirmed the important involvement of COX-2 in the modulation of spinal nociceptive information related to ephrinBs-EphBs signalling.

PKA is required for the modulation of spinal nociceptive information related to ephrinB-EphB signaling in mice.

EphB receptors and their ephrinB ligands are implicated in modulating of spinal nociceptive information processing. Here, we investigated whether protein kinase A (PKA), acts as a downstream effector, participates in the modulation spinal nociceptive information related to ephrinB-EphB signaling. Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal PKA catalytic subunit (PKAca) and phosphorylated cAMP-response element-binding protein (p-CREB). Pre-treatment with H89, a PKA inhibitor, prevented the activation of CREB by ephrinB2-Fc. Inhibition of spinal PKA signaling prevented and reversed pain behaviors induced by the intrathecal injection of ephrinB2-Fc. Furthermore, blockade of the EphB receptors by intrathecal injection of EphB2-Fc reduced formalin-induced inflammatory, chronic constrictive injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, which were accompanied by decreased expression of spinal PKAca and p-CREB. Overall, these results confirmed the important involvement of PKA in the modulation of spinal nociceptive information related to ephrinBs-EphBs signaling. This finding may have important implications for exploring the roles and mechanisms of ephrinB-EphB signaling in physiologic and pathologic pain.

Sevoflurane promotes the expansion of glioma stem cells through activation of hypoxia-inducible factors in vitro.

BACKGROUND: Growing evidences indicate that inhalational anaesthetics can enhance the growth and malignant potential of tumour cells and may affect tumour recurrence after surgery. Tumour stem cells play a vital role in tumour recurrence. This study investigates the effect of sevoflurane on glioma stem cells (GSCs) in vitro and the underlying molecular mechanisms in this process. METHODS: Cultured GSCs were exposed to clinically relevant concentrations and durations of sevoflurane exposure. Cell proliferation and self-renewal capacity were determined. Expression of the stem cell marker CD133, vascular endothelial growth factor (VEGF), hypoxia-inducible factors (HIFs), and phosphorylated Akt, which is a protein kinase invoved in multiple cellular processes, were measured using western blotting. Small interfering RNAs and an Akt inhibitor were used to investigate specific pathways. RESULTS: Compared with controls, cells exposed to 2% sevoflurane for 6 h induced a larger number of proliferated cells (31.2±7.6% vs 19.0±5.8%; P<0.01). Levels of CD133, VEGF, HIF-1α, HIF-2α, and p-Akt were up-regulated by sevoflurane in a time- and concentration-dependent manner. Small interfering RNA against HIFs decreased the percentage of proliferating GSCs after sevoflurane exposure and pre-treatment of cells with an Akt inhibitor abrogated the expression of HIFs induced by sevoflurane. CONCLUSIONS: Sevoflurane can promote the expansion of human GSCs through HIFs in vitro. Inhaled anaesthetics may enhance tumour growth through tumour stem cells.

Spinal ephrinB/EphB signalling contributed to remifentanil-induced hyperalgesia via NMDA receptor.

BACKGROUND: One of the major unresolved issues in treating pain is the paradoxical hyperalgesia produced by opiates, and accumulating evidence implicate that EphBs receptors and ephrinBs ligands are involved in mediation of spinal nociceptive information and central sensitization, but the manner in which ephrinB/EphB signalling acts on spinal nociceptive information networks to produce hyperalgesia remains enigmatic. The objective of this research was to investigate the role of ephrinB/EphB signalling in remifentanil-induced hyperalgesia (RIH) and its downstream effector. METHODS: We characterized the remifentanil-induced pain behaviours by evaluating thermal hyperalgesia and mechanical allodynia in a rat hind paw incisional model. Protein expression of EphB1 receptor and ephrinB1 ligand in spinal dorsal horn cord was determined by Western blotting, and Fos was determined by immunohistochemistry assay, respectively. To figure out the manner in which ephrinB/EphB signalling acts with N-methyl-d-aspartic acid (NMDA) receptor, we used MK-801, an antagonist of NMDA receptor, trying to suppressed the hyperalgesia induced by ephrinB1-Fc, an agonist of ephrinB/EphB. RESULTS: Continuing infusion of remifentanil produced a thermal hyperalgesia and mechanical allodynia, which was accompanied with increased protein expression of spinal-level EphB1 receptor, ephrinB1 ligand and Fos; what appeared above was suppressed by pretreatment with EphB1-Fc, an antagonist of ephrinB/EphB or MK-801, and increased pain behaviours induced by intrathecal injection of ephrinB1-Fc, an agonist of ephrinB/EphB, were suppressed by MK-801. CONCLUSIONS: Our findings indicated that ephrinB/EphB signalling is involved in RIH. EphrinB/EphB signalling might be the upstream of NMDA receptor.

Cardioprotective effects of cyclosporine A in an in vivo model of myocardial ischemia and reperfusion.

BACKGROUND: Recent evidence indicates that reperfusion of the heart after a period of ischemia leads to the opening of the mitochondrial permeability transition pore (MPTP). The aim of this study was to investigate cardioprotective effects of cyclosporine A (CsA), an inhibitor of the MPTP, in an in vivo model of myocardial ischemia and reperfusion. METHODS: Male Sprague-Dawley rats were subjected to occlusion of the left anterior descending coronary artery for 30 min followed by 180 min of reperfusion. CsA (10 mg/kg) or vehicle was given 10 min prior to ischemia via the femoral vein. Sham myocardial ischemia-reperfusion rats (sham-operation group) were used as controls. Infarct size was measured using the staining agent TTC (2,3,5-triphenyl tetrazolium chloride) and myocardial apoptosis by caspase-3 activity was determined by fluorescent assay. The myocardium mitochondria ultrastructure was observed through a transmission electron microscope. RESULTS: CsA significantly reduced infarct size (48.8 +/- 5.8% of left ventricle in vehicle + I/R group and 30.3 +/- 2.7% of left ventricle in CsA + I/R, respectively) and decreased caspase-3 activity in the myocardium [(0.62 +/- 0.17)/microg of protein and (0.42 +/- 0.15)/microg of protein, respectively] and relieved the injury of mitochondria. CONCLUSION: CsA reduced the cardiac damage associated with ischemia-reperfusion injury of the heart. The cardioprotective effects of CsA might be associated with the protection of mitochondria and the inhibition of caspase-3 activity. It also suggests that the MPTP might play an important role in cardiomyocytes death after ischemia-reperfusion injury.

[HPCE determination of trimebutine maleate in rat plasma and its pharmacokinetics].

AIM: To develop a method for the determination of trimebutine maleate in rat plasma by using high performance capillary electrophoresis. The method was employed to pharmacokinetic analysis of trimebutine maleate. METHODS: Plasma samples were deproteinized with acetonitrile (containing ephedrine hydrochloride as internal standard) and the supernatant was dried under N2 stream at 50 degrees C. The residue was dissolved with methanol-water (1:1) and injected into the capillary by siphon. The electrophoresis was performed in uncoated fused-silica capillary and the voltage was 10 kV. The running buffer was 0.03 mol.L-1 NaH2PO4(pH 6.0). The eluate was detected at 214 nm by UV detection. RESULTS: The recovery for trimebutine maleate in rat plasma was 72.8%-87.9%. The calibration curve in plasma was linear over the range 5-200 micrograms.L-1. The limit of quantitation was 5 micrograms.L-1. The intraday relative standard deviation (n = 6) and the interday relative standard deviation (n = 18) were less than 14%. The highest concentration in plasma was observed at 30 min after ig trimebutine maleate to rats. The pharmacokinetic results were AUC0-infinity = 8 micrograms.min.mL-1, T1/2(Ke) = 173 min and Ke = 5.6 x 10(-3) min-1. CONCLUSION: The method is accurate, sensitive and suitable for pharmacokinetic study of trimebutine maleate.

[3D geometric simulation of mandible with dental arch from CT data].

OBJECTIVE: A 3D geometric simulation of mandible with dental arch from CT has been obtained in this study. METHODS: This process uses automatic system assisted with interactive action to get 2D contour data from CT images,then 3D wireframe model and solid model were obtained by using CAD/CAM software Pro-E(USA) and DELCAM(UK). RESULTS: 3D solid model of mandible with dental arch were presented which can be fully edited. CONCLUSION: This model can be applied to further educational and clinical researches such as RP,biomechanics simulation in prosthetic dentistry.The processes of simulation has wide applications in clinical practice of dentistry and dental education.

[3D solid model of mandible with dental arch via LOM method].

OBJECTIVE: Based on 3D reconstruction data from CT scanning, a solid model of mandible with dental arch is obtained via a rapid prototype machine using LOM method. METHODS: 3D reconstruction data is transferred to STL file using software of Delcam(UK),which will be fed to Magics RP software for detection and rebuilding. Reproduction of the papery model of mandible with dental arch is then performed with highly geometric similarity. RESULTS: The RP model of mandible with dental arch is obtained. CONCLUSION: Accuracy of the reproduction model meets the demands of students in prosthetic dentistry,which gives the possibility of computer aided design of prosthetic dentistry based on 3D solid model.

Enhanced eosinophil luminol-dependent chemiluminescence and complement receptor expression by platelet-activating factor and interleukin-5.

The cytokine interleukin-5 (IL-5) and the lipid mediator platelet-activating factor (PAF) have both been shown to be involved in eosinophil differentiation and activation. We have measured and compared the effect of PAF and IL-5 on human eosinophils in terms of their luminol-dependent chemiluminescence (CL) response and their expression of complement receptors, CR1 and CR3. Both IL-5 and PAF enhanced the eosinophil CL response. The optimal concentrations were 40 U/ml for IL-5, and 10(-6) M for PAF. The priming effect of IL-5 was slow and reached a maximal response after 90 minutes incubation. In contrast, the effect of PAF peaked early and declined during incubation. In the complement receptor study, only PAF was able to enhance CR3 expression (p < 0.05) while the effect of IL-5 on eosinophil complement receptor expression was negligible. These results provide evidence that both inflammatory mediator (PAF) and cytokine (IL-5) can activate eosinophils but the effects of IL-5 and PAF on eosinophil CL response appear to be distinct. The activation of eosinophils by PAF and IL-5 may occur through different mechanisms.

The identification of an eosinophil differentiation factor in culture supernatant of mononuclear cells from asthmatics.

Although peripheral eosinophilia is a common feature of bronchial asthma, the precise mechanism of its production is still unknown. It has recently been reported that murine interleukin-5 (IL-5) can cross-react with human cells and selectively stimulates the proliferation and differentiation of eosinophils. This study identified the IL-5-like activity in culture supernatants of peripheral blood mononuclear cells (MNCs) from asthmatic patients. Murine recombinant IL-5 (rIL-5) was used as a positive control; the number of eosinophils was determined by both Wright's stain and eosinophil peroxidase measurement. Time course and dose response studies showed that rIL-5 at 40 U/ml induced a maximal eosinophil differentiation after a three week incubation with cord blood MNCs. Unstimulated MNC supernatants obtained from asthmatics possessed a higher eosinophil differentiation activity (OD490, 0.09 +/- 0.02, n = 23) than those obtained from the normals (0.03 +/- 0.01) (P < 0.02). This activity in unstimulated MNC supernatant can be neutralized by anti-IL-5 antibodies. Neither Bermuda grass- nor phytohemagglutinin-stimulated MNC supernatant showed a statistical significance between these two groups. The IL-5-like activity was associated with a protein of MW around 30kD as determined by Superose-12 PG gel filtration. In conclusion, MNC culture supernatants derived from asthmatics contained an eosinophil differentiation activity, which might be important for regulation of eosinophil generation and thus contribute to the asthma-related eosinophilia.

The effects of asthmatic mononuclear cell cultured supernatant on human eosinophils-chemiluminescence response, complement receptor expression and cellular density alteration.

We investigated the contributing effect of mononuclear cell (MNC) on granulocyte function in asthmatics. MNC, obtained from either normal subjects or asthmatics, were cultured with or without phytohaemagglutinin. The supernatants were tested on granulocyte luminol-dependent chemiluminescence (CL) response. The optimal incubation time was 30 min for neutrophils and 90 min for eosinophils. There was a dose-dependent enhancement of CL response by MNC supernatant on both neutrophils and eosinophils. When comparing the activities of MNC supernatants between asthmatics and normal, there was a significant enhancement of eosinophil CL response by MNC supernatants of asthmatics (70 +/- 24% from 29 +/- 28%, P = 0.0043). However, the degree of enhancement in neutrophil CL response was similar between the MNC supernatants of asthmatics and normal subjects. MNC supernatant not only enhanced eosinophil CL response, but also induced eosinophil hypodensity change (12 +/- 4% in RPMI, 26 +/- 10% in MNC supernatant), and enhanced complement receptor CR3 expression (102.4 +/- 25.5 in PRMI, 111.7 +/- 17.7 in MNC supernatant). These results provide evidence that MNC activation might contribute to the eosinophil activation in asthmatics.

The identification of PAF-release-enhancing activity in culture supernatant of mononuclear cells from asthmatics. Correlation between lymphocyte responses and immediate type hypersensitivity.

Cytokines from asthmatic mononuclear cells (MNC) cultured with Bermuda Grass pollen extract (BG), were tested for their effect on leukotriene B4 (LTB4) and platelet-activating factor (PAF) generation by neutrophil under calcium ionophore stimulation. Three groups were enrolled in this study: 9 allergic asthmatics showing skin intradermal test positive to BG, 12 non-allergic asthmatics and 5 normal individuals. Results showed that the stimulation index of BG on MNC and the mediator-release enhancing activity from both allergic and non-allergic asthmatics were similar; neither showed significant increase when compared to that of normal individuals. However, there was a significant increase in PAF-release-enhancing activity in unstimulated MNC supernatant from both allergic and non-allergic asthmatics (618 +/- 75 pg/10(6) neutrophils and 569 +/- 60 pg/10(6) neutrophils) when compared to that of normal individuals (460 +/- 40 pg/10(6) neutrophils). This PAF-release-enhancing activity did not correlate with either skin test reactivity or the amount of BG specific IgE antibodies. The PAF-release-enhancing activity in unstimulated MNC supernatants from asthmatics suggesting that there is a pre-activation of MNC which might contribute to the asthmatic reaction through generation of PAF by neutrophils.